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Responses of Bermudagrass and Seashore Paspalum to Sting and Spiral Nematodes

Permanent Link: http://ufdc.ufl.edu/UFE0042436/00001

Material Information

Title: Responses of Bermudagrass and Seashore Paspalum to Sting and Spiral Nematodes
Physical Description: 1 online resource (121 p.)
Language: english
Creator: Pang, Wenjing
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: bermudagrass, nematodes, paspalum, seashore, spiral, sting
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Bermudagrass (Cynodon spp.) and seashore paspalum (Paspalum vaginatum Swartz) are commonly used warm-season turfgrasses on golf courses in Florida. Belonolaimus longicaudatus is the most serious nematode pests on turfgrass and Helicotylenchus spp. are often found to have high population densities in seashore paspalum in Florida. Greenhouse and field experiments were conducted to test 17 bermudagrass cultivars and seven seashore paspalum cultivars for their resistance and tolerance to Belonolaimus longicaudatus or Helicotylenchus pseudorobustus. Greenhouse studies showed that no dwarf bermudagrass cultivars were both tolerant and resistant to B. longicaudatus. ?Tifdwarf? and ?Emerald Dwarf? were tolerant and ?Champion?, ?TifEagle?, and ?Floradwarf? were damaged by B. longicaudatus. Non-dwarf cultivars ?TifSport? and ?Riviera? were both tolerant and resistant to B. longicaudatus. Differences in tolerance among seashore paspalum were found although all cultivars were susceptible to B. longicaudatus but resistant to H. pseudorobustus. ?Salam?, ?SeaDwarf?, and ?SeaIsle Supreme? were tolerant to B. longicaudatus, and ?SeaSpray? and ?SeaIsle 2000? were tolerant to H. pseudorobustus. Field studies indicate that bermudagrass is a better host to B. longicaudatus, and seashore paspalum is a better host to H. pseudorobustus. A negative logarithmic relationship was found between the population densities of B. longicaudatus and H. pseudorobustus in two bermudagrass and three seashore paspalum cultivars. TifSport bermudagrass and SeaDwarf seashore paspalum were the most resistant to B. longicaudatus in field. Flow cytometry was used to determine the nuclear DNA content and ploidy levels of 47 University of Florida bermudagrass germplasm accessions. Twenty triploid, 24 tetraploid, one pentaploid, and three hexaploid accessions were identified. Most of these, along with 12 diploid African bermudagrass (Cynodon transvaalensis Burtt-Davy) accessions were further screened for nematode responses under greenhouse conditions. Results indicated that four African and seven common bermudagrass accessions were tolerant to B. longicaudatus, and eight African and 22 common bermudagrass accessions were resistant to B. longicaudatus, all of which could be used for future cultivar breeding for nematode tolerance or resistance. Accessions that are both resistant and tolerant to B. longicaudatus were identified in both common and African bermudagrass accessions, which would aid in increasing the genetic diversity of future bermudagrass cultivars on golf courses.
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 Wenjing Pang.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Crow, William T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042436:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042436/00001

Material Information

Title: Responses of Bermudagrass and Seashore Paspalum to Sting and Spiral Nematodes
Physical Description: 1 online resource (121 p.)
Language: english
Creator: Pang, Wenjing
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: bermudagrass, nematodes, paspalum, seashore, spiral, sting
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Bermudagrass (Cynodon spp.) and seashore paspalum (Paspalum vaginatum Swartz) are commonly used warm-season turfgrasses on golf courses in Florida. Belonolaimus longicaudatus is the most serious nematode pests on turfgrass and Helicotylenchus spp. are often found to have high population densities in seashore paspalum in Florida. Greenhouse and field experiments were conducted to test 17 bermudagrass cultivars and seven seashore paspalum cultivars for their resistance and tolerance to Belonolaimus longicaudatus or Helicotylenchus pseudorobustus. Greenhouse studies showed that no dwarf bermudagrass cultivars were both tolerant and resistant to B. longicaudatus. ?Tifdwarf? and ?Emerald Dwarf? were tolerant and ?Champion?, ?TifEagle?, and ?Floradwarf? were damaged by B. longicaudatus. Non-dwarf cultivars ?TifSport? and ?Riviera? were both tolerant and resistant to B. longicaudatus. Differences in tolerance among seashore paspalum were found although all cultivars were susceptible to B. longicaudatus but resistant to H. pseudorobustus. ?Salam?, ?SeaDwarf?, and ?SeaIsle Supreme? were tolerant to B. longicaudatus, and ?SeaSpray? and ?SeaIsle 2000? were tolerant to H. pseudorobustus. Field studies indicate that bermudagrass is a better host to B. longicaudatus, and seashore paspalum is a better host to H. pseudorobustus. A negative logarithmic relationship was found between the population densities of B. longicaudatus and H. pseudorobustus in two bermudagrass and three seashore paspalum cultivars. TifSport bermudagrass and SeaDwarf seashore paspalum were the most resistant to B. longicaudatus in field. Flow cytometry was used to determine the nuclear DNA content and ploidy levels of 47 University of Florida bermudagrass germplasm accessions. Twenty triploid, 24 tetraploid, one pentaploid, and three hexaploid accessions were identified. Most of these, along with 12 diploid African bermudagrass (Cynodon transvaalensis Burtt-Davy) accessions were further screened for nematode responses under greenhouse conditions. Results indicated that four African and seven common bermudagrass accessions were tolerant to B. longicaudatus, and eight African and 22 common bermudagrass accessions were resistant to B. longicaudatus, all of which could be used for future cultivar breeding for nematode tolerance or resistance. Accessions that are both resistant and tolerant to B. longicaudatus were identified in both common and African bermudagrass accessions, which would aid in increasing the genetic diversity of future bermudagrass cultivars on golf courses.
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 Wenjing Pang.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Crow, William T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042436:00001


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1 RESPONSES OF BERMUDAGRASS AND SEASHORE PASPALUM TO STING AND SPIRAL NEMATODES By WENJING PANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Wenjing Pang

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3 To my mother and father and who have helped me in my education throughout my lifetime, making this miracle possible

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4 ACKNOWLEDGMENTS I would like to thank my major professor Dr. William T. Crow and supervisory committee members Drs. Kevin E. Kenworthy, Robert McSorley, Robin M. Giblin Davis, and Jason K. Kruse for their support, guidance and advice during my pursuit of a PhD degree. I would also like to thank the U.S.G.A. and F.T.G.A. for their funding to this project. Also, I would like to show my appreciation to Dr. John E. Luc, who has taught me a lot both in work and life. I thank my lab mates including Tom, Jaycee, Nick, Frank and Yun, who have supported m e during my studies. Last but not the least, I would like to acknowledge my parents for their understanding and support of my education throughout my life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ....................... 10 ABSTRACT ................................ ................................ ................................ ................................ ... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 14 2 LITERATURE REVIEW ................................ ................................ ................................ ....... 18 Cynodon spp. ................................ ................................ ................................ .......................... 18 Distribution and Utility ................................ ................................ ................................ .... 18 Taxonomy and Species ................................ ................................ ................................ .... 18 Growth Habit and Establishment ................................ ................................ ..................... 19 Adaptation and Cultivation ................................ ................................ .............................. 19 Cultivars ................................ ................................ ................................ .......................... 20 Paspalum vaginatum ................................ ................................ ................................ .............. 21 Distribution and Utility ................................ ................................ ................................ .... 21 Growth Habit and Establishment ................................ ................................ ..................... 22 Adaptation and Cultivation ................................ ................................ .............................. 22 Cultivars ................................ ................................ ................................ .......................... 24 Belonolaimus longicaudatus ................................ ................................ ................................ ... 24 Taxonomy ................................ ................................ ................................ ........................ 24 Distribution and Hosts ................................ ................................ ................................ ..... 24 Life Cycle and Biology ................................ ................................ ................................ ... 25 Damage and Symptoms ................................ ................................ ................................ ... 25 Management ................................ ................................ ................................ .................... 25 Helicotylenchus spp. ................................ ................................ ................................ ............... 26 Taxonomy ................................ ................................ ................................ ........................ 26 Distribution and Hosts ................................ ................................ ................................ ..... 27 Life Cycle and Biology ................................ ................................ ................................ ... 27 Damage and Symptoms ................................ ................................ ................................ ... 28 Management ................................ ................................ ................................ .................... 28 3 BERMUDAGRASS CULTIVAR RESPONSES TO STING NEMATODES ...................... 29 Introduction ................................ ................................ ................................ ............................. 29 Materials and Methods ................................ ................................ ................................ ........... 31 Plant Materials ................................ ................................ ................................ ................. 31 Inoculum Preparation ................................ ................................ ................................ ...... 32

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6 Ne matode rDNA Analysis ................................ ................................ ............................... 32 Cultivar Nematode Responses ................................ ................................ ......................... 33 Data Analysis ................................ ................................ ................................ ................... 35 R esults ................................ ................................ ................................ ................................ ..... 35 Discussion ................................ ................................ ................................ ............................... 38 4 TOLERANCE AND RESISTANCE OF SEASHORE PASPALUM CULTIVARS TO STING AND SPIRAL NEMATODES ................................ ................................ .................. 46 Introduction ................................ ................................ ................................ ............................. 46 Materials and Methods ................................ ................................ ................................ ........... 47 Plant Materials ................................ ................................ ................................ ................. 47 Inoculum Preparation ................................ ................................ ................................ ...... 48 Nematode Species Identification ................................ ................................ ..................... 48 Greenhouse Studies ................................ ................................ ................................ ......... 48 Data Analysis ................................ ................................ ................................ ................... 50 Results ................................ ................................ ................................ ................................ ..... 51 Responses to Belonolaimus longicaudatus ................................ ................................ ..... 51 Responses to Helicotylenchus pseudorobustus ................................ ............................... 52 Discussion ................................ ................................ ................................ ............................... 52 5 FIELD RESPONSES OF BERMUDAGRASS AND SEASHORE PASPALUM CULTIVARS TO STING AND SPIRAL NEMATODES ................................ ..................... 57 Introduction ................................ ................................ ................................ ............................. 57 Materials and Methods ................................ ................................ ................................ ........... 59 Two year Field Study ................................ ................................ ................................ ...... 59 One year Field Study ................................ ................................ ................................ ....... 61 Statistical Analysis ................................ ................................ ................................ .......... 62 Results ................................ ................................ ................................ ................................ ..... 63 Two year Field Study ................................ ................................ ................................ ...... 63 Bermudagrass cultivars ................................ ................................ ............................ 63 Seashore paspalum cultivars ................................ ................................ .................... 65 One year Field Study ................................ ................................ ................................ ....... 66 Bermudagrass cultivars ................................ ................................ ............................ 66 Seashore paspalum cultivars ................................ ................................ .................... 66 Discussion ................................ ................................ ................................ ............................... 66 6 DNA CONTENT OF BERMUDAGRASS ACCESSIONS IN FLORIDA ........................... 84 Introduction ................................ ................................ ................................ ............................. 84 Materials and Methods ................................ ................................ ................................ ........... 86 Plant Materials ................................ ................................ ................................ ................. 86 Flow Cytometry ................................ ................................ ................................ ............... 86 Results and Discussion ................................ ................................ ................................ ........... 87

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7 7 SCREENING BERMUDAGRASS GERMPLASM ACCESSIONS FOR RE SPONES TO STING NEMATODES ................................ ................................ ................................ ..... 96 Introduction ................................ ................................ ................................ ............................. 96 Materials and Methods ................................ ................................ ................................ ........... 97 Pla nt Materials ................................ ................................ ................................ ................. 97 Inoculum Preparation ................................ ................................ ................................ ...... 98 Nematode Responses of Germplasm Accessions ................................ ............................ 98 Data Analysis ................................ ................................ ................................ ................. 100 Results ................................ ................................ ................................ ................................ ... 100 Discussion ................................ ................................ ................................ ............................. 102 Conclusions ................................ ................................ ................................ ........................... 103 8 SUMMARY ................................ ................................ ................................ .......................... 109 LIST OF REFERENCES ................................ ................................ ................................ ............. 112 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 121

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8 LIST OF TABLES Table page 3 1 Mean total root length of 17 bermudagrass cultivars 90 days after inoculation with Belonolaimus longicaudatus ................................ ................................ ............................. 41 3 2 Mean fine root length of 17 bermudagrass cultivars 90 days after inoculation with Belonolaimus longicaudatus ................................ ................................ ............................. 42 3 3 Mean final population density (P f) and reproductive factor (Rf) of Belonolaimus longicaudatus on 17 bermudagrass cultivars 90. ................................ ............................... 43 4 1 Mean total root length of seven seashore paspalum cultivars 90 days after inoculation with Be lonolaimus longicaudatus ................................ ................................ ..................... 55 4 2 Final mean population density (Pf) and reproduction factor (Rf) of Belonolaimus longicaudatus on seven seashore paspalum cultivars. ................................ ....................... 55 4 3 Mean total root length of seven seashore paspalum cultivars 90 days after inoculation with Helicotylenchus pseudorobustus ................................ ................................ ............... 56 4 4 Mean population density (Pf ) and reproduction factor (Rf) of Helicotylenchus pseudorobustus on seven seashore paspalum cultivars. ................................ .................... 56 5 1 Population density of Belonolaimus longicaudatus on eight bermudagrass cultivars. ...... 71 5 2 Population density of Helicotylenchus pseudorobustus on eight bermudagrass cultivars. ................................ ................................ ................................ ............................. 71 5 3 Total root length of eight bermudagra ss cultivars in nematode infested field plots. ......... 72 5 4 Aboveground percent green cover of eight bermudagrass cultivars in nematode infested field plots. ................................ ................................ ................................ ............. 72 5 5 Population density of Belonolaimus longicaudatus on three seashore paspalum cultivars. ................................ ................................ ................................ ............................. 73 5 6 Population density of Helicotylenchus pseudorobustus on three seashore paspa lum cultivars. ................................ ................................ ................................ ............................. 73 5 7 Total root length of three seashore paspalum cultivars in nematode infested field plots. ................................ ................................ ................................ ................................ ... 73 5 8 Aboveground perc ent green cover of three seashore paspalum cultivars in nematode infested field plots. ................................ ................................ ................................ ............. 74 5 9 Population density of Belonolaimus longicaudatus and Helicotylenchus pseudorobustus on bermudagrass a nd seashore paspalum. ................................ ............... 74

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9 5 10 Total root length and aboveground percent green cover of bermudagrass and seashore paspalum in nematode infested field plots. ................................ ......................... 74 5 11 Aboveground percent green cover of two bermudagrass cultivars in nematode infested field plots. ................................ ................................ ................................ ............. 75 5 12 Nematode population density of Belonolaimus longicaudatus and Helicotylenchus pseudorobustus on two bermudagrass cultivars. ................................ ............................... 75 5 13 Aboveground percent green cover of seven seashore paspalum cultivars in nematode infested field plots. ................................ ................................ ................................ ............. 75 5 14 Nematode population density of Belonolaimus longicaudatus on seven seashore paspalum cultivars. ................................ ................................ ................................ ............ 76 5 15 Nematode population density of Helicotylenc hus pseudorobustus on seven seashore paspalum cultivars. ................................ ................................ ................................ ............ 76 6 1 Nuclear DNA content and ploidy level of 47 University of Florida bermudagrass accessions and three commercial cultivars. ................................ ................................ ....... 91 7 1 Mean total root length of five cultivars and 46 germplasm accessions of bermudagrass 90 days after inoculation with Belonolaimus longicaudatus ................... 105 7 2 Final mean population density (Pf) and reproductive factor (Rf) of Belonolaimus longicaudatus on five cultivars and 46 germplasm accessions of bermudagrass. ........... 107

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10 LIST OF FIGURES Figure page 3 1 Regression relationship between the root volumes of bermudagrass measured by scanning method and graduated cylinder method in test one. ................................ ........... 44 3 2 Regression relationship between the root volumes of bermudagrass measured by scanning method and graduated cylinder method in test two. ................................ ........... 4 5 5 1 Regression relationship between population de nsity of Helicotylenchus pseudorobustus and Belonolaimus longicaudatus bermudagrass. ................................ ................................ ................................ .................... 77 5 2 bermudagrass and population density of Belonolaimus longicaudatus ................................ ................... 78 5 3 he population densities of Belonolaimus longicaudatus ................................ ................................ ............................. 79 5 4 Regression relationship between the total root length of bermudagrass and the population densities of Hel icotylenchus pseudorobustus ............. 80 5 5 bermudagrass and the population density of Helicotylenchus pseudorobustus ................ 81 5 6 Regression relationship between population density of Helicotylenchus pseudorobustus and Belonolaimus longicaudatus ................................ ................................ .......................... 82 6 1 Flow cytometric histogram of diploid, triploid, and tetraploid bermudagrass and trout erythrocyte nuclei.. ................................ ................................ ................................ ............. 92 6 2 Flow cytometric histogram o f diploid and triploid bermudagrass and trout erythrocyte nuclei.. ................................ ................................ ................................ ............. 92 6 3 Flow cytometric histogram of diploid and tetraploid bermudagrass and trout erythrocyte nuclei.. ................................ ................................ ................................ ............. 93 6 4 Flow cytometric histogram of diploid bermudagrass accession and trout erythrocyte nuclei.. ................................ ................................ ................................ ................................ 93 6 5 Flow cytometric histogram of triploid bermudagrass cultivar control and trout erythrocyte nuclei.. ................................ ................................ ................................ ............. 94 6 6 Flow cytometric histogram of tetraploid bermudagrass and trout erythrocyte nuclei.. ..... 94

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11 6 7 Flow cytometric histogram of hexaploid bermudagrass cultivar control and trout erythrocyte nuclei.. ................................ ................................ ................................ ............. 95

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Pa rtial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RESPONSES OF BERMUDAGRASS AND SEASHORE PASPALUM TO STING AND SPIRAL NEMATODES By Wenjing Pang December 2010 Chair: William T. Crow Major: Entomology and Nematology Bermudagra ss ( Cynodon spp.) and seashore paspalum ( Paspalum vaginatum Swartz) are commonly used warm season turfgrasses on golf courses in Florida. Belonolaimus longicaudatus is the most serious nematode pests on turfgrass and Helicotylenchus spp. are often found to have high population densities in seashore paspalum in Florida. Greenhouse and field experiments were conducted to test 17 bermudagrass cultivars and seven seashore paspalum cultivars for their resistance and tolerance to Belonolaimus longicaudatus or Hel icotylenchus pseudorobustus Greenhouse studies showed that no dwarf bermudagrass cultivars were both tolerant and resistant to B. longicaudatus B. longicaudatus Non B. longicaudatus Differences in tolerance among seashore paspalum were found although all cultivars were susceptible to B. longicaudatus but resistant to H. ps eudorobustus B. longicaudatus H. pseudorobustus Field studies indicate that bermudagrass is a better host to B. longicaudatus and seashore p aspalum is a better host to H. pseudorobustus A negative logarithmic relationship was found

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13 between the population densities of B. longicaudatus and H. pseudorobustus in two bermudagrass and three seashore paspalum cultivars. TifSport bermudagrass and Sea Dwarf seashore paspalum were the most resistant to B. longicaudatus in field. Flow cytometry was used to determine the nuclear DNA content and ploidy levels of 47 University of Florida bermudagrass germplasm accessions. Twenty triploid, 24 tetraploid, one pentaploid, and three hexaploid accessions were identified. Most of these, along with 12 diploid African bermudagrass ( Cynodon transvaalensis Burtt Davy) accessions were further screened for nematode responses under greenhouse conditions. Results indicate d that four African and seven common bermudagrass accessions were tolerant to B. longicaudatus and eight African and 22 common bermudagrass accessions were resistant to B. longicaudatus all of which could be used for future cultivar breeding for nematode tolerance or resistance. Accessions that are both resistant and tolerant to B. longicaudatus were identified in both common and African bermudagrass accessions, which would aid in increasing the genetic diversity of future bermudagrass cultivars on golf c ourses.

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14 CHAPTER 1 INTRODUCTION were $4.44 billion in Florida (Haydu and Hodges, 2002). The most widely used warm season turfgrasses on putting greens, tees, and fa irways of golf courses in Florida are bermudagrass ( Cynodon spp.) and seashore paspalum ( Paspalum vaginatum Swartz). Hybrid bermudagrass cultivars ( C. dactylon [L.] Pers. var. dactylon C. transvaalensis Burtt Davy) that produce fine textured and dense tu rf have become the standards for use on golf courses in Florida and other regions where warm season turfgrasses are utilized. As the most salt tolerant warm season turfgrass, seashore paspalum is increasingly used in the southern coastal areas. Based on cu rrent estimates, 87% of Florida golf courses are under the risk of nematode damage (Crow, 2005a). Sting ( Belonolaimus longicaudatus Rau ) and spiral nematodes ( Helicotylenchus spp.) are common nematode pests occurring on turf in Florida. Sting nematodes are frequently found in the sandy coastal soil of the southeastern areas of the United States and are considered as the most damaging plant parasitic nematode on bermudagrass in Florida (Crow, 2005a; Luc et al ., 2007). Sting nematodes are ectoparasites that f eed from the outside of the roots by inserting the stylet into the roots. Previous studies showed that sting nematode could lead to reduced drought tolerance (Trenholm et al ., 2005) and increased potential for nitrate leaching in turf (Luc et al ., 2006). S ting nematodes damage turf roots and their feeding usually causes the root tip to stop growing and results in stunted roots. Aboveground symptoms include wilting, chlorosis, and thinning of the turf, and these symptoms typically occur in irregular patches in the field. Spiral nematodes were found on >84% of golf courses (Crow, 2005a) and 85% of seashore paspalum lawns in Florida (Hixson and Crow, 2004). Population densities of spiral nematodes in

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15 the moderate or high risk categories (more than 500 nematode s/100 cm soil) in seashore paspalum were often found in Florida (Hixson and Crow, 2004). Spiral nematodes are found throughout the southeastern United States on turfgrasses and cause more damage on sandy soils (UOIE, 2000). Helicotylenchus spp. is smaller in size than B. longicaudatus and the body is spiral shaped when dead. Helicotylenchus spp. are ectoparasitic or semi endoparasitic on plant roots depending on the species. The underground symptoms include shortened and discolored roots, with necrotic le sions, and the aboveground symptoms on turf include a delayed spring green up, chlorosis and necrosis of leaf blades, stunting, reduced vigor, and gradual thinning of the turf (UOIE, 2000). Nematode management on golf courses can be challenging. Warm seas on turfgrasses are perennial, so use of crop rotation or cover crops is not available. Recent cancellation of fenamiphos (Nemacur, Bayer CropScience, Research Triangle Park, NC) has resulted in the need for alternative nematode management tactics. Current ly, 1,3 dichloropropene (Curfew Soil Fumigant, Dow AgroSciences, Indianapolis, IN) is the most effective chemical management option for plant parasitic nematodes on turf. However, Curfew provides only short term control, is expensive, and environmental re strictions highlight the need for alternative options. A bionematicide with Pasteuria Alachua, FL) is being used for management of B. longicaudatus on turf, but to date it has not been found to be effective in field trials (Crow, W. T., personal communication). Utilization of resistant or tolerant grass cultivars is the most efficient and least costly practice with minimal ecological effects on non targeted species (Giblin Davis et al ., 1992b). I nformation about the resistance and tolerance to B. longicaudatus of several newer bermudagrass cultivars used on

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16 cultivars used on greens share a common gene tic background. Therefore, there is intense environmental pressure for pest development on greens type bermudagrass cultivars due to a lack of genetic diversity (K. E. Kenworthy, personal communication). This highlights the need to select new sources of ge netically superior bermudagrass. Most bermudagrass cultivars that are widely used on golf courses are triploid bermudagrass ( C. dactylon var. dactylon C. transvaalensis ) resulting from hybridizations between tetraploid, common bermudagrass and diploid, A frican bermudagrass (Burton, 1974). Previous development of hybrids tended to focus on the selection of a superior common bermudagrass parent. This was because it had previously been assumed that there was limited genetic variation among available accessio ns of African bermudagrass. However, Kenworthy et al (2006) provided evidence that variation does exist for many traits in African bermudagrass. This necessitates the screening of both African and common bermudagrass for nematode responses. Not much is kn own about the range of responses of commercial seashore paspalum cultivars to B. longicaudatus or Helicotylenchus spp., but this information is needed by golf course superintendents or consumers to make decisions on which cultivar should be planted in a ne matode infested site. The objectives of this project were: 1) to determine the range of resistance and tolerance of newer commercial bermudagrass cultivars to B. longicaudatus and to compare these to standards mine the range of responses of seashore paspalum cultivars to B. longicaudatus and Helicotylenchus spp., respectively; 3) to determine if an alternative method proposed by Quesenberry and Dunn (1977) for assessing sting nematode damage on bermudagrass root s is as effective, or more efficient, than conventional methods; 4) to conduct field evaluations of bermudagrass and seashore paspalum cultivars to identify cultivars with superior turf characters and nematode responses; 5) to compare the relative

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17 resistan ce and tolerance of seashore paspalum to bermudagrass; 6) to confirm the ploidy level of bermudagrass germplasm accessions with good turf characters in the field so that ploidy levels can be grouped for nematode response screening and breeding; 7) to scree n selected bermudagrass germplasm accessions from the ploidy level tests for their resistance and tolerance to B. longicaudatus

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18 CHAPTER 2 LITERATURE REVIEW Cynodon spp. Distribution and Utility Bermudagrass ( Cynodon spp.) originated in Africa and was in troduced to the United States in 1751 (Hanson, 1972). It is now distributed in over 100 countries throughout the tropical and subtropical areas of the world, and is well adapted to Asia, Africa, Australia, India, South America and the southern region of th e United States (Harlan et al ., 1970). In the United States, bermudagrass is distributed throughout warmer regions: from Florida northward to Maryland and New Jersey along the east coast, and westward along the southern border to California (Harlan et al ., 1970). Bermudagrass is not only a forage grass but also a major turfgrass used on golf courses, sport fields, lawns, and parks. Taxonomy and Species Bermudagrass belongs to kingdom Plantae, subkingdom Tracheobionta, superdivision spermatophyta, division M agnoliophyta, class Liliopsida, subclass Commelinidae, order Cyperales, family Poaceae, subfamily Eragrostoideae, tribe Chloridea, and genus Cynodon (USDA, 2010 ). There are nine species in the genus Cynodon and the basic chromosome number is nine (Harlan a nd de Wet, 1969; Wu et al ., 2006). Tetraploid Cynodon dactylon [L.] Pers. var dactylon is known as common bermudagrass (2n = 4x = 36) and is the most widespread species (de Silva and Snaydon, 1995). Cynodon transvaalensis Burtt Davy (2n = 2x = 18), known a s African bermudagrass, is a diploid species (Forbes and Burton, 1963; Wu et al ., 2006). Triploid bermudagrass ( C. dactylon [L.] Pers. var. dactylon C. transvaalensis Burtt Davy) (2n = 3x = 27) produces fine textured, dense bermudagrass cultivars that ha ve become the standards for use

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19 on golf courses in regions where warm season turfgrasses are utilized. Pentaploid (2n = 5x = 45) and hexaploid (2n = 6x = 54) plants have been previously identified (Johnston, 1975; Hanna et al ., 1990; Burton et al ., 1993; W u et al ., 2006; Kang et al cultivar, has been used on golf courses, athletic fields, and home lawns (Hanna et al ., 1990). Growth Habit and Establishment Bermudagrass is a sod forming grass, which establishes quickly and s preads by stolons, rhizomes and seed. Most commercial cultivars are vegetatively propagated by planting of sprigs, sod, or plugs. Bermudagrass has a perennial root system with vigorous rhizomes. Roots are produced at the nodes of stolons after new leaves o r tillers are produced (Burton and Hanna, 1995). Adaptation and Cultivation Bermudagrass is well adapted to tropical and subtropical climates. It grows best under high temperatures, moderate to high rainfall, and mild winters (Turgeon, 2005). Temperature is the key factor that limits its adaptation in the world. Optimum daytime temperature for bermudagrass is between 35 and 38C and soil temperature for root growth is near 27C (Turgeon, 2005). Bermudagrass prefers moist climates and needs more than 50 c m of rainfall per year (Turgeon, 2005). During a long drought, bermudagrass can go dormant, but will restart growth with sufficient irrigation or rainfall (Trenholm, et al ., 2003). Bermudagrass grows well on a wide variety of soils. All fertilization shoul d be based on a soil test, but Florida soils are high in phosphorous, and typically require little if any phosphorus input (Trenholm, et al ., 2003). It was reported that 489 to 733 g N/100 m 2 per month is usually applied to bermudagrass in the field during the growing season (Trenholm, et al ., 2003). Bermudagrass on lawns is usually mowed at the height of 1.9 to 3.8 cm, but can be maintained at a lower mowing height of 1.3 cm with a high level of management if no more than 1/3 of the

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20 total blade is mowed (T renholm, et al ., 2003). While bermudagrass used on golf courses can be mowed low as 3 mm (Beard, 2002). Higher mowing heights could encourage a deeper root system and reduce weeds and pest problems. Bermudagrass has good drought and salt tolerance, but poo r cold and shade tolerance. It is poorly tolerant to many diseases, insects, and nematode pests (Trenholm, et al ., 2003). Arthropods such as mole crickets ( Scapteriscus spp.) are major pests on bermudagrass (Trenholm, et al ., 2003). However, several genera of nematodes are among the most serious pests of bermudagrass in Florida (Crow, 2005b). Nematodes cause yellowing, wilting, and stunting of growth in turf, and symptoms usually appear as patches in the field. Nematode damage is more serious on sandy soils (Crow, 2005b). Cultivars are non dwarf cultivars usually used on fairways and tees of golf courses. These cultivars are usually maint ained at a height of 0.6 to 2.5 cm (Beard, 2002). Tifway is the most popular bermudagrass cultivar used on golf course fairways and sports fields in the southern United States (Burton, 1966). This cultivar has good disease resistance and a very dark green color compared with other cultivars (Burton, 1966). TifSport is an induced mutant from cold tolerant Midiron and it produces better quality turf than Midiron and has improved cold tolerance compared to Tifway (Bouton et al ., 1997). Patriot has excellent co ld hardiness compared with other hybrid bermudagrasses ( Wu et al ., 2009 ) Midiron and Midlawn are cold tolerant triploid cultivar s with dark color and medium texture that are used on golf course fairways and lawns ( Polomski et al ., 2010) Tifton 10 is a ve getatively propagated hexaploid cultivar released in 1988 It has moderate salt toleran ce and better turf quality than Midiron, and has good winter survival in Ge orgia (Hanna et al ., 1990). Princess 77 and Riviera are improved

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21 seeded common bermudagrass cu ltivars. Princess 77 was released in 1995 with the characteristics of dark green color, good turf quality and density and fine leaf texture (Morris, 2002 ; Rodgers and Baltensperger 2005 ) It is now used on home lawns, parks, athletic fields, and golf cou rses Riviera is a cold tolerant, medium dark green, medium textured, and traffic tolerant cultivar, which forms a traffic tolerant turf similar to Tifway ( Wu et al ., 2009 ). Champion, MiniVerde, TifEagle, and Floradwarf are ultra dwarf cultivars that can tolerate mowing height as low as 3 mm ( Beard, 2002 ). Tifgreen was the first cul tivar released on golf course putting greens in 1956, and its offtype Tifdwarf, with darker green color and smaller leaves was released in 1965 (Burton, 1966). TifEagle, and induced mutant from Tifway II, was released in August 1997. It produces better qua lity turf than Tifdwarf under mowing height of 4 mm or less. TifEagle is vegetatively propagated and produces more stolons and thatch than Tifdwarf (Hanna and Elsner, 1999). Floradwarf is a natural mutant from Tifgreen and released in 1995. It is a dwarf, dense, fine textured cultivar that has greater turf density than Tifdwarf due to its short stolons and internodes (Dudeck and Murduch, 1998). Emerald Dwarf bermudagrass was selected in 1992, and it has greater shoot density and rhizome development than Tif green (CTF, 2010) Its shoot density and leaf morphology are similar to that of Tifdwarf, but forms deeper roots. Emerald Dwarf is usually used on golf course putting greens and high quality tees (CTF, 2010) Paspalum vaginatum Distribution and Utility Sea shore paspalum ( Paspalum vaginatum Swartz) is originally from tropical and subtropical areas of North and South America (Duncan and Carrow, 2000; Morton, 1973). This

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22 grass is widely used for turf in South Africa, Australia and New Zealand. In the United St ates, seashore paspalum is distributed from Florida northward to North Carolina along the east coast, and westward along the southern coast border from Florida to Texas (Duncan and Carrow, 2000; Morton, 1973). This grass is mainly adapted for turf use, and improved cultivars have been developed for golf courses, sports fields, and other landscape uses. Growth Habit and Establishment Similar to bermudagrass, seashore paspalum is also a creeping grass that establishes and spreads by stolons and rhizomes. Most commercial cultivars are vegetatively propagated by planting of sprigs, sod, stolons, or plugs. Seashore paspalum grows vigorously and the leaves expand more rapidly than most bermudagrass cultivars, but it roots less deeply than bermudagrass (Beard et al ., 1991). Adaptation and Cultivation Seashore paspalum has excellent salt tolerance and can grow in soils with salt levels as high as 54 dSm 1 at which most horticultural crops cannot survive ( Lee et al ., 2004 ). This character makes it well adapted to coa sta l areas subjected to salt spray and poor water quality (Duncan and Carrow, 2000) Seashore paspalum grows well in a wide range of soil types, including heavy and poorly drained soils ( (Duncan and Carrow, 2000 ). Compared with bermudagrass, seashore paspa lum is highly tolerant to various environmental stresses. It can maintain acceptable turf quality with less nitrogen fertilizer. It forms a good quality turf in soils ranging in pH from 3.6 to 10.2 and in waterlogged soils ( Duncan, 1999b; Duncan and Carrow 2000 ). Seashore paspalum grows best under warm temperatures and long day length conditions. It is watered on an as needed basis due to its good drought tolerance, and over watering can reduce its stress tolerance and predispose it to diseases ( Duncan and Carrow, 2000 ). Actively growing

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23 seashore paspalum cultivars require 2.5 to 3.8 cm of water per week ( Duncan, 1999b ). To promote root development, deep, infrequent irrigation is usually applied for established seashore paspalum. Watering during the early m orning will reduce the chance of disease development. Optimum annual nitrogen fertilizer rates range from 24 to 39 g/m 2 for golf courses, athletic fields, and landscape areas (Trenholm et al ., 2001). Florida soil is rich in phosphorous, so little or no sup plemental phosphorous is typically needed. An equal amount of potassium and nitrogen is best for seashore paspalum (Trenholm and Unruh, 2002). Seashore paspalum should be mowed below 2.5 cm and reductions in mowing height will produce denser turf. Golf cou rse putting greens are maintained between 3 and 5 mm, while tees and fairways kept between 1.3 and 1.9 cm and athletic fields are maintained between 1.3 and 2.5 cm (Duncan, 1999b; Trenholm and Unruh, 2003) Beard et al (1991) reported that the best mowin g height for seashore paspalum is 1.3 cm, which results in the best turf quality, shoot density, and competitiveness against weeds. Various turfgrass pests can affect seashore paspalum. Insects such as spittlebugs ( Aphrophora saratogensis Fitch) sod webwo rms ( Herpetogramma phaeopteralis Guene ), billbugs ( Sphenophorus venatus vestitus Chittenden ) and mole crickets ( Scapteriscus spp.) are usually observed on seashore paspalum (Duncan and Carrow, 2000; McCarty and Miller, 2002). Susceptibility to dollar spot ( Sclerotinia homoeocarpa Benn ), leaf spot diseases ( Helminthosporium spp., Bipolaris spp., Drechslera spp.) and fairy ring has been observed in seashore paspalum cultivars (McCarty and Miller, 2002) Incidences of fusarium blight ( Fusarium sp.) and take a ll patch ( Gaeumannomyces graminis var. graminis [Sacc.] Arx and D. L. Olivier) in seashore paspalum have been reported in Florida (Trenholm and Unruh, 2003) However, nematodes also have been shown to damage this grass (Hixson et al ., 2004).

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24 Cultivars to 20 years. SeaSpray is the only seeded cultivar. SeaIsle 1 is a fine leaved, de nse growing cultivar used on golf course fairways (Trenholm and Unruh, 2002). SeaIsle 2000 was the first cultivar of seashore paspalum recommended for use on greens. SeaDwarf is a dwarf cultivar for putting greens and fairway on golf courses (McCarty and M iller, 2002). Belonolaimus longicaudatus Taxonomy Sting nematode ( Belonolaimus longicaudatus Rau) belongs to the kingdom Animalia, subkingdom Metazoa, branch Eumetazoa, division Bilateralia, subdivision Protostomia, section Pseudocoelomata, superphylum As chelminthes, phylum Nematoda, class Secernentea, subclass Tylenchia, order Tylenchida, suborder Tylenchina, superfamily Dolichodoroidea, family Belonolaimidae, subfamily Belonolaiminae, genus Belonolaimus species longicaudatus (Siddiqi, 2000). Based on SS U rDNA, De Ley and Blaxter (2004) classified sting nematodes into class Chromadorea, subclass Chromadoria, order Rhabditida, suborder Tylenchina, infraorder Tylenchomorpha, superfamily Tylenchoidea, family Dolichodoridae, subfamily Belonolaiminae, genus Be lonolaimus and species longicaudatus Distribution and Hosts Belonolaimus longicaudatus is not only distributed in the sandy coastal plains of the Atlantic and gulf coasts but also occurs naturally in sandy areas of Kansas and Nebraska in the United State s. Through infested turf sod, B. longicaudatus has been introduced to California and internationally to the Caribbean islands, Puerto Rico, and Bermuda ( Perry and Rhoades, 1982 ). Sting nematodes are found in sandy soils and prefer at least 80% sand content to survive

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25 (Robbins and Barker, 1974). Sting nematodes have a wide host range including grains, turf and forage grasses, vegetables, and fruits. Plants such as beans, cabbage, carrots, millet, oat, rye, strawberry, and tomatoes are often attacked by sting nematodes in Florida (Crow and Han, 2005). Life Cycle and Biology Sting nematodes are large in size and adults are 2 to 3 mm long and 29 to 34 m wide (Rau, 1958; Mai et al ., 1996; Luc, 2004). They are ectoparasites that feed from the outside of the roots by use of a protracted stylet. The reproduction style of sting nematodes is amphimixis. Sting nematodes are bisexual and their life cycle is simple: second stage juveniles hatch from the eggs and feed on the roots and molt through the third and fourth s tage juveniles to become adults. Eggs are laid in pairs and about 128 eggs were laid by one female in 90 days (Huang and Becker, 1999). The total life cycle from egg to egg is about 24 days at 28 C (Huang and Becker, 1999). Damage and Symptoms Sting nemato des damage turf roots and their feeding usually causes the root tip to stop growing. They also affect the roots photosynthesis, and finally affect the plant biomass (Crow and Han, 2005). It was also reported that sting nematodes lead to reduced drought tolerance (Trenholm et al ., 2005) and increased nitrate leaching in turf (Luc et al ., 2006). Aboveground symptoms include wilting, chlorosis, and thinning of the turf, and these symptoms appear as irregular pat ches in the field. High population densities of sting nematodes may cause plant death (Crow and Han, 2005). Management Management of sting nematodes on golf courses is difficult. The nematicide 1, 3 dichloropropene (Curfew Soil Fumigant Dow AgroScience s Indianapolis, IN) is the only

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26 effective and currently available chemical product for nematode management on turfgrass. However, the nematode population densities may rebound following an application because this chemical does not have residual activity (C row, 2010). A bionematicide with the active ingredient Pasteuria sp. which parasitizes and kills B. longicaudatus ( BioScience, Alachua, FL) has been labeled for use on turfgrasses in Florida (Crow, 2010) However, consistent efficacy on sting nematodes in field studies has not been shown (W. T. Crow, unpublished data). Cultural practices could also help reduce the damage of sting nematodes on turf. Frequent and light irrigation and fertilization could keep the damaged grass (Crow, 2010). Maintenance of a healthy root system is essential to improve the nematode tolerance or resistance of turfgrass. Cultural practices such as soil amendments and aeration encourages a health y root system and enhances tolerance to nematodes (Crow, 2010). Little information is available about the efficacy of utilization of resistant or tolerant turfgrass cultivars for B. longicaudatus management. Helicotylenchus spp. Taxonomy Helicotylenchus spp. (spiral nematode) belongs to the kingdom Animalia, subkingdom Metazoa, branch Eumetazoa, division Bilateralia, subdivision Protostomia, section Pseudocoelomata, superphylum Aschelminthes, phylum Nematoda, class Secernentea, subclass Tylenchia, order T ylenchida, suborder Tylenchina, superfamily Tylenchoidea, family Hoplolaimidae, subfamily Hoplolaiminae, genus Helicotylenchus ( Steiner, 1914; Golden, 1956 ). However, based on SSU rDNA, De Ley and Blaxter (2004) recently classified it into class Chromadore a, subclass Chromadoria, order Rhabditida, suborder Tylenchina, infraorder

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27 Tylenchomorpha, superfamily Tylenchoidea, family Hoplolaimidae, subfamily Rotylenchoidinae, and genus Helicotylenchus Distribution and Hosts Spiral nematodes are found throughout t he southeastern United States on turfgrasses but cause more damage on sandy soils They are worldwide in distribution and well adapted to tropical and subtropical climates. Helicotylenchus multicinctus (Cobb, 1893) Golden, 1956 causes a serious decline of bananas; H. dihystera (Cobb, 1893) Sher, 1961 and H. digonicus ( Perry, 1959) have been reported to damage Kentucky bluegrass in Australia ( ; H. pseudorobustus (Steiner, 1914) Golden, 1956 can cause da mage to a lot of crops including corn, grasses, and some vegetables ( Inserra, 1989) Helicotylenchus spp. also can attack cocoa, sugarcane, coffee, corn, tea, turfgrasses, and weeds ( Wouts and Yeates, 1994). Life Cycle and Biology Spiral nema todes are smaller in size than sting nematodes The female and male body lengths range from 0.47 to 0.53 mm and 0.43 to 0.55 mm, respectively. The dead body of this nematode is generally spiral in shape, and this character is very helpful for identificatio n. Their feeding habits are ectoparasitic or semi endoparasitic depending on the species. Helicotylenchus pseudorobustus is a migratory semi endoparasite, whose anterior penetrates into the roots with the posterior remaining outside 1989) Helicotylenchus multicinctus is Ectoparasitic species feed only in the outer cortex of roots but do not migrate through cortex (Wouts and Yeates, 1994). Helic otylenchus multicinctus is bisexual and reproduces by cross fertilization, but for some Helicotylenchus species, males are rare and they reproduce by

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28 parthenogenesis ( ). The life cycle of Helicotylenchus spp. is similar to that of B. longicaudatus but it takes 30 to 45 days to go from egg to egg (Ferris, 2010). Damage and Symptoms There are mainly four species of Helicotylenchus spp. that cause economic damage in plants: H. multicinctus H. pseudorobustus H. digonicus and H. dih ystera ( Inserra, 1989 ) Helicotylenchus dihystera H. digonicus and H. pseudorobustus have been reported to damage turf or pasture in United States, New Zealand, and Australia ( Inserra, 1989; Davis et al ., 2004 ). In Florida, H. p seudorobustus occurs on turf and can cause damage to turfgrass (W. T. Crow, personal communication). The aboveground symptoms of spiral nematode damage on turfgrass include a slow green up in the spring, chlorosis and dieback of the grass blades, stunting, reduction in vigor, and gradual thinning of the turf (UOIE, 2000). However, symptoms may not be obvious in other crops. Parasitism by H. multicinctus can cause shortened and discolored roots with necrotic lesions and dieback, and sometimes can lead to the stress of the entire host plant Management Similar chemical and cultural strategies as used for sting nematode management can be used for spiral nematode management on turfgrass. As one of the most cost effective, as well as environmentally friendly practices, development and utilization of nematode resistant or tolerant turfgrass cultivars is worthwhile to evaluate as a method for managing spiral nematodes.

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29 CHAPTER 3 BERMUDAGRASS CULTIVAR RESPONSES TO STING NEMATODES Intro duction revenues were $4.44 billion in Florida (Haydu and Hodges, 2002). Bermudagrass is the most widely used warm season turfgrass on putting greens, tees, and fairw ays of golf. Crow (2005a) found that 87% golf courses in the state of Florida were at risk for nematode damage, with the most damaging species being the sting nematode ( Belonolaimus longicaudatus Rau ). Research has shown that sting nematodes lead to reduce d drought tolerance (Trenholm et al ., 2005) and increased potential for nitrate leaching in turf (Luc et al ., 2006). Furthermore, their damage can result in premature wilt, chlorosis, and even death of turfgrass when found in combination with other biotic or abiotic stresses (Johnson, 1970; Busey et al ., 1991). Nematode damaged turfgrass requires increased irrigation and fertilizer, which may result in waste of valuable water resources and increased risk of groundwater c ontamination with nitrates (Luc et al ., 200 6 ; Crow, 2005b). In recent years, environmental concerns have focused on the overuse of water, fertilizer, and pesticides (Haydu and Hodges, 2002). The recent cancellation of fenamiphos (Nemacur, Bayer CropScience, Research Triangle Park, NC) has r esulted in the need for new nematode management tactics. Currently, 1,3 dichloropropene (Curfew Soil Fumigant, Dow AgroSciences, Indianapolis, IN) is the only nematicide available for nematode management on turfgrass with established efficacy (Crow, 2010) However, it provides only short term control, is expensive, and alternative options are needed due to environmental restrictions (Crow, 2010). A biopesticide containing Pasteuria sp. ( labeled for sting nematode management on turfgrasses in Florida (Crow, 2010) However, it has not been effective in University of Florida turfgrass field trials (Crow, W. T., personal

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30 communication). Uti lization of resistant or tolerant cultivars could be one of the most environmentally friendly and least costly practices for nematode management on turf. Research has been conducted to test the responses of bermudagrass to sting nematodes. The University of Florida Cooperative Extension Service classifies that moderate and high risk of damage from sting nematode on bermudagrass occurs at 10 and 25 sting nematodes/100 cm 3 soil, respectively (Crow, 2010). Giblin Davis et al (1992b) tested the host status of 37 bermudagrass accessions to sting nematodes and recommended an arbitrary standard of tested, 26 were sensitive and suitable hosts to B. longicaudatus and comme rcial cultivars B. longicaudatus and were sensitive to their infection. Tifway had better tolerance but also supported the reproduction of B. longicaudatus Severe damage to Tifway caused by B. longicaudatus in the field is common (W. T. Crow, personal communication). Johnson (1970) tested the pathogenicity and interaction of Criconemoides ornatus Raski (now Mesocriconema ornatum (Raski, 1952) Loof and De Gr isse, 1989), Tylenchorhynchus martini Fielding (now T. annulatus (Cassidy, 1930) Golden, 1971 ), and Belonolaimus longicaudatus Tifdwar f ). He found that the number of f ibrous roots decreased as the number of nematode species and total number of nematodes increased. All bermudagrass cultivars supported high population densities of the three nematode species but B. longicaudatus was a better competitor than C. ornatus and T. martini since population densities of C. ornatus and T. martini were suppressed more by B. longicaudatus than B. longicaudatus was by the other nematode species. Good et al (1965) also reported that coastal bermudagrass supported the

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31 reproduction of B. longicaudatus However, other studies showed that the forage bermudagrass Nematologists have used root growth parameters to assess nematode damage in plant roots. Quesenberry and Dunn (1977) used a graduated cylinder method to measure root volume to evaluate nematode damage in plant roots. With the development of technology in recent years, WinRhizo root scanning equipment and software (Regent Instruments Inc., Ottawa, Canada) has been develop ed and used to measure root length, diameter, volume, surface area, root tips and crossings, etc. (Bauhus and Messier, 1999) However, the efficiency of the graduated cylinder and root scanning methods have not been compared, and is warranted to identify t he method most efficient for screening a large number of diverse genotypes. Although a range in responses of bermudagrass to sting nematodes has been studied, information about the responses of several more recently released cultivars is lacking. One objec tive of this research was to evaluate the relative resistance or tolerance of commercial bermudagrass cultivars to B. longicaudatus to help growers and turfgrass managers with cultivar selections where B. longicaudatus is present. Another objective was to determine if a proposed alternative method used by Quesenberry and Dunn (1977) for assessing sting nematode damage on bermudagrass roots is as effective as or more efficient than the WinRhizo root scanning method. Materials and Methods Plant Materials Eigh t dwarf and nine non dwarf bermudagrass cultivars were tested in two sequential experimental trials in 2009 in a glasshouse at the University of Florida Turfgrass Envirotron in Gainesville, FL. The dwarf cultivars were dwarf cultivars included

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32 Tifway Inoculum Preparation Belonolaimus longica udatus ( Stenotaphrum secundatum Kuntze ) grown in clay pots filled with United States Golf Association (USGA) specification putting green sand under greenhouse conditions (Giblin Davis et al ., 1992a; USGA, 1993) sieving technique (Cobb, 1918; Flegg, 1967) All stages of nematodes including juveniles and adults were collected. Nematode suspensions were concentrated using a 25 m (500 mesh) sieve. The a verage number of juveniles and adults were counted from five replicates of 1 ml aliquots and results were extrapolated to the total volume of the suspension. Suspensions were stored in a refrigerator until needed. Nematode rDNA Analysis Molecular analysis was conducted to aid in sting nematode species identification. Isohair extraction kit (Nippon Gene Co. LTD., Toyama, Japan) was used to extract DNA from individual females. Ribosomal DNA of the ITS (Internal Transcribed Spacer) was amplified by PCR using t TTG ATT ACG TCC CTG CCC TTT TTT CAC TCG CCG TTA CTA AGG Master Mix (Promega Corp. Labo ratories, Inc., Hercules, CA), with the cycling sequence as: one cycle of 94C for 7 min, 35 cycles of 94C for 1 min, 55C for 1 min, 72C for 1 min, and 72C for 10 min. Montage PCR

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33 centrifugal filter kit (Millipore Corp., Billerica, MA) was used to pur ify the PCR products. All products were sequenced on Perkin Elmer/Applied Biosystems automated DNA sequencers at the University of Florida ICBR sequencing core facility, and the same primers were used for sequencing as for PCR amplification. Sequences were edited using Sequencher (4.1.2 Gene Codes Corporation). Using the default parameters of Clustal X 1.83 (Thompson et al. 1997), the sequences gained in this study were aligned to each other and the outgroup taxon Pratylenchus coffeae (Zimmermann) Filipjev & Schuurmans Stekhoven (LSU GenBank accession #AF170443), and the alignments were adjusted manually in MacClade 4.0 (Maddison and Maddison, 2000). Cultivar Nematode Responses Nematode free aerial stolons of each cultivar were vegetatively propagated int o 3.8 cm diameter 21 cm deep (volume = 150 cm 3 ) UV Stuewe & Sons, Inc., Tangent, OR) filled with 100% USGA specification greens sand The bottom of the conetainers was filled with Poly fil (Fairfield Processin g Corporation, Danbury, CT) to prevent sand from escaping from the drainage holes. Two pieces of terminal aerial stolons with one node each were planted into each conetainer. Two minutes of overhead irrigation mist was applied six times daily for two weeks to allow the sprigs to establish roots. Beginning at the third week, the irrigation was reduced to once a day in the morning for six minutes, and three minutes a day from the fifth week. Six weeks after establishment, cultivars were inoculated with 0 or 5 0 B. longicaudatus per conetainer. Before inoculation, suspensions of B. longicaudatus were taken out of the refrigerator, concentrated to 10 nematodes/ml, and set at room temperature for three hours. A total of 5 ml of the suspension was divided into two 3 cm deep holes made 1 cm from the center of the pot in the inoculated treatments, and the uninoculated controls receive no solution. The holes were covered with a light layer of sand, and moistened with a light mist.

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34 The experiment was arranged as a rando mized complete block design with six replications. To provide insulation from temperature extremes, conetainers were placed in (60 35 15 cm) (Stuewe & Sons, Inc., Tangent, OR). The experiments were conducted under a temperat ure range of 24 to 34 o C with natural daylight in a greenhouse at the University of Florida in Gainesville, FL. Treatments were fertilized once a week using 24 8 16 (N P 2 O 5 K 2 O) at a rate of 0.5 kg N/100 m 2 per growing month and clipped once a week at 2.5 c m. Bermudagrass mites ( Eriophyes cynodoniensis Sayed) and rhodesgrass mealybugs ( Antonina graminis Maskell) were monitored and sprayed with bifenthrin (Bifen I/I Insecticide/Termiticide, Control Solutions, Inc., Pasadena, TX) when needed, and all conetaine rs were treated equally. Experiments were harvested 90 days after the inoculation of nematodes. Root and nematode samples were collected from each conetainer. Roots were collected by removing the shoots and Poly fil. The roots were washed free of soil on an 853 ml plastic tube, and submerged with water. Finer roots were separated from soil and collected into the plastic tube by submerging and shaking the 853 using WinRhizo root scanning equi pment and software (Regent Instruments Inc., Ottawa, diameter), length of all roots, and root volume were measured from the scanned images. After scanning, roots in each sample were collected and dried by rolling the m into a paper towel. For the graduated cylinder method the roots were then submerged into a 10 ml graduated cylinder prefilled with 5 ml water. The volumes of the water in the cylinder were recorded before and after submerging the roots into the cylinder. The volume of roots was calculated as follows: root volume = volume of the water after submerging the roots into the cylinder volume of the water before submerging the roots. Percent reduction in the root length or volume of inoculated plants compared w ith the non inoculated control plants

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35 was calculated by the following formula: (root measurement of non inoculated control root measurement of inoculated plant) / root measurement of non inoculated control 100. Nematodes were extracted from the tota l soil in the conetainer using the modified centrifugal flotation technique (Jenkins, 1964). The final nematode population densities (Pf) were counted under an inverted light microscope. The reproductive factor (Rf), which is an indicator of resistance (Oo stenbrink, 1966 ) was calculated by the following formula: Rf = final nematode density (Pf) / initial inoculum density (Pi). Data Analysis Data of the two trials were analyzed separately. Total root lengths and fine root lengths of the inoculated treatmen t and the non inoculated control of each cultivar were compared by a linear contrast at P < 0.1. Tolerance was determined by the difference in root length between the two treatments. A cultivar was considered tolerant if there was no difference ( P < 0.1) i n total root length between the two treatments; otherwise, the cultivar was classified as intolerant. Resistance was determined by the nematode reproductive factor (Rf) at harvest. A cultivar was designated as resistant if Rf < 1 and susceptible if Rf > 1. Final nematode population densities were subjected to analysis of variance (ANOVA), and the differences among cultivars were compared by the Fishers protected least significant difference test at P < 0.05. The root volumes were measured digitally using r oot scans and by water displacement (graduated cylinder). Regression analysis was conducted to compare the two methods. All statistical analyses were conducted using SAS programs (SAS Institute, Cary, NC). Results The sting nematode species used in this st udy was identified as B. longicaudatus by the rDNA analysis. Differences in responses to sting nematodes were observed among the cultivars

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36 evaluated (Table 3 1). A significant interaction was found between the experimental trials and turfgrass cultivars, s o the root lengths of each trial were analyzed separately. The total root length reductions for individual cultivars ranged from 1 to 40% and 3 to 46% in trials one and two, respectively. Linear contrasts showed that dwarf cultivars Champion, MiniVerde, Ti fEagle, Jones Dwarf, Floradwarf, and Tifgreen had significant reductions in total root length from B. longicaudatus compared with the non inoculated controls in both trials. The non dwarf cultivars, Princess 77, Midlawn, Celebration, and Midiron had signif icant reductions in the total root length in both trials. However, Tifton10 and Tifway showed significant reductions in root length from B. longicaudatus in trial two, but not in trial one. Based on both tests, the dwarf cultivars Tifdwarf and Emerald Dwar f did not suffer significant reductions in root length, therefore, they were considered tolerant to B. longicaudatus Among the non dwarf cultivars, TifSport, Patriot, and Riviera were consistently tolerant to B. longicaudatus in both trials. The fine root lengths of the cultivars were also compared and similar damage of B. longicaudatus as in the total root length was found. Differences in the fine root lengths between the inoculated and uninoculated treatments were identified (Table 3 2). The linear contr asts indicated that Champion, MiniVerde, TifEagle, Jones Dwarf, Floradwarf, and Tifgreen had significant reductions in fine root length from B. longicaudatus Treatment differences in fine root lengths also were detected among the non dwarf cultivars (Tabl e 3 2). The reductions were in the range of 1 to 29% and 5 to 37%, respectively for the two trials. We found that Midlawn, Celebration, and Midiron had significant reductions in the fine root length in both trials. Tifton 10, Tifway, and Riviera suffered s ignificant reductions in fine root length from B. longicaudatus only in trial two.

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37 Population densities of B. longicaudatus increased on some bermudagrass cultivars and decreased on others. Final nematode population densities per conetainer of B. longicau datus were in the range of 22 to 343 for trial one and 20 to 220 for trial two (Table 3 3). In trial one, compared to the initial inoculum density of 50 nematodes/conetainer, the population densities of B. longicaudatus increased on Jones Dwarf, Tifdwarf, and Emerald Dwarf with the corresponding Rf values of 2.5, 4.8, and 4.7, respectively. The most susceptible cultivar was the non dwarf cultivar Princess 77 with a 6.9 fold increase in the population density. In trial two, more cultivars showed susceptibili ty. MiniVerde, Jones Dwarf, Tifgreen, Tifdwarf, Emerald Dwarf, Princess 77, Celebration, and Patriot all supported the reproduction of B. longicaudatus As in trial one, the highest Rf of 4.4 was on Princess 77, and this cultivar served as a very good host to B. longicaudatus Some moderate hosts with Rf values close to one were identified, for example Tifway and TifSport. Overall, the dwarf cultivars Tifdwarf and Emerald Dwarf were tolerant but susceptible to B. longicaudatus Champion, TifEagle and Florad warf were resistant but intolerant to B. longicaudatus No dwarf cultivars were both tolerant and resistant to B. longicaudatus Non dwarf cultivars TifSport, Patriot and Riviera were tolerant to B. longicaudatus however, only TifSport and Riviera showed resistance. Most non dwarf cultivars did not support the reproduction of B. longicaudatus for example, Midlawn, Midiron, TifSport, Tifton 10, Tifway, and Riviera. The root volume of each sample was compared by the root scanning method and the graduated c ylinder method. Results from both methods were similar and produced a highly significant linear regression relationship between the two methods in trial one (r 2 = 0.9968, P < 0.0001) (Figure 3 1) and trial two (r 2 = 0.9988, P < 0.0001) (Figure 3 2). The resu lts provided by

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38 the two methods were extremely consistent with similar lines and slopes near 1.0 (Figure 3 1, Figure 3 2), which indicated that both methods can provide consistent root volume measurements. Discussion Although both methods mentioned above m ight give easy measurements of root volume, for roots with very small volume (less than 1 cm 3 ), the estimated error brought by reading the graduated cylinder (the minimum unit is 0.1 cm 3 ) could make the measurements less accurate than the root scanning met hod. Moreover, it took about the same amount of time using either method to do the root measurement. The graduated cylinder method can only measure the total volume of a sample, but the root scanning method also can measure root length, root surface area, and root volume in different diameter ranges. Additionally, the root scanning method also can measure the number of root tips and crossings. Therefore, the root scanning method provided more details in root measurement than the graduated cylinder method i n the same amount of time. However, the root scanning method is initially more expensive since special software and equipment are needed. Therefore, the root scanning method is preferred for root measurement when the facilities are available and the gradua ted cylinder method is an economical method to gain limited information, if root scanning equipment and software are not available. Among the dwarf cultivars, those with shorter roots might support a lower nematode population density. The average total ro ot lengths of the inoculated treatment in two trials were as low as 938, 1076, and 1094 cm, respectively for Champion, TifEagle, and Floradwarf; while in these cultivars the final nematode population densities fell to half of the initial inoculum level. On the other hand, cultivars Jones Dwarf, Tifgreen, Tifdwarf, and Emerald Dwarf all supported higher reproduction of B. longicaudatus and had root lengths of 1474, 1969, 2241, and 1959 cm,

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39 respectively. The greater amount of roots probably provided more food source and feeding sites, which could support more nematodes. The most susceptible cultivar Princess 77 with an average Rf of 5.7 in two trials also had the largest root length of 2226 cm for the inoculated treatment. rather an indicator of differences in carrying capacity among cultivars. The carrying capacity is the population level at which the nematodes decrease root production to the extent that they limit nematode reproduction. The damage function model for B. longicaudatus on cotton was determined to be quadratic, where high inoculation densities resulted in fewer nematodes due to the reduction in feeding sites (Crow et al ., 2000). The inoculation density used in the current experiment (50 B. longicaudatus /conetainer) may have resulted in population levels that were too high, limiting root production and further nematode reproduction. Cultivars defined as resistant might be defined susceptible if a lower i noculation density was used. Further research should use multiple inoculation densities to verify this hypothesis. The average percent reduction of root length in TifEagle was 32%, which was similar to the previously reported results (Schwartz et al ., 2010 a). Another previous study (Giblin Davis et al ., 1992b) found that Tifgreen, Tifdwarf, Midiron, and Tifway were susceptible cultivars to B. longicaudatus under controlled conditions at 140 days after inoculation. In this study Tifgreen and Tifdwarf were ob served to support the reproduction of B. longicaudatus 90 days after inoculation. Giblin Davis et al (1992b) found that Tifway was relatively more tolerant than other bermudagrass cultivars listed above, with a mean root dry weight reduction of 4%. Result s of this study indicated an 8% reduction in total root length in Tifway in the first trial and a 24% reduction in the second trial.

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40 Tifdwarf is the most widely used bermudagrass cultivars in golf course putting greens and Tifway is the most widely used on fairways. Tifdwarf and Tifgreen were very suitable hosts to B. longicaudatus and Tifgreen had significant reductions in root length when inoculated with sting nematodes. These results suggest that where B. longicaudatus is present, Tifdwarf and Emerald Dw arf might be good cultivar choices for putting greens because they were more tolerant of nematode damage. Similarly, TifSport and Riviera are non dwarf cultivars that were both resistant and tolerant to B. longicaudatus suggesting that they might be good cultivar selections for B. longicaudatus infested tees and fairways. Future studies will be needed to verify results under field conditions on golf courses and with different geographical and host isolates of B. longicaudatus The results of our study are preliminary but the screening methods and some of these resistant or tolerant cultivars can be used as standards for future turfgrass cultivar and germplasm screening.

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41 Table 3 1. Mean total root length of 17 bermudagrass cultivars 90 da ys after inoculation with Belonolaimus longicaudatus in two experiment trials. U = uninoculated and I = inoculated with 50 B. longicaudatus /conetainer. Total root length (cm) Trial 1 Trial 2 Cultivar U I % reduction U I % reduction Dwarf cultivars C hampion 1373* 810 c ab 40 a 1524 # 1066 de 31 ab MiniVerde 2219* 1413 b 35 ab 1995* 1096 de 34 ab TifEagle 2017* 1320 b 26 abc 1509* 832 e 37 ab Jones Dwarf 2258 # 1557 b 21 abc 1976 # 1391 cd 25 ab Floradwarf 1871 # 1365 b 24 abc 1972* 822 e 46 a Ti fgreen 2178 # 1635 b 20 abc 2650* 2302 a 13 b Tifdwarf 2876 2527 a 10 bc 2454 1954 ab 21 ab Emerald Dwarf 2387 2173 a 5 c 2089 1744 bc 16 ab Mean 2147 1600 23 2021 1401 28 Non dwarf cultivars Princess 77 2637 # 2139 a 15 2568 # 2312 a 10 c Midlawn 20 06* 1252 de 28 1551* 1201 c 20 Celebration 2786* 1992 ab 28 2267 # 1908 b 14 Midiron 1930* 1452 de 22 1882* 1263 c 26 TifSport 2173 1931 abc 7 2204 1952 ab 3 Tifton 10 1272 1189 e 1 1938* 1195 c 36 Tifway 1622 1548 cde 8 1764 # 1332 c 24 Patrio t 1379 1322 de 12 2265 2093 ab 4 Riviera 1683 1625 bcd 7 1604 1219 c 18 Mean 1943 1606 14 2005 1608 17 # *Uninoculated treatments significantly different from inoculated treatments at P < 0.1, and P < 0.05, respectively, according to the linear con trast analysis. a Data are means of six replications. b For each cultivar type (dwarf, non dwarf) means within a column followed by the same letter or by no letter are not different ( P < 0.05), by Fishers protected least significant difference test.

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42 Table 3 2. Mean fine root length of 17 bermudagrass cultivars 90 days after inoculation with Belonolaimus longicaudatus in two experiment trials. U = uninoculated and I = inoculated with 50 B. longicaudatus /conetainer. Fine root length (cm) Trial 1 T rial 2 Cultivar U I % reduction U I % reduction Dwarf cultivars Champion 1273* 750 c ab 40 a 1335 # 944 cd 30 ab MiniVerde 2001* 1262 b 35 a 1736* 994 cd 30 ab TifEagle 1819* 1194 b 26 ab 1328* 765 d 35 ab Jones Dwarf 1910* 1301 b 24 ab 1643* 1181 bc 23 ab Floradwarf 1698* 1252 b 24 abc 1694* 757 d 43 a Tifgreen 1944 # 1463 b 19 abc 2051 1807 a 10 b Tifdwarf 2215 1937 a 10 bc 2052 1640 a 20 ab Emerald Dwarf 1977 1842 a 2 c 1769 1484 ab 16 ab Mean 1855 1375 23 1701 1197 26 Non dwarf cu ltivars Princess 77 2093 1764 a 10 1984 1899 a 6 Midlawn 1768* 1104 cd 29 1383 # 1064 c 20 Celebration 2328* 1646 ab 28 1822 # 1556 b 12 Midiron 1706* 1267 cd 23 1585* 1112 c 25 TifSport 1870 1633 ab 8 1634 1591 b 13 Tifton 10 1113 1039 d 1 1721* 1058 c 37 Tifway 1449 1309 cd 13 1534* 1135 c 25 Patriot 1280 1204 cd 14 1767 1652 ab 5 Riviera 1543 1411 bc 3 1437 # 1025 c 23 Mean 1683 1375 14 1652 1344 18 # *Uninoculated treatments significantly different from inoculated treatments at P < 0. 1, and P < 0.05, respectively, according to the linear contrast analysis. a Data are means of six replications. b For each cultivar type (dwarf, non dwarf) means within a column followed by the same letter or by no letter are not different ( P < 0.05), by Fis hers protected least significant difference test.

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43 Table 3 3. Mean final population density (Pf) and reproductive factor (Rf) of Belonolaimus longicaudatus on 17 bermudagrass cultivars 90 days after inoculation with 50 B. longicaudatus /conetainer in two experimental trials. Final population density (Pf) a Reproductive factor (Rf) Cultivar Trial 1 Trial 2 Trial 1 Trial 2 Dwarf cultivars Champion 22 c bc 23 c 0.4 c a 0.5 c MiniVerde 49 c 56 bc 1.0 c 1.1 bc TifEagle 25 c 49 bc 0.5 c 1 .0 bc Jones Dwarf 124 b 170 a 2.5 b 3.4 a Floradwarf 31 c 32 c 0.6 c 0.6 c Tifgreen 43 c 176 a 0.9 c 3.5 a Tifdwarf 240 a 192 a 4.8 a 3.8 a Emerald Dwarf 237 a 89 b 4.7 a 1.8 b Mean 96 99 1.9 2.0 Non dwarf cultivars Princess 77 343 a 2 20 a 6.9 a 4.4 a Midlawn 44 b 47 c 0.9 b 0.9 c Celebration 47 b 130 b 0.9 b 2.6 b Midiron 37 b 50 c 0.7 b 1.0 c TifSport 43 b 52 c 0.9 b 1.0 c Tifton 10 29 b 29 c 0.6 b 0.6 c Tifway 50 b 44 c 1.0 b 0.9 c Patriot 39 b 135 b 0. 8 b 2.7 b Riviera 40 b 35 c 0.8 b 0.7 c Mean 75 82 1.5 1.6 a Numbers represent numbers of nematodes recovered from the whole conetainer. b Data are means of six replications. c For each cultivar type (dwarf, non dwarf) means within a column followe d by the same letter are not different ( P < 0.05), by Fishers protected least significant difference test.

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44 Figure 3 1. Regression relationship between the root volumes of bermudagrass measured by scanning method and graduated cylinder method in test one.

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45 Figure 3 2. Regression relationship between the root volumes of bermudagrass measured by scanning method and graduated cylinder method in test two.

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46 CHAPTER 4 TOLERANCE AND RESISTANCE OF SEASHORE PASPALUM CULTIVARS TO STING AND SPIRAL NEMA TODES Introduction Seashore paspalum ( Paspalum vaginatum Swartz) is a widely used warm season turfgrass on golf courses. It is well adapted to the sandy soil and humid climate of Florida. Compared with bermudagrass ( Cynodon spp.), seashore paspalum is high ly tolerant of different environmental stresses. It can form a high quality turf in waterlogged soils, high salinity soils, and lower fertility soils (Duncan and Carrow, 2000). Several commercial cultivars have recently been developed and released for golf course fairways and putting greens. A survey of golf courses in Florida found that 87% of Florida golf courses were under risk of nematode damage (Crow, 2005a), two of the common nematode genera reported in that survey were Belonolaimus spp. and Helicotyl enchus spp Belonolaimus longicaudatus Rau is frequently found in the sandy coastal soil of the southeastern areas of the United States, and was reported as the most damaging plant parasitic nematode on turfgrasses (Crow, 2005a; Luc et al ., 2007). Belonol aimus longicaudatus was reported on 50% of seashore paspalum golf courses and 40% of seashore paspalum lawns damaged by B. longicaudatus in greenhouse trials, but repro duction of the nematode on seashore paspalum was less than on bermudagrass (Hixson et al ., 2004). Helicotylenchus spp. are found throughout the southeastern United States on turfgrasses but cause more damage on sandy soils U nlike B. longicaudatus Helicotylenchus spp. are generally considered only minor pests on most turfgrasses, despite being commonly associated with them. Hixson and Crow (2004) reported that Helicotylenchus spp. were found on 88% of seashore paspalum golf c ourses and 85% of seashore paspalum home

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47 lawns in Florida. Similarly, Crow (2005a) reported that Helicotylenchus spp. were found on over 85% of bermudagrass golf courses. However, high population densities (more than 500 Helicotylenchus spp./100 cm soil) were common on seashore paspalum (Hixson and Crow, 2004), but high numbers were rare on bermudagrass (Crow, 2005a) in Florida. It has been hypothesized that seashore paspalum might be more susceptible to Helicotylenchus spp. than is bermudagrass (W. T. Cro w, personal communication). Helicotylenchus pseudorobustus (Steiner, 1914) Golden, 1956 is the most commonly encountered spiral nematode on turfgrasses in Florida (W. T. Crow, personal communication). Helicotylenchus pseudorobustus is a semi endoparasitic nematode and its feeding causes shortened and discolored roots with necrotic lesions and Inserra, 1989) The aboveground symptoms of H. pseudorobustus damage on turf include a slow green up in the spring, chlorosis and dieback of the leaf blades, stunting, reduction in vigor, and gradual thinning of the turf (UOIE, 2000). Seashore paspalum is primarily used in coastal areas ( Duncan and Carrow, 2000 ). However, improved cultivars are a recent development, and information about their resistance and t olerance to B. longicaudatus or H. pseudorobustus is limited. Most commercial cultivars have not been tested for nematode responses. Therefore, the objectives of this study were to evaluate the tolerance and resistance of commonly used seashore paspalum cu ltivars in Florida to B. longicaudatus and H. pseudorobustus respectively. Materials and Methods Plant Materials Seven commercial cultivars of Paspalum vaginatum ( were tested in two sequential experimental trials in 2009.

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48 Inoculum Preparation Helicotylenchus pseudorobustus was maintained on three seashore paspalum cultivars Aloha, SeaDwarf, and SeaIsle 1, and B. longicaudatus was maintained on Augusti negrass ( Stenotaphrum secundatum Kuntze ) grown in clay pots filled with pure sand sieving technique (Cobb, 1918; Flegg, 1967) All stages of nematodes including the juveniles and adults were collected. The nematode suspensions were concentrated using a 25 m sieve. The average number of juveniles and adults from five 1 ml aliquots were extrapolated to the total volume of the suspension. Suspensions were kept in a refr igerator until used. Nematode Species Identification Morphometric and molecular analysis were conducted to identify Helicotylenchus to species used in this study Twenty five H. pseudorobustus females were randomly hand picked and measured at 400 magnifi cation under a compound light microscope with the aid of a drawing tube. The key and description of Helicotylenchus (Sher, 1966) were used to match with the measurements of the nematode body characters in this study for species identification. The methods for molecular analysis were the same as that of Belonolaimus as described in Chapter 3. Specimens of Helicotylenchus were also sent to Division of Plant Industry Florida, Department of Agriculture and Consumer Services, Gainesville, FL for further identifi cation confirmation. Greenhouse Studies Nematode free aerial stolons of each cultivar were vegetatively propagated into (3.8 cm diameter 21 cm deep, volume = 150 cm 3 ) Stuewe & Sons, Inc., Tangent, OR) filled with 100% USGA specification greens sand The bottom of conetainers were lined with Poly fil (Fairfield Processing Corporation, Danbury, CT) to prevent sand from escaping through the drainage holes. Two pieces of terminal aerial stolons

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49 with one node each were planted into each conetainer. Two minutes of overhead irrigation mist was applied six times daily for two weeks to allow the sprigs to establish. From the third week, the irrigation was reduced to once a day in the morning for six minutes, and three minutes a day at the beginning of the fifth week. Six weeks after establishment, the grass was inoculated with no nematodes, 50 B. longicaudatus or 500 H. pseudorobustus per conetainer. Before inoculation, nematode suspensions were taken out of the refrige rator, concentrated to 10 or 25 nematodes/ml, respectively for B. longicaudatus and H. pseudorobustus which were then set at room temperature for three hours. Five milliliter of B. longicaudatus suspension or 20 ml of the H. pseudorobustus suspension was evenly distributed into three 3 cm deep holes made 1 cm from the surface center of the conetainer for the treatment inoculated with B. longicaudatus or with H. pseudorobustus respectively. The holes were covered with a light layer of sand, and moistened with a light mist. No holes were made or solutions were inoculated to the uninoculated controls. The experiment was arranged as a randomized complete block design with five replications for each cultivar. To provide insulation, conetainers were placed in ( 60 35 15 cm) Beaver (Stuewe & Sons, Inc., Tangent, OR). The experiments were set under a temperature range of 24 to 34 o C with natural daylight in an Envirotron greenhouse at University of Florida, Gainesville, FL. Grasses were fert ilized once a week using 24 8 16 (N P 2 O 5 K 2 O) at a rate of 0.5 kg N/100 m 2 per growing month. Turfgrass was clipped once a week at the mowing height of 2.5 cm. Experiments were harvested 90 days after the inoculation of nematodes. Root and soil samples we re collected from each conetainer. Roots were collected by removing the shoots and Poly fil from each conetainer. Roots were then washed free of soil on an 853 (20 mesh) sieve and placed into a 50 ml plastic tube submerged with water. Finer roots were separated from

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50 soil and collected into the plastic tube by submerging and shaking the 853 Ro ots were scanned into the computer and root lengths measured using WinRhizo root scanning equipment and software (Regent Instruments Inc., Ottawa, Canada). The percent root length reduction that indicates the difference between the nematode inoculated and non inoculated treatments was calculated by the following formula: root length % reduction = (root length of non inoculated control root length of inoculated plant) / root length of non inoculated control 100. Nematodes were extracted from the total soil in the conetainer by using the modified centrifugal flotation technique (Jenkins, 1964). The final nematode population densities (Pf) were counted under a microscope. The reproductive factor (Rf), which is an indicator of resistance (Oostenbrink, 196 6 ), was calculated by the following formula: Rf = final nematode density (Pf) / initial inoculum density (Pi). Data Analysis Conetainers inoculated with B. longicaudatus or with H. pseudorobustus were compared to a common set of uninoculated control conet ainers. The two sets of nematode data were not directly comparable to each other because the inoculation densities were different for each nematode. Therefore, the data were analyzed as two separate experiments, a B. longicaudatus experiment with seven cul tivars and two treatments, and a H. pseudorobustus experiment with seven cultivars and two treatments. Total root lengths of the inoculated treatment and the non inoculated control of each cultivar were compared by a linear single degree of freedom contras t at P < 0.10. Tolerance was determined by the difference in root length between the two treatments. A turfgrass cultivar was considered tolerant if there was no difference in total root length between the two treatments; otherwise, the cultivar was intole rant. The root lengths of

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51 inoculated treatments were subjected to analysis of variance (ANOVA), and when significant, the differences among cultivars were compared by the Fishers protected least significant difference test at P < 0.05. Resistance was deter mined by the nematode reproductive factor (Rf) at harvest. A cultivar was considered resistant if Rf < 1 and susceptible if Rf > 1. Final nematode population densities were subjected to analysis of variance (ANOVA), and the differences among cultivars were compared by the Fishers protected least significant difference test at P < 0.05. Statistical analysis was conducted by using the SAS program (SAS Institute, Cary, NC). Results Responses to Belonolaimus longicaudatus Interactions between trials and cultiva rs were found, so results of the two trials were presented separately. The root lengths of four seashore paspalum cultivars were reduced by B. longicaudatus in at least one trial (Table 4 1). No differences were found for the inoculated root lengths or for the percent reductions between the cultivars in both trials ( Table 4 1) Root reductions were less in trial two than in trial one. In trial one, four cultivars (SeaSpray, SeaIsle 1, Aloha, and SeaIsle 2000) had significant reductions in root length ( P < 0 .10), while only SeaSpray had significant damage from B. longicaudatus in trial two. Consistent for both trials, SeaIsle Supreme exhibited the least reduction in root length (5% average ), while SeaSpray had the greatest reduction (24% average). Aloha, Sala m, SeaDwarf, and SeaIsle Supreme had variable responses. Nematode population densities at harvest increased for all cultivars during both trials (Table 4 2). The final nematode population densities of B. longicaudatus were higher in trial one than trial tw o. All cultivars were excellent hosts, since the lowest Rf value obtained for both trials was 2.7. The highest population densities were for SeaIsle 2000 with a Rf of 9.5 and 5.6,

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52 respectively in trials one and trial two. Aloha had the lowest nematode Rf v alues, 2.7 and 2.8 for the two trials. Responses to Helicotylenchus pseudorobustus M orphometric analysis indicated that the spiral nematode species in this study was H. pseudorobustus However, molecular sequences obtained were not consistent with those fo r H. pseudorobustus in Genbank. There fore, the nematode in question will be concidered H. pseudorobustus sensu lato. Significant interactions between trials and cultivars also were found for H. pseudorobustus data, so the data for each trial was analyzed s eparately Root lengths were reduced only in trial one for Aloha and SeaDwarf; however, both trials resulted in significant root reductions for Salam, SeaIsle I and SeaIsle Supreme (Table 4 3). Similar to results with B. longicaudatus less reduction in roo t length was observed in trial two than in trial one. Differences among cultivars in root length of the inoculated treatment were found in trial two. The root length of the inoculated treatment was the highest for SeaIsle 2000, which was considered toleran t to H. pseudorobustus infection. Root length reductions were greatest for SeaIsle 1 and SeaIsle Supreme. In trial two, the root length of SeaIsle Supreme was significantly lower than SeaSpray and SeaIsle 2000 for the inoculated treatments. Based on both t rials, SeaSpray and SeaIsle 2000 were consistently tolerant to H. pseudorobustus The Rf values for H. pseudorobustus were <1 on all cultivars, indicating that these were poor hosts (Table 4 4). Final population densities of H. pseudorobustus were the high est in SeaIsle Supreme and lowest in SeaSpray and SeaIsle 2000; therefore, these latter two would be considered resistant. Discussion Damage of B. longicaudatus in seashore paspalum SeaIsle 1 has been previously reported. Hixson et al (2004) found tha t B. longicaudatus caused significant reductions in root length of

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53 SeaIsle 1 at 60 and 120 days after the inoculation of nematodes, and likewise in trial one of our study we also found significant reductions in root length of this cultivar 90 days after th e inoculation of B. longicaudatus Hixson et al (2004) found that population densities of B. longicaudatus increased on SeaIsle 1 at 120 days after inoculation, which is corroborated by our studies showing that SeaIsle 1 was a good host to B. longicaudatu s (Rf values averaged 5.3 and 2.9 in the two trials). In comparison of these two trials, lower population densities of B. longicaudatus as well as less root damage were observed in trial two than in trial one, suggesting that less root damage was associate d with the lower nematode population densities. For B. longicaudatus the Rf was >1 for all seashore paspalum cultivars, but this was not the case for bermudagrass cultivars (Chapter 3). One hypothesis is that the relatively dense and robust root systems o f seashore paspalum can support higher population densities of B. longicaudatus This would indicate that the carrying capacity for B. longicaudatus on seashore paspalum is higher than for bermudagrass and, therefore, seashore paspalum may be more suscepti ble to this nematode. Although population densities of Helicotylenchus spp. >500/100 cm 3 of soil were found on seashore paspalum in the field (Hixson and Crow, 2004), the nematode population densities were much lower in the conetainers. The smaller soil an d root volumes from the conetainers may limit the total nematode population densities that can be supported. Furthermore, when the nematodes were inoculated, the grass root systems were not fully established, and the carrying capacity on young developing r oots is likely lower than for established roots in the field. The initial inoculum level of H. pseudorobustus of 500 nematodes/conetainer might have been too high. For future reproduction studies, inoculum densities could be reduced to 50 nematodes/conetai ner. However,

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54 the fact that root reductions occurred in one or both trials for several cultivars inoculated with H. pseudorobustus indicates that this nematode is a potential pathogen of seashore paspalum. Salam, SeaDwarf, and SeaIsle Supreme were tolerant to B. longicaudatus SeaSpray and SeaIsle 2000 were tolerant to H. pseudorobustus No seashore paspalum cultivar was tolerant to both B. longicaudatus and H. pseudorobustus Therefore it is unlikely that any one cultivar will be free of the threat of nema tode problems in the field, especially considering that there are dozens of nematode species that are parasites of turfgrasses. Future field studies should be conducted to verify these findings. For future screening and development of nematode resistance in seashore paspalum, SeaIsle 2000 and Aloha can be used as standards for B. longicaudatus and SeaIsle Supreme and SeaSpray could be used for H. pseudorobustus Furthermore, interaction studies between B. longicaudatus and H. pseudorobustus could be condu cted under greenhouse conditions since they often coexist in the field.

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55 Table 4 1. Mean total root length of seven seashore paspalum cultivars 90 days after inoculation with Belonolaimus longicaudatus in two experiment trials. U = uninoculate d and I = inoculated with 50 B. longicaudatus /conetainer. Total root length (cm) Trial 1 Trial 2 Cultivar U I % reduction U I % reduction SeaSpray 2609* 1668 a 36 2070* 1818 12 SeaIsle 1 2299* 1647 28 1792 1791 0 Aloha 1790 # 1450 19 1837 1696 8 SeaIsle 2000 2016* 1645 18 1816 1738 4 Salam 1807 1515 16 1884 1643 13 SeaDwarf 1898 1666 12 1838 1711 7 SeaIsle Supreme 1806 1635 9 1801 1695 6 # *Uninoculated treatments significantly different from inoculated treatments at P < 0.10, and P < 0.05, respectively, according to the linear contrast analysis. a Data are means of five replications, and data within a column followed by no letter are not statistically different ( P < 0.05). Table 4 2. Final mean population density (Pf) and r eproduction factor (Rf) of Belonolaimus longicaudatus on seven seashore paspalum cultivars 90 days after inoculation with 50 B. longicaudatus /conetainer in two experimental trials. Final population density (Pf) a Reproductive factor (Rf) Cultivar Trial 1 Trial 2 Trial 1 Trial 2 SeaSpray 169 de bc 143 c 3.4 de 2.9 c SeaIsle 1 266 bc 145 bc 5.3 bc 2.9 bc Aloha 137 e 141 c 2.7 e 2.8 c SeaIsle 2000 477 a 281 a 9.5 a 5.6 a Salam 271 bc 197 abc 5.4 bc 3.9 abc SeaDwarf 229 cd 231 ab 4.6 cd 4.6 ab SeaIsle Su preme 335 b 213 abc 6.7 b 4.3 abc a Numbers represent numbers of nematodes recovered from the whole conetainer. b Data are means of five replications. c Means within a column followed by the same letter are not different ( P < 0.05), by Fishers protected leas t significant difference test.

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56 Table 4 3. Mean total root length of seven seashore paspalum cultivars 90 days after inoculation with Helicotylenchus pseudorobustus in two experiment trials. U = uninoculated and I = inoculated with 500 H. pseudorobustus /con etainer. Total root length (cm) Trial 1 Trial 2 Cultivar U I % reduction U I % reduction SeaSpray 2090 1903 a 9 2364 2350 ab 1 SeaIsle 1 2543* 2010 21 2222 # 1755 c 21 Aloha 2296 # 1842 20 2391 2145 abc 10 SeaIsle 2000 2309 1967 15 2598 2592 a 0 Salam 2509* 1740 31 2425* 2024 bc 17 SeaDwarf 2527 # 2074 18 2475 2441 ab 1 SeaIsle Supreme 2426* 1918 21 2256* 1864 c 17 # *Uninoculated treatments significantly different from inoculated treatments at P < 0.10, and P < 0.05, respectively, according to the linear contrast analysis. a Data are means of five replications, and m eans within a column followed by the same letter or no letter are not different ( P < 0.05), by Fishers protected least significant difference test. Table 4 4. Mean populat ion density (Pf) and reproduction factor (Rf) of Helicotylenchus pseudorobustus on seven seashore paspalum cultivars 90 days after inoculation with 500 H. pseudorobustus /conetainer in two experimental trials. Final population density (Pf) a Reproductive fa ctor (Rf) Cultivar Trial 1 Trial 2 Trial 1 Trial 2 SeaSpray 72 b bc 94 cd 0.1 b 0.2 d SeaIsle 1 237 a 291 b 0.5 a 0.6 b Aloha 209 a 147 cd 0.4 a 0.3 cd SeaIsle 2000 99 b 85 d 0.2 b 0.2 d Salam 250 a 270 b 0.5 a 0.5 b SeaDwarf 221 a 210 bc 0. 4 a 0.4 bc SeaIsle Supreme 265 a 422 a 0.5 a 0.8 a a Numbers represent numbers of nematodes recovered from the whole conetainer. b Data are means of five replications. c Means within a column followed by the same letter are not different ( P < 0.05), by Fish ers protected least significant difference test.

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57 CHAPTER 5 FIELD RESPONSES OF BERMUDAGRASS AND SEASHORE PASPALUM CULTIVARS TO STING AND SPIRAL NEMATODES Introduction Bermudagrass ( Cynodon spp.) is a warm season turfgrass widely used on golf courses, spor ts fields, and home lawns in tropical and subtropical regions. Use of seashore paspalum ( Paspalum vaginatum Swartz ) has increased primarily in coastal areas with the development of cultivars having finer leaf texture, high turf quality and excellent salini ty tolerance (Dudeck and Peacock, 1985; Duncan, 1999a). However, a major limitation of planting turfgrasses in the sandy soils of the southeastern United States is the destruction of roots by plant parasitic nematodes (Perry and Rhoades, 1982, Hixson et al ., 2004). Belonolaimus longicaudatus Rau and Helicotylenchus spp. are found in the southeastern United States on turfgrasses and are prevalent in sandy soils (Holdeman, 1955; Christie, 1959; Robbins and Barker, 1974). Belonolaimus longicaudatus is the most damaging nematode species on turfgrasses in Florida (Crow, 2005a). The feeding of B. longicaudatus can cause stunted root growth, decreased plant water and nutrient uptake, and decreased rates of plant evapotranspiration (Johnson, 1970; Perry and Rhoades, 1982; Busey et al ., 1991, 1993; Giblin Davis et al ., 1992a; Luc et al ., 2006). Helicotylenchus spp. have been found in turfgrasses on golf courses and home lawns in Canada and the United States (Yu, et al ., 1998; Hixson and Crow, 2004). Jordan and Mitkows ki (2006) found that Helicotylenchus spp. occurred in all 38 golf courses and 110 putting greens (98.2%) surveyed in New England. A survey of seashore paspalum golf courses and lawns found that 50% of golf courses and 40% of home lawns in Florida were infe sted with Belonolaimus longicaudatus ; while 88% of golf courses and 85% of home lawns were infested with Helicotylenchus spp. (Hixson and Crow, 2004). High densities of Helicotylenchus spp. (> 500 nematodes/100 cm 3 soil) were often found associated with sea shore paspalum in Florida (Hixson

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58 and Crow, 2004). Belonolaimus longicaudatus was the most common nematode found at damaging numbers in a survey of bermudagrass golf courses in Florida, present on 84% of golf courses, 60% of fairways, and 52% of greens in the golf courses surveyed by Crow (2005a). High population densities of B. longicaudatus that could cause damage to turfgrass were present on 60% of the golf courses, 25% of individual fairways, and 21% of individual greens surveyed (Crow, 2005a). The feed ing by Helicotylenchus spp. causes necrotic lesions and dieback of roots, which could further lead to the decline of the entire host plant Although several commercial bermudagrass cultivars and germplasm have been tested for t heir responses to B. longicaudatus or Helicotylenchus spp. under greenhouse conditions (Nign 1963; Johnson, 1970; Giblin Davis et al ., 1992b), information about their performance under field conditions is lacking. Davis et al (2004) evaluated the host sta tus of 15 commonly used forage grass species to H. pseudorobustus (Steiner, 1914) Golden, 1956 They found that tall fescue ( Festuca arundinacea Schreb.), annual bluegrass ( Poa annua L.), paspalum ( Paspalum spp. ), and perennial ryegrass ( Lolium perenne L.) were all good hosts to H. pseudorobustus under controlled conditions. Mixed populations of B. longicaudatus and Helicotylenchus spp. in the field have been reported ( Sasser et al ., 1975; Lewis et al ., 1993; Sikora et al ., 2001 ); however, little informatio n is available about the responses of bermudagrass or seashore paspalum to both nematode species under field conditions. Information also is lacking for individual cultivar responses when multiple nematode species coexist in the field. Therefore, the objec tives of this study were: to evaluate the responses of several commercial bermudagrass cultivars to B. longicaudatus under field conditions; to evaluate the responses of several commercial seashore paspalum cultivars to B. longicaudatus and H. pseudorobust us under field conditions; to determine if there is a correlation between the population densities of different

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59 nematode species in field; and to evaluate turf health of the different grasses while growing in a nematode infested environment. Materials and Methods Two year Field Study A field experiment was conducted from May 2008 to June 2010 at the IFAS Agronomy Forage Research Unit (29 o o The soil type was a flatwood soil (2% clay, 6% slit, and 92% sand) with 5% organic matter and a pH value of 5.9. A field was selected for this experiment that was naturally infested with B. longicaudatus and H. pseudorobustus along with small numbers of Meloidogyne sp. Paratrichodorus sp. Mesocriconema ornatum Hemicycliophor a sp. Hemicriconemoides sp. Pratylenchus sp., and Tylenchorhynchus sp Fifty five 1.5 m 1.5 m square plots were laid out, with 0.3 m wide non planted border areas Treatments consisted of 11 turfgrass cultivars: five dwarf bermudagrass cultivars Tifgr een Champion MiniVerde TifEagle Floradwarf dwarf Tifway Celebration TifSport and three seashore paspalum Aloha Sea Isle and SeaDwarf The experiment was a randomized complete block desi gn with five replications. Grasses were propagated by planting nematode free aerial stolons into 15 cm diameter clay plots in a glasshouse at the University of Florida under a temperature range of 24 to 34 o C with natural daylight. Grasses were fertilized once a week using 24 8 16 ( N P 2 O 5 K 2 O ) at a rate of 0.5 kg N/100 m 2 per growing month. Turfgrass was watered by an overhead irrigation for 6 min everyday and clipped once a week. Ten pots were maintained for each cultivar. Well established nematode free g rasses were transplanted from the greenhouse to the field plots on May 26, 2008 For planting each pot was divided in half that provided four plugs for each plot. Plugs were planted into the corners of 0.6 m 0.6 m squares in the plot center s Grasses we re watered for

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60 15 min three times a day for the first 20 days, and irrigated as needed thereafter. Fertilizer 10 10 10 ( N P 2 O 5 K 2 O ) was applied biweekly at a rate of 0.5 kg N/100 m 2 per growing month from May to October Nematode population densities in e ach plot were assayed just prior to planting Nine soil cores (2.5 cm diam. 10 cm deep) were randomly collected using a 2.5 cm diam. cone shaped sampler to form a representative sample for each plot. Sample holes were then refilled with air dried natural field soil from the same field. The soil in each sample was well mixed and nematodes were extracted from 100 cm 3 subsample of soil using a modified centrifugal flotation technique (Jenkins, 1964). The plant parasitic nematodes were identified to genus and counted under an inverted light microscope at 32 magnification. Nematode population densities were assayed similarly every three months throughout the study. Grasses were mowed at 2.5 cm by a reel mower and gramineous w eeds were hand picked when needed Pesticide applications included 2,4 D and dicamba mixture (Outlaw TM Helena Chemical Company, Collierville, TN) at a rate of 1.8 L/ha for control of weeds, azoxystrobin (Heritage, Syngenta, Wilmington, DE) at a rate of 0.6 kg/ha to prevent fungal diseas e, Bacillus thuringiensis, subsp. kurstaki (DiPel, Valent BioSciences, Libertyville, IL) at a rate of 1.1 kg/ha and fipronil (Top C hoice, Bayer CropScience, Research Triangle Park, NC) at a rate of 5.6 kg/ha to control caterpillar (order Lepidoptera) and mole crickets ( Scapteriscus spp.) were applied when needed. The rates are product rates. Turfgrass health was determined by evaluating root lengths and percent green cover every three months throughout the growing season. Digital images of each plot were a nalyzed using the SigmaScan Pro software (SPSS, Inc., Chicago, IL) to determine the percentage of green pixels in the image (PGC) (Karcher and Richardson, 2005). Turfgrasses were dormant during

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61 March of both years; therefore, these images were not included in the analysis. Root samples were collected from two soil cores (4 cm diam. 15 cm deep) taken randomly in each plot to form a representative sample, and the holes were then filled with air dried natural field soil from the same field. Samples were stor ed in a cooler until processing. Roots were collected by removing the shoots and thatch, and washing free of soil on an 853 then placed into a 50 ml plastic tube and submerged with water. Finer roots were further separated from soil and collected into the plastic tube by submerging and shaking the 853 sieve in tap water Roots were digitally scanned using WinRhizo root scanning equipment and software (Regent Instruments, Ottawa, Ontario, CA). Total root length of each sample was quantified. One year Field Study A separate field experiment was conducted from April 2009 to July 2010 in a nearby field at the same facility (Agronomy Forage Research Unit). The soil type was the same as the previous study. This field was naturally infested with low numbers of B. longicaudatus H. pseudorobustus Meloidogyne sp., Paratrichodorus sp., Mesocriconema sp., Hemicycliophora sp., Hemicriconemoides sp., and Tylenchorhynchus sp. One hundred plots were laid out with the same dimensions as those of the 2008 study. Four dwarf bermudagrass cultivars TifEagle dwarf cultivars Tifway, TifSport, Celebration seashore paspalum cultivars were tested from 2009 to 2010. Cultivars were propagated and maintained using the same protocol as the two year field study. Well established nematode free grasses were transplanted from the greenhouse to the field plots on April 24, 2009. T wo pieces of grass for each cultivar were planted into two corners in the center of each plot. Turfgrasses were

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62 maintained and cultivated in the same way as the field studies in 2008, except that the fertilizer rate was half of that applied to the 2008 plo ts. Nematode populations were assayed at the beginning of this study in April, as well as July and October of 2009 and January, April, and July of 2010. Percent green cover was evaluated October 2009, April, and July 2010. Nematode population densities in the plots and percent green cover were recorded accordingly. In spring 2010 it became evident that some plots were contaminated with different grass cultivars. Also, a number of the dwarf bermudagrasses did not fill in. The contaminated plots were killed w ith glyphosate (Roundup, Monsanto, St Louis, MO) in April 2010 and subsequently these plots, along with those that had not filled in, were replanted. Data from these plots were not included in the analysis. Grasses were watered for 15 min three times a day after transplanting for 30 days, and then fertilizer was applied as the field studies in 2008. Statistical Analysis Percent green cover, total root length, and nematode population densities at each sample date were subjected to analysis of variance (ANOVA ), and the differences among cultivars were compared using Fishers protected least significant difference test at P < 0.05. Data for bermudagrass dwarf cultivars, non dwarf cultivars, and seashore paspalum cultivars were analyzed separately. Means of all b ermudagrass cultivars as a group were compared with that of all seashore paspalum cultivars. A logarithmic regression analysis was conducted to determine the relationship between the soil population densities of B. longicaudatus and H. pseudorobustus in ea ch individual cultivar; separate linear regression analyses were conducted to determine the relationship between 1) the soil population densities of B. longicaudatus or H. pseudorobustus and turfgrass total root length; 2) and the population densities of B longicaudatus or H. pseudorobustus and percent green cover in each individual cultivar. Statistical analysis was conducted by using the SAS software (SAS Institute, Cary, NC).

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63 Results Two year Field Study Bermudagrass cultivars There was no difference in the population densities of B. longicaudatus among all dwarf cultivars throughout the two years (Table 5 1). By the end of the study, densities of B. longicaudatus increased for Champion and MiniVerde, and decreased for Floradwarf, Tifgreen, TifEagle comp ared to their respective initial population densities in May 2008. In December 2008, the population density of B. longicaudatus was the highest for Tifway and lowest for TifSport, and in June 2009, the nematode population density associated with TifSport w as significantly lower than densities observed Tifway and Celebration (Table 5 1). Mean population densities of B. longicaudatus were not different between the dwarf and non dwarf cultivars. Population densities of H. pseudorobustus were not different amon g the dwarf cultivars for the duration of the study (Table 5 2). Population densities of H. pseudorobustus increased in non dwarf bermudagrass cultivars from 6 to 270 nematodes/100 cm 3 of soil during the experiment. In June and December 2009, and June 2010 population densities of H. pseudorobustus were significantly higher in TifSport than the other two cultivars. Over the course of this experiment, population density increased from 6 to 744 H. pseudorobustus /100 cm 3 (124 fold) on TifSport. A negative regr ession relationship between the population densities of H. pseudorobustus and B. longicaudatus was observed for Celebration (Figure 5 1 A) and TifSport bermudagrass (Figure 5 1 B). Mean population densities of H. pseudorobustus were not different between t he dwarf and non dwarf cultivars. Total Root Lengths were not different for the dwarf bermudagrass cultivars in this study (Table 5 3). In September 2009, the root length of non dwarf cultivar Celebration was significantly greater than both Tifway and TifS port root lengths. Differences were not identified

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64 among the non dwarf cultivars for the remaining root length sampling dates. There was no difference in percent green cover among the dwarf cultivars from June 2009 to June 2010 except for December 2009 (Ta ble 5 4). In December 2009, the PGC of Tifgreen was significantly higher than that of Floradwarf, TifEagle and MiniVerde; and Champion had greater PGC than MiniVerde. No other dates provided significant differences for PGC among the dwarf cultivars nor wer e differences found (at any dates) between the non dwarf cultivars. The mean root lengths or turf densities of dwarf cultivars were not different from those of the non dwarf cultivars throughout the study. A negative linear relationship was found between t he total root length and the population density of B. longicaudatus (Figure 5 2) as well as PGC (Figure 5 3 B) in Celebration bermudagrass. Regression showed that for each B. longicaudatus in 100 cm 3 of soil, there was a corresponding reduction of 5 cm in total root length and 0.14% in PGC of Celebration A negative linear relationship between the population densities of H. pseudorobustus and the total root length (Figure 5 4 A) in Floradwarf bermudagrass was observed A negative linear relationship between the population densities of H. pseudorobustus and PGC was observed in Floradwarf (Figure 5 5 A). For each H. pseudorobustus in 100 cm 3 of soil, there was a corresponding reduction of 1 cm in total root length and 0.20% in PGC. Results from the regression analysis also showed that H. pseudorobustus reduced the root length of Tifgreen bermudagrass. For each H. pseudorobustus in 100 cm 3 of soil, there was a corresponding reduction of 0.6 cm in total root length (Figure 5 4 B). In addition, H. pseudorobustus affected TifEagle. Figure 5 5 B indicates that the PGC of TifEagle was reduced by 0.26% for each H. pseudorobustus / 100 cm 3 of soil

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65 Seashore paspalum cultivars Population densities of B. longicaudatus declined in all seashore paspalum cultivars at the end of the study compared with the initial (May 2008) population densities (Table 5 5). Population densities of B. longicaudatus were the highest in SeaIsle 1 (130 nematodes/100 cm 3 of soil) and lowest in Sea Dwarf (75 nematodes/100 cm 3 of soil) in December of 2008 and June 2009, respectively. However, there were no treatment differences at other dates. All the seashore paspalum cultivars were good hosts to H. pseudorobustus Population densities of H. pseudorobustus increased from less than 13 to as high as 1 414 nematodes/100 cm 3 of soil during the study (Table 5 6). However, there was no difference among the three cultivars throughout the two years. The total root lengths of the three cultivars varied from 373 to 993 cm, but were not different throughout the study (Table 5 7). No association was found between the population densities of B. longicaudatus or H. pseudorobustus and the root length of seashore paspalum. Percent green cover ranged from 44 to 80%. In June, September 2009, and June 2010, PGC of SeaDw arf was significantly higher than Aloha and SeaIsle 1 (Table 5 8). A negative linear relationship was found between the PGC of Aloha and the population density of B. longicaudatus (Figure 5 3 A). The PGC of Aloha was reduced by 0.94% for each B. longicauda tus in 100 cm 3 of soil (Figure 5 3 A). As with bermudagrass, associations between B. longicaudatus and H. pseudorobustus were found in seashore paspalum. A negative logarithmic relationship was found between the population densities of H. pseudorobustus an d B. longicaudatus in Aloha (Figure 5 6 A), Sea Dwarf (Figure 5 6 B), and Sea Isle 1 (Figure 5 6 C). When all bermudagrass cultivars were compared as a group to the seashore paspalum cultivars, population densities of B. longicaudatus were significantly h igher in bermudagrass than seashore paspalum on several sampling dates (Table 5 9). The root lengths of bermudagrass

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66 and seashore paspalum were not different throughout the study (Table 5 10). However, PGC was different between the two species for two of f our sampling dates (Table 5 10). One year Field Study Bermudagrass cultivars Although 13 bermudagrass cultivars were planted in the field, many were not included in the analysis due to contamination and poor establishment. Therefore, only Celebration and Tifway were included. Aboveground PGC was measured in October 2009, April and July of 2010, respectively. These turf densities ranged from 17 to 60%, but no difference was detected between the two cultivars at any time (Table 5 11). From April 2009 to Apr il 2010, the population densities of B. longicaudatus increased continuously in both cultivars through April of 2010. However, in July 2010, the nematode population densities dropped below 10 B. longicaudatus /100 cm 3 of soil (Table 5 12). No difference in population densities of B. longicaudatus or H. pseudorobustus was detected between these two cultivars throughout the study (Table 5 12). Seashore paspalum cultivars No differences in PGC among the seven cultivars of seashore paspalum were detected (Table 5 13). Population densities of B. longicaudatus were <10 nematodes/100 cm 3 of soil in all months except for January 2010 (Table 5 14). Population densities of H. pseudorobustus increased greatly from <6 to 495 nematodes/100 cm 3 of soil in seashore paspalu m cultivars from April 2009 to July 2010 (Table 5 15). Discussion All bermudagrass cultivars except TifSport were good hosts to B. longicaudatus During the two year study, population densities of B. longicaudatus increased on Champion and MiniVerde, but slightly dropped in Tifgreen, TifEagle, and Celebration, and dropped by 32%,

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67 33%, and 93% respectively in Floradwarf, Tifway and TifSport. Population densities of B. longicaudatus on TifSport continuously declined and were the lowest among the cultivars ev aluated (Table 5 1), indicating that TifSport might have some level of resistance to B. longicaudatus Conversely, TifSport was a good host for H. pseudorobustus and supported a 124 fold increase in reproduction. Therefore, TifSport was the most resistan t to B. longicaudatus and the most susceptible to H. pseudorobustus This difference in response of TifSport to two nematode species might be of great significance to consider when selecting a turfgrass for use or when designing a breeding program for imp roving nematode responses. The population densities of B. longicaudatus were two to five and a half times higher in bermudagrass than in seashore paspalum, which might indicate that bermudagrass is a better host to B. longicaudatus than seashore paspalum (Table 5 9). Contrastingly, population densities of H. pseudorobustus were significantly higher in seashore paspalum than in bermudagrass from September 2008 to June 2010 (Table 5 9). Population densities of H. pseudorobustus in seashore paspalum varied f rom two to 13 times higher than in bermudagrass, which indicated that seashore paspalum could be a better host to H. pseudorobustus than bermudagrass. Different from most bermudagrass cultivars, the population densities of B. longicaudatus in seashore pasp alum declined from near 109 to 13 nematodes/100 cm 3 soil. Potential explanations for this decline include: 1) Seashore paspalum might be a non host to B. longicaudatus under field conditions, although this would contradict the results of greenhouse experim ents (Chapter 4; Hixson et al ., 2005) and general field observations (W. T. Crow, personal communication); 2) Seashore paspalum might be intolerant to B. longicaudatus and have a low carrying capacity, agreeing with observations by Hixson et al (2005); or 3) Field interactions between B. longicaudatus and H. pseudorobustus may have limited reproduction of B. longicaudatus

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68 Seashore paspalum was a better host for H. pseudorobustus than for B. longicaudatus Population densities of H. pseudorobustus increase d 100 to 200 fold while those of B. longicaudatus decreased by 70 to 96% in the seashore paspalum cultivars Aloha, Sea Dwarf, and Sea Isle 1. This observation and the inverse regression equations suggest that competition or interactions exist between B. lo ngicaudatus and H. pseudorobustus and that H. pseudorobustus might suppress the reproduction of B. longicaudatus in seashore paspalum and for TifSport and Celebration bermudagrass (Figure 5 1, Figure 5 6). However, the suppressive effect was not obvious i n other bermudagrass cultivars. Therefore, the interaction between the two nematode species was host dependent, and could vary between genotypes and species. Although in April and July of 2010 the population densities of H. pseudorobustus in Celebration an d Tifway were numerically higher than those in Tifway, no statistical difference was observed (Table 5 12), which could be due to the high variability of the nematode densities among the plots (91 to 237 and 2 to 17 nematodes/100 cm 3 of soil in April 2010 in Celebration and Tifway, and 23 to 485 and 0 to 10 nematodes/100 cm 3 of soil in July 2010). Although B. longicaudatus and Helicotylenchus spp. were reported to coexist in the field ( Sasser et al ., 1975; Lewis et al ., 1993; Sikora et al ., 2001 ) very few studies have been conducted to test the interaction or competition between them. Only Sasser et al (1975) reported that there was a significant positive correlation between the population densities of H. dihystera and B. longicaudatus 60 days (r = 0.37, P < 0.01), 90 (r = 0.28, P < 0.01) and 120 days (r = 0.38, P < 0.01) after planting peanut in the field. This does not conflict with our results since their sampling dates were relatively soon after planting the crop, when both the crop and nematode population densities were growing, which could result in a positive correlation. Johnson (1970) tested the pathogenicity and interaction of Criconemoides ornatus (syn. Mesocriconema

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69 ornatum ), Tylenchorhynchus martini (syn. T. annulatus ), and B. longicaudatus on six bermudagrass cultivars. He found that all bermudagrass cultivars supported high population densities of the three nematode species but B. longicaudatus was a better competitor than C. ornatus and T. martini The population densities of C. ornatus and T. ma rtini were suppressed more than B. longicaudatus by other nematode species. Similar to our study, we also found that B. longicaudatus was a better competitor than H. pseudorobustus in most (seven) bermudagrass cultivars. In the future, greenhouse studies c ould be conducted to study the interactions and pathogenicity between B. longicaudatus and H. pseudorobustus on different turfgrass hosts. A negative linear relationship between the population densities of B. longicaudatus and total root length or abovegro und PGC were observed for Celebration. Greenhouse studies (Chapter 3) had shown that B. longicaudatus caused significant root length reductions in Celebration, confirming that Celebration was not tolerant to B. longicaudatus The negative relationships bet ween nematode population density and turfgrass growth parameters could indicate that Floradwarf, Tifgreen, and TifEagle may not be tolerant to H. pseudorobustus A negative linear relationship between the population densities of B. longicaudatus and aboveg round PGC of seashore paspalum Aloha was observed. Considering these field results and that root length was reduced in a controlled greenhouse trial (Chapter 4), it appears that Aloha may be intolerant to B. longicaudatus infestation Based on the ability to maintain greater root lengths and PGC in nematode infested soil, SeaDwarf might be a tolerant cultivar (Table 5 7, Table 5 8). A previous greenhouse study also indicated that SeaDwarf was tolerant to B. longicaudatus damage (Chapter 4). Compared with gr eenhouse studies, field studies more closely approximated a real life situation where multiple nematode species or pathogens coexist.

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70 Furthermore, these cultivars could be tested under different populations or isolates of B. longicaudatus and H. pseudorobu stus in the future. Generally speaking, bermudagrass is a better host to B. longicaudatus than H. pseudorobustus and B. longicaudatus is more damaging than H. pseudorobustus to bermudagrass. The exception was TifSport, which was resistant to B. longicauda tus but an excellent host to H. pseudorobustus The seashore paspalum cultivars evaluated were better hosts to H. pseudorobustus than B. longicaudatus and H. pseudorobustus appeared more damaging than B. longicaudatus to seashore paspalum. Seashore paspa lum had higher PGC than bermudagrass in the nematode infested field studies. TifSport bermudagrass might be a good choice for tees and fairways infested with B. longicaudatus SeaDwarf might be a good seashore paspalum cultivar to use to use on greens infe sted with B. longicaudatus

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71 Table 5 1. Population density of Belonolaimus longicaudatus on eight bermudagrass cultivars for the two year field study at Hague, FL. Cultivar Nematode population density a May 2008 Sep 2008 Dec 2008 Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Dwarf cultivars Champion 86 b 66 276 355 178 20 40 116 118 Floradwarf 98 49 327 310 170 14 31 101 67 Tifgreen 113 92 301 273 162 33 85 225 109 MiniVerde 94 78 327 255 137 13 68 70 132 TifEagle 96 76 248 248 186 7 30 84 79 Mean 97 72 296 288 167 17 51 119 101 Non dwarf cultivars Tifway 80 43 206 a 197 61 a 8 21 44 54 Celebration 96 26 144 ab 73 69 a 5 26 25 70 TifSport 102 46 71 b 40 26 b 3 20 17 7 Mean 93 38 140 103 52 5 22 29 44 a Numbers represent numbers of nematodes recovered from 100 cm 3 of soil. b Data are means of five replications; and for each cultivar type, means within a column followed by the sa me letter or no letter are not different ( P < 0.05), Fishers protected least significant difference test. Table 5 2. Population density of Helicotylenchus pseudorobustus on eight bermudagrass cultivars for the two year field study at Hague, FL. Cultivar Nematode population density a May 2008 Sep 2008 Dec 2008 Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Dwarf cultivars Champion 5 b 5 13 6 20 13 76 74 100 Floradwarf 11 7 15 5 11 61 65 84 117 Tifgreen 11 16 20 25 30 84 175 137 187 MiniVerde 4 6 4 4 10 22 14 25 48 TifEagle 22 17 17 31 19 7 66 64 120 Mean 11 10 14 14 18 37 79 77 114 Non dwarf cultivars Tifway 9 4 5 3 3 b 5 8 b 10 47 b Celebration 2 4 2 2 3 b 40 23 b 114 20 b TifSport 6 16 130 227 262 a 198 592 a 333 744 a Mean 6 8 46 77 89 81 208 152 270 a Numbers represent numbers of nematodes re covered from 100 cm 3 of soil. b Data are means of five replications; and for each cultivar type, means within a column followed by the same letter or no letter are not different ( P < 0.05), Fishers protected least significant difference test.

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72 Table 5 3. Tot al root length of eight bermudagrass cultivars in nematode infested field plots for the two year field study at Hague, FL. Cultivar Total root length (cm) Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Dwarf cultivars Champion 540 a 405 47 3 539 436 337 Floradwarf 498 486 447 732 402 438 Tifgreen 634 707 801 842 671 583 MiniVerde 553 328 357 370 540 273 TifEagle 619 539 611 710 493 465 Mean 569 493 538 639 508 419 Non dwarf cultivars Ti fway 659 723 566 b 831 808 739 Celebration 572 558 1054 a 1042 895 1003 TifSport 528 620 605 b 748 809 848 Mean 586 634 742 874 837 863 a Data are means of five replications; and for each cultivar type, means within a column foll owed by the same letter or no letter are not different ( P < 0.05), Fishers protected least significant difference test. Table 5 4. Aboveground percent green cover of eight bermudagrass cultivars in nematode infested field plots for the two year field study at Hague, FL. Cultivar Percent green cover (%) Jun 2009 Sep 2009 Dec 2009 Jun 2010 Dwarf cultivars Champion 61 a 76 34 ab 57 Floradwarf 70 73 28 bc 39 Tifgreen 65 75 44 a 59 MiniVerde 72 68 22 c 33 TifEagle 73 55 26 bc 28 Mean 6 8 69 31 43 Non dwarf cultivars Tifway 69 79 47 64 Celebration 71 80 54 63 TifSport 72 76 57 61 Mean 71 78 53 63 a Data are means of five replications; and for each cultivar type, means within a column followed by the same letter or no lette r are not different ( P < 0.05), Fishers protected least significant difference test.

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73 Table 5 5. Population density of Belonolaimus longicaudatus on three seashore paspalum cultivars for the two year field study at Hague, FL. Cultivar Nematode population de nsity a May 2008 Sep 2008 Dec 2008 Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Aloha 87 b 54 104 ab 116 28 ab 7 24 23 17 SeaDwarf 134 33 75 b 115 25 b 6 21 10 6 SeaIsle 1 105 51 130 a 128 39 a 8 16 26 15 a Numbers represent num bers of nematodes recovered from 100 cm 3 of soil. b Data are means of five replications; and means within a column followed by the same letter or no letter are not different ( P < 0.05), Fishers protected least significant difference test. Table 5 6. Population density of Helicotylenchus pseudorobustus on three seashore paspalum cultivars for the two year field study at Hague, FL. Cultivar Nematode population density a May 2008 Sep 2008 Dec 2008 Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Aloha 8 b 10 162 282 611 1029 1066 1045 1414 SeaDwarf 13 33 161 254 451 585 939 515 1377 SeaIsle 1 5 15 84 226 482 564 1282 894 1070 a Numbers represent numbers of nematodes recovered from 100 cm 3 of soil. b Data are means of five rep lications, and data within a column followed by no letter are not statistically different ( P < 0.05). Table 5 7. Total root length of three seashore paspalum cultivars in nematode infested field plots for the two year field study at Hague, FL. Culti var Total root length (cm) Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Aloha 506 a 438 413 647 457 373 SeaDwarf 622 651 558 807 760 768 SeaIsle 1 552 365 577 993 514 572 a Data are means of five replications, and data within a column foll owed by no letter are not statistically different ( P < 0.05).

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74 Table 5 8. Aboveground percent green cover of three seashore paspalum cultivars in nematode infested field plots for the two year field study at Hague, FL. Cultivar Percent green cover (%) Jun 2009 Sep 2009 Dec 2009 Jun 2010 Aloha 69 b 92 b 76 a 44 b SeaDwarf 71 a 96 a 82 64 a SeaIsle 1 64 b 91 b 75 44 b a Data are means of five replications; and means within a column followed by the same letter or no letter are not different ( P < 0.05) Fishers protected least significant difference test. Table 5 9. Population density of Belonolaimus longicaudatus and Helicotylenchus pseudorobustus on bermudagrass and seashore paspalum for the two year field study at Hague, FL. Cultivar May 2008 Sep 2008 Dec 2008 Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Population density of Belonolaimus longicaudatus a Bermudagrass 96 b 60 237 a 219 a 124 a 13 40 85 72 a Seashore paspalum 109 46 103 b 119 b 31 b 7 20 20 13 b Population density of Helicotylenchus pseudorobustus a Bermudagrass 9 9 b 26 b 38 b 45 b 54 b 127 b 105 b 173 b Seashore paspalum 9 20 a 136 a 254 a 515 a 726 a 1096 a 818 a 1287 a a Numbers represent numbers of nematodes reco vered from 100 cm 3 of soil. b Data are means of five replications; and means within a column followed by the same letter or no letter are not different ( P < 0.05), Fishers protected least significant difference test. Table 5 10. Total root length and abo veground percent green cover of bermudagrass and seashore paspalum in nematode infested field plots for the two year field study at Hague, FL. Cultivar Mar 2009 Jun 2009 Sep 2009 Dec 2009 Mar 2010 Jun 2010 Total root length (cm) Bermudagrass 576 a 54 6 614 727 632 586 Seashore paspalum 560 484 516 815 577 571 Percent green cover (%) Bermudagrass ---69 73 39 b 51 ---Seashore paspalum ---65 93 78 a 51 ---a Data are means of five replications; and means within a column followe d by the same letter or no letter are not different ( P < 0.05), Fishers protected least significant difference test. ---Data were not collected.

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75 Table 5 11. Aboveground percent green cover of two bermudagrass cultivars in nematode infested field plots for the one year field study at Hague, FL. Cultivar Percent green cover (%) Oct 2009 Apr 2010 Jul 2010 Celebration 54 a 51 55 Tifway 34 60 17 a Data are means of five replications, and data within a column followed by no letter are not statistically diff erent ( P < 0.05). Table 5 12. Nematode population density of Belonolaimus longicaudatus and Helicotylenchus pseudorobustus on two bermudagrass cultivars for the one year field study at Hague, FL. Cultivar Apr 2009 Jul 2009 Oct 2009 Jan 2010 Apr 201 0 Jul 2010 Population density of Belonolaimus longicaudatus a Celebration 3 b 10 17 74 93 10 Tifway 2 5 3 41 103 5 Population density of Helicotylenchus pseudorobustus a Celebration 4 1 7 35 165 251 Tifway 5 12 3 18 7 3 a Num bers represent numbers of nematodes recovered from 100 cm 3 of soil. b Data are means of five replications, and data within a column followed by no letter are not statistically different ( P < 0.05). Table 5 13. Aboveground percent green cover of seven sea shore paspalum cultivars in nematode infested field plots for the one year field study at Hague, FL. Cultivar Percent green cover (%) Oct 2009 Apr 2010 Jul 2010 SeaSpray 59 a 77 34 SeaIsle 1 52 70 41 Aloha 57 67 48 SeaIsle 2000 53 77 54 Salam 48 6 1 31 SeaDwarf 56 75 61 SeaIsle Supreme 56 62 53 a Data are means of five replications, and data within a column followed by no letter are not statistically different ( P < 0.05).

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76 Table 5 14. Nematode population density of Belonolaimus longicaudatus on sev en seashore paspalum cultivars for the one year field study at Hague, FL. Cultivar Nematode population density a Apr 2009 Jul 2009 Oct 2009 Jan 2010 Apr 2010 Jul 2010 SeaSpray 10 b 2 2 4 10 2 SeaIsle 1 1 2 1 16 9 2 Aloha 1 3 3 16 8 4 SeaIsle 2000 2 4 2 15 8 1 Salam 1 3 6 16 8 4 SeaDwarf 1 2 1 8 5 1 SeaIsle Supreme 2 5 6 30 10 5 a Numbers represent numbers of nematodes recovered from 100 cm 3 of soil. b Data are means of five replications, and data within a column followed by no letter are not statistically different ( P < 0.05). Table 5 15. Nematode population density of Helicotylenchus pseudorobustus on seven seashore paspalum cultivars for the one year field study at Hague, FL. Cultivar Nematode population density a Apr 2009 Jul 2009 Oct 2009 Jan 2010 Apr 2010 Jul 2010 SeaSpray 10 b 89 40 340 368 443 SeaIsle 1 16 59 79 279 346 362 Aloha 9 19 96 199 286 495 SeaIsle 2000 11 21 107 183 163 163 Salam 12 59 77 195 339 240 SeaDwarf 9 71 134 315 241 428 SeaIsle Sup reme 5 17 122 339 179 324 a Numbers represent numbers of nematodes recovered from 100 cm 3 of soil. b Data are means of five replications, and data within a column followed by no letter are not statistically different ( P < 0.05).

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77 Figure 5 1. Regress ion relationship between population density of Helicotylenchus pseudorobustus and Belonolaimus longicaudatus bermudagrass for the two

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78 Figure 5 2. Regress and population density of Belonolaimus longicaudatus for the two year field study at Hague, Florida.

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79 Figure 5 3. Regression relationship between the percent green co Belonolaimus longicaudatus respectively for the two year field study at Hague,

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80 Figure 5 4. Regression relationship b etween the total root length of bermudagrass and the population densities of Helicotylenchus pseudorobustus for the two

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81 Figure 5 5. Regression relationshi bermudagrass and the population density of Helicotylenchus pseudorobustus for the two

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82 Figure 5 6. Regression relationship b etween population density of Helicotylenchus pseudorobustus and Belonolaimus longicaudatus year field study at Hague, Florida. A:

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83 Figure 5 6. Co ntinued.

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84 CHAPTER 6 DNA CONTENT OF BERMUDAGRASS ACCESSIONS IN FLORIDA Introduction Bermudagrass ( Cynodon spp.) is widely distributed in China, India, Africa, Australia, South America and the southern region of the United States (Abulaiti and Yang, 1998; Wu et al ., 2006). In the United States, it is distributed throughout the warmer regions: from Florida northward to Maryland and New Jersey along the east coast, and westward along the southern border to California (Harlan et al ., 1970; Abu laiti and Yang, 1998). In Florida, bermudagrass is one of the most widely used warm season grasses. Improved fine textured cultivars produce a vigorous and dense turf that are widely used on golf courses, sports fields, lawns and parks (Trenholm et al ., 20 03). There are nine species in the genus Cynodon and the basic chromosome number is nine (Harlan and de Wet, 1969; Wu et al ., 2006). Tetraploid Cynodon dactylon L. (Pers.) var dactylon (2n = 4x = 36), known as common bermudagrass is characterized as havi ng medium to coarse leaf blades, an aggressive growth rate and its wide adaptability to differing climates and soil conditions. Cynodon transvaalensis Burtt Davy (2n = 2x = 18), African bermudagrass, is a diploid species (Forbes and Burton, 1963; Wu et al 2006) described as having a fine leaf texture and overall poor color. It is not widely adapted and is only found in a small geographic region in the countries of South Africa and Lesotho. Common bermudagrass is used extensively as a turfgrass, while Afri can bermudagrass has had only very limited turfgrass use as a stand alone species. Triploid (2n = 3x = 27) hybrids ( C. dactylon var. dactylon C. transvaalensis ) from these two species have become some of the most widely used turfgrasses in the world and are standards for use on golf courses where warm season turfgrasses are utilized. Pentaploid (2n = 5x = 45) and hexaploid (2n = 6x = 54) plants have been previously reported (Johnston, 1975; Hanna et al ., 1990; Burton et al ., 1993; Wu et al ., 2006; Kang e t al ., 2007).

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85 home lawns (Hanna et al ., 1990). Flow cytometry (FCM) was originally utilized for analyzing animal cells, and was subsequently adapted for ana lysis of plant cells (Galbraith, 1990). FCM has provided a rapid and accurate DNA content analysis and ploidy level determination for plant breeding programs (Dolezel et al ., 1989; Arumuganathan and Earle, 1991; Schwartz et al ., 2010b ). FCM also has been u sed in plant cell cycles analysis (Galbraith et al ., 1983) and sex identification for dioecious plants (Costich et al ., 1991). FCM has been used in genome analysis for cool season grass species such as Kentucky bluegrass ( Poa pratensis L.) (Barcaccia et al ., 1997), fine fescue ( Festuca spp.) (Huff and Palazzo, 1998), ryegrass ( Lolium spp.) (Barker et al ., 2001), and bentgrass ( Agrostis spp.) (Bonos et al ., 2002). Further, FCM has been used to determine genome sizes and ploidy levels for warm season grass sp ecies such as buffalograss [ Buchloe dactyloides (Nutt.) Engelm.], Paspalum spp., Zoysia spp., and Cynodon spp. (Jarret et al ., 1995; Taliaferro et al ., 1997; Johnson et al ., 1998; Vaio et al ., 2007; Schwartz et al ., 2010b ). Arumuganathan et al (1999) repo rted the nuclear genome sizes of diploid, triploid, and tetraploid bermudagrass genotypes. Triploid, tetraploid, pentaploid, and hexaploid genotypes were identified in Chinese and Korean bermudagrass species, respectively by Wu et al (2006) and Kang et al (2007). Information is lacking regarding the DNA content or ploidy level of bermudagrass accessions collected in Florida. Bermudagrass is the most widely used species on golf courses in Florida (Trenholm et al ., 2003). Existing cultivars are susceptible to several pests that result in the use of pesticides for maintenance of high quality turf required for golf courses and sports fields. Therefore, the screening and breeding of bermudagrass germplasm is warranted to develop pest resistant cultivars for us e in Florida and lower latitudes. Prior to making crosses it is

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86 essential to determine the ploidy level of identified superior performing lines that might be utilized in a breeding program. Ploidy level identification of Florida adapted germplasm would all ow for the correct pairing of elite lines as parents to develop superior cultivars well adapted to Florida with improved levels of tolerance/resistance to common pest problems that currently require pesticide applications. The objective of this study was t o determine the nuclear DNA content and ploidy level of selected superior (Florida adapted) UF bermudagrass germplasm accessions. Materials and Methods Plant Materials Forty seven Cynodon accessions selected for having superior turfgrass performance in Gai known ploidy levels or nuclear DNA contents were included in this test (Table 6 1). A known diploid African bermudagrass accession was also included as a reference. Ea ch genotype was vegetatively propagated into 15 cm diam pots filled with 100% USGA specification greens sand (USGA, 1993) and grown at the University of Florida Envirotron greenhouse facility. The accessions were maintained at a temperature range of 24 to 34 o C under natural daylight, watered for six min a day by an overhead automatic irrigation system and fertilized once every other week using 24 8 16 ( N P 2 O 5 K 2 O ) at a rate of 0.5 kg N/100 m 2 per growing month. Top growth was clipped once a week leaving ae rial stolons growing over the edge of the pots to be used for FCM. Flow Cytometry Flow cytometry analyses were conducted in the forage evaluation support laboratory (FESL) at the University of Florida on a Partec PA, one parameter flow cytometer (Partec Gm bH, Otto Hahn Str. 32, D 48161 Munter, Germany) with a 100 watt HBO short arc lamp

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87 emitting UV light at 420 nm to excite fluorescence. Nuclear DNA content was measured by procedures modified from Arumuganathan and Earle (1991). When at least 10 aerial stol ons were present in each pot, flow cytometry analysis was initiated. A CyStain PI Absolute P (05 5002, Partec North America, Inc., Mt. Laurel, NJ) nuclei extraction and DNA staining buffer kit was used to prepare samples. Triploid trout erythrocyte nucle i (BioSure Inc., Grass Valley, CA) with a nuclear DNA content of 7.2 pg/2C nucleus 1 (Hardie and Hebert, 2003; 2004) were used as an internal standard. Approximately 50 mg of fresh nodal or stolon tip tissue from each accession was chopped with a razor bl ade on a petri minute, the solution was transferred into a 5 mL test tube through a 50 m Partec CellTrics monofil nylon filter. After adding 1.6 mL DNA staining so lution and incubating for another 10 minutes at room temperature, five drops of triploid trout erythrocyte nuclei were added into the test tube and well mixed with the solution. The test tube with the solution was put into the flow cytometer and DNA conten t of each plant sample was measured based on at least 10,000 scanned nuclei per sample. For each accession, three replications were measured on three different days. Sample DNA content was calculated by the following formula: sample nuclear DNA content = [ (mean position of sample peak) / (mean position of the control peak)] DNA content of the control (Arumuganathan et al ., 1999). The mean and standard deviation of the genome size were calculated for each genotype. The ploidy levels of the genotypes were t hen determined by the relative comparisons with genome size ranges of previously bermudagrass ploidy levels (Taliaferro et al ., 1997; Arumuganathan et al ., 1999; Wu et al ., 2006; Kang et al ., 2007). Results and Discussion Mean nuclear DNA contents and plo idy levels for the 47 bermudagrass accessions and three reference cultivars are presented in Table 6 1. Representative histograms of the flow

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88 cytometry peaks are shown in Figures 6 1 to 6 7. The peaks of the trout erythrocyte nuclei were relatively smaller than those of plant samples, because there were fewer cells in the triploid trout erythrocyte nuclei than the plant samples, but this did not affect the results. Clear and consistent peaks were obtained for all genotypes, and the cells in the G2 phase wer e also observed. Nuclear DNA content of UF bermudagrass accessions ranged from 1.38 to 2.75 pg/2C nucleus 1 (Table 6 1), which was lower in value than those reported for Chinese and Korean accessions (Wu et al ., 2006; Kang et al ., 2007). Using previously r eported ploidy levels associated with bermudagrass nuclear DNA content (Taliaferro et al ., 1997; Arumuganathan et al ., 1999; Wu et al ., 2006; Kang et al ., 2007), the ploidy levels of the Florida adapted bermudagrass accessions were determined. Ploidy level s and genome size ranges included: 19 (41%) triploid accessions with a genome size range of 1.38 to 1.61 pg/2C nucleus 1 24 (51%) tetraploid accessions with a genome size of 1.94 to 2.24 pg/2C nucleus 1 one (2%) pentaploid accession with the genome size of 2.47 pg/2C nucleus 1 and three (6%) hexaploid accessions with the genome size of 2.64 to 2.75 pg/2C nucleus 1 The genome sizes of all accessions were inside the previously reported ploidy ranges (Table 6 1). The nuclear DNA contents of cultivars Tifw ay, Tifgreen, and Tifton 10 in this study were very close to the values previously reported (Arumuganathan et al ., 1999; Wu et al ., 2006), which corroborated the accuracy of this assay. Compared with the Korean and Chinese accessions (Wu et al ., 2006; Kang et al ., 2007), a lower percentage of tetraploid genotypes were identified among these superior UF accessions. Relatively more triploid accessions (42%) were identified, which are likely mutants of commercial triploid cultivars. The 47 bermudagrass accessi ons evaluated were selected, based on their mult year performance, from a larger germplasm collection of 180 accessions. The 180 accessions were collected from primarily

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89 managed turf sites in the state of Florida and many from golf courses that were likely planted with a triploid bermudagrass cultivar. Because triploids are known for having superior turfgrass performance it is very probable that inadvertently collected triploids would have been selected as part of the group of 47 accessions that represent t hose genotypes with the best overall multi year performance. If the entire collection of 180 genotypes had been evaluated it is that the percentage of triploids would have likely reduced and the percentage of tetraploids increased. Plant leaves were used f or nuclear extraction and staining, but the results were not as consistent as those of terminal nodes or stolon tips. The same problems were observed in Zoysia spp. (Schwartz et al ., 2010b). Plant tissues under stress or with disease were also used; howeve r, they yielded variable results. Therefore, healthy, non stressed nodal plant tissue should be used for flow cytometry studies. Plants have been used as internal controls for grasses in previous FCM studies. Diploid barley ( Hordeum vulgare L.), hexaploid wheat ( Triticum aestivum L.), and tobacco ( Nicotiana tabacum L.) were used to test nuclear DNA contents of 13 turfgrass species ( Arumuganathan et al ., 1999). standard for bermudagrass in FCM studie s because its nuclear content was similar to other genotypes tested (Kang et al ., 2007). In this study, triploid Tifway, with known DNA nuclear content (Arumuganathan et al ., 1999; Wu et al ., 2006), was used as an internal control. However, clear and repea table peaks were not obtained for all genotypes, especially for those with a nuclear DNA content similar to Tifway. Interactions could occur between plant samples and the control, which might counteract the peaks of the samples. Plant control with a DNA co ntent overlapping those of the samples was not a good internal standard in this study. However, with a larger nuclear DNA content (7.2 pg/2C nucleus 1 ), no interaction was found between trout erythrocyte nuclei and bermudagrass cells. Clear, consistent, an d repeatable peaks were obtained

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90 for all genotypes. Therefore, trout erythrocyte nuclei were a very good internal standard for bermudagrass FCM analysis. Other animal blood cells such as those of channel catfish ( Ictalurus punctatus Rafinesque ) also have b een reported as good standard for bermudagrass nuclear DNA content measurement (Wu et al ., 2006). Using this modified method, the CVs for the peaks of all genotypes were less than 6.0%. The standard deviations of nuclear DNA content ranged from 0.01 to 0.1 6 pg/2C nucleus 1 which agreed with the previous studies by Arumuganathan et al (1999), Wu et al (2006) and Kang et al (2007), in that flow cytometry was a very precise method for bermudagrass genome size measurement. All tetraploid and hexaploid acces sions could be used to further the University of Florida bermudagrass breeding program. Superior collected triploid accessions should be compared with current commercial triploid standards for biotic and abiotic stress responses. A triploid with improved p est responses could be considered for release with no further breeding required.

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91 Table 6 1. Nuclear DNA content and ploidy level of 47 University of Florida bermudagrass accessions and three commercial cultivars. Accession DNA content mean SD Inferred ploidy (2n) Accession DNA content mean SD Inferred ploidy (2n) (pg/2C) (pg/2C) 102 1.61 0.06 a 3x 355 2.24 0.12 4x 131 2.02 0.12 4x 445 2.05 0.05 4x 132 2.20 0.12 4x 481 1.94 0.08 4x 157 2.00 0.03 4x 489 1.38 0.16 3x 17 1 2.09 0.09 4x 490 1.50 0.15 3x 173 1.45 0.08 3x 525 1.54 0.07 3x 227 2.20 0.14 4x 528 2.08 0.16 4x 282 1.49 0.06 3x PI 289922 2.47 0.07 5x 283 1.54 0.08 3x PI 290868 2.08 0.06 4x 285 1.48 0.09 3x PI 290872 2.00 0.03 4x 286 1. 49 0.08 3x PI 290895 1.51 0.02 3x 291 1.47 0.08 3x PI 291590 1.94 0.09 4x 293 1.52 0.08 3x UFC03 2.08 0.15 4x 295 2.70 0.01 6x UFC06 2.19 0.06 4x 296 1.48 0.11 3x UFC07 2.75 0.09 6x 297 1.63 0.03 3x UFC11 2.16 0.06 4x 299 1.98 0.05 4x UFC12 2.06 0.01 4x 301 1.98 0.10 4x UFC25 1.59 0.05 3x 304 2.16 0.03 4x UFC26 1.56 0.03 3x 319 1.44 0.02 3x UFC29 2.00 0.11 4x 320 1.60 0.10 3x UFC30 1.97 0.06 4x 334 2.03 0.02 4x Tifway 1.53 0.05 3x 343 2.10 0.06 4 x Tifgreen 1.58 0.05 3x 344 2.64 0.06 6x Tifton 10 3.06 0.01 6x 347 2.06 0.09 4x AB b 1.17 0.07 2x 352 1.50 0.01 3x a Means and standard deviations of three replications. b AB is a African bermudagrass accession.

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92 Figure 6 1. Flow cytomet ric histogram of diploid, triploid, and tetraploid bermudagrass and trout erythrocyte nuclei. Peak 1 = diploid accession, Peak 2 = triploid cultivar (Tifgreen) control, Peak 3 = tetraploid accession, Peak 4 = G2 phase of diploid accession, Peak 5 = G2 phas e of triploid cultivar control, Peak 6 = G2 phase of tetraploid accession, Peak 7 = trout erythrocyte nuclei (control). Figure 6 2. Flow cytometric histogram of diploid and triploid bermudagrass and trout erythrocyte nuclei. Peak 1 = diploid accession Peak 2 = triploid cultivar (Tifgreen) control, Peak 3 = G2 phase of diploid accession, Peak 4 = G2 phase of triploid cultivar control, Peak 5 = trout erythrocyte nuclei (control).

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93 Figure 6 3. Flow cytometric histogram of diploid and tetraploid bermudag rass and trout erythrocyte nuclei. Peak 1 = diploid accession, Peak 2 = tetraploid accession, Peak 3 = G2 phase of diploid accession, Peak 4 = G2 phase of tetraploid accession, Peak 5 = trout erythrocyte nuclei (control). Figure 6 4. Flow cytometric his togram of diploid bermudagrass accession and trout erythrocyte nuclei. Peak 1 = diploid accession, Peak 2 = G2 phase of diploid accession, Peak 3 = trout erythrocyte nuclei (control).

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94 Figure 6 5. Flow cytometric histogram of triploid bermudagrass cultiva r control and trout erythrocyte nuclei. Peak 1 = triploid cultivar (Tifway) control, Peak 2 = G2 phase of triploid cultivar control, Peak 3 = trout erythrocyte nuclei (control). Figure 6 6. Flow cytometric histogram of tetraploid bermudagrass and trout erythrocyte nuclei. Peak 1 = tetraploid accession, Peak 2 = G2 phase of tetraploid accession, Peak 3 = trout erythrocyte nuclei (control).

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95 Figure 6 7. Flow cytometric histogram of hexaploid bermudagrass cultivar control and trout erythrocyte nuclei. Pea k 1 = hexaploid cultivar (Tifton 10) control, Peak 2 = G2 phase of hexaploid cultivar control, Peak 3 = trout erythrocyte nuclei (control).

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96 CHAPTER 7 SCREENING BERMUDAGRASS GERMPLASM ACCESSIONS FOR RESPONES TO STING NEMATODES Introduct ion B ermudagrass ( Cynodon spp.) is the predominant turfgrass used in the southern United States and other warm regions in the world. A limitation for the utilization of bermudagrass in the southeastern United States is the s ting nematode ( Belonolaimus long icaudatus Rau ), which is frequently found in sandy coastal soils. It has been considered the most damaging plant parasitic nematode on bermudagrass in Florida (Crow, 2005a; Luc et al ., 2007). Sting nematode causes damage to greens, fairways, and rough area s on golf courses (Crow and Han, 2005). With the cancellation of fenamiphos (Nemacur, Bayer CropScience, Research Triangle Park, NC) turfgrass managers are in need of new nematode management strategies. Utilization of resistant or tolerant cultivars would be the most desirable, least costly nematode management practice with the minimum number of ecological effects on non target species (Giblin Davis et al ., 1992b). Breeding and improvement of new bermudagrass cultivars with superior nematode responses are e ssential. Giblin Davis et al (1992b) tested the nematode tolerance and resistance of seven commercial bermudagrass cultivars and 30 germplasm accessions. They found that 26 accessions showed a significant reduction in root dry weights compared with the un inoculated controls and that 25 of them supported the reproduction of B. longicaudatus Currently, there are few known sting nematode resistant or tolerant bermudagrass genotypes available. Except for are related to each other (K. E. Kenworthy, personal communication); therefore, environmental pressure exists for the development of a significant pest problem on bermudagrass greens. This highlights the need to select new sources of genetically superior b ermudagrass accessions for use in a breeding program. Most bermudagrass cultivars that have been widely used on golf courses are triploids ( Cynodon

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97 dactylon [L.] Pers. var. dactylo n C. transvaalensis Burtt Davy) derived from hybridizations of tetraploid common bermudagrass and diploid African bermudagrass. Previous development of sterile, triploid hybrids has focused primarily on the selection of a superior common bermudagrass parent. This was due, in part, to a lack of knowledge regarding the genetic div ersity and potential of improvement for African bermudagrass. Information is now available that indicates that improvement of African bermudagrass is possible for several turfgrass performance traits (Kenworthy et al ., 2006). This necessitates the screenin g of both African and common bermudagrass. The University of Florida bermudagrass breeding program has through multi year evaluations identified superior, Florida adapted, experimental accessions of common and African bermudagrass to utilize in crosses to develop new sterile bermudagrass hybrids for use on golf courses (K. E. Kenworthy, personal communication). Sting nematode responses to these bermudagrass accessions remain uncharacterized and could provide valuable information in the selection of parents to develop progeny and cultivars resistant to this serious turfgrass pest. The objectives of this study were to test the range of damage caused by sting nematodes on superior UF accessions of common and African bermudagrass, and to select germplasm access ions with superior nematode responses for future cultivar breeding and development. Materials and Methods Plant Materials Five commercial cultivars ( and 46 germplasm accessions of bermudagrass were tested in two sequential experimental trials in 2009 in a greenhouse at the University of Florida Turfgrass Envirotron in Gainesville, FL. The bermudagrass accessions tested are listed in Table 7 1.

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98 Inoculum Preparation Belonolaimus longicaudatus was ( Stenotaphrum secundatum Kuntze ) grown in clay pots filled with pure sand under greenhouse conditions (Giblin Davis et al decanting and sieving technique (Cobb, 1918; Flegg, 1967) All stages of nematodes including the juveniles and adults were collected. The nematode suspensions were concentrated using a 25 m (500 mesh) sieve. The average number of juveniles and adults were counted from five 1 ml aliquot s and extrapolated to the total volume of the suspension. Suspensions were kept in a refrigerator until used. Nematode Responses of Germplasm Accessions Nematode free aerial stolons of each cultivar/accession was vegetatively propagated into (3.8 cm diamet er 21 cm deep, volume = 150 cm 3 ) (SC10, Stuewe & Sons, Inc., Tangent, OR) filled with 100% USGA specification greens sand (USGA, 1993) The bottom of conetainers was filled with Poly fil (Fairfield Processing Corporation, Danbury, CT) to prevent san d from escaping from the drainage holes. Two pieces of terminal aerial stolons with one node each were planted into each conetainer. Two minutes of overhead mist irrigation was applied six times daily for two weeks to allow the sprigs to establish. From th e third week, the irrigation was reduced to once a day in the morning for six minutes, and three minutes a day from the beginning of the fifth week. Six weeks after establishment, grass was inoculated with no nematodes or 50 B. longicaudatus per conetainer Before inoculation, suspensions of B. longicaudatus were taken out of the refrigerator, concentrated to 10 nematodes/ml, and set at room temperature for three hours. None or a total of 5 ml of the suspensions were inoculated into two 3 cm deep holes made 1 cm from the base of the grass near the root zone in the uninoculated and inoculated treatments, respectively. The

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99 holes were covered with a light layer of sand and moistened with a light mist. Turfgrasses were maintained in a randomized complete block d esign with six replications for each genotype with a total of 51 bermudagrass genotypes. To provide insulation from temperature fluctuation, Inc., Tangent, OR). The ex periments were set under a temperature range of 24 to 34 o C with natural daylight in a greenhouse at the Envirotron at the University of Florida, Gainesville, FL. Grasses were fertilized once a week using 24 8 16 (N P 2 O 5 K 2 O) at a rate of 0.5 kg N/100 m 2 pe r growing month. Turfgrass was mowed once a week at a mowing height of 2.5 cm. Experiments were harvested 90 days after the inoculation of nematodes. Root and soil samples were collected from each conetainer. Roots were collected by removing the shoots and Poly fil. Roots were washed free of soil on an 853 (20 mesh) sieve and put into a 50 ml plastic tube submerged with water. Finer roots were separated from soil and collected into the plastic tube by submerging and shaking the 853 Roots were digitally scanned using WinRhizo root scanning equipment and software (Regent Instruments, Ottawa, Ontario, Canada). Root length was measured from the scanned images. Percent reduction in the root length of inoculated plants compared with the n on inoculated control was calculated by the following formula: (root length of non inoculated control root length of inoculated plant) / root length of non inoculated control 100. Nematodes were extracted from the whole soil in the conetainer by usi ng the modified centrifugal flotation technique (Jenkins, 1964). The final nematode population densities (Pf) were counted under a microscope. The reproductive factor (Rf), which is an indicator of resistance (Oostenbrink, 1966 ), was calculated by the foll owing formula: Rf = final nematode density (Pf) / initial inoculum density (Pi), where Pi = 50 nematodes per conetainer.

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100 Data Analysis Data from the two trials were analyzed separately. Total root lengths of the inoculated treatment and the non inoculated control of each cultivar were compared by a linear contrast with the P values at P < 0.1. Tolerance was determined by the difference in root length between the two treatments. A turfgrass cultivar/accession was tolerant if there was no difference in total root length between the two treatments; otherwise, the cultivar/accession was intolerant. Resistance was determined by the nematode reproductive factor (Rf) at harvest. A cultivar/accession was considered resistant if Rf < 1 and susceptible if Rf > 1. Fina l nematode population densities were subjected to analysis of variance (ANOVA), and the differences among cultivar/accessions were compared by the Fishers protected least significant difference test at P < 0.05. Statistical analysis was conducted by using the SAS program (SAS Institute, Cary, NC). Results In trial one, differences in root length reductions caused by sting nematodes were observed among accessions (Table 7 2). Among the accessions, the root lengths varied from 689 to 1625 cm for the uninocul ated controls, and 423 to 1453 cm for the inoculated treatments. The root length reductions of the inoculated treatment were ranged from 1 to 55%. Minimum reductions (<15%) in root length were observed for 17 accessions (Table 7 2). For these 17 accessions there was no difference in root length between the two treatments; therefore, these accessions were labeled as tolerant to B. longicaudatus in trial one. Cultivars Celebration, TifGrand, Tifway and 17 accessions had a root length reduction of 16 to 30% ( Table 7 2). Among them, Celebration and nine accessions had significant reductions in root length by B. longicaudatus which was an indication of susceptible response. Reductions in the range of 30 to 40% were found for TifSport and eight bermudagrass acce

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101 B. longicaudatus infection (Table 7 2). The rest of the tested accessions AB4, UFC12, UFC25, and UFC29 all had a root length reduction of more than 40%, and were considered intoleran t to B. longicaudatus (Table 7 2). Results were not consistent between the two trials for all accessions. Roots were relatively longer in trial two than trial one (Table 7 2). In trial two, the total root length of the non inoculated controls and inoculate d treatments varied from 920 to 2358 cm, and 426 to 1817 cm, respectively (Table 7 2). Compared with the uninoculated treatments, the root length reductions ranged from 2 to 55%. In trial two, less than 15% reduction in root length occurred in 13 bermudag rass accessions, all of which were considered tolerant to B. longicaudatus (Table 7 2). reduction of more than 40%, and were classified as susceptible to B. longicaudat us (Table 7 2). Reductions in the range of 30 to 40% were found for TifGrand and eight accessions, and all of them were susceptible to B. longicaudatus infection (Table 7 2). The rest of the accessions had moderate damage of 16 to 30% reductions in root le ngth, which was significant for cultivars Celebration and TifEagle and nine accessions (Table 7 2). Accessions that were consistently tolerant to B. longicaudatus in both tests were Accessions also showed differences in host status to B. longicaudatus (Table 7 3). reproduction of B. longicaudatus The highest Rf of 2.4 wa s in 355. We considered accessions

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102 that supported the reproduction of B. longicaudatus even in one trial as susceptible accessions, and those that suppressed its reproduction in both trials as resistant accessions. Based on both trials, all accessions list ed above were susceptible, while the rest were considered resistant to B. longicaudatus Based on both trials, accessions that were both tolerant and resistant to B. longicaudatus included: 132, 171, 173, 295, 296, 343, PI 290868, UFC11 UFC26, AB1, AB33, a nd AB37. Among them, 132, 171, 173, 343, PI 290868, and UFC11 are common bermudagrass accessions (4x), 296 and UFC26 are triploid accessions (3x), 295 is a hexaploid bermudagrass (6x), and AB1, AB33, as well as AB37 were African bermudagrass accessions (2x ). Resistance and tolerance to B. longicaudatus were identified in accessions with diverse genetic backgrounds (diploid, triploid, tetraploid, and hexaploid), which should provide valuable material for future cultivar breeding to increase the variability o f the cultivars. Discussion This study was consistent with the previous cultivar tests under greenhouse conditions (Chapter 3) in that Celebration and TifEagle were consistently intolerant to B. longicaudatus infection in both trials. This study agreed wit h the previous greenhouse studies (Chapter 3) in that TifEagle did not support the reproduction of B. longicaudatus In the previous greenhouse study (Chapter 3) we found that TifSport was consistently tolerant to B. longicaudatus damage, and the same res ults were obtained in this study. However, one of the trials in the previous studies (Chapter 3) showed that Tifway suffered significant root length reductions, but in this study, no significant damage in roots was observed in either trial. Generally, thes e four cultivars performed consistently during these trials, and they should be useful as good standards for bermudagrass germplasm screening.

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103 TifGrand is a newly released triploid bermudagrass cultivar with good shade tolerance (equal to Celebration). It was reported that this cultivar could tolerate up to high shade levels of 60% to 70% (USGA, 2009) and it has excellent mole cricket non preference resistance (USGA, 2009). However, this cultivar did not have good nematode resistance or tolerance as shown i n this study. In both trials, TifGrand supported reproduction of B. longicaudatus and had significant reductions in root length in one trial. Therefore, this cultivar was considered as both susceptible and intolerant to B. longicaudatus damage. The results of this study should not be considered definitive since multiple nematode species or pathogens exist in natural fields, and the nematode or pest responses of these genotypes need to be assessed with future field studies. Conclusions This study indicated that bermudagrass germplasm accessions respond differently in host suitability and susceptibility to B. longicaudatus and that the selection of genotypes with improved responses to commercial cultivars is possible. This study showed that sting nematodes di d not reduce the total root length in four African bermudagrass accessions and seven common bermudagrass accessions ( P > 0.1). These genotypes showed a tolerant response to sting nematode damage, which could be useful for future cultivar breeding for nemat ode tolerance. On the other hand, several genotypes did not support the reproduction of sting nematodes (Rf <1), a good indicator of resistance. Eight African bermudagrass accessions and 22 common bermudagrass accessions were considered resistant to B. lon gicaudatus which might be potentially useful for future cultivar breeding for nematode resistance. Accessions that are both resistant and tolerant to B. longicaudatus were identified with variable ploidy levels. This is the first reporting of sting nemato de responses on African bermudagrass and variable responses were observed. The use of diploid and tetraploid genotypes identified in this research may prove to be

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104 highly valuable genetic resources for the future improvement of bermudagrass and serve to red uce the dependence on nematicides.

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105 Table 7 1. Mean total root length of five cultivars and 46 germplasm accessions of bermudagrass 90 days after inoculation with Belonolaimus longicaudatus in two experiment trials. U = u ninoculated and I = inoculated with 50 B. longicaudatus /conetainer. Total Root Length (cm) Trial 1 Trial 2 Genotype Ploidy (2n) U I % reduction U I % reduction Celebration 3x 1535* 1143 23 abcd ab 2358 # 1810 22 abcd TifGrand 3x 1561 1197 17 abcd 1635* 1046 33 abcd TifEagle 3x 1219 # 670 31 abcd 1641 # 1007 24 bcd TifSport 3x 1311 901 37 abcd 1614 1354 16 abcd Tifway 3x 1101 776 27 abcd 1624 1221 26 abcd 131 4x 1146* 716 36 abcd 1906 1683 8 d 132 4x 1367 1133 16 bcd 2042 1817 12 bcd 157 4x 1405 1268 8 cd 2115 # 1649 21 abcd 171 4x 1198 1086 6 cd 1650 1546 6 d 173 3x 1126 991 6 cd 1572 1444 8 d 227 4x 1258 1070 15 d 1739* 1145 32 abcd 295 6x 1322 1026 22 abcd 1594 1525 2 d 296 3x 1062 805 32 abcd 1482 1187 20 abcd 299 4x 912 782 10 bcd 1862* 1028 37 abcd 301 4x 689 # 463 27 abcd 1168 1042 7 d 304 4x 1049 # 651 28 abcd 1832* 1237 31 abcd 334 4x 1435* 950 32 abcd 1640 1369 17 abcd 343 4x 1255 993 6 cd 1486 1403 11 bcd 344 6x 792 587 22 abcd 1339* 927 28 abcd 347 4x 1292 839 21 abcd 1952 # 1305 30 abcd 355 4x 1443 1167 33 abcd 1823 1247 26 abcd 445 4x 1263 # 946 22 abcd 2175* 1560 28 abcd 481 4x 1577* 1064 29 abcd 2078 1664 17 abcd 525 3x 777 745 3 d 1787 # 1307 27 abcd 528 4x 1392 # 114 2 17 bcd 1483* 930 23 abcd PI 289922 5x 650 528 17 abcd 1406* 618 55 a PI 290868 4x 757 706 18 abcd 920 717 18 abcd PI 290872 4x 872 747 6 cd 1336* 643 49 abc PI 290895 3x 798 691 35 abcd 1210 # 426 43 abcd PI 291590 4x 15 99* 1061 33 abcd 2182 # 1701 22 abcd AB1 2x 1124 910 31 abcd 1580 1167 14 abcd AB2 2x 1043 # 776 24 abcd 1008 927 10 cd AB21 2x 1605 1453 7 cd 2220* 1325 39 abcd AB3 2x 1563 1172 23 abcd 1844 1714 6 d AB33 2x 1106 1063 2 d 1880 1607 16 abcd AB37 2x 1327 1184 10 bcd 1550 1501 13 bcd AB38 2x 1051 751 24 abcd 1420 # 1035 22 abcd AB39 2x 1625 1434 6 cd 2014* 1312 34 abcd AB4 2x 1034* 578 42 abc 1231 1029 18 abcd

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106 Table 7 1. Continued. Total Root Length (cm) Trial 1 Trial 2 Genotype P loidy (2n) U I % reduction U I % reduction AB42 2x 1553* 1052 31 abcd 2506* 1561 37 abcd AB43 2x 1126 # 744 27 abcd 1353 1347 26 abcd AB7 2x 1464* 1040 27 abcd 1789 1465 16 abcd UFC03 4x 1010 # 730 28 abcd 1232 801 25 abcd UFC06 4x 1279 1126 15 bc d 1867* 1306 29 abcd UFC07 6x 1277 1201 2 d 1450 # 1091 25 abcd UFC11 4x 841 669 8 cd 1093 780 30 abcd UFC12 4x 1001* 423 55 a 1115* 725 31 abcd UFC25 3x 1104* 579 47 ab 1306 1143 2 d UFC26 3x 863 781 13 bcd 1214 1080 5 d UFC29 4x 1023* 539 43 abc 1428* 870 37 abcd UFC30 4x 939 821 11 bcd 1071* 484 52 ab # *Uninoculated treatments significantly different from inoculated treatments at P < 0.1, and P < 0.05, respectively, according to the linear contrast analysis. a Da ta are means of six replications. b Means within a column followed by the same letter are not different ( P < 0.05), Fishers protected least significant difference test.

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107 Table 7 2. Final mean population density (Pf) and reproductive fac tor (Rf) of Belonolaimus longicaudatus on five cultivars and 46 germplasm accessions of bermudagrass, 90 days after inoculation with 50 B. longicaudatus /conetainer in two experimental trials. Genotype Ploidy (2n) Final Population Density (Pf) a Reproductive Factor (Rf) Trial 1 Trial 2 Trial 1 Trial 2 Celebration 3x 61 bcd bc 8 j 1.2 bcd 0.2 j TifGrand 3x 111 a 80 b 2.2 a 1.6 b TifEagle 3x 21 de 25 defghij 0.4 de 0.5 defghij TifSport 3x 25 cde 30 cdefghij 0.5 cde 0.6 cdefghij Tifway 3x 15 de 53 bcdefghi 0.3 de 1.1 bcdefghi 131 4x 40 bcde 14 j 0.8 bcde 0.3 j 132 4x 29 cde 16 ij 0.6 cde 0.3 ij 157 4x 72 abc 62 bcde 1.4 abc 1.2 bcde 171 4x 21 de 7 j 0.4 de 0.1 j 173 3x 32 cde 13 j 0.6 cde 0.3 j 227 4x 35 c de 19 hij 0.7 cde 0.4 hij 295 6x 23 cde 25 defghij 0.5 cde 0.5 defghij 296 3x 49 bcde 27 defghij 1.0 bcde 0.5 defghij 299 4x 39 bcde 10 j 0.8 bcde 0.2 j 301 4x 22 de 21 hij 0.4 de 0.4 hij 304 4x 21 de 9 j 0.4 de 0.2 j 334 4x 8 e 5 j 0.2 e 0.1 j 343 4x 14 de 24 efghij 0.3 de 0.5 efghij 344 6x 29 cde 35 cdefghij 0.6 cde 0.7 cdefghij 347 4x 49 bcde 60 bcdefg 1.0 bcde 1.2 bcdefg 355 4x 40 bcde 120 a 0.8 bcde 2.4 a 445 4x 86 ab 53 cdefghi 1.7 ab 1.1 c defghi 481 4x 50 bcde 60 bcdef 1.0 bcde 1.2 bcdef 525 3x 4 e 38 cdefghij 0.1 e 0.8 cdefghij 528 4x 43 bcde 23 fghij 0.9 bcde 0.5 fghij PI 289922 5x 15 de 14 j 0.3 de 0.3 j PI 290868 4x 6 e 17 ij 0.1 e 0.3 ij PI 290872 4x 5 e 19 hij 0.1 e 0.4 hij PI 290895 3x 16 de 2 j 0.3 de 0.0 cdefghij PI 291590 4x 36 cde 32 cdefghij 0.7 cde 0.6 ij AB1 2x 17 de 15 ij 0.3 de 0.3 ij AB2 2x 2 e 17 ij 0.0 e 0.3 ij AB21 2x 112 a 68 bc 2.2 a 1.4 bc AB3 2x 31 cde 63 bcd 0.6 cde 1.3 bcd AB33 2x 11 e 15 ij 0.2 e 0.3 ij AB37 2x 26 cde 17 ij 0.5 cde 0.3 ij AB38 2x 33 cde 6 j 0.7 cde 0.1 j AB39 2x 114 a 17 ij 2.3 a 0.3 ij AB4 2x 9 e 19 hij 0.2 e 0.4 hij AB42 2x 43 bcde 82 ab 0.9 bcde 1. 6 ab AB43 2x 25 cde 30 bcdefghij 0.5 cde 0.6 bcdefghij

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108 Table 7 2. Continued. Genotype Ploidy (2n) Final Population Density (Pf) Reproductive Factor (Rf) Trial 1 Trial 2 Trial 1 Trial 2 AB7 2x 24 cde 16 ij 0.5 cde 0.3 ij UFC03 4x 10 e 55 bcdefgh 0.2 e 1.1 bcdefgh UFC06 4x 20 de 24 defghij 0.4 de 0.5 defghij UFC07 6x 8 e 8 j 0.2 e 0.2 j UFC11 4x 12 de 17 ij 0.2 de 0.3 ij UFC12 4x 14 de 17 ij 0.3 de 0.3 ij UFC25 3x 7 e 18 hij 0.1 e 0.4 hij UFC26 3x 21 de 2 2 ghij 0.4 de 0.4 ghij UFC29 4x 43 bcde 37 cdefghij 0.9 bcde 0.7 cdefghij UFC30 4x 30 cde 17 ij 0.6 cde 0.3 ij a Numbers represent numbers of nematodes recovered from the whole conetainer. b Data are means of six replications. c Means within a colu mn followed by the same letter are not different ( P < 0.05), Fishers protected least significant difference test.

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109 CHAPTER 8 SUMMARY In recent years, management of nematodes in turf has become more and more challenging due to environmental concerns and resulting cancellation of nematicides. Utilization of nematode resistant and tolerant turfgrass cultivars was tested in this study as an environmentally safe nematode management practice. Resistant and tolerant bermudagrass ( Cynodon spp.) and seashore pasp alum ( Paspalum vaginatum Swartz ) cultivars were identified in greenhouse and field studies to sting nematode ( Belonolaimus longicaudatus Rau ) and spiral nematode ( Helicotylenchus pseudorobustus (Steiner, 1914) Golden 1945 ). The greenhouse studies indicated B. longicaudatus B. longicaudatus No dwarf cultivars were both tolerant and resistant to B. longicaudatus Non tolerant to B. longicaudatus while damaged by B. longicaudatus in both trials. TifSport and Riviera might be both res istant and tolerant to B. longicaudatus Non dwarf cultivar Princess 77 was the best bermudagrass host to B. longicaudatus with population increases of 6.9 fold and 4.4 fold in two trials. Under greenhouse conditions, all seashore paspalum cultivars suppor ted the reproduction of B. longicaudatus Rfs ranged from 2.7 to 9.5 in the two trials. However, population densities of H. pseudorobustus declined in all seashore paspalum cultivars due to the high initial inoculum density of 500 nematodes/conetainer. Sea shore paspalum cultivars showed differences in root tolerant to B. longicaudatus H. pseudorobustus in both trials. No cultivar was tolerant to both nematode species.

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110 The field study showed that bermudagrass was a better host for B. longicaudatus whereas seashore paspalum was a better host for H. pseudorobustus A negative logarithmic relationship was found between the population densities of B. longicaudatus and H. pseudorobustus in B. longicaudatus remained above 54 nematodes/100 cm 3 soil by the end of the study in all bermudagrass cultivars except for TifSport, in which the nematode population density declined to 7 B. longicaudatus /100 cm 3 of soil, a 93% reduction compared with the beginning of the study. Population densities of B. longicaudatus and H. pseudorobu stus decreased by 88% and increased 14300%, respectively, in seashore paspalum. TifSport and Sea Dwarf were the best bermudagrass and seashore paspalum cultivars, respectively, to use in this site infested with B. longicaudatus All seashore paspalum culti vars evaluated were good hosts of H. pseudorobustus in the field. A negative linear relationship was found between the population density of B. longicaudatus and the root length or percent green cover of the bermudagrass Celebration and the seashore paspal um Aloha, which might indicate their intolerance to B. longicaudatus damage. The regression analysis showed that the total root length increasing population dens ities of H. pseudorobustus in the soil, which may indicate their intolerance to H. pseudorobustus The nuclear DNA content and ploidy level of 48 University of Florida bermudagrass germplasm accessions that had good turfgrass characters under field conditi ons were determined by the flow cytometry method. Twenty triploid, 24 tetraploid, one pentaploid, and three hexaploid accessions were identified. All tetraploid, pentaploid, hexaploid, some triploid accessions, as well as 12 African accessions were further screened for nematode responses under

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111 greenhouse conditions. We found that four African and seven common bermudagrass accessions were tolerant to B. longicaudatus and eight African and 22 common bermudagrass accessions were resistant to B. longicaudatus Three diploid, two triploid, six tetraploid, and one hexaploid bermudagrass accessions were found to be both resistant and tolerant to B. longicaudatus Nematode resistance and tolerance were identified in different ploidy levels, which could aid in the t urfgrass breeding program by increasing the genetic diversity for breeding future bermudagrass cultivars for golf course cultivation. Greenhouse studies should be conducted in the future to test the interaction and competition between B. longicaudatus and H. pseudorobustus in turfgrass. Bermudagrass and seashore paspalum could be inoculated with B. longicaudatus alone, H. pseudorobustus alone, or both nematode species together, with non inoculated controls as a comparison. The interaction relationship could be tested under different hosts or turfgrass cultivars. This screening of nematode resistant and tolerant bermudagrass germplasm accessions under greenhouse conditions was preliminary, and they should be further tested under field conditions with multiple nematode species or other pathogens coexisting in soil. Furthermore, the grass cultivars and germplasm accessions should eventually be tested with different populations and geographical isolates of B. longicaudatus or H. pseudorobustus to more broadly sel ect for resistance.

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11 2 LIST OF REFERENCES Abulaiti, D. S. Shi, and G. Yang. 1998. A preliminary survey of native Xinjiang bermudagrass. Journal of Xinjiang Agricultural University 21:124 127. Arumuganathan, K., and E. D. Earle. 1991. Estimation of nuclear DNA content of plants by flow cytometry. Plant Molecular Biology Reporter 9:229 233. Arumuganathan, K., S. P. Tallury, M. L. Fraser, A. H. Bruneau, and R. Qu. 1999. Nuclear DNA content of thirteen turfgrass species by flow cytometry. Crop Science 39:1518 1521. Barcaccia, G., A. Mazzucato, A. Belardinelli, M. Pezzotti, S. Lucretti, and M. Falcinelli. 1997. Inheritance of parental genomes in progenies of Poa pratensis L. from sexual and apomictic genotypes as assessed by RAPD markers and flow cytometry. Th eoretical and Applied Genetics 95:516 524. Barker, R. E., J. A. Kilgore, R. L. Cook, A. E. Garay, and S. E. Warnke. 2001. Use of flow cytometry to determine ploidy level of ryegrass. Seed Science and Technology 29:493 502. Bauhus, J., and C. Messier. 19 99. Evaluation of fine root length and diameter measurements obtained using RHIZO image analysis. Agronomy Journal 91:142 147. Beard, J. B. 2002. Turf M anagement for G olf C ourses. 2nd ed. United States Golf Association. Chelsea, Michigan: Ann Arbor Press Beard, J. B., S. I. Sifers, and W. G. Menn. 1991. Cultural strategies for seashore paspalum. Grounds Maintenance 26:32. Bonos, S. A., K. A. Plumley, and W. A. Meyer. 2002. Ploidy determination in Agrostis using flow cytometry and morphological traits. Crop Science 42:192 196. Bouton, J. H., R. R. Duncan, R. N. Gates, C. S. Hoveland, and D. T. Wood. 1997. Registration of Burton, G. W. 1966. Registration of crop varieties. Crop Science 6:93 94. Burton, G. W 1972. Registration of 'Coastcross 1' bermudagrass. Crop Science 12:125. Burton, G. W. 1974. Breeding bermudagrass for turf. Pp. 18 22. in E. C. Roberts ed. Proceedings of the Second International Turfgrass Research Conference. Madison: WI. ASA and CSSA Crop Science 33:644 645.

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121 BIOGRAPHICAL SKETCH Wenjing Pang earned her B.S. degree in agronomy (plant breeding and genetics) from China Agricultural University, Beijing, China. She then came to the United States for graduate studies. She received her M.S. in plant science (nematology) from the University of Idaho. Her nematology. She then became a graduate s tudent at the Entomology and Nematology Department, University of Florida. Upon graduation from the University of Florida with a PhD, she will continue to work in nematology to start and expand her career.