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Evaluation of Amino Acids against Belonolaimus Longicaudatus

Permanent Link: http://ufdc.ufl.edu/UFE0042619/00001

Material Information

Title: Evaluation of Amino Acids against Belonolaimus Longicaudatus
Physical Description: 1 online resource (65 p.)
Language: english
Creator: Zhang, Yun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 112, 224, 448, amino, analog, belonolaimus, lysine, meloidogyne, methionate, nematicide, post, turf
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS Due to the cancellation of fenamiphos, the most commonly used post-plant nematicide on turf, management of plant-parasitic nematodes has become more difficult. The use of amino acids provides a potential environmentally friendly and effective method of nematode management. Experiments were conducted to determine if DL-methionine, L-threonine, lysine, Na-methionate, K-methionate and methionine hydroxyl analog were effective against Belonolaimus longicaudatus (sting nematode). Meanwhile, petri dish trials evaluated if DL-methionine solution had direct effects on the activity of B. longicaudatus. Bench screen experiments revealed that DL-methionine, Na-methionate, K-methionate and methionine hydroxyl analog at rates of 224 and 448 kg amino acid/ha reduced the number of B. longicaudatus and M. incognita (southern root-knot nematode) J2 recovered, whereas threonine and lysine were not effective in reducing the number of either nematode. Futhermore, greenhouse experiments showed that DL-methionine, Na-methionate, K-methionate and methionine hydroxyl analog were equally effective against B. longicaudatus at the rates of 112, 224 and 448 kg amino acid/ha, and the highest rate (448 kg amino acid/ha) of all amino acids was more effective in reducing the number of B. longicaudatus than the two lower rates. However, phytotoxic response was observed on creeping bentgrass treated with 448 kg amino acid/ha of methionine hydroxyl analog and DL methionine. In addition, bermudagrass field experiments indicated that turf density was increased and the number of B. longicaudatus was significantly reduced by high rates (224 kg amino acid/ha) of DL-methionine with or without a surfactant and K-methionate with or without a surfactant compared to untreated plots at one site, but no treatment differences were observed at another site. A petri dish study showed that DL-methionine solutions have direct effect on the activity of B. longicaudatus at concentrations of greater or equal to 0.01%.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yun Zhang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Crow, William T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042619:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042619/00001

Material Information

Title: Evaluation of Amino Acids against Belonolaimus Longicaudatus
Physical Description: 1 online resource (65 p.)
Language: english
Creator: Zhang, Yun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 112, 224, 448, amino, analog, belonolaimus, lysine, meloidogyne, methionate, nematicide, post, turf
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS Due to the cancellation of fenamiphos, the most commonly used post-plant nematicide on turf, management of plant-parasitic nematodes has become more difficult. The use of amino acids provides a potential environmentally friendly and effective method of nematode management. Experiments were conducted to determine if DL-methionine, L-threonine, lysine, Na-methionate, K-methionate and methionine hydroxyl analog were effective against Belonolaimus longicaudatus (sting nematode). Meanwhile, petri dish trials evaluated if DL-methionine solution had direct effects on the activity of B. longicaudatus. Bench screen experiments revealed that DL-methionine, Na-methionate, K-methionate and methionine hydroxyl analog at rates of 224 and 448 kg amino acid/ha reduced the number of B. longicaudatus and M. incognita (southern root-knot nematode) J2 recovered, whereas threonine and lysine were not effective in reducing the number of either nematode. Futhermore, greenhouse experiments showed that DL-methionine, Na-methionate, K-methionate and methionine hydroxyl analog were equally effective against B. longicaudatus at the rates of 112, 224 and 448 kg amino acid/ha, and the highest rate (448 kg amino acid/ha) of all amino acids was more effective in reducing the number of B. longicaudatus than the two lower rates. However, phytotoxic response was observed on creeping bentgrass treated with 448 kg amino acid/ha of methionine hydroxyl analog and DL methionine. In addition, bermudagrass field experiments indicated that turf density was increased and the number of B. longicaudatus was significantly reduced by high rates (224 kg amino acid/ha) of DL-methionine with or without a surfactant and K-methionate with or without a surfactant compared to untreated plots at one site, but no treatment differences were observed at another site. A petri dish study showed that DL-methionine solutions have direct effect on the activity of B. longicaudatus at concentrations of greater or equal to 0.01%.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yun Zhang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Crow, William T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042619:00001


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1 EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICA UDATUS By Yun Zhang 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 FLORID A 2010

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2 2010 Yun Zhang

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3 T o my beloved mother and father, we transformed a dream into reality

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4 ACKNOWLEDGMENTS I would like to express my heartfelt thanks to my major professor Dr. William T. Crow. Special thanks are extended for his extreme patience with me that helped to cultivate a strong personal interest in nematology. Thank you for enthusiastically believing in me, even after a slow st work ethic and his pursuit of good science, along with his attitude towards life will remain with me for the rest of my life. Thank you Dr. Crow! I would also like to thank my committee members, Drs. Cuda and Giblin Davis. Their guidance of my course work and research, their broad and profound knowledge, and their patience with me in the past two and half years has helped to bring me to where I am today. Thanks to them for motivating me to pursue knowledge and an academic stance towards science. Thank you! Thanks also to my parents, for giving me a foundation of love and support on which to begin to build my dreams. dedication, hard work and friendship. I have learned so much from you, best of luck to both of you. Last but not least, I give my sincere thanks to Nick, Jaycee and Wenjing. Thanks for your selflessness and warmth, and help with my projects. I wish you all the best that life has to offer. May our true friendship last forever.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .............. 4 LIST OF TABLES ................................ ................................ ................................ .......................... 7 LIST OF FIGURES ................................ ................................ ................................ ........................ 9 ABSTRACT ................................ ................................ ................................ ................................ .. 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ................. 12 Belonolaimus longicaudatus ................................ ................................ ................................ .. 13 Taxonomy ................................ ................................ ................................ ....................... 13 Systematics and Phylogeny ................................ ................................ ............................ 14 Morphology and Anatomy ................................ ................................ .............................. 15 Life Cycle and Distribution ................................ ................................ ............................ 16 Nematicides for Managing Nematode Problems ................................ ................................ ... 19 Amino Acid ................................ ................................ ................................ .................... 19 Methionine ................................ ................................ ................................ ...................... 20 2 EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS IN A BENCH SCREEN STUDY ................................ ....................... 23 Introduction ................................ ................................ ................................ ............................ 23 Materials and Methods ................................ ................................ ................................ .......... 24 Results ................................ ................................ ................................ ................................ .... 26 Discussion ................................ ................................ ................................ .............................. 27 3 GREENHOUSE EVALUATION OF AMINO ACIDS AGAINS T BELONOLAIMUS LONGICAUDATUS ................................ ................................ ................................ ............... 31 Introduction ................................ ................................ ................................ ............................ 31 Materials and Methods ................................ ................................ ................................ .......... 32 Results ................................ ................................ ................................ ................................ .... 34 Discussion ................................ ................................ ................................ .............................. 34 4 EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS IN FIELD TESTS ................................ ................................ ................................ .................. 39 Introduction ................................ ................................ ................................ ............................ 39 Materials and Methods ................................ ................................ ................................ .......... 41 Results ................................ ................................ ................................ ................................ .... 43 Discussion ................................ ................................ ................................ .............................. 44

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6 5 EVALUATION OF DL METHIONINE SOLUTIONS AGAINST B. LONGICAUDATUS IN PETRI DISH STUDY ................................ ................................ ..... 51 Introduction ................................ ................................ ................................ ............................ 51 Materials and Methods ................................ ................................ ................................ .......... 52 Resul ts ................................ ................................ ................................ ................................ .... 53 Discussion ................................ ................................ ................................ .............................. 53 LIST OF REFERENCES ................................ ................................ ................................ .............. 59 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ........ 65

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7 LIST OF TABLES Table page 2 1 Effects of DL methionine (DL), L threonine (L t), lysine, Na methionate (Na), K methionate (K), and methionine hydroxy analog (MHA), applied at 224 or 448 kg amino a cid/ha on M. incognita after three days of expo sure during trial 1 and trial 2 ...... 30 2 2 Effects of DL methionine, L threonine, lysin e, Na methionate, K methionate, and methionine hydroxy analog, applied at 224 or 448 kg amino acid/ha on B. longicaudatus after three days of exposure during trial 1 and trial 2. ............................... 30 3 1 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), across rates on B. longicaudatus two weeks after application ................................ ................................ ................................ .......................... 38 3 2 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (N a), applied at 112, 224, and 448 kg amino acid/ha to bentgrass ................................ ................................ ................................ ............................ 38 4 1 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on Belonolaimus longicaudatus ................................ ................................ .............................. 47 4 2 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on turf density of Tifdwarf bermudagrass. ................................ ................................ .................... 47 4 3 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on turf dens ity of Celebration bermudagrass ................................ ................................ ................. 48 4 4 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 1 1.2 kg a.i./ha on the root lengths and number of root tips from two 58 cm 3 cores per plot from the Tifdwarf and Celebration bermudagrass trials ................................ ................................ .................. 48 4 5 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 222 factorial experiment on Belonolaimus longicaudatus for both trials ................................ 49 4 6 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were anal yzed as a 222 factorial experiment on root lengt h and root tips for both trials ................................ ........ 49

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8 4 7 Effects of amino acid formulation, amino a cid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 222 factorial experiment on turf density for the Tifdwarf trial ................................ ................. 50 4 8 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 222 factorial experiment on turf density for the Celebration trial. ................................ ........... 50

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9 LIST OF FIGURES Figure page 2 1 Effects of DL methionine (DL), L threonine (L t), lysine, Na methionate (Na), K methionate (K), and methionine hydroxy analog (MHA), applied at 224 or 448 kg amino acid/ha on B. longicaudatus after three days of exposure during trial 1 and trial 2 ................................ ................................ ................................ ................................ .. 28 2 2 Effects of DL methionine (DL), L threonine (L t), lysine, Na methionate (Na), K methionate (K), and methionine hydroxy analog (MHA), applied at 224 or 448 kg amino acid/ha on M. incognita after three days of exposure during trial 1 and trial 2 ...... 29 3 1 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), applied at112, 224 and 448 kg amino acid/ha on B. longicaudatus two weeks after application. Each bar is the m ean of ten replications ....... 36 3 2 Ef fects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), applied at112, 224 and 448 kg amino acid/ha on so il pH two weeks after treatment ................................ ................................ ........................... 36 3 3 Effects of methionine hydroxy analog (MHA), DL methion ine (DL), K methionate (K), and Na methionate (Na), applied at 112, 224, and 448 kg amino acid/ha to bentgrass ................................ ................................ ................................ ............................ 37 5 1 Percent mortality of Belonolaimus longicaudatus exposed to increasing % DL methionine solutions (DL) over time during trial 1 and trial 2 ................................ .......... 55 5 2 Effects of increasing concentrations of DL methionine solutions (0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%) on Belonolaimus longicaudatus in petri dishes, A) 3, B) 4, and C) 6 days after treatments du ring trial 1 ................................ ............................. 56 5 3 Effects of increasing concentrations of DL methionine solutions (0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%) on Belonolaimus longicaudatus in petri dishes, A) 3, B) 4, and C) 6 days after treatments during trial 2 ................................ ............................. 57

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10 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 EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICA UDATUS By Yun Zhang December 2010 Chair: William T. Crow Major: Entomology and Nematology Due to the cancellation of fenamiphos the most common ly used post plant nematicide on turf, management of plant parasitic nematodes has become more difficult. The use of amino acids provides a potential environmental ly friendly and effective method of nematode manageme n t Experiments were conducted to determ ine if DL methionine, L threonine, l ysine, Na methionate, K methionate and methionine hydroxyl analog were effective against Belonolaimus longicaudatus (sting nematode) Meanwhile, petri dish trials evaluated if DL methionine solution had direct effect s on the activity of B. longicaudatus Bench screen experiments revealed that DL methionine, Na methionate, K methionate and methionine hydroxyl analog at rates of 224 and 448 kg amino acid/ha reduced the number of B. longicaudatus and M. incognita (southern root knot nematode) J2 recovered, whereas threonine and lysine were not effective in reducing the num ber of either nematode. Futhermore, green house experiments showed that DL methionine, Na methionate, K methionate and methionine hydroxyl analo g were equally effective against B. longicaudatus at the rates of 112, 224 and 448 kg amino acid/ha, and the highest rate (448 kg amino acid/ha) of all amino acids was more effective in reducing the number of B. longicaudatus than the two low er rates. Howe ver, phytotoxic response was observed on creeping bentgrass treated with 448 kg amino

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11 acid/ha of methionine hydroxyl analog and DL methionine. In addition, bermudagrass field experiments indicated that turf density was increased and the number of B. longic audatus was significantly reduced by high rates (224 kg amino acid /ha) of DL methionine with or without a surfactant and K methionate with or without a surfactant compared to untreated plots at one site, but no treatment differences were observed at anothe r site. A p etri dish study showed that DL methionine solutions have direct effect on the activity of B. longicaudatus at concentration s o 0 .01%.

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12 CHAPTER 1 INTRODUCTION Plant parasitic nematodes are viewed as important pathogens, causing significant damage on many plants. Among these nematodes, Belonolaimus longicaudatus Rau (sting nematode) is commonly found causing plant damage in the southeastern United States (Perry a nd Rhoades 1982; Smart and Nguyen 1991). An ectoparasitic nematode, B. longicaudatus has an extensive host range, which includes many vegetable, agronomic, horticultural, and ornamental crops. Important hosts include high bush blueberry ( Vaccinium corym bosum ), Chinese elm ( Ulmus parvifolia ), muscadine grape ( Vitia rotundifolia ), pecan ( Carya illinoensis ), potato ( Solanum tuberosum ), soybean ( Glycine max ), wheat ( Triticum aestivum ), pearl millet ( Pennisetum glaucum ), barley ( Hordeum vulgare ), corn ( Zea mays ), turfgrasses, cotton ( Gossypium hirsutum ), peanut ( Arachis hypogaea ), citrus, strawberry ( Fragaria virginiana ) celery ( Apium graveolens ), sweet corn ( Zea mays ) and most vegetables (Christie et al., 195 2; Abu Gharbieh and Perry, 1970; Robbins and Bar ker, 1973). In Florida, B. longicaudatus is viewed as the most destructive plant parasitic nematode to turf (Crow, 2005 a ). million hectares of turfgrasses were gr own in 1991 1992 in Florida, and this acreage is still increasing annually (Hodges et al., 1994) Between 1991 and 1992, the turf industry contributed $4.44 b illion in Florida (Haydu and Hodges, 2002). The turf industry continues to be an Sting nematode is an important economic pathogen on turfgrasses in the southeastern United States. Due to its rela tively large body (2 3 mm length), it is confined to soils with > 80% sand content and <10% clay with minimal organic matter (Robbins and Barker, 1974; Rhoades, 1980).

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13 The sandy soil texture in the southeastern coastal plains provides an optimal environmen t for sting nematode, which has a very extensive host range, including most of the grasses, such as perennial ryegrass ( Lolium perenne ) bahiagrass ( Paspalum notatum ), seashore paspalum ( Paspalum vaginatum ), creeping bentgrass ( Agrostis palustris ), bermuda grass ( Cynodon dactylon ), centipedegrass ( Eremochloa ophiuroides ), St. Augustinegrass ( Stenotaphrum secundatum ), and zoysia ( Zoysia japonica ) (Crow, 2007). It was reported that sting nematode was found at damaging levels on 60% of golf courses surveyed in Florida (Crow, 2005 b ). Turf parasitized by sting nematode often shows severe decline and even death (Crow, 2007). Luc et al. (2007) also found B. longicaudatus can cause severe root reduction, which reduces water and nutrient uptake from soil and increase s nitrate leaching in bermudagrass. McGroary et al., (2009) concluded that B. longicaudatus can decrease evapotranspiration rates resulting in reduced root length, turf quality, color, and density on bermudagrass. Plants damaged by sting nematode are more susceptible to drought stress, heat stress, and malnutrition (Lucas, 1982; Trenholm et al., 2005). While sting nematode can cause considerable damage to turf, it has been shown to be one of the most responsive nematodes to nematicide treatments and can be effectively managed with either contact or systemic nematicides. Belonolaimus longicaudatus Taxonomy The genus Belonolaimus was first established by Steiner (1949), who found and descri b ed Belonolaimus gracilis from the rhizosphere of slash pine ( Pinus elliotii ) and longleaf pine ( Pinus palustris ) in the Ocala National Forest near Ocala, FL. Subsequently, several nematologists reported B. gracilis damaging crops such as celery ( Apium graveolens ), strawberry and corn in Florida sorghum ( Sorghum bicolor ) millet ( Sorghum halepense ), peanut in Virginia and cotton, soybean and cowpea ( Vigna unguiculata ) in South Carolina. (Owens,

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14 1951; Christie et al. 1952; Graham and Holdeman, 1953 ; Christie, 1953). However, most of these early crop damage reports were later attributed to B. longicaudatus and not B. gracilus Ten years later, Belonolaimus longicaudatus Rau a second species of the genus Belonolaimus, was observed and described by Rau (1958) Compared to B. gracilis, B. longicaudatus has a longer stylet a nd shorter tail. Subsequently, Rau (1963) added three additional species of Belonolaimus : B. euthychilus B. maritimus, and B. nortoni Perry and Rhoades (1982) concluded that B. longicaudatus was the more commonly found destructive species to agricultural crops and turfgrasses in the southeastern United States. Systematics and Phylogeny Siddiqi ( 2000 ) established taxonomic placement of B. longicaudatus within the domain Eukaryota which encomp asses the kingdom Animalia, subkingdom Metazoa, branch Eumetazoa, division Bilateralia, subdivision Protostomia, section Pseudocoelomata, superphylum Aschelminthes, phylum Nematoda, class Secernentea, subclass Tylenchia, order Tylenchida, suborder Tylenchi na, superfamily Dolichodoroidea, family Belonolaimidae, subfamily Belonolaiminae, genus Belonolaimus species Belonolaimus longicaudatus From the 1950s, researchers began to report morphological differences among B. longicaudatus populations. Several pop ulation differences have been confirmed, involving the following charact ers: average stylet length, gubernaculum length, tail, head shape, number of annules in the lip region, lip constriction degree, presence or absence of sclerotized pieces in the vagina and number of tail annules; as well as a, b, and c ratios (a = ratio of total length to maximum width; b = ratio of total length to esophagus length; c = ratio of total length to length of tail)(Owens, 1951; Rau, 1958; 1961; 1963; Rau and Fassuliotis, 19 70; Gray and Miller, 196 2; Abu Gharbieh and Perry, 1970; Robbins and Hirschmann, 1974). Recently, Han et al. (2006) collected isolates of B. longicaudatus from different hosts and regions of the southern United

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15 States, including Gainesville FL; Hastings, FL; Lake Alfred, FL; Tifton, GA; and Scotland County, NC. They found several morphological differences between B. longicaudatus populations, including body length and width; stylet cone and shaft lengths; knob shape and length; distance of excretory pore to anterior end; head, esophagus, and tail lengths However, these morphological differences were not reflected by phylogenetic analysis of the ITS1 region of rDNA. In the same year, Gozel et al. (2006) observed similar morphological differences from 33 B. longicaudatus populations collected throughout Florida. I n this study however, three clades of B. longicaudatus were observed by phylogenetic analysis with overlapping geographical regions moving east to west across the peninsula of Florida using combined molecular sequences of the D2 D3 expansion segments of LSU (large ribosomal subunit) and the ITS regions of SSU (small ribosomal subunit). Morphology and A natomy The body length and width of B. longicaudatus adults range from 2000 to 3000 m long and 29 to 34 m wide Stylet length ranges from 107 to 115 m long with well developed, round knobs (Rau, 1958). However, different isol ates of B. longicaudatus exhibit morphological variation. It was reported that the stylet length of California and central Florida isolates was longer than other Florida isolates, while the body and stylet length of the North Carolina and Georgia isolates were totally different (Robbins and Hirshmann, 1974; Duncan et al., 1996; Han et al., 2006). Duncan et al (1998) concluded that environmental conditions, including nutrient availability and population age structure, could result in these morphological va riations. The general description of B. longicaudatus is as follows: a single deep incisure marks the lateral field, which extends from the base of the lip region to near terminus. Lip region composed of four rounded sub lateral lips, with a single conspic uous papilla on each of them. Lip region is set off by a conspicuous constriction. The lips have deep lateral grooves that align with those of

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16 the lateral field. The amphid apertures are located near the margin of the head in the grooves. Lateral indentati ons are more conspicuous than dorsal and ventral. The lip region appears quadrangular in face view. The median esophageal bulb is more than half the width of the neck, while the isthmus is shorter than the neck width. The nerve ring is located next to the median bulb. Esophageal glands are lobe like and extend over anterior end of the intestine. The excretory pore is located behind the median bulb. The hemizonid is slightly anterior to the excretory pore. The vulva is a transverse slit. Ovaries are outstret ched with each uterus having a spermatheca. The intestine extends to the tail, with the rectum located ventrally. The tail is about four to five times the anal body diameter width. The male has a caudal bursa, which extends from slightly anterior of the sp icula to the tail. Phasmids lie in the base of the busa (Rau, 1958) Life C ycle and Distribution Belonolaimus longicaudatus exhibits a high damage potential at relatively low population densities. It feeds predominantly on the root tips of young plants (P erry and Rhoades, 1982). Upon locating the roots of a host plant, B. longicaudatus will insert its long stylet into the root tip and subsequently injects digestive enzymes into the roots. Feeding by B. longicaudatus can cause root tips to stop growing, and distress to young plants with a developing root system (Crow et al., 1997; Huang and Becker, 1997; Crow et al., 2003). Belonolaimus longicaudatus is a bisexual species which reproduces sexually, adult populations are generally composed of approximately 4 0 percent males (Perry and Rhoades, 1982). Robbins and Barker (1973) suggested that B. longicaudatus can complete its life cycle in 28 days under suitable conditions. Huang and Becker (1999) conducted experiments with B. longicaudatus on axenic corn root c ultures at 28 C and provided a detailed description of the life cycle. Males and females of B. longicaudatus mate soon after the females complete their last molt. After mating, 124 16 eggs were laid, as long as adequate food source was available, for

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17 ea ch female. Following egg deposition, the first stage juvenile was visible in the egg after 3 to 4 days and the second stage juvenile (J2) hatched on the fifth day. J2 approached root tips and began feeding for 12 to 24 hours, while keeping the body shape l second molt started on day 7 and ended on day 9. The third stage juvenile (J3) began feeding again after the molt and after 2 days molted into the fourth stage juvenile (J4). After a fourth molt into adult nematodes, the s exes can be distinguished. Males molted from J4 from day 18 to 20. Female nematodes molted from J4 from day 19 to 22. The whole life cycle of B. longicaudatus from J2 to J4 was finished in 24 days. However, the optimum reproduction temperature for differe nt populations of B. longicaudatus is different. Florida populations of B. longicaudatus reproduced best at 29.4 C, whereas Georgia populations reproduced well at 30 C and North Carolina populations began to decline at 30 C (Boyd and Perry, 1971; Perry, 1964; Robbins and Barker, 1974). Robbins and Barker (1974) also reported that B. longicaudatus reproduced better at 7% moisture level than at 2% and 30%. Belonolaimus longicaudatus is native to the southeastern United States where it is found in soils with > 80% sand content and <10% clay with minimal organic matter (Robbins and Barker, 1974; Rhoades, 1980). The sandy coastal plains of the southeastern United States provide an optimum soil texture for B. longicaudatus However, B. longicaudatus has been spread widely in contaminated planting material. Belonolainus longicaudatus has been found in Arkansas, California, Connecticut, Kansas, Oklahoma, Nebraska, New Jersey, Missouri and Texas (Robbins and Barker, 1974; Rhoades, 1980; Perry and Rhoades, 1982; Huang and Becker, 1999). Perry and Rhoades (1982) also found that B. longicaudatus has been reported from Costa Rica, Bermuda, and several other island nations in the Caribbean. The vertical distribution patterns of nematodes in soil are greatly influence d by root

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18 distribution, soil texture, temperature, rainfall, and subsoil depth (Ferris and Bernard, 1971; soil particle size acts as a decisive factor that influen ces the movement of nematodes. The relatively large body length of B. longicaudatus (2 3 mm) influences its optimum living environment. As the diameter and length of a nematode increase, the optimum pore and particle size of soil for the maximum movement o f the nematode also increases. However, when soil pore diameter is too large, the lateral movement will be restricted (Brodie, 1976). Soil moisture also affects nematode population density. For B. longicaudatus the optimum soil moisture is about 7%, where as 2 to 30% soil moisture is sufficient for some reproduction (Robbins and Barker, 1974). According to Wallace (1963; 1971), the capacity to replenish oxygen in saturated soils is much slower than in well drained soil. As Van Gundy et al. (1962) concluded, the activity of ectoparasitic nematodes can be reduced in low oxygen environments. Soil type, moisture, and aeration are interrelated in determining the movement of nematodes. Movement through soil directly influences the reproduction of amphimictic nem atodes like B. longicaudatus because movement is essential for locating food and finding mates (Robbins and Barker, 1974). Root depth of host plants is another factor which can influence the distribution of nematodes. The greatest population densities of B. longicaudatus are found in the top 30 cm of soil profile, whereas some individuals are found as deep as 75 cm (Brodie, 1976; Todd, 1989; McSorley and Dickson, 1990). The vertical distribution of B. longicaudatus is greatly affected by the host plant. Th e greatest population density was found in the top few cm of soil on turfgrass (Giblin Davis et al., 1991), whereas high populations can be found below 30 cm soil depth in corn and citrus (Brodie, 1976; Todd, 1989; Noling, 1993). However, the distribution of nematodes is always erratic even when sampling within the same field site (Todd, 1989).

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19 Nematicides for Managing Nematode P roblems Even though plant parasitic nematodes can cause economic damage to turf, managing these pathogens is difficult due to the lack of tolerant turfgrass species, cultivars and effective treatments (Johnson, 1970; Busey et al., 1991; Crow et al, 2009). Until recently, the post plant organophosphate nematicide fenamiphos (Nemacur, Bayer CropScience, Research Triangle Park, NC) was the most commonly used nematicide on turf, but it is no longer manufactured or sold in United States (Anonymous, 2002). Currently, 1,3 dichloropropene (1, 3 D; Curfew Soil Fumigant Dow AgroSciences, Indianapolis, IN) is an effective product against B. longicaudatus and certain other plant parasitic nematodes on turf in some states (Crow et al., 2003; 2005). However, the shortcoming s of this product include high application costs, invasive injection methods, lengthy re entry interval, and geological restrictions, all of which limit the application of 1, 3 D. Although there are many other nematode management products available in the turf market, most of them lack established efficacy (Crow et al. 2005). With a lack of effective treatmen ts, there is a vital need for effective, safe, environmentally friendly, and economical product s for nematode management on turfgrass. Amino A cid In 1 960, Owens and Novotny reported that when plants were infested by endoparasitic nematodes, amino acids accumulated in the giant cells of diseased plants. Overman and Woltz (1962) found that reproduction of Tricho dorus christiei (now Paratrichodorus minor ) and Helicotylenchus nannus and galling of tomato roots by M eloi dogyne incognita were inhibited by certain amino acids. Peacock (1966) and Andel (1966) suggested that amino acids could be used as nematicides because of their chemotherapeutic effects on some diseased plants. Prasad and Webster (1967) began to test the effectiveness of DL methionine, DL alanine, DL ethionine and DL amino butyric acid against three nematode species, Heterodera avenae, Ditylenchus dipsaci,

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20 and Aphlenchoides ritzemabosi They found that DL methionine and DL alanine reduced the numbers of H. avenae DL alani ne, and DL ethionine decreased the numbers of D. dipsaci and DL amino butyric acid reduced the numbers of A. ritzemabosi Evans and Trudgill (1971) tested the effectiveness of DL amino n butyric acid, DL ethionine, DL methionine, and hydroxy L proline a gainst the potato cyst nematode ( Globodera rostochiensis ). Among these amino acids, DL methionine was found to be the most effective compound either in pots or in field tests. Similarly, Epstein (1973) reported that DL methionine increased the top weight o f Bur marigold ( Bidens cernua ) plants infected by Longidorus africanus In plants, amino acids are always in the form of L isomers, while D isomers sometimes serve as antimetabolites. The suggested mechanism of amino acids against nematodes was that they a re similar to essential compounds in structure, and compete with these essential compounds on enzymes (Evans and Trudgill, 1971). Methionine Methionine, as an essential amino acid, is produced in plants solely in the form of L methionine and is widely pre sent in organisms as a protein component or as substrate for methyl transaction reactions (Crow et al., 2009). When produced artificially, equal parts of the L and D chiral forms of methionine occur, which have no known harmful effects on humans or the env ironment. Currently, methionine has been classified by the United States Environmental has sufficient information to reasonably conclude that the current use patte rn in pesticide methionine, such as D methionine or DL re with essential Overman and Woltz hypothesized that nematodes are incapable of feeding when exposed to

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21 antimetabolities such as methionine. Later, experiments were c onducted to determine the effectiveness of DL methionine on nematodes. Davies (1966) noted that turnip storage tissue accumulates S adenosylmethionine (metabolically inactive and accumulating in the cell vacuoles) after incubating with methionine. The oxid ative phosphorylation and respiration of the root cells become slow after removing adenosine, and thus influence the nematodes. In 1967, Prasad and Webster found that DL methionine reduced the number of H avenae females in wheat. Similarly, Evans and Trud gill (1971) showed that DL methionine could reduce numbers of G. rostochiensis on potato and speculated that amino acids might accumulate in the giant cells of plants infected by nematodes and that nematodes that feed on this extra methionine might have re duced oxygen metabolism. Hasegawa et al. (2003) noted that D methionine and L methionine resulted in increasing root hair numbers in Brassica rapa In 2005, DL methionine was found to inhibit hatching of eggs and mobility of M. incognita in laboratory expe riments (Talavera and Mizukubo, 2005). More recently, Crow et al. (2009) conducted field effectiveness trials of DL methionine against B. longicaudatus and Mesocriconema ornatum on bermudagrass and zoysiagrass. Parameters evaluated were nematode counts, tu rf density, and root lengths after DL for development as a turfgrass nematicide, but further research is needed to determine how it can Due to the hi gh cost of amino acids, the use of methionine as a nematicide was not considered practical in years past (Talavera and Mizukubo, 2005). However, large amounts of DL methionine are currently being produced and used in animal feed as amino acid supplements. Because of increased production, the retail price of DL methionine has been greatly reduced. At present, methionine prices are < US $4/kg (K. Riley, personal communication) which, depending

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22 on rate, may be economically feasible for high value crops like tu rfgrass. Concerns have arisen that the limited solubility of DL methionine might reduce its movement in to soil, thereby reducing efficacy and increasing potential for phytotoxicity (W. T. Crow, personal communication). Th e objectives of this research we re to: 1. Conduct a preliminary evaluation of two alternative amino acids (lysine and threonine) and several liquid methionine analogues for efficacy against the nematode B. longicaudatus on turf. 2. Evaluate if methionine and liquid methionine analogues change soil pH significantly in a greenhouse study. 3. Test the influence of methionine and liquid methionine analogues on nematode population densities and health of turf infested with B. longicaudatus in the field.

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23 CHAPTER 2 EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS IN A BENCH SCREEN STUDY Introduction Plant parasitic nematodes have been viewed as one of the most important pathogen groups, causing significant damage on many types of crops, including turf. Sting nematode ( Belonolaimus longica udatus ), and southern root knot nematode ( Meloidogyne incognita ) are considered the most damaging nematodes on many crops in Florida, USA (Crow, 2005 a ; Handoo et al., 2004). Until now, the main methods for managing nematodes are still agricultural chemical s, such as fumigants and organophosphate nematicides (Talavera and Mizukubo, 2005 ). The negative effects of these chemical nematicides on the environment and humans are of increasing concern (Talavera and Mizukubo, 2005). Recently, the organophosphate nema ticide fenamiph os, which was the most commonly used nematicide on turfgrasses, is no longer manufactured or sold in United States (Anonymous, 2002). Therefore, alternative methods for managing nematode problems are urgently needed. Previous studies have re ported that amino acids can act as nematicides, due to their chemotherapeutic effects on some diseased plants (Overman and Woltz, 1962; Peacock, 1966; and Andel, 1966; Evans and Trudgill, 1971). DL methionine has been shown to reduce the number of Heterode ra avenae females on wheat and Globodera rostochiensis on potato in lab tests (Prasad and Webster, 1967; Evans and Trudgill, 1971). As an essential amino acid, methionine is produced in plants solely in the form of L methionine and is widely present in or ganisms as a protein component or as a substrate for methyl transaction reactions (Crow et al., 2009). No known harmful effects on human beings or the environment have been reported. Currently, methionine has been classified by the United States Environmen

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24 ingredients for which EPA has sufficient information to reasonably conclude that the current use pattern in pesticide products will not adversely affect public health or the environment (Anonymous, 2004). Based upon economic and environmental considerations, DL methionine has the potential to be used as a nematicide, at least on high value crops like turfgrasses. Crow et al. (2009) found that DL methionine could be effectively used for managing B. longicaudatus in the field. However, phytotoxicity was observed in one of the two trials and the rates used (224 and 1140 kg/ha) were high. Both of these problems may hav e been the result of the low solubility of DL methionine (30g/liter at 20 C). Because the methionine must be transported into the ground via irrigation water in order to contact the nematodes, low solubility could cause reduced infiltration thereby increas ing the use rate. This also would lead to accumulation of methionine at the surface thereby causing phytotoxicity. Several liquid methionine analogues are available that could potentially move into soil better than DL methionine lowering rates with less r isk to plants. These include Na methionate, K methionate, and methionine hydroxyl analog. In addition to methionine, several other amino acids are being produced commercially on a large scale as animal feed supplements, including threonine and lysine. If e ffective, these amino acids also could be economically viable nematode management treatments for high value crops like turf. The objective of this study was to compare the effectiveness against B. longicaudatus and M. incognita of threonine and lysine, and several liquid methioni ne analogues, to DL methionine These experiments were conducted using bench screens with nematodes in soil. M aterials and Methods In December 2009, bench trials were conducted using a RCB design with 8 replications in the Entomolog y and Nematology Department at the University of Florida, Gainesville, FL. In these experiments the following materials were evaluated: DL methionine (Evonik Degussa,

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25 Theodore, AL ), L threonine (Evonik Degussa, Theodore, AL ), Lysine (Biolys ; Evonik Deguss a, Theodore, AL ), Na methionate (Na Liquimeth ; Evonik Degussa, Theodore, AL ), K methionate (K Liquimeth ; Evonik Degussa, Theodore, AL ), and methionine hydroxyl analog (Alimet ; Novus International, St. Charles, MO). Among these amino acids, DL methionine, L threonine, and Biolys are solid materials, whereas Na Liquimeth K Liquimeth and Alimet are liquid materials. The percentage amino acid content (w/w) of each treatment was: DL methionine (99%), L threonine (98.5%), Biolys (50.7%), Na Liquimeth (46%), K Liquimeth (35%), and Alimet (88%). Application rates of each treatment were based on the weight of amino acid and not the formulation. All of the amino acids were evaluated at two rates (224 and 448 kg amino acid /ha), for their ability to affect B. longicaudatus (juveniles and adults) or M. incognita second stage juveniles (J2) in soil. The soil infested with B. longicaudatus was collected from greenhouse pot cultures or an infested field site, for trials 1 and 2, respectively. The green house soil was United States Golf Association (USGA) putting green specification sand (Anonymus, 1993) that had been inoculated with a B. longicaudatus isolate originating s ( Stenotaphrum secundatum ). The B. longicaudatus field soil was collected from an infested Cynodon dactylon ). Both M. incognita trials used infested field soil from an alyce clover ( Alysicarpus ovalifolius ) field in Sumter County, FL. One hundred cubic centimeters of dry soil was used for analyzing soil texture by hydrometer (Bouyoucos, 1936). Another t wo gram s of dry soil was heated at 500 C for three hours for ignition of organic matter and then weighed again to calculate the percentage of organic matter. After soil analysis, all the soil samp les from the field were found to

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26 have >90% sand content. However, soil from the field in Spring Hill contain ed 5% organic matter, whereas so il from Sumter County had <2% organic matter. The soil was hand mixed to make sure the nematodes were evenly distributed in the soil. Next, 200 cm 3 aliquots were placed into small plastic pots. Solid treatments were applied topically followed with 50 ml of water to carry the materials into the soil. Liquid materials were mixed into water and then 50 ml of solution was poured onto the soil. Untreated controls had 50 ml of water poured onto the soil. Th ree days after treatments were applied, the soil was removed from the pots and placed onto modified B aermann funnels (McSorley and Frederick, 1991) Nematodes were counted using an inverted light micro scope at 40 magnification. Data were subjected to anal range test ( P 0.05) for each nematode using SAS software (SAS Institute, Cary, NC). Additionally, the methionine and methionine analog dat a were further evaluated as a 4 2 factorial wit h 4 amino acid treatments and 2 rate treatments. Results Both rates of DL methionine and liquid methionine analogues reduced M. incognita recovered compared with the water control in Trial 1 ( P 1). However, only the liquid formulations r educed the number of B. longicaudatus in Trial 2 Both levels of methionine treatments and methionine analogues were effective ( P 0.05) in reducing the number of M. incognita recovered (Fig. 2 2). However, Alimet wa s not as effective as the other methionine products against M. incognita in both trail s (Figure 2 2, Table 2 1). T hreonine and lysine were not effective in reducing the number of B. longicaudatus or M. incognita J2 recovered in any trial ( P 0.05) (Figure 2 1, Figure 2 2, Table 2 1 and Table 2 2)

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27 Discussion Amino acids have been studied for several years for their negative effects on nematodes. In these experiments, the number of B. longicaudatus (juveniles and adults) and M. incognita J2 recovered were reduced by methionine and all m ethionine analog ues but not by threonine or lysine Two rates (224 and 448 kg amino acid /ha) of DL methio nine Na Liquimeth, K Liquimeth were equally effective, showing no differences against M. incognita in both trials of this study. Even though Alimet r educed the number of M. incognita J2 recovered compared with the water control, it w as not as effective as DL methionine and the other methionine analogues. These results indicate that methionine is much more promising as a nematicide than either threonin e or lysine. In trial two of the B. longicaudatus experiment DL methionine showed poor performance against B. longicaudatus There are several possible reasons for this finding First, USGA specification sand might allow for greater infiltration and therefore increased movement of DL methionine than does the field soil Also, the field soil had more organic matter (5%) than the USGA specification sand, which might inhibit DL methionine m oving into the soil. Talavera and Mizukubo (2005) reported that M. incognita can be directly affected by methionine. In our experiments, no plants were used, only nematodes in soil. Therefore, chemotherapeutic effects can be ruled out, corroborating the di rect effects observed by Talavera and Mizukubo (2005) In conclusion, liquid methionine analogues had effects equal to or better than DL methionine on B. longicaudatus K Methionate and Na methionate also were equal to DL methionine against M. i ncognita J2 but methionine hydroxy analog was not Threonine and lysine did not reduce the number of either nematode recovered from soil at the rates evaluated. Therefore, only DL methionine and its liquid analogues will be evaluated further in greenhouse and field trials.

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28 Figure 2 1 Effects of DL methionine (DL), L threonine (L t), lysine, Na methionate (Na), K methionate (K), and methionine hydroxy analog (MHA), applied at 224 or 448 kg amino acid/ha on B. longicaudatus after three days of exposure during trial 1 and trial 2 Each bar is the mean of eight replications. Means with common letters are not different ( P range test. 0 1 2 3 4 5 6 7 8 9 10 B. longicaudatus / 100 cm 3 of soil 0 5 10 15 20 25 30 35 40 45 B. longicaudatus / 100 cm 3 of soil Trial 1 Trial 2

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29 Figure 2 2 Effects of DL methionine (DL), L threonine (L t) lysine, Na methionate (Na), K methionate (K), and methionine hydroxy analog (MHA), applied at 224 or 448 kg amino acid/ha on M. incognita after three days of exposure during trial 1 and trial 2. Each bar is the mean of eight replications. Means with common letters are not different ( P range test. 0 50 100 150 200 250 300 350 M. incognita / 100 cm 3 of soil 0 50 100 150 200 250 300 350 M. incognita / 100 cm 3 of soil Trial 1 Trial 2

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30 Table 2 1 Effects of DL methionine (DL), L threonine (L t), lysine, Na methionate (Na), K methionate (K), and methionine hydroxy analog (MHA), applied at 224 or 448 kg amino acid/ha on M. incognita after three days of exposure during trial 1 and trial 2. M. incog nita / 100 cm 3 of soil Variable Trial 1 Trial 2 Formulation L t 235 a 123 a lysine 186 b 112 a MHA 83 c 45 b DL 26 d 20 c K 21 d 18 c Na 13 d 17 c Rate 224 kg amino acid /ha 99 54 448 kg amino acid /ha 89 58 Means with common letters are not different ( P range test. Table 2 2. Effects of DL methionine, L threonine, lysine, Na methionate, K methionate, and methionine hydroxy analog, applied at 224 or 448 kg amino acid/ha on B longicaudatus after three days of exposure during trial 1 and trial 2. B. longicaudatus / 100 cm 3 of soil Variable Trial 1 Trial 2 Formulation L t 4 a 30 a lysine 4 a 32 a MHA 1 b 1 c DL 0 b 23 b K 0 b 1 c Na 0 b 1 c Rate 224 kg amino acid /ha 2 14 448 kg amino acid /ha 2 15 Means with common letters are not different ( P range test.

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31 CHAPTER 3 GREENHOUSE EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS Introduction Belonolaimus longicaudatus (sting nematode) is the most destructive plant parasitic turf nematode in Florida (Crow, 2005 a ). The host range of B. longicaudatus is extensive, including most of the grasses i.e., perennial ryegras s, bahiagrass, seashore paspalum, creeping bentgrass, bermudagrass, centipedegrass, St. Augustinegrass, and zoysia (Crow, 2007). Sting nematode was found at damaging levels on 60% of golf courses surveyed in Florida (Crow, 2005 b ). Turf parasitized by sting nematode often shows severe decline and even death (Crow, 2007). Due to the lack of effective post plant nematicides, damage caused by plant parasitic nematodes has been of great concern to the turf industry (Luc et al, 2007). Following the EPA cancellat ion of the organophosphate nematicide fenamiphos (Nemacur Bayer CropScience, Research Triangle Park, NC) (Anonymous, 2002), more economical, effective, and environmentally friendly nematode management tools are needed for turfgrasses. Methionine is an e ssential amino acid produced commercially primarily as an animal feed supplement (Crow, 2009), yet also has shown nematicidal potential in previous research (Overman and Woltz, 1962; Peacock, 1966; Andel, 1966; Evans and Trudgill, 1971). Solutions containi ng DL methionine have been shown to inhibit the activity of Meloidogyne incognita (root knot nematode) juveniles (Talavera and Mizukubo, 2005). Applications of DL methionine to soil has been found to reduce the number of Globodera rostochiensis in soil an d potato roots (Evans and Trudgill, 1971), and the number of Heterodera avenae females in wheat (Prasad and Webster, 1967). Field applications of high rates of DL methionine to turf were effective at reducing numbers of B. longicaudatus but also caused p hytotoxicity (Crow et al. 2009). The low

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32 solubility of DL methionine (30g/liter at 20 C) may inhibit its movement into soil and thereby increase the effective use rate and potential for phytotoxicity, which could affect its utility as a nematicide. Ther e also is concern that application of large amounts of amino acids could have an impact on soil pH. A previous experiment found that several liquid methionine analogues had activity equal to or better than DL methionine in bench screens (Chapter 2). Thes e analogues could potentially move into soil better than DL methionine and avoid some of its shortcomings. The objective s of this experiment w ere to: 1. Compare the efficacy against B. longicaudatus and phytotoxicity of several liquid methionine analogues of DL methionine. 2. Determine if applications of DL methionine and liquid methionine analogues have a detrimental effect on soil pH. Materials and Methods Two sequential identical trials were conducted in a growth room on the campus of the University of Florida in Gainesville, FL. These trials compared the effects of several liquid methionine analogues to DL methionine in pots inoculated with B. longicaudatu s and planted with creeping bentgrass ( Agrostis palustris ). Parameters evaluated were treatments effects on B. longicaudatus plant phytotoxicity, and on soil pH. Treatments were: 1) Untreated control, 2) DL methionine (Evonik Degussa, Theodore, AL ), 3) Na methionate (Na Liquimeth ; Evonik Degussa, Theodore, AL ), 4) K methionate (K Liquimeth ; Evonik Degussa, Theodore, AL ), and 5) methionine hydroxyl analog (Alimet ; Novus International, St. Charles, MO). Each of the amino acid treatments was evaluated at three rates; 112, 224, and 448 kg amino acid/ha. One hundred and thirty 10.16 cm diam. pots were filled with 400 cm 3 of USGA (United States Golf Association) specification greens sand (Anonymous, 1993), and seeded with 08 g per pot. The seed was then irrigated for 2 minutes with a

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33 mister irrigation system with subsequent irrigation every 4 hours for 1 minute. Inoculum of B. longicaudatus was from greenhouse cultures of an isolate originating in Sun City, FL and maintaine Stenotaphrum secundatum ). After seed germination, B. longicaudatus was collected using a decant and sieve technique (Cobb, 1918), and 100 mixed life stages were inoculated into each pot and allowed to reproduce for 4 to 6 weeks. Following turf and nematode establishment, treatments were applied. The liquid materials were mixed with water and 50 ml of solution was applied topically to the pots as a drench. Dry DL methionine was applied topically and then 50 ml water added t o the surface to move it into the soil. The untreated control treatment received 50 ml of water. Turfgrass was monitored visually for phytotoxic response daily. A visual damage rating scale was used to evaluate phytotoxicity, where 0 = no phytotoxicity, 1 = slight phytotoxicity, 2 = moderate phytotoxicity, and 3 = severe phytotoxicity. The experiment was terminated 2 weeks after treatment and the soil was analyzed for treatment effects on nematodes and soil pH. Soil was removed from each pot, mixed, and a 100 cm 3 subsample was removed for quantification of nematodes. Nematodes were extracted using the sugar floatation centrifugation method (Jenkins, 1964). The remaining soil was used to determine soil pH following treatments. A 20 cm 3 subsample was ta ken from the remaining soil and placed into a 100 ml plastic cup with 40 ml deionized water. A pH meter (AR10 pH/mV/degC Meter, Standardize accumet Research, Fisher Scientific, USA) was used to measure the soil pH. Data were subjected to analysis of varia nce and means were separated range test ( P NC). Additionally, the methionine and liquid methionine analog da ta was further evaluated as a 3 4 factorial with three rate treatmen ts and 4 amino acid treatments. The results of both trails

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34 were analyzed for heterogeneity and the results did not differ among the trials except for the phytotoxicity measurements. Therefore, data other than the phytotoxicity data were combined from both trials prior to analysis. Results The effects of all treatments on B. longicaudatus are shown in Figure 3 1 and Table 3 1. All amino acid treatments were equally effective against B. longicaudatus (Table 3 1). All rates of DL methionine and liquid methi onine analogues reduced population densities of B. longicaudatus compared to the water controls ( P 1), but the highest rate (448 kg amino acid/ha) of all amino acids was more effective in reducing the number of B. longicaudatus than the two lower rates (Table 3 1). The phytotoxicity effects on grass after treatments are shown in Figure 3 3. Three days after application, the grass treated with 448 kg amino acid/ha of methionine hydroxyl analog and DL methionine began to exhibit p hytotoxicity that remained throughout the rest of the trial (Figure 3 3 and Table 3 2). Symptoms included chlorosis, turning into decline, and finally wilt by the end of the experiment. The phytotoxicity was more severe from the methionine hydroxyl analog than from DL methionine (Table 3 2). The effects of treatments on soil pH are shown in Figure 3 2 and Table 3 2. The higher rates (224 and 448 kg amino acid/ha) of methionine hydroxyl analog and DL methionine reduced soil pH compared to the untreated cont rol ( P 2 and Table 3 2). However, these reductions were only a few tenths on the pH scale and are therefore not considered biologically significant. Discussion In these experiments, all three rates (112, 224, and 448 kg amino acid/ha) o f DL methionine and liquid methionine analogues reduced the number of B. longicaudatus even

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35 though the highest rate (448 kg amino acid /ha) of them performed the best (Fig. 3 1). However, Crow et al. (2009) concluded that two applications of 112 kg/ha of D L methionine were required to reduce population densities of B. longicaudatus in field trials. The difference in outcome may be caused by the different soil textures in these two experiments. In growth room pot experiments, USGA specification greens sand w as used, and there was no thatch present. This might allow for greater infiltration of the methionine in the pot experiment versus the field trials. Future trials should evaluate liquid methionine analogues in the field to determine if enhanced infiltrati on into the soil might reduce their effective use rate compared to DL methionine. In this experiment, the higher rates of DL methionine and the methionine hydroxyl analog resulted in phytotoxicity. Phytotoxicity from DL methionine also was reported by Cro w (2009). All the amino acids tested were equal in their ability to reduce population densities of B. longicaudatus However, both DL methionine and methionine hydroxyl analog have shown potential to cause phytotoxicity to turf in this experiment, and i n others (W. T. Crow, unpublished data). Therefore, K and Na methionate appear to have greater potential for turfgrass therapeutic nematode treatments. Of these two, Na methionate is 6% sodium and K methionate is 7% potassium. Sodium accumulation is con sidered a detriment to turf growth, whereas potassium is a plant nutrient that is a component of most turfgrass fertility programs. Therefore, K methionate appears to be the best candidate for development as a turfgrass nematicide. Field evaluation of K methionate should be conducted to verify the results of these greenhouse experiments.

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36 Figure 3 3 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), applied at112, 224 a nd 448 kg amino acid/ha on B. longicaudatus two weeks after application. Each bar is the mean of ten replications. multiple range test. Figure 3 4 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), applied at112, 224 and 448 kg amino acid/ha on soil pH two weeks after treatment Each bar is the mean of ten replications. Means with common letter s are not different ( P range test. 0 50 100 150 200 250 B. longicaudatus / 100 cm 3 of soil 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 Soil pH

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37 Figure 3 5 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), applied at 112, 224, and 44 8 kg amino acid/ha to bentgrass using a phytotoxicity scale. 0 = no phytotoxicity, 1 = mild phytotoxicity, 2 = moderate phytotoxicity, 3 = severe phytotoxicity. 3 2.5 2 1.5 1 0.5 0 0 4 8 12 16 Phytotoxicity (0 to 3) Days of exposure MHA 112 kg/ha MHA 224 kg/ha MHA 448 kg/ha DL 112 kg/ha DL 224 kg/ha DL 448 kg/ha K 112 kg/ha K 224 kg/ha K 448 kg/ha Na 112 kg/ha Trial 1 3 2.5 2 1.5 1 0.5 0 0 4 8 12 16 Phytotoxicity (0 to 3) Days of exposure MHA 112 kg/ha MHA 224 kg/ha MHA 448 kg/ha DL 112 kg/ha DL 224 kg/ha DL 448 kg/ha K 112 kg/ha K 224 kg/ha K 448 kg/ha Na 112 kg/ha Trial 2

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38 Table 3 3 Effects of methionine hydroxy analog (MHA), DL m ethionine (DL), K methionate (K), and Na methionate (Na), across rates on B. longicaudatus two weeks after application. Data was analyzed as a 4 3 factorial experiment. B. longicaudatus/100 cm 3 of soil Variable Formulation K methionate 33 a Na methionate 32 a DL methionine 28 a MHA 21 a Rate 112 kg/ha 42 a 224 kg/ha 28 b 448 kg/ha 15 c Means with common letters are not different ( P range test. Table 3 4 Effects of methionine hydroxy analog (MHA), DL methionine (DL), K methionate (K), and Na methionate (Na), applied at 112, 224, and 448 kg amino acid/ha to bentgrass using a phytotoxicity scale 0 = no phytotoxicity, 1 = mild phytotoxicity, 2 = moderate phytotoxici ty, 3 = severe phytotoxicity. Phytotoxicity (0 to 3) Trail 1 Trail 2 Treatment Day 3 Day 8 Day 14 Day 3 Day 8 Day 14 MHA 112 kg/ha 0a 0a 0a 0a 0a 0a MHA 224 kg/ha 0a 0a 0a 0a 0a 0a MHA 448 kg/ha 1b 2c 2.2c 1b 2c 2.8c DL 112 kg/ha 0a 0a 0a 0a 0a 0a DL 224 kg/ha 0a 0a 0a 0a 0a 0a DL 448 kg/ha 0a 1b 1.2b 0a 1b 1.2b K 112 kg/ha 0a 0a 0a 0a 0a 0a K 224 kg/ha 0a 0a 0a 0a 0a 0a K 448 kg/ha 0a 0a 0a 0a 0a 0a Na 112 kg/ha 0a 0a 0a 0a 0a 0a Na 224 kg/ha 0a 0a 0a 0a 0a 0a Na 448 kg/ha 0a 0a 0a 0a 0a 0a WATER 0a 0a 0a 0a 0a 0a Means with common letters are not different ( P range test.

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39 CHAPTER 4 EVALUATION OF AMINO ACIDS AGAINST BELONOLAIMUS LONGICAUDATUS IN FIELD TESTS I ntroduction The turf industry is an important component to the economy of Florida. More than four million acres of turfgrasses were grown in 1991 1992 in Florida, and this acreage is still increasing (Hodges et al. 1994) Between 1991 an d 1992, the turf industry contributed $7.5 $4.44 billion in the state of Florida (Haydu and Hodges, 2002). As one of the most destructive pathogens of many crop pl ants, Belonolaimus longicaudatus (sting nematode) also causes a great deal of damage to turfgrasses. Belonolaimus longicaudatus was found at damaging levels on 60% of golf courses surveyed in Florida (Crow, 2005 b ). Turf parasitized by sting nematode often shows severe decline and even death (Crow, 2007). On bermudagrass ( Cynodon dactylon C. transvaalensis ), B. longicaudatus can cause severe root reduction, which reduces water and nutrient uptake from soil, increases the potential for nit rate leaching as w ell as reducing turf quality, color, and density (Luc et al, 2006 ; 2007; McGroary et al., 2009). Production of the organophosphate nematicide fenamiphos (Nemacur Bayer CropScience, Research Triangle Park, NC), which was t he most commonly used nematicide on turfgrasses, was terminated in the United States in 2007 (Anonymous, 2002). 1, 3 Dichloropropene (1, 3 D; Curfew Soil Fumigant Dow AgroSciences, Indianapolis, IN) is currently the only effective nematicide labeled for use on turfgrasses in the southe astern United States. However, the potential negative effects of 1, 3 D and other chemical nematicides on the environment and humans are of concern. DL methionine has no known harmful effects on humans or the environment and has been suggested as a potent ial turfgrass nematicide (Crow et al. 2009). Talavera and Mizukubo

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40 (2005) found that 0.25 g liter 1 DL methionine solutions effectively inhibited egg hatching and reduced the juvenile mobility of Meloidogyne incognita Crow et al. (2009) concluded that DL methionine could be effectively used for managing B. longicaudatus on turf in the field. However, phytotoxicity was observed in one of two trials, and the rates tested (224 and 1140 kg/ha) were high. The low solubility of DL methionine (30g/liter at 20 C) may be the main reason for the observed phytotoxicity. Low solubility could reduce infiltration of DL methionine into the ground where the nematodes are located and thereby increas e the rate use, and also lead to accumulation of methionine at the surface of the grass thereby resulting in phytotoxicity. One potential way to overcome some of the negative effects of methionine that limit its utility as a turfgrass nematicide is to use liquid methionine analogs that might have better movement into the soil. In laboratory and greenhouse experiments, K methionate had equivalent activity against sting nematode as DL methionine and caused no phytotoxicity to turf when applied at the same rates of amino acid (Chapters 2, 3). K methionate is a liquid salt formulat ion of methionine that has nutrie nt value in addition to nematicide or nematostatic effects. A second option that might improve movement of DL methionine into the soil is the use of emulsifiers. Evonik Industries ine products, has a division that specializes in surfactants and emulsifiers. Their internal research has identified a surfactant/emulsifier that might be ideally suited for this purpose. The objectives of this study were to: 1. Compare the efficacy of DL methionine and K methionate and a standard nematicide against B. longicaudatus in the field. 2. Compare the effects on turf health of DL methionine K methionate and a standard nematicide in the field. 3. Evaluate use of a surfactant/e mulsifier to improve efficacy of DL methionine against B. longicaudatus and reduce turf phytotoxicity

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41 Materials and Methods In 2010, two field trials were conducted to compare the nematode and turf health effects of DL methionine and K methionate, and to determine if use of an emulsifier enhanced these effects. Two sites were used for this experiment; both were located at the University of Florida Plant Science Research Unit (PSRU) in Citra, FL. One site was pla nted with bermudagrass, and the another site was planted with infested with potentially damaging numbers of B. longicaudatus The Tifdwarf site was managed under putting green conditions, whereas the Celebration site was managed as a tee box. The experimental design was randomized block with five replications. Fifty plots were used at each site. Plots were 1.5 m 2 with 0.6 m untreated borders between plots. In order to minimize initial treatment variance, treatments were applied to the plots b ased on initial number of B. longicaudatus The ten t reatments included DL methionine (Evonik Degussa, Theodore, AL) at rates of 112 and 224 kg/ha with or without a surfactant/emulsifier (Break Thru S240, Evonik Goldschmidt, Hopewell, VA) at 1.3 liter/ha, K methionate (Evonik Degussa, Theodore, AL) at rates of 112 and 224 kg amino acid/ha with or without a surfactant/emulsifier, fenamiphos (Nemacur 10G, Bayer CropScience, Research Triangle Park, NC) applied at 11.2 kg a.i./ha, and an untreated control. DL methionine and fenamiphos treatments were applied topically using a drop spreader (Gandy, Owatonna, MN). K methionate and the surfactant/emusifier were each mixed with water and sprayed topically on the plots using a CO 2 powered backpack sprayer (Weed Sys tems, Hawthorne, FL). F enamiphos was applied only once; all treatments receiving DL methionine or K methionate were applied twice, on a 4 week interval. After treatment, each plot (including the untreated controls) was irrigated with 15 liters of water fr om a sprinkler can. An additional 0.6 cm of water was applied to the entire field after all the treatments were applied on the first application date only. The turf was maintained by the PSRU staff using standard

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42 maintenance practices. Turf was maintained at a 0.45 c m mowing height on the Tifdwarf site, and 1.4 cm on the Celebration site. Nematode samples consisting of nine cores ( 10 cm deep1.9 cm diameter ) were collected from each plot before treatm ent and 2 weeks after each treatment application date. L eaves, stolons, rhizomes, and organic thatch layer were discarded and the soil was mixed by hand. A 100 cm 3 subsample of soil was used for extracting nematodes by a centrifugal flotation method (Jenkins, 1964). Plant parasitic nematodes were identified and counted using a n inverted light microscope at 40 magnification. Another 100 cm 3 dry soil was used for analyzing soil texture by hydrometer (Bouyoucos, 1936) Two gram s of dry soil was heated at 500 C for three hours for ignition of organic matter and then wei ghed again to calculate the percentage of organic matt er. After soil analysis, soil at both sites was found to have >90% sand and <2% organic matter. To evaluate turf health ; turf density root length and numbe r of root tips were used. Turf density is a measurement of the percentage of the plo t surface covered by green turf A digital photo was taken of the center m 2 of each plot. The percentage of the pixels in each photo that g a macro developed for SigmaScan Pro5 software (SPSS Inc., Chicago, IL) (Richardson et al., 2001). P ercentage of the total pixels in the image that were green was the measure of turf density. To determine root lengths, two cores ( 3.8 cm diameter15.25 cm deep ) were collected from each plot. The top thatch layer was removed from each core. The remaining soil and roots were wash ed across a 1 mm mesh sieve. R oots caught on the sieve were removed and placed into a 50 ml centrifuge tube containing water for st orage. The roots were placed into a clear plastic tray and scanned using a desktop scanner (Epson perfection 4990, Epson America, Long Beach, CA) to obtained bitmap images of the root system. The images

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43 were imported into WinRHIZO software (Regent Instrum ents, Quebec, Canada) for analysis. WinRHIZO processes the images to calculate root lengths and number of root tips in the sample. Treatments were applied on 16 June and 15 July at the Tifdwarf site and 23 June and 20 July at the Celebration site. Nemat ode samples were collected on 8 June, 30 June and 28 July 2010 at the Tifdwarf site, and 8 June, 7 July, and 4 August 2010 at the Celebration site. Root samples were collected on 11 August and 18 August 2010 at the Tifdwarf and Celebration sites, respectively. Turf density was measured on 15 June, 1 July, 15 July, 28 July, 10 August, and 24 August 2010 at the Tifdwarf site. Turf density was measured on 23 June, 7 July, 20 July, 4 August, and 18 August 2010 at the Celebration site. Data were subjected to analysis of variance and treatment means were separated according to a range test. Additionally, the DL methionine and K methionate d ata was further analyzed as a 22 2 factorial with two amino acids, two rates, and two surfactant/emulsifier treatments. Results The effects of treatments on B. longicaudatus at both sites are shown in Table 4 1. The number of B. longicaudatus was significantly reduced by high rates (224 kg amino acid /ha) of DL methionine with or without surfactant and K methionate with o r without surfactant compared with the untreated control ( P application sampling dates (Table 4 1). However, no effects on B. longicaudatus from a n y of the treatments compared with the untreated control were observed at the Tifdwarf site on either post application sampling date (Table 4 1). Fenamiphos did not affect population densities of B. longicaudatus in either trial (Table 4 1) When the amin o acid treatments were analyzed as a factorial experiment, DL methionine and K methionate were not different from each other (Table 4 5). The use of the surfactant/emulsifier only had an effect on one date of the Tifdwarf trial whe re the

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44 surfactant/emulsi fier treatment was less effective than the no n surfactant/emulsifier treatment (Table 4 5). Rate was significant at the first post application sampling of the Tifdwarf trial, and both post application samplings of the Celebration trial (Table 4 5), in th ese cases the 224 kg amino acid /ha rate was more effective than 112 kg amino acid /ha against B. longicaudatus None of the treatments increased root length or number of root tips compared to the untreated controls in either trial (Table 4 4). However, when the amino acid treatments were analyzed as a factorial experiment, the 224 kg amino acid /ha rate of amino acid had greater root length than the 112 kg amino acid /ha rate in the Celebration trial (Table 4 6). None of the treatment s had an effect on turf density in the Tifdwarf trial (Table 4 2). However, all of the treatments except for fenamiphos and K methionate at 112 kg amino acid/ha without surfactant/emuls ifier improved turf density on 4 August of the Celebration trial (Table 4 3). On 18 Augu st, Celebration bermudagrass turf density was improved by all treatments r eceiving 224 kg amino acid/ha (T able 4 3). When amino acid treatments were analyzed as a factorial experiment there were no differences for any variable in the Tifdwarf trial (Tab le 4 8). In the Celebration trial, 224 kg amino acid/ha had greater turf density than the 112 kg amino acid /ha rate (Table 4 7). Discussion In this experiment, DL methionine and K methionate were equally effective in reducing numbers of B. longicauda tus and improving turf health. Therefore, there is no evidence that the liquid methionine analog moved into the ground better than DL methionine. The higher rate of either methionine formulation (224 kg amino acid/ha) was better than fenamiphos in reduc ing population density of B. longicaudatus in soil and improving turf density. No phytotoxicity was observed from any of the treatments evaluated in these trials, making it impossible to determine if K methionate had less potential for causing turf damage than DL methionine. Further research

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45 creating ideal conditions for phytotoxicity, such as increasing use rates or applying less water, should focus on these comparisons. If methionine is to be developed as a commercial nematicide K methionate has several advantages over DL methionine that were noted during this experiment. These advantages include: 1) DL methionine has a strong odor that lasts for a long time, whereas K methionate is relatively odor free. 2) K methionate is easily applied using standar d spray equipment whereas DL methionine is a light powder which makes it difficult to apply. 3) Both DL methionine and K methionate contain nitrogen and sulfur, however, K methionate contains an additional plant nutrient component, i.e. potassium. Use of a surfactant /emulsifier did not improve efficacy of methionine against B. longicaudatus. Because phytotoxicity was not observed in either trial, it is unknown if the surfactant/emulsifier reduced phytotoxicity. Further research is required to make any conclusions as to the benefits, if any, of including a surfactant/emulsifier with methionine treatments to turf. Hasegawa et al. (2003) noted that D methionine and L methionine resulted in increased root tip spiraling and root hair numbers in Brassic a rapa and Crow et al. (2009) found a root length increase of bermudagrass treated with 1120 kg/ha of DL methionine At the Celebration site of our experiment, root lengths were enhanced by 224 kg amino acid/ha compared with 112 kg amino acid/ha. However, no amino acid treatment had greater root lengths than the untreated control. Therefore, we cannot make any meaningful statements about the effects of the methionine on turf roots from this experiment. The turf nutrient component of the methionine treatm ents can make it difficult to separate out turf density effects caused by increased fertility and those related to nematode reduction. Turf density was improved by both rates of either me thionine formulation compared with the

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46 untreated control in the Cele bration trial. This same trial was the one where these treatments had significant nematode reductions. In the Tifdwarf experiment there was no t urf density improvement and no nematode reductions associated with methionine treatments. This suggests that turf density improvement could be, at least in part, a result of nematode e ffects. It is unknown why the methionine treatments worked better on the Celebration trial than the Tifdwarf trial. One possibility is that unknown biotic factors such as disease or pests influenced the results. Meanwhile differences among the grasses at the two sites and their response to B. longicaudatus may have influenced the outcome. Further research should evaluate the effects of methionine across a range of environmental conditions and turf species and cultivars. In conclusion, DL methionine and K methionate exhibited the same or better performance as fenamiphos as a turfgrass nematicide against B. longicaudatus However, use rates of 224 kg amino acid/ha appear to be nee ded to achieve a successful treatment. Further research is still needed to compare the full benefits and liabilities of DL methionine and K methionate.

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47 Table 4 5 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on Belonolaimus longicaudatus a Nematode samples were collected before treatment, and two weeks after each of two amino acid application dates. b Pi=Nematode samples were collected before treatment. c Means with common letters are not different ( P range test. Table 4 6 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on turf density of Tifdwarf bermudagrass. No differences ( P B. longicaudatus / 100 cm 3 of soil a Tifdwarf bermudagrass Celebration bermudagrass Treatment Pi b Jun. 30 Jul. 28 Pi Jul. 07 Aug. 04 DL 112 kg/ha 42 17 ab c 8 ab 37 26 a 41 ab DL 112 kg/ha with surfactant 40 21 a 13 ab 37 17 ab 63 a DL 224 kg/ha 38 9 ab 13 ab 36 6 b 17 bc DL 224 kg/ha with surfactant 42 7 ab 15 ab 37 6 b 11 c K 112 kg/ha 38 14 ab 9 ab 37 20 ab 34 abc K 112 kg/ha with surfactant 38 17 ab 16 a 37 20 ab 54 a K 224 kg/ha 41 7 ab 3 b 37 7 b 8 c K 224 kg/ha with surfactant 37 9 ab 13 ab 37 5 b 8 c Fenamiphos 41 6 b 18 a 38 18 ab 41 ab Untreated 37 13 ab 12 ab 36 25 a 53 a Turf density (0 1 00%) of Tifdwarf bermudagrass Treatment 15.Jun 1.Jul 15.Jul 28.Jul 10.Aug 24.Aug DL 112 kg/ha 71 94 80 69 72 80 DL 112 kg/ha with surfactant 68 92 75 60 73 86 DL 224 kg/ha 75 94 82 69 79 90 DL 224 kg/ha with surfactant 63 92 75 65 72 80 K 112 kg/ha 58 91 71 61 76 83 K 112 kg/ha with surfactant 62 92 75 55 73 84 K 224 kg/ha 70 94 76 64 65 69 K 224 kg/ha with surfactant 66 92 75 66 75 8 Fenamiphos 64 92 79 69 70 70 Untreated 67 91 78 69 74 87

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48 Table 4 7 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on turf density of Celebration bermudagrass. a Means with common letters are not different ( P range test. Table 4 8 Effects of DL methionine (DL) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, K methionate (K) with or without surfactant/emulsifier at rates of 112 and 224 kg amino acid/ha, and fenamiphos at 11.2 kg a.i./ha on the root lengths and nu mber of root tips from two 58 cm 3 cores per plot from the Tifdwarf and Celebration bermudagrass trials. Root length and root tips a Tifdwarf Celebration Treatment Length (cm) Tips Length (cm) Tips DL 112 kg/ha 363 1562 575 3651 DL 112 kg/ha with surfactant 350 1628 516 3138 DL 224 kg/ha 537 2359 575 3651 DL 224 kg/ha with surfactant 318 1522 516 3138 K 112 kg/ha 359 1702 629 3397 K 112 kg/ha with surfactant 352 1701 556 3114 K 224 kg/ha 286 1455 787 4699 K 224 kg/ha with surfactant 309 1514 693 3985 Fenamiphos 546 2704 629 3397 Untreated 405 1768 556 3114 a Root lengths and number of root tips from two 58 cm 3 cores per plot from the Tifdwarf and Celebration bermudagrass trials. No differences ( P Turf density (0 100%) of 'Celebration' bermudagrass Treatment Jun. 23 Jul. 7 Jul. 20 Aug. 4 Aug. 18 DL 112 kg/ha 64 74 67 79 a a 89 abc DL 112 kg/ha with surfactant 73 76 68 80 a 90 abc DL 224 kg/ha 67 70 65 82 a 92 ab DL 224 kg/ha with surfactant 67 72 71 80 a 92 ab K 112 kg/ha 66 72 59 75 ab 87 bc K 112 kg/ha with surfactant 67 71 65 82 a 90 abc K 224 kg/ha 72 76 66 82 a 92 ab K 224 kg/ha with surfactant 69 73 67 84 a 93 a Fenamiphos 74 68 59 65 b 85 c Untreated 69 58 62 69 b 85 c

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49 Table 4 9 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 2 2 2 factorial experiment on Belonolaimus longicaudatus for both trials. B. longicaudatus / 100 cm 3 of soil Tifdwarf Celebration Variable Jun. 30 Jul. 28 Jul. 07 Aug. 04 Formulation DL m ethionine 14 12 14 33 K m ethionate 12 10 13 25 Surfactant With 13 14 12 34 Without 12 8* 15 24 Rate 112 kg amino acid /ha 17 12 21 48 224 kg amino acid /ha 8* 11 6* 11* *Variable comparison is significant ( P Table 4 10 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 22 2 factorial experiment on root length and root tips for both trials. Root length and root tips Tifdwarf Celebration Variable Length (cm) Tips Length (cm) Tips Formulation DL m ethionine 392 1767 666 3754 K m ethionate 326 1593 624 3799 Surfactant With 332 15 91 616 3564 Without 386 1769 674 3990 Rate 112 kg amino acid /ha 356 1648 569 3325 224 kg amino acid /ha 362 1712 721 4229 *Variable comparison is significant ( P

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50 Table 4 11 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 22 2 factorial experiment on turf density for the Tifdwarf trial Tifdwarf bermudagrass turf density (0 100%) Variable Jun. 15 Jul. 1 Jul. 15 Jul. 28 Aug. 10 Aug. 24 Formulation DL methionine 69* 93 78 66 74 83 K methionate 63 92 74 62 72 81 Surfactant With 65 92 75 62 73 80 Without 68 93 77 66 73 85 Rate 112 kg amino acid/ha 65 92 75 62 73 82 224 kg amino acid/ha 69 93 76 66 74 83 *Variable comparison is significant ( P Table 4 12 Effects of amino acid formulation, amino acid rate, and addition of a surfactant/emulsifier when all amino acid treatments were analyzed as a 22 2 factorial experiment on turf density for the Celebration trial. Celebration bermudagrass turf density (0 100%) Variable Jun. 23 Jul. 7 Jul. 20 Aug. 4 Aug. 18 Formulation DL methionine 68 73 68 80 91 K methionate 68 73 64 81 90 Surfactant With 69 73 68 81 91 Without 67 73 64 80 90 Rate 112 kg amino acid/ha 68 73 65 79 88 224 kg amino acid/ha 69 73 67 82 92* *Variable comparison is significant ( P

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51 CHAPTER 5 EVALUATION OF DL METHIONINE SOLUTIONS AGAINST B. LONGICAUDATUS IN PETRI DISH STUDY Introduction The amino acid methionine is produced in the form of L methionine in plants and is widely present in organisms as a protein component or as a substrate for methyl transaction reactions (Crow et al., 2009). Recently, methionine has been viewed as an insecticide due to the ability t o block ion flow through the cation anion modulated amino acid transporter with channel properties (CAATCH1) system, which is an insect membrane protein from the midgut nutritive absorptive epithelium (Feldman et al., 2000; Quick and Stevens, 2001 ; Stevens et al., 2002 ) Methionine has been studied for many years as a potential nematicide. In particular, it has been shown to reduce the number of certain plant parasitic nematodes in crops, such as wheat, potato, and turfgrass (Prasad and Webster, 1967; Evans and Trudgill, 1 971; Crow et al., 2009). However, the mode of action of amino acids against nematodes is still unclear. Overman and Woltz (1962) hypothesized that nematodes are incapable of feeding when exposed to antimetabolities such as D methionine. Davies (1966) note d that turnip storage tissue accumulates S adenosylmethionine (metabolically inactive and accumulating in the cell vacuoles) after incubation with methionine. The oxidative phosphorylation and respiration of the root cells become decreases after removing a denosine, and thus might affect plant parasitic nematode survivals. Evans and Trudgill (1971) suggested that amino acids are always in the form of L isomers, whereas D isomers sometimes serve as antimetabolites in nematodes, which could compete with these essential compounds on enzymes. However, re production of Globodera rostochiensis was prevented equally by D, L, and DL forms of methionine in pot studies (Evans and Trudgill, 1971)

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52 Most of the previous hypotheses were based on pot or field studies, and in cluded effects on plants as a potential mode of action on nematodes. Evans and Trudgill (1971) noted that G. rostochiensis juveniles were not affected by methionine solutions (0.6, 3, 15 g litre 1 ). Conversely, Talavera and Mizukubo (2005) found that DL me thionine solutions had direct effects on Meloidogyne incognita and they concluded that a 0.25 g litre 1 solution of DL methionine prevented egg hatching and immobilized juvenile M. incognita. Bench screen experiments with Belonolaimus longicaudatus and M. incognita second stage juveniles in soil (Chapter 2) showed that DL methionine had a direct impact on the ability of these nematodes to move through a Baermann funnel. This indicated that the mode of action is not plant related and that the methionine was not ingested, as there were no plants in the system and the nematodes were supposedly not feeding in this experiment. Because the Baermann funnel extraction technique requires nematodes to be mobile, it is unknown if the methionine killed the nematode s or caused paralysis, as either mode of action would give the same results. To ascertain the mode of action of methionine on B. longicaudatus a Petri dish experiment was conducted. The objectives of this experiment were to: 1. Determine if exposure to m ethionine solution causes mortality or paralysis of B. longicaudatus 2. Determine the solution concentration of DL methionine required to directly affect B. longicaudatus. Materials and Methods In July and September 2010, t wo trials of a petri dish exp eriment were conducted in the Entomology and Nematology Department at the University of Florida, Gainesville, FL. DL methionine solutions were prepared at concentrations of 0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, and 2% (w/w), the latter being the maximum co ncentration of methionine that would stay in solution at room temperature. In total, 20 ml of each solution was pour ed into Petri dishes, with

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53 five replications of each concentration. The room temperature was maintained at 26.7 C Belonolaimus longicauda tus maintained on St. Augustinegrass in the greenhouse was extracted from soil by modified Baermann funnels to insure nematodes were active. Ten B. longicaudatus were picked into each dish and observed for activity and symptoms on 1, 2, 3, 5, and 6 days th ereafter using an inverted light microscope at 40 magnification. When nematodes were observed as immobile, they were counted as dead. Percent mortality B. longicaudatus was recorded in each solution on each date. P ercent mortality of B. longicaudatus was regressed on DL methionine concentration for data collected on days 3, 5, and 6 to determine solution concentration effects on B. longicaudatus using SAS software (SAS Institute, Cary, NC). Results The effects of different concentrations of DL methion ine solutions on B. longicaudatus mortality are shown in Fig ure 5 1, 5 2, and 5 3 The mortality of B. longicaudatus increased significantly at the concentrations of 0.01%, 0.1%, 1%, and 2% when compared with B. longicaudatus kept in water ( P re 5 1 5 2, 5 3 ). However, no differences in mortality were observed until three days after exposure. After 6 days, there was almost 90% mortality in the 1% and 2% solutions. Discussion These results show that DL methionine can have a direct effect on B. longicaudatus Unfortunately, the methodology used in this experiment did not allow us to distinguish between paralyzed and dead nematodes. Therefore, we were unable to answer the question posed in the first objective.

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54 Talavera and Mizukubo (2005) reported that 0.25 g litre 1 (2.5%) DL methionine solution immobilized M. incognita juveniles and inhibited egg hatching of M. incognita after 4 days of exposure. Our studies showed similar results on B. longicaudatus In conclusion, DL methionine soluti ons have a direct effect on the activity of B. longicaudatus. There was a concomitant increase in B. longicaudatus immobility with increases in the methionine conc entration of solutions These effects were not immediate, but took several days to occur.

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55 Figure 5 6 Percent mortality of Belonolaimus longicaudatus exposed to increasing % DL methionine solutions (DL) over time during trial 1 and trial 2. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 % Mortility of B. longicaudatus Days of exposure 0.0001% DL 0.001% DL 0.01% DL 0.1% DL 1% DL 2% DL 0% DL 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 % Mortility of B. longicaudatus Days of exposure 0.0001% DL 0.001% DL 0.01% 0.1% DL 1% DL 2% DL 0% DL Trail 1 Trail 2

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56 Figure 5 7 Effects of increasing concentrations of DL methionine solutions (0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%) on Belonolaimus longicaudatus in petri dishes, A) 3, B) 4, and C) 6 days after treatments during trial 1 Each concentration level consists of five replications. y = 14.424x + 6.447 R = 0.3115 CV=123.6 P =0.0005 0 10 20 30 40 50 60 70 80 90 100 0 1 2 % Mortality of B. longicaudatus Concentration of DL methionine A y = 36.254x + 17.316 R = 0.727 CV=49.3 P 0 10 20 30 40 50 60 70 80 90 100 0 1 2 % Mortality of B. longicaudatus Concentration of DL methionine B

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57 Figure 5 2 Continued. Figure 5 8 Effects of increasing concentrations of DL methionine solutions (0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%) on Belonolaimus longicaudatus in petri dishes, A) 3, B) 4, an d C) 6 days after treatments during trial 2 Each concentration level consists of five replications. y = 35.234x + 33.769 R = 0.651 CV=38.7 P 0 10 20 30 40 50 60 70 80 90 100 0 1 2 % Mortality of B. longicaudatus Concentration of DL methionine C y = 10.553x + 7.596 R = 0.3596 CV=84.9 P =0.0001 0 10 20 30 40 50 60 70 80 90 100 0 1 2 % Mortality of B. longicaudatus Concentration of DL methionine A

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58 Figure 5 3. Continued. y = 28.877x + 22.594 R = 0.5546 CV=54.1 P 0 10 20 30 40 50 60 70 80 90 100 0 1 2 % Mortality of B. longicaudatus Concentration of DL methionine B y = 31.161x + 34.722 R = 0.5498 CV=43.0 P 0 10 20 30 40 50 60 70 80 90 100 0 1 2 % Mortality of B. longicaudatus Concentration of DL methionine C

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59 LIST OF R EFER E NCES Abu Gharbieh, W. I., and Perry, V. G. 1970. Host differences among Florida population of Belonolaimus longicaudatus R au. Journal of Nematology 2(3): 209 216. Andel, O. M. V. 1966. Amino acids and plant diseases. Annual Review Phytopathology 4:349 368. Anonymous. 1993. USGA recommendation for a method of putting green construction: The 1993 revision. USGA Gr een Section Record 31:1 3. Anonymous. 2002. Fenamiphos: Notice of receipt of request to voluntarily cancel all product registrations. Federal Register 67(188):61098 61099. Anonymous. 2004. List of inert pesticide ingredients l ist 4b. U. S. Envronmental Pro tection Agency. www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf. Bouyoucos, G. J. 1936. Directions for making mechanical analysis of soil by the hydrometer method. Soil Sc ience 42 (3):225 230. Boyd, F. T., and Perry, V. G. 1971. Effects of seasonal temper atures and certain cultural treatments on sting nematodes in forage grass. Proceedings of the Soil and Crop Science Society of Florida 30:360 365. Brodie, B. B. 1976. Vertical distribution of three nematode species in relation to certain soil properti es. J ournal of Nematology 8(3): 243 247 Busey, P., Giblin Davis, R. M., Riger, C. W., and Zaenker, E. I. 1991. Susceptibility of diploid St. Augustinegrass es to Belonolaimus longicaudatus Supplement to the Journal of Nematology 23 (4S) :604 610. Christie, J. R. 1953. The sting nematode can be controlled by soil fumigation. Down to Earth 9:8 9. Christie, J. R., Brooks, A. N., and Perry, V. G. 1952. The sting nematode, Belonolaimus gracilis a parasite of major importance on strawberries, celery and s weet corn in F lorida. Phytopath ology 42:173 176. 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 Ty lencholaimus aequalis n. sp. Washington D.C.: Government Printing Office Crow, W. T. 2005 a Alternatives to fenamiphos for management of plant parasitic nematodes on bermudagr asses. Journal of Nematology 37 (4):477 482.

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60 Crow, W. T. 2005b How bad are Turf Digest 22:10 12. Crow, W. T. 2007. Understanding and managing plant parasitic nematodes on turfgrasses. Pp. 351 374 In M. Pessarakli, ed. Handbook of Turfgrass Management and Physiology. CRC Press, Boca Raton, FL. Crow, W. T., Cuda, J. P., and Stevens B. R. 2009. Efficacy of methionine against ectoparasitic nematodes on golf course turf. Journal of Nematology 41 (3):217 220. Crow, W. T., Dickson, D. W., and Weingartner, D. P. 1997. Stubby root sympto ms on cotton induced by Belonolaimus longicaudatus Journal of Nematology 29:574 (Abstr ) Crow, W. T., Giblin Davis, R. M., and Lickfeldt, D. W. 2003. Slit injection of 1,3 d ichloropropene for management of Belonolaimus longicaudatus on established bermud a grass. Journal of Nematology 35 (3):302 305. Crow, W. T., Lickfeldt, D. W., and Unruh J. B. 2005. Management of sting nematode ( Belonolaimus longicaudatus ) on bermudagrass putting greens with 1,3 dichloropropene. International Turfgrass Society Research Jo urnal 10:734 741. Davies, D. D. 1966. The control of respiration of turnip disks by L methionine. Journal of Experimental Botany 17:320 331. Duncan, L. W., Inserra, R. N., and Dunn, D. 1998. Seasonal changes in citrus root starch concentration and body length of female Pratylenchus coffeae Nematropica 28 (2) :263 266. Duncan, L. W., Noling, J. W., Inserra, R. N., and Dunn, D. 1996. Spatial patte rns of Belonolaimus spp. among and withi idg e. Journal of Nematology 28(3): 352 359. Epstein, E. 1973. Effect of pretreatment with some amino acids and amino acid antimetabolites on Longidorus africanus infected and no n infected B idens tripartita Nematologica 18 (4) :555 562. Evans, K., and Trudgill, D. L. 1971. Effects of amino acids on the reproduction of Heterodera rostochiensis Nematologica 17 (4) : 495 500. Feldman, D. H., Harvey, W. R., and Stevens B. R. 2000. A novel electrogenic amino acid transporter is activated by K + or Na + is alkaline pH dependent, and is Cl independent. The Journal of Biological Chemistry 275:24518 24526. Ferris, V. R., and Bernard, R. L. 1971. Effect of soil type on population densities o f nematodes in soybean rotation fields. Journal of Nematology 3(2):123 128. Giblin Davis, R. M., Cisar, J. L., Bilz, F. G., and Williams, K. E. 1991. Management bermudagrass. Nematropica 2(1): 59 69

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61 Gozel, U., Adams, B. J., Nguyen, K. B., Inserra, R. N., Giblin Davis, R. M., and Duncan, L. W. 2006. A phylogeny of Belonolaimus populations in Florida inferred from DN A sequences. Nematropica 36(2): 155 161. Graham, T. W., and Holdeman, Q. J. 1953. The s ting nematode, Belonolaimus gracilis Steiner: A para site on cotton and other crops i n South Carolina. Phytopathology 43:434 439. Gray, B. J., and Miller, L. I. 1962. Gross morphology of the Virginia population of the sting nematode. Virginia Journal of Sci ence 13:212 213. Han, H R., Jeyaprakash, A., Weingartner, D. P., and Dickson, D. W. 2006. Morphological and molecular biological characterization of Belonolaimus longicaudatus Nematropica 36(1): 37 52 Hasegawa, N., Yamaji, Y., Minoda, M., and Kubo, M. 200 3. Effects of D methionine or L methionine on root hair s of Brassica rapa Journal of Bioscience and Bioengineering 95(4):419 420. Haydu, J., and Hodges, A. 2002. Economic dimensions of the Florida golf course industry. Florida Cooperative Extension Servic e Fact Sheet FE 344. Department of Food and Resource Economics, University of Florida, Gainesville, FL. Hodge s A.W., Haydu, J. J., Van Blokland, P. J., and Bell, A. P. 1994. Contribution of the 92: A value add ed approach. Economics Report 94 1. Institute of Food and Agricultural Science Food and Resource Ecomomics Department. University of Florida, Gainesville, FL. Huang, X., and Becker, J. O. 1997. In vitro culture and feeding behavior of Belonolaimus longicau datus on excised Zea mays roots. Jou rnal of Nematology 29(3): 411 415 Huang, X., and Becker, J. O. 1999. Life cycle and mating behavior of Belonolaimus longicaudatus in Gnotobiotic cultur e. Journal of Nematology 31(1): 70 74 Jenkins, W. R 1964. A rapid centrifugal flo tation technique for separating nematodes from soil. Plant Disease Reporter 48:692. Johnson, W. R. 1970. Pathogenicity and interactions of three nematode species on six bermudagrasses. Journal of Nematology 2 (1) :36 41. Luc, J. E., Crow, W. T., Stimac, J. L., Sartain, J. B., and Giblin Davis, R. M. 2006. Influence of Belonolaimus longicaudatus on nitrate leaching in tur f. Journal of Nematology 38(4): 461 465. Luc, J. E., Crow, W. T., Stimac, J. L., Sartain, J. B., and Giblin Davis, R. M. 2007 Effects of Belonolaimus longicaudatus m anagement and nitrogen fertility on turf quality of golf course fairway s. Journal of Nematolog y 39(1): 62 66.

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62 Lucas, L. T. 1982. Population dynamics of Belonolaimus longicaudatus and Criconemella ornata and growth re sponse of bermudagrass and overseeded grass on golf greens following treatments with nematicides. Journal of Nematology 14 (3) :358 363 McGroary, P., Crow, W. T., Mc Sorley, R., Giblin Davis, R. M., and Cisar, J. L. 2009. Seasonal fluctuations of Belonolaimu s longicaudatus in bermudagrass. Nematropica 39 (1): 99 110. McSorley, R., and Dickson, D. W. 1990. Vertical distribution of plant parasitic nematodes in sandy soil under soybean. Journal of Nematology 22(1):90 96. McSorley, R., and Frederick, J. J. 1991. Extraction efficiency of Belonolaimus longicaudatus from sandy soil. Journal of Nematology 23(4):511 518. Miller, L. I. 1960. The influence of soil components on the survival and development of the sting nematode, Belonolaimus longicaudatus Rau. Virginia Journal of Science 11: 160 (Abstr ). Miller, L. I. 1972. The influence of soil texture on the survival of Belonolaimus longicaudatus Phytopathology 62: 670 671 (Abstr ). Noling, J. W. 1993. Citrus root growth and soi l pest management practices. Florida Cooperative Extension Service Fact Sheet ENY 617. University of Florida, Gainesville, FL. three parasitic nematodes in Florida citru s. Journal of Nematology 4 (3) :194 199. Overman, A. J., and Woltz, S. S. 1962. Effects of amino acid antimetabolites upon nematodes and tomatoes. Florida State Horticultural Society Proceedings 10:734 741 Owens, J. V. 1951. The pathological effects of Belo nolaimus gracilis on peanuts in Virginia. Phytpathology 41: 29 (Abstr ) Owens, R. G., and Novotny, H. M. 1960. Physiological and biochemical studies on nematode galls. Phytopathology 50:650 Peacock, F. C. 1966. Nematode control by plant chemotherapy. Nema tologica 12 (1) :70 86. Perry, V. G. 1964. Nematode host parasitic relationships. Florida Agricultural Experiment Station Annual Report 1964:115 116 Perry, V. G., and Rhoades, H. 1982. The genus Belonolaimus Pp. 144 149 in R. D. Riggs, ed. Nematology in t he southern region of the United States, South Cooperative Series Bulletin 276. Fayetteville, AR: Arkansas Agricultural Experiment Station, University of Arkansas.

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63 Potter, J. M. 1967. Vertical distribution of sting, ring, and stunt nematodes in a Norfolk s andy loam following peanuts. Nematologica 13 (1) :150 (Abstr ) Prasad, S. K., and Webster, J. M. 1967. The eff ect of amino acid antimetabolit es on four nematode species and their host plants. Nematologica 13 (2) :318 323. Quick, M., and Stevens B. R. 2001. Amino acid transporter CAATCH1 is also an amino acid gated cation channel. The Jour nal of Biological Chemistry 276 (36):33143 33418. Rau, G. J. 1958. A new species of sting nematode. Proceedings of the Helminthologic al Society of Washington 25(2): 95 98. Rau G. J. 1961. Amended descriptions of Belonolaimus gracilis Steiner, 1949 and B. longicaudatus Rau, 1958 (Nematoda: Tylenchida). Proceedings of the Helminthologic al Society of Washington 28(2): 198 200. Rau, G. J. 1963. Three New species of Belonolaimus (Ne matoda: Tylenchida) with additional data on B. longicaudatus and B. gracilis Proceedings of the Helminthologic al Society of Washington 30(1): 119 128 Rau, G. J., and Fassuliotis, G. 1970. Equal frequency tolerance ellipses for population studies of Belono laimus longicaudatus Journal of Nematology 2(1): 84 92 Rhoades, H. L. 1980. Reproduction of Belonolaimus longicaudatus in treated and untreated muck soil. Nematropica 10 (2) :139 140. Richardson, M. D., Karcher, D. E., and Purcell, L. C. 2001. Quantifying t urfgrass cover using digital image analysis. Crop Science 41:1884 1888. Robbins, R. T., and Barker, K. R. 1973. Comparison of host range and reproduction among populations of Belonolaimus longicaudatus from North Carolina and Georgia. Plant Disease Reporte r 57:750 754. Robbins, R. T., and Barker, K. R. 1974. The effects of soil type, particle size, temperature, and moisture on reproduction of Belonolaimus longicaudatus Journal of Nematology 6 (1) :1 6. Robbins, R. T., and Hirschmann, H. 1974. Variation among Populations of Belonolaimus longicaudatus Journal of Nematology 6(2): 87 94. Siddiqi, M. R. 2000. Tylenchida: parasites of plant and insects 2 nd ed. New York: CABI Publishing. Smart, G. C., Jr., and Nguyen, K.B. 1991. Sting and awl nematodes: Belonolaimus spp. and Dolichodorus spp. Pp. 627 667 in W. R. Nickle, ed. Manual of Agricultural N ematology New York: Marcel Dekker.

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64 Steiner, G. 1949. Plant nematodes the grower should know. Soil and Crop Science Society of Florida Proceedings 4:72 117. Stevens, B. R., Feldman, D. H., Liu, Z., and Harvey, W. R. 2002. Conserved tyrosine 147 plays a critical role in the ligand gated current of the epithelial cation/anion acid transporter/channel CAATCH1. Journal of Experimental Biology 205:2545 2553. Talavera, M., and Miz ukubo, T. 2005. Effects of DL methionine on hatching and activity of Meloidogyne incognita eggs and juvenil es. Pest Management Science 61: 413 416. Todd, T. C. 1989. Population dynamics and damage p otential of Belonolaimus sp. on c orn. Supplement to Journal of Nematology 21(4): 697 702. Trenholm, L. E., Lick feldt, D. W., an d Crow, W. T. 2005. Use of 1,3 d ichloropropene to reduce irrigation requirements of sting nematode infested bermudagrass. Hortscience 40(5):1543 1548. Van Blokland, P.J., Hodges, A.W., and Haydu, J. J. 1997. Measuring the economic contribution of Florid a's turfgrass industry using a value added methodology. International Turfgr ass Society Research Journal 8: 1127 1133 Van Gundy, S. D., Stolzy, L. H., Szuszkiewicz, T. E., and Rackham, R. L. 1962. Influence of oxygen supply on survival of plant parasitic nematodes in soil. Phytopathology 52:628 632. Wallace, H. R. 1963. The biology of plant parasitic nematodes. Edward Arnold. Ltd., London. Wallace, H. R. 1971. Abiotic influence in the soil en vironment. Pages 257 280 in Zuckerman, B. M., Mai, W. F., and Rohde, R. A., eds. Plant Parasitic Nematodes vol. 1, Academic Press, New York.

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65 BIOGRAPHICAL SKETCH Yun Zhang was born in Benxi, Liaoning, China to Wenjia Zhang and Jingrong Zhao. He gradua ted from Shenyang Agricultural University with a Bachelor of Agriculture degree in Plant Protection in 2006. In summer 2005, he practiced in Human Genome Project Beijing Center in China as an intern, where international scientific research experience was g ained. Between November 2005 and May 2006, he assisted Professor Yuxi Duan and Vice Professor Lijie Chen to classify the n ematode s of China in Shenyang Agricultural University, where a love of nematology was born. Since nematology education was in adequate in China, he decided to come to study in the United States. He was self employed while he was preparing for the GRE and TOFEL tests in 2007. He relocated to Gainesville FL to attend the University of Florida pursuing a Master of Science degree in entomol ogy and n ematology in 2008. He works for Dr. William T. Crow, Landscape Nematologist, University of Florida, Gainesville, Florida as a Acids against Plant Paras He graduated from the University of Florida in December 2010 with a Master of Science degree in entomology and nematology.