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Effects of Plant Parasitic Nematodes and Nitrogen Fertility Management on Hybrid Bermudagrass


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EFFECTS OF PLANT PARASITIC NEMATODES AND NITROGEN FERTILITY MANAGEMENT ON HYBRID BERMUDAGRASS By JOHN ERIC LUC 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 2004

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Copyright 2004 by John Eric Luc

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iii ACKNOWLEDGMENTS I would like to thank my chairman and committee members, William T. Crow, Jerry B. Sartain, Jerry L. Stimac, and Robin M. Giblin-Davis, for their guidance and patience during my pursuit of a Master of Science degree. I have gained a great deal of knowledge from each of them during my undergraduate and graduate education. Their dedication to the advancement and excellence of graduate education is unyielding. I hope to have the same patience and dedication that they have exhibited during all my endeavors. Also I would like to acknowledge Augustus Porter Alden, my grandfather, who never wavered in his belief of me, even when I did not believe in myself. On July 27, 1998, he left our world and continued his journey. His passing was the catalyst for my return to academics. I dedicate this degree to him with all my heart. John T. and Lynda A. Luc, my parents, deserve my sincere appreciation. Their guidance, patience, love, and support have carried me a long way. Without it I would have never reached this point in my life. However, my journey has not concluded. I will have to draw on them again, as I continue my education and pursuit of a doctor of philosophy degree. I would also like to thank, Joseph C. Parker “Buck,” Amy Parker, and Joseph C. Parker II, my best friends and their son, for the emotional and financial support they have shown. I have known Buck for 22 years; during that time our friendship has never been broken. I wish him and his family all the love and happiness in the world. To the staff of the nematode assay lab and particularly Matthew R. Coon, this degree is as much theirs as it is mine. Without their dedication and hard work this project would not have been successful.

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iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS..............................................................................................................ii i LIST OF TABLES................................................................................................................. ........vii LIST OF FIGURES................................................................................................................ ........ix ABSTRACT....................................................................................................................... .............xi CHAPTER 1 INTRODUCTION............................................................................................................ .......1 2 LITERATURE REVIEW........................................................................................................4 Belonolaimus longicaudatus ....................................................................................................4 Taxonomy....................................................................................................................... .4 Morphology and Anatomy...............................................................................................5 Biology and Distribution..................................................................................................6 Soil Fertility................................................................................................................. ............8 History........................................................................................................................ ......8 Nitrogen Fertilizers..........................................................................................................9 Nitrogen Use in the Soil and Turfgrass System.....................................................................11 Mineralization................................................................................................................1 1 Leaching....................................................................................................................... ..13 Erosion and Run off.......................................................................................................14 Plant Uptake................................................................................................................... 15 Adsorption..................................................................................................................... .16 Immobilization...............................................................................................................16 Volatilization................................................................................................................. .16 Denitrification................................................................................................................ 17 Turfgrass Cultivars............................................................................................................ .....18 ‘Tifdwarf’ Bermudagrass...............................................................................................18 ‘Tifway 419’ Bermudagrass...........................................................................................18 Root Systems................................................................................................................... .......19 History........................................................................................................................ ....19 Root Development and Nutrients...................................................................................19 3 INFLUENCE OF PLANT-PARASITIC NEMATODES ON NITRATE LEACHING IN TURF........................................................................................................................... ..........21 Introduction................................................................................................................... .........21 Materials and Methods.......................................................................................................... .22

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v Trial 1........................................................................................................................ .............22 Establishment of experimental units..............................................................................22 Nematode inoculum.......................................................................................................23 Turf maintenance............................................................................................................23 Evaluation and sampling techniques..............................................................................23 Trial 2........................................................................................................................ .............25 Establishment of experimental units..............................................................................25 Nematode inoculum.......................................................................................................25 Turf maintenance............................................................................................................26 Evaluation and sampling techniques..............................................................................26 Data Analysis.................................................................................................................2 6 Results........................................................................................................................ ............27 Discussion..................................................................................................................... .........27 4 EFFECT OF NEMATODE MANAGEMENT AND NITROGEN FERTILITY ON FAIRWAY TURF QUALITY...............................................................................................36 Introduction................................................................................................................... .........36 Materials and Methods.......................................................................................................... .37 Experimental Sites..........................................................................................................37 Pathogens...................................................................................................................... .37 Insects and weeds...........................................................................................................38 Turf........................................................................................................................... ......38 Soil properties................................................................................................................ 38 Experimental Design............................................................................................................ ..38 Nematicide Treatments..................................................................................................39 Fertilization.................................................................................................................. ..40 General Production Practices.................................................................................................40 Turf Maintenance...........................................................................................................40 Pesticides..................................................................................................................... ...41 Sampling and Evaluations......................................................................................................4 1 Turf Evaluations.............................................................................................................41 Nematodes...................................................................................................................... 41 Roots.......................................................................................................................... ....42 Turf Tissue.................................................................................................................... .42 Data Analysis.................................................................................................................. .......42 Results........................................................................................................................ ............43 5 SUMMARY...................................................................................................................... .....51 APPENDIX A DETAILED MATERIALS AND METHODS USED IN THE GREENHOUSE STUDY...54 Introduction................................................................................................................... .........54 Experimental Materials......................................................................................................... .54 Lysimeters..................................................................................................................... .54 Turf........................................................................................................................... ......54 Soil Properties................................................................................................................ 56 Nematode Inoculum.......................................................................................................56 Experimental Design............................................................................................................ ..57

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vi General Production Practices.................................................................................................58 Turf Establishment.........................................................................................................58 Nematode Establishment................................................................................................58 Temperature...................................................................................................................5 9 Watering....................................................................................................................... ..59 Fertilization.................................................................................................................. ..59 Pesticides..................................................................................................................... ...60 Sampling and Evaluations......................................................................................................6 0 Turf Evaluations.............................................................................................................60 Nematodes and Roots.....................................................................................................60 Leachate....................................................................................................................... ..62 Turf Tissue.................................................................................................................... .63 Data Analysis.................................................................................................................6 3 B DETAILED MATERIALS AND METHODS USED IN THE FIELD STUDY..................66 Introduction................................................................................................................... .........66 Experimental Sites............................................................................................................. ....66 Pathogens...................................................................................................................... .66 Insects and weeds...........................................................................................................67 Turf........................................................................................................................... ......67 Soil Properties................................................................................................................ 67 Experimental Design............................................................................................................ ..67 Nematicide Treatments..................................................................................................68 Fertilization.................................................................................................................. ..71 General Production Practices.................................................................................................71 Turf Maintenance...........................................................................................................71 Pesticides..................................................................................................................... ...72 Sampling and Evaluations......................................................................................................7 2 Turf Evaluations.............................................................................................................72 Nematodes...................................................................................................................... 72 Roots.......................................................................................................................... ....73 Turf Tissue.................................................................................................................... .73 Data Analysis.................................................................................................................. .......73 C SUPPLEMENTAL FIGURES AND TABLES.....................................................................74 LIST OF REFERENCES............................................................................................................. ..90 BIOGRAPHICAL SKETCH.........................................................................................................95

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vii LIST OF TABLES Table page 3-1. Effects of inoculating turf with Belonolaimus longicaudatus on ‘Tifdwarf’ bermudagrass root length, surface area, and dry weight at 6, 12, and 18 weeks after turf and nematode establishment during trial 1.................................................................................................30 3-2. Effects of inoculating turf with Belonolaimus longicaudatus on‘Tifdwarf’ bermudagrass root length, surface area, and dry weight at 6, 12, and 18 weeks after turf and nematode establishment during trial 2.................................................................................................31 3-3. Linear regression analysis conducted to determine relationships between nematode populations, root length, nitrogen uptake, and nitrate leached during both trials................32 4-1. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a ‘Tifway 419’ bermudagrass fairway at 0 to 16 weeks after treatment during trial 1................................................................................................................. .......45 4-2. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a ‘Tifway 419’ bermudagrass fairway at 0 to 16 weeks after treatment during trial 2................................................................................................................. .......46 4-3. Total root lengths observed in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a ‘Tifway 419’ bermudagrass fairway at 0, 6, and 16 weeks after treatment during both trials.........................................................................................47 C-1. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 6 weeks after turf and nematodes establishment during trial 1...........................................75 C-2. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 12 weeks after turf and nematodes establishment during trial 1.........................................76 C-3. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 18 weeks after turf and nematodes establishment during trial 1.........................................77 C-4. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 6 weeks after turf and nematodes establishment during trial 2...........................................78

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viii C-5. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 12 weeks after turf and nematodes establishment during trial 2.........................................79 C-6. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 18 weeks after turf and nematodes establishment during trial 2.........................................80 C-7. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 6 weeks after turf and nematodes establishment during trial 1..............................................81 C-8. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 12 weeks after turf and nematodes establishment during trial 1..............................................82 C-9. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 18 weeks after turf and nematodes establishment during trial 1..............................................83 C-10. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 6 weeks after turf and nematodes establishment during trial 2..............................................84 C-11. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 12 weeks after turf and nematodes establishment during trial 2..............................................85 C-12. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 18 weeks after turf and nematodes establishment during trial 2..............................................86 C-13. Root dry weights of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths for both trials.................................................................................................................... .........87 C-14. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month in untreated control, mechanical, and nematode management plots throughout trial 2............................................................................................................. ....88 C-15. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month in untreated control and nematode management plots through out trial 2........................................................................................................................ ............89

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ix LIST OF FIGURES Figure page 2-1. Nitrogen cycle............................................................................................................ .............12 3-1. Effects of inoculating turf with Belonolaimus longicaudatus on mg nitrate leached at 3, 6, 9, 12, 15, and 18 or 6, 12, and 18 weeks after turf and nematode establishment during trial 1 (A) and trial 2 (B) respectively. Inoculated plants received 138 (trial 1) and 300 20 B. longicaudatus (trial 2), while uninoculated plants received no nematodes. Error bars indicate standard error of individual population means......................................................28 3-2. Effects of inoculating turf with Belonolaimus longicaudatus on cumulative nitrate leached at 3, 6, 9, 12, 15, and 18 weeks after turf and nematode establishment during trial 1 (A) and trial 2 (B) respectively. Inoculated plants received 138 (trial 1) and 300 40 B. longicaudatus (trial 2), while uninoculated plants received no nematodes. Error bars indicate standard error of individual population means......................................................29 4-1. Means of Belonolaimus longicaudatus per 100 cm3 of soil sampled from 6 to16 weeks after nematode management tactics were applied during trial 1 (A) and trial 2 (B) respectively. Error bars indicate standard error of individual population means.....................................44 A-1. A lysimeter used as an experimental unit during glasshouse trials at the University of Florida Turfgrass Envirotron from 29 January 2002 to 16 April 2003............................................55 A-2. Screen placed within a lysimeter after assembly. The screen holds the soil profile away from drainage hole preventing drain blockage.............................................................................55 A-3. A threaded bushing screwed into a lysimeter. The bushing enables leachate to be collected.56 A-4. Glass imaging pan with white background placed on a flat bed scanner to obtain bitmap images of root systems........................................................................................................6 1 A-5. After washing, turf tissue samples were placed on a paper plate and dried in a 1000-W microwave oven for two to six minutes depending on sample size....................................64 A-6. Cyclone sample mill used to grind turf tissue for analysis. Dried tissue is placed into the yellow cone, top center, and is retrieved from the glass jar, bottom center........................64 A-7. Near infrared reflectance spectroscopy (NIRS) scanning instrument, left. Spectral data determined by NIRS was imported into a laptop computer with Toro Diagnostic software program for analysis, right. Sampling cells waiting to be analyzed, top left, sampling cells previously analyzed, bottom left.........................................................................................65

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x B-1. Plot plan of the field study undertaken at Citrus County, FL during 2002. Plots marked with X were not used as experimental units due to nematode counts being below damaging threshold for Belonolaimus longicaudatus ..........................................................................69 B-2. Plot plan of the field study undertaken at Pasco County, FL during 2003. Plots marked with X were not used as experimental units due to nematode counts being below damaging threshold for Belonolaimus longicaudatus ..........................................................................70 C-1. Regression models of turf quality response to untreated control, mechanical disruption with slit injection equipment without nematicide, and slit injection of 1,3-dichloropropene at a rate of 46.76 liter per hectare at N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month through out trial 1 (A) and trial 2 (B)..................................................................................74

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xi 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 EFFECTS OF PLANT PARASITIC NEMATODES AND NITROGEN FERTILITY MANAGEMENT ON HYBRID BERMUDAGRASS By John Eric Luc May 2004 Chair: William T. Crow Major Department: Entomology and Nematology Damage caused by B. longicaudatus to bermudagrass root systems can cause decreased water and nutrient uptake, reduced plant growth, and predispose turf to other adverse conditions such as drought stress, heat stress, and malnutrition which can reduce turf quality. Glasshouse experiments were conducted to determine the relationships between nematode damage to ‘Tifdwarf’ bermudagrass roots and nitrogen uptake and nitrate leached. Forty lysimeters were sprigged with ‘Tifdwarf’ bermudagrass to simulate a greens soil profile, of which 20 were inoculated with B. longicaudatus using a random complete block design. Leaching events were simulated at 21 and 42 day intervals for trial 1 and 2 respectively. Turf was fertilized every 21 days with potassium nitrate at 92 and 109 kg/ha N for trial 1 and 2. Turf quality, color, and density were evaluated every 21 days. Nematode counts and root lengths were assessed 6, 12, and 18 weeks after turf and nematode establishment. Differences ( P 0.05) were observed for total root length at 6, 12, and 18 weeks and milligrams of nitrate (NO3 -) leached at 18 weeks during both trials. When amount of cumulative nitrate leached was compared between treatments, root systems inoculated with B. longicaudatus leached more NO3 than did uninoculated root systems

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xii ( P 0.05) in trial 1, but not in trial 2. Nematode feeding reduced root density by 30 to 94 percent and increased the amount of nitrate leached by as much as 429 percent. No differences ( P 0.05) were observed for turf quality, color, density, tissue nitrogen levels, dry matter production, or total nitrogen uptake. Field experiments were conducted during 2002 and 2003 to describe relationships between nematode management and N fertility in terms of turf quality and root lengths on golf course fairways. Treatments were untreated control, mechanical, and 1,3-dichloropropene with N fertility levels of 0, 36, 73, and 110 kg/ha/month. Treatments were randomized within blocks with four replications. Reduced ( P 0.05) B. longicaudatus counts were observed in plots treated with the nematicide 1,3-dichloropropene compared to untreated control plots at 2, 4, and 6 weeks after treatment during both trials. Differences ( P 0.05) were observed between untreated control and nematicide treated plots within individual N fertility levels at specific sampling dates with respect to turf quality, color, and density during both trials. Differences ( P 0.05) in turf quality were observed between untreated control and nematode management within each N fertility level at 2 and 4 weeks and 8 to 16 weeks after treatment at N fertility levels of 73.30 and 109.95 kg N/ha/month during trial 2. No differences ( P 0.05) in root length and surface area of specified root diameters, total root length, total surface area, or root weight were observed between nematode management tactics at individual N fertility levels for 0, 6, and 16 weeks after treatment during either trial. In conclusion, glasshouse studies indicated that nematode damage to turf roots can increase nitrate leaching, thereby adding to water quality concerns. Field experiments indicate that increased N fertility on nematode infested sites without nematode management could be detrimental to turf quality, especially when the turf experiences stress. These studies reveal that nematode and N fertility management are equally important to providing a quality turf and minimizing environmental impacts.

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1 CHAPTER 1 INTRODUCTION Nitrogen (N) is frequently the limiting nutrient for most plants. In the United States of America from 1955 to 1990, annual N fertilizer inputs by agriculturalists increased from 2 million tons to over 12 million tons. Each year Florida growers apply about 2.0 million tons of fertilizer at a cost of over 250 million dollars to grow in excess of 3 billion dollars worth of crops (Bottcher and Rhue, 2000). In 2000, total acreage dedicated to Florida golf facilities was 207,582 acres with 147,927 acres maintained as golf playing areas. This represents an increase of 12.66% in maintained turf area since 1991. Hybrid bermudagrasses ( Cynodon spp.) are the prevalent grasses used in maintained turf areas on golf courses due to good drought and wear tolerance, accounting for 92% of land use (Haydu and Hodges, 2002). Bermudagrass requires ample nutrient inputs for optimal growth and turf quality. Due to Florida’s long growing season, high annual rainfall, and sandy soils, golf course turfgrass requires higher fertility inputs than other areas in the United States (Unruh et al., 1999). Between 1995 and 2000, fertilizer use per acre has increased on 29% of golf courses in Florida (Haydu and Hodges, 2002). In recent years, heightened environmental awareness has brought water quality and consumption to the forefront of public concern, focusing attention on heavy users of water, fertilizers, and pesticides (Haydu and Hodges, 2002). The intensive use of N fertilizers on golf courses has added to these concerns and spurred questions as to the fate of N following application. Nitrogen leaching into groundwater is one concern. The possibility of groundwater contamination is increased on sandy soils with low levels of organic matter receiving intensive rainfall or irrigation. Of the shallow wells tested in northern Florida, approximately 21% in agricultural areas and 7% in urban area were found to have nitrate levels in excess of the

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2 maximum contaminant level (10 mg/liter) set by the Federal Environmental Protection Agency. The highest level of nitrate contamination (12 mg/liter) in an urban area was found in Ocala, FL (Berndt et al., 1998). In the near future, golf courses may be exposed to much more rigorous monitoring of water and fertilizer practices. Studies have revealed that nitrate leaching from healthy turf is minimal (Sartain and Gooding, 2000; Snyder et al., 1984). While these research efforts are valuable for a better understanding of turfgrass processes, they are normally performed under optimal conditions and without pest infestations. However, rarely on golf courses do these optimal and pest free conditions exist. How much fertilizer is needed when the turf is not healthy? How much of this resource is lost due to poor turf vigor? Plant-parasitic nematodes are root-feeding pests that greatly reduce the development of turf roots. Damaged root systems are less efficient at water and nutrient uptake (Crow et al., 2003). Consequently, turf that suffers nematode damage may require more water and fertilizer to maintain an acceptable appearance. Hypothetically, damaged roots in conjunction with increased fertilization and watering could lead to increased nitrate leaching into the groundwater. The question was recently raised, “If golf course managers had no products to control nematodes couldn’t they increase their water and fertility usage and get by?” A short time ago, ethoprop (Mocap) was pulled off the market for use on turfgrass and fenamiphos (Nemacur) is being phased out in the next few years. We are reaching a point where we may have to “get by.” As mandated by the Food Quality Protection Act of 1996 a review of pesticides is ongoing. When nematicides are reviewed for turfgrass use, there is a perception that governmental agencies do not see this as a critical need. However, if nematode management can be shown to decrease nitrate leaching, more consideration may be given to development and registration of new nematicides for turf uses.

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3 The objectives of these studies were to: 1) determine the relationships between nematode damage to ‘Tifdwarf’ bermudagrass [ Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy] roots and nitrogen uptake and nitrate leached and 2) describe relationships between nematode population counts and nitrogen fertility in terms of turf quality and root lengths on golf course fairways.

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4 CHAPTER 2 LITERATURE REVIEW Belonolaimus longicaudatus Taxonomy Steiner (1949) first established the genus Belonolaimus with the discovery and description of Belonolaimus gracilis. This first observation of B. gracilis was on the roots of slash pine ( Pinus elliotii Engelm) and longleaf pine ( Pinus palustris P. Mill.) in the Ocala National Forest near Ocala, FL. Over the next few years, B. gracilis was reported to damage crops such as celery ( Apium graveolens L.), corn ( Zea mays L.), sorghum ( Sorghum bicolor L. Moench), millet ( Sorghum halepense L. Pers.), peanut ( Arachis hypogaea L.), cotton ( Gossypium hirsutum L.), soybean ( Glycine max L. Merr.), and cowpea ( Vigna unguiculata L. Walp.) (Christie, 1952; Christie et al., 1952; Christie, 1953; Owens, 1951). Rau (1958) described Belonolaimus longicaudatus adding a second species to the genus Belonolaimus The major morphological differences separating these species are that B. longicaudatus has a longer stylet and shorter tail than B. gracilis Belonolaimus longicaudatus was initially found on crops such as bermudagrass ( Cynodon dactylon L. Pers.), corn, citrus ( Citrus spp.), soybean, peanut, and other crops (Rau, 1958). Furthermore, Rau stated that B. longicaudatus was the more commonly encountered species. In 1963, Rau described three additional species of Belonolaimus : B. euthychilus B. maritimus and B. nortoni Since then, species have been added and the genus has been moved several times. Belonolaimus longicaudatus current taxonomic placement within the kingdom Animalia is: subkingdom Metazoa, branch Eumetazoa, division Bilateralia, subdivision Protostomia, section Pseudocoelomata, superphylum Aschelminthes, phylum Nematoda, class Secernentea, order Tylenchida, suborder Tylenchina, superfamily Tylenchoidea, family Belonolaimidae, subfamily

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5 Belonolaiminae, genus Belonolaimus species longicaudatus (Fortuner and Luc, 1987; Smart and Nguyen, 1988). Morphology and Anatomy Belonolaimus longicaudatus adults are 2 to 3 mm long and 29 to 34 m wide (Mai et al., 1996; Rau, 1958). The lip region has four major lobes with two smaller lobes having amphids present and a constriction just below it, setting it off from the rest of the body. The lateral field has one incisure running most of the body length. The stylet is thin and flexible ranging in size from 100 to 140 m long with rounded knobs. When retracted, the stylet causes the esophageal tube to be convoluted (Ferris, 1999). The median bulb is well developed and elongated. Esophageal glands overlap the anterior end of the intestines on the ventral side. The intestine can be found slightly posterior of the median bulb extending almost to the terminus. Lateral canals are prominent and serpentine along the intestine, becoming visible near the esophageal glands and extending to the terminus. The vulva is a transverse slit found near the middle of the female, with lips not protruding. The vagina nearly always has two sclerotized pieces that can be observed in lateral view. The reproductive system is didelphic, amphidelphic, and outstretched. Spermathecae are present which store sperm after copulation for fertilization of eggs over time. The male reproductive system is found posteriorly, with testis prodelphic and outstretched. Spicules and gubernaculum are well-developed averaging 44 m and 16 m long, respectively. The female tail is 115 to 189 m long with a rounded terminus. The male tail tapers to a more pointed terminus and is enveloped by a long and narrow bursa, which may aid during copulation. (Ferris, 1999; Mai et al., 1996; Robbins and Barker, 1973). It should be noted, that populations of B. longicaudatus from North Carolina and Georgia have been shown to exhibit differing morphological characteristics such as stylet length, stylet cone and shaft length, knob shape and length, distance of excretory pore to anterior end, tail length, and spicule length. Interpopulation mating of these two populations resulted in a few offspring, which were sterile, while intrapopulation offspring reproduced normally. All populations investigated possessed eight

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6 haploid chromosomes suggesting they are closely related, but may be different species (Robbins and Hirschmann, 1974). Biology and Distribution Belonolaimus longicaudatus has an extensive host range and has been recognized as a pathogen of many agronomic, horticultural, and ornamental crops (Abu-Gharbieh and Perry, 1970). Belonolaimus longicaudatus is an ectoparasite, meaning it feeds from the outside of the plant root. The long stylet is used to penetrate deep into the roots where digestive enzymes can be injected (Huang and Becker, 1997). Feeding usually causes the root tips to stop growing; this can be devastating to young plants with a developing root system (Crow et al., 2003; Huang and Becker, 1997; Perry and Rhoades, 1982). Belonolaimus longicaudatus is a bisexual species, which reproduces exclusively through amphimixis (sexually) with males accounting for 40% of the population (Huang and Becker, 1999). Experiments conducted on corn root cultures at 28 C by Huang and Becker (1997; 1999) have given a detailed description of the life cycle of B. longicaudatus After mating, females lay eggs in pairs as long as a food source is available, with each female laying about 128 13 eggs in 90 days (Huang and Becker, 1999). Following egg deposition (day 0) the first stage juvenile (J1) molted in the egg during day 4 and the second stage juvenile (J2) hatched from the egg during day 5. The J2 must quickly find root tips to feed on or die. Once a food source was found, the J2 fed for 12 to 24 hours before becoming immobile and the second molt began during day 7 and ended during day 9. Third stage juveniles (J3) began feeding again and then entered the third molt on day 12 which lasted 2 days. Fourth stage juveniles (J4) began feeding once more, but depending on the final sex of the nematodes, molting began at different times. Males entered the fourth molt during day 18 and by day 20 were fully functional males and began feeding again. Females entered the fourth molt during day 19, and by day 22 were considered virgin females ready for mating. The life cycle from J2 to J2 took 24 days at 28 C (Huang and Becker, 1999).

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7 Belonolaimus longicaudatus is found predominately in sandy costal areas of the southeastern United States; however it has been observed in localized areas of the northeastern states of Connecticut and New Jersey, and in the midwestern states of Arkansas, Kansas, Oklahoma and Nebraska (Rhoades, 1980; Robbins and Barker, 1974). Belonolaimus longicaudatus has been found in California, Puerto Rico, Bermuda, Australia, and some of the Caribbean islands, usually in resort areas where sod or sprigs were sent from the southeastern United States to establish fairways, greens, tees, or commercial turf (Mundo-Ocampo et al., 1994; Perry and Rhoades, 1982). Soil texture, soil particle size, soil moisture, root depth, and movement of nematodes are factors which influence distribution of nematodes within the soil profile as well as geographically. Soil texture influences nematode movement and distribution as a function of nematode size to soil pore and particle size. As the diameter and length of the nematode increase, the pore and particle size of the soil must increase or movement can be hindered (Wallace, 1971). However, if pore diameter becomes too large, lateral movement can be hindered (Brodie, 1976). Soils with less than 80% sand and more than 10% clay hinder movement of B. longicaudatus Furthermore, medium and course sand also have been shown to hinder their movement. Optimum soil moisture for B. longicaudatus is about 7%, however populations have been recovered from soils ranging from 2 to 30% soil moisture (Robbins and Barker, 1974). Saturated soils replenish oxygen (O2) much slower than well-drained soils, which can reduce activity and ultimately be detrimental to certain nematode species. Conversely, soils with excessively low soil moisture hinder movement by reducing the water films encircling soil particles used by nematodes to move through the soil. Soil texture, particle size, moisture, and aeration are interrelated factors, which effect movement. Movement is essential for species that reproduce exclusively by amphimixis ( B. longicaudatus ) because they must search for food as well as a mate to reproduce, otherwise the population will die (Robbins and Barker, 1974). Root depth of the host plant also influences where B. longicaudatus will occur, generally they are found within the top 75 cm of the soil

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8 profile, with greatest population densities found in the top 30 cm of the soil profile (Brodie, 1976; McSorley and Dickson, 1990; Todd, 1989). In sites where turf is the host crop, greatest population densities occur within the top few cm of soil. Conversely, in corn and citrus, which possess deeper root systems, high populations can be found below 30 cm depth when other soil conditions are favorable for nematode movement (Brodie, 1976; Noling, 1993; Todd, 1989). Nematode populations are frequently erratically distributed at standard sampling depths even within the same fields site (Todd, 1989). Erratic distribution of nematodes can be a result of previously mentioned factors, as well as the lack of active movement of nematodes from one location to another. When consideration is given to the previously discussed factors, golf greens represent ideal habitat for B. longicaudatus due to a high content of fine to medium sand that hold approximately 12 to 15% water at field capacity (Anonymous, 1993). Soil Fertility History The first known use of amendments to increase productivity of the soil was in 2500 B.C. in Mesopotamia. Greek writings also suggest the use of manures to increase vegetable and olive production. In the early 1600’s, water was considered to be the principle nourishment to plants and soil was just a media to hold the plant. In the 1700’s it was believed soil particles were ingested into the plant through the root system and then circulated throughout the plant (Sartain, 2000). In 1862, Justus Von Liebig, known as The Father of Soil Chemistry, was first to publish the concept that plant production can be no greater than that level allowed by the growth factor present in the lowest amount relative to the optimum amount for that factor (limiting factor), whether it is temperature, water, or nutrient supply (Brady and Weil, 2000). This concept is known as the “Law of the Minimum” (Sartain, 2000). In the 1820’s early fertilizer production began with a bi-product of soap production, ammonium sulfate, which was utilized as an inorganic synthetic source of nitrogen (N). On the same day in 1842, James Murray and John Bennet Lawes each patented the process for

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9 production of superphosphate. By the 1850’s, the fertilizer industry was born. In 1867, the phosphorous (P) fertilizer industry flourished with the opening of phosphate mining in South Carolina. In 1921, atmospheric N was directly converted to ammonia and less than ten years later the process for urea production was discovered. Discovery of potassium (K) deposits in Carlsbad, NM in 1931 were badly needed at the time for production of gunpowder. In 1933, the Tennessee Valley Authority (TVA) and National Fertilizer Development Center (NFDC) were created to provide power and to conduct research related to fertilizer materials. In 1945, TVA developed a method for solid ammonium nitrate (NH4NO3), and the cone mixer, which increased production of concentrated superphosphates, wet-process phosphoric acid, and ammonium phosphates. By the 1950’s, the fertilizer industry was expanding rapidly with increases in granular fertilizer production, specialty fertilizers, introduction of slow release fertilizers, and wider recognition for the need of micronutrient fertilizers (Sartain, 2000). Nitrogen Fertilizers Nitrogen fertilizers are categorized as soluble or slow-release. Soluble fertilizers such as ammonium sulfate [(NH4)2SO4], ammonium nitrate (NH4NO3), urea [CO(NH2)2], and potassium nitrate (KNO3), readily release near 100% of their nutrient load into soil solution immediately upon application. While immediate availability of N imparts a sudden increase in growth, soluble forms of N also may be more prone to leaching into the groundwater (Wang and Alva, 1996). Slow-release fertilizers (SRF) such as sulfur coated urea (SCU), polymer/sulfur coated fertilizers (PSCF), urea-formaldehyde (UF), and isobutylidene diurea (IBDU), release their N over a longer duration, and may reduce the incidence of N leaching into the groundwater (Sartain, 2000). Turfgrass in general responds well to N, and SRF are well suited for turfgrass management. However, understanding the factors that release N: temperature, soil moisture, soil pH, microbial activity, coating thickness, material particle size, hydrolysis and diffusion rate is essential to forming an efficient fertility program.

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10 The turfgrass industry uses a wide variety of N fertilizers, the more frequently used N sources include: NH4NO3, (NH4)2SO4, and SCU. All these N sources vary in N release rates, and in turf response. Ammonium nitrate is white, crystalline solid, containing 33.5% N. Because this material is very hydroscopic, the prill can be coated with MgCl2 or clay to prevent water absorption. Ammonium nitrate is highly soluble in water and releases equal amounts of NH4 + and NO3 -. Ammonium nitrate has a salt index of 2.99, and is therefore considered to be moderately to highly damaging to turf (Sartain, 2000). In studies conducted by Wang and Alva (1996), N leaching on sandy soils was found to be between 88 to 100% of the applied N when using NH4NO3. While ammonium nitrate imparts a rapid green response, the longevity of the response is usually less than 30 days (Sartain and Kruse, 2001). Ammonium sulfate is grayish, angular solid, containing 21% N and 24% sulfur (S). Ammonium sulfate solubility is low, making this material ideal to produce fertilizer blends. Ammonium sulfate is mainly produced as a by-product of the Bessemer process: 2 NH3 + H2SO4 (NH4)2SO4. With a salt index of 3.25, it will cause phytotoxicity at higher rates (Sartain, 2000). It has the highest acid forming potential of N sources through a chemical reaction with oxygen and water: [(NH4)2SO4 + O2 + H2O 2H2O + 4H+ + SO4 + 2NO3 -]. This can be a benefit for crops which prefer acidic conditions, especially in Florida were high pH soils are common (Sartain, 2000). Ammonium sulfate provides a dark green color, which tends to last longer than 30 days when applied at recommended rates (Sartain and Kruse, 2001). Sulfur coated urea consists of urea particles coated with a S shell. First urea is formed by reacting carbon dioxide with ammonia: CO2 + 2NH3 CO(NH2)2 + H2O. Then technology developed by the Tennessee Valley Authority in the 1960’s and 1970’s is used to coat the urea with an S shell. Sulfur was chosen as the coating material due to its low cost and nutrient value. Depending on the urea source and sealant used to strengthen the sulfur coat, SCU can range in color from brown to yellow. Nitrogen content of SCU can range from 30 to 40% (Sartain, 2000).

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11 The mechanism for N release from SCU is water penetration through micropores, and cracks in the S shell. Shell thickness and quality directly affect the rate of release. Once water penetrates the shell a rapid release on N from the core occurs. However, if a wax sealant is used, microbial degradation must occur to reveal the imperfection of the S shell. Microbial activity is directly affected by soil temperature, producing and uneven release and severe mottling of turfgrasses during cooler periods. Turf response to SCU lags in comparison to soluble materials, however duration of response can last from 6 to 16 weeks. Nitrogen Use in the Soil and Turfgrass System Nitrogen is normally the limiting nutrient in the turfgrass system (Unruh et al., 1999). Nitrogen is a highly mobile nutrient within the plant. When plant uptake is inadequate, N supplies will be transferred from older tissue to the newest tissue for production of chlorophyll, amino acids, proteins, enzymes, and nucleic acids. All are vital for plant processes throughout the plant (Brady and Weil, 2000). The atmosphere consists of 78% N, unfortunately turfgrasses cannot assimilate N2 gas. Therefore, it must be converted to a plant available form. Whether by lightning and rain (arc process): N2 + O2 2 NO + O2 2NO2; 3 NO2 + H20 2HNO3 + NO, or through biological fixation: N2 + 8H+ 2NH3 + H2. Once in a plant available form, the nitrogen can have many different fates (Figure. 2-1) (Brady and Weil, 2000; Sartain, 2000). Nitrogen can be leached to groundwater, lost to runoff into lakes and streams, assimilated into plant material and animals, adsorbed to the soil, or lost to the atmosphere (Brady and Weil, 2000). Mineralization A large portion of soil nitrogen (95 to 99%) is held in organic compounds protecting it from loss, but that is not available to plants. Mineralization is a three-step process consisting of aminization, ammonification, and nitrification, which converts organic forms of N to inorganic forms of N (Brady and Weil, 2000; Sartain, 2000). Aminization is the breakdown of proteins by heterotrophic organisms (bacteria and fungi) to amino acids. Ammonification is the breakdown of amino acids by heterotrophic organisms into ammonia (NH3) and ammonium (NH4+). In these

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12 Figure 2-1. Nitrogen cycle. (Brady and Weil, 2000). forms N can be converted to nitrate, used by plants, adsorbed to clay particles, or lost to the atmosphere. Nitrification is a two-step oxidation process, which occurs when autotrophic bacteria (Nitrosomonas and Nitrobacter) convert NH4+ to nitrite (NO2-) and then nitrate (NO3-). Protein heterotrophic organisms amino acid + CO2 + energy Amino acids + H2O heterotrophic organisms NH3 + R-OH + energy H2O NH4 + + OH2NH4 + + 3O2 Nitrosomonas 2NO2 + 2H2O + 4H+ 2NO2 + O2 Nitrobacter 2NO3

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13 The rate of conversion of organic N or inorganic N is greatly dependant upon the bacteria population and environment factors that effect these bacteria such as availability of NH4 +, soil pH, soil aeration, soil moisture, and soil temperature. Availability of NH4 + is directly related to the C:N ratio within the soil. When C:N ratios are high no NH4 + will be released, unless N is added to the system to lower the C:N ratio (Sartain, 2000). Ultimately, nitrification will be slowed or possibly halted. However, excessive NH4 + can be toxic to Nitrobacter bacteria and should be avoided (Brady and Weil, 2000). Nitrification occurs between a pH range of 5.0 to 8.5, with optimal conversion at pH 8.0. Inhibition of nitrification occurs below pH 4.6, due to the solubility of aluminum, which can become toxic to the bacteria. Aerobic conditions in the soil are essential for the nitrification to proceed with a minimum of 2% O2 and optimal nitrification occurring at about 20% O2 (Sartain, 2000). Optimum soil moisture is about 60% of the pore space filled with water (Brady and Weil, 2000). Soil temperatures for these bacteria range between 5 and 35 C and are optimal between 30 and 35 C. Leaching Leaching can occur when N fertilizers are applied to well drained soils. Leaching is considered to occur when the soil solution N has passed the root zone. Differing sources of N have the potential to leach at differing rates. Ammonium, a cation, tends to adsorb to soil particles that are negatively charged, while NO2 and NO3 tend to move through the soil profile more quickly (Taiz and Zeiger, 1998). The rates of mineralization and immobilization can affect the amount of inorganic N available for leaching. Soil pH can affect nitrification as previously discussed, increasing the amount of N adsorption of NH4 + sources. However, Sartain (2000) conducted an experiment studying the response of bermudagrass to controlled-release N sources over a 112 day period, illustrating losses of NH4 + can occur just as readily as NO3 -. The amount and intensity of rainfall can influence infiltration, which directly relates to the amount of N leached. Leaching studies were conducted by Shurman (2002) using soluble 20-20-20 at two N rates (1.22 g/m2 and 2.44 g/m2) applied every other week in conjunction with irrigation applied at

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14 three rates: level of evapotransportation, 0.64 cm/day, and 1.25 cm/day. These studies revealed NO3 leaching between 8 to 13% from the higher irrigation. Snyder et al. (1984) studied the effects of irrigation on seasonal leaching from ‘Tifgreen’ bermudagrass [ Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy]. The percent of N leached ranged from 0.3 to 56% and was highly influenced by time of year. Greatest leaching occurred in late winter to early spring and less occurred during mid summer. Soil slope and soil moisture level when rainfall/irrigation is applied affects the amount of N leaching. Saturated soils without a slope will tend to have higher N leaching potential than drier soils without a slope. Soil texture and structure affect permeability of the soil to water movement, through soil pore and particle size (Sartain, 2000). Soil texture can dramatically affect N leaching due to differences in the cation exchange capacity (CEC). Sandy soils tend to have very low CEC (7 cmol/kg), while organic soils tend to have very high CEC (200 cmol/kg) (Brady and Weil, 2000). Erosion and Run off Adsorption of nutrients to soil particles in conjunction with heavy rainfall can lead to erosion and surface water contamination in some cropping systems. However, this is not the case in established turfgrass stands. The larger concern is with runoff from turfgrass. When N sources are surface applied, there is a possibility for N to be lost to runoff. Runoff occurs when water input exceeds the infiltration rate of the soil. Typically runoff occurs only when high rainfall/irrigation are applied to a sloped, saturated soil, over a short period of time. Shurman (2002) conducted studies on fairway runoff of N and phosphorus (P). The soil profile at this site was a Cecil sandy loam consisting of 49.8% sand, 18% silt, and 32.2% clay. Ammonium phosphate was applied in early February; then on 22 February, during a 3.8 cm rain event, samples were collected every hour over a 24-hour period. Nitrate levels ranged from 1 to 2 mg/liter per observation, while NH4 + ranged from 1 to 200 mg/liter per observation, with 75% of the observations above 100 mg/liter. Soil moisture at the time of rainfall has a direct effect on runoff volume. In studies conducted by Shurman (2002) a positive linear relationship between

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15 soil moisture and runoff volume was observed with 5 cm simulated rainfall events. Likewise, Cole et al. (1997) found that runoff volumes from fairways with Kirkland silt loam soil were greater when soil moisture was higher prior to rainfall. Plant uptake For nutrient uptake to occur N must be in a plant available form. Portions of the root systems are frequently in direct contact with soil particles, allowing for direct exchange of nutrients. However, this supply is quickly depleted. Three basic methods are used to maintain nutrient concentration around the root system: root intercept, mass flow, and diffusion. Root intercept is the growth of roots into new, undepleted soils. Mass flow occurs when nutrients are carried in water that is being drawn toward plants for use. Diffusion is the random movement of ions in all directions from an area of high concentration to low concentration, regardless of mass flow (Brady and Weil, 2000). It should be noted that plant membranes are soluble to some ions under certain conditions. However, most nutrients only enter the roots by active transport, for this reason plants can accumulate higher concentrations of nutrients inside the root compared to the soil solution (Taiz and Zeiger, 1998). With specific carriers for each nutrient, plants can exert some control over amounts and proportions of the nutrients taken up (Brady and Weil, 2000). Typically, when N applications are made the goal is plant uptake. Due to losses of N discussed in previous and later sections, plant uptake of N ranges between 30 to 50%, and rarely exceeds 60% of applied N. Tissue dry matter of most turfgrasses consists of between 2 to 6% N (Unruh et al., 1999). Most plants can take up N as NH4 + and NO3 forms, but due to soil processes NO3 is the more prevalent form (Brady and Weil, 2000; Sartain, 2000). Soil moisture and air content can affect nutrient uptake. If soil moisture is excessively low, mass flow cannot take place. Likewise, low soil moisture will tend cause stomatas to close, reducing CO2 intake, and over time reducing plant growth. Conversely, saturated soils may produce anaerobic conditions that can decrease metabolic activity of the roots (Jackson and Drew, 1984). Likewise, anaerobic conditions in the soil can reduce available N through denitrification of

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16 NO3 -. Field capacity, or the amount of water in a soil after free drainage due to gravity has ceased, normally provides adequate amounts of soil moisture and air for optimal yields. Soil pH affects nutrient uptake in two ways: 1) by oxidizing cations and 2) by altering microbial activity. High pH levels will be conducive to oxidation of NH4 + to NH3, increasing volatilization and reducing available N for plant uptake. Conversely, as soil pH levels decrease below pH 8.0 the rate of microbial activity decreases, lowering the amount of NO3 available to plants. Adsorption Adsorption of N to soil particles, largely occurs with NH4 + and directly relates to soil texture and CEC of that soil. Soil humus, illite, and vermiculite clays have high CEC and surface areas enabling them to hold onto large amounts of nutrients. Inversely, kaolinite clay and sand have low CEC and do not retain nutrients well. Depending on rainfall, percolation rate, and soil type, adsorption may or may not occur (Sartain, 2000). Soil and water pH greatly influence nutrient movement into and out of soil solution, due the solubility of ions found in them. Likewise, soil solution losses to plant uptake, must be replenished from soil reserves (Brady and Weil, 2000). Immobilization Immobilization is the conversion of inorganic N to organic N. As microorganisms feed upon organic matter it may become necessary to incorporate mineral nitrogen ions into their cellular components. This temporarily leaves the soil solution impoverished of NH4 + and NO3 (Brady and Weil, 2000). When the organisms die, NH4 + and NO3 are returned to the soil solution and the rest becomes part of the organic matter found in the soil for later use in the nitrogen cycle. Volatilization Volatilization is the conversion of NH4 + to NH3, which can be lost to the atmosphere. Ammonia gas can be released from mineralization, applications of anhydrous ammonia, application of ammonium to calcareous soils, and surface applications of urea. Ammonia and

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17 NH4 + are in equilibrium depending on soil pH levels (Brady and Weil, 2000). Mineralization releases NH3 that is converted to NH4 + unless soil pH is high, then volatilization is more likely to occur. Soil texture, soil moisture, and depth of application affect the retention of anhydrous ammonia. Therefore, if these factors are miscalculated retention in the soil may be short term. When NH4 + is surface applied to calcareous soil or liming occurs with fertilization, calcium carbonate in soil water will bond with ammonium to form ammonium bicarbonate. Through a double decomposition reaction NH3 and CO2 will be lost. Surface application of urea in conjunction with high temperature, alkaline soils, and minimal soil moisture, can have losses of 60 to 90% of the urea N applied. However, these losses can be avoided through timely irrigation or using another source of N (Sartain, 2000). gas NH4 + + OHH2O + NH3 2NH4 + + Ca(HCO3)2 2NH4HCO3 + Ca(NO3)2 gas gas 2NH3 + CO2 + 2H2O gas CO(NH2)2 + H2O UREASE (NH4)2CO3 HEAT 2NH3 + CO2 + H2O Denitrification Denitrification is a complex microbial process conducted under anaerobic conditions in a saturated soil that converts NO3 to NO2 -, N2O, NO, and N2 (Brady and Weil, 2000). Losses of N due to denitrification can range between 10 to 20% of available N (Sartain, 2000). The proportions of gaseous products produced rely heavily upon pH, temperature, oxygen depletion,

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18 and amount of nitrate and nitrite ions available. Heterotrophic and autotrophic bacteria involved in the process are: Psuedomonas spp., Bacillus spp., Micrococcus spp., Achromobacter spp., and Thiobacillus denitrificans The process can occur at temperatures between 2 to 50 C, but the optimum range is between 25 to 35 C (Brady and Weil, 2000). Soil aeration is a major controlling factor of this process. Denitrification can occur at soil oxygen levels below 10%, but progresses faster when soil oxygen levels are below 2% (Brady and Weil, 2000; Sartain, 2000). Optimum performance occurs at a neutral pH. During denitrification N2O is the dominant gas produced at a soil pH range of 4.9 to 5.6, whereas N2 is produced in abundance at a pH range of 7.3 to 7.9. Turfgrass Cultivars ‘Tifdwarf’ Bermudagrass In 1965, ‘Tifdwarf’ bermudagrass [ Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy] was released for use on golf greens. It is a dwarf mutation selected originally from ‘Tifgreen’ bermudagrass. ‘Tifdwarf’ bermudagrass is a warm season grass, predominantly used on greens in the southeastern United States. Greens tend to be sodded or sprigged with vegetative material in late April to early May. Mowing height for healthy greens ranges from 0.5 to 0.75 cm. However, most greens today are mowed at cutting heights between 0.3 and 0.4 cm, and are prone to problems with diseases, drought stress, heat stress, and scalping issues (Morris, 2003). ‘Tifway 419’ Bermudagrass ‘Tifway 419’ bermudagrass [ Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy] is a warm season grass predominantly used on golf course fairways, athletic fields, and other areas throughout the southern United States. Internode length is relatively short compared to common bermudagrass, and some seeded bermudagrass cultivars. Shortened internode length allows for a dense turf stand, which is resistant to wear. This cultivar is also propagated from sprigs in late spring, but rarely sodded in fairways.

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19 Root Systems History As early as 1873, German botanist Julius Von Sachs studied root systems directly by using simple soil filled boxes with a glass wall. Now, facilities for the study of roots in the soil have large underground chambers for observation and analysis of roots, while aerial portions off the plant are exposed to field conditions. These facilities are called rhizotrons (rhizo meaning root; tron meaning a device for studying) (Klepper and Kasper, 1994). Details about root morphology (size and distribution) can be determined as well as root growth over time through time-lapse photography. The use of root periscopes has become a popular alternative for some scientists due to the enormous cost of rhizotrons. Root periscopes are transparent plastic tubes that are buried near roots to be observed. A miniature video camera is inserted into the tube to make observations about the affects of nutrient and water inputs on root density and root growth (Taiz and Zeiger, 1998). A study conducted by Dittmer (1937) examining root systems of individual winter rye plants after 16 weeks of growth. Primary and lateral root axes were estimated at 13 x 106 with root lengths of 500 km which provided approximately 200 m2 of surface area. These plants also had root hair estimations of 1010 providing approximately another 300 m2 of surface area. Root Development and Nutrients In monocots, root development begins with the emergence of three to six primary (seminal) root axes from the germinating seed. In vegetatively propagated species the seminal root axes emerge at the nodes. With the uptake of nutrients additional root growth of brace roots (nodal) occurs. Over time, the primary and brace root grow into a complex fibrous root system. In the fibrous root system, the main root axes are generally the same root diameter. However newly forming roots and lateral roots can vary extensively in root diameter. There are three zones of development found close to the root tips, which are: meristematic zone, elongation zone, and maturation zone. The meristematic zone produces cells in two

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20 directions, toward the root base and apex. Root cells produced toward the root base will develop during elongation and maturation. Root cells directed toward the apex will help to form and maintain the root cap, which continually loses cells while pushing through soil (Taiz and Zeiger, 1998). Mucigel, a gelatinous matrix produced by the root cap, has been hypothesized to aid in root movement through soil, prevent desiccation, nutrient transfer to the root, or affect root/microorganism interactions (Russell, 1977). The elongation zone is where cells undergo the last few cell divisions forming the endodermis, Casparian strip, cortex, and stele. The maturation zone is the portion of the root where root hairs are formed and xylem fully develops, increasing the ability of the root to take up water and solutes (Taiz and Zeiger, 1998). Depending on the nutrient in question, many opinions exist about the areas where nutrients can be taken up. Potassium (K), NO3 -, NH4 +, and P can be freely taken up by corn along the entire root surface (Clarkson and Hanson, 1980). However, in corn the elongation zone has been shown to be the area for maximum uptake of NO3 and P. Likewise, in corn and rice, the root apex has been shown to take up NH4 + more readily than the elongation zone (Colmer and Bloom, 1998). At this time it appears more research is need to determine nutrient uptake over a wider range of species.

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21 CHAPTER 3 INFLUENCE OF PLANT-PARASITIC NEMATODES ON NITRATE LEACHING IN TURF Introduction Belonolaimus longicaudatus Rau, the sting nematode, was initially found on several crops including bermudagrass ( Cynodon dactylon L. Pers.)(Rau, 1958). Belonolaimus longicaudatus has an extensive host range and has been recognized as a pathogen of many agronomic, horticultural, and ornamental crops (Abu-Gharbieh and Perry, 1970). While it can be devastating to a wide range of crops, B. longicaudatus is found predominately in sandy coastal areas of the southeastern United States. Soil texture has a major influence on the distribution of B. longicaudatus which is most frequently found in soils consisting of >80% sand and <10% clay with minimal organic matter (Rhoades, 1980; Robbins and Barker, 1974). Feeding by B. longicaudatus usually causes the root tips to stop growing; this is particularly devastating to young plants with a developing root system (Crow et al., 1997; Crow et al., 2003; Huang and Becker, 1997; Perry and Rhoades, 1982). Damage caused by B. longicaudatus to bermudagrass root systems can cause decreased water and nutrient uptake, and reduced plant growth (Johnson, 1970). In recent years, heightened environmental awareness has focused attention on heavy users of water, fertilizers and pesticides. This has brought water quality and consumption to the forefront of public concern (Haydu and Hodges, 2002). Nitrogen (N) is normally the limiting nutrient in the turfgrass system (Unruh et al., 1999). The intensive use of N fertilizers on golf courses coupled with a 12.7% increase in maintained turf area over the past 5 years has added to these concerns and spurred questions to the fate of N following application. Nitrogen leaching is considered to occur when the soil solution N has passed the root zone. Leaching can

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22 occur when N fertilizers are applied to well drained soils coupled with increased rainfall or irrigation. Nitrate (NO3 -) is the most leachable form of nitrogen. Because plant-parasitic nematodes cause reductions to turf root systems they might increase the amount of NO3 leaching in turfgrass systems. The objective of this study was to determine if damage caused by B. longicaudatus to turfgrass roots increases nitrate leaching and reduces nitrogen uptake by the turfgrass plant. Materials and Methods A 40-lysimeter greenhouse experiment consisting of two trials was conducted in a glass house at the University of Florida Turfgrass Envirotron in Gainesville, Florida from 29 January 2002 to 13 September 2002 and from 1 November 2002 to 16 April 2003. Data were collected over a 126-day period for each trial. Trial 1 Establishment of experimental units Forty lysimeters (15-cm-diam.; 45.75-cm-high; 8,339-cm3-volume) were used to simulate a putting green soil profile. In the bottom of the lysimeters was placed 15 cm of gravel (2-mm-diam.) covered with an additional 30 cm of nematode-free U.S. Golf Association (USGA) specification root-zone sand (Anonymous, 1993). The lysimeters were brought to field capacity and weighed. Aerial sprigs of 'Tifdwarf' bermudagrass were planted at a rate of 218 kg/ha (0.4g/lysimeter) and top dressed with approximately 0.3 cm of nematode-free sand. During establishment turf was watered six times a day starting at 0700 hours at 2-hour intervals with 8 ml of water from a mister irrigation system. Turf was fertilized once, five days after sprigging with 20-20-20 (N-P2O5-K2O) fertilizer (United Industries Corp., St. Louis, MO). Nutrient inputs were 91.96 kg/ha N, 40.46 kg/ha P, 76.33 kg/ha K, and trace amounts of essential micronutrients. The turf was allowed to grow-in and establish a root system for six weeks before being inoculated with nematodes.

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23 Nematode inoculum Following turf establishment, 20 lysimeters each were inoculated with B. longicaudatus or remained uninoculated, using a completely randomized design. Soil samples collected a week earlier from a golf course green in Palatka, FL were used to extract nematode inoculum using a modified Baermann method (McSorley and Frederick, 1991). Inoculum consisted of mixed life stages of B. longicaudatus that were hand picked into 10 ml of water. Nematode inoculum was poured into four holes (1-cm-diam. x 2.5-cm-deep) in the soil at a rate of 138 nematodes/lysimeter and allowed to reproduce for a period of eight weeks. Turf maintenance Following turf and nematode establishment, turf was watered three times a week. The first and second watering were 150 ml of water per application, followed by a third where measured amounts of water were added until prerecorded field capacity weights were achieved and values of water added were recorded. Turf was fertilized every three weeks with Potassium Nitrate 14-0-46 (N-P2O5-K2O) at a rate of 668.81 kg/ha/application. Nutrient inputs were 91.96 kg/ha N and 255.35 kg/ha K. Evaluation and sampling techniques Turf evaluations were conducted every three weeks after turf and nematode establishment. Turf quality and color were evaluated on a 1 to 9 scale (1 being poor and 9 being excellent). Turf density was evaluated on percent of live cover (PLC). Nematode population counts and root lengths were assessed 6, 12, and 18 weeks after turf and nematode establishment. Nematode population counts and root lengths were measured from four, five and eleven samples of each treatment selected at random at the 6, 12, and 18 week evaluations, respectively. Nematode and root samples were obtained by removing the soil profile (15-cm-diam.) from each lysimeter. The sample extended from the soil surface to the rock layer (30.48 0.5 cm). The sample was cut in 7.62 0.1 cm lengths to determine nematode counts and root lengths at four soil depths (0 to 7.62 cm, 7.62 to 15.24 cm, 15.24 to

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24 22.86 cm, and 22.86 to 30.48 cm). Each sub sample was placed onto a 135 m sieve. The roots were rinsed with water and the sand and nematodes were collected. Rinsates were agitated with water and poured into a 25 m sieve to catch any B. longicaudatus present (Cobb, 1918). Nematodes were collected and counted using an inverted light microscope at 30. Roots were collected, stained with methylene blue, and refrigerated for at least 24 hours. The stained roots were placed into a glass-bottom tray and scanned with an HP Scanjet 2cx desktop scanner (Hewlett Packard, Boise, ID) to obtain bitmap images of the root system (Kaspar and Ewing, 1997; Pan and Bolton, 1991). The bitmap images were imported into the GSRoot (Louisiana State University, Baton Rouge, LA) software program for analysis. This program is designed to determine root length and surface areas in millimeters for specified root diameters. Root diameters in mm specified for this analysis were: < 0.05, 0.05 to 0.1, 0.1 to 0.2, 0.2 to 0.3, 0.3 to 0.4, 0.4 to 0.5, and > 0.5. Following root scanning, samples were dried at 70 C for at least 48 hours and then weighed. Leaching events were simulated using three soil pore volumes of water at 21 1-day intervals. The leaching technique requires the lysimeters be brought to field capacity and then water added that is equal to 3 times the pore space of the soil (3,750 ml). The leachate from each lysimeter was collected and a 20 ml sub sample was taken for analysis; the remaining volume of leachate was measured. Samples were analyzed for NO3 per liter of water using an air segmented continuous flow auto spectrometer (Flow Solution IV, O.I. Analytical, College Station, TX). The equation (mg NO3 -/liter volume of leachate) was used to determine the mg of NO3 leached from each lysimeter during leaching events. Turf tissue was collected from each lysimeter at 3-week intervals until destructive root and nematode sampling. Turf was trimmed to 0.95 cm height and collected. Samples were placed into a 75 m sieve washed and then spread evenly on a paper plate. Each sample was placed in a 1000-W microwave oven and dried for two to six minutes depending on sample size. Following drying, each sample was weighed. If sufficient dry matter (1.75 g 0.25 g) was

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25 obtained the tissue was ground in a cyclone sample mill (Sample Mill, Udy Corporation, Fort Collins, CO) to pass through a 1.0-mm screen, placed into a sampling cell, and loaded into a near infrared reflectance spectroscopy (NIRS) scanning instrument (Model 5000, Foss NIRSystems, Silver Springs, MD). Spectral data was imported into the Toro Diagnostic software program (Version 2.4, The Toro Company, Bloomington, MN) for analysis and values recorded (Rodriguez and Miller, 2000). The equation [tissue percent N tissue dry weight] was used to determine mg of N uptake. However, if sufficient tissue was not collected during a particular three-week interval the tissue was washed, dried, and stored until sufficient tissue was collected. Trial 2 Establishment of experimental units Soil profiles, turf, and irrigation were established as previously stated for trial 1. Turf was fertilized once, five days after sprigging with 20-20-20 (N-P2O5-K2O) fertilizer. Nutrient inputs were 109.3 kg/ha N, 48.09 kg/ha P, 90.72 kg/ha K, and trace amounts of essential micronutrients. The turf was allowed to grow-in and establish a root systems for three weeks prior to nematode inoculation. Nematode inoculum Belonolaimus longicaudatus cultures were established from inoculum obtained from R.M. Giblin-Davis, which originated from Sanford, FL (Giblin-Davis et al., 1992). The cultures were maintained on ‘Tifdwarf’ bermudagrass grown on nematode free USGA specification putting green sand mix for several months. Following turf establishment, four lysimeters within each block were inoculated with B. longicaudatus using a random complete block design. Belonolaimus longicaudatus were extracted by decant and sieve method, collected into a beaker, and the volume brought up to 500 ml (Cobb, 1918). One ml of water and nematodes was placed onto a counting slide (Hawksley and Sons Limited, Lancing, Sussex, United Kingdom) to determine the number of nematodes per ml. Nematode counts were

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26 replicated five times with 15 2 nematodes per ml. Nematode inoculum was pipetted into four holes (1-cm-diam. x 2.5-cm-deep) in the soil at a rate of 300 40 nematodes/lysimeter and allowed to reproduce for a period of three weeks. Turf maintenance Following turf and nematode establishment, the turf was watered twice daily with 25 3 ml of water from an overhead mister irrigation system. Turf was fertilized every three weeks following leaching events with Potassium Nitrate 14-0-46 (N-P2O5-K2O) at a rate of 794.9 kg/ha/application. Nutrient inputs were 109.3 kg/ha N and 303.5 kg/ha K. Evaluation and sampling techniques The sampling and evaluation process were the same as in trial 1, with the following exceptions: Nematode population counts and root lengths were measured from one inoculated and one uninoculated lysimeter selected at random from each block at the six-week and twelve week evaluation, leaving two inoculated and two uninoculated lysimeters from each block at the end of the study. Nematode and root samples were obtained by removing one core sample (5-cm-diam.) from the middle of each lysimeter. Leaching events were simulated at 42 1-day intervals. Stolons and leaf tissue were collected and added to the turf tissue samples for analysis when destructive root and nematode sampling occurred. Data Analysis Nitrate leached data collected at 6 weeks after turf and nematode establishment was square root transformed (x +1) to normalize the data. T tests were performed to compare uninoculated and inoculated turf for quality, color, density, root lengths, root surface area, root weight, NO3 leached, cumulative NO3 leached, tissue dry weights, tissue percent N, and N uptake at individual sampling dates. Regression analysis was used to characterize relationships between nematode population counts, root length, nitrogen uptake, and nitrate leached. These T tests were performed using SAS software (SAS Institute, Cary, NC) while regression analysis was performed using Minitab software (State College, PA)

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27 Results Differences ( P 0.05) in amount of NO3 leached between uninoculated and inoculated turf at specific sampling dates were observed at 18 weeks after turf and nematode establishment during both trials (Figure 3-1 A, 3-1 B). When amount of cumulative NO3 leached was compared between treatments at week 18, T tests revealed root systems inoculated with B. longicaudatus leached more NO3 than did uninoculated root systems ( P 0.05) in trial 1, but not in trial 2 (Figure 3-2 A, 3-2 B). Differences ( P 0.05) in total root length and total root weight were observed between uninoculated and inoculated turf at 6, 12, and 18 weeks after turf and nematode establishment for both trials. Differences ( P 0.05) in total root surface area were observed at 6, 12, and 18 weeks during trial 1. However, differences in total surface area ( P 0.05) were only observed at 12 and 18 weeks during trial 2 (Table 3-1, 3-2). No differences ( P 0.05) were observed between uninoculated and inoculated turf systems with respects to turf quality, turf color, turf density, tissue dry weight, tissue percent N, or N uptake following turf and nematode establishment during either trial. Although several models each year were statistically significant ( P 0.05) correlations were low or unrepeated between years (Table 3-3).Regression analysis provided no good predictive models to characterize relationships between nematode counts, root densities, nitrogen uptake, and nitrate leached. Discussion Differences ( P 0.05) in amount of NO3 leached between uninoculated and inoculated turf at specific sampling dates were observed 18 weeks after turf and nematode establishment during both trials (Figure 3-1 A, 3-1 B). However, differences in root growth, N assimilation, and feeding by B. longicaudatus influenced the way these outcomes were expressed in each trail. During trial 1, actively growing root systems were relatively well established prior to

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28 Trial One Milligrams of Nitrate Leached0 20 40 60 80 100 120Weeks after Turf and Nematode EstablishmentMeans of Nitrate Leached (mg) Uninoculated Inoculated 3 6 9 12 15 18 Trial Two Milligrams of Nitrate Leached0 20 40 60 80 100 120 Weeks after Turf and Nematode EstablishmentMeans of Nitrate Leached (mg) Uninoculated Inoculated 6 12 18 Figure 3-1. Effects of inoculating turf with Belonolaimus longicaudatus on mg nitrate leached at 3, 6, 9, 12, 15, and 18 or 6, 12, and 18 weeks after turf and nematode establishment during trial 1 (A) and trial 2 (B), respectively. Inoculated plants received 138 (trial 1) and 300 20 B. longicaudatus (trial 2), while uninoculated plants received no nematodes. Error bars indicate standard error of individual population means. ** Inoculated different from uninoculated at ( P 0.01). ** ** A B

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29 Trial One Cumulative Nitrate Leached0 50 100 150 200 250 300 350 369121518 Weeks after Turf and Nematode EstablishmentMeans of Nitrates Leached (mg) uninoculated inoculated Trial Two Cumulative Nitrate Leached0 50 100 150 200 250 300 35061218Weeks after Turf and Nematode EstablishmentMeans of Nitrate Leached (mg) uninoculated inoculated Figure 3-2. Effects of inoculating turf with Belonolaimus longicaudatus on cumulative nitrate leached at 3, 6, 9, 12, 15, and 18 weeks after turf and nematode establishment during trial 1 (A) and trial 2 (B), respectively. Inoculated plants received 138 (trial 1) and 300 40 B. longicaudatus (trial 2), while uninoculated plants received no nematodes. Error bars indicate standard error of individual population means. A B

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30 Table 3-1. Effects of inoculating with Belonolaimus longicaudatus on ‘Tifdwarf’ bermudagrass root length, surface area, and dry weight at 6, 12, and 18 weeks after turf and nematode establishment during trial 1. Lysimeter Depthsa Root Lengths (mm) Root Surface Area (mm2) Root Dry Weights (mg) 6 weeks 0-7.62 (U)b 7,104 210 c 2,352 218 1,213 63 (I) 4,570 608 ** 1,594 151 896 167 7.62-15.24 (U) 4,335 597 1,470 173 571 77 (I) 3,564 268 1,359 164 477 44 15.24-22.86 (U) 3,043 61 1,092 83 356 32 (I) 1,832 132 *** 523 94 ** 188 18 ** 22.86-30.48 (U) 2,528 110 777 68 340 48 (I) 1,991 173 650 113 194 16 All Depths (U) 17,011 528 5,691 279 2,480 140 (I) 11,957 359 *** 4,126 389 1,755 159 12 weeks 0-7.62 (U) 6,028 151 2,534 204 1,020 106 (I) 3,994 566 ** 1,633 83 ** 666 124 7.62-15.24 (U) 4,385 335 1,747 159 730 73 (I) 2,908 408 957 111 ** 388 48 ** 15.24-22.86 (U) 3,195 78 1,250 84 406 23 (I) 1,445 306 372 99 *** 170 34 *** 22.86-30.48 (U) 3,083 157 1,189 137 319 26 (I) 1,399 120 *** 301 49 ** 136 12 *** All Depths (U) 16,691 297 6,720 242 2,476 126 (I) 9,746 1,221 ** 3,263 263 *** 1,359 208 ** 18 weeks 0-7.62 (U) 6,809 522 2,788 202 1,063 60 (I) 3,170 329 *** 1,305 156 *** 651 5 *** 7.62-15.24 (U) 3,299 48 1,333 41 555 35 (I) 2,459 132 *** 912 80 *** 324 32 *** 15.24-22.86 (U) 2,565 59 1,056 47 380 29 (I) 1,759 157 *** 539 75 *** 142 18 *** 22.86-30.48 (U) 2,476 76 945 75 305 21 (I) 1,209 123 *** 433 139 ** 107 10 *** All Depths (U) 15,151 591 6,121 275 2,302 110 (I) 8,597 472 *** 3,188 229 *** 1,224 71 *** *, **, *** Inoculated different from uninoculated at a specified depth. a Soil profile depths are reported in centimeters b (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. c Means and mean standard error for replications.

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31 Table 3-2. Effects of inoculating with Belonolaimus longicaudatus on ‘Tifdwarf’ bermudagrass root length, surface area, and dry weight at 6, 12, and 18 weeks after turf and nematode establishment during trial 2. Lysimeter Depthsa Root Lengths (mm) Root Surface Area (mm2) Root Dry Weights (mg) 6 weeks 0-7.62 (U)b 475 73 c 88 20 28.2 3.3 (I) 216 84 60 25 12.0 4.4 7.62-15.24 (U) 164 44 28 8 5.3 2.2 (I) 66 19 10 3 2.3 1.0 15.24-22.86 (U) 89 30 16 5 3.0 1.6 (I) 74 29 11 4 1.3 0.5 22.86-30.48 (U) 77 32 11 4 1.6 0.8 (I) 47 26 8 5 1.3 0.9 All Depths (U) 807 92 143 18 38.1 6.3 (I) 404 107 89 29 16.9 5.5 12 weeks 0-7.62 (U) 479 128 110 39 27.6 7.9 (I) 138 48 31 13 10.1 5.3 7.62-15.24 (U) 223 47 35 9 9.8 2.5 (I) 111 30 13 4 3.4 0.9 15.24-22.86 (U) 144 41 22 8 5.5 1.6 (I) 86 36 9 4 2.7 1.2 22.86-30.48 (U) 331 118 67 24 11.2 3.1 (I) 71 31 10 4 3.5 1.9 All Depths (U) 1,176 232 234 60 54.0 12.5 (I) 406 85 64 13 19.7 4.0 18 weeks 0-7.62 (U) 1,801 441 229 117 67.9 12.4 (I) 99 10 ** 26 5 10.1 2.2 *** 7.62-15.24 (U) 1,228 266 77 27 35.2 4.4 (I) 77 9 ** 15 3 5.2 0.7 *** 15.24-22.86 (U) 777 197 53 17 22.1 3.1 (I) 48 12 ** 11 5 3.6 1.3 *** 22.86-30.48 (U) 443 115 34 13 12.6 3.3 (I) 26 11 ** 5 3 1.7 0.7 ** All Depths (U) 4,249 958 393 141 137.7 20.8 (I) 250 25 ** 58 10 20.6 3.3 *** *, **, *** Inoculated different from uninoculated at a specified depth. a Soil profile depths are reported in centimeters b (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. c Means and mean standard error for replications.

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32 Table 3-3. Linear regression analysis conducted to determine relationships between nematode populations, root length, nitrogen uptake, and nitrate leached during both trials. Dependant Variable Independent Variable Y = r2 P Trail 1 Nematodes Root length (total)a 7311.25 + 3.19x 0.269 0.023 Nematodes Root length (small)b 3342.34 + 1.62x 0.290 0.017 Nematodes N uptake 437.50 – 0.16x 0.321 0.011 Nematodes N leached 408.27 – 0.26x 0.493 0.001 Root Length (total) N uptake 374.47 – 0.00x 0.020 0.381 Root Length (total) N leached 336.45 – 0.01x 0.143 0.016 Root Length (small) N uptake 409.91 – 0.01x 0.058 0.135 Root Length (small) N leached 362.50 – 0.03x 0.172 0.008 N uptake N leached 139.10 – 0.42x 0.161 0.324 Trial 2 Nematodes Root length (total) 458.19 – 0.16x 0.018 0.570 Nematodes Root length (small) 471.26 – 0.32x 0.031 0.458 Nematodes N uptake 31.02 + 0.18x 0.501 0.000 Nematodes N leached 90.85 + 0.18x 0.055 0.319 Root Length (total) N uptake 98.09 + 0.01x 0.083 0.071 Root Length (total) N leached 151.05 – 0.02x 0.064 0.114 Root Length (small) N uptake 109.14 + 0.01x 0.001 0.886 Root Length (small) N leached 176.41 – 0.12x 0.065 0.113 N uptake N leached 79.43 + 0.16x 0.216 0.039 a Total = root lengths for all root diameters throughout the entire soil profile. b Small = root lengths for root diameters 0.2 mm throughout the entire soil profile.

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33 inoculation, and B. longicaudatus reduced the root systems over time. A short-term solution for actively growing root systems may have been to compensate for root reductions by B. longicaudatus with increased N assimilation throughout the remaining root system. However, increasing root reduction by B. longicaudatus overwhelmed the plants ability to assimilate N, which led to the differences observed in NO3 leached. Conversely, during trial 2 root systems were slow to establish and develop due to limited light intensity and duration during winter months. As the experiment progressed, light intensity and duration improved, which led to increased root growth and N assimilation in uninoculated root systems, but feeding by B. longicaudatus still retarded root development. Total root lengths in inoculated root systems were reduced 30 %, 42 %, and 43 % at 6, 12, and 18 weeks after turf and nematodes establishment during trial 1 and 50 %, 66 %, and 94 % during trial 2. Differences ( P 0.05) in nitrate leached were not observed until reductions in root lengths and surface area were observed in a large portion of root diameters at all rooting depths in actively growing root systems (Table C-1 to Table C-13). This directly relates to the ability of the entire root system of actively growing turf to assimilate N (Clarkson and Hanson, 1980). Differences ( P 0.05) in nitrate leached may occur so1r in mature stands of turf with increased suberization of large diameter roots which can shift the burden of N assimilation to root tips, root hairs, and finer lateral roots were nematode feeding typically occurs. When amount of cumulative NO3 leached was compared between treatments, root systems inoculated with B. longicaudatus leached more NO3 than did uninoculated root systems ( P 0.05) in trial 1, but not in trial 2 (Figure 3-2 A, 3-2 B). Upon reviewing the amount of NO3 leached for each treatment at specific sampling dates, 1 might expect differences. However, root growth and root reductions by B. longicaudatus are highly variable and not evenly distributed throughout experimental units (Table 3-1, 3-2). When these factors were combined the resulting variability within treatment was greater than variability between treatments at specific sampling dates until 18 weeks after turf and nematode establishment

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34 during both trials. Furthermore, cumulative nitrate leached incorporates variability within and between treatments from all dates, causing an additive effect to the variability within treatment. Differences in the amount of nitrate leached between uninoculated and inoculated root systems at 18 weeks after turf and nematode establishment were sufficient to overcome the additive effect to the variability within treatment during trial 1, but not trial 2 at ( P 0.05). During trial 2, light intensity and duration began to improve at 12 weeks after turf and nematode establishment, which increased NO3 and root differences between treatments. Given more time differences in cumulative nitrate leached would likely have continued to increase as well. Feeding by B. longicaudatus can cause varying degrees of damage to root systems depending on plant type and age of the plant when its root system is first attacked. Rarely does nematode feeding al1 kill a plant (Christie, 1959). Typically, nematode feeding will predispose turf to other adverse conditions such as drought stress, heat stress, and malnutrition, which could lead to reduced turf quality, color, and density. Furthermore, reductions in turf density can reduce the leaf surface area hindering evapotransportation, subsequently reducing water and nutrient uptake needed for photosynthesis and tissue production. In the glasshouse, no differences ( P 0.05) were observed between uninoculated and inoculated turf systems following turf and nematode establishment during either trial with respects to turf quality, turf color, turf density, tissue dry weight, tissue percent N, or N uptake. This may be to a lack of persistent adverse conditions. In conclusion, whether turfgrass root systems were well established or newly forming, damage caused by B. longicaudatus to the entire turfgrass root system can increase nitrate leaching. However, the rates of root growth, N assimilation, and feeding by B. longicaudatus can determine the amount of time needed to observe differences in NO 3 leached. Nitrogen uptake was not hindered during either trial. However differences ( P 0.05) in nitrate leaching were not observed until the end of both trials. Nitrogen uptake may have been slowly declining

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35 during the last six weeks of the study but due to N dilution within the plant, no differences were detected.

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36 CHAPTER 4 EFFECT OF NEMATODE MANAGEMENT AND NITROGEN FERTILITY ON FAIRWAY TURF QUALITY Introduction Belonolaimus longicaudatus Rau, the sting nematode, was initially found on several crops including bermudagrass ( Cynodon dactylon L. Pers.) (Rau, 1958). Belonolaimus longicaudatus has an extensive host range and has been recognized as a pathogen of many agronomic, horticultural, and ornamental crops (Abu-Gharbieh and Perry, 1970). Belonolaimus longicaudatus is found predominately in sandy coastal areas of the southeastern United States. Soil texture has a major influence on the distribution of B. longicaudatus which is most frequently found in soils consisting of >80% sand and <10% clay with minimal organic matter (Rhoades, 1982; Robbins and Barker, 1974). Belonolaimus longicaudatus is considered to be the most damaging plant-parasitic nematode on turfgrasses in Florida. Feeding by B. longicaudatus can cause varying degrees of damage to root systems depending on plant type, and age when its root system is first attacked. Damage caused by B. longicaudatus to bermudagrass root systems can cause decreased water and nutrient uptake, and reduced plant growth, but rarely does nematode feeding al1 kill a plant (Christie, 1959; Johnson, 1970). Typically, nematode feeding will predispose turf to other adverse conditions such as drought stress, heat stress, malnutrition, arthropods, pathogens, and weeds which could lead to reduced turf quality, color, and density (Lucas, 1982). Recently, nematode management has been perceived by the turf industry as a growing problem due to fewer effective nematicides being available. If nematode management is not effective, the typical response to decreasing turf quality, color, and density by golf course managers is to increase water and nitrogen (N) fertility levels. When N fertilizers are applied to

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37 well-drained soils leaching can occur. Reductions of the turfgrass root system by B. longicaudatus could increase the leaching potential. In recent years, heightened environmental awareness has focused attention on heavy users of water, fertilizers and pesticides. This has brought water quality and consumption to the forefront of public concern (Haydu and Hodges, 2002). The intensive use of N fertilizers on golf courses coupled with a 12.7% increase in maintained turf area over the past 5 years has added to these concerns and spurred questions to the fate of N following application. The objective of this study was to describe relationships between nematode management and nitrogen fertility in terms of turf quality and root lengths on golf course fairways. Materials and Methods A 2-year field study consisting of two trials was conducted in West central Florida on golf course fairways infested with B. longicaudatus (Rau, 1958). Trial 1 was conducted in Citrus County, Florida, from 12 March 2002 to 29 August 2002, while trial 2 was conducted in Pasco County, Florida from 13 March 2003 to 29 August 2003. Data were collected over a 112-day period during each trial. Experimental Sites Pathogens Plant-parasitic nematodes present at the Citrus County site included B. longicaudatus, Hopolaimus galeatus (Cobb Thorne) Helicotylenchus sp., Peltamigratus sp., Trichodorus sp., Paratrichodorus sp., Hemicycliophora sp., Hemicric1moides sp., and Mesocric1ma sp. Fungal diseases previously treated for at this site were Bermudagrass Decline ( Gaeumannomyces graminis var. graminis Sacc. Arx. and D.L. Olivier), Brown Patch ( Rhizoctonia solani J. G. Kohn), and Fairy Ring ( Chlorophyllum, Marasmius, or Lepiota spp.). Plant-parasitic nematodes present at the Pasco County site included B. longicaudatus H. galeatus Helicotylenchus sp., Trichodorus sp., and Mesocric1ma sp. Fungal diseases previously

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38 treated for at this site were Bermudagrass Decline, Brown Patch, and Damping-Off ( Pythium spp.). Insects and weeds Pest insects observed during these trials were southern mole cricket ( Scapteriscus borrellii Giglo-Tos), tawny mole cricket ( Scapteriscus vicinus Shudder), fall armyworm ( Spodoptera frugiperda J.E. Smith), red imported fire ant ( Solenopsis invicta Buren), ringlegged earwig ( Euborellia annulipes Lucas), and two lined spittlebug ( Propsapia bicincta L.). Weeds observed during these trials were goosegrass ( Eluesine indica L. Gaertn.), crabgrass ( Digitaria spp. ), crowfootgrass ( Dactyloctenium aegyptium L. Willd.), carpetgrass ( Axonopus affinis Chase), creeping signalgrass ( Brachiaria plantaginea L. A. S. Hitchc.), doveweed ( Murdannia nudiflora L. Brenan), and spotted spurge ( Euphorbia maculata L.). Turf In both trials, golf course fairways had mature stands (15 to 20 years old) of ‘Tifway 419’ bermudagrass [ Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy]. Turf at both locations was maintained at 1.3 cm cutting height and watered as needed. Both fairways had histories of nematode damage over the last few years, requiring more attention to cultural practices and inputs. Soil properties Soil texture at a depth of 10 to 15 cm was analyzed using the hydrometer method (Bouyoucos, 1936). Soil at the Citrus County site was Tavares fine sand with a composition of 92% sand, 4.5% silt, 3.5% clay; < 1% organic matter and pH 5.8 (USDA, 1982). Soil at the Pasco County site was Millhopper-Candler Variant soil with a composition of 97% sand, 0% silt, 3% clay; < 1% organic matter and pH 6.0 (USDA, 1985). Experimental Design The experimental design varied from 2002 to 2003. In 2002, the experimental design was arranged as a split plot design. Whole plots were three nematode management tactics: 1,3-

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39 dichloropropene (1,3-D) applied by slit-injection (Crow et al., 2003), a mechanical slit treatment with no chemical applied, and untreated control. Each whole plot was replicated four times. Sub plots consisted of four N rates 0, 36.65, 73.30, and 109.95 kg/ha /month. Main plots were 3.7-mwide and 15.2-m-long, with sub plots being 0.9-m-wide and 15.2-m-long. Main plots were separated by border areas (1.5 m on the sides and 3.0 m at each end), which were only mowed and watered. In 2003, the experimental design was arranged in a randomized complete block. Eight treatments were two nematode management tactics: 1,3-dichloropropene and untreated control with four N rates of 0, 36.65, 73.30, and 109.95 kg/ha /month. Treatments were replicated four times. These plots were 3.7-m-long and 3.7-m-wide. Plots were separated by border areas (1.5 m wide on all sides), which were maintained as previously stated. The change in experimental design was d1 to reduce the incidents of fertility runoff from 1 sub plot into another. In both trials, nematode samples were collected six weeks prior to nematicide treatments. Nematodes were extracted from the soil using a modified centrifugal-flotation technique and counted (Jenkins, 1964). Plots with counts below 30 B. longicaudatus per 100 cm3 of soil were excluded from the study. The remaining plots were assigned to blocks according to B. longicaudatus population counts. Treatments were randomized within each block. Nematicide Treatments Nematicide treatments for the 2002 trial were 1,3-dichloropropene, mechanical, and untreated control. In 2003, the mechanical treatment was eliminated after no differences ( P 0.05) were observed for nematode populations counts or visual performance between mechanical and control plots in 2002. Nematicide treatments were applied once per trail during the first week of May. 1,3-dichloropropene was injected at a rate of 46.76 liters/ha with a nitrogen gas pressurized application rig. The application rig had straight coulters placed on 30.5 cm centers, followed by a chisel with a metal drip line attached which placed the material at a depth of 13 to

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40 17 cm. A steel roller wheel followed each chisel to close the soil. Mechanical treatments consisted of running the application rig through the soil as preformed with the 1,3dichloropropene treatments, without the chemical being applied. Immediately after nematode management tactics were concluded, approximately 1.25 cm of water was applied, which assisted in holding the 1,3-dichloropropene in the soil. Fertilization The turf fertilization program varied from 2002 to 2003. In 2002, fertilization began two weeks prior to nematicide treatment and continued at two-week intervals until the end of the study. Turf was fertilized with Potassium Nitrate 14-0-46 (N-P2O5-K2O) at N rates of 0, 36.65, 73.30, and 109.95 kg/ha /month using a drop spreader. During the 2002 trial, when fertilizer was applied without the turf being watered, salt induced phytotoxicity occurred. Portions of subplots fertilized with N rates of 36.65, 73.30, and 109.95 kg/ha/month showed proportional damage depending on fertilizer rates. This problem was corrected in 2003 by using a slow release fertilizer, which consisted of Sulfur Coated Urea, Sulfur Coated Ammonium Phosphate, Sulfur Coated Sulfate of Potash, Iron Oxide, and Manganese Sucrate. In 2003, fertilization began four weeks prior to nematode management treatments being applied and continued at two-week intervals until the end of the study. Turf was fertilized with a 14-14-14 (N-P2O5-K2O) sulfur coated blend at N rates of 0, 36.65, 73.30, and 109.95 kg/ha /month using a hand-held rotary spreader. In 2003, an unscheduled fertilizer application occurred during week 11 with a slow release blend of 21-0-18 (N-P2O5-K2O) at an N rate of 70.68 kg/ha (broadcast). General Production Practices Turf Maintenance In both trails, turf was mowed by the golf courses staff three times a week at a cutting height of 1.3 cm. However, on several occasions the turf was not mowed due to rain. Cultural practices conducted by golf course staff such as aerification, slicing, and vertical mowing were

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41 halted for this experiment. In 2002, turf was irrigated with 0.64 cm of water as needed. Conversely, in 2003 turf was irrigated once a day with 0.64 cm of water until week 3 when the irrigation system failed causing the turf to go without watering for 3 to 4 days. Thereafter, turf was irrigated twice a day with 0.64 cm of water except during week 9 when the irrigation system failed again causing the turf to go without watering for 3 to 4 days. Pesticides In 2002, no additional pesticides were used, except for the experimental treatment. Subsequently, MSMA at 2.25 kg a.i./ha and metribuzin at 0.035 kg a.i./ha were tank mixed and applied as a spot treatment to control Eluesine indica on 14 July 2003 in trial 2. Sampling and Evaluations Turf Evaluations Turf evaluations were conducted every two weeks, beginning with the first N fertility treatment each year. Turf quality and color were evaluated on a 1 to 9 scale (1 being poor, 6.5 acceptable, and 9 being excellent). Turf density was evaluated on percent of live cover (PLC). In 2002, each subplot (0.91 m x 15.24 m) was evaluated as a whole, which made evaluations difficult. Subsequently, in 2003 each plot was divided into four equal quadrants, each quadrant was evaluated for turf quality, color, and density. Nematodes In both trials, twelve cores (2.5-cm-diam and 10.2-cm-depth) were obtained from each plot using a c1 sampler to determine nematode population counts. A 15-cm buffer z1 was established inside the parameter of each treatment plot to ensure accurate treatment results. Samples were taken twice prior to nematicide treatment (six weeks, and 1 day prior to nematicide treatments), and at two-week intervals following nematicide treatments. Each sample was mixed thoroughly and a 100-cm3 sub sample was obtained. Nematodes were extracted from the soil using a modified centrifugal-flotation technique (Jenkins, 1964). Traditionally, the extraction process requires the soil to be passed through a 2 mm sieve to remove debris, however this step

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42 was omitted to prevent B. longicaudatus from being lodged in the mesh of the sieve (McSorley and Fredrick, 1991). Following, extraction all plant-parasitic nematodes were counted using an inverted light microscope at a magnification of 20 x. Roots Root samples (3.5-cm-diam and 15-cm-depth) were obtained inside the buffer z1 with a tee sampler. In 2002, following nematicide treatments, two root cores were collected at 0, 6, and 16 weeks from each treatment plot and combined into a single sample with no differences observed ( P 0.05). Previous studies had shown increases in root length when plant-parasitic nematodes were being managed, so an increase in sample size was suggested. In 2003, following nematicide treatments, three root cores were collected at 0, 6, and 16 weeks from each treatment plot. In both trials, roots were processed, analyzed, and weighed as described in Appendix A. Turf Tissue Turf tissue was collected every two-weeks, beginning with the first N fertility treatment each year. In 2002, tissue samples were collected from three 30 cm x 30 cm areas within each treatment plot. However, in 2003 tissue was collected from the entire treatment plot (3.7 m x 3.7 m). Turf was trimmed to 0.95 cm height during both trails. Tissue samples were processed and analyzed as described in Appendix A. However, since large amounts of tissue were collected from each treatment plot, following the grinding step each sample was thoroughly mixed and a 2.0 g 0.5 g sub sample was obtained for analysis. Data Analysis ANOVA were performed to compare turf quality, color, density, root lengths, root surface area, root weight, tissue dry weights, tissue percent N, and N uptake between treatments at individual sampling dates. Due to interactions between nematode management tactics and N fertility, general linear models and orthogonal contrasts were performed at individual N fertility levels to compare between treatments. Regression analysis was used to characterize relationships between nematode population counts and fertility in terms of turf quality and root lengths on golf

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43 course fairways. ANOVA, general linear models, and orthogonal contrasts were performed using SAS software (SAS Institute, Cary, NC) while regression analysis was performed using Minitab software (State College, PA). Results Differences ( P 0.05) were observed in B. longicaudatus population means between untreated control and nematode management at 2, 4, and 6 weeks after treatment during both trials, respectively (Figure 4-1A, 4-1B). Differences ( P 0.05) in turf quality were observed between untreated control and nematode management at individual N fertility levels at 2, 8, 10, and 12 weeks after treatment during trial 1 and 2, 4, 8, 10, 12, 14, and 16 weeks after treatment during trial 2 (Table 4-1, 4-2). During trial 2, turf quality was not evaluated at week 6 due to excessive leaf material being left across the research area. No differences ( P 0.05) in root length and surface area of specified root diameters, total root length, total surface area, or root weight were observed between nematode management tactics at individual N fertility levels at 0, 6, and 16 weeks after treatment during either trial (Table 4-3). Confounding issues with respects to collection of tissue samples precluded an unbiased analysis of tissue dry weight, tissue percent N, or N uptake during either trial. During trial 1, scheduling conflicts precluded the continued use of mowing equipment for collection of tissue samples. During trial 2, on numerous occasions the turf was mowed just prior to arrival, thereby removing differences in vertical leaf growth that may have occurred between treatments. In both incidents actions beyond the experimenters control precluded collection of accurate samples. Regression analysis provided no good predictive models to characterize relationships between nematode management and nitrogen fertility in terms of turf quality and root lengths.

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44 Nematode Population Differences among Treatments during Trial One0 20 40 60 80 100 120 140 160Weeks after Nematode Management TacticAverage Nematode Count per 100cc of Soil Control Mechanical Nematicide -6 0 2 4 6 8 10 12 14 16 A Nematode Population Differences between Treatments during Trial Two0 20 40 60 80 100 120Weeks after Nematode Management TacticAverage Nematode Count per 100cc of Soil Control Nematicide -6 0 2 4 6 8 10 12 14 16B Figure 4-1. Means of Belonolaimus longicaudatus per 100 cm3 of soil sampled from 6 to16 weeks after nematode management tactics were applied during trial 1 (A) and trial 2 (B), respectively. Error bars indicate standard error of individual population means. Control = no added soil disturbance or nematicide treatment; Mechanical = soil disturbance without nematicide; Nematicide = Injection of 1-3, dichloropropene at a rate of 46.76 liters per hectare at 13 to 17 cm of soil depth. indicates differences ( P 0.05) between Control and Nematicide treatments. * * *

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45Table 4-1. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a ‘Tifway 419’ bermudagrass fairway at 0 to 16 weeks after treatment during trial 1. Data are means and mean standard error of four replications. *, **, *** Untreated control different from nematicide treated using orthogonal contrast at a specified nitrogen (N) fertility level. a Kilograms of N per hectare per month. b (CO) Untreated control = no added soil disturbance or nematicide treatment (NT) Nematicide = Injection of 1-3, dichloropropene at a rate of 46.76 liters per hectare at 13 to 17 cm of soil depth. c Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as maximum turf quality, and 6.5 as the threshold for acceptability. N Fertilitya Weeks Treatment 0 36.65 73.30 109.95 0 COb 4.25 0.14c 4.38 0.13 4.63 0.24 4.25 0.32 NT 4.25 0.14 4.50 0.00 4.75 0.14 4.88 0.24 2 CO 4.25 0.14 4.63 0.24 4.38 0.24 4.63 0.38 NT 4.75 0.14 5.00 0.20 5.25 0.32 5.75 0.60 4 CO 4.88 0.24 5.25 0.25 4.63 0.13 4.75 0.25 NT 5.38 0.55 5.50 0.29 5.63 0.24 6.00 0.50 6 CO 5.38 0.13 4.75 0.14 4.63 0.13 4.75 0.14 NT 5.13 0.24 5.25 0.14 5.13 0.31 5.13 0.24 8 CO 4.63 0.24 4.50 0.20 4.63 0.24 4.88 0.31 NT 5.38 0.24 5.38 0.13 5.25 0.32 5.50 0.41 10 CO 4.75 0.25 4.50 0.20 ** 4.56 0.21 4.75 0.32 NT 5.31 0.19 5.25 0.10 5.69 0.31 5.19 0.61 12 CO 4.38 0.13 4.38 0.13 ** 4.38 0.13 4.44 0.26 NT 5.13 0.13 5.38 0.13 5.25 0.25 4.88 0.43 14 CO 4.63 0.24 4.50 0.35 4.25 0.48 4.50 0.50 NT 5.00 0.20 4.88 0.43 5.00 0.20 4.63 0.72 16 CO 4.81 0.24 4.94 0.21 4.13 0.33 5.19 0.31 NT 5.44 0.39 5.44 0.39 5.56 0.33 5.38 0.43

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46Table 4-2. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a ‘Tifway 419’ bermudagrass fairway at 0 to 16 weeks after treatment during trial 2. N Fertilitya Weeks Treatment 0 36.65 73.30 109.95 0 COb 6.28 0.09c 6.69 0.12 6.16 0.25 7.20 0.16 NT 6.31 0.14 6.47 0.27 6.39 0.24 7.28 0.12 2 CO 6.34 0.16 *** 6.25 0.17 *** 6.02 0.19 *** 6.89 0.18 *** NT 7.14 0.09 7.34 0.29 7.23 0.06 7.48 0.13 4 CO 5.91 0.23 5.55 0.42 5.33 0.43 *** 5.52 0.32 *** NT 6.77 0.25 6.44 0.24 6.86 0.14 7.20 0.17 8 CO 6.53 0.18 6.20 0.34 6.00 0.34 ** 6.05 0.32 *** NT 5.98 0.21 6.72 0.24 7.31 0.21 7.36 0.30 10 CO 6.08 0.23 6.39 0.27 5.09 0.39 *** 5.50 0.35 *** NT 6.58 0.15 6.64 0.24 7.22 0.20 7.45 0.33 12d CO 6.59 0.12 6.77 0.09 5.92 0.12 *** 6.61 0.14 *** NT 6.72 0.15 6.66 0.17 7.17 0.11 7.81 0.09 14 CO 6.42 0.14 6.64 0.14 5.56 0.32 *** 6.64 0.22 *** NT 6.72 0.17 6.81 0.24 7.28 0.15 7.72 0.09 16 CO 7.20 0.17 7.02 0.11 *** 6.22 0.35 6.48 0.11 *** NT 7.42 0.14 5.73 0.34 6.22 0.15 7.19 0.11 Data are means and mean standard error of 16 replications. *, **, *** Untreated control different from nematicide treated using orthogonal contrast at a specified nitrogen (N) fertility level. a Kilograms of N per hectare per month. b (CO) Untreated control = no added soil disturbance or nematicide treatment (NT) Nematicide= Injection of 1,3-dichloropropene at a rate of 46.76 liters per hectare at 13 to 17 cm of soil depth. c Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as maximum turf quality, and 6.5 as acc eptable. d Between weeks 10 and 12 an unscheduled fertilizer application of a slow release blend of 21-0-18 (N-P2O5-K2O) was evenly applied across all experimental plots at a rate of 336.58 kg/ha; Nutrient rates were 70.68 kg N/ha and 50.28 kg K (broadcast).

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47Table 4-3. Total root lengths observed in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertili ty levels on a ‘Tifway 419’ bermudagrass fairway at 0, 6, and 16 weeks after treatment during both trials. N Fertilitya Weeks Treatment 0 36.65 73.30 109.95 Trial 1 0 COb 580.90 153.89c 681.08 142.63 577.04 111.78 499.88 118.34 NT 704.31 265.99 420.56 141.51 526.59 121.89 847.31 253.25 6 CO 263.24 66.89 321.59 64.72 363.19 59.81 308.23 84.39 NT 359.71 48.42 401.58 74.96 434.20 88.89 443.85 85.96 16 CO 217.82 84.89 217.29 99.83 187.68 69.85 200.69 37.42 NT 199.32 55.16 264.74 37.38 249.33 40.47 299.66 60.05 Trial 2 0 CO 154.77 33.84 85.84 22.70 85.63 33.20 171.47 52.58 NT 134.14 36.75 84.51 30.25 148.08 52.78 158.76 27.69 6 CO 173.08 28.43 91.43 19.75 129.45 53.83 157.14 39.15 NT 226.14 46.43 240.79 81.22 281.20 72.85 234.86 43.08 16 CO 170.54 42.64 94.51 24.42 85.16 30.65 126.84 26.83 NT 187.18 29.99 191.80 45.53 159.91 44.48 203.00 64.23 Data are means and mean standard error of 8 and 12 replications for trial 1 and 2, respectively. *, **, *** Untreated control different from nematicide treated using orthogonal contrast at a specified nitrogen (N) fertility level. a Kilograms of N per hectare per month. b (CO) Untreated control = no added soil disturbance or nematicide treatment (NT) Nematicide = Injection of 1,3-dichloropropene at a rate of 46.76 liters per hectare at 13 to 17 cm of soil depth. c Root length are presented as millimeters.

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48 Some models each year were statistically significant ( P 0.05). However, correlations were low, preventing further comparisons (Figure C-1A, C-1B) (Table C-14, C-15). Discussion Reduced ( P 0.05) B. longicaudatus populations counts were observed in nematicide treated plots compared to untreated controls at 2, 4, and 6 weeks after treatment during both trials. These reductions in B. longicaudatus populations counts indicate adequate coverage and rates of nematicide treatments for B. longicaudatus control during these trails. Likewise, a general decline in nematode populations over time was observed during both trails, which may indicate a seasonal reduction of nematode populations from March through August. Further studies of B. longicaudatus population dynamics may be warranted to determine when best to apply nematicide treatments for a maximum benefit to turf stands. No differences ( P 0.05) were observed between control and mechanical treatments, indicating the slit-injection process by itself has minimal effect on B. longicaudatus populations (Figure 4-1A, 4-1B). Turf quality is a function of turf color and density, which is generally used to infer turf health status. Therefore turf quality was focused upon during these studies. Differences ( P 0.05) were observed between untreated control and nematicide treated plots within individual N fertility levels with respect to turf quality, color, and density at some dates during both trials (Table 4-1, 4-2). Differences ( P 0.05) in turf quality were observed between untreated control and nematicide treated plots within each N fertility level at 2 and 4 weeks after treatment and 8 to 16 weeks after treatment at the higher N fertility levels of 73.30 and 109.95 kg N/ha/month during trial 2. The irregular turf quality, color, and density response to nematode management and increased N fertility during trial 1 may have been caused by salt induced phytotoxicity that occurred from high rates of potassium nitrate that were not irrigated properly. Similarly, lack of root development may have hindered the ability of the turf to take up water and nutrients for plant development (Christie, 1959; Johnson, 1970). During trial 2, differences ( P 0.05) were observed in turf quality at 2 and 4 weeks after treatment at each N fertility level, which

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49 corresponded with reduced B. longicaudatus populations counts. Even with root development hindered, reduced parasitism by B. longicaudatus may have allowed more water and nutrients to reach leaf tissue leading to increased production, storage, and use of photosynthates which could be utilized for plant development or during times of drought stress. Furthermore, reduced parasitism coupled with possible increased reserves of photosynthates may explain differences ( P 0.05) in turf quality that were observed between untreated control and nematicide treatments following each of the irrigation system failures. While reduced turf quality was observed in both the untreated control and nematicide treated plots following irrigation system failures, turf quality losses were minimized in nematicide treated plots. Upon reviewing turf quality following the first irrigation failure, drought damage was greatest in untreated control plots at N fertility levels 73.30 and 109.95 kg N/ha/month. The drought damage continued to hinder turf quality in untreated control plots at N fertility levels 73.30 and 109.95 kg N/ha/month for several weeks and was reinforced during week 9 with a second irrigation failure. This situation illustrates that increased watering and N fertility can improve turf quality in the short term, however if adverse conditions such as drought stress, heat stress, or improper mowing occur on turf stands suffering nematode infestations, then turf quality can be reduced for an extended period of time (Lucas, 1982). No differences ( P 0.05) in root length and surface area of specified root diameters, total root length, total surface area, or root weight were observed between nematode management tactics at individual N fertility levels at 0, 6, and 16 weeks after treatment during either trial. These results differ from results reported by Crow et al. (2003), which revealed increases ( P 0.05) in total root length following slit injections of 1,3-dichloropropene. However during both trials, root systems possessed necrotic tissue, which may indicate another factor in addition to nematodes could have been suppressing root development. In conclusion, nematode management has been shown to improve turf quality under some conditions, most notably nematode management helps to minimize turf quality loses when turf experiences stress under field conditions (Crow et al., 2003). Likewise, increasing N fertility has

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50 also been shown to improve turf quality. While this experiment was unable to determine whether nematode management with increasing N fertility levels improved turf quality, it indicates that increased N fertility without nematode management could be detrimental to turf quality, especially when the turf experiences stress. No inferences can be made with respect to nematode management and N fertility in relation to root length. Lack of root development during both trials illustrates that nematode management is but 1 concern that must be addressed with respects to turf root systems.

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51 CHAPTER 5 SUMMARY These experiments confirm that Belonolaimus longicaudatus is a pathogen on bermudagrasses in Florida. Research revealed that feeding by plant parasitic nematodes can reduce turf root systems, allowing increased amounts of nitrates to be leached. Furthermore, this research is a foundation for later projects concerning: timing of nematicide applications, turf reductions by B. longicaudatus in the presences of nematode antagonists, turf reductions and nitrate leaching with minimal water and N fertility inputs. Glasshouse experiments revealed differences ( P 0.05) between uninoculated and inoculated turf in total root length at 6, 12, and 18 weeks and milligrams of nitrate leached at 18 weeks during both trials. Belonolaimus longicaudatus feeding reduced total root length by 30 to 94 percent, and increased the amount of nitrate leaching as much as 429 percent (Chapter 3). However, differences ( P 0.05) between uninoculated and inoculated turf in amount of nitrate leached were not observed until reductions in root length and surface area were observed in a large portion of root diameters at all rooting depths in actively growing root systems (Appendix C). No differences ( P 0.05) were observed between nematode inoculated and uninoculated lysimeters in tissue nitrogen levels, dry matter production, or total nitrogen uptake (Chapter 3). This directly relates to the ability of the entire root system of actively growing turf to assimilate N (Clarkson and Hanson, 1980). When the amount of cumulative nitrate leached was compared between treatments, root systems inoculated with B. longicaudatus leached more nitrate than did uninoculated root systems ( P 0.05) in trial 1, but not in trial 2. No differences ( P 0.05) were observed for turf quality, color, or density during either trial, confirming that through intensive management of water, nutrients, pests, and pathogens; turf appearance can be maintained even with root damage from plant parasitic nematodes. Subsequently, the intensive management of turf

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52 to maintain turf quality, color, and density coupled with nematode damage to turf roots may increase nitrate leaching, thereby adding to water quality concerns. In field experiments, reduced ( P 0.05) B. longicaudatus populations counts were observed between untreated control and 1,3-dichloropropene treatments at 2, 4, and 6 weeks after treatment during both trials. No differences ( P 0.05) were observed between control and mechanical treatments, indicating the slit-injection process has no detectable effect on B. longicaudatus populations (Figure 4-1A, 4-1B). Furthermore, a general decline in nematode populations over time was observed during both trails, which may indicate further study of B. longicaudatus population dynamics is warranted to determine when best to apply nematicide treatments for a maximum benefit to turf stands. Differences ( P 0.05) were observed between untreated control and nematicide treated plots within individual N fertility levels with respect to turf quality, color, and density at some dates during both trials. During trial 2, differences ( P 0.05) were observed in turf quality at 2 and 4 weeks after treatment which corresponded with reduced B. longicaudatus counts. Reduced parasitism by B. longicaudatus may have allowed more water and nutrients to reach leaf tissue leading to increased production, storage, and use of photosynthates which could be utilized for plant development or during times of drought stress. Differences ( P 0.05) were observed in turf quality from 8 to 16 weeks after treatment at N fertility levels of 73.30 and 109.95 kg N/ha/month. Reduced parasitism coupled with possible increased reserves of photosynthates may explain differences ( P 0.05) in turf quality that were observed between untreated control and nematicide treated plots following each of the irrigation system failures. While reduced turf quality was observed in both the untreated control and nematicide treated plots following irrigation system failures, turf quality losses were minimized in nematicide treated plots. Upon reviewing turf quality following the first irrigation failure, drought damage was greatest in untreated control plots at N fertility levels 73.30 and 109.95 kg N/ha/month. The drought damage continued to hinder turf quality in untreated control plots at N

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53 fertility levels 73.30 and 109.95 kg N/ha/month for several weeks and was reinforced during week 9 with a second irrigation failure. No differences ( P 0.05) in root length and surface area of specified root diameters, total root length, total surface area, or root weight were observed between untreated control and nematicide treated plots within individual N fertility levels at 0, 6, and 16 weeks after treatment during either trial. These results differ from results reported by Crow et al. (2003), which revealed increases ( P 0.05) in total root length following slit injections of 1,3-dichloropropene. However, root systems possessed necrotic tissue, which may indicate another factor other than nematodes could have been suppressing root development. Lack of root development illustrates that nematode management is but 1 concern that must be addressed with respects to turf root systems. In conclusion, glasshouse studies indicated that nematode damage to turf roots can increase nitrate leaching, thereby adding to water quality concerns. Field experiments indicate that increased N fertility without nematode management could be detrimental to turf quality, especially when the turf experiences stress. These studies indicate that nematode and N fertility management are equally important to providing a quality turf and minimizing environmental impacts.

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54 APPENDIX A DETAILED MATERIALS AND METHODS USED IN THE GREENHOUSE STUDY Introduction A 40-lysimeter greenhouse experiment consisting of two trials was conducted over a 16month period at the University of Florida Turfgrass Envirotron glass houses in Gainesville, Florida from 29 January 2002 to 13 September 2002, and from 1 November 2002 to 16 April, 2003. Data were collected over a 126-day period during each trial. The objective of this study was to determine if damage caused by Belonolaimus longicaudatus (Rau, 1958) to turfgrass roots could increase nitrate leaching and reduce nitrogen uptake by the turfgrass plant. Experimental Materials Lysimeters Lysimeters were constructed of 15-cm-diam. polyvinyl chloride (PVC) pipe cut in 45.75 cm lengths. Heavy-duty window screen was cut in 23 cm x 23 cm squares and placed over 1 open end of the pipe held on by a rubber band. Then a 1.27 cm hole was drilled into the center of a 15cm-diam. PVC end cap. A threaded brass reducer bushing (1.27-cm-exterior-diam. male to 0.95cm-interior-diam. male) was threaded into the predrilled hole. The PVC end cap was then carefully hammered onto the end of pipe with a rubber mallet and excess screen was cut away. A bead of white bathtub caulking was placed around the exterior junction of the pipe and cap. The caulking was allowed to dry at least 12 hours and then the lysimeters were checked for leaks (Figure A-1, A-2, A-3). Turf A stand of ‘Tifdwarf’ bermudagrass was obtained from Dr. G. L. Miller at the University of Florida Turfgrass Envirotron in September, 2001. Forty clay pots measuring 10.16-cm-diam.

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55 Figure A-1. A lysimeter used as an experimental unit during glasshouse trials at the University of Florida Turfgrass Envirotron from 29 January 2002 to 16 April 2003. Figure A-2. Screen placed within a lysimeter after assembly. The screen holds the soil profile away from drainage hole preventing drain blockage.

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56 Figure A-3. A threaded bushing screwed into a lysimeter. The bushing enables leachate to be collected. were filled with nematode free sand and sprigged with aerial stolons of ‘Tifdwarf’ bermudagrass. During January 2002 and November 2002 aerial stolons were cut at least 7.62 cm 0.1 cm from the pot, collected, weighed, and sprigged into the lysimeters. Soil Properties The United States Golf Association (USGA) sand mix was tested for soil particle requirements established by the USGA for greens construction (Anonymous, 1993). A 2,000 cm3 soil sample was obtained from the sand being placed into the lysimeters. The sample was spread out onto an oven rack covered with newspaper, then placed into a drying oven for approximately 48 hours at 70 C. The soil sample was run through a series of sieves stacked 1 on top of another to separate soil particles by size. Particle sizes in mm of interest were: < 0.15, 0.15 to 0.25, 0.25 to 0.5, 0.5 to 1.0, 1.0 to 2.0, and > 2.0 mm. Sand mix used in these trials met USGA specifications. Nematode Inoculum Trail 1: A mixed population of nematodes was collected from a golf course green in Palatka, FL. Nematodes were extracted from the soil using a modified Baermann method

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57 (McSorley and Frederick, 1991). Belonolaimus longicaudatus were hand picked from the mixed population of nematodes and placed into a small glass beaker containing 10 ml of water until proper inoculum levels were reached. Trail 2: A pure population of B. longicaudatus was obtained from Dr. R.M. Giblin-Davis, which originated from Sanford, FL (Giblin-Davis et al., 1992). Eight clay pots measuring 15-cmdiam. were filled with nematode free sand and then sprigged with ‘Tifdwarf’ bermudagrass. After turf establishment, nematode inoculum suspended in 20 ml of water was pipetted into four holes (1-cm-diam. x 2.5-cm-deep) in the soil at a rate of 100 nematodes/pot and allowed to reproduce. Five months later, soil and roots from each pot were separated into four sub samples for convenience. Each sub sample was placed onto a 120-mesh sieve. The roots were firmly rinsed with water, collecting the sand and nematodes in a stainless steel container below. The sand and nematodes were then agitated with water to separate the nematodes from the soil. The water and nematodes were then poured into a 25 m sieve and collected into a 1,000 ml beaker (Cobb, 1918). After all the sub samples had been run, the volume in the beaker was brought up to 500 ml. 1 ml of water and nematodes was placed onto a counting slide to determine the number of nematodes per ml of water. This process was replicated five times with an average of 15 nematodes per ml. Twenty ml of water and nematode solution were required to deliver proper inoculum levels. Experimental Design The experimental design varied from trial 1 to trial 2. The experimental design in trial 1 was arranged in a completely randomized design with 20 lysimeters inoculated with B. longicaudatus and 20 uninoculated control lysimeters. Trial 2 was arranged in a randomized complete block design. Lysimeters were assigned to 1 of five blocks with eight lysimeters per block (40 total), within each block four lysimeters (20 total) were selected at random to be inoculated with B. longicaudatus

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58 General Production Practices Turf Establishment Trial 1: Forty lysimeters were set up with 15 cm of gravel placed in the bottom of the lysimeters covered with an additional 30 cm of nematode-free sand. Water was run through the lysimeters to remove air pockets and the sand was brought back to 30 cm depth. Each lysimeter was brought to field capacity and weights recorded. Lysimeters were sprigged with nematodefree 'Tifdwarf' bermudagrass at a rate of 218 kg/ha (0.4 g/lysimeter) and top dressed with approximately 0.32 cm of nematode-free sand. Turf was watered six times a day starting at 0700 hours at 2-hour intervals with 8 ml of water from a mister irrigation system during establishment. Turf in each lysimeter was fertilized once, five days after sprigging with 20-20-20 (N-P2O5-K2O) fertilizer (United Industries Corp., St. Louis, MO) at a rate of 8.4 g/liter of water. Nutrient inputs were 91.96 kg/ha N, 40.46 kg/ha P, 76.33 kg/ha K, and trace amounts of essential micronutrients. The turf was allowed to grow-in and establish a root system for six weeks. Trial 2: Forty lysimeters were filled and planted as described in trial 1 except field capacity was not determined. Turf was watered six times a day with 8 ml of water from a mister irrigation system to prevent desiccation during the establishment period. Turf in each lysimeter was fertilized once, five days after sprigging with 20-20-20 (N-P2O5-K2O) fertilizer at a rate of 9.97 g/liter of water. Nutrient inputs were 109.3 kg/ha N, 48.09 kg/ha P, 90.72 kg/ha K, and trace amounts of essential micronutrients. The turf was allowed to grow-in and establish a root system for three weeks. Nematode Establishment Trial 1: Following six weeks of root establishment, 20 lysimeters were inoculated with B. longicaudatus Nematode inoculum suspended in 10 ml of water was poured into four holes (1cm-diam. x 2.5-cm-deep) in the soil at a rate of 138 nematodes/lysimeter and allowed to reproduce for a period of eight weeks.

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59 Trail 2: Following three weeks of root establishment, 20 lysimeters were inoculated with B. longicaudatus Nematode inoculum suspended in 20 ml of water was pipetted into four holes (1cm-diam. x 2.5-cm-deep) in the soil at a rate of 300 nematodes/lysimeter and allowed to reproduce for a period of three weeks. Temperature Trial 1: Monthly average high and low air temperature in the glasshouse ranged from 22 C to 33 C and 18 C to 25C, respectively. Trial 2: Monthly average high and low air temperature in the glass house ranged from 21 C to 26 C and 18 C to 20C, respectively. Watering Trial 1: Following turf and nematode establishment, the turf was watered between 09001000 hours, three times a week. The first and second watering consisted of 150 ml of water per application, followed by a third watering which required adding measured amounts of water until the lysimeters were brought back to field capacity using the prerecorded weights. Total water requirements were recorded for each lysimeter. Trial 2: Following turf and nematode establishment, the turf was watered at 1000 and 1500 hours, daily with 25 3 ml of water from an overhead mister irrigation system. Fertilization Trial 1: Following turf and nematode establishment, turf was fertilized with Potassium Nitrate 14-0-46 (N-P2O5-K2O) at a rate of 668.8 kg/ha/application. Nutrient inputs were 91.96 kg/ha N and 255.35 kg/ha K. Applications were made at three-week intervals immediately after leaching events. Trial 2: Following turf and nematode establishment, turf was fertilized with Potassium Nitrate 14-0-46 (N-P2O5-K2O) at a rate of 794.9 kg/ha/application. Nutrient inputs were 109.3 kg/ha N and 303.5 kg/ha K. Applications were made at three-week intervals immediately after leaching events.

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60 Pesticides The primary arthropod pest species found attacking ‘Tifdwarf’ bermudagrass in these trials was bermudagrass mite ( Eriophyes cynodoniensis ). This pest was managed with two applications of fluvalinate over a seven-day period. Treatments were repeated as needed. Sampling and Evaluations Turf Evaluations Turf evaluations were conducted every three weeks after turf and nematode establishment. Turf quality and color were evaluated on a 1 to 9 scale (1 being poor and 9 being excellent). Turf density was evaluated on percent of live cover (PLC). Nematodes and Roots Trial 1: Nematode population counts and root lengths were assessed 6, 12, and 18 weeks after turf and nematode establishment. Nematode population counts and root lengths were measured from four inoculated and four uninoculated lysimeters selected at random at the sixweek evaluation, five inoculated and five uninoculated lysimeters at the twelve-week evaluations, and 11 inoculated and 11 uninoculated lysimeters at the end of the study. Nematode and root samples were obtained by removing the entire soil profile (15-cm-diam.) of each lysimeter from the soil surface to the rock layer (30.48 0.5-cm-deep). The sample was cut in 7.62 0.1 cm lengths to determine nematode counts and root lengths at four differing depths (0 to 7.62 cm, 7.62 to 15.24 cm, 15.24 to 22.86 cm, and 22.86 to 30.48 cm). Each sub sample was placed into a 135 m sieve. The roots were firmly rinsed with water, collecting the sand and nematodes in a stainless steel container below. The sand and nematodes were then agitated with water to separate the nematodes from the soil. The water and nematodes were poured into a 25m sieve and collected for counting (Cobb, 1918). Roots were collected from the 135 m sieve, placed into a glass container, stained with methylene blue, and refrigerated for at least 24 hours. The equipment was washed at length to prevent contamination between samples. After root staining, root samples were individually placed onto a glass-imaging pan. The roots were spread apart to

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61 prevent overlap and a white background was placed over the roots. The glass-imaging pan was then placed onto a flat bed scanner to obtain a black and white bitmap image of the roots (Figure. A-4) (Kaspar and Ewing, 1997; Pan and Bolton, 1991). The bitmap images were imported into the GSRoot (Louisiana State University, Baton Rouge, LA) software program for analysis. This program is designed to determine root length and surface areas in millimeters for specified root diameters. Root diameters in mm specified for this analysis were: < 0.05, 0.05 to 0.1, 0.1 to 0.2, 0.2 to 0.3, 0.3 to 0.4, 0.4 to 0.5, and > 0.5. Figure A-4. Glass imaging pan with white background placed on a flat bed scanner to obtain bitmap images of root systems. Following root scanning, each root sample was collected from the glass-imaging pan, placed into a small paper bag, and labeled appropriately. The samples were placed into a drying oven for approximately 48 hours at 70 C and then weighed. Trial 2: Nematode population counts and root lengths were assessed 6, 12, and 18 weeks after turf and nematode establishment. Nematode population counts and root lengths were measured from 1 inoculated and 1 uninoculated lysimeter selected at random from each block

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62 (total of ten) at the six and twelve-week evaluations. Nematode and root measurements were collected from the remaining 20 lysimeters (two inoculated and two uninoculated from each block) at the end of the study. Nematode and root samples were obtained by removing 1 core sample (5-cm-diam.) from the middle of each lysimeter. The core sample extended from the soil surface to the rock layer (30.48 0.5 cm). The sample was cut in 7.62 0.1 cm lengths to determine nematode population density and root length and surface area at four differing depths (0 to 7.62 cm, 7.62 to 15.24 cm, 15.24 to 22.86 cm, and 22.86 to 30.48 cm). Each sub sample was placed into a 135m sieve. The roots were firmly rinsed with water collecting the sand and nematodes in a stainless steel container below. The sand and nematodes were then agitated with water to separate the nematodes from the soil. The water and nematodes were then poured into a 25m sieve and collected for counting (Cobb, 1918). Root lengths and dry weights were obtained as described previously. Leachate Trial 1: Leaching events were simulated using three soil pore volumes of water at 21 1day intervals. The leaching technique requires the lysimeters be brought to field capacity and then water added that is equal to 3 times the pore space of the soil (3,750 ml). The leachate from each lysimeter was collected in a separate bucket placed under each lysimeter during the leaching events. Each bucket was brought to a work area and stirred with a plastic utensil. A 20 ml sample was taken from the bucket and the remaining volume of leachate was measured. Samples were analyzed using an air segmented continuous flow auto spectrometer (Flow Solution IV, O.I. Analytical, College Station, TX). The equation [mg NO3/liter volume of leachate recovered] was used to determine the mg of nitrate leached from each lysimeter. Trial 2: Leaching events were simulated as previously discussed, however at 42 1-day intervals.

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63 Turf Tissue Following turf and nematode establishment, turf tissue was collected from each lysimeter separately at 3-week intervals until destructive root and nematode sampling. Each lysimeter was lifted out of the rack and laid on its side on a workbench. Turf was trimmed to 0.95 cm height and collected into small paper bags. Later, each tissue sample was placed into a 75 m sieve washed to remove soil and debris, and then spread evenly on a paper plate. Each sample was placed in a 1000-W microwave oven and dried for two to six minutes depending on sample size (Figure. A5). Following the drying process, each sample was weighed. If sufficient dry matter (1.75 g 0.25 g) was obtained, the tissue was ground in a cycl1 sample mill (Figure. A-6) (Sample Mill, Udy Corporation, Fort Collins, CO) to pass through a 1.0-mm screen, placed into a sampling cell, and loaded into a near infrared reflectance spectroscopy (NIRS) scanning instrument (Model 5000, Foss NIRSystems, Silver Springs, MD). Spectral data was imported into the Toro Diagnostic software program (Version 2.4, The Toro Company, Bloomington, MN) for analysis and values recorded (Figure A-7). The equation [tissue percent N tissue dry weight] was used to determine mg of N uptake. However, if sufficient tissue was not collected during a particular three-week interval the tissue was washed, dried, and stored until sufficient tissue was collected. During trail 2, when destructive root and nematode sampling occurred, stolons and leaf tissue were collected for each lysimeter separately, processed, and analyzed as well. Data Analysis Nitrate leached data collected at 6 weeks after turf and nematode establishment was square root transformed (x +1) to normalize the data. Milligrams of NO3 leached were compared between treatments across all sampling dates using analysis of variance. These T tests were performed to compare turf quality, color, density, root lengths, root surface area, root weight, NO3 leached, tissue dry weights, tissue percent N, and N uptake between treatments at individual sampling dates. Regression analysis was used to characterize relationships between nematode population counts, root length, nitrogen uptake, and nitrate leached. Analysis of variance and T

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64 tests were performed using SAS software (SAS Institute, Cary, NC) while regression analysis was performed using Minitab software (State College, PA). Figure A-5. After washing, turf tissue samples were placed on a paper plate and dried in a 1000W microwave oven for two to six minutes depending on sample size. Figure A-6. Cycl1 sample mill used to grind turf tissue for analysis. Dried tissue is placed into the yellow cone, top center, and is retrieved from the glass jar, bottom center.

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65 Figure A-7. Near infrared reflectance spectroscopy (NIRS) scanning instrument, left. Spectral data determined by NIRS was imported into a laptop computer with Toro Diagnostic software program for analysis, right. Sampling cells waiting to be analyzed, top left, sampling cells previously analyzed, bottom left.

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66 APPENDIX B DETAILED MATERIALS AND METHODS USED IN THE FIELD STUDY Introduction A 2-year field study consisting of two trials was conducted in west central Florida on golf course fairways infested with Belonolaimus longicaudatus (Rau, 1958). Trial 1 was conducted in Citrus County, Florida, from 12 March 2002 to 29 August 2002, while trial 2 was conducted in Pasco County, Florida from 13 March 2003 to 29 August 2003. Data were collected over a 112day period during each trial. The objective of this study was to describe relationships between nematode management and nitrogen fertility in terms of turf quality and root lengths on golf course fairways. Experimental Sites Pathogens Plant-parasitic nematodes present at the Citrus County site included B. longicaudatus, Hopolaimus galeatus (Cobb Thorne) Helicotylenchus sp., Peltamigratus sp., Trichodorus sp., Paratrichodorus sp., Hemicycliophora sp., Hemicric1moides sp., and Mesocric1ma sp. Fungal diseases previously treated for at this site were Bermudagrass Decline ( Gaeumannomyces graminis var. graminis Sacc. Arx. and D.L. Olivier), Brown Patch ( Rhizoctonia solani J. G. Kohn), and Fairy Ring ( Chlorophyllum, Marasmius, or Lepiota spp.). Plant-parasitic nematodes present at the Pasco County site included B. longicaudatus H. galeatus Helicotylenchus sp., Trichodorus sp., and Mesocric1ma sp. Fungal diseases previously treated for at this site were Bermudagrass Decline, Brown Patch, and Damping-Off ( Pythium spp.).

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67 Insects and weeds Pest insects observed during these trials were southern mole cricket ( Scapteriscus borrellii Giglo-Tos), tawny mole cricket ( Scapteriscus vicinus Shudder), fall armyworm ( Spodoptera frugiperda J.E. Smith), red imported fire ant ( Solenopsis invicta Buren), ringlegged earwig ( Euborellia annulipes Lucas), and two lined spittlebug ( Propsapia bicincta L.). Weeds observed during these trials were goosegrass ( Eluesine indica L. Gaertn.), crabgrass ( Digitaria spp. ), crowfootgrass ( Dactyloctenium aegyptium L. Willd.), carpetgrass ( Axonopus affinis Chase), creeping signalgrass ( Brachiaria plantaginea L. A. S. Hitchc.), doveweed ( Murdannia nudiflora L. Brenan), and spotted spurge ( Euphorbia maculata L.). Turf In both trials, golf course fairways had mature stands (15 to 20 years old) of ‘Tifway 419’ bermudagrass [ Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy]. Turf at both locations was maintained at 1.3 cm cutting height and watered as needed. Both fairways had histories of nematode damage over the last few years, requiring more attention to cultural practices and inputs. Soil Properties Soil texture at a depth of 10 to 15 cm was analyzed using the hydrometer method (Bouyoucos, 1936). Soil at the Citrus County site was Tavares fine sand with a composition of 92% sand, 4.5% silt, 3.5% clay; < 1% organic matter, and pH 5.8. Soil at the Pasco County site was Millhopper-Candler Variant soil with a composition of 97% sand, 0% silt, 3% clay; < 1% organic matter, and pH 6.0. Experimental Design The experimental design varied from 2002 to 2003. In 2002, the experimental design was arranged as a split plot design. Whole plots were three nematode management tactics: 1,3dichloropropene (1,3-D) applied by slit-injection (Crow et al., 2003), a mechanical slit treatment with no chemical applied, and untreated control. Each whole plot was replicated four times. Sub

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68 plots consisted of four N rates 0, 36.65, 73.30, 109.95 kg/ha/application. Main plots were 3.7-mwide and 15.2-m-long, with sub plots being 0.9-m-wide and 15.2-m-long. Main plots were separated by border areas (1.5 m on the sides and 3.0 m at each end), which were only mowed and watered. A plot plan illustrating the experimental layout is shown in Fig. B-1. In 2003, the experimental design was arranged in a randomized complete block. Eight treatments were two nematode management tactics: 1,3-dichloropropene and untreated control with four N rates of 0, 36.65, 73.30, 109.95 kg/ha/application. Treatments were replicated four times. These plots were 3.7-m-long and 3.7-m-wide. Plots were separated by border areas (1.5-mwide on all sides), which were maintained as previously stated. A plot plan illustrating the experimental layout is shown in Fig. B-2. The change in experimental design was d1 to reduce the incidents of fertility runoff from 1 sub plot into another. In both trials, nematode samples were collected six weeks prior to nematicide treatments. Plots were assigned to blocks according to B. longicaudatus population counts. Treatments were randomized within each block. Nematicide Treatments Nematicide treatments for the 2002 trial were 1,3-dichloropropene, mechanical, and control. In 2003, the mechanical treatment was eliminated after no differences ( P 0.05) were observed for nematode populations counts or visual performance between mechanical and control plots. Nematicide treatments were applied once per trail during the first week of May. 1,3-dichloropropene was injected at a rate of 46.76 liters/ha with nitrogen gas pressurized application rig. The application rig had straight coulters placed on 30.5 cm centers, followed by a chisel with a metal drip line attached which placed the material at a depth of 13 to 17 cm. A steel roller wheel followed each chisel to close the soil. Mechanical treatments consisted of running the application rig through the soil as preformed with the 1,3-dichloropropene treatment, but without the chemical being applied. Immediately after nematode management tactics were concluded,

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69 approximately 1.25 cm of water was applied, which assisted in holding the 1,3-dichloropropene in the soil. Figure B-1. Plot plan of the field study undertaken at Citrus County, FL during 2002. Plots marked with X were not used as experimental units due to nematode counts being below the action threshold for Belonolaimus longicaudatus 1 2 310 11 12 13 14 15 CU CUCU M M CO CO 0123 0123 0123 120 3 120 3 120 3 2301 2301 23013012 3012 3012X X X X X X4 5 6 7 8 9 16 17 18 CU M M CO CO 1.5 m 3 m 14.0 m 3.7 m 0.9 m 15.2 m 106.7 m NCO = Control M = Mechanical (No nematicide) CU = 1-3, Dichloropropene 0 = No N added experimentally 1 = 36.65 kg N/ha/month 2 = 73.30 kg N/ha/month 3 = 109.95 kg N/ha/month

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70 Figure B-2. Plot plan of the field study undertaken at Pasco County, FL during 2003. Plots marked with X were not used as experimental units due to nematode counts being below the action threshold for Belonolaimus longicaudatus 10 31 11 1 30 40 CO0 CO0 CO0 CO0X X X X X XCO1 CO2 CO3 CO1 CO1 CO1 CO2 CO3 CO3 CO3 CU0 CU2 CU3 CU3 CU3 CU2 CU2 CU1 CU1 CU1 CU0 CU0 CU3 CO2 CO2 CU1 CU2 21 20X XCU0 19.2 m 53.3 m 3.66 m 3.66 m 1.5 m NCO = Control CU = 1-3, Dichloropropene 0 = No N added experimentally 1 = 36.65 kg N/ha/month 2 = 73.30 kg N/ha/month 3 = 109.95 kg N/ha/month

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71 Fertilization The turf fertilization program varied from 2002 to 2003. In 2002, fertilization began two weeks prior to nematode management treatments being applied and continued at two-week intervals until the end of the study. Turf was fertilized with Potassium Nitrate 14-0-46 (N-P2O5K2O) at N rates of 0, 36.65, 73.30, and 109.95 kg/ha /month using a drop spreader. During the 2002 trial, when fertilizer was applied without the turf being watered, salt induced phytotoxicity occurred. Portions of subplots fertilized with N rates of 36.65, 73.30, and 109.95 kg/ha/month showed proportional damage depending on fertilizer rates. This problem was corrected in 2003 by using a slow release fertilizer, which consisted of Sulfur Coated Urea, Sulfur Coated Ammonium Phosphate, Sulfur Coated Sulfate of Potash, Iron Oxide, and Manganese Sucrate. In 2003, fertilization began four weeks prior to nematode management treatments being applied and continued at two-week intervals until the end of the study. Turf was fertilized with a 14-14-14 (N-P2O5-K2O) sulfur coated blend at N rates of 0, 36.65, 73.30, 109.95 kg/ha /month (broadcast). In 2003, an unscheduled fertilizer application occurred during week 11 with a slow release blend of 21-0-18 (N-P2O5-K2O) at an N rate of 70.68 kg/ha (broadcast). General Production Practices Turf Maintenance In both trails, turf was mowed by the golf courses staff three times a week at a cutting height of 1.3 cm. However, on several occasions the turf was not mowed due to rain. Cultural practices conducted by golf course staff such as aerification, slicing, and vertical mowing were halted for this experiment. In 2002, turf was irrigated with 0.64 cm of water as needed. Conversely, in 2003 turf was irrigated once a day with 0.64 cm of water until week 3 when the irrigation system failed causing the turf to go without watering for 3 to 4 days. Thereafter, turf was irrigated twice a day with 0.64 cm of water except during week 9 when the irrigation system failed again causing the turf to go without watering for 3 to 4 days.

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72 Pesticides In 2002, no additional management tactics were warranted to control insects, weeds, or pathogens, except experimental treatment. 1 management tactic was implemented on 14 July 2003 (week 10 of trail 2), when MSMA 2.25 kg a.i./ha and metribuzin 0.035 kg a.i./ha were tank mixed and applied as a spot treatment to control Eluesine indica Sampling and Evaluations Turf Evaluations Turf evaluations were conducted every two weeks, beginning with the first N fertility treatment each year. Turf quality and color were evaluated on a 1-9 scale (1 being poor, 6.5 acceptable, and 9 being excellent). Turf density was evaluated on percent of live cover (PLC). In 2002, each subplot (0.91m x 15.24 m) was evaluated as a whole, which made evaluations difficult. Subsequently, in 2003 each plot was divided into four equal quadrants, each quadrant was evaluated for turf quality, color, and density. Nematodes In both trials, twelve cores (2.5-cm-diam and 10.2-cm-depth) were obtained from each plot using a c1 sampler to determine nematode population counts. A 15 cm buffer z1 was established inside the parameter of each treatment plot to ensure accurate treatment results. Samples were taken twice prior to nematicide treatment (six weeks and 1 day prior to nematicide treatments), and at two-week intervals following nematicide treatments. Each sample was mixed thoroughly and a 100-cm3 sub sample was obtained. Nematodes were extracted from the soil using a modified centrifugal-flotation technique (Jenkins, 1964). Traditionally, the extraction process requires the soil to be passed through a 2 mm sieve to remove debris, however this step was omitted to prevent B. longicaudatus from being lodged in the mesh of the sieve (McSorley and Fredrick, 1991). Following, extraction all plant-parasitic nematodes were counted using an inverted light microscope at a magnification of 20 x.

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73 Roots Root samples (3.5-cm-diam and 15-cm-depth) were obtained inside the buffer z1 with a tee sampler. In 2002, two root cores were collected at 0, 6, and 16 weeks after nematicide treatment from each treatment plot and combined into a single sample with no differences observed ( P 0.05). Previous studies had shown increases in root length when plant-parasitic nematodes were being managed, so an increase in sample size was suggested. In 2003, three root cores were collected at 0, 6, and 16 weeks after nematicide treatment from each treatment plot. In both trials, roots were processed, analyzed, and weighted as described in Appendix A. Turf Tissue Turf tissue was collected every two-weeks, beginning with the first N fertility treatment each year. In 2002, tissue samples were collected from three 30 cm x 30 cm areas within each treatment plot, however in 2003 tissue was collected from the entire treatment plot (3.7 m x 3.7 m). Turf was trimmed to 0.95 cm height during both trails. Tissue samples were processed and analyzed as described in Appendix A. However, since large amounts of tissue were collected from each treatment plot, following the grinding step each sample was thoroughly mixed and a 2.0 g 0.5 g sub sample was obtained for analysis. Data Analysis ANOVA were performed to compare turf quality, color, density, root lengths, root surface area, root weight, tissue dry weights, tissue percent N, and N uptake among treatments at individual sampling dates. Due to interactions between nematode management tactics and N fertility, orthogonal contrasts were performed at individual N fertility levels to compare individual treatments. Regression analysis was used to characterize relationships between nematode population counts and fertility in terms of turf quality and root lengths on golf course fairways. ANOVA, general linear models, and orthogonal contrasts were performed using SAS software (SAS Institute, Cary, NC) while regression analysis was performed using Minitab software (State College, PA).

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74 APPENDIX C SUPPLEMENTAL FIGURES AND TABLES Effects of Nematode and N Fertility Management during Trial Oney = -0.0001x + 4.68 r2 = 7E-05 y = 0.0014x + 4.55 r2 = 0.014 y = 0.003x + 5.16 r2 = 0.0444 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 020406080100120 kg N/ha/monthTurf Quality Control Mechanical Nematicide Effects of Nematode Management and N Fertility during Trial Twoy = 0.0079x + 6.41 r2 = 0.154 y = 0.0018x + 6.39 r2 = 0.0076 6.2 6.4 6.6 6.8 7 7.2 7.4 020406080100120 kg N/ha/monthTurf Qaulity Nematicide Control Figure C-1. Regression models of turf quality response to Untreated Control, Mechanical = disruption with slit injection equipment without nematicide, and Nematicide = slit injection of 1,3-dichloropropene at a rate of 46.76 liter per hectare at N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month through out trial 1 (A) and trial 2 (B). A B

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75Table C-1. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 6 weeks after turf and nematodes establishment during trial 1. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 214e 1,526 1,606 901 585 443 1,828 7,104 3,347 1 I 122 ** 936 1,018 594 413 290 1,229 4,570 ** 2,075 2 U 131 913 967 557 356 266 1,145 4,335 2,011 2 I 90 697 737 453 301 233 1,054 3,564 1,524 3 U 84 622 636 368 259 198 875 3,043 1,342 3 I 70 420 *** 438 244 ** 157 *** 113 ** 390 ** 1,832 *** 928 ** 4 U 69 544 569 327 226 172 621 2,528 1,183 4 I 62 442 446 256 160 129 496 1,991 949 All U 499 3,606 3,779 2,153 1,426 1,078 4,470 17,011 7,883 All I 345 ** 2,494 ** 2,638 ** 1,547 ** 1,032 ** 764 *** 3,170 11,957 *** 5,476 ** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. e Mean values of root length in millimeters for replications.

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76Table C-2. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 12 weeks after turf and nematodes establishment during trial 1. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 166e 1,164 1159 726 500 362 1,952 6,028 2,488 1 I 102 732 801 491 352 279 1,237 ** 3,994 ** 1,635 2 U 108 841 797 488 333 302 1,516 4,385 1,746 2 I 85 623 646 357 246 200 751 ** 2,908 1,354 3 U 88 639 638 372 239 210 1,008 3,195 1,366 3 I 55 360 ** 356 ** 179 ** 117 ** 93 ** 284 *** 1,445 *** 772 *** 4 U 70 583 614 403 237 222 953 3,083 1,267 4 I 65 414 *** 364 *** 163 *** 105 *** 85 ** 202 *** 1,399 *** 844 *** All U 432 3,227 3,208 1,989 1,309 1,097 5,430 16,691 6,867 All I 308 2,129 ** 2,167 1,190 ** 820 ** 658 *** 2,474 *** 9,746 ** 4,604 ** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. e Mean values of root length in millimeters for replications.

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77Table C-3. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 18 weeks after turf and nematodes establishment during trial 1. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 183e 1,347 1334 783 578 417 2,166 6,809 2,864 1 I 87 *** 655 *** 613 *** 358 *** 254 *** 197 *** 1,007 *** 3,170 *** 1,355 *** 2 U 101 652 653 371 268 197 1,057 3,299 1,407 2 I 67 *** 494 *** 510 *** 296 *** 210 *** 161 ** 721 *** 2,459 *** 1,071 *** 3 U 71 502 487 291 209 160 847 2,565 1,060 3 I 56 *** 416 399 ** 224 143 *** 100 *** 422 *** 1,759 *** 871 ** 4 U 75 520 495 284 199 160 743 2,476 1,090 4 I 53 *** 351 *** 335 *** 159 *** 104 *** 73 *** 220 *** 1,209 *** 739 *** All U 430 3,022 2,969 1,729 1,254 934 4,812 15,151 6,420 All I 263 *** 1,916 *** 1,857 *** 1,037 *** 711 *** 531 *** 2,369 *** 8,597 *** 4,036 *** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. e Mean values of root length in millimeters for replications.

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78Table C-4. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 6 weeks after turf and nematodes establishment during trial 2. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 15.14e 122.56 166.71 74.20 42.57 20.97 33.34 475 304 1 I 6.65 37.76 ** 54.05 ** 41.43 25.42 17.85 33.05 216 98 ** 2 U 6.70 51.21 53.08 24.10 11.73 6.68 11.02 165 111 2 I 4.08 22.50 21.24 8.48 4.27 2.34 2.65 66 48 3 U 4.06 24.99 34.20 11.91 6.19 2.31 5.39 89 63 3 I 5.40 27.22 21.84 9.29 4.22 2.78 3.94 75 55 4 U 3.37 27.94 24.42 10.06 6.33 2.32 3.17 78 56 4 I 3.56 14.53 12.60 8.00 3.81 2.17 3.01 48 31 All U 29.27 226.70 278.41 120.27 66.82 32.27 52.92 807 534 All I 19.68 102.00 109.74 ** 67.20 37.71 25.14 42.65 404 231 ** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. e Mean values of root length in millimeters for replications.

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79Table C-5. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 12 weeks after turf and nematodes establishment during trial 2. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 14.84e 111.36 139.34 78.75 47.13 31.98 55.16 479 266 1 I 5.82 34.86 38.17 19.47 12.31 8.79 18.16 138 79 2 U 9.38 70.21 79.48 27.70 14.44 9.38 12.17 223 159 2 I 5.79 44.08 41.91 10.48 2.42 2.41 3.76 111 92 3 U 6.72 49.74 46.67 19.71 8.28 5.50 7.17 144 103 3 I 6.60 35.08 31.89 6.48 2.31 2.16 1.64 86 74 4 U 10.79 91.84 94.57 56.07 24.09 17.53 36.19 331 197 4 I 2.63 24.19 27.64 9.91 2.18 2.25 2.20 71 54 All U 41.73 323.15 360.07 182.23 93.93 64.39 110.70 1,176 725 All I 20.84 138.21 ** 139.61 46.51 19.22 15.61 25.76 406 299 *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. e Mean values of root length in millimeters for replications.

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80Table C-6. Root lengths of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges 18 weeks after turf and nematodes establishment during trial 2. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 23.46e 64.65 85.82 99.47 300.15 125.78 1,101.50 1,801 174 1 I 3.94 *** 23.81 25.48 ** 13.25 *** 9.56 ** 5.62 ** 17.37 ** 99 ** 53 ** 2 U 22.52 60.84 69.27 90.22 274.64 104.38 606.06 1,228 153 2 I 4.19 *** 24.59 19.75 ** 8.51 *** 5.88** 4.20 ** 9.72 ** 77 ** 48 ** 3 U 15.29 47.65 53.39 59.27 140.38 64.70 396.60 777 116 3 I 2.13 *** 14.31 15.19 6.22 *** 2.75 1.71 ** 5.54 48 ** 32 ** 4 U 10.45 37.22 39.91 37.26 91.58 38.86 187.57 443 88 4 I 1.15 ** 8.10 7.54 3.12 ** 1.69 1.43 ** 3.10 26 ** 17 All U 71.72 210.35 248.38 286.22 806.75 333.73 2,291.70 4,249 530 All I 11.41 *** 70.81 67.96 *** 31.09 *** 19.87 ** 12.96 *** 35.73 ** 250 ** 150 ** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. e Mean values of root length in millimeters for replications.

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81Table C-7. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 6 weeks after turf and nematodes establishment during trial 1. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 0.92e 60 215 210 186 175 1,505 2,352 276 1 I 0.59 34 ** 137 141 134 116 1,030 1,594 172 2 U 0.52 31 124 121 113 104 976 1,470 156 2 I 0.51 23 96 102 95 94 948 1,359 120 3 U 0.34 20 81 81 82 80 748 1,092 102 3 I 0.22 14 ** 55 56 50 *** 41 *** 308 523 ** 69 4 U 0.34 18 74 71 70 70 474 777 92 4 I 0.20 15 56 ** 57 52 48 422 650 71 ** All U 2.12 129 495 483 450 428 3,703 5,691 626 All I 1.51 87 ** 345 ** 356 ** 330 ** 300 *** 2,707 4,126 433 ** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. e Mean values of root surface area in square millimeters for replications.

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82Table C-8. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 12 weeks after turf and nematodes establishment during trial 1. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 0.73e 42 152 168 161 146 1,865 2,534 195 1 I 0.51 27 108 117 109 106 1,165 1633 ** 136 2 U 0.36 29 106 123 110 110 1,269 1,747 136 2 I 0.31 21 83 85* 74 75 620 ** 957 ** 104 3 U 0.32 20 82 88 74 81 904 1,250 103 3 I 0.20 12 45 ** 43 ** 35 *** 34 *** 203 *** 372 *** 57 ** 4 U 0.33 20 81 110 72 65 841 1,189 101 4 I 0.17 14 ** 44 *** 38 *** 32 *** 32 ** 141 ** 301 ** 58 *** All U 1.74 112 420 488 416 402 4,878 6,720 534 All I 1.20 74 281 283 ** 250 ** 246 *** 2,128 *** 3,263 *** 356 *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. e Mean values of root surface area in square millimeters for replications.

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83Table C-9. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 18 weeks after turf and nematodes establishment during trial 1. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 0.52e 47 169 188 165 168 2,039 2,788 215 1 I 0.31 23 *** 79 *** 85 *** 79 *** 77 *** 961 *** 1,305 *** 103 *** 2 U 0.26 25 78 85 81 82 981 1,333 103 2 I 0.37 17 *** 65 ** 68 *** 67 ** 64 ** 630 *** 912 *** 83 *** 3 U 0.28 18 63 67 67 66 776 1,056 81 3 I 0.25 14 50 ** 51 45 *** 40 *** 340 *** 539 *** 64 ** 4 U 0.28 18 62 65 63 65 672 945 81 4 I 0.18 12 *** 41 *** 36 *** 30 *** 46 256 ** 433 ** 54 *** All U 1.34 107 372 405 376 380 4,467 6,121 480 All I 1.11 67 *** 235 *** 239 *** 221 *** 227 *** 2,186 *** 3,188 *** 303 *** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter. e Mean values of root surface area in square millimeters for replications.

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84Table C-10. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 6 weeks after turf and nematodes establishment during trial 2. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 0.06e 5.14 22.73 18.25 13.78 8.93 19.20 88 28 1 I 0.01 1.80 ** 8.23 ** 10.64 8.93 8.01 21.89 60 10 ** 2 U 0.02 2.04 6.97 5.47 3.43 2.51 7.33 28 9 2 I 0.00 0.94 3.13 1.92 1.56 0.84 1.71 10 4 3 U 0.00 1.11 4.57 2.92 6.06 1.04 3.73 16 6 3 I 0.03 1.30 2.91 1.73 1.44 0.99 2.50 11 4 4 U 0.00 0.93 3.41 2.19 1.99 1.02 1.58 11 4 4 I 0.00 0.57 1.69 2.05 1.34 1.00 1.73 8 2 All U 0.09 9.22 37.68 28.82 25.27 13.50 31.84 143 47 All I 0.04 4.62 15.95 ** 16.33 13.28 10.83 27.83 89 21 ** *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. e Mean values of root surface area in square millimeters for replications.

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85Table C-11. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 12 weeks after turf and nematodes establishment during trial 2. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 0.15e 4.54 20.32 18.76 16.39 14.13 35.89 110 25 1 I 0.02 1.48 5.08 5.14 3.78 2.84 12.59 31 7 2 U 0.01 2.77 10.42 6.38 4.49 4.51 6.15 35 13 2 I 0.03 1.95 4.87 2.47 0.87 0.62 2.49 13 7 3 U 0.00 2.04 6.28 4.91 2.47 2.66 3.46 22 8 3 I 0.02 1.76 3.74 1.39 0.84 0.77 0.91 9 6 4 U 0.08 3.51 12.56 12.68 7.97 7.40 22.95 67 16 4 I 0.00 1.41 3.68 2.27 0.60 0.03 1.24 10 5 All U 0.24 12.86 49.58 42.73 31.31 28.70 68.44 234 63 All I 0.07 6.61 17.37 11.27 6.09 5.07 17.23 64 24 *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. e Mean values of root surface area in square millimeters for replications.

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86Table C-12. Root surface areas of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 18 weeks after turf and nematodes establishment during trial 2. Root Diameters (mm) Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb 1c Ud 0.02e 2.29 8.03 9.50 9.82 7.88 81.44 229 10 1 I 0.02 1.03 3.57 3.11 3.32 1.93 13.38 26 5 2 U 0.02 2.01 7.01 7.14 6.65 6.15 47.86 77 9 2 I 0.01 0.95 2.51 2.15 1.82 1.92 5.67 15 3 3 U 0.06 1.75 6.18 7.65 6.85 6.46 24.30 53 8 3 I 0.00 0.66 2.06 1.45 0.76 0.57 5.91 11 3 4 U 0.02 1.32 4.33 4.90 3.69 3.35 16.60 34 6 4 I 0.00 0.37 0.95 0.75 0.63 0.43 2.23 5 1 All U 0.12 7.36 25.55 29.18 27.00 23.84 170.19 393 33 All I 0.04 3.00 9.09 7.46 6.53 4.86 27.18 58 12 *, **, *** Inoculated different from uninoculated at a specified depth. a Total roots consist of cumulative root lengths for all diameters for a specified depth. b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth. c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. d (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. e Mean values of root surface area in square millimeters for replications.

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87Table C-13. Root dry weights of ‘Tifdwarf’ bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths for both trials. Root wt (mg) Trial 1 Trial 2 Depths 6weeka 12 week 18 week 6 week 12 week 18 week 1b Uc 1,214d 1,020 1,063 28 28 67 1 I 896 666 651 *** 12 10 10 *** 2 U 571 730 555 5 9 35 2 I 477 388 ** 324 *** 3 3 5 *** 3 U 355 406 380 3 6 22 3 I 188 ** 170 *** 142 *** 1 3 4 *** 4 U 340 319 305 2 11 13 4 I 194 136 *** 107 *** 1 4 2 ** All U 2,480 2,476 2,302 38 54 137 All I 1,755 1,359 ** 1,224 *** 17 20 21 *** *, **, *** Inoculated different from uninoculated at a specified depth. a Weeks after turf and nematode establishment period. b Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm. c (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per lysimeter. d Means values for replications

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88 Table C-14. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month in untreated control, mechanical, and nematode management plots throughout trial 2. Week Nematode Tactic Y = r2 P 0a Controlb 4.34 + 0.00068x 0.01 0.804 Mechanical 4.14 + 0.00546x 0.33 0.020 Nematicide 4.28 + 0.00580x 0.43 0.006 2 Control 4.34 + 0.00239x 0.04 0.452 Mechanical 4.26 + 0.00375x 0.17 0.113 Nematicide 4.70 + 0.00887x 0.25 0.048 4 Control 5.03 # 0.00273x 0.06 0.354 Mechanical 4.69 + 0.00512x 0.17 0.117 Nematicide 5.33 + 0.00546x 0.09 0.269 6 Control 5.18 # 0.00546x 0.36 0.015 Mechanical 4.85 # 0.00409x 0.16 0.120 Nematicide 5.18 # 0.00034x 0.00 0.917 8 Control 4.53 + 0.00239x 0.04 0.460 Mechanical 4.65 + 0.00068x 0.01 0.810 Nematicide 5.34 + 0.00068x 0.01 0.842 10 Control 4.63 + 0.00017x 0.00 0.955 Mechanical 4.67 # 0.00051x 0.00 0.841 Nematicide 5.35 + 0.00017x 0.00 0.968 12 Control 4.36 + 0.00051x 0.01 0.792 Mechanical 4.36 # 0.00034x 0.01 0.880 Nematicide 5.29 # 0.00239x 0.04 0.460 14 Control 4.56 # 0.00171x 0.01 0.720 Mechanical 4.16 + 0.00443x 0.07 0.324 Nematicide 5.03 # 0.00273x 0.02 0.597 16 Control 4.82 + 0.00358x 0.09 0.274 Mechanical 4.74 + 0.00222x 0.12 0.193 Nematicide 5.46 # 0.00017x 0.00 0.969 Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as maximum turf quality, and 6.5 as the threshold for acceptability. Fertilization began four weeks prior to nematicide treatment. a Weeks after nematicide treatment b Control = no added soil disturbance or nematicide treatment Mechanical = soil disruption with slit injection equipment without nematicide treatment. Management = Injection of 1-3, dichloropropene at a rate of 46.76 liters/ha at 13 to 17 cm of soil depth.

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89 Table C-15. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month in untreated control and nematicide treated plots through out trial 2. Week Nematode Tactic Y = r2 P 0a Controlb 6.25 + 0.00610x 0.10 0.009 Nematicide 5.79 + 0.01040x 0.18 0.001 2 Control 6.16 + 0.00384x 0.04 0.097 Nematicide 6.76 + 0.00524x 0.08 0.027 4 Control 5.78 # 0.00379x 0.01 0.386 Nematicide 6.56 + 0.00473x 0.05 0.066 6 Control 6.75 + 0.00205x 0.01 0.383 Nematicide 6.72 + 0.00328x 0.07 0.043 8 Control 6.44 # 0.00452x 0.02 0.221 Nematicide 6.14 + 0.01290x 0.23 0.000 10 Control 6.22 # 0.00827x 0.07 0.042 Nematicide 6.49 + 0.00874x 0.13 0.004 12 Control 6.59 # 0.00217x 0.02 0.217 Nematicide 6.52 + 0.01040x 0.37 0.000 14 Control 6.38 # 0.00115x 0.00 0.700 Nematicide 6.61 + 0.00946x 0.25 0.000 16 Control 7.17 # 0.00806x 0.14 0.003 Nematicide 6.67 # 0.00056x 0.00 0.865 Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as maximum turf quality, and 6.5 as the threshold for acceptability. Fertilization began four weeks prior to nematicide treatment. a Weeks after nematicide treatment b Control = no added soil disturbance or nematicide treatment Management = Injection of 1-3, dichloropropene at a rate of 46.76 liters/ha at 13 to 17 cm of soil depth.

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90 LIST OF REFERENCES Abu-Gharbieh, W. I., and V. G. Perry. 1970. Host differences among Florida populations of Belonolaimus longicaudatus. Journal of Nematology 2:209-216. Anonymous. 1993. USGA recommendations for a method of putting green construction. USGA Green Section Record 31:1-3. Berndt, M.P., H. H. Hatzell, C. A. Crandall, M. Turtora, J. R. Pittman, and E. T. Oaksford. 1998. Water quality in the Georgia-Florida Coastal Plain. . Date last visited: March 19, 2004. Bottcher, D. and D. Rhue. 2000. Fertilizer management – key to a sound water quality program. Florida Cooperative Extension Service Circular-816. Soil and Water Science Department, University of Florida, Gainesville, FL. Bouyoucos, G. J. 1936. Directions for making mechanical analyses of soils by the hydrometer method. Soil Science 42:225-229. Brady, N. C., and R. R. Weil. 2000. Elements of the nature and properties of soil. Upper Saddle River, NJ: Prentice-Hall. Brodie, B. B. 1976. Vertical distribution of three nematode species in relation to certain soil properties. Journal of Nematology 8:243-247. Christie, J. R. 1952. Some new nematode species of critical importance to Florida growers. Soil and Crop Science Society of Florida Proceedings 12:1-15. Christie, J. R. 1953. Ectoparasitic nematodes of plants. Phytopathology 43:295-297. Christie, J. R. 1959. Plant nematodes: their bionomics and control. Jacksonville, FL: H. and W. B. Drew. 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. Clarkson, D. T., and J. B. Hanson. 1980. The mineral nutrition of higher plants. Annual Review of Plant Physiology 31:239-298. Cobb, N. A. 1918. Estimating the nema population of soil with special reference to the sugar beet and root gall nematodes Heterodera schachtii Schmidt and Heterodera radicicola (Greef) Mller, and with a description of Tylencholaimus aequalis n. sp. Washington D.C.: Government Printing Office.

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91 Cole, J. T., J. H. Baird, N. T. Basta, R. L. Huhnke, D. E. Storm, G. V. Johnson, M. E. Payton, M. D. Smolen, D. L. Martin, and J. C. Cole. 1997. Influence of buffers on pesticide and nutrient runoff from bermudagrass turf. Journal of Environmental Quality 26:1589-1598. Colmer, T. D., and A. J. Bloom. 1998. A comparison of net NH4 + and NO3 fluxes along roots of rice and maize. Plant Cell Environment 21:240-246. Crow, W. T., D. W. Dickson, and D. P. Weingartner. 1997. Stubby-root symptoms on cotton induced by Belonolaimus longicaudatus Journal of Nematology 29:574. Crow, W. T., R. M. Giblin-Davis, and D. W. Lickfeldt. 2003. Slit injection of 1,3dichloropropene for management of Belonolaimus longicaudatus on established bermudagrass. Journal of Nematology 35:302-305. Crow, W. T., J. W. Noling, R. A. Kinloch, J. R. Rich, and R. A. Dunn. 2003. Florida Nematode Management Guide. Entomology and Nematology Department. University of Florida, Gainesville, FL. Dittmer, H. J. 1937. A quantitative study of the roots and root hairs of a winter rye plant ( Secale cereale ). American Journal of Botany 24:417-420. Ferris, H. 1999. Belonolaimus longicaudatus . Date last visited: March 19, 2004. Fortuner, R., and M. Luc. 1987. A reappraisal of Tylenchina (Nemata). The family Belonolaimidae Whitehead, 1960. Revue de Nematologie 10:183-202. Giblin-Davis, R. M., J. L. Cisar, F. G. Bilz, and K. E. Williams. 1992. Host status of different bermudagrasses ( Cynodon spp.) for the sting nematode, Belonolaimus longicaudatus Supplement to Journal of Nematology 24:749-756. Haydu, J., and A. Hodges. 2002. Economic dimensions of the Florida golf course industry. Florida Cooperative Extension Service Fact Sheet FE-344. Department of Food and Resource Economics, University of Florida, Gainesville, FL. 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. Jackson, M. B., and M. C. Drew. 1984. Effects of flooding on growth and metabolism of herbaceous plants. Pp. 47-128 in T.T. Kozlowski, ed. Flooding and Plant Growth. New York, NY: Academic Press. Jenkins, W. R. 1964. A rapid centrifugal-floatation technique for separating nematodes from soil. Plant Disease Reporter 48:692. Johnson, A. W. 1970. Pathogenicity and interaction of three nematode species on six bermudagrasses. Journal of Nematology 2:36-41.

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92 Kaspar, T. C., and R. P. Ewing. 1997. ROOTEDGE: Software for measuring root length from desktop scanner images. Agronomy Journal 89:932-940. Klepper, B., and T. C. Kaspar. 1994. Rhizotrons–Their development and use in agricultural research. Agronomy Journal 86:745-753. Lucas, L. T. 1982. Population dynamics of Belonolaimus longicaudatus and Cric1mella ornata and growth response of bermudagrass and overseeded grasses on golf greens following treatments with nematicides. Journal of Nematology 14:358-363. Mai, W. F., P. G. Mullins, H. H. Lyon, and K. Loeffler. 1996. Plant-parasitic nematodes: A pictorial key to genera. Ithaca, NY: Cornell University Press. McSorley, R., and D. W. Dickson. 1990. Vertical distribution of plant-parasitic nematodes in sandy soil under soybean. Journal of Nematology 22:90-96. McSorley, R., and J. J. Frederick. 1991. Extraction efficiency of Belonolaimus longicaudatus from sandy soil. Journal of Nematology 23:511-518. Morris, K. 2003. Bentgrass and bermudagrass for today’s putting green. USGA Green Section Record 41:8-12. 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. Noling, J. W. 1993. Citrus root growth and soil pest management practices. Florida Cooperative Extension Service Fact Sheet ENY-617. Entomology and Nematology Department, University of Florida, Gainesville, FL. Owens, J. V. 1951. The pathological effects of Belonolaimus gracilis on peanuts in Virginia. Phytopathology 41:29. Pan, W. L., and R. P. Bolton. 1991. Root quantification by edge discrimination using a desktop scanner. Agronomy Journal 83:1047-1052. Perry, V. G., and H. L. 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. University of Arkansas Agricultural Publications: Fayetteville, AR. Rau, G. J. 1958. A new species of sting nematode. Proceedings of the Helminthological Society 25:95-98. 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 30:119128. Rhoades, H. L. 1980. Reproduction of Belonolaimus longicaudatus in treated and untreated muck soil. Nematropica 10:139-140.

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93 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, I. R., and G. L. Miller. 2000. Using near infrared reflectance spectroscopy to schedule nitrogen applications on dwarf-type bermudagrasses. Agronomy Journal 92:423-427. Russell, R. S. 1977. Plant root systems: their function and interaction with the soil. London, England: McGraw Hill. Sartain, J. B. 2000. SOS 4115 Fertilizer and soil fertility (Class notes from Spring, 2001). Gainesville, FL: University Copy Shop. Sartain, J. B., and H. D. Gooding. 2000. Reducing nitrate leaching during green grow-in. Golf Course Maintenance 2:70-73. Sartain, J. B., and J. K. Kruse. 2001. Selected fertilizers used in turfgrass fertilization. Florida Cooperative Extension Service Circular-1262. Soil and Water Science Department, University of Florida, Gainesville, FL. Shuman, L. M. 2002. Nutrient leaching and runoff from golf courses. USGA Turfgrass and Environmental Research Online 1:1-11. Smart, G. C., and K. B. Nguyen. 1988. Illustrated key for the identification of common nematodes in Florida. Entomology and Nematology Department, University of Florida, Gainesville, FL. Snyder, G. H., G. J. Augustin, and J. M. Cavison. 1984. Moisture sensor-controlled irrigation for reducing N leaching in bermudagrass turf. Agronomy Journal 76:964-969. Steiner, G. 1949. Plant nematodes the grower should know. Soil and Crop Science Society of Florida Proceedings 4:72-117. Taiz, L., and E. Zeiger. 1998 Plant physiology 2nd ed. Sutherland, MA: Sinauer Associates. Todd, T. C., 1989. Population dynamics and damage potential of Belonolaimus sp. on corn. Supplement to Journal of Nematology 21:697-702. Unruh, J. B., M. L. Elliott, G. L. Miller, J. L. Cisar, A. E. Dudeck, J. B. Sartain, G. H. Snyder, P. Busey, J. H. Frank, R. A. Dunn, and B. J. Brecke. 1999. Best management practices for Florida Golf Courses. 2nd edition. University of Florida, Gainesville, FL. USDA Soil Conservation Service. 1982. Soil Survey of Citrus County Florida. Washington, D.C.: U.S. Government Printing Office.

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94 USDA Soil Conservation Service. 1985. Soil Survey of Pasco County Florida. Washington, D.C.: U.S. Government Printing Office. Wang, F. L., and A. K. Alva. 1996. Leaching of nitrogen from slow-release urea sources in sandy soil. Soil Science Society of America Journal 60:1454-1458. Wallace, H. R. 1971. Abiotic influence in the soil environment. Pp. 257-280 in B. M. Zuckerman, W. F. Mai, and R. A. Rohde, eds. Plant parasitic nematodes. Vol. 1. New York, NY: Academic Press.

PAGE 107

95 BIOGRAPHICAL SKETCH John Eric Luc was born October 16, 1971, in Hialeah, Florida to John Theodore and Lynda Alden Luc. They moved to Citrus County, Florida in 1972 where he held residency for 31 years. Due to his father being in the military, he lived throughout the Eastern United States, returning to Florida in 1988. He graduated from Citrus High School in 1990. After a couple of unsuccessful years in college, he decided to leave college in 1992. He worked numerous jobs, finally settling down with a position at Citrus County Parks and Recreation where a love of turfgrass was born. However, his thirst for knowledge could not be quenched there. Through the advice of Jeff Hayden, he sought out Dr. Grady L. Miller, assistant professor of turfgrass science and later mentor. Upon entrance to the University of Florida, he relocated to Gainesville and obtained employment with SNAP (Student Nighttime Auxiliary Patrol). Later, he felt the need to seek out employment in an agricultural field, to assist with learning and to obtain experience in research. Currently, he is a graduate student and a research assistant conducting master’s research with Dr. William T. Crow, Landscape Nematologist, University of Florida, Gainesville, Florida. His thesis title is “Effects of Plant-Parasitic Nematodes and Nitrogen Fertility Management on Hybrid Bermudagrass.” He hopes to begin studies for the doctor of philosophy degree in plant pathology at Clemson University, Clemson, South Carolina, in May 2004, under Dr. Bruce Martin. His research will examine ascomycetes responsible for Bermudagrass decline as well as patch diseases on Bentgrass.


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EFFECTS OF PLANT PARASITIC NEMATODES AND NITROGEN FERTILITY
MANAGEMENT ON
HYBRID BERMUDAGRASS
















By

JOHN ERIC LUC


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


2004



































Copyright 2004

by

John Eric Luc
















ACKNOWLEDGMENTS

I would like to thank my chairman and committee members, William T. Crow, Jerry B.

Sartain, Jerry L. Stimac, and Robin M. Giblin-Davis, for their guidance and patience during my

pursuit of a Master of Science degree. I have gained a great deal of knowledge from each of them

during my undergraduate and graduate education. Their dedication to the advancement and

excellence of graduate education is unyielding. I hope to have the same patience and dedication

that they have exhibited during all my endeavors.

Also I would like to acknowledge Augustus Porter Alden, my grandfather, who never

wavered in his belief of me, even when I did not believe in myself. On July 27, 1998, he left our

world and continued his journey. His passing was the catalyst for my return to academics. I

dedicate this degree to him with all my heart.

John T. and Lynda A. Luc, my parents, deserve my sincere appreciation. Their guidance,

patience, love, and support have carried me a long way. Without it I would have never reached

this point in my life. However, my journey has not concluded. I will have to draw on them again,

as I continue my education and pursuit of a doctor of philosophy degree.

I would also like to thank, Joseph C. Parker "Buck," Amy Parker, and Joseph C. Parker

II, my best friends and their son, for the emotional and financial support they have shown. I have

known Buck for 22 years; during that time our friendship has never been broken. I wish him and

his family all the love and happiness in the world.

To the staff of the nematode assay lab and particularly Matthew R. Coon, this degree is as

much theirs as it is mine. Without their dedication and hard work this project would not have

been successful.

















TABLE OF CONTENTS
Page

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

L IST O F TA B LE S .................... ....... .. ............................................ .............. .. .....vii

LIST O F FIG U RES ................. ............................................. ............ ....................... .... ix

A B STR A CT ........................ ................................................................. ...................... xi

CHAPTER

1 IN T R O D U C T IO N .............. ........................................................... .. .......... ...... .............. 1

2 L IT E R A T U R E R EV IEW ............................................... ................................................... 4

B elonolaim us longicaudatus ............................................. ................................................. 4
T ax on om y ............................................................................. .............................. . 4
M orphology and A natom y ....................................... .................................................. 5
B biology and D distribution ............................................................................................. ... 6
Soil Fertility ................................................................... 8
H isto ry ......................................................... ...... .......... ....... 8
N itrogen Fertilizers ................ ................... ... ........ .......... ............ 9
Nitrogen Use in the Soil and Turfgrass System............................... ................................. 11
M in eralization ........ ........... ................ ................ .. .............. ......... .. ... ............. 11
L teaching ......... ............................................................. ...................... ............... 13
E rosion and R un off..................... ................................................ ........................... 14
P lant U ptak e ................................................................. 15
A dsorption .............................. .............. ....... 16
Im m mobilization ............. ........................... .... ............ 16
V olatilization ............. .. .............. ........... .................................................. .... ...... 16
D enitrification .. ........ .......... .......................................... .................... .. .. .. ............ 17
T urfgrass C ultivars............................................................... .................... ......... .. .. 18
'T ifdw arf B erm udagrass ........................................ ................................................. 18
'Tifw ay 419' B erm udagrass......... ............................................... ......... ... ................ 18
Root Systems........................................................... 19
History...................... ................................... ......... 19
Root Development and Nutrients........................................................ 19

3 INFLUENCE OF PLANT-PARASITIC NEMATODES ON NITRATE LEACHING IN
TURF ....... .......................... ................ 21

In tro d u ctio n ............................................. ...................... ................. .................. ................ 2 1
M materials and M methods ..................................................................................... ................. 22










T ria l 1 ........................................ ................... 2 2
Establishment of experimental units .......... ........................................................... 22
N em ato d e in ocu lu m ....................................................................................................... 2 3
T urf m aintenance.......................... .... .. ........ .. .................... .............. ............ .. 23
Evaluation and sam pling techniques ................ ................................................... 23
T rial 2 ............................................... ................................ .................. .... 2 5
Establishm ent of experim ental units .............................. ......................... ................. 25
N em atode inoculum ................................................. ................................................. 25
Turf m aintenance.......................... .... .. ........ .. ................... ............ ... ............ .. 26
Evaluation and sampling techniques ........................................ .................... 26
Data A analysis ....... ................. .. .................. 26
R results .................................................... ................ .... ..... ..... ................ 27
D iscu ssion ................................ ................ ......... .............. .. ..................... ......... .. ....... .. 2 7

4 EFFECT OF NEMATODE MANAGEMENT AND NITROGEN FERTILITY ON
FAIRW AY TURF QUALITY .............................................................................................. 36

In tro d u ctio n ...................................................................................... .................................. .. 3 6
M materials and M ethods ........................................................................................................... 37
E x p erim ental S ites............................................................................... ........................ 3 7
P ath o g en s ....................... ............................................................................................... 3 7
Insects and w eeds .............. ........ ..... .......... .. ............. ... .... .......... ...... 38
T urf ......................... ............. ........................... ...................... ......... ....... 38
Soil properties ............................. .... ............ 38
Experim mental D design ............... ................. .................................... ..... .................. 38
N em aticide Treatments .............. ............................................. ................. 39
F fertilization .... .............................................................................. ............. ....... 4 0
G general Production Practices ............................................ ................................................ 40
T urf M maintenance ............ .... ................................... ... ..................... 40
Pesticides ...................... ....... ......... ... ................................................... 41
Sam pling and E valuations............. .................... ... .. .............. ................... .......... ............. 4 1
Turf Evaluations .......................... ...... .......... .... ..................... 41
N em atodes ........................ ........ .................... ... ......... ......... ............... 41
Roots ................................................................ 42
T urf T issue ................. ................................................................................................... 42
Data Analysis .................................................................. 42
R e su lts ..................................................................................................................................... 4 3

5 SU M M A R Y ............................. .............. ...... ...................................................... ........... 51

APPENDIX

A DETAILED MATERIALS AND METHODS USED IN THE GREENHOUSE STUDY... 54

In tro d u ctio n .......................................................................................................................... 5 4
E xperim mental M materials ................................................... .................................................. 54
Lysim eters ........................................................................... 54
T urf....................................................................................... ...... ..... ................ 54
Soil Properties ....................... .. .................. 56
N em atode Inoculum ................................................. ................................................. 56
Experim mental D design ........... ............. ....... ......... ......... ......................... ... ......... ...... 57



v










G general Production Practices ............................................ ................................................ 58
Turf Establishment ........... ....... ...................................................... .... .................. .. 58
Nematode Establishment...................................................................... ................ 58
Temperature ............................ .. .......... ............... 59
W ate rin g ...................................... ................................................. .................. ......... 5 9
Fertilization .................................................................. 59
Pesticides .................. ...... .. ................................ ....................... ....... .. 60
Sampling and Evaluations............................................................................................... 60
Turf Evaluations ................ ...................................... ................... 60
N em atodes and R oots..................................................................................... ................ 60
Leachate .................................................................. 62
Turf Tissue .................................. ............................................................................... 63
D ata A n aly sis ................................................................................................................. 6 3

B DETAILED MATERIALS AND METHODS USED IN THE FIELD STUDY ............... 66

Introduction ................................... .................................... ................................... .............. 66
E x p erim en tal S ite s ................................................................................................................. 6 6
P ath o g en s ....................... ............................................................................................... 6 6
Insects and weeds .............................. ...... .... .................................... ................. 67
Turf ................................................................................. ..................... 67
S o il P ro p ertie s ................................................................................................................ 6 7
E xperim mental D design .................................... ............................................ ................. 67
Nematicide Treatments .............. ............................................. ................ 68
F fertilization ............................................................ ........................ .. ............. 7 1
G general Production Practices ................ ...................................... ........................................ 7 1
Turf Maintenance ................................. ................ ................................................ 71
P e stic id e s ............................................................................................ ................. ........ 7 2
Sam pling and E valuations ...................................................................................................... 72
T urf Evaluations ................................. .. ........ ............ .. .......... .. .................... 72
N em atodes ................................... .............................................................................. 72
Roots ................................................................ 73
T u rf T issue e .................................. ................................................... ........................... 7 3
Data Analysis .................................................................. 73

C SUPPLEMENTAL FIGURES AND TABLES ................................... 74

L IST O F R E FE R E N C E S ............................................................................................................... 90

BIOGRAPHICAL SKETCH ................................................................. 95
















LIST OF TABLES


Table page

3-1. Effects of inoculating turf with Belonolaimus longicaudatus on 'Tifdwarf bermudagrass root
length, surface area, and dry weight at 6, 12, and 18 weeks after turf and nematode
establish ent during trial 1 ................................... ............................................. 30

3-2. Effects of inoculating turf with Belonolaimus longicaudatus on'Tifdwarf bermudagrass root
length, surface area, and dry weight at 6, 12, and 18 weeks after turf and nematode
establish ent during trial 2 ............................................ .............................................. 3 1

3-3. Linear regression analysis conducted to determine relationships between nematode
populations, root length, nitrogen uptake, and nitrate leached during both trials............. 32

4-1. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N
fertility levels on a 'Tifway 419' bermudagrass fairway at 0 to 16 weeks after treatment
during trial 1 .............................................................................. 45

4-2. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N
fertility levels on a 'Tifway 419' bermudagrass fairway at 0 to 16 weeks after treatment
during trial 2 .............................................................................. 46

4-3. Total root lengths observed in plots treated with 1,3-dichloropropene and in untreated plots at
individual N fertility levels on a 'Tifway 419' bermudagrass fairway at 0, 6, and 16 weeks
after treatm ent during both trials. ............................................. ............................... 47

C-1. Root lengths of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated
or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges
6 weeks after turf and nematodes establishment during trial 1.........................................75

C-2. Root lengths of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated
or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges
12 weeks after turf and nematodes establishment during trial 1..................................... 76

C-3. Root lengths of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated
or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges
18 weeks after turf and nematodes establishment during trial 1..................................... 77

C-4. Root lengths of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated
or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges
6 weeks after turf and nematodes establishment during trial 2. .......................................78










C-5. Root lengths of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated
or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges
12 weeks after turf and nematodes establishment during trial 2. ................................. 79

C-6. Root lengths of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated
or inoculated with Belonolaimus longicaudatus at specified soil depths and diameter ranges
18 weeks after turf and nematodes establishment during trial 2. ................................. 80

C-7. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 6
weeks after turf and nematodes establishment during trial 1........................................... 81

C-8. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 12
weeks after turf and nematodes establishment during trial 1........................................... 82

C-9. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 18
weeks after turf and nematodes establishment during trial 1............................................. 83

C-10. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 6
weeks after turf and nematodes establishment during trial 2. ........................................... 84

C-11. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 12
weeks after turf and nematodes establishment during trial 2. ........................................... 85

C-12. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths 18
weeks after turf and nematodes establishment during trial 2. ........................................... 86

C-13. Root dry weights of 'Tifdwarf bermudagrass grown in lysimeters that were either
uninoculated or inoculated with Belonolaimus longicaudatus at specified soil depths for
both trials ..................................... .................. .... ..... ............ 87

C-14. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30, and
109.95 kg N/ha/month in untreated control, mechanical, and nematode management plots
throughout trial 2...... .......... .... ...................... .. 88

C-15. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30, and
109.95 kg N/ha/month in untreated control and nematode management plots through out
trial 2 ........................................................................................... 8 9
















LIST OF FIGURES


Figure page

2-1. N nitrogen cycle.. .................. ............................................ ...................... ........ ............. 12

3-1. Effects of inoculating turf with Belonolaimus longicaudatus on mg nitrate leached at 3, 6, 9,
12, 15, and 18 or 6, 12, and 18 weeks after turf and nematode establishment during trial 1
(A) and trial 2 (B) respectively. Inoculated plants received 138 (trial 1) and 300 20 B.
longicaudatus (trial 2), while uninoculated plants received no nematodes. Error bars
indicate standard error of individual population means. ................. ............... ............. 28

3-2. Effects of inoculating turf with Belonolaimus longicaudatus on cumulative nitrate leached at
3, 6, 9, 12, 15, and 18 weeks after turf and nematode establishment during trial 1 (A) and
trial 2 (B) respectively. Inoculated plants received 138 (trial 1) and 300 40 B.
longicaudatus (trial 2), while uninoculated plants received no nematodes. Error bars
indicate standard error of individual population means. .............. ............................... 29

4-1. Means of Belonolaimus longicaudatus per 100 cm3 of soil sampled from 6 to16 weeks after
nematode management tactics were applied during trial 1 (A) and trial 2 (B) respectively.
Error bars indicate standard error of individual population means. ............................... 44

A-1. A lysimeter used as an experimental unit during glasshouse trials at the University of Florida
Turfgrass Envirotron from 29 January 2002 to 16 April 2003.......................................... 55

A-2. Screen placed within a lysimeter after assembly. The screen holds the soil profile away from
drainage hole preventing drain blockage ................................................ ................. 55

A-3. A threaded bushing screwed into a lysimeter. The bushing enables leachate to be collected.56

A-4. Glass imaging pan with white background placed on a flat bed scanner to obtain bitmap
im ag es o f ro ot sy stem s. ................................................ ................................................. 6 1

A-5. After washing, turf tissue samples were placed on a paper plate and dried in a 1000-W
microwave oven for two to six minutes depending on sample size. .................................. 64

A-6. Cyclone sample mill used to grind turf tissue for analysis. Dried tissue is placed into the
yellow cone, top center, and is retrieved from the glass jar, bottom center .................... 64

A-7. Near infrared reflectance spectroscopy (NIRS) scanning instrument, left. Spectral data
determined by NIRS was imported into a laptop computer with Toro Diagnostic software
program for analysis, right. Sampling cells waiting to be analyzed, top left, sampling cells
previously analyzed, bottom left. ....................................................................... 65










B-1. Plot plan of the field study undertaken at Citrus County, FL during 2002. Plots marked with
X were not used as experimental units due to nematode counts being below damaging
threshold for Belonolaimus longicaudatus..................................... ........................ 69

B-2. Plot plan of the field study undertaken at Pasco County, FL during 2003. Plots marked with
X were not used as experimental units due to nematode counts being below damaging
threshold for Belonolaimus longicaudatus..................................... ........................ 70

C-1. Regression models of turf quality response to untreated control, mechanical disruption with
slit injection equipment without nematicide, and slit injection of 1,3-dichloropropene at a
rate of 46.76 liter per hectare at N rates of 0, 36.65, 73.30, and 109.95 kg N/ha/month
through out trial 1 (A) and trial 2 (B). ........................................ ............................ 74
















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

EFFECTS OF PLANT PARASITIC NEMATODES AND NITROGEN FERTILITY
MANAGEMENT ON
HYBRID BERMUDAGRASS

By

John Eric Luc

May 2004

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

Damage caused by B. longicaudatus to bermudagrass root systems can cause decreased

water and nutrient uptake, reduced plant growth, and predispose turf to other adverse conditions

such as drought stress, heat stress, and malnutrition which can reduce turf quality. Glasshouse

experiments were conducted to determine the relationships between nematode damage to

'Tifdwarf' bermudagrass roots and nitrogen uptake and nitrate leached. Forty lysimeters were

sprigged with 'Tifdwarf bermudagrass to simulate a greens soil profile, of which 20 were

inoculated with B. longicaudatus using a random complete block design. Leaching events were

simulated at 21 and 42 day intervals for trial 1 and 2 respectively. Turf was fertilized every 21

days with potassium nitrate at 92 and 109 kg/ha N for trial 1 and 2. Turf quality, color, and

density were evaluated every 21 days. Nematode counts and root lengths were assessed 6, 12, and

18 weeks after turf and nematode establishment. Differences (P < 0.05) were observed for total

root length at 6, 12, and 18 weeks and milligrams of nitrate (NO3-) leached at 18 weeks during

both trials. When amount of cumulative nitrate leached was compared between treatments, root

systems inoculated with B. longicaudatus leached more NO3- than did uninoculated root systems










(P < 0.05) in trial 1, but not in trial 2. Nematode feeding reduced root density by 30 to 94 percent

and increased the amount of nitrate leached by as much as 429 percent. No differences (P < 0.05)

were observed for turf quality, color, density, tissue nitrogen levels, dry matter production, or

total nitrogen uptake.

Field experiments were conducted during 2002 and 2003 to describe relationships between

nematode management and N fertility in terms of turf quality and root lengths on golf course

fairways. Treatments were untreated control, mechanical, and 1,3-dichloropropene with N

fertility levels of 0, 36, 73, and 110 kg/ha/month. Treatments were randomized within blocks with

four replications. Reduced (P < 0.05) B. longicaudatus counts were observed in plots treated with

the nematicide 1,3-dichloropropene compared to untreated control plots at 2, 4, and 6 weeks after

treatment during both trials. Differences (P < 0.05) were observed between untreated control and

nematicide treated plots within individual N fertility levels at specific sampling dates with respect

to turf quality, color, and density during both trials. Differences (P < 0.05) in turf quality were

observed between untreated control and nematode management within each N fertility level at 2

and 4 weeks and 8 to 16 weeks after treatment at N fertility levels of 73.30 and 109.95 kg

N/ha/month during trial 2. No differences (P < 0.05) in root length and surface area of specified

root diameters, total root length, total surface area, or root weight were observed between

nematode management tactics at individual N fertility levels for 0, 6, and 16 weeks after

treatment during either trial.

In conclusion, glasshouse studies indicated that nematode damage to turf roots can increase

nitrate leaching, thereby adding to water quality concerns. Field experiments indicate that

increased N fertility on nematode infested sites without nematode management could be

detrimental to turf quality, especially when the turf experiences stress. These studies reveal that

nematode and N fertility management are equally important to providing a quality turf and

minimizing environmental impacts.















CHAPTER 1
INTRODUCTION

Nitrogen (N) is frequently the limiting nutrient for most plants. In the United States of

America from 1955 to 1990, annual N fertilizer inputs by agriculturalists increased from 2 million

tons to over 12 million tons. Each year Florida growers apply about 2.0 million tons of fertilizer

at a cost of over 250 million dollars to grow in excess of 3 billion dollars worth of crops (Bottcher

and Rhue, 2000).

In 2000, total acreage dedicated to Florida golf facilities was 207,582 acres with 147,927

acres maintained as golf playing areas. This represents an increase of 12.66% in maintained turf

area since 1991. Hybrid bermudagrasses (Cynodon spp.) are the prevalent grasses used in

maintained turf areas on golf courses due to good drought and wear tolerance, accounting for

92% of land use (Haydu and Hodges, 2002). Bermudagrass requires ample nutrient inputs for

optimal growth and turf quality. Due to Florida's long growing season, high annual rainfall, and

sandy soils, golf course turfgrass requires higher fertility inputs than other areas in the United

States (Unruh et al., 1999). Between 1995 and 2000, fertilizer use per acre has increased on 29%

of golf courses in Florida (Haydu and Hodges, 2002).

In recent years, heightened environmental awareness has brought water quality and

consumption to the forefront of public concern, focusing attention on heavy users of water,

fertilizers, and pesticides (Haydu and Hodges, 2002). The intensive use of N fertilizers on golf

courses has added to these concerns and spurred questions as to the fate of N following

application. Nitrogen leaching into groundwater is one concern. The possibility of groundwater

contamination is increased on sandy soils with low levels of organic matter receiving intensive

rainfall or irrigation. Of the shallow wells tested in northern Florida, approximately 21% in

agricultural areas and 7% in urban area were found to have nitrate levels in excess of the










maximum contaminant level (10 mg/liter) set by the Federal Environmental Protection Agency.

The highest level of nitrate contamination (12 mg/liter) in an urban area was found in Ocala, FL

(Berndt et al., 1998). In the near future, golf courses may be exposed to much more rigorous

monitoring of water and fertilizer practices.

Studies have revealed that nitrate leaching from healthy turf is minimal (Sartain and

Gooding, 2000; Snyder et al., 1984). While these research efforts are valuable for a better

understanding ofturfgrass processes, they are normally performed under optimal conditions and

without pest infestations. However, rarely on golf courses do these optimal and pest free

conditions exist. How much fertilizer is needed when the turf is not healthy? How much of this

resource is lost due to poor turf vigor?

Plant-parasitic nematodes are root-feeding pests that greatly reduce the development of turf

roots. Damaged root systems are less efficient at water and nutrient uptake (Crow et al., 2003).

Consequently, turf that suffers nematode damage may require more water and fertilizer to

maintain an acceptable appearance. Hypothetically, damaged roots in conjunction with increased

fertilization and watering could lead to increased nitrate leaching into the groundwater.

The question was recently raised, "If golf course managers had no products to control

nematodes couldn't they increase their water and fertility usage and get by?" A short time ago,

ethoprop (Mocap) was pulled off the market for use on turfgrass and fenamiphos (Nemacur) is

being phased out in the next few years. We are reaching a point where we may have to "get by."

As mandated by the Food Quality Protection Act of 1996 a review of pesticides is ongoing.

When nematicides are reviewed for turfgrass use, there is a perception that governmental

agencies do not see this as a critical need. However, if nematode management can be shown to

decrease nitrate leaching, more consideration may be given to development and registration of

new nematicides for turf uses.







3


The objectives of these studies were to: 1) determine the relationships between nematode

damage to 'Tifdwarf bermudagrass [Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt

Davy] roots and nitrogen uptake and nitrate leached and 2) describe relationships between

nematode population counts and nitrogen fertility in terms of turf quality and root lengths on golf

course fairways.















CHAPTER 2
LITERATURE REVIEW

Belonolaimus longicaudatus

Taxonomy

Steiner (1949) first established the genus Belonolaimus with the discovery and description

of Belonolaimus gracilis. This first observation of B. gracilis was on the roots of slash pine

(Pinus elliotii Engelm) and longleaf pine (Pinus palustris P. Mill.) in the Ocala National Forest

near Ocala, FL. Over the next few years, B. gracilis was reported to damage crops such as celery

(Apium graveolens L.), corn (Zea mays L.), sorghum (Sorghum bicolor L. Moench), millet

(Sorghum halepense L. Pers.), peanut (Arachis hypogaea L.), cotton (Gossypium hirsutum L.),

soybean (Glycine max L. Merr.), and cowpea (Vigna unguiculata L. Walp.) (Christie, 1952;

Christie et al., 1952; Christie, 1953; Owens, 1951).

Rau (1958) described Belonolaimus longicaudatus, adding a second species to the genus

Belonolaimus. The major morphological differences separating these species are that B.

longicaudatus has a longer stylet and shorter tail than B. gracilis. Belonolaimus longicaudatus

was initially found on crops such as bermudagrass (Cynodon dactylon L. Pers.), corn, citrus

(Citrus spp.), soybean, peanut, and other crops (Rau, 1958). Furthermore, Rau stated that B.

longicaudatus was the more commonly encountered species. In 1963, Rau described three

additional species of Belonolaimus: B. euthychilus, B. maritimus, and B. nortoni. Since then,

species have been added and the genus has been moved several times. Belonolaimus

longicaudatus current taxonomic placement within the kingdom Animalia is: subkingdom

Metazoa, branch Eumetazoa, division Bilateralia, subdivision Protostomia, section

Pseudocoelomata, superphylum Aschelminthes, phylum Nematoda, class Secerentea, order

Tylenchida, suborder Tylenchina, superfamily Tylenchoidea, family Belonolaimidae, subfamily










Belonolaiminae, genus Belonolaimus, species longicaudatus (Fortuner and Luc, 1987; Smart and

Nguyen, 1988).

Morphology and Anatomy

Belonolaimus longicaudatus adults are 2 to 3 mm long and 29 to 34 .im wide (Mai et al.,

1996; Rau, 1958). The lip region has four major lobes with two smaller lobes having amphids

present and a constriction just below it, setting it off from the rest of the body. The lateral field

has one incisure running most of the body length. The stylet is thin and flexible ranging in size

from 100 to 140 mm long with rounded knobs. When retracted, the stylet causes the esophageal

tube to be convoluted (Ferris, 1999). The median bulb is well developed and elongated.

Esophageal glands overlap the anterior end of the intestines on the ventral side. The intestine can

be found slightly posterior of the median bulb extending almost to the terminus. Lateral canals are

prominent and serpentine along the intestine, becoming visible near the esophageal glands and

extending to the terminus. The vulva is a transverse slit found near the middle of the female, with

lips not protruding. The vagina nearly always has two sclerotized pieces that can be observed in

lateral view. The reproductive system is didelphic, amphidelphic, and outstretched. Spermathecae

are present which store sperm after copulation for fertilization of eggs over time. The male

reproductive system is found posteriorly, with testis prodelphic and outstretched. Spicules and

gubemaculum are well-developed averaging 44 .im and 16 .im long, respectively. The female tail

is 115 to 189 mm long with a rounded terminus. The male tail tapers to a more pointed terminus

and is enveloped by a long and narrow bursa, which may aid during copulation. (Ferris, 1999;

Mai et al., 1996; Robbins and Barker, 1973). It should be noted, that populations of B.

longicaudatus from North Carolina and Georgia have been shown to exhibit differing

morphological characteristics such as stylet length, stylet cone and shaft length, knob shape and

length, distance of excretory pore to anterior end, tail length, and spicule length. Interpopulation

mating of these two populations resulted in a few offspring, which were sterile, while

intrapopulation offspring reproduced normally. All populations investigated possessed eight









haploid chromosomes suggesting they are closely related, but may be different species (Robbins

and Hirschmann, 1974).

Biology and Distribution

Belonolaimus longicaudatus has an extensive host range and has been recognized as a

pathogen of many agronomic, horticultural, and ornamental crops (Abu-Gharbieh and Perry,

1970). Belonolaimus longicaudatus is an ectoparasite, meaning it feeds from the outside of the

plant root. The long stylet is used to penetrate deep into the roots where digestive enzymes can be

injected (Huang and Becker, 1997). Feeding usually causes the root tips to stop growing; this can

be devastating to young plants with a developing root system (Crow et al., 2003; Huang and

Becker, 1997; Perry and Rhoades, 1982).

Belonolaimus longicaudatus is a bisexual species, which reproduces exclusively through

amphimixis (sexually) with males accounting for 40% of the population (Huang and Becker,

1999). Experiments conducted on corn root cultures at 28 C by Huang and Becker (1997; 1999)

have given a detailed description of the life cycle of B. longicaudatus. After mating, females lay

eggs in pairs as long as a food source is available, with each female laying about 128 + 13 eggs in

90 days (Huang and Becker, 1999). Following egg deposition (day 0) the first stage juvenile (J1)

molted in the egg during day 4 and the second stage juvenile (J2) hatched from the egg during

day 5. The J2 must quickly find root tips to feed on or die. Once a food source was found, the J2

fed for 12 to 24 hours before becoming immobile and the second molt began during day 7 and

ended during day 9. Third stage juveniles (J3) began feeding again and then entered the third molt

on day 12 which lasted 2 days. Fourth stage juveniles (J4) began feeding once more, but

depending on the final sex of the nematodes, molting began at different times. Males entered the

fourth molt during day 18 and by day 20 were fully functional males and began feeding again.

Females entered the fourth molt during day 19, and by day 22 were considered virgin females

ready for mating. The life cycle from J2 to J2 took 24 days at 28 C (Huang and Becker, 1999).










Belonolaimus longicaudatus is found predominately in sandy costal areas of the

southeastern United States; however it has been observed in localized areas of the northeastern

states of Connecticut and New Jersey, and in the midwestern states of Arkansas, Kansas,

Oklahoma and Nebraska (Rhoades, 1980; Robbins and Barker, 1974). Belonolaimus

longicaudatus has been found in California, Puerto Rico, Bermuda, Australia, and some of the

Caribbean islands, usually in resort areas where sod or sprigs were sent from the southeastern

United States to establish fairways, greens, tees, or commercial turf (Mundo-Ocampo et al., 1994;

Perry and Rhoades, 1982).

Soil texture, soil particle size, soil moisture, root depth, and movement of nematodes are

factors which influence distribution of nematodes within the soil profile as well as

geographically. Soil texture influences nematode movement and distribution as a function of

nematode size to soil pore and particle size. As the diameter and length of the nematode increase,

the pore and particle size of the soil must increase or movement can be hindered (Wallace, 1971).

However, if pore diameter becomes too large, lateral movement can be hindered (Brodie, 1976).

Soils with less than 80% sand and more than 10% clay hinder movement of B. longicaudatus.

Furthermore, medium and course sand also have been shown to hinder their movement. Optimum

soil moisture for B. longicaudatus is about 7%, however populations have been recovered from

soils ranging from 2 to 30% soil moisture (Robbins and Barker, 1974). Saturated soils replenish

oxygen (02) much slower than well-drained soils, which can reduce activity and ultimately be

detrimental to certain nematode species. Conversely, soils with excessively low soil moisture

hinder movement by reducing the water films encircling soil particles used by nematodes to move

through the soil. Soil texture, particle size, moisture, and aeration are interrelated factors, which

effect movement. Movement is essential for species that reproduce exclusively by amphimixis (B.

longicaudatus) because they must search for food as well as a mate to reproduce, otherwise the

population will die (Robbins and Barker, 1974). Root depth of the host plant also influences

where B. longicaudatus will occur, generally they are found within the top 75 cm of the soil










profile, with greatest population densities found in the top 30 cm of the soil profile (Brodie, 1976;

McSorley and Dickson, 1990; Todd, 1989). In sites where turf is the host crop, greatest

population densities occur within the top few cm of soil. Conversely, in corn and citrus, which

possess deeper root systems, high populations can be found below 30 cm depth when other soil

conditions are favorable for nematode movement (Brodie, 1976; Noling, 1993; Todd, 1989).

Nematode populations are frequently erratically distributed at standard sampling depths even

within the same fields site (Todd, 1989). Erratic distribution ofnematodes can be a result of

previously mentioned factors, as well as the lack of active movement of nematodes from one

location to another. When consideration is given to the previously discussed factors, golf greens

represent ideal habitat for B. longicaudatus due to a high content of fine to medium sand that hold

approximately 12 to 15% water at field capacity (Anonymous, 1993).

Soil Fertility

History

The first known use of amendments to increase productivity of the soil was in 2500 B.C. in

Mesopotamia. Greek writings also suggest the use of manures to increase vegetable and olive

production. In the early 1600's, water was considered to be the principle nourishment to plants

and soil was just a media to hold the plant. In the 1700's it was believed soil particles were

ingested into the plant through the root system and then circulated throughout the plant (Sartain,

2000). In 1862, Justus Von Liebig, known as The Father of Soil Chemistry, was first to publish

the concept that plant production can be no greater than that level allowed by the growth factor

present in the lowest amount relative to the optimum amount for that factor (limiting factor),

whether it is temperature, water, or nutrient supply (Brady and Weil, 2000). This concept is

known as the "Law of the Minimum" (Sartain, 2000).

In the 1820's early fertilizer production began with a bi-product of soap production,

ammonium sulfate, which was utilized as an inorganic synthetic source of nitrogen (N). On the

same day in 1842, James Murray and John Bennet Lawes each patented the process for










production of superphosphate. By the 1850's, the fertilizer industry was born. In 1867, the

phosphorous (P) fertilizer industry flourished with the opening of phosphate mining in South

Carolina. In 1921, atmospheric N was directly converted to ammonia and less than ten years later

the process for urea production was discovered. Discovery of potassium (K) deposits in Carlsbad,

NM in 1931 were badly needed at the time for production of gunpowder. In 1933, the Tennessee

Valley Authority (TVA) and National Fertilizer Development Center (NFDC) were created to

provide power and to conduct research related to fertilizer materials. In 1945, TVA developed a

method for solid ammonium nitrate (NH4NO3), and the cone mixer, which increased production

of concentrated superphosphates, wet-process phosphoric acid, and ammonium phosphates. By

the 1950's, the fertilizer industry was expanding rapidly with increases in granular fertilizer

production, specialty fertilizers, introduction of slow release fertilizers, and wider recognition for

the need of micronutrient fertilizers (Sartain, 2000).

Nitrogen Fertilizers

Nitrogen fertilizers are categorized as soluble or slow-release. Soluble fertilizers such as

ammonium sulfate [(NH4)2SO4], ammonium nitrate (NH4NO3), urea [CO(NH2)2], and potassium

nitrate (KNO3), readily release near 100% of their nutrient load into soil solution immediately

upon application. While immediate availability of N imparts a sudden increase in growth, soluble

forms of N also may be more prone to leaching into the groundwater (Wang and Alva, 1996).

Slow-release fertilizers (SRF) such as sulfur coated urea (SCU), polymer/sulfur coated fertilizers

(PSCF), urea-formaldehyde (UF), and isobutylidene diurea (IBDU), release their N over a longer

duration, and may reduce the incidence of N leaching into the groundwater (Sartain, 2000).

Turfgrass in general responds well to N, and SRF are well suited for turfgrass management.

However, understanding the factors that release N: temperature, soil moisture, soil pH, microbial

activity, coating thickness, material particle size, hydrolysis and diffusion rate is essential to

forming an efficient fertility program.









The turfgrass industry uses a wide variety of N fertilizers, the more frequently used N

sources include: NH4NO3, (NH4)2SO4, and SCU. All these N sources vary in N release rates, and

in turf response.

Ammonium nitrate is white, crystalline solid, containing 33.5% N. Because this material is

very hydroscopic, the prill can be coated with MgCl2 or clay to prevent water absorption.

Ammonium nitrate is highly soluble in water and releases equal amounts of NH4 and NO3s.

Ammonium nitrate has a salt index of 2.99, and is therefore considered to be moderately to highly

damaging to turf (Sartain, 2000). In studies conducted by Wang and Alva (1996), N leaching on

sandy soils was found to be between 88 to 100% of the applied N when using NH4NO3. While

ammonium nitrate imparts a rapid green response, the longevity of the response is usually less

than 30 days (Sartain and Kruse, 2001).

Ammonium sulfate is grayish, angular solid, containing 21% N and 24% sulfur (S).

Ammonium sulfate solubility is low, making this material ideal to produce fertilizer blends.

Ammonium sulfate is mainly produced as a by-product of the Bessemer process: 2 NH3 + H2S04

-* (NH4)2SO4. With a salt index of 3.25, it will cause phytotoxicity at higher rates (Sartain,

2000). It has the highest acid forming potential of N sources through a chemical reaction with

oxygen and water: [(NH4)2S04 + 02 + H20 -2 2H20 + 4H+ + SO4 + 2N03-]. This can be a benefit

for crops which prefer acidic conditions, especially in Florida were high pH soils are common

(Sartain, 2000). Ammonium sulfate provides a dark green color, which tends to last longer than

30 days when applied at recommended rates (Sartain and Kruse, 2001).

Sulfur coated urea consists of urea particles coated with a S shell. First urea is formed by

reacting carbon dioxide with ammonia: CO2 + 2NH3 CO(NH2)2 + H20. Then technology

developed by the Tennessee Valley Authority in the 1960's and 1970's is used to coat the urea

with an S shell. Sulfur was chosen as the coating material due to its low cost and nutrient value.

Depending on the urea source and sealant used to strengthen the sulfur coat, SCU can range in

color from brown to yellow. Nitrogen content of SCU can range from 30 to 40% (Sartain, 2000).










The mechanism for N release from SCU is water penetration through micropores, and cracks in

the S shell. Shell thickness and quality directly affect the rate of release. Once water penetrates

the shell a rapid release on N from the core occurs. However, if a wax sealant is used, microbial

degradation must occur to reveal the imperfection of the S shell. Microbial activity is directly

affected by soil temperature, producing and uneven release and severe mottling ofturfgrasses

during cooler periods. Turf response to SCU lags in comparison to soluble materials, however

duration of response can last from 6 to 16 weeks.

Nitrogen Use in the Soil and Turfgrass System

Nitrogen is normally the limiting nutrient in the turfgrass system (Unruh et al., 1999).

Nitrogen is a highly mobile nutrient within the plant. When plant uptake is inadequate, N

supplies will be transferred from older tissue to the newest tissue for production of chlorophyll,

amino acids, proteins, enzymes, and nucleic acids. All are vital for plant processes throughout the

plant (Brady and Weil, 2000). The atmosphere consists of 78% N, unfortunately turfgrasses

cannot assimilate N2 gas. Therefore, it must be converted to a plant available form. Whether by

lightning and rain (arc process): N2 + 02 2 NO + 02 2NO2; 3 NO2 + H20 2HNO3 + NO,

or through biological fixation: N2 + 8HF 2NH3 + H2. Once in a plant available form, the

nitrogen can have many different fates (Figure. 2-1) (Brady and Weil, 2000; Sartain, 2000).

Nitrogen can be leached to groundwater, lost to runoff into lakes and streams, assimilated into

plant material and animals, adsorbed to the soil, or lost to the atmosphere (Brady and Weil, 2000).

Mineralization

A large portion of soil nitrogen (95 to 99%) is held in organic compounds protecting it

from loss, but that is not available to plants. Mineralization is a three-step process consisting of

aminization, ammonification, and nitrification, which converts organic forms ofN to inorganic

forms of N (Brady and Weil, 2000; Sartain, 2000). Aminization is the breakdown of proteins by

heterotrophic organisms (bacteria and fungi) to amino acids. Ammonification is the breakdown of

amino acids by heterotrophic organisms into ammonia (NH3) and ammonium (NH4+). In these








































Figure 2-1. Nitrogen cycle. (Brady and Weil, 2000).

forms N can be converted to nitrate, used by plants, adsorbed to clay particles, or lost to the

atmosphere. Nitrification is a two-step oxidation process, which occurs when autotrophic bacteria

(Nitrosomonas and Nitrobacter) convert NH4+ to nitrite (NO2-) and then nitrate (NO3-).

Protein heterotrophic organisms amino acid + CO energy


Amino acids + H20 heterotrophic organisms NH3 + R-OH + energy

H20

SNH4 + OH

2NH4 + 302 Nitrosomonas 2N02 + 2H20+ 4H

2N02 + 02 Nltrobacter 2N03









The rate of conversion of organic N or inorganic N is greatly dependant upon the bacteria

population and environment factors that effect these bacteria such as availability of NH4, soil

pH, soil aeration, soil moisture, and soil temperature. Availability of NH4 is directly related to

the C:N ratio within the soil. When C:N ratios are high no NH4 will be released, unless N is

added to the system to lower the C:N ratio (Sartain, 2000). Ultimately, nitrification will be

slowed or possibly halted. However, excessive NH4 can be toxic to Nitrobacter bacteria and

should be avoided (Brady and Weil, 2000). Nitrification occurs between a pH range of 5.0 to 8.5,

with optimal conversion at pH 8.0. Inhibition of nitrification occurs below pH 4.6, due to the

solubility of aluminum, which can become toxic to the bacteria. Aerobic conditions in the soil are

essential for the nitrification to proceed with a minimum of 2% 02 and optimal nitrification

occurring at about 20% 02 (Sartain, 2000). Optimum soil moisture is about 60% of the pore space

filled with water (Brady and Weil, 2000). Soil temperatures for these bacteria range between 5

and 35 C and are optimal between 30 and 35 C.

Leaching

Leaching can occur when N fertilizers are applied to well drained soils. Leaching is

considered to occur when the soil solution N has passed the root zone. Differing sources of N

have the potential to leach at differing rates. Ammonium, a cation, tends to adsorb to soil particles

that are negatively charged, while NO2- and NO3 tend to move through the soil profile more

quickly (Taiz and Zeiger, 1998). The rates of mineralization and immobilization can affect the

amount of inorganic N available for leaching. Soil pH can affect nitrification as previously

discussed, increasing the amount of N adsorption ofNH4+ sources. However, Sartain (2000)

conducted an experiment studying the response of bermudagrass to controlled-release N sources

over a 112 day period, illustrating losses ofNH4+ can occur just as readily as NOs.3 The amount

and intensity of rainfall can influence infiltration, which directly relates to the amount of N

leached. Leaching studies were conducted by Shurman (2002) using soluble 20-20-20 at two N

rates (1.22 g/m2 and 2.44 g/m2) applied every other week in conjunction with irrigation applied at









three rates: level of evapotransportation, 0.64 cm/day, and 1.25 cm/day. These studies revealed

NO3- leaching between 8 to 13% from the higher irrigation. Snyder et al. (1984) studied the

effects of irrigation on seasonal leaching from 'Tifgreen' bermudagrass [Cynodon dactylon (L.)

Pers. X C. transvaalensis Burtt Davy]. The percent of N leached ranged from 0.3 to 56% and was

highly influenced by time of year. Greatest leaching occurred in late winter to early spring and

less occurred during mid summer. Soil slope and soil moisture level when rainfall/irrigation is

applied affects the amount of N leaching. Saturated soils without a slope will tend to have higher

N leaching potential than drier soils without a slope. Soil texture and structure affect permeability

of the soil to water movement, through soil pore and particle size (Sartain, 2000). Soil texture can

dramatically affect N leaching due to differences in the cation exchange capacity (CEC). Sandy

soils tend to have very low CEC (7 cmol/kg), while organic soils tend to have very high CEC

(200 cmol/kg) (Brady and Weil, 2000).

Erosion and Run off

Adsorption of nutrients to soil particles in conjunction with heavy rainfall can lead to

erosion and surface water contamination in some cropping systems. However, this is not the case

in established turfgrass stands. The larger concern is with runoff from turfgrass. When N sources

are surface applied, there is a possibility for N to be lost to runoff. Runoff occurs when water

input exceeds the infiltration rate of the soil. Typically runoff occurs only when high

rainfall/irrigation are applied to a sloped, saturated soil, over a short period of time. Shurman

(2002) conducted studies on fairway runoff of N and phosphorus (P). The soil profile at this site

was a Cecil sandy loam consisting of 49.8% sand, 18% silt, and 32.2% clay. Ammonium

phosphate was applied in early February; then on 22 February, during a 3.8 cm rain event,

samples were collected every hour over a 24-hour period. Nitrate levels ranged from 1 to 2

mg/liter per observation, while NH4+ ranged from 1 to 200 mg/liter per observation, with 75% of

the observations above 100 mg/liter. Soil moisture at the time of rainfall has a direct effect on

runoff volume. In studies conducted by Shurman (2002) a positive linear relationship between










soil moisture and runoff volume was observed with 5 cm simulated rainfall events. Likewise,

Cole et al. (1997) found that runoff volumes from fairways with Kirkland silt loam soil were

greater when soil moisture was higher prior to rainfall.

Plant uptake

For nutrient uptake to occur N must be in a plant available form. Portions of the root

systems are frequently in direct contact with soil particles, allowing for direct exchange of

nutrients. However, this supply is quickly depleted. Three basic methods are used to maintain

nutrient concentration around the root system: root intercept, mass flow, and diffusion. Root

intercept is the growth of roots into new, undepleted soils. Mass flow occurs when nutrients are

carried in water that is being drawn toward plants for use. Diffusion is the random movement of

ions in all directions from an area of high concentration to low concentration, regardless of mass

flow (Brady and Weil, 2000). It should be noted that plant membranes are soluble to some ions

under certain conditions. However, most nutrients only enter the roots by active transport, for this

reason plants can accumulate higher concentrations of nutrients inside the root compared to the

soil solution (Taiz and Zeiger, 1998). With specific carriers for each nutrient, plants can exert

some control over amounts and proportions of the nutrients taken up (Brady and Weil, 2000).

Typically, when N applications are made the goal is plant uptake. Due to losses of N discussed in

previous and later sections, plant uptake of N ranges between 30 to 50%, and rarely exceeds 60%

of applied N. Tissue dry matter of most turfgrasses consists of between 2 to 6% N (Unruh et al.,

1999). Most plants can take up N as NH4 and NO3 forms, but due to soil processes NO3 is the

more prevalent form (Brady and Weil, 2000; Sartain, 2000).

Soil moisture and air content can affect nutrient uptake. If soil moisture is excessively low,

mass flow cannot take place. Likewise, low soil moisture will tend cause stomatas to close,

reducing CO2 intake, and over time reducing plant growth. Conversely, saturated soils may

produce anaerobic conditions that can decrease metabolic activity of the roots (Jackson and Drew,

1984). Likewise, anaerobic conditions in the soil can reduce available N through denitrification of










NO3-. Field capacity, or the amount of water in a soil after free drainage due to gravity has ceased,

normally provides adequate amounts of soil moisture and air for optimal yields.

Soil pH affects nutrient uptake in two ways: 1) by oxidizing cations and 2) by altering

microbial activity. High pH levels will be conducive to oxidation of NH4 to NH3, increasing

volatilization and reducing available N for plant uptake. Conversely, as soil pH levels decrease

below pH 8.0 the rate of microbial activity decreases, lowering the amount of NO3 available to

plants.

Adsorption

Adsorption of N to soil particles, largely occurs with NH4+ and directly relates to soil

texture and CEC of that soil. Soil humus, illite, and vermiculite clays have high CEC and surface

areas enabling them to hold onto large amounts of nutrients. Inversely, kaolinite clay and sand

have low CEC and do not retain nutrients well. Depending on rainfall, percolation rate, and soil

type, adsorption may or may not occur (Sartain, 2000). Soil and water pH greatly influence

nutrient movement into and out of soil solution, due the solubility of ions found in them.

Likewise, soil solution losses to plant uptake, must be replenished from soil reserves (Brady and

Weil, 2000).

Immobilization

Immobilization is the conversion of inorganic N to organic N. As microorganisms feed

upon organic matter it may become necessary to incorporate mineral nitrogen ions into their

cellular components. This temporarily leaves the soil solution impoverished ofNH4+ and NO3

(Brady and Weil, 2000). When the organisms die, NH4+ and NO3- are returned to the soil solution

and the rest becomes part of the organic matter found in the soil for later use in the nitrogen cycle.

Volatilization

Volatilization is the conversion of NH4 to NH3, which can be lost to the atmosphere.

Ammonia gas can be released from mineralization, applications of anhydrous ammonia,

application of ammonium to calcareous soils, and surface applications of urea. Ammonia and










NH4 are in equilibrium depending on soil pH levels (Brady and Weil, 2000). Mineralization

releases NH3 that is converted to NH4 unless soil pH is high, then volatilization is more likely to

occur. Soil texture, soil moisture, and depth of application affect the retention of anhydrous

ammonia. Therefore, if these factors are miscalculated retention in the soil may be short term.

When NH4 is surface applied to calcareous soil or liming occurs with fertilization, calcium

carbonate in soil water will bond with ammonium to form ammonium bicarbonate. Through a

double decomposition reaction NH3 and CO2 will be lost. Surface application of urea in

conjunction with high temperature, alkaline soils, and minimal soil moisture, can have losses of

60 to 90% of the urea N applied. However, these losses can be avoided through timely irrigation

or using another source ofN (Sartain, 2000).



gas

NH4 +OH H20 + NH3



2NH4 + Ca(HCO3)2 2NH4HCO3 + Ca(NO3)2

gas gas

-- 2NH3 +CO2 + 2H20



gas

CO(NH2)2 + H20 UREASE (N)2CO3 HEAT 2NH3 + C2 + H20



Denitrification

Denitrification is a complex microbial process conducted under anaerobic conditions in a

saturated soil that converts NO3- to NO2-, N20, NO, and N2 (Brady and Weil, 2000). Losses of N

due to denitrification can range between 10 to 20% of available N (Sartain, 2000). The

proportions of gaseous products produced rely heavily upon pH, temperature, oxygen depletion,










and amount of nitrate and nitrite ions available. Heterotrophic and autotrophic bacteria involved

in the process are: Psuedomonas spp., Bacillus spp., Micrococcus spp., Achromobacter spp., and

Thiobacillus denitrificans. The process can occur at temperatures between 2 to 50 OC, but the

optimum range is between 25 to 35 C (Brady and Weil, 2000). Soil aeration is a major

controlling factor of this process. Denitrification can occur at soil oxygen levels below 10%, but

progresses faster when soil oxygen levels are below 2% (Brady and Weil, 2000; Sartain, 2000).

Optimum performance occurs at a neutral pH. During denitrification N20 is the dominant gas

produced at a soil pH range of 4.9 to 5.6, whereas N2 is produced in abundance at a pH range of

7.3 to 7.9.

Turfgrass Cultivars

'Tifdwarf Bermudagrass

In 1965, 'Tifdwarf bermudagrass [Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt

Davy] was released for use on golf greens. It is a dwarf mutation selected originally from

'Tifgreen' bermudagrass. 'Tifdwarf bermudagrass is a warm season grass, predominantly used

on greens in the southeastern United States. Greens tend to be sodded or sprigged with vegetative

material in late April to early May. Mowing height for healthy greens ranges from 0.5 to 0.75 cm.

However, most greens today are mowed at cutting heights between 0.3 and 0.4 cm, and are prone

to problems with diseases, drought stress, heat stress, and scalping issues (Morris, 2003).

'Tifway 419' Bermudagrass

'Tifway 419' bermudagrass [Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy]

is a warm season grass predominantly used on golf course fairways, athletic fields, and other

areas throughout the southern United States. Intemode length is relatively short compared to

common bermudagrass, and some seeded bermudagrass cultivars. Shortened intemode length

allows for a dense turf stand, which is resistant to wear. This cultivar is also propagated from

sprigs in late spring, but rarely sodded in fairways.










Root Systems

History

As early as 1873, German botanist Julius Von Sachs studied root systems directly by using

simple soil filled boxes with a glass wall. Now, facilities for the study of roots in the soil have

large underground chambers for observation and analysis of roots, while aerial portions off the

plant are exposed to field conditions. These facilities are called rhizotrons (rhizo meaning root;

tron meaning a device for studying) (Klepper and Kasper, 1994). Details about root morphology

(size and distribution) can be determined as well as root growth over time through time-lapse

photography. The use of root periscopes has become a popular alternative for some scientists due

to the enormous cost of rhizotrons. Root periscopes are transparent plastic tubes that are buried

near roots to be observed. A miniature video camera is inserted into the tube to make

observations about the affects of nutrient and water inputs on root density and root growth (Taiz

and Zeiger, 1998). A study conducted by Dittmer (1937) examining root systems of individual

winter rye plants after 16 weeks of growth. Primary and lateral root axes were estimated at 13 x

106 with root lengths of 500 km which provided approximately 200 m2 of surface area. These

plants also had root hair estimations of 1010 providing approximately another 300 m2 of surface

area.

Root Development and Nutrients

In monocots, root development begins with the emergence of three to six primary

(seminal) root axes from the germinating seed. In vegetatively propagated species the seminal

root axes emerge at the nodes. With the uptake of nutrients additional root growth of brace roots

nodall) occurs. Over time, the primary and brace root grow into a complex fibrous root system. In

the fibrous root system, the main root axes are generally the same root diameter. However newly

forming roots and lateral roots can vary extensively in root diameter.

There are three zones of development found close to the root tips, which are: meristematic

zone, elongation zone, and maturation zone. The meristematic zone produces cells in two










directions, toward the root base and apex. Root cells produced toward the root base will develop

during elongation and maturation. Root cells directed toward the apex will help to form and

maintain the root cap, which continually loses cells while pushing through soil (Taiz and Zeiger,

1998). Mucigel, a gelatinous matrix produced by the root cap, has been hypothesized to aid in

root movement through soil, prevent desiccation, nutrient transfer to the root, or affect

root/microorganism interactions (Russell, 1977). The elongation zone is where cells undergo the

last few cell divisions forming the endodermis, Casparian strip, cortex, and stele. The maturation

zone is the portion of the root where root hairs are formed and xylem fully develops, increasing

the ability of the root to take up water and solutes (Taiz and Zeiger, 1998).

Depending on the nutrient in question, many opinions exist about the areas where nutrients

can be taken up. Potassium (K), NOs3, NH4, and P can be freely taken up by corn along the entire

root surface (Clarkson and Hanson, 1980). However, in corn the elongation zone has been shown

to be the area for maximum uptake of NO3 and P. Likewise, in corn and rice, the root apex has

been shown to take up NH4 more readily than the elongation zone (Colmer and Bloom, 1998).

At this time it appears more research is need to determine nutrient uptake over a wider range of

species.















CHAPTER 3
INFLUENCE OF PLANT-PARASITIC NEMATODES ON
NITRATE LEACHING IN TURF

Introduction

Belonolaimus longicaudatus Rau, the sting nematode, was initially found on several

crops including bermudagrass (Cynodon dactylon L. Pers.)(Rau, 1958). Belonolaimus

longicaudatus has an extensive host range and has been recognized as a pathogen of many

agronomic, horticultural, and ornamental crops (Abu-Gharbieh and Perry, 1970). While it can

be devastating to a wide range of crops, B. longicaudatus is found predominately in sandy

coastal areas of the southeastern United States. Soil texture has a major influence on the

distribution of B. longicaudatus, which is most frequently found in soils consisting of >80%

sand and <10% clay with minimal organic matter (Rhoades, 1980; Robbins and Barker, 1974).

Feeding by B. longicaudatus usually causes the root tips to stop growing; this is

particularly devastating to young plants with a developing root system (Crow et al., 1997; Crow

et al., 2003; Huang and Becker, 1997; Perry and Rhoades, 1982). Damage caused by B.

longicaudatus to bermudagrass root systems can cause decreased water and nutrient uptake,

and reduced plant growth (Johnson, 1970).

In recent years, heightened environmental awareness has focused attention on heavy

users of water, fertilizers and pesticides. This has brought water quality and consumption to the

forefront of public concern (Haydu and Hodges, 2002). Nitrogen (N) is normally the limiting

nutrient in the turfgrass system (Unruh et al., 1999). The intensive use of N fertilizers on golf

courses coupled with a 12.7% increase in maintained turf area over the past 5 years has added

to these concerns and spurred questions to the fate of N following application. Nitrogen

leaching is considered to occur when the soil solution N has passed the root zone. Leaching can










occur when N fertilizers are applied to well drained soils coupled with increased rainfall or

irrigation. Nitrate (NO3) is the most leachable form of nitrogen. Because plant-parasitic

nematodes cause reductions to turf root systems they might increase the amount of NO3

leaching in turfgrass systems. The objective of this study was to determine if damage caused by

B. longicaudatus to turfgrass roots increases nitrate leaching and reduces nitrogen uptake by the

turfgrass plant.

Materials and Methods

A 40-lysimeter greenhouse experiment consisting of two trials was conducted in a glass

house at the University of Florida Turfgrass Envirotron in Gainesville, Florida from 29 January

2002 to 13 September 2002 and from 1 November 2002 to 16 April 2003. Data were collected

over a 126-day period for each trial.

Trial 1

Establishment of experimental units

Forty lysimeters (15-cm-diam.; 45.75-cm-high; 8,339-cm3-volume) were used to

simulate a putting green soil profile. In the bottom of the lysimeters was placed 15 cm of gravel

(2-mm-diam.) covered with an additional 30 cm of nematode-free U.S. Golf Association

(USGA) specification root-zone sand (Anonymous, 1993). The lysimeters were brought to field

capacity and weighed. Aerial sprigs of'Tifdwarf bermudagrass were planted at a rate of 218

kg/ha (0.4g/lysimeter) and top dressed with approximately 0.3 cm of nematode-free sand.

During establishment turf was watered six times a day starting at 0700 hours at 2-hour intervals

with 8 ml of water from a mister irrigation system. Turf was fertilized once, five days after

sprigging with 20-20-20 (N-P205-K20) fertilizer (United Industries Corp., St. Louis, MO).

Nutrient inputs were 91.96 kg/ha N, 40.46 kg/ha P, 76.33 kg/ha K, and trace amounts of

essential micronutrients. The turf was allowed to grow-in and establish a root system for six

weeks before being inoculated with nematodes.










Nematode inoculum

Following turf establishment, 20 lysimeters each were inoculated with B. longicaudatus,

or remained uninoculated, using a completely randomized design. Soil samples collected a

week earlier from a golf course green in Palatka, FL were used to extract nematode inoculum

using a modified Baermann method (McSorley and Frederick, 1991). Inoculum consisted of

mixed life stages of B. longicaudatus that were hand picked into 10 ml of water. Nematode

inoculum was poured into four holes (1-cm-diam. x 2.5-cm-deep) in the soil at a rate of 138

nematodes/lysimeter and allowed to reproduce for a period of eight weeks.

Turf maintenance

Following turf and nematode establishment, turf was watered three times a week. The

first and second watering were 150 ml of water per application, followed by a third where

measured amounts of water were added until prerecorded field capacity weights were achieved

and values of water added were recorded. Turf was fertilized every three weeks with Potassium

Nitrate 14-0-46 (N-P205-K20) at a rate of 668.81 kg/ha/application. Nutrient inputs were 91.96

kg/ha N and 255.35 kg/ha K.

Evaluation and sampling techniques

Turf evaluations were conducted every three weeks after turf and nematode

establishment. Turf quality and color were evaluated on a 1 to 9 scale (1 being poor and 9 being

excellent). Turf density was evaluated on percent of live cover (PLC).

Nematode population counts and root lengths were assessed 6, 12, and 18 weeks after

turf and nematode establishment. Nematode population counts and root lengths were measured

from four, five and eleven samples of each treatment selected at random at the 6, 12, and 18

week evaluations, respectively. Nematode and root samples were obtained by removing the soil

profile (15-cm-diam.) from each lysimeter. The sample extended from the soil surface to the

rock layer (30.48 0.5 cm). The sample was cut in 7.62 0.1 cm lengths to determine

nematode counts and root lengths at four soil depths (0 to 7.62 cm, 7.62 to 15.24 cm, 15.24 to










22.86 cm, and 22.86 to 30.48 cm). Each sub sample was placed onto a 135 .im sieve. The roots

were rinsed with water and the sand and nematodes were collected. Rinsates were agitated with

water and poured into a 25 .im sieve to catch any B. longicaudatus present (Cobb, 1918).

Nematodes were collected and counted using an inverted light microscope at x 30. Roots were

collected, stained with methylene blue, and refrigerated for at least 24 hours. The stained roots

were placed into a glass-bottom tray and scanned with an HP Scanjet 2cx desktop scanner

(Hewlett Packard, Boise, ID) to obtain bitmap images of the root system (Kaspar and Ewing,

1997; Pan and Bolton, 1991). The bitmap images were imported into the GSRoot (Louisiana

State University, Baton Rouge, LA) software program for analysis. This program is designed to

determine root length and surface areas in millimeters for specified root diameters. Root

diameters in mm specified for this analysis were: < 0.05, 0.05 to 0.1, 0.1 to 0.2, 0.2 to 0.3, 0.3

to 0.4, 0.4 to 0.5, and > 0.5. Following root scanning, samples were dried at 70 OC for at least

48 hours and then weighed.

Leaching events were simulated using three soil pore volumes of water at 21 + 1-day

intervals. The leaching technique requires the lysimeters be brought to field capacity and then

water added that is equal to 3 times the pore space of the soil (3,750 ml). The leachate from

each lysimeter was collected and a 20 ml sub sample was taken for analysis; the remaining

volume of leachate was measured. Samples were analyzed for NO3 per liter of water using an

air segmented continuous flow auto spectrometer (Flow Solution IV, O.I. Analytical, College

Station, TX). The equation (mg NO3-/liter x volume of leachate) was used to determine the mg

of NO3 leached from each lysimeter during leaching events.

Turf tissue was collected from each lysimeter at 3-week intervals until destructive root

and nematode sampling. Turf was trimmed to 0.95 cm height and collected. Samples were

placed into a 75 .im sieve washed and then spread evenly on a paper plate. Each sample was

placed in a 1000-W microwave oven and dried for two to six minutes depending on sample

size. Following drying, each sample was weighed. If sufficient dry matter (1.75 g 0.25 g) was










obtained the tissue was ground in a cyclone sample mill (Sample Mill, Udy Corporation, Fort

Collins, CO) to pass through a 1.0-mm screen, placed into a sampling cell, and loaded into a

near infrared reflectance spectroscopy (NIRS) scanning instrument (Model 5000, Foss

NIRSystems, Silver Springs, MD). Spectral data was imported into the Toro Diagnostic

software program (Version 2.4, The Toro Company, Bloomington, MN) for analysis and values

recorded (Rodriguez and Miller, 2000). The equation [tissue percent N x tissue dry weight] was

used to determine mg of N uptake. However, if sufficient tissue was not collected during a

particular three-week interval the tissue was washed, dried, and stored until sufficient tissue

was collected.

Trial 2

Establishment of experimental units

Soil profiles, turf, and irrigation were established as previously stated for trial 1. Turf

was fertilized once, five days after sprigging with 20-20-20 (N-P205-K20) fertilizer. Nutrient

inputs were 109.3 kg/ha N, 48.09 kg/haP, 90.72 kg/ha K, and trace amounts of essential

micronutrients. The turf was allowed to grow-in and establish a root systems for three weeks

prior to nematode inoculation.

Nematode inoculum

Belonolaimus longicaudatus cultures were established from inoculum obtained from

R.M. Giblin-Davis, which originated from Sanford, FL (Giblin-Davis et al., 1992). The

cultures were maintained on 'Tifdwarf' bermudagrass grown on nematode free USGA

specification putting green sand mix for several months. Following turf establishment, four

lysimeters within each block were inoculated with B. longicaudatus using a random complete

block design. Belonolaimus longicaudatus were extracted by decant and sieve method,

collected into a beaker, and the volume brought up to 500 ml (Cobb, 1918). One ml of water

and nematodes was placed onto a counting slide (Hawksley and Sons Limited, Lancing, Sussex,

United Kingdom) to determine the number of nematodes per ml. Nematode counts were










replicated five times with 15 2 nematodes per ml. Nematode inoculum was pipetted into four

holes (1-cm-diam. x 2.5-cm-deep) in the soil at a rate of 300 40 nematodes/lysimeter and

allowed to reproduce for a period of three weeks.

Turf maintenance

Following turf and nematode establishment, the turf was watered twice daily with 25 3

ml of water from an overhead mister irrigation system. Turf was fertilized every three weeks

following leaching events with Potassium Nitrate 14-0-46 (N-P205-K20) at a rate of 794.9

kg/ha/application. Nutrient inputs were 109.3 kg/ha N and 303.5 kg/ha K.

Evaluation and sampling techniques

The sampling and evaluation process were the same as in trial 1, with the following

exceptions: Nematode population counts and root lengths were measured from one inoculated

and one uninoculated lysimeter selected at random from each block at the six-week and twelve

week evaluation, leaving two inoculated and two uninoculated lysimeters from each block at

the end of the study. Nematode and root samples were obtained by removing one core sample

(5-cm-diam.) from the middle of each lysimeter. Leaching events were simulated at 42 1-day

intervals. Stolons and leaf tissue were collected and added to the turf tissue samples for analysis

when destructive root and nematode sampling occurred.

Data Analysis

Nitrate leached data collected at 6 weeks after turf and nematode establishment was

square root transformed (x +1) to normalize the data. T tests were performed to compare

uninoculated and inoculated turf for quality, color, density, root lengths, root surface area, root

weight, NO3 leached, cumulative NO3 leached, tissue dry weights, tissue percent N, and N

uptake at individual sampling dates. Regression analysis was used to characterize relationships

between nematode population counts, root length, nitrogen uptake, and nitrate leached. These T

tests were performed using SAS software (SAS Institute, Cary, NC) while regression analysis

was performed using Minitab software (State College, PA)










Results

Differences (P < 0.05) in amount of NO3- leached between uninoculated and inoculated

turf at specific sampling dates were observed at 18 weeks after turf and nematode establishment

during both trials (Figure 3-1 A, 3-1 B). When amount of cumulative NO3- leached was

compared between treatments at week 18, T tests revealed root systems inoculated with B.

longicaudatus leached more NO3- than did uninoculated root systems (P < 0.05) in trial 1, but

not in trial 2 (Figure 3-2 A, 3-2 B).

Differences (P < 0.05) in total root length and total root weight were observed between

uninoculated and inoculated turf at 6, 12, and 18 weeks after turf and nematode establishment

for both trials. Differences (P < 0.05) in total root surface area were observed at 6, 12, and 18

weeks during trial 1. However, differences in total surface area (P < 0.05) were only observed

at 12 and 18 weeks during trial 2 (Table 3-1, 3-2).

No differences (P < 0.05) were observed between uninoculated and inoculated turf

systems with respects to turf quality, turf color, turf density, tissue dry weight, tissue percent N,

or N uptake following turf and nematode establishment during either trial.

Although several models each year were statistically significant (P < 0.05) correlations

were low or unrepeated between years (Table 3-3).Regression analysis provided no good

predictive models to characterize relationships between nematode counts, root densities,

nitrogen uptake, and nitrate leached.

Discussion

Differences (P < 0.05) in amount of NO3- leached between uninoculated and inoculated

turf at specific sampling dates were observed 18 weeks after turf and nematode establishment

during both trials (Figure 3-1 A, 3-1 B). However, differences in root growth, N assimilation,

and feeding by B. longicaudatus influenced the way these outcomes were expressed in each

trail. During trial 1, actively growing root systems were relatively well established prior to











Trial One Milligrams of Nitrate Leached


O Uninoculated
* Inoculated


3 6 9 12 15 18
Weeks after Turf and Nematode Establishment




Trial Two Milligrams of Nitrate Leached


E Uninoculated
* Inoculated


12 18
Weeks after Turf and Nematode
Establishment


Figure 3-1. Effects of inoculating turf with Belonolaimus longicaudatus on mg nitrate leached
at 3, 6, 9, 12, 15, and 18 or 6, 12, and 18 weeks after turf and nematode
establishment during trial 1 (A) and trial 2 (B), respectively. Inoculated plants
received 138 (trial 1) and 300 + 20 B. longicaudatus (trial 2), while uninoculated
plants received no nematodes. Error bars indicate standard error of individual
population means. ** Inoculated different from uninoculated at (P < 0.01).


E
100

80
1 80
-j
2 60

z 40
0
20

g


S120

S100
c
a 80
-1
60

z 40

w 20

| 0










Trial One Cumulative Nitrate Leached


- uninoculated
- inoculated


3 6 9 12 15 18
Weeks after Turf and Nematode
Establishment






Trial Two Cumulative Nitrate Leached


--- uninoculated
- o -inoculated


6 12 18


Weeks after Turf and Nematode
Establishment


Figure 3-2. Effects of inoculating turf with Belonolaimus longicaudatus on cumulative nitrate
leached at 3, 6, 9, 12, 15, and 18 weeks after turf and nematode establishment
during trial 1 (A) and trial 2 (B), respectively. Inoculated plants received 138 (trial
1) and 300 + 40 B. longicaudatus (trial 2), while uninoculated plants received no
nematodes. Error bars indicate standard error of individual population means.


350
300
250
200
150
100
50
0


350
300
250
200
150
100
50
0










Table 3-1. Effects of inoculating with Belonolaimus longicaudatus on 'Tifdwarf bermudagrass
root length, surface area, and dry weight at 6, 12, and 18 weeks after turf and
nematode establishment during trial 1.

Lysimeter Root Lengths Root Surface Area Root Dry Weights
Depthsa (mm) (mm2) (mg)


0-7.62 (U)b
(I)
7.62-15.24 (U)
(I)
15.24-22.86 (U)
(I)
22.86-30.48 (U)
(I)
All Depths (U)
(I)


0-7.62 (U)
(I)
7.62-15.24 (U)
(I)
15.24-22.86 (U)
(I)
22.86-30.48 (U)
(I)
All Depths (U)
(I)


0-7.62 (U)
(I)
7.62-15.24 (U)
(I)
15.24-22.86 (U)
(I)
22.86-30.48 (U)
(I)
All Depths (U)
(I)


7,104
4,570
4,335
3,564
3,043
1,832
2,528
1,991 +
17,011 +
11,957


210
608 **
597
268
61
132 ***
110
173*
528
359 ***


6,028 151
3,994 566**
4,385 335
2,908 408 *
3,195 78
1,445 306
3,083 157
1,399 120***
16,691 297
9,746 1,221 **


6,809
3,170
3,299
2,459
2,565
1,759
2,476
1,209
15,151
8,597


522
329 ***
48
132 ***
59
157 ***
76
123 ***
591
472 ***


6 weeks
2,352 + 218
1,594 151 *
1,470 173
1,359 164
1,092 83
523 94**
777 68
650 113
5,691 + 279
4,126 + 389 *

12 weeks
2,534 204
1,633 83 **
1,747 159
957+ 111 **
1,250 + 84
372 99***
1,189 137
301 49**
6,720 242
3,263 + 263 ***


18 weeks
2,788 202
1,305 156 ***
1,333 41
912 80***
1,056 47
539 75***
945 75
433 139 **
6,121 275
3,188 229 ***


1,213 63
896 167
571 77
477 44
356 32
188 18**
340 48
194 16*
2,480 140
1,755 159 *


1,020 106
666 124
730 73
388 48**
406 23
170 34***
319 26
136 12***
2,476 126
1,359 208 **


1,063
651
555
324
380
142
305
107
2,302
1,224


60
5 ***
35
32 ***
29
18 ***
21
10 ***
110
71 ***


*, **, *** Inoculated different from uninoculated at a specified depth.
a Soil profile depths are reported in centimeters
b (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
c Means and mean standard error for replications.










Table 3-2. Effects of inoculating with Belonolaimus longicaudatus on 'Tifdwarf bermudagrass
root length, surface area, and dry weight at 6, 12, and 18 weeks after turf and
nematode establishment during trial 2.

Lysimeter Root Lengths Root Surface Area Root Dry Weights
Depthsa (mm) (mm2) (mg)


0-7.62 (U)b
(I)
7.62-15.24 (U)
(I)
15.24-22.86 (U)
(I)
22.86-30.48 (U)
(I)
All Depths (U)
(I)


0-7.62 (U)
(I)
7.62-15.24 (U)
(I)
15.24-22.86 (U)
(I)
22.86-30.48 (U)
(I)
All Depths (U)
(I)


0-7.62 (U)
(I)
7.62-15.24 (U)
(I)
15.24-22.86 (U)
(I)
22.86-30.48 (U)
(I)
All Depths (U)
(I)


475
216
164
66
89
74
77
47
807
404


73
84*
44
19
30
29
32
26
92
107*


479 128
138 48*
223 47
111 30
144 41
86 36
331 + 118
71 31
1,176 + 232
406 85 *


1,801 + 441
99 10**
1,228 266
77 9**
777 197
48 12**
443 115
26 11**
4,249 + 958
250 25**


6 weeks
88 20
60 25
28 8
10 3
16 5
11 4
11 4
8+ 5
143 18
89+ 29

12 weeks
110 39
31+ 13
35 9
13 4
22 8
9+ 4
67 24
10+ 4
234 60
64 13 *


18 weeks
229 117
26 5
77 27
15 3*
53 17
11 5*
34 13
5+ 3*
393 141
58 10*


28.2
12.0
5.3
2.3
3.0
1.3
1.6
1.3
38.1
16.9


3.3
4.4*
2.2
1.0
1.6
0.5
0.8
0.9
6.3
5.5 *


27.6 + 7.9
10.1 5.3
9.8 2.5
3.4 0.9 *
5.5 1.6
2.7 1.2
11.2 3.1
3.5 1.9
54.0 + 12.5
19.7 4.0 *


67.9 12.4
10.1 2.2***
35.2 4.4
5.2 0.7***
22.1 3.1
3.6 1.3 ***
12.6 3.3
1.7 0.7**
137.7 + 20.8
20.6 3.3***


*, **, *** Inoculated different from uninoculated at a specified depth.
a Soil profile depths are reported in centimeters
b (U) = uninoculated (I)= inoculated plants received 300 40 B. longicaudatus per
lysimeter.
c Means and mean standard error for replications.










Table 3-3. Linear regression analysis conducted to determine relationships between nematode
populations, root length, nitrogen uptake, and nitrate leached during both trials.

Dependant Independent
Variable Variable Y= r2 P<


Trail 1

Nematodes Root length 7311.25 + 3.19x 0.269 0.023
(total)a
Nematodes Root length 3342.34 + 1.62x 0.290 0.017
(small)b
Nematodes N uptake 437.50 0.16x 0.321 0.011
Nematodes N leached 408.27 0.26x 0.493 0.001
Root Length N uptake 374.47 0.OOx 0.020 0.381
(total)
Root Length N leached 336.45 0.01x 0.143 0.016
(total)
Root Length N uptake 409.91 0.01x 0.058 0.135
(small)
Root Length N leached 362.50 0.03x 0.172 0.008
(small)
N uptake N leached 139.10 0.42x 0.161 0.324

Trial 2

Nematodes Root length 458.19 0.16x 0.018 0.570
(total)
Nematodes Root length 471.26 0.32x 0.031 0.458
(small)
Nematodes N uptake 31.02 + 0.18x 0.501 0.000
Nematodes N leached 90.85 + 0.18x 0.055 0.319
Root Length N uptake 98.09 + 0.01x 0.083 0.071
(total)
Root Length N leached 151.05 0.02x 0.064 0.114
(total)
Root Length N uptake 109.14 + 0.01x 0.001 0.886
(small)
Root Length N leached 176.41 0.12x 0.065 0.113
(small)
N uptake N leached 79.43 + 0.16x 0.216 0.039
a Total = root lengths for all root diameters throughout the entire soil profile.
b Small = root lengths for root diameters < 0.2 mm throughout the entire soil profile.










inoculation, and B. longicaudatus reduced the root systems over time. A short-term solution for

actively growing root systems may have been to compensate for root reductions by B.

longicaudatus with increased N assimilation throughout the remaining root system. However,

increasing root reduction by B. longicaudatus overwhelmed the plants ability to assimilate N,

which led to the differences observed in NO3- leached. Conversely, during trial 2 root systems

were slow to establish and develop due to limited light intensity and duration during winter

months. As the experiment progressed, light intensity and duration improved, which led to

increased root growth and N assimilation in uninoculated root systems, but feeding by B.

longicaudatus still retarded root development. Total root lengths in inoculated root systems

were reduced 30 %, 42 %, and 43 % at 6, 12, and 18 weeks after turf and nematodes

establishment during trial 1 and 50 %, 66 %, and 94 % during trial 2. Differences (P < 0.05) in

nitrate leached were not observed until reductions in root lengths and surface area were

observed in a large portion of root diameters at all rooting depths in actively growing root

systems (Table C-l to Table C-13). This directly relates to the ability of the entire root system

of actively growing turf to assimilate N (Clarkson and Hanson, 1980). Differences (P < 0.05) in

nitrate leached may occur so r in mature stands of turf with increased suberization of large

diameter roots which can shift the burden of N assimilation to root tips, root hairs, and finer

lateral roots were nematode feeding typically occurs.

When amount of cumulative NO3- leached was compared between treatments, root

systems inoculated with B. longicaudatus leached more NO3- than did uninoculated root

systems (P < 0.05) in trial 1, but not in trial 2 (Figure 3-2 A, 3-2 B). Upon reviewing the

amount of NO3- leached for each treatment at specific sampling dates, 1 might expect

differences. However, root growth and root reductions by B. longicaudatus are highly variable

and not evenly distributed throughout experimental units (Table 3-1, 3-2). When these factors

were combined the resulting variability within treatment was greater than variability between

treatments at specific sampling dates until 18 weeks after turf and nematode establishment










during both trials. Furthermore, cumulative nitrate leached incorporates variability within and

between treatments from all dates, causing an additive effect to the variability within treatment.

Differences in the amount of nitrate leached between uninoculated and inoculated root systems

at 18 weeks after turf and nematode establishment were sufficient to overcome the additive

effect to the variability within treatment during trial 1, but not trial 2 at (P < 0.05). During trial

2, light intensity and duration began to improve at 12 weeks after turf and nematode

establishment, which increased NO3- and root differences between treatments. Given more time

differences in cumulative nitrate leached would likely have continued to increase as well.

Feeding by B. longicaudatus can cause varying degrees of damage to root systems

depending on plant type and age of the plant when its root system is first attacked. Rarely does

nematode feeding all kill a plant (Christie, 1959). Typically, nematode feeding will predispose

turf to other adverse conditions such as drought stress, heat stress, and malnutrition, which

could lead to reduced turf quality, color, and density. Furthermore, reductions in turf density

can reduce the leaf surface area hindering evapotransportation, subsequently reducing water

and nutrient uptake needed for photosynthesis and tissue production. In the glasshouse, no

differences (P < 0.05) were observed between uninoculated and inoculated turf systems

following turf and nematode establishment during either trial with respects to turf quality, turf

color, turf density, tissue dry weight, tissue percent N, or N uptake. This may be to a lack of

persistent adverse conditions.

In conclusion, whether turfgrass root systems were well established or newly forming,

damage caused by B. longicaudatus to the entire turfgrass root system can increase nitrate

leaching. However, the rates of root growth, N assimilation, and feeding by B. longicaudatus

can determine the amount of time needed to observe differences in NO 3- leached. Nitrogen

uptake was not hindered during either trial. However differences (P < 0.05) in nitrate leaching

were not observed until the end of both trials. Nitrogen uptake may have been slowly declining







35


during the last six weeks of the study but due to N dilution within the plant, no differences were

detected.















CHAPTER 4
EFFECT OF NEMATODE MANAGEMENT AND NITROGEN FERTILITY ON
FAIRWAY TURF QUALITY

Introduction

Belonolaimus longicaudatus Rau, the sting nematode, was initially found on several crops

including bermudagrass (Cynodon dactylon L. Pers.) (Rau, 1958). Belonolaimus longicaudatus

has an extensive host range and has been recognized as a pathogen of many agronomic,

horticultural, and ornamental crops (Abu-Gharbieh and Perry, 1970). Belonolaimus longicaudatus

is found predominately in sandy coastal areas of the southeastern United States. Soil texture has a

major influence on the distribution of B. longicaudatus, which is most frequently found in soils

consisting of >80% sand and <10% clay with minimal organic matter (Rhoades, 1982; Robbins

and Barker, 1974).

Belonolaimus longicaudatus is considered to be the most damaging plant-parasitic

nematode on turfgrasses in Florida. Feeding by B. longicaudatus can cause varying degrees of

damage to root systems depending on plant type, and age when its root system is first attacked.

Damage caused by B. longicaudatus to bermudagrass root systems can cause decreased water and

nutrient uptake, and reduced plant growth, but rarely does nematode feeding all kill a plant

(Christie, 1959; Johnson, 1970). Typically, nematode feeding will predispose turf to other adverse

conditions such as drought stress, heat stress, malnutrition, arthropods, pathogens, and weeds

which could lead to reduced turf quality, color, and density (Lucas, 1982).

Recently, nematode management has been perceived by the turf industry as a growing

problem due to fewer effective nematicides being available. If nematode management is not

effective, the typical response to decreasing turf quality, color, and density by golf course

managers is to increase water and nitrogen (N) fertility levels. When N fertilizers are applied to










well-drained soils leaching can occur. Reductions of the turfgrass root system by B.

longicaudatus, could increase the leaching potential.

In recent years, heightened environmental awareness has focused attention on heavy users

of water, fertilizers and pesticides. This has brought water quality and consumption to the

forefront of public concern (Haydu and Hodges, 2002). The intensive use of N fertilizers on golf

courses coupled with a 12.7% increase in maintained turf area over the past 5 years has added to

these concerns and spurred questions to the fate of N following application. The objective of this

study was to describe relationships between nematode management and nitrogen fertility in terms

of turf quality and root lengths on golf course fairways.

Materials and Methods

A 2-year field study consisting of two trials was conducted in West central Florida on golf

course fairways infested with B. longicaudatus (Rau, 1958). Trial 1 was conducted in Citrus

County, Florida, from 12 March 2002 to 29 August 2002, while trial 2 was conducted in Pasco

County, Florida from 13 March 2003 to 29 August 2003. Data were collected over a 112-day

period during each trial.

Experimental Sites

Pathogens

Plant-parasitic nematodes present at the Citrus County site included B. longicaudatus,

Hopolaimus galeatus (Cobb,Thome) Helicotylenchus sp., Peltamigratus sp., Trichodorus sp.,

Paratrichodorus sp., Hemicycliophora sp., Hemicriclmoides sp., and Mesocriclma sp. Fungal

diseases previously treated for at this site were Bermudagrass Decline (Gaeumannomyces

graminis var. graminis Sacc. Arx. and D.L. Olivier), Brown Patch (Rhizoctonia solani J. G.

Kohn), and Fairy Ring (Chlorophyllum, Marasmius, or Lepiota spp.).

Plant-parasitic nematodes present at the Pasco County site included B. longicaudatus, H.

galeatus, Helicotylenchus sp., Trichodorus sp., and Mesocriclma sp. Fungal diseases previously










treated for at this site were Bermudagrass Decline, Brown Patch, and Damping-Off (Pythium

spp.).

Insects and weeds

Pest insects observed during these trials were southern mole cricket (Scapteriscus

borrellii Giglo-Tos), tawny mole cricket (Scapteriscus vicinus Shudder), fall armyworm

(Spodopterafrugiperda J.E. Smith), red imported fire ant (Solenopsis invicta Buren), ringlegged

earwig (Euborellia annulipes Lucas), and two lined spittlebug (Propsapia bicincta L.).

Weeds observed during these trials were goosegrass (Eluesine indica L. Gaertn.), crabgrass

(Digitaria spp.), crowfootgrass (Dactyloctenium aegi'ptiunm L. Willd.), carpetgrass (Axonopus

affinis Chase), creeping signalgrass (Brachiaria plantaginea L. A. S. Hitchc.), doveweed

(Murdannia nudiflora L. Brenan), and spotted spurge (Euphorbia maculata L.).

Turf

In both trials, golf course fairways had mature stands (15 to 20 years old) of 'Tifway 419'

bermudagrass [Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy]. Turf at both

locations was maintained at 1.3 cm cutting height and watered as needed. Both fairways had

histories of nematode damage over the last few years, requiring more attention to cultural

practices and inputs.

Soil properties

Soil texture at a depth of 10 to 15 cm was analyzed using the hydrometer method

(Bouyoucos, 1936). Soil at the Citrus County site was Tavares fine sand with a composition of

92% sand, 4.5% silt, 3.5% clay; < 1% organic matter and pH 5.8 (USDA, 1982). Soil at the Pasco

County site was Millhopper-Candler Variant soil with a composition of 97% sand, 0% silt, 3%

clay; < 1% organic matter and pH 6.0 (USDA, 1985).

Experimental Design

The experimental design varied from 2002 to 2003. In 2002, the experimental design was

arranged as a split plot design. Whole plots were three nematode management tactics: 1,3-










dichloropropene (1,3-D) applied by slit-injection (Crow et al., 2003), a mechanical slit treatment

with no chemical applied, and untreated control. Each whole plot was replicated four times. Sub

plots consisted of four N rates 0, 36.65, 73.30, and 109.95 kg/ha/month. Main plots were 3.7-m-

wide and 15.2-m-long, with sub plots being 0.9-m-wide and 15.2-m-long. Main plots were

separated by border areas (1.5 m on the sides and 3.0 m at each end), which were only mowed

and watered.

In 2003, the experimental design was arranged in a randomized complete block. Eight

treatments were two nematode management tactics: 1,3-dichloropropene and untreated control

with four N rates of 0, 36.65, 73.30, and 109.95 kg/ha/month. Treatments were replicated four

times. These plots were 3.7-m-long and 3.7-m-wide. Plots were separated by border areas (1.5 m

wide on all sides), which were maintained as previously stated. The change in experimental

design was dl to reduce the incidents of fertility runoff from 1 sub plot into another.

In both trials, nematode samples were collected six weeks prior to nematicide treatments.

Nematodes were extracted from the soil using a modified centrifugal-flotation technique and

counted (Jenkins, 1964). Plots with counts below 30 B. longicaudatus per 100 cm3 of soil were

excluded from the study. The remaining plots were assigned to blocks according to B.

longicaudatus population counts. Treatments were randomized within each block.

Nematicide Treatments

Nematicide treatments for the 2002 trial were 1,3-dichloropropene, mechanical, and

untreated control. In 2003, the mechanical treatment was eliminated after no differences (P <

0.05) were observed for nematode populations counts or visual performance between mechanical

and control plots in 2002. Nematicide treatments were applied once per trail during the first week

of May.

1,3-dichloropropene was injected at a rate of 46.76 liters/ha with a nitrogen gas

pressurized application rig. The application rig had straight coulters placed on 30.5 cm centers,

followed by a chisel with a metal drip line attached which placed the material at a depth of 13 to










17 cm. A steel roller wheel followed each chisel to close the soil. Mechanical treatments

consisted of running the application rig through the soil as preformed with the 1,3-

dichloropropene treatments, without the chemical being applied. Immediately after nematode

management tactics were concluded, approximately 1.25 cm of water was applied, which assisted

in holding the 1,3-dichloropropene in the soil.

Fertilization

The turf fertilization program varied from 2002 to 2003. In 2002, fertilization began two

weeks prior to nematicide treatment and continued at two-week intervals until the end of the

study. Turf was fertilized with Potassium Nitrate 14-0-46 (N-P205-K20) at N rates of 0, 36.65,

73.30, and 109.95 kg/ha/month using a drop spreader.

During the 2002 trial, when fertilizer was applied without the turf being watered, salt

induced phytotoxicity occurred. Portions of subplots fertilized with N rates of 36.65, 73.30, and

109.95 kg/ha/month showed proportional damage depending on fertilizer rates. This problem was

corrected in 2003 by using a slow release fertilizer, which consisted of Sulfur Coated Urea, Sulfur

Coated Ammonium Phosphate, Sulfur Coated Sulfate of Potash, Iron Oxide, and Manganese

Sucrate. In 2003, fertilization began four weeks prior to nematode management treatments being

applied and continued at two-week intervals until the end of the study. Turf was fertilized with a

14-14-14 (N-P205-K20) sulfur coated blend at N rates of 0, 36.65, 73.30, and 109.95 kg/ha

/month using a hand-held rotary spreader. In 2003, an unscheduled fertilizer application occurred

during week 11 with a slow release blend of 21-0-18 (N-P205-K20) at an N rate of 70.68 kg/ha

(broadcast).

General Production Practices

Turf Maintenance

In both trails, turf was mowed by the golf courses staff three times a week at a cutting

height of 1.3 cm. However, on several occasions the turf was not mowed due to rain. Cultural

practices conducted by golf course staff such as aerification, slicing, and vertical mowing were










halted for this experiment. In 2002, turf was irrigated with 0.64 cm of water as needed.

Conversely, in 2003 turf was irrigated once a day with 0.64 cm of water until week 3 when the

irrigation system failed causing the turf to go without watering for 3 to 4 days. Thereafter, turf

was irrigated twice a day with 0.64 cm of water except during week 9 when the irrigation system

failed again causing the turf to go without watering for 3 to 4 days.

Pesticides

In 2002, no additional pesticides were used, except for the experimental treatment.

Subsequently, MSMA at 2.25 kg a.i./ha and metribuzin at 0.035 kg a.i./ha were tank mixed and

applied as a spot treatment to control Eluesine indica on 14 July 2003 in trial 2.

Sampling and Evaluations

Turf Evaluations

Turf evaluations were conducted every two weeks, beginning with the first N fertility

treatment each year. Turf quality and color were evaluated on a 1 to 9 scale (1 being poor, 6.5

acceptable, and 9 being excellent). Turf density was evaluated on percent of live cover (PLC). In

2002, each subplot (0.91 m x 15.24 m) was evaluated as a whole, which made evaluations

difficult. Subsequently, in 2003 each plot was divided into four equal quadrants, each quadrant

was evaluated for turf quality, color, and density.

Nematodes

In both trials, twelve cores (2.5-cm-diam and 10.2-cm-depth) were obtained from each

plot using a cl sampler to determine nematode population counts. A 15-cm buffer zl was

established inside the parameter of each treatment plot to ensure accurate treatment results.

Samples were taken twice prior to nematicide treatment (six weeks, and 1 day prior to nematicide

treatments), and at two-week intervals following nematicide treatments. Each sample was mixed

thoroughly and a 100-cm3 sub sample was obtained. Nematodes were extracted from the soil

using a modified centrifugal-flotation technique (Jenkins, 1964). Traditionally, the extraction

process requires the soil to be passed through a 2 mm sieve to remove debris, however this step










was omitted to prevent B. longicaudatus from being lodged in the mesh of the sieve (McSorley

and Fredrick, 1991). Following, extraction all plant-parasitic nematodes were counted using an

inverted light microscope at a magnification of 20 x.

Roots

Root samples (3.5-cm-diam and 15-cm-depth) were obtained inside the buffer zl with a

tee sampler. In 2002, following nematicide treatments, two root cores were collected at 0, 6, and

16 weeks from each treatment plot and combined into a single sample with no differences

observed (P < 0.05). Previous studies had shown increases in root length when plant-parasitic

nematodes were being managed, so an increase in sample size was suggested. In 2003, following

nematicide treatments, three root cores were collected at 0, 6, and 16 weeks from each treatment

plot. In both trials, roots were processed, analyzed, and weighed as described in Appendix A.

Turf Tissue

Turf tissue was collected every two-weeks, beginning with the first N fertility treatment

each year. In 2002, tissue samples were collected from three 30 cm x 30 cm areas within each

treatment plot. However, in 2003 tissue was collected from the entire treatment plot (3.7 m x 3.7

m). Turf was trimmed to 0.95 cm height during both trails.

Tissue samples were processed and analyzed as described in Appendix A. However, since

large amounts of tissue were collected from each treatment plot, following the grinding step each

sample was thoroughly mixed and a 2.0 g + 0.5 g sub sample was obtained for analysis.

Data Analysis

ANOVA were performed to compare turf quality, color, density, root lengths, root surface

area, root weight, tissue dry weights, tissue percent N, and N uptake between treatments at

individual sampling dates. Due to interactions between nematode management tactics and N

fertility, general linear models and orthogonal contrasts were performed at individual N fertility

levels to compare between treatments. Regression analysis was used to characterize relationships

between nematode population counts and fertility in terms of turf quality and root lengths on golf










course fairways. ANOVA, general linear models, and orthogonal contrasts were performed using

SAS software (SAS Institute, Cary, NC) while regression analysis was performed using Minitab

software (State College, PA).

Results

Differences (P < 0.05) were observed in B. longicaudatus population means between

untreated control and nematode management at 2, 4, and 6 weeks after treatment during both

trials, respectively (Figure 4-1A, 4-1B).

Differences (P < 0.05) in turf quality were observed between untreated control and

nematode management at individual N fertility levels at 2, 8, 10, and 12 weeks after treatment

during trial 1 and 2, 4, 8, 10, 12, 14, and 16 weeks after treatment during trial 2 (Table 4-1, 4-2).

During trial 2, turf quality was not evaluated at week 6 due to excessive leaf material being left

across the research area.

No differences (P < 0.05) in root length and surface area of specified root diameters, total

root length, total surface area, or root weight were observed between nematode management

tactics at individual N fertility levels at 0, 6, and 16 weeks after treatment during either trial

(Table 4-3).

Confounding issues with respects to collection of tissue samples precluded an unbiased

analysis of tissue dry weight, tissue percent N, or N uptake during either trial. During trial 1,

scheduling conflicts precluded the continued use of mowing equipment for collection of tissue

samples. During trial 2, on numerous occasions the turf was mowed just prior to arrival, thereby

removing differences in vertical leaf growth that may have occurred between treatments. In both

incidents actions beyond the experimenters control precluded collection of accurate samples.

Regression analysis provided no good predictive models to characterize relationships

between nematode management and nitrogen fertility in terms of turf quality and root lengths.















160
140
120
100
80
60
40


44



Nematode Population Differences among
Treatments during Trial One







SControl
A Mechanical
o Nematicide




17h3--D0-- 0--D


-6 0 2 4 6 8 10 12 14 16
Weeks after Nematode Management Tactic


B
Nematode Population Differences between
Treatments during Trial Two

w 120
C.
'I--
S 100
o 80
0 80

o 60
IO 60 Control

E| o --o--Nematicide
z 40
Qr 20 -

0
> 0---------------------
-6 0 2 4 6 8 10 12 14 16
Weeks after Nematode Management
Tactic


Figure 4-1. Means of Belonolaimus longicaudatus per 100 cm3 of soil sampled from 6 tol6 weeks
after nematode management tactics were applied during trial 1 (A) and trial 2 (B),
respectively. Error bars indicate standard error of individual population means.
Control = no added soil disturbance or nematicide treatment; Mechanical = soil
disturbance without nematicide; Nematicide = Injection of 1-3, dichloropropene at a
rate of 46.76 liters per hectare at 13 to 17 cm of soil depth. indicates differences (P
< 0.05) between Control and Nematicide treatments.













Table 4-1. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a 'Tifway 419'
bermudagrass fairway at 0 to 16 weeks after treatment during trial 1.

N Fertility


Weeks Treatment
0 COb
NT
2 CO
NT
4 CO
NT
6 CO
NT
8 CO
NT
10 CO
NT
12 CO
NT
14 CO
NT
16 CO
NT


36.65


4.25 + 0.14c
4.25 + 0.14
4.25 + 0.14
4.75 + 0.14
4.88 + 0.24
5.38 + 0.55
5.38 0.13
5.13 + 0.24
4.63 0.24
5.38 + 0.24
4.75 0.25
5.31 + 0.19
4.38 0.13
5.13 0.13
4.63 0.24
5.00 + 0.20
4.81 + 0.24
5.44 + 0.39


4.38 + 0.13
4.50 + 0.00
4.63 0.24
5.00 + 0.20
5.25 + 0.25
5.50 + 0.29
4.75 + 0.14
5.25 + 0.14
4.50 + 0.20 *
5.38 + 0.13
4.50 + 0.20 **
5.25 + 0.10
4.38 + 0.13 **
5.38 + 0.13
4.50 + 0.35
4.88 + 0.43
4.94 + 0.21
5.44 + 0.39


73.30


4.63 0.24
4.75 + 0.14
4.38 + 0.24 *
5.25 + 0.32
4.63 + 0.13
5.63 + 0.24
4.63 + 0.13
5.13 0.31
4.63 0.24
5.25 + 0.32
4.56 + 0.21 *
5.69 + 0.31
4.38 + 0.13 *
5.25 + 0.25
4.25 0.48
5.00 + 0.20
4.13 0.33
5.56 + 0.33


Data are means and mean standard error of four replications.
*, **, *** Untreated control different from nematicide treated using orthogonal contrast at a specified nitrogen (N) fertility level.
a Kilograms of N per hectare per month.
b (CO) Untreated control = no added soil disturbance or nematicide treatment (NT) Nematicide = Injection of 1-3, dichloropropene
at a rate of 46.76 liters per hectare at 13 to 17 cm of soil depth.
c Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as maximum turf quality, and 6.5 as the threshold for
acceptability.


109.95


4.25 + 0.32
4.88 + 0.24
4.63 + 0.38
5.75 + 0.60
4.75 0.25
6.00 + 0.50
4.75 + 0.14
5.13 + 0.24
4.88 0.31
5.50 + 0.41
4.75 + 0.32
5.19 0.61
4.44 + 0.26
4.88 + 0.43
4.50 + 0.50
4.63 0.72
5.19 0.31
5.38 + 0.43













Table 4-2. Turf quality in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a 'Tifway 419'
bermudagrass fairway at 0 to 16 weeks after treatment during trial 2.

N Fertility


Weeks Treatment


36.65


73.30


109.95


0 COb
NT
2 CO
NT
4 CO
NT
8 CO
NT
10 CO
NT
12d CO
NT
14 CO
NT
16 CO
NT
Data are means and mean


6.28 + 0.09c
6.31 + 0.14
6.34 + 0.16 ***
7.14 + 0.09
5.91 + 0.23 *
6.77 + 0.25
6.53 + 0.18
5.98 + 0.21
6.08 + 0.23
6.58 + 0.15
6.59 + 0.12
6.72 + 0.15
6.42 + 0.14
6.72 + 0.17
7.20 + 0.17
7.42 + 0.14
standard error of 16 replications.


6.69 + 0.12
6.47 + 0.27
6.25 + 0.17 ***
7.34 + 0.29
5.55 + 0.42 *
6.44 0.24
6.20 + 0.34
6.72 + 0.24
6.39 + 0.27
6.64 + 0.24
6.77 + 0.09
6.66 + 0.17
6.64 + 0.14
6.81 + 0.24
7.02 0.11 ***
5.73 + 0.34


6.16 + 0.25
6.39 + 0.24
6.02 + 0.19 ***
7.23 0.06
5.33 + 0.43 ***
6.86 + 0.14
6.00 + 0.34 **
7.31 + 0.21
5.09 0.39***
7.22 + 0.20
5.92 + 0.12 ***
7.17 0.11
5.56 + 0.32***
7.28 + 0.15
6.22 + 0.35
6.22 + 0.15


7.20 + 0.16
7.28 + 0.12
6.89 + 0.18 ***
7.48 + 0.13
5.52 0.32***
7.20 + 0.17
6.05 + 0.32 ***
7.36 + 0.30
5.50 + 0.35 ***
7.45 + 0.33
6.61 + 0.14 ***
7.81 + 0.09
6.64 0.22***
7.72 + 0.09
6.48 0.11 ***
7.19 0.11


*, **, Untreated control different from nematicide treated using orthogonal contrast at a specified nitrogen (N) fertility level.
a Kilograms of N per hectare per month.
b (CO) Untreated control = no added soil disturbance or nematicide treatment (NT) Nematicide= Injection of 1,3-dichloropropene
at a rate of 46.76 liters per hectare at 13 to 17 cm of soil depth.
c Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as maximum turf quality, and 6.5 as acceptable.
d Between weeks 10 and 12 an unscheduled fertilizer application of a slow release blend of 21-0-18 (N-P205-K20) was evenly applied across all
experimental plots at a rate of 336.58 kg/ha; Nutrient rates were 70.68 kg N/haand 50.28 kg K (broadcast).













Table 4-3. Total root lengths observed in plots treated with 1,3-dichloropropene and in untreated plots at individual N fertility levels on a 'Tifway
419' bermudagrass fairway at 0, 6, and 16 weeks after treatment during both trials.

N Fertility


Weeks Treatment


36.65


73.30


109.95


580.90 153.89c
704.31 265.99
263.24 66.89
359.71 48.42
217.82 84.89
199.32 55.16


154.77 33.84
134.14 36.75
173.08 28.43
226.14 46.43
170.54 42.64
187.18 29.99


Trial 1
681.08
420.56
321.59 +
401.58
217.29
264.74


142.63
141.51
64.72
74.96
99.83
37.38


Trial 2
85.84 22.70
84.51 30.25
91.43 19.75
240.79 81.22
94.51 24.42
191.80 45.53


577.04
526.59
363.19
434.20
187.68
249.33


111.78
121.89
59.81
88.89
69.85
40.47


85.63 33.20
148.08 52.78
129.45 53.83
281.20 72.85
85.16 30.65
159.91 44.48


499.88 118.34
847.31 253.25
308.23 84.39
443.85 85.96
200.69 37.42
299.66 60.05


171.47 52.58
158.76 27.69
157.14 39.15
234.86 43.08
126.84 26.83
203.00 64.23


Data are means and mean standard error of 8 and 12 replications for trial 1 and 2, respectively.
*, **, *** Untreated control different from nematicide treated using orthogonal contrast at a specified nitrogen (N) fertility level.
a Kilograms of N per hectare per month.
b (CO) Untreated control = no added soil disturbance or nematicide treatment (NT) Nematicide = Injection of 1,3-dichloropropene at a rate of
46.76 liters per hectare at 13 to 17 cm of soil depth.
c Root length are presented as millimeters.


COb
NT
CO
NT
CO
NT










Some models each year were statistically significant (P < 0.05). However, correlations were low,

preventing further comparisons (Figure C-1A, C-1B) (Table C-14, C-15).

Discussion

Reduced (P < 0.05) B. longicaudatus populations counts were observed in nematicide

treated plots compared to untreated controls at 2, 4, and 6 weeks after treatment during both trials.

These reductions in B. longicaudatus populations counts indicate adequate coverage and rates of

nematicide treatments for B. longicaudatus control during these trails. Likewise, a general decline

in nematode populations over time was observed during both trails, which may indicate a

seasonal reduction of nematode populations from March through August. Further studies of B.

longicaudatus population dynamics may be warranted to determine when best to apply

nematicide treatments for a maximum benefit to turf stands. No differences (P < 0.05) were

observed between control and mechanical treatments, indicating the slit-injection process by itself

has minimal effect on B. longicaudatus populations (Figure 4-1A, 4-1B).

Turf quality is a function of turf color and density, which is generally used to infer turf

health status. Therefore turf quality was focused upon during these studies. Differences (P < 0.05)

were observed between untreated control and nematicide treated plots within individual N fertility

levels with respect to turf quality, color, and density at some dates during both trials (Table 4-1,

4-2). Differences (P < 0.05) in turf quality were observed between untreated control and

nematicide treated plots within each N fertility level at 2 and 4 weeks after treatment and 8 to 16

weeks after treatment at the higher N fertility levels of 73.30 and 109.95 kg N/ha/month during

trial 2. The irregular turf quality, color, and density response to nematode management and

increased N fertility during trial 1 may have been caused by salt induced phytotoxicity that

occurred from high rates of potassium nitrate that were not irrigated properly. Similarly, lack of

root development may have hindered the ability of the turf to take up water and nutrients for plant

development (Christie, 1959; Johnson, 1970). During trial 2, differences (P < 0.05) were

observed in turf quality at 2 and 4 weeks after treatment at each N fertility level, which










corresponded with reduced B. longicaudatus populations counts. Even with root development

hindered, reduced parasitism by B. longicaudatus may have allowed more water and nutrients to

reach leaf tissue leading to increased production, storage, and use of photosynthates which could

be utilized for plant development or during times of drought stress. Furthermore, reduced

parasitism coupled with possible increased reserves ofphotosynthates may explain differences (P

< 0.05) in turf quality that were observed between untreated control and nematicide treatments

following each of the irrigation system failures. While reduced turf quality was observed in both

the untreated control and nematicide treated plots following irrigation system failures, turf quality

losses were minimized in nematicide treated plots. Upon reviewing turf quality following the first

irrigation failure, drought damage was greatest in untreated control plots at N fertility levels 73.30

and 109.95 kg N/ha/month. The drought damage continued to hinder turf quality in untreated

control plots at N fertility levels 73.30 and 109.95 kg N/ha/month for several weeks and was

reinforced during week 9 with a second irrigation failure. This situation illustrates that increased

watering and N fertility can improve turf quality in the short term, however if adverse conditions

such as drought stress, heat stress, or improper mowing occur on turf stands suffering nematode

infestations, then turf quality can be reduced for an extended period of time (Lucas, 1982).

No differences (P < 0.05) in root length and surface area of specified root diameters, total

root length, total surface area, or root weight were observed between nematode management

tactics at individual N fertility levels at 0, 6, and 16 weeks after treatment during either trial.

These results differ from results reported by Crow et al. (2003), which revealed increases (P <

0.05) in total root length following slit injections of 1,3-dichloropropene. However during both

trials, root systems possessed necrotic tissue, which may indicate another factor in addition to

nematodes could have been suppressing root development.

In conclusion, nematode management has been shown to improve turf quality under some

conditions, most notably nematode management helps to minimize turf quality loses when turf

experiences stress under field conditions (Crow et al., 2003). Likewise, increasing N fertility has







50


also been shown to improve turf quality. While this experiment was unable to determine whether

nematode management with increasing N fertility levels improved turf quality, it indicates that

increased N fertility without nematode management could be detrimental to turf quality,

especially when the turf experiences stress. No inferences can be made with respect to nematode

management and N fertility in relation to root length. Lack of root development during both trials

illustrates that nematode management is but 1 concern that must be addressed with respects to

turf root systems.















CHAPTER 5
SUMMARY

These experiments confirm that Belonolaimus longicaudatus is a pathogen on

bermudagrasses in Florida. Research revealed that feeding by plant parasitic nematodes can

reduce turf root systems, allowing increased amounts of nitrates to be leached. Furthermore, this

research is a foundation for later projects concerning: timing of nematicide applications, turf

reductions by B. longicaudatus in the presence of nematode antagonists, turf reductions and

nitrate leaching with minimal water and N fertility inputs.

Glasshouse experiments revealed differences (P < 0.05) between uninoculated and

inoculated turf in total root length at 6, 12, and 18 weeks and milligrams of nitrate leached at 18

weeks during both trials. Belonolaimus longicaudatus feeding reduced total root length by 30 to

94 percent, and increased the amount of nitrate leaching as much as 429 percent (Chapter 3).

However, differences (P < 0.05) between uninoculated and inoculated turf in amount of nitrate

leached were not observed until reductions in root length and surface area were observed in a

large portion of root diameters at all rooting depths in actively growing root systems (Appendix

C). No differences (P < 0.05) were observed between nematode inoculated and uninoculated

lysimeters in tissue nitrogen levels, dry matter production, or total nitrogen uptake (Chapter 3).

This directly relates to the ability of the entire root system of actively growing turf to assimilate N

(Clarkson and Hanson, 1980). When the amount of cumulative nitrate leached was compared

between treatments, root systems inoculated with B. longicaudatus leached more nitrate than did

uninoculated root systems (P < 0.05) in trial 1, but not in trial 2. No differences (P < 0.05) were

observed for turf quality, color, or density during either trial, confirming that through intensive

management of water, nutrients, pests, and pathogens; turf appearance can be maintained even

with root damage from plant parasitic nematodes. Subsequently, the intensive management of turf










to maintain turf quality, color, and density coupled with nematode damage to turf roots may

increase nitrate leaching, thereby adding to water quality concerns.

In field experiments, reduced (P < 0.05) B. longicaudatus populations counts were

observed between untreated control and 1,3-dichloropropene treatments at 2, 4, and 6 weeks after

treatment during both trials. No differences (P < 0.05) were observed between control and

mechanical treatments, indicating the slit-injection process has no detectable effect on B.

longicaudatus populations (Figure 4-1A, 4-1B). Furthermore, a general decline in nematode

populations over time was observed during both trails, which may indicate further study of B.

longicaudatus population dynamics is warranted to determine when best to apply nematicide

treatments for a maximum benefit to turf stands. Differences (P < 0.05) were observed between

untreated control and nematicide treated plots within individual N fertility levels with respect to

turf quality, color, and density at some dates during both trials. During trial 2, differences (P <

0.05) were observed in turf quality at 2 and 4 weeks after treatment which corresponded with

reduced B. longicaudatus counts. Reduced parasitism by B. longicaudatus may have allowed

more water and nutrients to reach leaf tissue leading to increased production, storage, and use of

photosynthates which could be utilized for plant development or during times of drought stress.

Differences (P < 0.05) were observed in turf quality from 8 to 16 weeks after treatment at N

fertility levels of 73.30 and 109.95 kg N/ha/month. Reduced parasitism coupled with possible

increased reserves of photosynthates may explain differences (P < 0.05) in turf quality that were

observed between untreated control and nematicide treated plots following each of the irrigation

system failures. While reduced turf quality was observed in both the untreated control and

nematicide treated plots following irrigation system failures, turf quality losses were minimized

in nematicide treated plots. Upon reviewing turf quality following the first irrigation failure,

drought damage was greatest in untreated control plots at N fertility levels 73.30 and 109.95 kg

N/ha/month. The drought damage continued to hinder turf quality in untreated control plots at N










fertility levels 73.30 and 109.95 kg N/ha/month for several weeks and was reinforced during

week 9 with a second irrigation failure.

No differences (P < 0.05) in root length and surface area of specified root diameters, total

root length, total surface area, or root weight were observed between untreated control and

nematicide treated plots within individual N fertility levels at 0, 6, and 16 weeks after treatment

during either trial. These results differ from results reported by Crow et al. (2003), which revealed

increases (P < 0.05) in total root length following slit injections of 1,3-dichloropropene.

However, root systems possessed necrotic tissue, which may indicate another factor other than

nematodes could have been suppressing root development. Lack of root development illustrates

that nematode management is but 1 concern that must be addressed with respects to turf root

systems.

In conclusion, glasshouse studies indicated that nematode damage to turf roots can increase

nitrate leaching, thereby adding to water quality concerns. Field experiments indicate that

increased N fertility without nematode management could be detrimental to turf quality,

especially when the turf experiences stress. These studies indicate that nematode and N fertility

management are equally important to providing a quality turf and minimizing environmental

impacts.















APPENDIX A
DETAILED MATERIALS AND METHODS USED IN THE GREENHOUSE STUDY

Introduction

A 40-lysimeter greenhouse experiment consisting of two trials was conducted over a 16-

month period at the University of Florida Turfgrass Envirotron glass houses in Gainesville,

Florida from 29 January 2002 to 13 September 2002, and from 1 November 2002 to 16 April,

2003. Data were collected over a 126-day period during each trial. The objective of this study was

to determine if damage caused by Belonolaimus longicaudatus (Rau, 1958) to turfgrass roots

could increase nitrate leaching and reduce nitrogen uptake by the turfgrass plant.

Experimental Materials

Lysimeters

Lysimeters were constructed of 15-cm-diam. polyvinyl chloride (PVC) pipe cut in 45.75

cm lengths. Heavy-duty window screen was cut in 23 cm x 23 cm squares and placed over 1 open

end of the pipe held on by a rubber band. Then a 1.27 cm hole was drilled into the center of a 15-

cm-diam. PVC end cap. A threaded brass reducer bushing (1.27-cm-exterior-diam. male to 0.95-

cm-interior-diam. male) was threaded into the predrilled hole. The PVC end cap was then

carefully hammered onto the end of pipe with a rubber mallet and excess screen was cut away. A

bead of white bathtub caulking was placed around the exterior junction of the pipe and cap. The

caulking was allowed to dry at least 12 hours and then the lysimeters were checked for leaks

(Figure A-i, A-2, A-3).

Turf

A stand of 'Tifdwarf' bermudagrass was obtained from Dr. G. L. Miller at the University

of Florida Turfgrass Envirotron in September, 2001. Forty clay pots measuring 10.16-cm-diam.






































Figure A-1. A lysimeter used as an experimental unit during glasshouse trials at the University of
Florida Turfgrass Envirotron from 29 January 2002 to 16 April 2003.


Figure A-2. Screen placed within a lysimeter after assembly. The screen holds the soil profile
away from drainage hole preventing drain blockage.





























Figure A-3. A threaded bushing screwed into a lysimeter. The bushing enables leachate to be
collected.

were filled with nematode free sand and sprigged with aerial stolons of 'Tifdwarf bermudagrass.

During January 2002 and November 2002 aerial stolons were cut at least 7.62 cm + 0.1 cm from

the pot, collected, weighed, and sprigged into the lysimeters.

Soil Properties

The United States Golf Association (USGA) sand mix was tested for soil particle

requirements established by the USGA for greens construction (Anonymous, 1993). A 2,000 cm3

soil sample was obtained from the sand being placed into the lysimeters. The sample was spread

out onto an oven rack covered with newspaper, then placed into a drying oven for approximately

48 hours at 70 OC. The soil sample was run through a series of sieves stacked 1 on top of another

to separate soil particles by size. Particle sizes in mm of interest were: < 0.15, 0.15 to 0.25, 0.25

to 0.5, 0.5 to 1.0, 1.0 to 2.0, and > 2.0 mm. Sand mix used in these trials met USGA

specifications.

Nematode Inoculum

Trail 1: A mixed population of nematodes was collected from a golf course green in

Palatka, FL. Nematodes were extracted from the soil using a modified Baermann method










(McSorley and Frederick, 1991). Belonolaimus longicaudatus were hand picked from the mixed

population of nematodes and placed into a small glass beaker containing 10 ml of water until

proper inoculum levels were reached.

Trail 2: A pure population ofB. longicaudatus was obtained from Dr. R.M. Giblin-Davis,

which originated from Sanford, FL (Giblin-Davis et al., 1992). Eight clay pots measuring 15-cm-

diam. were filled with nematode free sand and then sprigged with 'Tifdwarf' bermudagrass. After

turf establishment, nematode inoculum suspended in 20 ml of water was pipetted into four holes

(1-cm-diam. x 2.5-cm-deep) in the soil at a rate of 100 nematodes/pot and allowed to reproduce.

Five months later, soil and roots from each pot were separated into four sub samples for

convenience. Each sub sample was placed onto a 120-mesh sieve. The roots were firmly rinsed

with water, collecting the sand and nematodes in a stainless steel container below. The sand and

nematodes were then agitated with water to separate the nematodes from the soil. The water and

nematodes were then poured into a 25 .im sieve and collected into a 1,000 ml beaker (Cobb,

1918). After all the sub samples had been run, the volume in the beaker was brought up to 500

ml. 1 ml of water and nematodes was placed onto a counting slide to determine the number of

nematodes per ml of water. This process was replicated five times with an average of 15

nematodes per ml. Twenty ml of water and nematode solution were required to deliver proper

inoculum levels.

Experimental Design

The experimental design varied from trial 1 to trial 2. The experimental design in trial 1

was arranged in a completely randomized design with 20 lysimeters inoculated with B.

longicaudatus and 20 uninoculated control lysimeters. Trial 2 was arranged in a randomized

complete block design. Lysimeters were assigned to 1 of five blocks with eight lysimeters per

block (40 total), within each block four lysimeters (20 total) were selected at random to be

inoculated with B. longicaudatus.










General Production Practices

Turf Establishment

Trial 1: Forty lysimeters were set up with 15 cm of gravel placed in the bottom of the

lysimeters covered with an additional 30 cm of nematode-free sand. Water was run through the

lysimeters to remove air pockets and the sand was brought back to 30 cm depth. Each lysimeter

was brought to field capacity and weights recorded. Lysimeters were sprigged with nematode-

free 'Tifdwarf bermudagrass at a rate of 218 kg/ha (0.4 g/lysimeter) and top dressed with

approximately 0.32 cm of nematode-free sand. Turf was watered six times a day starting at 0700

hours at 2-hour intervals with 8 ml of water from a mister irrigation system during establishment.

Turf in each lysimeter was fertilized once, five days after sprigging with 20-20-20 (N-P205-K20)

fertilizer (United Industries Corp., St. Louis, MO) at a rate of 8.4 g/liter of water. Nutrient inputs

were 91.96 kg/ha N, 40.46 kg/ha P, 76.33 kg/ha K, and trace amounts of essential micronutrients.

The turf was allowed to grow-in and establish a root system for six weeks.

Trial 2: Forty lysimeters were filled and planted as described in trial 1 except field capacity

was not determined. Turf was watered six times a day with 8 ml of water from a mister irrigation

system to prevent desiccation during the establishment period. Turf in each lysimeter was

fertilized once, five days after sprigging with 20-20-20 (N-P205-K20) fertilizer at a rate of 9.97

g/liter of water. Nutrient inputs were 109.3 kg/ha N, 48.09 kg/haP, 90.72 kg/ha K, and trace

amounts of essential micronutrients. The turf was allowed to grow-in and establish a root system

for three weeks.

Nematode Establishment

Trial 1: Following six weeks of root establishment, 20 lysimeters were inoculated with B.

longicaudatus. Nematode inoculum suspended in 10 ml of water was poured into four holes (1-

cm-diam. x 2.5-cm-deep) in the soil at a rate of 138 nematodes/lysimeter and allowed to

reproduce for a period of eight weeks.










Trail 2: Following three weeks of root establishment, 20 lysimeters were inoculated with B.

longicaudatus. Nematode inoculum suspended in 20 ml of water was pipetted into four holes (1-

cm-diam. x 2.5-cm-deep) in the soil at a rate of 300 nematodes/lysimeter and allowed to

reproduce for a period of three weeks.

Temperature

Trial 1: Monthly average high and low air temperature in the glasshouse ranged from 22 C

to 33 C and 18 C to 250C, respectively.

Trial 2: Monthly average high and low air temperature in the glass house ranged from 21

C to 26 C and 18 C to 200C, respectively.

Watering

Trial 1: Following turf and nematode establishment, the turf was watered between 0900-

1000 hours, three times a week. The first and second watering consisted of 150 ml of water per

application, followed by a third watering which required adding measured amounts of water until

the lysimeters were brought back to field capacity using the prerecorded weights. Total water

requirements were recorded for each lysimeter.

Trial 2: Following turf and nematode establishment, the turf was watered at 1000 and 1500

hours, daily with 25 + 3 ml of water from an overhead mister irrigation system.

Fertilization

Trial 1: Following turf and nematode establishment, turf was fertilized with Potassium

Nitrate 14-0-46 (N-P205-K20) at a rate of 668.8 kg/ha/application. Nutrient inputs were 91.96

kg/ha N and 255.35 kg/ha K. Applications were made at three-week intervals immediately after

leaching events.

Trial 2: Following turf and nematode establishment, turf was fertilized with Potassium

Nitrate 14-0-46 (N-P205-K20) at a rate of 794.9 kg/ha/application. Nutrient inputs were 109.3

kg/ha N and 303.5 kg/ha K. Applications were made at three-week intervals immediately after

leaching events.










Pesticides

The primary arthropod pest species found attacking 'Tifdwarf bermudagrass in these trials

was bermudagrass mite (Eriophyes cynodoniensis). This pest was managed with two applications

of fluvalinate over a seven-day period. Treatments were repeated as needed.

Sampling and Evaluations

Turf Evaluations

Turf evaluations were conducted every three weeks after turf and nematode establishment.

Turf quality and color were evaluated on a 1 to 9 scale (1 being poor and 9 being excellent). Turf

density was evaluated on percent of live cover (PLC).

Nematodes and Roots

Trial 1: Nematode population counts and root lengths were assessed 6, 12, and 18 weeks

after turf and nematode establishment. Nematode population counts and root lengths were

measured from four inoculated and four uninoculated lysimeters selected at random at the six-

week evaluation, five inoculated and five uninoculated lysimeters at the twelve-week evaluations,

and 11 inoculated and 11 uninoculated lysimeters at the end of the study. Nematode and root

samples were obtained by removing the entire soil profile (15-cm-diam.) of each lysimeter from

the soil surface to the rock layer (30.48 0.5-cm-deep). The sample was cut in 7.62 0.1 cm

lengths to determine nematode counts and root lengths at four differing depths (0 to 7.62 cm, 7.62

to 15.24 cm, 15.24 to 22.86 cm, and 22.86 to 30.48 cm). Each sub sample was placed into a 135

mm sieve. The roots were firmly rinsed with water, collecting the sand and nematodes in a

stainless steel container below. The sand and nematodes were then agitated with water to separate

the nematodes from the soil. The water and nematodes were poured into a 25.m sieve and

collected for counting (Cobb, 1918). Roots were collected from the 135 tim sieve, placed into a

glass container, stained with methylene blue, and refrigerated for at least 24 hours. The

equipment was washed at length to prevent contamination between samples. After root staining,

root samples were individually placed onto a glass-imaging pan. The roots were spread apart to










prevent overlap and a white background was placed over the roots. The glass-imaging pan was

then placed onto a flat bed scanner to obtain a black and white bitmap image of the roots (Figure.

A-4) (Kaspar and Ewing, 1997; Pan and Bolton, 1991). The bitmap images were imported into

the GSRoot (Louisiana State University, Baton Rouge, LA) software program for analysis. This

program is designed to determine root length and surface areas in millimeters for specified root

diameters. Root diameters in mm specified for this analysis were: < 0.05, 0.05 to 0.1, 0.1 to 0.2,

0.2 to 0.3, 0.3 to 0.4, 0.4 to 0.5, and > 0.5.


r-























Figure A-4. Glass imaging pan with white background placed on a flat bed scanner to obtain
bitmap images of root systems.

Following root scanning, each root sample was collected from the glass-imaging pan,

placed into a small paper bag, and labeled appropriately. The samples were placed into a drying

oven for approximately 48 hours at 70 OC and then weighed.

Trial 2: Nematode population counts and root lengths were assessed 6, 12, and 18 weeks

after turf and nematode establishment. Nematode population counts and root lengths were

measured from 1 inoculated and 1 uninoculated lysimeter selected at random from each block










(total often) at the six and twelve-week evaluations. Nematode and root measurements were

collected from the remaining 20 lysimeters (two inoculated and two uninoculated from each

block) at the end of the study. Nematode and root samples were obtained by removing 1 core

sample (5-cm-diam.) from the middle of each lysimeter. The core sample extended from the soil

surface to the rock layer (30.48 0.5 cm). The sample was cut in 7.62 0.1 cm lengths to

determine nematode population density and root length and surface area at four differing depths

(0 to 7.62 cm, 7.62 to 15.24 cm, 15.24 to 22.86 cm, and 22.86 to 30.48 cm). Each sub sample was

placed into a 135.m sieve. The roots were firmly rinsed with water collecting the sand and

nematodes in a stainless steel container below. The sand and nematodes were then agitated with

water to separate the nematodes from the soil. The water and nematodes were then poured into a

25!m sieve and collected for counting (Cobb, 1918). Root lengths and dry weights were

obtained as described previously.

Leachate

Trial 1: Leaching events were simulated using three soil pore volumes of water at 21 + 1-

day intervals. The leaching technique requires the lysimeters be brought to field capacity and then

water added that is equal to 3 times the pore space of the soil (3,750 ml). The leachate from each

lysimeter was collected in a separate bucket placed under each lysimeter during the leaching

events. Each bucket was brought to a work area and stirred with a plastic utensil. A 20 ml sample

was taken from the bucket and the remaining volume of leachate was measured. Samples were

analyzed using an air segmented continuous flow auto spectrometer (Flow Solution IV, O.I.

Analytical, College Station, TX). The equation [mg NO3/liter x volume of leachate recovered]

was used to determine the mg of nitrate leached from each lysimeter.

Trial 2: Leaching events were simulated as previously discussed, however at 42 1-day

intervals.










Turf Tissue

Following turf and nematode establishment, turf tissue was collected from each lysimeter

separately at 3-week intervals until destructive root and nematode sampling. Each lysimeter was

lifted out of the rack and laid on its side on a workbench. Turf was trimmed to 0.95 cm height and

collected into small paper bags. Later, each tissue sample was placed into a 75 .im sieve washed

to remove soil and debris, and then spread evenly on a paper plate. Each sample was placed in a

1000-W microwave oven and dried for two to six minutes depending on sample size (Figure. A-

5). Following the drying process, each sample was weighed. If sufficient dry matter (1.75 g

0.25 g) was obtained, the tissue was ground in a cycll sample mill (Figure. A-6) (Sample Mill,

Udy Corporation, Fort Collins, CO) to pass through a 1.0-mm screen, placed into a sampling cell,

and loaded into a near infrared reflectance spectroscopy (NIRS) scanning instrument (Model

5000, Foss NIRSystems, Silver Springs, MD). Spectral data was imported into the Toro

Diagnostic software program (Version 2.4, The Toro Company, Bloomington, MN) for analysis

and values recorded (Figure A-7). The equation [tissue percent N x tissue dry weight] was used to

determine mg ofN uptake. However, if sufficient tissue was not collected during a particular

three-week interval the tissue was washed, dried, and stored until sufficient tissue was collected.

During trail 2, when destructive root and nematode sampling occurred, stolons and leaf tissue

were collected for each lysimeter separately, processed, and analyzed as well.

Data Analysis

Nitrate leached data collected at 6 weeks after turf and nematode establishment was square

root transformed (x +1) to normalize the data. Milligrams of NO3- leached were compared

between treatments across all sampling dates using analysis of variance. These T tests were

performed to compare turf quality, color, density, root lengths, root surface area, root weight,

NO3- leached, tissue dry weights, tissue percent N, and N uptake between treatments at individual

sampling dates. Regression analysis was used to characterize relationships between nematode

population counts, root length, nitrogen uptake, and nitrate leached. Analysis of variance and T










tests were performed using SAS software (SAS Institute, Cary, NC) while regression analysis

was performed using Minitab software (State College, PA).


Figure A-5. After washing, turf tissue samples were placed on a paper plate and dried in a 1000-
W microwave oven for two to six minutes depending on sample size.


Figure A-6. Cycll sample mill used to grind turf tissue for analysis. Dried tissue is placed into the
yellow cone, top center, and is retrieved from the glass jar, bottom center.




























Figure A-7. Near infrared reflectance spectroscopy (NIRS) scanning instrument, left. Spectral
data determined by NIRS was imported into a laptop computer with Toro Diagnostic
software program for analysis, right. Sampling cells waiting to be analyzed, top left,
sampling cells previously analyzed, bottom left.















APPENDIX B
DETAILED MATERIALS AND METHODS USED IN THE FIELD STUDY

Introduction

A 2-year field study consisting of two trials was conducted in west central Florida on golf

course fairways infested with Belonolaimus longicaudatus (Rau, 1958). Trial 1 was conducted in

Citrus County, Florida, from 12 March 2002 to 29 August 2002, while trial 2 was conducted in

Pasco County, Florida from 13 March 2003 to 29 August 2003. Data were collected over a 112-

day period during each trial. The objective of this study was to describe relationships between

nematode management and nitrogen fertility in terms of turf quality and root lengths on golf

course fairways.

Experimental Sites

Pathogens

Plant-parasitic nematodes present at the Citrus County site included B. longicaudatus,

Hopolaimus galeatus (Cobb,Thome) Helicotylenchus sp., Peltamigratus sp., Trichodorus sp.,

Paratrichodorus sp., Hemicycliophora sp., Hemicriclmoides sp., and Mesocriclma sp. Fungal

diseases previously treated for at this site were Bermudagrass Decline (Gaeumannomyces

graminis var. graminis Sacc. Arx. and D.L. Olivier), Brown Patch (Rhizoctonia solani J. G.

Kohn), and Fairy Ring (Chlorophyllum, Marasmius, or Lepiota spp.).

Plant-parasitic nematodes present at the Pasco County site included B. longicaudatus, H.

galeatus, Helicotylenchus sp., Trichodorus sp., and Mesocriclma sp. Fungal diseases previously

treated for at this site were Bermudagrass Decline, Brown Patch, and Damping-Off (Pythium

spp.).










Insects and weeds

Pest insects observed during these trials were southern mole cricket (Scapteriscus

borrellii Giglo-Tos), tawny mole cricket (Scapteriscus vicinus Shudder), fall armyworm

(Spodopterafrugiperda J.E. Smith), red imported fire ant (Solenopsis invicta Buren), ringlegged

earwig (Euborellia annulipes Lucas), and two lined spittlebug (Propsapia bicincta L.).

Weeds observed during these trials were goosegrass (Eluesine indica L. Gaertn.), crabgrass

(Digitaria spp.), crowfootgrass (Dactyloctenium Legi'ptimI L. Willd.), carpetgrass (Axonopus

affinis Chase), creeping signalgrass (Brachiaria plantaginea L. A. S. Hitchc.), doveweed

(Murdannia nudiflora L. Brenan), and spotted spurge (Euphorbia maculata L.).

Turf

In both trials, golf course fairways had mature stands (15 to 20 years old) of 'Tifway 419'

bermudagrass [Cynodon dactylon (L.) Pers. X C. transvaalensis Burtt Davy]. Turf at both

locations was maintained at 1.3 cm cutting height and watered as needed. Both fairways had

histories of nematode damage over the last few years, requiring more attention to cultural

practices and inputs.

Soil Properties

Soil texture at a depth of 10 to 15 cm was analyzed using the hydrometer method

(Bouyoucos, 1936). Soil at the Citrus County site was Tavares fine sand with a composition of

92% sand, 4.5% silt, 3.5% clay; < 1% organic matter, and pH 5.8. Soil at the Pasco County site

was Millhopper-Candler Variant soil with a composition of 97% sand, 0% silt, 3% clay; < 1%

organic matter, and pH 6.0.

Experimental Design

The experimental design varied from 2002 to 2003. In 2002, the experimental design was

arranged as a split plot design. Whole plots were three nematode management tactics: 1,3-

dichloropropene (1,3-D) applied by slit-injection (Crow et al., 2003), a mechanical slit treatment

with no chemical applied, and untreated control. Each whole plot was replicated four times. Sub










plots consisted of four N rates 0, 36.65, 73.30, 109.95 kg/ha/application. Main plots were 3.7-m-

wide and 15.2-m-long, with sub plots being 0.9-m-wide and 15.2-m-long. Main plots were

separated by border areas (1.5 m on the sides and 3.0 m at each end), which were only mowed

and watered. A plot plan illustrating the experimental layout is shown in Fig. B-1.

In 2003, the experimental design was arranged in a randomized complete block. Eight

treatments were two nematode management tactics: 1,3-dichloropropene and untreated control

with four N rates of 0, 36.65, 73.30, 109.95 kg/ha/application. Treatments were replicated four

times. These plots were 3.7-m-long and 3.7-m-wide. Plots were separated by border areas (1.5-m-

wide on all sides), which were maintained as previously stated. A plot plan illustrating the

experimental layout is shown in Fig. B-2. The change in experimental design was dl to reduce

the incidents of fertility runoff from 1 sub plot into another.

In both trials, nematode samples were collected six weeks prior to nematicide treatments.

Plots were assigned to blocks according to B. longicaudatus population counts. Treatments were

randomized within each block.

Nematicide Treatments

Nematicide treatments for the 2002 trial were 1,3-dichloropropene, mechanical, and

control. In 2003, the mechanical treatment was eliminated after no differences (P < 0.05) were

observed for nematode populations counts or visual performance between mechanical and control

plots. Nematicide treatments were applied once per trail during the first week of May.

1,3-dichloropropene was injected at a rate of 46.76 liters/ha with nitrogen gas pressurized

application rig. The application rig had straight coulters placed on 30.5 cm centers, followed by a

chisel with a metal drip line attached which placed the material at a depth of 13 to 17 cm. A steel

roller wheel followed each chisel to close the soil. Mechanical treatments consisted of running the

application rig through the soil as preformed with the 1,3-dichloropropene treatment, but without

the chemical being applied. Immediately after nematode management tactics were concluded,












approximately 1.25 cm of water was applied, which assisted in holding the 1,3-dichloropropene


in the soil.


14.0 m


5 8 17 cU





1 2 3 0


N



CO Control
M M=echanical (Nonematicide)
CU 1-3, Dichloropopene
0 = No N added experimentally
1 3665kgN/ha/month
2 7330kgN/ha/month
3 =109 95 kg N/ha/month









106.7 m


Figure B-1. Plot plan of the field study undertaken at Citrus County, FL during 2002. Plots
marked with X were not used as experimental units due to nematode counts being below the
action threshold for Belonolaimus longicaudatus.








192 m


1 20 21 40

CUl2 C02 CO1 CUO
1.5

X
C02 CU2 COO
3.66 m

X
CU3 CU0 COO


X
C03 COO C03


X
CU1 CU3 CO



CU2 C03 CU3 CU3


x








CU2

10 COO

COO


CO2



CUO


x


11

CO1


CU2



CUO



CO1

30

C02


CU1



CU1


X


31

C03


CO Control
CU 1-3, Dichloropropene
0 =No N added experimentally
1 = 36 65 kg N/ha/month
2 = 73 30 kg N/ha/month
3 109 95 kg N/ha/month






53.3 m


Figure B-2. Plot plan of the field study undertaken at Pasco County, FL during 2003. Plots
marked with X were not used as experimental units due to nematode counts being
below the action threshold for Belonolaimus longicaudatus.










Fertilization

The turf fertilization program varied from 2002 to 2003. In 2002, fertilization began two

weeks prior to nematode management treatments being applied and continued at two-week

intervals until the end of the study. Turf was fertilized with Potassium Nitrate 14-0-46 (N-P205-

K20) at N rates of 0, 36.65, 73.30, and 109.95 kg/ha/month using a drop spreader.

During the 2002 trial, when fertilizer was applied without the turf being watered, salt

induced phytotoxicity occurred. Portions of subplots fertilized with N rates of 36.65, 73.30, and

109.95 kg/ha/month showed proportional damage depending on fertilizer rates. This problem was

corrected in 2003 by using a slow release fertilizer, which consisted of Sulfur Coated Urea, Sulfur

Coated Ammonium Phosphate, Sulfur Coated Sulfate of Potash, Iron Oxide, and Manganese

Sucrate.

In 2003, fertilization began four weeks prior to nematode management treatments being

applied and continued at two-week intervals until the end of the study. Turf was fertilized with a

14-14-14 (N-P205-K20) sulfur coated blend at N rates of 0, 36.65, 73.30, 109.95 kg/ha/month

(broadcast). In 2003, an unscheduled fertilizer application occurred during week 11 with a slow

release blend of 21-0-18 (N-P205-K20) at an N rate of 70.68 kg/ha (broadcast).

General Production Practices

Turf Maintenance

In both trails, turf was mowed by the golf courses staff three times a week at a cutting

height of 1.3 cm. However, on several occasions the turf was not mowed due to rain. Cultural

practices conducted by golf course staff such as aerification, slicing, and vertical mowing were

halted for this experiment. In 2002, turf was irrigated with 0.64 cm of water as needed.

Conversely, in 2003 turf was irrigated once a day with 0.64 cm of water until week 3 when the

irrigation system failed causing the turf to go without watering for 3 to 4 days. Thereafter, turf

was irrigated twice a day with 0.64 cm of water except during week 9 when the irrigation system

failed again causing the turf to go without watering for 3 to 4 days.










Pesticides

In 2002, no additional management tactics were warranted to control insects, weeds, or

pathogens, except experimental treatment. 1 management tactic was implemented on 14 July

2003 (week 10 of trail 2), when MSMA 2.25 kg a.i./ha and metribuzin 0.035 kg a.i./ha were tank

mixed and applied as a spot treatment to control Eluesine indica.

Sampling and Evaluations

Turf Evaluations

Turf evaluations were conducted every two weeks, beginning with the first N fertility

treatment each year. Turf quality and color were evaluated on a 1-9 scale (1 being poor, 6.5

acceptable, and 9 being excellent). Turf density was evaluated on percent of live cover (PLC). In

2002, each subplot (0.91m x 15.24 m) was evaluated as a whole, which made evaluations

difficult. Subsequently, in 2003 each plot was divided into four equal quadrants, each quadrant

was evaluated for turf quality, color, and density.

Nematodes

In both trials, twelve cores (2.5-cm-diam and 10.2-cm-depth) were obtained from each

plot using a cl sampler to determine nematode population counts. A 15 cm buffer z was

established inside the parameter of each treatment plot to ensure accurate treatment results.

Samples were taken twice prior to nematicide treatment (six weeks and 1 day prior to nematicide

treatments), and at two-week intervals following nematicide treatments.

Each sample was mixed thoroughly and a 100-cm3 sub sample was obtained. Nematodes were

extracted from the soil using a modified centrifugal-flotation technique (Jenkins, 1964).

Traditionally, the extraction process requires the soil to be passed through a 2 mm sieve to

remove debris, however this step was omitted to prevent B. longicaudatus from being lodged in

the mesh of the sieve (McSorley and Fredrick, 1991). Following, extraction all plant-parasitic

nematodes were counted using an inverted light microscope at a magnification of 20 x.










Roots

Root samples (3.5-cm-diam and 15-cm-depth) were obtained inside the buffer zl with a tee

sampler. In 2002, two root cores were collected at 0, 6, and 16 weeks after nematicide treatment

from each treatment plot and combined into a single sample with no differences observed (P <

0.05). Previous studies had shown increases in root length when plant-parasitic nematodes were

being managed, so an increase in sample size was suggested. In 2003, three root cores were

collected at 0, 6, and 16 weeks after nematicide treatment from each treatment plot. In both trials,

roots were processed, analyzed, and weighted as described in Appendix A.

Turf Tissue

Turf tissue was collected every two-weeks, beginning with the first N fertility treatment

each year. In 2002, tissue samples were collected from three 30 cm x 30 cm areas within each

treatment plot, however in 2003 tissue was collected from the entire treatment plot (3.7 m x 3.7

m). Turf was trimmed to 0.95 cm height during both trails.

Tissue samples were processed and analyzed as described in Appendix A. However, since

large amounts of tissue were collected from each treatment plot, following the grinding step each

sample was thoroughly mixed and a 2.0 g + 0.5 g sub sample was obtained for analysis.

Data Analysis

ANOVA were performed to compare turf quality, color, density, root lengths, root

surface area, root weight, tissue dry weights, tissue percent N, and N uptake among treatments at

individual sampling dates. Due to interactions between nematode management tactics and N

fertility, orthogonal contrasts were performed at individual N fertility levels to compare

individual treatments. Regression analysis was used to characterize relationships between

nematode population counts and fertility in terms of turf quality and root lengths on golf course

fairways. ANOVA, general linear models, and orthogonal contrasts were performed using SAS

software (SAS Institute, Cary, NC) while regression analysis was performed using Minitab

software (State College, PA).

















APPENDIX C
SUPPLEMENTAL FIGURES AND TABLES


A Effects of Nematode and N Fertility Management
during Trial One


6
5.8 Y = 0.003x +5.16
C r2r2 = 0 044


y =-0.0001 x+ 4.68
r2 = 7E-05


y 0.0014x + 4.55
r 0.014


-Control
Mechanical
- Nematicide


B









ro
0
.4-
I-


0 20 40 60 80 100 120
kg N/ha/month




Effects of Nematode Management and N Fertility
during Trial Two



y = 0.0079x + 6.41
.r2 = 0.154


00"_ -Nematicide
,, -- Control

Sy = 0.00 1 8x + 6.39
r2 = n nn7


0 20 40 60 80 100 120
kg N/ha/month


Figure C-1. Regression models of turf quality response to Untreated Control, Mechanical =
disruption with slit injection equipment without nematicide, and Nematicide = slit
injection of 1,3-dichloropropene at a rate of 46.76 liter per hectare at N rates of 0,
36.65, 73.30, and 109.95 kg N/ha/month through out trial 1 (A) and trial 2 (B).













Table C-1. Root lengths of 'Tifdwarf' bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths and diameter ranges 6 weeks after turf and nematodes establishment during trial 1.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3


214e
122 **
131
90
84
70
69
62
499
345 **


1,526
936*
913
697
622
420 ***
544
442*
3,606
2,494 **


1,606
1,018*
967
737
636
438*
569
446*
3,779
2,638 **


901
594*
557
453
368
244 **
327
256*
2,153
1,547**


0.3 to 0.4 0.4 to 0.5 0.5 <


585
413*
356
301
259
157 ***
226
160*
1,426
1,032 **


443
290*
266
233
198
113 **
172
129
1,078
764 ***


1,828
1,229 *
1,145
1,054
875
390 **
621
496
4,470
3,170*


Total

7,104
4,570**
4,335
3,564
3,043
1,832 ***
2,528
1,991 *
17,011
11,957***


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
e Mean values of root length in millimeters for replications.


Smallb

3,347
2,075 *
2,011
1,524
1,342
928 **
1,183
949*
7,883
5,476 **













Table C-2. Root lengths of 'Tifdwarf' bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths and diameter ranges 12 weeks after turf and nematodes establishment during trial 1.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


1c Ud


166e
102*
108
85
88
55*
70
65
432
308*


1,164
732*
841
623
639
360**
583
414 ***
3,227
2,129**


1159
801
797
646
638
356**
614
364 ***
3,208
2,167*


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


491 *
488
357
372
179 **
403
163 ***
1,989
1,190**


500
352
333
246
239
117 **
237
105 ***
1,309
820**


362
279*
302
200*
210
93 **
222
85 **
1,097
658 ***


0.5 <


1,952
1,237**
1,516
751 **
1,008
284***
953
202 ***
5,430
2,474 ***


Total


6,028
3,994 **
4,385
2,908 *
3,195
1,445 ***
3,083
1,399 ***
16,691
9,746 **


*, **,** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
e Mean values of root length in millimeters for replications.


Smallb


2,488
1,635 *
1,746
1,354
1,366
772 ***
1,267
844 ***
6,867
4,604 **













Table C-3. Root lengths of 'Tifdwarf' bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths and diameter ranges 18 weeks after turf and nematodes establishment during trial 1.


Root Diameters (mm)


Depths <0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


1c Ud


183e
87 ***
101
67 ***
71
56 ***
75
53 ***
430
263 ***


1,347
655 ***
652
494 ***
502
416*
520
351 ***
3,022
1,916 ***


1334
613 ***
653
510 ***
487
399 **
495
335 ***
2,969
1,857***


358 ***
371
296 ***
291
224*
284
159 ***
1,729
1,037***


578
254 ***
268
210 ***
209
143 ***
199
104 ***
1,254
711 ***


417
197 ***
197
161 **
160
100 ***
160
73 ***
934
531 ***


0.5 <

2,166
1,007***
1,057
721 ***
847
422 ***
743
220 ***
4,812
2,369***


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
e Mean values of root length in millimeters for replications.


Total

6,809
3,170***
3,299
2,459 ***
2,565
1,759 ***
2,476
1,209***
15,151
8,597***


Smallb

2,864
1,355 ***
1,407
1,071 ***
1,060
871 **
1,090
739 ***
6,420
4,036 ***













Table C-4. Root lengths of 'Tifdwarf' bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths and diameter ranges 6 weeks after turf and nematodes establishment during trial 2.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


1 Ud
1 I
2 U
2 I
3 U
3 I
4 U
4 I
All U
All I


15.14e 122.56


6.65
6.70
4.08
4.06
5.40
3.37
3.56
29.27
19.68


37.76**
51.21
22.50
24.99
27.22
27.94
14.53
226.70
102.00 *


166.71
54.05 **
53.08
21.24
34.20
21.84
24.42
12.60
278.41
109.74 **


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


74.20
41.43
24.10
8.48
11.91
9.29
10.06
8.00
120.27
67.20


42.57
25.42
11.73
4.27
6.19
4.22
6.33
3.81
66.82
37.71


20.97
17.85
6.68
2.34
2.31
2.78
2.32
2.17
32.27
25.14


0.5 <

33.34
33.05
11.02
2.65
5.39
3.94
3.17
3.01
52.92
42.65


Total

475
216 *
165
66
89
75
78
48
807
404*


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
e Mean values of root length in millimeters for replications.


Smallb

304
98 **
111
48
63
55
56
31
534
231 **













Table C-5. Root lengths of 'Tifdwarf' bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths and diameter ranges 12 weeks after turf and nematodes establishment during trial 2.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


14.84e
5.82
9.38
5.79
6.72
6.60
10.79
2.63
41.73
20.84 *


111.36
34.86 *
70.21
44.08
49.74
35.08
91.84
24.19
323.15
138.21 **


139.34
38.17*
79.48
41.91
46.67
31.89
94.57
27.64
360.07
139.61 *


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


78.75
19.47*
27.70
10.48
19.71
6.48
56.07
9.91
182.23
46.51 *


47.13
12.31
14.44
2.42*
8.28
2.31
24.09
2.18
93.93
19.22 *


31.98
8.79
9.38
2.41
5.50
2.16
17.53
2.25
64.39
15.61 *


0.5 <

55.16
18.16
12.17
3.76
7.17
1.64
36.19
2.20*
110.70
25.76 *


Total

479
138 *
223
111
144
86
331
71
1,176
406*


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
e Mean values of root length in millimeters for replications.


Smallb

266
79*
159
92
103
74
197
54
725
299*













Table C-6. Root lengths of 'Tifdwarf' bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths and diameter ranges 18 weeks after turf and nematodes establishment during trial 2.


Root Diameters (mm)

Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb

1c Ud 23.46e 64.65 85.82 99.47 300.15 125.78 1,101.50 1,801 174
1 I 3.94*** 23.81 25.48** 13.25*** 9.56** 5.62** 17.37 ** 99** 53 **
2 U 22.52 60.84 69.27 90.22 274.64 104.38 606.06 1,228 153
2 I 4.19 *** 24.59 19.75 ** 8.51*** 5.88** 4.20** 9.72 ** 77** 48 **
3 U 15.29 47.65 53.39 59.27 140.38 64.70 396.60 777 116
3 I 2.13 *** 14.31 15.19* 6.22*** 2.75 1.71 ** 5.54* 48 ** 32**
4 U 10.45 37.22 39.91 37.26 91.58 38.86 187.57 443 88
4 I 1.15 ** 8.10 7.54* 3.12** 1.69* 1.43** 3.10* 26 ** 17*
All U 71.72 210.35 248.38 286.22 806.75 333.73 2,291.70 4,249 530
All I 11.41*** 70.81* 67.96*** 31.09*** 19.87** 12.96*** 35.73** 250** 150 **
*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
e Mean values of root length in millimeters for replications.













Table C-7. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths 6 weeks after turf and nematodes establishment during trial 1.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


0.92e
0.59
0.52
0.51
0.34
0.22
0.34
0.20*
2.12
1.51 *


60
34 **
31
23
20
14 **
18
15
129
87 **


215
137*
124
96
81
55*
74
56**
495
345 **


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


210
141 *
121
102
81
56*
71
57
483
356 **


186
134*
113
95
82
50 ***
70
52*
450
330 **


175
116*
104
94
80
41 ***
70
48*
428
300 ***


0.5 <


1,505
1,030
976
948
748
308*
474
422
3,703
2,707


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
e Mean values of root surface area in square millimeters for replications.


Total

2,352
1,594 *
1,470
1,359
1,092
523**
777
650
5,691
4,126*


Smallb

276
172 *
156
120
102
69*
92
71 **
626
433 **













Table C-8. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths 12 weeks after turf and nematodes establishment during trial 1.


Root Diameters (mm)

Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb

1c Ud 0.73e 42 152 168 161 146 1,865 2,534 195
1 I 0.51 27 108 117 109 106* 1,165* 1633** 136
2 U 0.36 29 106 123 110 110 1,269 1,747 136
2 I 0.31 21 83 85* 74 75 620** 957** 104
3 U 0.32 20 82 88 74 81 904 1,250 103
3 I 0.20 12* 45 ** 43 ** 35 *** 34 *** 203 *** 372 *** 57 **
4 U 0.33 20 81 110 72 65 841 1,189 101
4 I 0.17 14 ** 44** 38 *** 32** 32 ** 141** 301** 58 ***
All U 1.74 112 420 488 416 402 4,878 6,720 534 oo
All I 1.20 74 281 283 ** 250** 246*** 2,128*** 3,263 *** 356 *
, **, ** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
e Mean values of root surface area in square millimeters for replications.













Table C-9. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths 18 weeks after turf and nematodes establishment during trial 1.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


0.52e
0.31 *
0.26
0.37
0.28
0.25
0.28
0.18*
1.34
1.11 *


47
23 ***
25
17 ***
18
14*
18
12 ***
107
67 ***


169
79 ***
78
65 **
63
50**
62
41 ***
372
235 ***


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


188
85 ***
85
68 ***
67
51 *
65
36 ***
405
239***


165
79 ***
81
67**
67
45 ***
63
30 ***
376
221 ***


168
77 ***
82
64 **
66
40 ***
65
46
380
227 ***


0.5 <

2,039
961***
981
630***
776
340***
672
256**
4,467
2,186 ***


Total

2,788
1,305***
1,333
912 ***
1,056
539 ***
945
433**
6,121
3,188 ***


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 138 B. longicaudatus per lysimeter.
e Mean values of root surface area in square millimeters for replications.


Smallb

215
103 ***
103
83 ***
81
64**
81
54 ***
480
303 ***













Table C-10. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths 6 weeks after turf and nematodes establishment during trial 2.


Root Diameters (mm)

Depths < 0.05 0.05 to 0.1 0.1 to 0.2 0.2 to 0.3 0.3 to 0.4 0.4 to 0.5 0.5 < Totala Smallb

1c Ud 0.06e 5.14 22.73 18.25 13.78 8.93 19.20 88 28
1 I 0.01 1.80** 8.23** 10.64 8.93 8.01 21.89 60 10 **
2 U 0.02 2.04 6.97 5.47 3.43 2.51 7.33 28 9
2 I 0.00 0.94 3.13 1.92 1.56 0.84 1.71 10 4
3 U 0.00 1.11 4.57 2.92 6.06 1.04 3.73 16 6
3 I 0.03 1.30 2.91 1.73 1.44 0.99 2.50 11 4
4 U 0.00 0.93 3.41 2.19 1.99 1.02 1.58 11 4
4 I 0.00 0.57 1.69 2.05 1.34 1.00 1.73 8 2
All U 0.09 9.22 37.68 28.82 25.27 13.50 31.84 143 47
All I 0.04 4.62* 15.95** 16.33 13.28 10.83 27.83 89 21 **
, **, ** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
e Mean values of root surface area in square millimeters for replications.













Table C-11. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths 12 weeks after turf and nematodes establishment during trial 2.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


1c Ud


0.15e
0.02
0.01
0.03
0.00
0.02
0.08
0.00
0.24
0.07


4.54
1.48 *
2.77
1.95
2.04
1.76
3.51
1.41
12.86
6.61 *


20.32
5.08*
10.42
4.87
6.28
3.74
12.56
3.68
49.58
17.37*


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


18.76
5.14
6.38
2.47
4.91
1.39
12.68
2.27
42.73
11.27*


16.39
3.78
4.49
0.87*
2.47
0.84
7.97
0.60
31.31
6.09


14.13
2.84
4.51
0.62
2.66
0.77
7.40
0.03
28.70
5.07


0.5 <

35.89
12.59
6.15
2.49
3.46
0.91
22.95
1.24*
68.44
17.23


Total

110
31
35
13
22
9
67
10
234
64*


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
e Mean values of root surface area in square millimeters for replications.


Smallb

25
7*
13
7
8
6
16
5
63
24*













Table C-12. Root surface areas of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths 18 weeks after turf and nematodes establishment during trial 2.


Root Diameters (mm)


Depths < 0.05 0.05 to 0.1 0.1 to 0.2


0.02e
0.02
0.02
0.01
0.06
0.00*
0.02
0.00
0.12
0.04


2.29
1.03
2.01
0.95
1.75
0.66
1.32
0.37
7.36
3.00


8.03
3.57
7.01
2.51
6.18
2.06
4.33
0.95
25.55
9.09


0.2 to 0.3 0.3 to 0.4 0.4 to 0.5


9.50
3.11
7.14
2.15
7.65
1.45 *
4.90
0.75 *
29.18
7.46*


9.82
3.32
6.65
1.82*
6.85
0.76*
3.69
0.63
27.00
6.53 *


7.88
1.93
6.15
1.92
6.46
0.57*
3.35
0.43
23.84
4.86*


0.5 <

81.44
13.38*
47.86
5.67*
24.30
5.91
16.60
2.23*
170.19
27.18*


Total

229
26
77
15*
53
11
34
5*
393
58*


*, **, *** Inoculated different from uninoculated at a specified depth.
a Total roots consist of cumulative root lengths for all diameters for a specified depth.
b Small roots consist of cumulative root lengths for root diameters of < 0.2 mm for a specified depth.
c Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
d (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
e Mean values of root surface area in square millimeters for replications.


Smallb













Table C-13. Root dry weights of 'Tifdwarf bermudagrass grown in lysimeters that were either uninoculated or inoculated with Belonolaimus
longicaudatus at specified soil depths for both trials.


Root wt (mg)

Trial 1 Trial 2

Depths 6weeka 12 week 18 week 6 week 12 week 18 week

1b Uc 1,214d 1,020 1,063 28 28 67
1 I 896 666 651*** 12* 10 10***
2 U 571 730 555 5 9 35
2 I 477 388** 324*** 3 3* 5***
3 U 355 406 380 3 6 22
3 I 188** 170*** 142*** 1 3 4***
4 U 340 319 305 2 11 13 oo
4 I 194* 136*** 107*** 1 4 2**
All U 2,480 2,476 2,302 38 54 137
All I 1,755 1,359 ** 1,224 *** 17 20 21 ***
, **, ** Inoculated different from uninoculated at a specified depth.
a Weeks after turf and nematode establishment period.
b Soil profile depths: 1 = 0 to 7.62 cm, 2 = 7.62 to 15.24 cm, 3 = 15.24 to 22.86 cm, and 4 = 22.86 to 30.48 cm.
c (U) = uninoculated (I)= inoculated plants received 300 + 40 B. longicaudatus per lysimeter.
d Means values for replications










Table C-14. Regression models of biweekly turf quality in response to N rates of 0, 36.65, 73.30,
and 109.95 kg N/ha/month in untreated control, mechanical, and nematode
management plots throughout trial 2.


Nematode Tactic


4.34 + 0.00068x
4.14 + 0.00546x
4.28 + 0.00580x
4.34 + 0.00239x
4.26 + 0.00375x
4.70 + 0.00887x
5.03 -0.00273x
4.69 + 0.00512x
5.33 + 0.00546x
5.18 -0.00546x
4.85 -0.00409x
5.18 -0.00034x
4.53 + 0.00239x
4.65 + 0.00068x
5.34 + 0.00068x
4.63 + 0.00017x
4.67 0.0005 lx
5.35 + 0.00017x
4.36 + 0.0005 lx
4.36 0.00034x
5.29 0.00239x
4.56 0.00171x
4.16 + 0.00443x
5.03 0.00273x
4.82 + 0.00358x
4.74 + 0.00222x
5.46 0.00017x


0.01
0.33
0.43
0.04
0.17
0.25
0.06
0.17
0.09
0.36
0.16
0.00
0.04
0.01
0.01
0.00
0.00
0.00
0.01
0.01
0.04
0.01
0.07
0.02
0.09
0.12
0.00


0.804
0.020
0.006
0.452
0.113
0.048
0.354
0.117
0.269
0.015
0.120
0.917
0.460
0.810
0.842
0.955
0.841
0.968
0.792
0.880
0.460
0.720
0.324
0.597
0.274
0.193
0.969


Turf quality was rated on a subjective 1 to 9 scale, with 1 as completely dead turf, 9 as
maximum turf quality, and 6.5 as the threshold for acceptability. Fertilization began four weeks
prior to nematicide treatment.
a Weeks after nematicide treatment
b Control = no added soil disturbance or nematicide treatment Mechanical = soil
disruption with slit injection equipment without nematicide treatment. Management = Injection
of 1-3, dichloropropene at a rate of 46.76 liters/ha at 13 to 17 cm of soil depth.


Week


Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide
Control
Mechanical
Nematicide