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Phenotypic Differences in Root-Knot Nematode (Meloidogyne spp.) White Clover (Trifolium repens L.) Interactions and Comb...

Permanent Link: http://ufdc.ufl.edu/UFE0024143/00001

Material Information

Title: Phenotypic Differences in Root-Knot Nematode (Meloidogyne spp.) White Clover (Trifolium repens L.) Interactions and Combining Ability Analysis of Resistance
Physical Description: 1 online resource (131 p.)
Language: english
Creator: Acharya, Ananta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: ability, arenaria, clover, combining, gca, javanica, knot, meloidogyne, nematode, osceola, repens, rkn, root, sca, trifolium, ufwc5, white
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: White clover (Trifolium repens L.) is an important forage legume worldwide and also in the southeastern USA. Its higher crude protein and digestibility make it an important component in mixture with grasses to increase the overall nutritive value. Root-knot nematodes (RKN) (Meloidogyne spp.) can be a major factor limiting the production and persistence of white clover especially in the sandy soil condition of Florida. The purpose of this study was to compare the new cultivar UFWC5 released as tolerant to southern RKN with the commercial cultivar ?Osceola? for host-pathogen responses to different populations of RKN. A second objective was to estimate the magnitudes of general combining ability (GCA) and specific combining ability (SCA) for various RKN resistance responses in UFWC5 to better understand the genetics behind the RKN resistance responses. Our study found that UFWC5 was resistant to all four races of M. incognita with gall scores and egg mass scores less than 2.0 compared to Osceola which had egg mass scores and gall scores higher than 3.0. Similarly, eggs per plant were reduced by ca. 50% when inoculated with M. incognita race 1 and ca. 80 to 90% when inoculated with other the three races of M. incognita. The egg mass score and gall score for UFWC5 roots inoculated with M. arenaria race 1 and M. javanica was above the level for it to be classified as resistant (more than 2.0) but still much reduced compared to Osceola roots (above 3.0 and above 4.0 respectively for M. arenaria race 1 and M. javanica). Egg production as assessed by eggs per plant was reduced by ca. 70% when inoculated with M. arenaria race 1 and by ca. 80% when inoculated with M. javanica. This study pointed out the differences in the virulence of different RKN populations. This may suggest the involvement of different genes for resistance to the different populations of RKN. There were no significant differences between non-inoculated Osceola and UFWC5 for either root or shoot weights. This finding suggests that selecting for RKN resistance did not alter the yield potential of this newly selected white clover cultivar. Based on three different diallel analysis studies involving three RKN populations M. incognita race 4, M. arenaria race 1 and M. javanica, additive genetic variance appeared to be the principal type of gene action involved in selection for RKN resistance in UFWC5. All these genetic studies showed that additive variance was more important than non-additive variance in the inheritance of resistance to RKN. The plants which were resistant to M. incognita race 4 were not necessarily resistant in the same degree to M. arenaria or M. javanica and the degree of susceptibility was also different in these three populations. One parent that showed resistance to M. incognita race 4 was susceptible to M. javanica, which suggests that there may be differences in the genes that confer resistance to different populations of RKN. The importance of additive variance suggests that selection of a few superior parents for development of a synthetic variety would be the most appropriate breeding strategy. Based on our research, the clones R1, R4 and M3 would be superior parents for breeding resistance to M. incognita race 4. Only one parent in each case was outstanding for resistance to M. arenaria race 1 and M. javanica (R6 and R1, respectively).
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 Ananta Acharya.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Quesenberry, Kenneth H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024143:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024143/00001

Material Information

Title: Phenotypic Differences in Root-Knot Nematode (Meloidogyne spp.) White Clover (Trifolium repens L.) Interactions and Combining Ability Analysis of Resistance
Physical Description: 1 online resource (131 p.)
Language: english
Creator: Acharya, Ananta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: ability, arenaria, clover, combining, gca, javanica, knot, meloidogyne, nematode, osceola, repens, rkn, root, sca, trifolium, ufwc5, white
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: White clover (Trifolium repens L.) is an important forage legume worldwide and also in the southeastern USA. Its higher crude protein and digestibility make it an important component in mixture with grasses to increase the overall nutritive value. Root-knot nematodes (RKN) (Meloidogyne spp.) can be a major factor limiting the production and persistence of white clover especially in the sandy soil condition of Florida. The purpose of this study was to compare the new cultivar UFWC5 released as tolerant to southern RKN with the commercial cultivar ?Osceola? for host-pathogen responses to different populations of RKN. A second objective was to estimate the magnitudes of general combining ability (GCA) and specific combining ability (SCA) for various RKN resistance responses in UFWC5 to better understand the genetics behind the RKN resistance responses. Our study found that UFWC5 was resistant to all four races of M. incognita with gall scores and egg mass scores less than 2.0 compared to Osceola which had egg mass scores and gall scores higher than 3.0. Similarly, eggs per plant were reduced by ca. 50% when inoculated with M. incognita race 1 and ca. 80 to 90% when inoculated with other the three races of M. incognita. The egg mass score and gall score for UFWC5 roots inoculated with M. arenaria race 1 and M. javanica was above the level for it to be classified as resistant (more than 2.0) but still much reduced compared to Osceola roots (above 3.0 and above 4.0 respectively for M. arenaria race 1 and M. javanica). Egg production as assessed by eggs per plant was reduced by ca. 70% when inoculated with M. arenaria race 1 and by ca. 80% when inoculated with M. javanica. This study pointed out the differences in the virulence of different RKN populations. This may suggest the involvement of different genes for resistance to the different populations of RKN. There were no significant differences between non-inoculated Osceola and UFWC5 for either root or shoot weights. This finding suggests that selecting for RKN resistance did not alter the yield potential of this newly selected white clover cultivar. Based on three different diallel analysis studies involving three RKN populations M. incognita race 4, M. arenaria race 1 and M. javanica, additive genetic variance appeared to be the principal type of gene action involved in selection for RKN resistance in UFWC5. All these genetic studies showed that additive variance was more important than non-additive variance in the inheritance of resistance to RKN. The plants which were resistant to M. incognita race 4 were not necessarily resistant in the same degree to M. arenaria or M. javanica and the degree of susceptibility was also different in these three populations. One parent that showed resistance to M. incognita race 4 was susceptible to M. javanica, which suggests that there may be differences in the genes that confer resistance to different populations of RKN. The importance of additive variance suggests that selection of a few superior parents for development of a synthetic variety would be the most appropriate breeding strategy. Based on our research, the clones R1, R4 and M3 would be superior parents for breeding resistance to M. incognita race 4. Only one parent in each case was outstanding for resistance to M. arenaria race 1 and M. javanica (R6 and R1, respectively).
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 Ananta Acharya.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Quesenberry, Kenneth H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024143:00001


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PHENOTYPIC DIFFERENCES IN ROOT-KNOT NEMATODE ( Meloidogyne spp.) WHITE CLOVER ( Trifolium repens L.) INTERACTIONS AND COMB INING ABILITY ANALYSIS OF RESISTANCE By ANANTA RAJ ACHARYA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Ananta Raj Acharya 2

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To my late Grandparents, 3

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ACKNOWLEDGMENTS I would like to extend my a ppreciation to my committee chair Dr. Kenneth Quesenberry. His encouragements and insights into the s ubject matter always helped me throughout the research period. He always helped me with sugge stions in graduate work, research and even some tiny day to day matters. He has been an ex cellent guardian and friend at the same time. I would also like to thank Dr Kevin Kenworthy for his valuable suggestions in quantitative procedures involved in the research. I also want to thank Dr. Yoana Newman for her support and suggestions in my research. I want to appreciate Dr. David Wofford for his research ideas and thought provoking questions and suggestions. I want to thank technicians Eric Ostmark and Judith Dampier. Eric was the first to teach me every skill required to conduct the research and his wittiness and friendship always entertained during the laborious lab works. I want to thank Samantha, Kevin, Dylan, Jamie, Olubunmi, Carlos, Vivienne and Ka ren for their help with tedious and laborious lab works. I also want to thank Subodh, Shweta and Pr akash for a close friendship which often reminded me of my home. I would like to tha nk Smita for her encouragement, support and understanding. Last but not the least, I woul d like to thank and congratulate my parents Hari Acharya and Radha Acharya whose dream, direction and support br ought me to this stage. And I heartily want to thank my brother Kul Acharya and sister-inlaw Sulochana for their unselfish support. My brother is always my source of inspiration a nd his support since childhood brought me here, even when he himself needed support and might have ha d other priorities. Finally I want to remember all my family members; brothers, sisters, niece, nephew and everyone. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.......................................................................................................................12 ABSTRACT...................................................................................................................................13 CHAPTER 1 INTRODUCTION................................................................................................................. .15 2 LITERATURE REVIEW.......................................................................................................17 White Clover...........................................................................................................................17 Root-knot Nematode...............................................................................................................19 Root-knot Nematode Disease in White Clover......................................................................22 Root-knot Nematode Resistance Breeding.............................................................................25 Mode of Resistance.................................................................................................................27 Genetics of Resistance......................................................................................................... ...27 Statistics..................................................................................................................................29 3 COMPARISON OF OSCEOLA AND UFWC5 FOR RESPONSE TO DIFFERENT SPECIES/RACES OF ROOT-KNOT NEMATODE.............................................................33 Abstract....................................................................................................................... ............33 Introduction................................................................................................................... ..........34 Materials and Methods...........................................................................................................36 Nematode Egg Extraction and Inoculation.....................................................................36 Maintenance....................................................................................................................37 Data Collection...............................................................................................................38 Results and Discussion......................................................................................................... ..39 4 QUANTITATIVE GENETIC BASIS OF INHERITANCE OF RESISTANCE IN WHITE CLOVER TO SOUTHERN ROOT-KNOT NEMATODE......................................46 Abstract....................................................................................................................... ............46 Introduction................................................................................................................... ..........47 Materials and Methods...........................................................................................................49 Selection of Parents.........................................................................................................49 Crossing...........................................................................................................................50 Inoculation.......................................................................................................................50 Data Collection and Analysis..........................................................................................51 Results and Discussions........................................................................................................ ..52 5

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Egg Mass Score...............................................................................................................52 Gall Score..................................................................................................................... ...53 Eggs g-1 Dry Root Weight...............................................................................................54 Eggs Plant-1......................................................................................................................55 Root Weight.................................................................................................................... .56 Shoot Weight................................................................................................................... 57 Correlations.....................................................................................................................57 5 QUANTITATIVE GENETIC BASIS OF INHERITANCE OF RESISTANCE IN WHITE CLOVER TO PEANUT ROOT-KNOT NEMATODE............................................75 Abstract....................................................................................................................... ............75 Introduction................................................................................................................... ..........76 Materials and Methods...........................................................................................................78 Selection of Parents.........................................................................................................78 Crossing...........................................................................................................................78 Inoculation.......................................................................................................................79 Data Collection and Analysis..........................................................................................79 Results and Discussion......................................................................................................... ..80 Percentage Root System Galled (PRSG).........................................................................80 Egg Mass Score...............................................................................................................82 Gall Score..................................................................................................................... ...82 Eggs g-1 Dry Root Weight...............................................................................................83 Eggs Plant-1......................................................................................................................84 Correlation..............................................................................................................................84 6 QUANTITATIVE GENETIC BASIS OF INHERITANCE OF RESISTANCE IN WHITE CLOVER TO JAVANESE ROOT-KNOT NEMATODE.......................................99 Abstract....................................................................................................................... ............99 Introduction................................................................................................................... ........100 Materials and Methods.........................................................................................................102 Selection of Parents.......................................................................................................102 Crossing.........................................................................................................................103 Inoculation.....................................................................................................................103 Data Collection and Analysis........................................................................................104 Results and Discussions........................................................................................................ 105 Egg Mass Score.............................................................................................................105 Gall Score..................................................................................................................... .106 Eggs g-1 Dry Root Weight.............................................................................................108 Eggs Plant-1....................................................................................................................109 Correlation.....................................................................................................................110 7 CONCLUSIONS.................................................................................................................. 122 6

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REFERENCES............................................................................................................................125 BIOGRAPHICAL SKETCH.......................................................................................................131 7

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LIST OF TABLES Table page 3-1 Egg mass score, gall score and eggs plant-1 of Osceola and UFWC5 white clover when inoculated with six differe nt root-knot nematode populations.................................43 3-2 Correlations between gall scores and e gg mass scores of Osceola and UFWC5 white clover when inoculated w ith six RKN populations...........................................................44 3-3 Response in the shoot and root growth of Osceola and UFWC5 white clover when inoculated with six different popu lations of root-knot nematodes....................................45 4-1 Analysis of variance of combining abili ties of the variables egg mass score and gall score of selected white clove r clones inoculated with M. incognita race 4.......................59 4-2 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on egg mass scores of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4...........................................................60 4-3 Mean egg mass score of roots of thre e resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. .........................................61 4-4 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on gall scores of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. ....................................................................62 4-5 Mean gall score of roots of three resi stant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. ............................................................63 4-6 Analysis of variances of combin ing abilities of the variables Eggs g-1and Eggs plant-1 of selected white clover clones inoculated with M. incognita race 4................................64 4-7 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on log transformed eggs g-1 dry root weight of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. ...................65 4-8 Mean eggs g-1 dry root weight of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. .........................................66 4-9 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on log transformed eggs plant-1 of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. ...............................................67 4-10 Mean eggs plant-1 of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. ......................................................................68 8

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4-11 Analysis of variances of combining abili ties of the variables r oot weight and shoot weight of selected white cl over clones inoculated with M. incognita race 4 and noninoculated clones.............................................................................................................. ..69 4-12 Analysis of variances of combining abili ties of the variables egg mass score and gall score of selected white clove r clones inoculated with M. incognita race 4.......................69 4-13 General combining ability (GCA) and sp ecific combining ability (SCA) effects on root weights of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. ....................................................................70 4-14 Mean root weights of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. ......................................................................71 4-15 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on shoot weights of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. ....................................................................72 4-16 Mean shoot weights of th ree resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. ......................................................................73 5-1 Analysis of variance of combining abilitie s of the variables percentage root system galled (PRSG) egg mass score, and gall score of selected white clover clones inoculated with M. arenaria race 1....................................................................................86 5-2 General combining ability (GCA) and Sp ecific combining ability (SCA) effects for percentage root system galle d (PRSG) of three resistant, two intermediate and three susceptible white clover clones inoculated with M arenaria race 1. ................................87 5-3 Mean percentage root system galled (P RSG) of roots of three resistant, two intermediate and three susceptibl e white clover inoculated with M. arenaria race 1.......88 5-4 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on egg mass score of three resistant, two interm ediate and three susceptible white clover clones inoculated with M arenaria race 1..........................................................................89 5-5 Mean egg mass scores of roots of thr ee resistant, two intermediate and thee susceptible white clover inoculated with M. arenaria race 1............................................90 5-6 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on gall score of three resistant, two interm ediate and thee susceptible white clover clones inoculated with M arenaria race 1..........................................................................91 5-7 Means of gall score of root s of three resistant, two inte rmediate and thee susceptible white clover inoculated with M. arenaria race 1...............................................................92 9

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5-8 Analysis of variance of combini ng abilities for the variables eggs g-1 of dry root weight and egg splant-1 of selected white clover clones inoculated with M. arenaria race 1......................................................................................................................... .........93 5-9 General combining ability (GCA) and sp ecific combining ability (SCA) effects on eggs g-1 of dry root weight of three resistan t, two intermediate and thee susceptible white clover clones inoculated with M arenaria race 1.....................................................94 5-10 Means of eggs g-1 of dry root weight of three resistant, two intermediate and three susceptible white clover inoculated with M. arenaria race 1............................................95 5-11 General combining ability (GCA) and sp ecific combining ability (SCA) effects on eggs plant-1 of three resistant, two intermedia te and thee susceptible white clover clones inoculated with M arenaria race 1..........................................................................96 5-12 Means of eggs plant-1 of three resistant, two intermediate and three susceptible white clover clones inoculated with M. arenaria race 1..............................................................97 5-13 Correlations among egg mass score, gall sc ore and PRSG of eight clones of white clover inoculated with M. arenaria race 1.........................................................................98 6-1 Analysis of variance of egg mass scores and gall scores combining ability of progeny from crosses of selected white clover parents inoculated with M. javanica ....................112 6-2 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on egg mass score of three resistant, two inte rmediate and thee susceptible white clover clones inoculated with M javanica ..................................................................................113 6-3 Mean egg mass scores of roots of thr ee resistant, two intermediate and thee susceptible white clover inoculated with M. javanica .....................................................114 6-4 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on gall score of three resistant, two intermediate and thee susceptible white clover clones inoculated with M javanica ..................................................................................115 6-5 Means of gall score of root s of three resistant, two inte rmediate and thee susceptible white clover inoculated with M. javanica ........................................................................116 6-6 Analysis of variance of eggs g-1 of dry root weight and eggs plant-1 combining abilities of selected white cl over parents inoculated with M. javanica. ..........................117 6-7 General combining ability (GCA) and sp ecific combining ability (SCA) effects on eggs g-1 of dry root weight of three resistant, two intermediate and thee susceptible white clover clones inoculated with M. javanica .............................................................118 6-8 Means of eggs g-1 of dry root weight of three re sistant, two intermediate and thee susceptible white clover inoculated with M. javanica .....................................................119 10

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6-9 General combining ability (GCA) and Sp ecific combining ability (SCA) effects on eggs plant-1of three resistant, two intermedia te and thee susceptible white clover clones inoculated with M. javanica. ................................................................................120 6-10 Means of eggs plant-1of roots of three resistant, two intermediate and thee susceptible white clover inoculated with M. javanica ........................................................................121 11

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LIST OF FIGURES Figure page 4-1 General combining ability (GCA) effects on root weights of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita race 4......................................................................................................................... .........74 12

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHENOTYPIC DIFFERENCES IN ROOT-KNOT NEMATODE ( Meloidogyne spp) AND WHITE CLOVER ( Trifolium repens L.) INTERACTION AND COMBINING ABILITY ANALYSIS OF RESISTANCE By Ananta Raj Acharya May, 2009 Chair: Kenneth H. Quesenberry Major: Agronomy White clover ( Trifolium repens L.) is an important forage legume worldwide and also in the southeastern USA. Its higher crude protein a nd digestibility make it an important component in mixture with grasses to increase the overa ll nutritive value. Root -knot nematodes (RKN) ( Meloidogyne spp.) can be a major factor limiting the production and persistence of white clover especially in the sandy soil condition of Florida. The purpose of this study was to compare the new cultivar UFWC5 released as tolerant to southern RKN with the commercial cultivar Osceola for host-pathogen responses to diffe rent populations of RKN. A second objective was to estimate the magnitudes of general combini ng ability (GCA) and specific combining ability (SCA) for various RKN resistance responses in UFWC5 to better understand the genetics behind the RKN resistance responses. Our study found that UFWC5 was resi stant to all four races of M. incognita with gall scores and egg mass scores less than 2.0 compar ed to Osceola which had egg mass scores and gall scores higher than 3.0. Similarly, eggs per plant were reduced by ca. 50% when inoculated with M. incognita race 1 and ca. 80 to 90% when inoculated with other th e three races of M. incognita The egg mass score and gall score for UFWC5 roots inoculated with M. arenaria race 13

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14 1 and M. javanica was above the level for it to be classified as resistant (more than 2.0) but still much reduced compared to Osceola root s (above 3.0 and above 4.0 respectively for M. arenaria race 1 and M. javanica ). Egg production as assessed by eggs per plant was reduced by ca. 70% when inoculated with M. arenaria race 1 and by ca. 80% when inoculated with M. javanica This study pointed out the differences in the viru lence of different R KN populations. This may suggest the involvement of different genes for re sistance to the different populations of RKN. There were no significant di fferences between non-inocul ated Osceola and UFWC5 for either root or shoot weights. This finding suggest s that selecting for RKN resistance did not alter the yield potential of this newly selected white clover cultivar. Based on three different diallel analysis studies involving th ree RKN populations M. incognita race 4, M. arenaria race 1 and M. javanica additive genetic variance appeared to be the principal type of gene action involved in se lection for RKN resistance in UFWC5. All these genetic studies showed that a dditive variance was more importan t than non-additive variance in the inheritance of resistance to RKN. The plants which were resistant to M. incognita race 4 were not necessarily resistant in the same degree to M. arenaria or M. javanica and the degree of susceptibility was also different in these three po pulations. One parent that showed resistance to M. incognita race 4 was susceptible to M. javanica which suggests that there may be differences in the genes that confer resistance to different populations of RKN. The importance of additive vari ance suggests that selection of a few superior parents for development of a synthetic variety would be th e most appropriate bree ding strategy. Based on our research, the clones R1, R4 and M3 would be superior parents for breeding resistance to M. incognita race 4. Only one parent in each case was outstanding for resistance to M. arenaria race 1 and M. javanica (R6 and R1, respectively).

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CHAPTER 1 INTRODUCTION White clover ( Trifolium repens L.) is a major forage crop in many areas of the world including the southeastern USA. It is a cool season perennial legume but acts as a reseeding annual in Florida (Chambliss and Wofford, 2006 ). The warm season grasses that dominate Florida pastures generally have lower nutritive values. Thus, white clover can be an important component of Florida pasture. It generally has hi gher crude protein and dige stibility than tropical grasses and when grown in a mixture with grasse s will result in increased nutritive value of the overall diet. Despite the added benefit in terms of forage quality, under Florida conditions, there are many diseases and nematodes which decrease the persistence and yi eld of white clover. Root-knot nematodes (RKN, Meloidogyne spp.) may be one of the major problems of white clover in the southeastern region of the United States (UC SAREP, 2008). There are many kinds of nematodes that dama ge plant roots, but root-knot nematodes ( Meloidogyne spp.) cause about 75 percent of all nema tode damage to landscape ornamentals and annual crops in warm climates (Dunn and Sydenham, 1992). Root-Knot nematodes have a very wide host range, are favored by sandy soils with moist and warm soil conditions. They are very small (0.25 mm to 3 mm long) and a transp arent organism. They induce the formation of giant cells (hence, galls) and use these cells as feeding sites to parasiti ze the plant roots. Rootknot nematodes not only compete for nutrients but also open the door for other pathogens and pests to invade plant root s (Dunn and Sydenham, 1992). There have been several attempts to manage RKN disease in white clover. These methods have included chemical., cultural., biological and resistance breeding (Dropkin, 1989). Due to many factors limiting utilization of other techniques, developm ent and planting of cultivars resistant to RKN may be the best practical so lution for RKN management. Some resistant white 15

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clovers have been selected in an attempt to reduce the damage caused by RKN including SC-1 (Gibson, 1973), and MSNR4 (Pederson and Windham, 1995). A new cultivar UFWC5 was recently released as a result of five cycles of phenotypic recurrent selecti on for resistance to the southern RKN ( M. incognita ) from Osceola (Baltensperger et al., 1984). This cultivar has shown reduction in root gall rati ng and egg mass rating against M. incognita (Wofford and Ostmark, 2005). Depending on the species being evaluated, re sistance to RKN has been shown to be monogenic, oligogenic or quantitativ e in nature. Regardless of the resistance mechanism in white clover, it is important to know th e inheritance pattern of resistance to RKN. Partitioning of the genotypic variance into general and specific combining abilities will be even more important in breeding for resistance in synthetic varieties (Baker, 1978). Griffing (1956) gave the generalized model to estimate combining abilities using a di allel mating design that allows partitioning of total genetic variances into general and specific combining abilities. One objective of this research was to comp are the new cultivar UFWC5 and Osceola for resistance responses to four races of M. incognita and two other species, M. arenaria and M. javanica A second objective was to estimate the magn itudes of general and specific combining ability for various RKN resistance responses in UFWC5 to better understand the genetics behind the RKN host-pathogen responses. 16

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CHAPTER 2 LITERATURE REVIEW White Clover White clover ( Trifolim repens L.) is known to be a well adapted perennial legume in temperate climates, but it is also adapted to humid, subtropical climates. White clover is grown throughout the humid eastern USA and also in drie r areas of the western USA using irrigation. It grows well both on clay and silt soils in humid a nd irrigated areas and wh ite clover prefers a soil pH range of 5.5 to 7.0 (USDA, 2002). Although thought to be native to Eurasia, white clover is widely distributed around the world (Williams, 1978a). Although white clovers have been cultivated as ornamentals and cover crops, the main usage is as grazed forage. White clover is consid ered one of the most nutritious forages available and it is generally mixed with grasses to increas e the nutritive value of the available forage. White clover has good persistence fo r grazing and is also suitable for hay, silage and green chop. White clover is also known as a very good nitrogen fixing crop when inocul ated with appropriate symbiotic rhizobium bacteria. The amount of nitrogen fixation depends on the genotype, effective inoculation, growing season, a nd sward density (Gibson and Cope, 1985). White clover cultivars have been classified as small, intermediate and large (ladino) types. Most commercially available cultivars are the la dino type including Regal and Osceola or the intermediate type such as Lousiana S-1, Gra sslands Huia, and Durana. Intermediate types have more profuse and early flowering which results in sufficient seed production for reseeding (Gibson and Cope, 1985). White clover is a tetraploid (2n = 4x = 32) plan t. It has a gametophytic self incompatibility system based on multiple alleles at an S locus th at has been suggested to have more than 30 alleles (Williams, 1987). This system is also known to have the presence of a self fertility (Sf) 17

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allele partially dominant to the self incompatible alleles. Because of the high degree of outcrossing in white clover, in dividuals are highly heterozy gous and populations are highly heterogenous in nature. White clover breeding programs have b een conducted in different countries and different locations within the USA. The main goals of breeding have been for plant types, yield, seasonal yield, persis tence and resistance to physical stresses, nematodes, insects, viruses, fungi and bacteria. There also have been emphases on improving forage quality and nodulation characteristics (Williams, 1987). The main breeding method used in these efforts has been phenotypic recurrent selecti on. A white clover breeding progr am generally can be a 10 to 12 year long process with this method. One reason for the length of breeding programs is that heritability for many traits is low, e.g. yiel d as reported by Suzuki et al. (1958). Interspecific crosses with perennial and annual species relate d to white clover have been utilized to introduce desirable genes into the cultivated crops. Trifolium ambiguum a strong perennial species that has been hybridized w ith white clover has abundant rhizomes and resistance to many viruses. Trifolium uniflorum another perennial sp ecies that has been hybridized with white clover has larger seeds, shorter internodes and woody roots. The interspecific hybrids of { T. repens T. nigrescens (an annual species)} T. repens T. isthmocarpum (an annual species) T. repens and T. repens T. uniflorum were identified as good sources of southern RKN resist ance (Pederson and Windham, 1989). Several viral and fungal diseases, and nematode s can affect white clover stands. Pepper spot (casual organism, Leptosphaerulina trifoli (Rost.) Petr.) is one of the foliar disease prevalent in cool, wet weather. Soot y blotch (casual organism, Cymadothea trifoli (Pers.) Ex Fr.), cercospora leaf and stem spot (casual organism, Cercospora zebrine Pass.) are some other common foliar diseases that infest white clover. Root and stolon rot is also a profound problem 18

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caused by Fusarium, Rhizoctonia, Collectrotrichum, Mycoleptodiscus, Curvularia, Macrophomina, Scleretinia and Sclerotium (Gibson and Cope, 1985). Root-knot nematodes (RKN, Meloidogyne spp.) can be one of the most impor tant pathological problems of white clover persistence and produc tion (UC SAREP, 2008). Root-knot Nematode Root-knot nematodes (RKN) were first identi fied by Goeldi (1887 as cited by Taylor and Sasser, 1978) and named Meloidogyne exigua Later on, RKN was thought to be a species of Heterodora and synonimized with H. radicicola and H. marioni Chitwood (1949) again described them as a different genus Meloidogyne and identified four species, M. incognita (Kofoid and White) Chitwood, M. arenaria (Neal) Chitwood, M. javanica (Treub) Chitwood and M. hapla Chitwood (Taylon and Sasser, 1978). More than 100 species of the genus Meloidogyne have been reported. Among them, Southern RKN ( M. incognita ) accounts for 51% of the worl dwide population, Javanese RKN ( M. javanica ) accounts for 31% and M. arenaria and M. hapla each contribute 8%. These four species together account for more than 95% of RKN populations worldwide (Sasser et al., 1983). In some RKN species, there are host speci fic races that cannot be differentiated morphologically but only with host differentiation tests. These are known as physiological races. Four host races of M. incognita and two races of M. arenaria have been defined with their differential host specificity to a particular set of hosts (Sas ser et al., 1983). When a large number of M. incognita populations were subjected to North Carolina Host Differential Test (Hartman and Sasser, 1985), M. incognita race 1 comprised about 72% of all M. incognita populations, whereas M. incognita race 2, M. incognita race 3 and M. incognita race 4 accounted for 13%, 13% and 2%, respectively. While in M. arenaria, 16% of populations were race 1 (peanut race) 19

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and other 84% were race 2. M. javanica and M. hapla are not reported to show any host specificity (Sasser et al., 1983). These nematodes have a very extensive host rang e. They have been shown to attack almost every crop of agronomic or horticultural importa nce. But the species and race pathogenicity may be different for different hosts Their worldwide distribution a ppears to be affected by many ecological factors. Average minimum temperatures have been one important factor related to the distribution of all four major Meloidogyne species. Meloidogyne incognita M. arenaria and M. javanica were not found in areas whose average te mperature in cold months was below 3C while M. hapla occurred in cooler climates with mi nimum temperatures as low as -15C and average temperatures of about 24 to 27C. Meloidogyne javanica is best adapted in areas with distinct dry and wet seasons while M. incognita is less adapted to these conditions (Sasser et al., 1983) Root-knot nematodes are sexually dimorphic species. Adult females are pear shaped endoparasites measuring about 0.5 mm in length and 0.3 to 0.4 mm in width. They bear a 12 to 15 m long stylet which is of tylenculous type with a prominent basa l knob (Dropkin, 1989). The stylet has a continuous lumen from the tip to the basal knobs from which this lumen continues to the esophageal tube. They have an esophagus wi th a prominent spherical metacorpus. Muscles attached to the metacorpus serve as a pump fo r food intake (Taylor and Sasser, 1978). Uteri of two gonads join just anterior to the vulva. A distinct perennial patter n (striations surrounding vulva and anus) can be seen in RKNs which ofte n serve as an identifica tion tool for different species (Dropkin, 1989). The adult males are cylindrical., worm shaped, about 2 mm in length, and free living in the soil. Males possess a stylet but the esophagus is not developed as they apparently do not feed on plants (Taylor and Sasser, 1978).The RKN eggs are elongated and oval 20

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shaped. The embryogenesis of RKN is holoblasti c (whole egg dividing) and determinate. The division of cells ultimately leads to the formation of the first stage juvenile (J1) inside the egg. This juvenile stage remains coiled inside the e gg and molts to form a second stage juvenile (J2). The J2 hatches from the egg breaking it by its st ylet (Thorne, 1961). The J2 may remain in the egg mass for some time and then it can move in search of plant roots. The J2 are elongate measuring 400 to 500 m and are the infective stage wh ich bears a stylet, esophagus and esophageal glands. When a root tip is encountered, the J2 penetrates the root just above the root cap and moves intercellularly. It reaches the cort ex region and then pierces the cells in this region with its stylet and an esophageal secretion is injected. These secretions cause the formation of giant cells (synctia) both by larg e cell size (hypertrophy) and cell number due to intense cell multiplication (hyperplasia) (Thorne, 1961). Each infected host cell enlarges and the large cen tral vacuole is replaced by small vacuoles while the cytoplasm increases in volume and density. The cell wall is remodeled to form elaborate ingrowths which are the sites to meet the nutrient demand of nematodes (Hussey and Janssen, 2002). This process then may lead to the development of visi ble galls (Taylor and Sasser, 1978). Dropkin (1969) suggest ed that the ce llular reaction of plant cells to RKN was not a passive reaction to enzymes but an active host participation to some controlling force of the parasite. After the J2 establishes itself in th e plant root system, its width increases and the esophageal gland enlarges. The cell of genital primordium starts to differentiate either into a female form exhibiting fork shape or into a male form exhibiting cy lindrical growth. With continuous feeding the second stage juvenile becomes flask shaped and molts twice to form third stage juvenile (J3) and fourth st age juvenile (J4). Male and female adults start to differentiate 21

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with the third stage larvae. The female become s pyriform shaped while the male remains eel shaped (Taylor and Sasser, 1978). Except for some M. hapla populations (Triantaphyllou, 1966) all other species reproduce parthenogenetically. Oogoni a are formed in female reproductive system and divide mitotically. The most advanced oogonia then stops dividi ng and becomes an oocyte which ultimately becomes an egg after one mitotic division and is deposited in a gela tinous egg mass surrounding the posterior end of the female. The number of e ggs in an egg mass may va ry with an average of 200-300 (Taylor and Sasser, 1978). Larvae hatched from eggs move to nearby ro ot cells within the same gall/root system. When there is complete destruction of cell tissu es, the larvae move to nearby roots of the same plant or other plants. There are very low numbers of males in root galls and the number may vary according to the microclimatic conditions (Thorne, 1961). Since RKN invade and damage fine roots, the RKN infected plants wilt easily, become stunted and may die. Symptoms of chlorosis may also be seen. The RKN damage in infected fields often is manifested as patches of dead plants indicating localized areas of high infection. The clear sign of root-knot nemat odes is that the roots are swolle n due to galling and have a knot like appearance. Young small seedlings may die without any clear sign of galling (Thorne, 1961). One reason for the spotty app earance of field damage is th at RKN are sedentary parasites and do not move long distances laterally but move up and down according to the soil water table. Root-knot Nematode Disease in White Clover The association of root-knot nematodes and white cover is well documented. Root-knot nematodes on white clover have been found in a very wide geographic ar ea including the USA (Cook and Yeates, 1993), Australia (Mcleish et al., 1997), New Zealand (Skip and Christensen, 1983), and Europe (Cook et al., 1992). The presence of RKN in white clover has been shown to 22

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decrease yield and persistence of white clover. The economic loss due to infestation with M. incognita has been reported to limit persistence in white clover by 51 to 79% (Baxter and Gibson, 1959). As a forage crop, the whole top part of the plant is economically important. Brink and Windham (1990) reported a reduction in dry weight of stolons by 36 to 83% by infection of RKN in SC-1 and Regal white clover. The numbers of stolons also were decreased by 12 to 20% and yield was reduced by 6 to 17% in a study by Pederson et al., (1991) in Mississippi USA. About 58% of New Zealand white clover pastures were infected by Meloidogyne Spp (Skipp and Christensen, 1983), and about 77% of Aust ralian pastures (Mcleish et al., 1997). M. hapla appears to be the predominant root-knot nematode species in cool regions. In England and Wales 4% of white clover pastures were infect ed by this species (Cook et al., 1992). Root-knot nematodes have been shown to be one of the factors adversely affecting white clover growth, stolon density, persistence, seed ling vigor, nitrogen fixing ability, and phosphorus utilization (Zahid et al., 2001). In addition to thei r direct invasion effects, RKN root penetration can create wounds for secondary pathogens attac k. The secondary attack is caused due to the access to the root facilitated by nematodes, the change in rhizosphere, physiological changes and resistance break caused by interactions (Eva ns and Haydock, 1993). Nematodes interact with other pathogens including fungi, bacteria viruses, and even other nematodes. Fusarium Oxysporum and Verticilium spp are common wilt inducing para sites that interact with RKN (Francl and Wheeler, 1993). Rhizoctonia solani, Pythium ultimum, and Fusarium oxysporum are some other fungi that have been shown to inte ract with RKN to cause root rot (Evans and Haydock, 1993). The RKNs also interact with ot her ecto or sedentar y parasites such as Hoplolaimus galeatus H. columbus Tylenchorhynchus vulgaris Scutellononema brachyrum to stimulate their reproduction and penetrance (Eis enback, 1993). The interaction of RKN and 23

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Mycorrhizae can reduce plant yiel d (Francl, 1993). Nematodes can interact with bacteria by creating wounds for bacterial entr y and increase susceptibility by modifying plant cells, breaking resistance to bacteria, and sometimes act as a vector. Pseudomonas solanacearum and Meloidogyne spp interaction is one of the common bacterial-RKN interaction. The RKN also interact with Corynebacterium flaccumfaciens, Xanthomonas phaseoli, Erwinia carotovora (Sitaramaiah and Pathak, 1993). It has also been shown that RKN can reduce nodulation by interacting antagonistically w ith nodule inducing bacteria ( Rhizobium spp.) (Abd-El-Samie and Taha, 1993). When nematodes infect one part of a plan t host, the entire ph ysiological processes throughout the plant may be disrupted. These phys iological anomalities then may affect host plant yield and persistence (Melakeberhan and Webster, 1993). The control of RKN disease in white clover can be difficult. Although some nematicides have been shown to decrease RKN populations (Taylor and Sasser, 1978; Yeates et al., 1975), currently there are no registered nematicides for use in pasturelands. Crop rotations have been proposed to limit RKN infestation as juvenile s generally move no more than 50 cm. Crop rotations including host and non host plants may help reduce the nematode populations in the field (Taylor and Sasser, 1978). Other cultural practices including sa nitation, fallowing, dessication, and use of antagonistic plants have also been pract iced (Taylor and Sasser, 1978; Sasser et al., 1983). Biol ogical controls using Paecilomyces fungus (Sasser et al., 1983), Catenaria anguillulae Arthrobotrys Dactylella (Taylor and Sasser, 1978) had been utilized. Other predatory nematodes, arthropods, and wo rms can also be utilized but with little documented effect (Taylor and Sasser, 1978). Sasser et al. (1983) summarized that effective control would be the best combination of all available control measures, including resistant 24

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cultivars, crop rotation, nematic ides, and sanitary and cultura l practices used to develop integrated crop protection systems. Thus development of resistant varieties is likely the best solution to have a persistent produc tive forage crop in the field. Root-knot Nematode Resistance Breeding Selections have been conducted for decades to achieve the RKN resistance in white clover. Bain (1959) evaluated lines of white clover seedli ngs and selected genoty pes with tolerance to RKN. Gibson (1973) developed S C-1 white clover, which was reported to be tolerant to southern RKN. This population was a first generation recombination among 145 genetically diverse white clover clones selected for toleranc e to southern RKN. Those clones were screened from thousands of plants from white clover cu ltivars and foreign intr oductions. Studies from Windham and Pederson (1989) showed that SC-1 wa s only moderately tolera nt to two of eight populations of M. incognita This different response compared to the results of Gibson may be due to the fact that different races were used by Windham and Pederson that overcame the resistance in SC1. These results show that there is a need for selection and evaluation using all the predominant races and populatio ns of RKN. Mercer et al. ( 2000) gained some success in selecting white clover strains resistant to M. trifolia (Bernard & Eisenback), previously thought to be M. hapla This Meloidogyne isolate failed to reproduce in tomato and other plants of the North Carolina Host Differential Test but reproduced in white clover and as a consequence was taxonomically described as a new species, M. trifolia (Zahid et al., 2001). Pederson and Windham (1995) have released MSNR4 after four cycles of recurrent selection from a wide pool of white clover germplasm. This popul ation was shown to be resistant to M. incognita [percent root system galled (PRSG) score of 1.0, egg score of 2.3], M. arenaria (PRSG score of 0.9 and egg score of 2.2) and M. graminicola (Golden & Birchfield) (PRSG score of 0.9 and egg score of 1.9). The cultivar UFWC5 was recently developed by recurrent phenotypic selection 25

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using Osceola as the base population and southern root-knot nematode r ace 4 as the selective pathogen. This population was officially released by the University of Florida (Wofford and Ostmark, 2005). A standard greenhouse screening procedure as described by Quesenberry et al. (1993) was used for the selection process. The tw o week old seedlings were inoculated with ca. 1200 to 1500 eggs of M. incognita race 4 Eight weeks after inoculation, the seedlings were extracted from growth containers, washed and i mmersed in Phloxine-B to highlight egg masses. The root system of individual plants was evalua ted for gall and egg masses using the scale; 0 = no gall and/or egg mass, 1 = 1 to 2 galls and/or egg masses, 2 = 3 to 10 ga lls and/or egg masses, 3 = 11 to 30 galls and/or egg masses, 4 = 31 to 100 galls and/or egg masses and 5 = more than 100 galls and/or egg masses. Only elite plants with the lowest gall a nd egg mass scores were selected. It was field tested la ter and showed resistance to Southern RKN (Wofford and Ostmark, 2005). Nevertheless it is important to screen any cultivar for all th e predominant species/races of RKN because their resistance in teraction may be different. Other than recurrent selecti on, interspecific hybridization, ge netic transfer, and somaclonal variations can be other possible sources for re sistance breeding. The study from Pederson and Windham (1989) also showed that interspecific h ybrids utilizing T. nigrescens could be utilized in resistance breeding. Quesenberr y et al. (1997) and Koume et al (1998) have identified several native North American Trifolium species resistant to RKN. Two annual species, T. carolinianum and T. bejariense were found to be resistant and two perennial species, T. calccaricum and T. stoloniferum were highly resistant. But their lack of sexual compatibility with cultivated clovers has been a constraint to gene transfer. 26

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Although some sources of resistance have b een found and incorporated, without a proper understanding of the inheritance patterns and genetics behind the resistance to RKN, it is difficult to achieve success in resistance breeding. Mode of Resistance There are many mechanisms related to how plants defend against RKN. One of the mechanisms is non-preference where a re sistant plant allows entrance of Meloidogyne juveniles that subsequently leave the plant due to non-pr eference and seek an al ternative host. Another mechanism of resistance is hypersensitivity wher e cell are penetrated by nematodes die quickly blocking further development of nematodes. Reduced juvenile growth rate is another mode of resistance and the inhibition of female growth is also another mechanism which causes an increased sex ratio of males to females and reduces egg production (Dropkin, 1989). Genetics of Resistance The nature of resistance to RKN has been de scribed as varying from control by a single dominant gene to polygenic inhe ritance. Several dominant or semidominant resistance genes have been identified and mapped (Williamson and Hussy, 1996). Plum [ Prunus cerasifera (Ehrh.), Salses et al., 1998], peach [ P. persica (L.) Batsch Claverie et al., 2004], tomato [ Solanum lycopersicom (L.), Williamson, 1998], peach [P. persica (L.) Batsch] for M. javanica (Zhen-Xiang et al., 2000) are reported examples of a single dominant gene for resistance. Resistance in peach [ P. persica (L.) Batsch] to M. incognita was described as controlled by two dominant genes (Zhen-Xiang et al., 2000); whereas resistance in blackeye-type cowpea [ Vigna unguiculata (L.) Walp.] line H8-8R was controlled by a single recessive gene (Ehlers et al., 2000). Red clover [ Trifolium pratense (L.), Quesenberry et al., 1989] is an example of a number of legumes that have shown polygenic inheritance of resistance to RKN. In some cases, 27

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polygenic resistance has been resolved into major genes that are genetically dominant and minor genes that may modulate the response (Williamson and Hussey, 1996). Barett et al. (2005) have id entified a single dominant ge ne (designated TRKR) in Trifolium semipilosum which conferred resistant to clover root-knot nematode ( M. trifolia ) by screening with T. repens SSR markers. In tomato and some other cr ops, the Mi (Mi-1) gene was identified conferring resistance to M. incognita M. javanica and M. arenaria (Hussey and Janssen, 2002). Mi-3 (Tomato), Mi-9 (Tomato), Ma (Plum), Me3 (Pepper, Capsicum annuum L.), Rmc1 (Potato, Solanum tuberosum L.) are other mapped genes that confer resistance to one or more species of RKN (Williamson and Kumar, 2006). The RKN resi stance in soybean was identified as multigenic and quantitative and some Quantitativ e Trait Loci (QTL) have been identified. (Tamulonis et al., 1997). The resistance mode of inheritance to M. hapla is not as straightforward and is always under oligogenic or polygenic control (Bunte et al., 1997). Experiments by Van De Bosch and Mercer (1996) showed the variabilit y for resistance to an unidentified Meloidogyne species thought previously to be M. hapla, but more recently classified as M. trifolia had low repeatability (heritability). Broa d-sense heritability estimates also showed that breeding for resistance is possible, but that progress could be slow. Regardless of the number of genes involved in resistance, for breeding it is important to estimate the type of gene action involved. Pa rtitioning of the varian ces to additive and nonadditive sources of variation can be more important in the case of a quantitative mode of inheritance (Zhang et al., 2007). Partitioning variance components into General Combining Ability (GCA) and Specific Combining Ability (SCA) can be very useful in designing a breeding program. The GCA is defined as the averag e performance of a line in multiple hybrid 28

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combinations while the SCA is defined as the performance of a specific cross. Sprague and Tatum (1942) defined SCA as the deviation exp ected from the sum of the GCA of both the parents. This information will be helpful in development of synthetic varieties (Baker, 1978). Many authors have looked at GCA and SCA effe cts. Studies on white clover (Pederson and Windham, 1992), corn [ Zea mays L., Williams and Windham (1990)], cotton [Gossypium hirsutum L., Mcpherson et al. (1995); Zh ang et al., (2007)], and red clover (Call et al., 1997) are some examples that have identified GCA as more important than SCA for resistance to RKN. Pederson and Windham (1992) used three resistant and three susceptible plants for a diallel study and found that resistant parent s produced progeny with the least M. incognita reproduction while susceptible parents produced susceptibl e progeny. Progeny from two crosses performed worse than expected from the GCA effects of the parents but no crosses performed significantly better than expected. Although nonadditive gene actions such as dominance and epistasis might have been involved in some crosses, addi tive gene action was more significant. A diallel analysis of four resistant, thr ee intermediate, and two susceptible red clover parents performed by Call et al. (1997) also sh owed predominantly sign ificant GCA effects and non significant SCA effects. The crosses involvi ng a resistant parent (119) showed the least number of galls and egg masses while the cr osses involving suscepti ble parents (N1, K4) produced the highest number of galls and egg masses. This study also suggested the importance of additive gene action in breeding for RKN resistance in red clover. Statistics According to Sprague and Tatum (1942), aver age mean performance of a cross between two lines is expressed as eqauation (Eq. 2-1) ij = GCAi+ GCAj+SCAij (2-1) 29

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The differences due to GCA are due to addi tive genetic variance and additive additive epistasis while the differences due to SCA are due to non-additive variances (dominance, and dominance additive epista sis). The relative contribution of GCA and SCA would be determined by the magnitude of additive and non-additive variation. Griffing (1956) has postulated a model for th e estimation of GCA and SCA using diallel mating designs. He proposed eight different models according to the crosses included and fixed and random effects in the model. Griffings anal ysis method 4, model I is based on fixed effects and crosses that do not include pa rents and reciprocals. So there are n (n-1)/2 entries where n is equal to the number of parents. Statistically, the phenotypic variation is given by the equation. (Eq. 2-2) 2 P = 2 G + 2 E (2-2) where, 2 E = Environmental variation 2 G = Total genotypic variation this total genotypi c variation is given by Eq. 2-3 2 G = 2 A + 2 NA (2-3) where, 2 A = additive variance 2 NA = non-additive variance In the absence of epistasis non-additive vari ance is equivalent to dominance variance ( 2 D). In the case of completely inbred parents (F = 1), th e additive and dominance variance are equivalent as given in Eq. 2-4 a nd Eq. 2-5 respectively. 2 A = 2 2 GCA (2-4) 30

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2 D = 2 SCA (2-5) but in the absence of inbreeding (F = 0), the additive and dominance variance are equivalent as given in Eq. 2-6 and Eq. 2-7 respectively. 2 A = 4 2 GCA (2-6) 2 D = 4 2 SCA (2-7) The phenotypic value of any cross is also composed of the GCA and SCA effects. Statistically, the phenotypic value of ijth observation can be repr esented as equation 2-8. xij = +gi+gj+sij + (2-8) where, = population mean gi(j ) = GCA effect of ith (jth) line sij = SCA effect of cross of ith and jth line including reciprocals = Environmental error For the purpose of identifying the relativ e importance of GCA and SCA effects many authors have used the GCA:SCA variance ratio (Baker, 1978). The nearer the ratio is to unity, the greater will be the prediction of progeny based on a single parent. Due to the cumbersome calculations needed to conduct a diallel analysis, many authors have reported the use of statis tical analyses programs. One of the most popular programs for diallel analysis in crop species is DIALLELSAS written by Zhang and Kang (1997) and its successor DIALLEL-SAS05 (Zhang et al., 2005). Bo th of these programs are written in SAS utilizing the GLM procedure. Xiang and Li ( 2001) have also devel oped a program in SAS utilizing PROC MIXED. Some authors have also referenced a program written by Burrow and 31

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Coors (1994). Magari and Kang (1994) also re ported a program in BA SIC for analysis of Griffings models. Availability of root-knot nematode resistant cultivars can be very helpful to farmers who wish to incorporate legumes in grass dominate d pastureland. The availability of a new white clover cultivar showing toleran ce to southern RKN can be advantageous for producers. The existence of multiple populations of RKN requires the screening of this new cultivar to all those economically important RKN populations. The unders tanding of the inheri tance pattern of the resistance to RKN helps in the further breeding attempts. Thus the focus of this research was characterization of the response of UFWC5 to multiple RKN species/races, and study of the quantitative basis of inheritance of resistance to those populations. 32

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CHAPTER 3 COMPARISON OF OSCEOLA AND UFWC5 FOR RESPONSE TO DIFFERENT SPECIES/RACES OF ROOT-KNOT NEMATODE Abstract White clover ( Trifolium repens L.) is a major forage crop of the southeastern USA, including Florida. Although it is a cool season perennial legume it acts as an annual in Florida. White clover is one of the most nutritious forages available and is generally mixed with grasses to increase their nutritive valu e. There are many constraints to white clover production. Rootknot nematodes ( Meloidogyne spp., RKN) can be a factor adve rsely affecting the white clover growth, stolon density, persistence, seedling vigor, nitrogen fixing ability, and phosphorus utilization. Root-knot nematodes ar e endoparasites that have a dive rse host range. In addition to their direct invasion, they create wounds that can lead to in fection by secondary pathogens. No nematicides are labeled for pastures but even if available, it is likely their use would be cost prohibitive. Thus, development of resistant varieties appears to be the best solution to enhance field production and persistence of white cl over. The cultivar UFWC5 was developed by recurrent phenotypic selection for reduced RKN galling and was recently released primarily on the basis of improved tolerance to root-knot nema todes. This research compared UFWC5 and the commercial cultivar Osceola fo r response to six different RKN species and/or races (herein after called RKN populations). Nine ty-eight plants of UFWC5 and of Osceola were planted in Cone-tainers (Steuwe and Sons, Inc., Tangent, OR) in a randomized complete block design to access response to each RKN population. Three weeks after germination, 98 plants of UFWC5 and of Osceola were inoculated with ca. 500 eggs (ca. 3 eggs cm-3 of soil) of each RKN population. Nine weeks after inocul ation, data were collected for shoot growth, root growth, egg mass score, gall score and eggs per plant. Differe nces in response to a ll six RKN species/races were observed for egg mass score, gall score and eggs per plant with UFWC5 being lower than 33

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Osceola for all comparisons. The largest reduc tion in gall score and egg mass score between Osceola and UFWC5 were observed in response to the four races of M. incognita the species that was used in the selection process. Introduction Osceola is an established cultivar of white clover ( Trifolium repens L.). Although it has been planted for over 20 years and has many usef ul traits it lacks resistance to root-knot nematodes (RKN). Root-knot nematodes ( Meloidogyne spp.) may be a limiting factor to the growth and establishment of white clover in the southeastern US A. There have been previous selection efforts to breed for resistance to RKN in white clover. Bain (1959) evaluated lines of white clover seedlings and selected genotypes w ith tolerance to RKN. Gibson (1973) developed SC-1 white clover, which was reported to be tole rant to southern RKN. The SC-1 population was a first generation recombination among 145 genetically diverse white clover clones selected for tolerance to southern RKN. Those clones were sc reened from thousands of plants from white clover cultivars and fore ign introductions. Studies from Windham and Pederson (1989) showed that SC-1 was only moderately tolera nt to two of eight populations of M. incognita This different response compared to the results of Gibs on may be due to the fact that different races were used by Windham and Pederson that overc ame the resistance in SC1. These results show that there is a need for selection and evaluation using all the predominant races and populations of RKN. UFWC5 is a new cultivar derived fr om Osceola through five cycles of phenotypic recurrent selection for resistance to Southern RKN ( M. incognita) (Wofford and Ostmark, 2005). Among more than 100 species of Meloidogyne, four species account for more than 95% of worldwide RKN population. Southern RKN ( M. incognita ) accounts for 51% of the worldwide population, Javanese RKN ( M. javanica ) accounts for 31% and M. arenaria and M. hapla each contribute 8% (Sasser et al., 1983). In some specie s, host specific races are found that cannot be 34

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differentiated morphologically but can through host differentiation tests. These are known as physiological races. Four host races of M. incognita and two races of M. arenaria have been defined with their host specificity to a particular set of hosts (Sasser et al., 1983) when the populations were subjected to No rth Carolina Host Differential Te st (Hartman and Sasser, 1985). Meloidogyne incognita race 1 comprised about 72% of all M. incognita populations whereas M. incognita race 2, M. incognita race 3 and M. incognita race 4 accounted for 13%, 13% and 2%, respectively (Sasser et al., 1983). The plant response to the RKN can be assessed by the amount of galling and the host plant effects on the RKN lifecycle can be accessed by egg and egg mass production. Mani (as cited by Bird, 1979) described galls as the ph ysiologically developed cells, tissu es or organs of plants that mostly arise by hypertrophy and hyperplasia under th e influence of a parasitic organism. After a RKN infective juvenile (J2) establishes inside th e root, it will typically form galls. With normal life cycle progress, females reproduce by laying eggs in a gelatinous egg mass on the root surface (de Guiran and Ritte r, 1979). Thus galling can be viewed as a measure of the response of the plant to RKN infection, and egg mass producti on can be viewed as a measure of RKN ability to reproduce on a given host. A single egg mass may contain 200 to 300 nematode eggs (Taylor and Sasser, 1978). Therefore, egg counts are more representative of RKN reproduction than egg mass score alone, but egg extraction for counting is a labor and time consuming variable to determine. The cultivar UFWC5 was originally selected for resistance to M. incognita race 4. As discussed above regarding the existence of multiple economically important populations of RKN, it is necessary to screen any cultivar with as many of the RKN populations as possible. In this research, we tested the response of UFWC5 in comparison to Osceola to M. arenaria race 1 35

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(peanut RKN), M. javanica (Javanese RKN) and 4 races of M. incognita (M. incognita race 1, M. incognita race 2, M. incognita race 3 and M. incognita race 4). Materials and Methods To test the response of white clover cultivars to each isolate of RK N, 98 seeds of Osceola and an equal number of UFWC5 were planted in Ray Leach Cone-tainers (ca. 150 cm3 soil volume) (Stuewe & Sons Inc., Tangent, OR) a nd placed in RL98 trays (Stuewe & Sons Inc., Tangent, OR) for support. Before planting, the s eeds were gently scarified. The cone-tainers were filled with commercial building sand. Two week s after germination of the seeds, the plants were inoculated with ca. 500 eggs of the appropriate Meloidogyne populations. Just prior to inoculation, the cultivars UFWC 5 and Osceola were arranged in a randomized complete block design of 7 replications and 14 pl ants per replication. Each of the six different RKN populations used ( M. incognita race 1, M. incognita race 2, M. incognita race 3, M. incognita race 4, M. arenaria race 1, and M. javanica ) was treated as a separate randomized complete block experiment. Due to nematode containment issues, a second environment consisting of 7 replications of 7 plants each of Osceola and UF WC5 was planted at the same time and compared as a non-inoculated control for shoot and root wei ghts only. An extra flat of Osceola was planted and inoculated at the same time and evaluate d for nematode symptom progression to determine the appropriate time for termination of the e xperiment. The experiment was terminated when most of the check Osceola plants showed a root galling score and egg mass score between 3 and 5. Nematode Egg Extraction and Inoculation The six different RKN populations were mainta ined in a separate greenhouse from that used for the experiment. These nematodes were maintained on RKN susceptible Rutgers tomato ( Solanum lycopersicom ). Nematodes were extracted from the plants which had been 36

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inoculated on 9 to 10 weeks earlier. The tomato plant was uprooted gently and the root system was washed gently. After washing, the roots were cut into small pieces about 2 cm in length. Then roots were placed in a bl ender with 0.25% chlorine to brea k the proteinous gel of the egg mass and blended for 20 seconds. Sieves of 500, 200 and 50-mesh size were stacked together with 50-mesh on top and 500-mesh on bottom. The blended solution was poured through this sieve stack and washed for 2 to 3 minutes with tap water to remove most of the chlorine. The residue remaining on the 500-mesh sieve (primarily RKN eggs) wa s collected in a beaker and diluted. The concentration of the nematode eggs was determined using a hemocytometer slide (1 ml volume composed of 24 grids). Four ra ndom grids were counted at 40 on a compound microscope. This process was repeated three times and the counts were then averaged and multiplied by 24 to estimate the total nematode eggs in 1 ml solution. This number was then multiplied by the total volume of solution to obtain the total number of eggs extracted. The extracted egg solution was then diluted so that a 3 ml injection contained ca. 500 eggs and this volume was injected into each cone-t ainer containing two-week old seedlings. This solution was placed in a 3.5 L beaker with a magnetic stirrer inside it to keep the egg suspended in solution while inoculating. All the six races were inoculated by this procedure. The inoculum concentration of M. incognita race 1 was ca. 1000 eggs per plant. A tray with Osceola was also inoculated with the respective race of RKN as a susceptible control. Between any two subsequent extractions and inocul ations, all the apparatus were cl eaned using chlorine to kill the previous nematode eggs and pr event any cross inoculation. Maintenance These plants were regularly fertilized and irri gated until they were ready for data collection as determined by the root galling of th e Osceola. The fertilizer used was Peters 20:20:20 N:P2O5:K2O. A diluted solution of 1.5 g L-1 of N, P2O5, K2O was applied as irrigation weekly. 37

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The plants were also treated for thrips, mites, aphids, white flies, army worms and other worms, and snail. We used tank mixture of Avid (0.5 mL L-1), Parmethrin (0.5 mL L-1), Conserve (1.5 mL L-1), Mavrick (0.5 mL L-1) for mite control. We used tank mixture of Enstar (0.8 mL L-1), Parmethrin (0.5 mL L-1), Conserve (0.8 mL L-1) for aphids and white fly control. We used tank mixture of Enstar (0.8 mL L-1), Parmethrin (0.5 mL L-1), Conserve (1.5 mL L-1), Mavrick (0.5 mL L-1) for thrips control. We also used Xentari (2.5 mL L-1) for controlling army worm. Ortho (Bug-Geta bait) was used for snail and slug control. Data Collection Depending upon the nematode population, the plan ts were evaluated 8 to 10 weeks after inoculation. The root system of plants were car efully removed from container and washed. Roots were then immersed in a solution of 0.05% red food color (McCormik & Co, Hunt Valley, MD). Although other researchers have used Phloxine-B to stain egg masses (Holbrook et al., 1983), we found the red food color to be equally eff ective with a reduced le vel of toxicity than that of Phloxine-B. Individual plants were gi ven a score for egg mass and galls. They were scored as 0 = 0 galls/egg masses, 1 = 1 to 2 ga lls/egg masses, 2 = 3 to 10 galls/egg masses, 3 = 11 to 30 galls/egg masses, 4 = 31 to 100 galls/egg masses and 5 = more than 100 galls/egg masses (Taylor and Sasser, 1978). After scoring, the individual plants were separated into root and shoot. All shoots from a replication were plac ed in paper bag and drie d at 50C to constant weigh. Root parts of a replicati on were also collected in a plas tic bag and processed further for egg extraction. The root systems of a replication were cut into smaller pieces of about five centimeters and mixed with 0.5% chlorine solution. This was bl ended for 20 seconds and sieved through the stack of three sieves of 500, 200 and 50-mesh size. Residue from the bottom (500-mesh size) 38

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sieve containing the nematode eggs was collected in a tube for counting. The macerated roots were collected in a paper bag and also dried at 50C to constant weight. The egg solution collected in the tube was br ought to a fixed volume and counted using the same procedure described above for inoculat ion. The scoring of ga lls, egg masses and egg counting were done by different i ndividuals who were always asso ciated with replications. Data were analyzed as a randomized comple te block using the GLM procedure in SAS. The means were separated using Duncans critical range (CR). We compared the shoot and root weights of the inoculated vs non-inoculated Osceola and UFWC5 using a model of entries nested within inoculation treatments. Results and Discussion An analysis of variance showed that there were significant ( P < 0.01) differences in gall scores and egg mass scores between Osceola an d UFWC5 for all RKN populations (Table 3-1). Osceola showed a higher degree of susceptibil ity for both variables. Although the differences were statistically significant ( P < 0.01) in all races, there was a marked difference in the egg mass scores when these plants we re inoculated with any of the M. incognita races while less marked for plants inoculated with M. arenaria race 1 and M. javanica This is likely due to the fact that UFWC5 was originally selected using race 4 of M. incognita. For the gall scores, there was also marked difference between Osceola and UFWC5 for all races of M. incognita except race 1 although it was sta tistically significant ( P < 0.01). There was also a marked difference in the means gall score of Osceola and UFWC5 wh en these plants were inoculated with M. arenaria race 1 and M. javanica The plants with scores 0, 1 or 2 are categorized as resistant and 3, 4 and 5 are categorized as susceptible (T aylor and Sasser, 1978). If one follows this convention, UFWC5 would be categorized as re sistant to all the M. incognita races. For M. arenaria race 1 and M. javanica the mean scores are in betw een 2 and 3, so UFWC5 cannot be 39

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categorized as resistant to these species but they show significantly lower scores of gall and egg mass than Osceola. There was a strong correlation between the egg mass score and gall score for Osceola (r = 0.73, P < 0.001) while there is less correlati on between those tw o variables for UFWC5 (r = 0.35, P < 0.001) when inoculated with M. incognita race 1. This may signify for UFWC5 that although nematodes enter the plant r oot and initiate galling, they may not have matured to egg producing females. This differe nce between Osceola and UFWC5 was also found when plants were inoculated with M. arenaria race 1. While for the other races of M. incognita and for M. javanica the correlations between the gall scores and egg mass scores were similar for both Osceola and UFWC5 (Table 3.2). The differences in M. incognita race 1 may be due to the fact that the plants were inoculated with ca.1000 eggs per plant (about double than other populations). A higher amount of inoculum might ha ve lead to higher gal ling and reproduction in Osceola. Although the higher galling was achieve d in UFWC5, the nematodes might not have completed their lifecycle to produ ce eggs. The similar result of M. arenaria race 1 could be due to the reported aggressiveness of this species. The eggs plant-1 were significantly (P < 0.001) reduced in UFWC5 compared to Osceola when inoculated with any race. Eggs plant-1 were reduced by 50% in UFWC5 compared to Osceola when inoculated with M. incognita race 1 and was reduced by ca. 70% when inoculated with M. arenaria race 1. When inoculated with other four RKN populations reductions were ca. 80 to 90% (Table 3.1). Although the numbers of eggs per plant were still high enough to maintain the population in the soil, the reduced numbers on UFWC5 compared to Osceola should give improved stand persistence in UFWC5. As expected there were no galls, egg masses and nematode eggs in the non-inoculated Osceola or UFWC5. The shoot weig hts were not significantly diffe rent between the inoculated 40

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and non-inoculated plants of Osceola for all the races used except for M. incognita race 4 ( P = 0.03) (Table 3.3). Similarly, the shoot weights in UFWC5 were not si gnificantly different between the inoculated and non-inoculated for any RKN population used. But there was a significant difference in the root weights of Osceo la with higher weights in non-inoculated roots in comparison to those when inoculated with M. incognita race 2 ( P = 0.002) and M. incognita race 3 ( P < 0.001). For UFWC5, the roots showed a signi ficantly lower weight in the inoculated treatment when inoculated with M. incognita race 2 ( P = 0.0002), M. incognita race 3 ( P < 0.001), M. incognita race 4 ( P = 0.007) and M. javanica ( P = 0.01). Except for these, there was no significant difference between inoculated and non-inoculated UFWC5 for the other RKN populations although the roots tende d to weigh higher in the inoc ulated treatments (Table 3.3). The reason for higher root weight on non-inoculated treatments than inoculated is likely due to the decay of plant root system. The M. incognita race 2 and M. incognita race 3 were harvested at ten weeks versus eight weeks for other races Thus there may have been an opportunity for additional disease development and decay of root sy stem in the plants inoculated with these two races leading to the higher root we ights in the inoculated treatment. There were no significant di fferences between the shoot and root weights of noninoculated Osceola vs UFWC5 (T able 3.3). This fact leads us to believe that yield and production characteristics were not altered by th e selection for RKN resistance in Osceola. UFWC5 showed resistance to the races of M. incognita studied under these greenhouse condition. Although UFWC5 cannot be classified as resistant (score of 2.0 or less) to M. javanica and M. arenaria race 1, it did demonstrate reduced galls and egg masses comp ared to Osceola for these nematode populations. UFWC5 can be utilized with a high level of confidence in southern root-knot nematode in fested areas while there may be need for further cycles of 41

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selection for resistance to peanut and northern root-knot nematode populations. With very short growth period in the greenhouse and with good irri gation and fertilizatio n, the shoot and root growth was not observed to be impacted by ro ot-knot nematodes, but with a longer growth period and exposure to moisture stress the root system will ultimately decay or become less functional for translocation of minerals and wate r; and thus, will likely effect yield and stand persistence. Additional study of the mechanism(s) of reduction in root galling, egg mass production and egg number production would seem to be fruitf ul areas for research. Call (1997) showed preinfectional or early post-infectional resistance indicated by lower penetr ation (as measured by gall score) and post-penetration resistance showed by delayed maturation, lower fecundity rates and fewer adult females (as measured by egg mass sc ore) in red clover selected for resistance to RKN. Similar types of mechanisms may have been involved in expression of resistance RKN in UFWC5 white clover. Further resear ch on the mechanism(s) of resist ance should also be fruitful. 42

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Table 3-1. Egg mass score, gall score and eggs plant-1 of Osceola and UFWC5 white clover when inoculated with six different root-knot nematode populations. RKN population Cultivar Egg mass score Gall score Eggs plant-1 M. incognita race 1 Osceola 3.5a* 2.6a 17,600a UFWC5 0.9b 2.0b 9,200b CR 0.3 0.3 5,500 M. incognita race 2 Osceola 3.8a 3.8a 28,700a UFWC5 1.3b 1.7b 4,400b CR 0.2 0.3 9,000 M. incognita race 3 Osceola 3.7a 3.7a 22,300a UFWC5 0.6b 1.0b 2,300b CR 0.3 0.3 3,000 M. incognita race 4 Osceola 3.9a 3.3a 44,200a UFWC5 1.6b 1.0b 4,800b CR 0.3 0.3 12,000 M. arenaria race 1 Osceola 3.9a 3.5a 28,800a UFWC5 2.6b 2.4b 8,900b CR 0.2 0.2 3,600 M. javanica Osceola 4.0a 4.2a 71,900a UFWC5 3.0b 2.6b 14,600b CR 0.2 0.2 9,700 Means followed by differen t letters are different (P = 0.05). Egg masses and galls were rated on a 1 to 5 sc ale where 0 = 0 galls or egg masses, 1 = 1 to 2 galls or egg masses, 2 = 3 to 10 galls or egg ma sses, 3 = 11 to 30 galls or egg masses, 4 = 31 to 100 galls or egg masses and 5 = mo re than 100 galls or egg masses CR: Duncans critical range ( P = 0.05) 43

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Table 3-2. Correlations between gall scores a nd egg mass scores of Osceola and UFWC5 white clover when inoculated with six RKN populations. Combined Osceola UFWC5 M. incognita race 1 0.56 *** 0.73*** 0.35*** M. incognita race 2 0.87*** 0.61*** 0.69*** M. incognita race 3 0.86*** 0.65*** 0.69*** M. incognita race 4 0.89*** 0.65*** 0.67*** M. arenaria race 1 0.54*** 0.87*** 0.33*** M. javanica 0.69*** 0.43*** 0.46*** *** significance in < 0.001 probability level. 44

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Table 3-3. Response in the shoot and root grow th of Osceola and UFWC5 white clover when inoculated with six different popu lations of root-knot nematodes. Nematode population Varieties Shoot Root Inocula ted Noninoculat ed CR Inocula ted Noninoculat ed CR -g-g-g-gOsceola 1.05 1.15 0.21 0.40 0.31 0.15 M. incognita race 1 UFWC5 0.94 0.95 0.01 0.35 0.27 0.15 CR 0.16 0.14 0.14 0.15 Osceola 1.36 1.26 0.22 0.44 0.80 0.14 M. incognita race 2 UFWC5 1.29 1.27 0.19 0.39 0.83 0.01 CR 0.17 0.3 0.10 0.13 Osceola 1.22 1.26 0.22 0.40 0.80 0.15 M. incognita race 3 UFWC5 1.35 1.27 0.43 0.36 0.83 0.19 CR 0.26 0.30 0.16 0.13 Osceola 0.99 1.17 0.17 0.36 0.45 0.13 M. incognita race 4 UFWC5 1.00 1.16 0.24 0.34 0.47 0.08 CR 0.18 0.23 0.06 0.16 Osceola 1.21 1.15 0.14 0.44 0.31 0.12 M. arenaria race 1 UFWC5 0.91 0.95 0.07 0.33 0.27 0.07 CR 0.10 0.16 0.06 0.15 M. javanica Osceola 1.02 1.17 0.22 0.39 0.45 0.14 UFWC5 0.98 1.16 0.19 0.35 0.47 0.08 CR 0.19 0.22 0.08 0.16 CR: Duncans critical range ( P = 0.05) between inoculated and non-inoculated plants CR: Duncans critical range ( P = 0.05) between UFWC5 and Os ceola white clover plants 45

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CHAPTER 4 QUANTITATIVE GENETIC BASIS OF INHERITANCE OF RESISTANCE IN WHITE CLOVER TO SOUTHERN ROOT-KNOT NEMATODE Abstract White clover (T rifolium repens L.) is an important forage crop. Root-knot nematodes ( Meloidogyne spp.) can be a major factor limiting white clover production and persistence. This study was conducted to determine the genetic basis of inheritance of resistance to M. incognita race 4 on white clover. Eight parents composed of three resistant, two intermediate and three susceptible clones were crossed in partial dial lel design and progeny of those 28 crosses were evaluated for egg mass score, gall score, eggs g-1 dry root weight, eggs plant-1, shoot weight and root weight. Progeny from crosses were arranged in a randomized complete block design with 5 replications each consisting 14 individual plants Two weeks after germination, plants were inoculated with ca. 500 M. incognita race 4 eggs. To serve as a non-inoculate control, cross progeny were arranged in another randomized complete block design with 3 replications each consisting 7 plants. The analysis of egg mass score, gall score, eggs per gram dry root weight and eggs per plant showed that both general combin ing ability (GCA) and specific combining ability (SCA) were significant in the e xpression of those variables. W ith a very high GCA:SCA ratio, additive effects were more important than non-additive effects for the inheritance of the above traits. The GCA effects were related with previously classified resistance reaction to the southern RKN. Root weights in inoculated and non inocul ated plants were significantly different with inoculated roots being heavier. For root wei ghts, both GCA and SCA were significant, with a lower GCA:SCA ratio indicating the reduced import ance of additive effects. The GCA values of parents were not in the same direction as previo usly mentioned variables and also did not match the previously classified resist ance reaction. This suggested th at root weight is not a good 46

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variable to select for resistance to RKN in early growth stages. The shoo t weights did not show any significant differences between inoculated and non-inocul ated white clover parents. Introduction White clover ( Trifolium repens L.) is one of the major legume forage crops worldwide and also in the southeastern USA including Florida. Although it is a cool se ason perennial legume, it generally behaves as a reseeding annual in Florid a. It is suitable for hay, silage, green chop and importantly for grazed pastures. It has a higher cr ude protein and digestibility than grasses and can be an important component of Florida pastures (Chambliss and Wofford, 2006). Among several pathological problems that may hinder the production and persistence of white clover, root-knot nematodes (RKN, Meloidogyne spp.) can be an important factor, especially on light textured soils common in Flor ida. There are four predominant species of rootknot nematodes that account for more than 95% of the world distribution. They are M. incognita (Kofoid and White) Chitwood, M. arenaria (Neal) Chitwood, M. javanica (Treub) Chitwood and M. hapla Chitwood (Sasser et al., 1983). In some RKN species, host specific races are found and M. incognita is one such species with four races. These races cannot be differentiated morphologically but can be through host differentiation tests. These are known as physiological races. Four host races of M. incognita have been defined with their differential host sp ecificity when subjected to the North Carolina Host Differential Test (Hartman and Sasser, 1985) composed of a particular set of hosts. When a large number of M. incognita populations were subjected to the test, M. incognita race 1 comprised about 72% of all M. incognita populations whereas M. incognita race 2, M. incognita race 3 and M. incognita race 4 accounted for 13%, 13% and 2%, respectively (Sasser et al., 1983). Meloidogyne incognita race 4 is less aggressive than other races of this species which 47

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could be utilized to select for resistance to southern RKN in white clover (Windham and Pederson, 1989). Meloidogyne incognita is distributed worldwide in tropi cal and other warm regions. They also have a wide host range att acking nearly all cultivated pl ant species in warmer regions (Sasser et al., 1983). Since RKN invade and damage fine roots, the RKN infected plants wilt easily, become stunted and may die. Symptoms of chlorosis may also be seen. The RKN damage in infected fields often is manifested as patches of dead pl ants indicating localized areas of high infection. A clear sign of root-knot nematodes is that the roots are swollen due to galling and have a knot like appearance (Thorne, 1961). Control of RKN disease can be very difficult, nevertheless the most effective control will be the combination of all available control m easures including resistan t cultivars. Bain (1959) evaluated lines of white clover seedlings a nd selected for RKN tolerance. Gibson (1973) developed SC-1 from the selection of wide pool of white clover germplasms which was reported to be resistant to RKN. Mercer et al ., (2000) gained some success in selecting white clover strains resistant to M. trifolia (previously identified as M. hapla). Pederson and Windham (1995) released MSNR4 after four cycles of recurrent selection from a wide genetic base of white clover germplasm. This population was shown to be resistant to M. incognita, M. arenaria and M. graminicola. The cultivar UFWC5, which was re ported to be resistant to southern RKN, was also developed by recurrent phenotyp ic selection using Osceola as the base population and southern root-knot nematode ( M. incognita Race 4) as the selective pathogen (Wofford and Ostmark, 2005). 48

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Understanding the inheritance pattern of RKN resistance a nd understanding the importance of additive and non-additive effects in inherita nce of RKN resistance should improve progress from selection in a breeding program to enha nce RKN resistance. Partitioning the genetic variability to General Combining Ability (GCA ) and Specific Combining Ability (SCA) effects would help understand the genetics conditioning resistance. Such information should be helpful in development of synthetic varieties that are common in white clove r (Baker, 1978). The GCA provides a measure of the addi tive variation and SCA provide s a measure of the non-additive variations. Griffing (1956) has gi ven a procedure to differentiate these combining abilities using diallel crosses. This procedure has been uti lized in many crops to understand the inheritance pattern. The objective of this re search was to estimate the GC A and SCA effects on expression of host-pathogen interaction responses using a set of white clover diallel cross progeny inoculated with M. incognita race 4. Materials and Methods Selection of Parents Seeds of UFWC5 were pl anted in Cone-tainers (Stuewe and Sons, Inc., Tangent, OR) filled with fine commercial building sand. Two week s after germination, the seedling plants were inoculated with ca. 500 eggs of M. incognita race 4. Eight weeks later, these plants were carefully taken out from each container. The root systems were rinsed in water to remove sand and then immersed in a solution of 0.05% red food color (McCormik & Co., Hunt Valley, MD) to stain and highlight the egg masses. The numbe r of egg masses and galls were counted and the plants were classified. The plants with 0 to 5 galls or egg masses were classified as resistant, plants with 6 to 30 as intermediate and thos e with more than 30 galls or egg masses as susceptible (Call et al., 1997). Elev en resistant, eleven susceptibl e and nine intermediate plants were selected. These plants were then plante d in 15-cm diameter pots. Two to five clonal 49

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cuttings of each plant were produced and planted to other 15-cm diameter pots. These pots were maintained in a pollinator free greenhouse. Crossing Flowers were not emasculated prior to making crosses, since white cl over is known to be relatively self incompatible. Hand crosses were made with the aid of a toothpick and Emory paper glued to the flat surface of the toothpick as described by Taylor (1980). Attempts were made to complete all possible crosses within these 31 parents. As white clover is self incompatible, no selfs were made and attempts at selfing yielded only 6 seeds from about 100 flower heads, each head containing 30 to 40 flow ers (ca. 3000 to 4000 total flowers). Under short day conditions, artificial light was used to extend the dayl ength to 16 hours in the greenhouse to ensure the flowering in white clover as it is known to be a long day flowering plant. At 20 to 30 days after pollination, the flower heads were harvested and seeds were hand threshed. These seeds were collected in small pa per bags, labeled by crosses and replications and stored. The seeds of recipro cal crosses were combined. Although we attempted to complete all cro sses among the 31 white clover clones, only progeny from eight clones were used for this diallel experiment. The availability of enough seeds from every cross for a half diallel design wa s the major factor determining the number of parents. We also chose to use a larger number of progeny of each cross in each replication, rather than attempting analysis with a large number of crosses. Inoculation Eight parents, consisting of three resistant (R1, R4, R7), two intermediate (M1, M3) and three susceptible (S1, S3, S7), were used in this diallel experiment. Ninety-one plants of each cross, from a total of 28 crosses, were planted in the cone-tainers. Prio r to inoculation, plants were arranged in a randomized complete block design with 5 replications of 14 plants each for 50

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inoculation. Due to nematode containment issues non-inoculated controls were arranged in a different randomized complete block design with 3 replications of 7 plants each. We also planted and inoculated 98 Osceola plants as check plants to monitor the extent of galling and egg mass production on susceptible plants. After two week s of seedling growth, the progeny plants of 28 crosses arranged for inoculation were inoculated with ca. 500 eggs of M. incognita race 4 with the aid of a continuous flow syringe as descri bed previously in chapter three. The source inoculum was maintained in a separate greenhouse and eggs were extracted with the same method described in chapter three. Data Collection and Analysis The diallel experiment was terminated when most plants of check Osceola showed a gall score and egg mass score between 3 and 5. Variables evaluated were egg mass score, gall score, eggs g-1 dry root weight and eggs plant-1. Individual plants were scored for egg mass and gall numbers. The scores used were 0 = 0 galls or egg masses, 1 = 1 to 2 galls or egg masses, 2 = 3 to 10 galls or egg masses, 3 = 11 to 30 galls or e gg masses, 4 = 31 to 100 galls or egg masses and 5 = more than 100 galls or egg masses (Taylor and Sa sser, 1978). All plants in a replication were pooled for egg extraction and the eggs were coun ted on a replication basi s with the aid of a hemocytometer slide. Four grids on the hemocy tometer slide were counted, and 3 sub-samples from each replication were counted and averaged to calculate to tal egg numbers extracted from each replication of each progeny. The egg counts we re then divided by the dry root weight to obtain eggs g-1 dry root weight. Although the experiment was initiated with 14 plants in each replication, all did not survive. Thus at the time of termination and we divided the egg count by the number of surviving plants to obtain the eggs plant-1 variable. The data collection procedure was as described in chapter three. Individuals were asso ciated with replications for counting egg masses and gall numbers and for counting egg numbers with the microscope. 51

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The data analysis was conducted based on Griffings method 4 model I (Griffing, 1956) using the SAS code as described by Zhang et al. (2005). Results and Discussions Egg Mass Score An analysis of variance for the variable egg mass score showed that there were significant ( P < 0.001) differences both due to the replication and crosses (Table 4-1). The replication effects may be due to environmental effects in side the greenhouse or to differences in how individuals visualized and scored egg masses. Any effects due to individuals may also contribute to significant replicati on effects seen for other response variables. The cross effects were partitioned into the GCA effect and the SCA effect Both effects were a significant source of variation with P < 0.001 (Table 4-1). The contribution due to GCA effect (or GCA:SCA ratio) was 0.9. A GCA:SCA ratio closer to unity signifi es the higher importance of additive effects than SCA effects, additive effects were more important in the expressi on of egg mass production in white clover roots inoculated with M. incognita race 4. All the parents had significant ( P < 0.001) GCA effects and thir teen out of twenty eight SCA effects were significant (Tab le 4-2). Seven out of thirteen significant SCA effects were negative indicating those cross combinations re duced the egg masses from the expected from their GCA effects. The progeny from cross combination of resistant parent R1 (GCA = -0.7) with other resistant parent R7 (GCA = -0.6) produced higher egg masses (SCA = 0.2) than expected from GCA of those parents. The progeny from re sistant parent R4 (GCA = -0.6) produced more egg masses (SCA = 0.2) when combined with a susceptible parent M1 (GCA = 0.2) while the same parent produced less egg masses (SCA = -0.3) when combined with other susceptible parent S1 (GCA = 0.3). Progeny from the most of the resistant by susceptible crosses (R4S1, R4S3, R7M1, M3S3, and M3S7) tended to produ ce less egg masses than expected from their 52

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GCA. This result may suggest the dominance or partial dominance of resistance genes over the susceptible ones. The average egg mass score from all the proge ny of each resistant parent was less than 2.0 (Table 4-3) while the egg mass scores from a ll the progeny from two susceptible parents S3 and S7 produced an average of mo re than 3.0. Based on the averages from the progeny, the best individual cross combination was R1R4 (average egg score = 0.9) and the worst cross combination was S3S7 with average egg score 4-2. Gall Score An analysis of variance for the variable gall score showed that th ere were significant ( P < 0.001) differences both due to the replication and crosses (Table 4-1). Th e cross effects were partitioned into the GCA effect and SCA effect. Bo th effects were significant source of variation with P < 0.001 (Table 4-1). The contribution due to GCA effect (or GCA:SCA ratio) was 0.88. The GCA:SCA ratio closer to unity signifies that the additive effects were more important in the expression of gall production. The analysis for the individual GCA and SCA effects resulted in significant ( P < 0.001) GCA effects of all eight parents (Table 4-4). Progeny from only six out of nine significant cross combinations produced significantly fewer galls. As we discussed for egg mass score, progeny from most of the resistant by susceptible cros s combinations also produced fewer galls. No progeny of susceptible by susceptible or resist ant by resistant crosses had the significant SCA effects. This suggests that the behavior of ga ll production in progeny can be well described with the GCA effects alone. This suggests the importance of additive effects in the inheritance of gall production in white clover. The average gall scores of all the progeny fr om resistant parents R1 and R4 was 2.1 each, while the average gall scores of all the progeny from susceptible parents S3 and S7 was 3.3 each 53

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(Table 4-5). The best cross combination producin g the least gall scores was R1R4 (mean gall score = 1.4) while the cross comb ination producing the highest gall score (mean gall score = 4.3) was S3S7 which was also true ba sed on the egg mass score variable. Eggs g-1 Dry Root Weight The variable eggs g-1 dry root weight was log transformed to meet the normality requirements for the analysis. Both the replicatio n effect and cross effects were significant ( P < 0.001) source of variation (Table 4-6). The parti tion of the cross effect variance resulted in significant GCA effects (P < 0.001) and significant SCA effects (P < 0.001). The GCA:SCA ratio was 0.8 signifying the relatively higher import ance of additive effects in the inheritance of egg production in white clover. All eight parents had significant GCA effects ( P < 0.001) (Table 4-7). All three parents classified as resistant had negative GCA effects a nd all three parents classi fied as susceptible had positive GCA effects. One of the parents (M1) previ ously classified as intermediately resistant to M. incognita race 4 had positive GCA score (0.22) and another (M3) had negative GCA score (0.39). Progeny from seventeen indi vidual crosses showed the significant SCA score on which seven were on desirable direction (reduced egg). The progeny from the resistant parent R1 (GCA = -0.85) crossed with susceptible parent S1 (G CA = 0.46) reduced the egg production from the expected while the same parent R1 resulted in progeny with increased egg production when crossed with another su sceptible parent S3 (GCA = 0.88). The progeny from cross combination R1R4 resulted in higher (SCA = 0.24) egg produc tion than expected from the parents GCA effects. The mean eggs g-1 dry root weight for the progeny of cross R7M3 was least (2,700) while it was highest (60,000) for the progeny of S3S7 (Table 4-8). The average eggs g-1 dry root weight of all progeny from R1 crossed with othe r parents was least (6,900) showing highest level 54

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of resistance while the average eggs g-1 dry root weight of all pr ogeny from S7 crossed with other parents showed the highest (28,200) level of susceptibility. Eggs Plant-1 The analysis of variance showed that ther e was a significant diffe rence in eggs plant-1 both in between the replications ( P < 0.0001) and in between the crosses ( P < 0.0001) (Table 4-6). The variance due to cross effect was again pa rtitioned into GCA and SCA. Both the GCA and SCA showed their significance ( P < 0.0001) in the expression of th e resistance to RKN (Table 46). The higher GCA:SCA ratio was high (0.86) indicating the hi gher importance of additive effects in the inheritance of RKN egg production in white clover. The effects due to GCA were significant for all parents while SCA were significant only for nineteen crosses (Table 4-9) Again the GCA effects were as expected from the previously classified resistance reaction, R1, R4 and R7 show ing negative score suggesting decrease in eggs plant-1 and S1, S3 and S7 showing positive score suggesting increased eggs plant-1. Ten out of those nineteen crosses with significant SCA had negative SCA effect indicating that these combinations produced less eggs plant-1 than expected from their GCA. The progeny from the cross of two resistant parents R1 (GCA = -0.87) and R7 (GCA = -0.75) produced a more resistant parent (SCA = -0.19) than the expected from GCA effects while the cross of R1 with resistant parent R4 (GCA = -0.5) produced higher eggs plant-1 (SCA = 0.25) than expected from GCA. The same resistant parent R1 pr oduced fewer (SCA = -0.58) eggs plant-1 when crossed with one susceptible parent S1 (GCA = 0.38) while the same parent produced as higher (SCA = 0.66) eggs plant-1 when crossed with anot her susceptible parent S3 (SCA = 0.66). But, this susceptible parent S3 produced lower (SCA = -0.46) eggs plant-1 when crossed with another resistant parent R4 (GCA = -0.5). This difference mi ght suggest that some ep istatic effect is also 55

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involved along with additive and do minance effect in the inheritan ce of RKN resistance in white clover as quantified by eggs plant-1. The progeny from cross combinations R1R4, R1R7 and R4R7 each produced a mean eggs plant-1 less than 1,000 while the cross S3S7 produced ca. 20,000 eggs plant-1 (Table 4-10). The best parents for resistance to RKN ware R1 and R7 as indicated by their mean eggs plant-1 from all crosses while the most susceptible parent was S7. Root Weight An analysis for mean separation between the inoculated and non-inoc ulated white clover suggested that there was significan t difference in the root weights the inoculated plants weighing more (Table 4-11). The mean root weight of inoculated plants was 0.3 g and the mean root weight of non-inoculated plan ts was 0.26 g and the Duncans critical difference for mean separation was 0.02. Although this wa s a statistically si gnificant difference, it is questionable whether this translates to a bi ologically important difference. Further analysis with only inoc ulated plants resulted in si gnificant difference both due to replication and cross effects (Table 4-12). The cross effects were partitioned into GCA and SCA effects which were both signifi cant. The GCA:SCA ratio was onl y 0.16 indicating that only the additive effects cannot predict the root weight of white clover plan ts inoculated with M. incognita race 4 but non-additive effects are also involved in the i nheritance of expression of root weight. All the parents except one resistant (R1) and one susceptible (S3) showed significant GCA effects (Table 4-13). One parent classified as resistant (R4) showed a negative GCA effect (0.012) and another parent classified as resistant (R7) showed a positive GCA effect (0.017). Similarly, one parent classified as susceptible (S1) showed a negativ e GCA effect (-0.031) and another parent classified as susceptible (S7) showed a positive GCA effect (0.059). Both the 56

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intermediate parents showed negative GCA effects. These effects are not consistent with their previous classification and are al so not consistent with the results from other variables egg mass score, gall score, eggs g-1 dry root weight and eggs plant-1. Our result suggested that root weight is not a good variable to select for resistance to M. incognita race 4 in white clover. The mean root weights shown in Table 4-14 reflect the above disparities. A likely cause is the fact that roots of susceptible plants with a high amount of galling may weigh more than roots from resistant plants that are mostly fine fibrous roots. Shoot Weight An analysis for mean separation between the inoculated and non-inoc ulated white clover suggested that there was no signi ficant difference in the shoot weights (Table 4-11). Further analysis with only inoculated pl ants resulted in significant difference both due to replication and cross effects (Table 4-12). The cross effects were partitioned into GCA and SCA effects which were both significant. The GCA:SCA ratio was only 0.005 indicating that additive effect alone cannot predict the inheritance of shoot weight in inoculated white clover. Five of eight parents showed significant GCA effect s and twenty one out of twenty eight cross combinations showed significant SCA (Table 4-15). These GCA effects were not consistent with the previous cl assification and also not consistent with the results from other variables egg mass score, gall score, eggs g-1 dry root weight and eggs plant-1. The mean shoot weights shown in Table 4-16 refl ect the above disparities. Thes e results suggested that along with root weight, shoot weight is also not a good variable to select for resistance to M. incognita race 4 in white clover. Correlations There was a high degree of correlation (r = 0.82, P < 0.0001) between the egg mass score and gall score. This signifies the interrelation of the galls and egg masses produced. There was 57

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also intermediate correlation betw een egg mass score and eggs plant-1 (r = 0.55, P < 0.0001) and between gall score and eggs plant-1 (r = 0.45, P < 0.0001). The higher correlation with egg mass is obvious because the eggs are inside egg ma ss. There could be a hi gher correlation between eggs plant-1 and egg mass or gall scores if the act ual numbers of egg masses or galls were counted instead of using the 0 to 5 scale that leads to subj ective variability. The overall results from this diallel study are similar to those of Pederson and Windham (1992) who found that selected resistant parents produced proge ny with the least M. incognita reproduction in a diallel study of three resistant and three susceptible plants. Their study also found that additive genetic effects were of mu ch greater importance in inheritance of RKN resistance in white clover than non-additive ge netic effects although some degree of epistasis may be involved. A different dial lel analysis by Call et al. (1997) using four resistant, three intermediate and two susceptible red clover pare nts also showed predominantly significant GCA effects and non-significant SCA effects. Some ot her diallel studies have also identified GCA effects as more important than SCA effects in resistance to RKN (Williams and Windham, 1990; Mcpherson et al., 1995; Zhang et al., 2007). 58

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Table 4-1. Analysis of variance of combining ab ilities of the variable s egg mass score and gall score of selected white clove r clones inoculated with M. incognita race 4. Source DF Egg Mass Score Gall Score REP 4 7.83*** 25.48*** Cross 27 47.17*** 34.34*** GCA 7 168.13*** 117.60*** SCA 20 3.89*** 3.41*** Error 0.80 0.80 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively Egg masses and galls were rated on a 1 to 5 sc ale where 0 = 0 galls or egg masses, 1 = 1 to 2 galls or egg masses, 2 = 3 to 10 galls or egg ma sses, 3 = 11 to 30 galls or egg masses, 4 = 31 to 100 galls or egg masses and 5 = mo re than 100 galls or egg masses 59

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Table 4-2. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on egg mass scores of three resistant, two inte rmediate and three susceptible white clover clones inoculated with M. incognita Race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.7*** -0.1 0.2* -0.1 0.1 -0.2* 0.0 0.1 R4 -0.6*** 0.0 0.2* 0.3*** -0.3*** -0.2* 0.1 R7 -0.6*** -0.5*** -0.2* 0.3*** 0.1 0.2* M1 0.2*** 0.3*** 0.1 0.0 0.0 M3 -0.3*** 0.0 -0.2* -0.3*** S1 0.3*** 0.2 -0.1 S3 0.8*** 0.1 S7 1.0*** GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the egg mass score from the mean and the positive value means it increased. Egg masses were rated on a 1 to 5 scale where 0 = 0 egg masses, 1 = 1 to 2 egg masses, 2 = 3 to 10 egg masses, 3 = 11 to 30 egg masses, 4 = 31 to 100 egg masses and 5 = more than 100 egg masses 60

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Table 4-3. Mean egg mass score of roots of three re sistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 1.7 0.9 1.1 1.7 1.4 1.7 2.4 2.6 R4 1.8 1.1 2.2 1.7 1.7 2.4 2.8 R7 1.8 1.3 1.1 2.2 2.6 2.9 M1 2.5 2.5 2.9 3.4 3.5 M3 2.0 2.3 2.6 2.7 S1 2.5 3.6 3.5 S3 3.0 4.2 S7 3.2 The bold in the diagonal are means of that parent crossed with others. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Egg masses were rated on a 1 to 5 scale where 0 = 0 egg masses, 1 = 1 to 2 egg masses, 2 = 3 to 10 egg masses, 3 = 11 to 30 egg masses, 4 = 31 to 100 egg masses and 5 = more than 100 egg masses 61

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Table 4-4. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on gall scores of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.7*** 0.1 0.1 -0.3** 0.0 0.1 0.0 0.1 R4 -0.6*** 0.0 0.2* 0.1 -0.3*** -0.2* 0.1 R7 -0.4*** -0.3*** -0.3*** 0.3*** 0.0 0.2* M1 0.3*** 0.4 0.0 0.1 -0.1 M3 -0.2*** 0.1 0.0 -0.3*** S1 0.1** 0.0 -0.1 S3 0.8*** 0.1 S7 0.7*** GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative value of GCA and/or SCA indicate that this particular clone and/or cross decreased the gall score from the mean and the positive value means it increased. Galls were rated on a 1 to 5 scale where 0 = 0 ga lls, 1 = 1 to 2 galls, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls and 5 = more than 100 galls 62

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Table 4-5. Mean gall score of r oots of three resistant, two inte rmediate and thee susceptible white clover inoculated with M. incognita race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 2.1 1.4 1.7 2.0 1.8 2.2 2.7 2.8 R4 2.1 1.7 2.5 2.0 1.9 2.6 2.9 R7 2.4 2.2 1.9 2.8 3.1 3.2 M1 2.9 3.2 3.0 3.8 3.6 M3 2.5 2.7 3.3 2.9 S1 2.8 3.5 3.4 S3 3.3 4.3 S7 3.3 The bold in the diagonal are means of that parent crossed with others. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Galls were rated on a 1 to 5 scale where 0 = 0 ga lls, 1 = 1 to 2 galls, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls and 5 = more than 100 galls 63

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Table 4-6. Analysis of variances of combining abilities of the variables Eggs g-1 and Eggs plant-1 of selected white clover clones inoculated with M. incognita race 4. Source DF Eggs g-1 Eggs plant-1 REP 4 118.51*** 62.50*** Cross 27 59.78*** 63.05*** GCA 7 201.70*** 220.64*** SCA 20 8.37*** 5.79*** Error 0.21 0.17 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively 64

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Table 4-7. General combining abili ty (GCA) and Specific combini ng ability (SCA) effects on log transformed eggs g-1 dry root weight of three resist ant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.85*** 0.24** -0.04 0.02 -0.09 -0.66*** 0.66*** -0.14* R4 -0.47*** 0.01 -0.12 0.19** -0.23** -0.52*** 0.42*** R7 -0.80*** -0.42*** -0.10 0.32*** 0.09 0.14* M1 0.22*** 0.24*** 0.18* -0.11 0.22** M3 -0.39*** 0.43*** -0.15* -0.52*** S1 0.46*** 0.06 -0.09 S3 0.88*** -0.03 S7 0.94*** GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative value of GCA and/or SCA indicate that this particular clone and/or cross decreased the eggs g-1 dry root weight from the mean and the positive value means it increased. 65

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Table 4-8. Mean eggs g-1 dry root weight of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 6,900 3,100 3,100 5,500 2,800 3,900 20,800 8,800 R4 9,200 3,100 7,600 5,100 8,400 12,800 24,200 R7 7,200 3,700 2,700 9,700 12,800 15,600 M1 16,700 10,700 21,900 26,300 41,300 M3 9,300 17,100 13,700 13,000 S1 19,700 42,900 34,000 S3 27,100 60,600 S7 28,200 The bold in the diagonal are means of that parent crossed with others. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 66

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Table 4-9. General combining abili ty (GCA) and Specific combini ng ability (SCA) effects on log transformed eggs plant-1 of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.87*** 0.25*** -0.19** -0.13* 0.03 -0.58*** 0.66*** -0.04 R4 -0.50*** 0.07 -0.14* 0.14* -0.09 -0.46*** 0.24*** R7 -0.75*** -0.10 -0.26*** 0.31*** 0.02 0.16** M1 0.19*** 0.28*** 0.18** -0.14* 0.05 M3 -0.47*** 0.24*** -0.14* -0.28*** S1 0.38*** 0.08 -0.12* S3 0.89*** -0.01 S7 1.12*** GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 intermediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative value of GCA and/or SCA indicate that this particular clone and/or cross decreased the eggs plant-1 from the mean and the positive value means it increased. 67

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Table 4-10. Mean eggs plant-1 of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. R1 R4 R7 M1 M3 S1 S3 S7 R1 1,900 800 700 1,300 700 1,000 5,600 3,100 R4 2,400 900 1,800 1,200 2,300 3,300 6,200 R7 2,000 1,500 600 2,600 3,300 4,800 M1 4,400 2,700 5,400 7,000 10,800 M3 2,300 3,100 3,700 4,300 S1 5,100 11,100 10,000 S3 7,700 19,900 S7 8,500 The bold in the diagonal are means of that parent crossed with others. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 68

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69 Table 4-11. Analysis of variances of combining ab ilities of the variables root weight and shoot weight of selected white cl over clones inoculated with M. incognita race 4 and noninoculated clones. Source DF Root weight Shoot weight REP 4 0.11*** 0.03 Inoculation 1 0.29*** 0.02 Error 0.01 0.05 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively Table 4-12. Analysis of variances of combining abilities of the variable s egg mass score and gall score of selected white clove r clones inoculated with M. incognita race 4. Source DF Root weight Shoot weight REP 4 1.54*** 0.86*** Cross 27 0.19*** 0.77*** SCA 7 0.31*** 0.79*** GCA 20 0.15*** 0.77*** Error 0.01 0.02 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively

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Table 4-13. General combining ability (GCA) and specific combining ability (SCA) effects on root wei ghts of three resistant, tw o intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. R1 R4 R7 M1 M3 S1 S3 S7 -gR1 -0.004 0.006 -0.049*** -0.041*** 0.037*** 0.017* -0.009 0.039*** R4 -0.012** 0.010 -0.004 -0.020* 0.041*** 0.028*** -0.062*** R7 0.017*** 0.103*** -0.056*** -0.004 -0.022** 0.018* M1 -0.011** 0.003 0.003 -0.001 -0.063*** M3 -0.018*** -0.046*** -0.003 0.085*** S1 -0.031*** 0.007 -0.018* S3 0.000 0.000 S 7 0.059*** GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative value of GCA and/or SCA indicate that this particular clone and/or cross de creased the root weight from the mean and the positive value means it increased. 70

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Table 4-14. Mean root weights of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. R1 R4 R7 M1 M3 S1 S3 S7 -gR1 0.30 0.29 0.26 0.24 0.31 0.28 0.29 0.39 R4 0.29 0.32 0.27 0.25 0.30 0.32 0.29 R7 0.31 0.41 0.24 0.28 0.29 0.39 M1 0.29 0.27 0.26 0.29 0.29 M3 0.28 0.20 0.28 0.43 S1 0.27 0.28 0.31 S3 0.30 0.36 S7 0.35 The bold in the diagonal are means of that parent crossed with others. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 71

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Table 4-15. General combining ability (GCA) and Specific combining ability (SCA) effect s on shoot weights of three resistant, t wo intermediate and three susceptible white clover clones inoculated with M. incognita Race 4. R1 R4 R7 M1 M3 S1 S3 S7 -gR1 -0.009 0.012 -0.109*** -0.031* -0.005 0.001 0.095*** 0.036** R4 -0.003 0.210*** -0.017 -0.073*** 0.072*** -0.086*** -0.118*** R7 -0.016** 0.061*** -0.079*** 0.051*** -0.132*** -0.003 M1 0.020** 0.062*** -0.037** 0.001 -0.040** M3 -0.082*** -0.190*** 0.113*** 0.171*** S1 0.028*** 0.079*** 0.023 S3 0.071*** -0.070*** S 7 -0.010 GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative value of GCA and/or SCA indicate that this particular clone and/or cross de creased the shoot weight from the mean an d the positive value means it increased. 72

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Table 4-16. Mean shoot weights of three resistant, two intermediate and thee susceptible white clover inoculated with M. incognita race 4. R1 R4 R7 M1 M3 S1 S3 S7 -gR1 0.78 0.79 0.65 0.77 0.69 0.81 0.94 0.80 R4 0.78 0.98 0.79 0.63 0.88 0.77 0.66 R7 0.77 0.85 0.61 0.85 0.71 0.76 M1 0.80 0.79 0.80 0.88 0.76 M3 0.72 0.54 0.89 0.87 S1 0.81 0.96 0.83 S3 0.85 0.78 S7 0.78 The bold in the diagonal are means of that parent crossed with others. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 73

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Fig 4-1. General combining ability (GCA) effect s on root weights of three resistant, two intermediate and three susceptible white clover clones inoculated with M. incognita race 4. Root weight (g) Parents R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative value of GCA indicates that this partic ular clone decreased the root weight from the mean and the positive value means it increased. 74

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CHAPTER 5 QUANTITATIVE GENETIC BASIS OF INHERITANCE OF RESISTANCE IN WHITE CLOVER TO PEANUT ROOT-KNOT NEMATODE Abstract White clover (T rifolium repens L.) is an important forage crop worldwide. It can also be an important component of pastures on light textured soil found in much of Florida. Root-knot nematodes (Meloidogyne spp.) can be a major limiting factor in white clover production and persistence especially on sandy so ils. This study was conducted to determine the genetic basis of inheritance of resistance to M. arenaria race 1 in a selected group of white clover clones. Eight parents composed of three resist ant, two intermediate and three susceptible clones were crossed in a partial diallel design and t hose 28 crosses were evaluated fo r percentage root system galled (PRSG), egg mass score, gall score, eggs per gram of dry root weight and eggs per plant. This original resistant/intermediate/susceptibility cl assification of parents was based on their reaction to M. incognita race 4. The progeny evaluation experi ment was arranged in a randomized complete block design with 7 rep lications of 14 plants of each cr oss grown in a greenhouse. At two weeks after germination, seedlings we re inoculated with ca. 500 eggs of M. arenaria race 1. Eight weeks after inoculation, the plant root s were washed and evaluated for the above mentioned variables. A diallel analysis (Griffi ngs method 4 model I) of the variables PRSG, gall score and egg mass score showed that both General Combining Ability (GCA) effects and Specific Combining Ability (SCA) effects were significant. An analysis for the variables eggs per gram of dry root and eggs per plant s howed only GCA effects were significant. A high GCA:SCA ratio for all variables indicated that additive effects were more important than nonadditive effects. The GCA effect s were correlated with resistance reaction to the nematodes. The GCA effects of resistant clones vari ed in magnitude from each other and that was also true in the case of susceptible clones. Only a few of the SCA effects were significant. The cross of R6 with 75

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S3 gave more resistance than expected from GCA effects of parents while the cross of R6 with other susceptible clones gave less resistance than predicted. This outcome suggests a more complicated inheritanc e of resistance to M. arenaria race 1 in white clover than for resistance to M. incognita. Introduction White clover ( Trifolium repens L.) is one of the major forage legume crops of the southeastern USA. Although the spec ies is considered perennial, individual plants usually persist for only one year in Florida; and thus it behave s as a reseeding annual. White clover generally has been shown to have high nutritive value which will increase the overall feed value of the diet when mixed with grasses. Alt hough very well adapted for the southern USA, white clover suffers from many pathological problems, one of them being root-knot nematode ( Meloidogyne spp) infestation. Root-knot nematodes (RKN) create galling, compete for food and reduce crop yield, vigor and persistence. Due to the lack of any registered nematicides for use on pasturelands, RKN resistant cultivars are the only and best solution. Meloidogyne arenaria is one of the four major species of Meloidogyne contributing 8% of the worldwide population with the other three being M. incognita, M. javanica and M. hapla Meloidogyne arenaria shows differential host specificity (r aces) that cannot be distinguished morphologically but can be differe ntiated physiologically and by hos t differential tests. Sixteen percent of the worldwide M. arenaria population is contributed by race 1 and the remaining percent by race 2 (Sasser et al., 1983). The control of RKN disease can best be achieved through the combination of all available control measure including resistan t cultivars, chemical and cultu ral practices. Bain (1959) first reported selection for tole rance and/or resistance to RKN in white clover by evaluating lines of white clover seedlings and selected for RKN to lerance. Gibson (1973) developed SC-1 white 76

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clover which was reported to be resistant to RKN. Mercer et al., (2000) gained some success in selecting white clover strains resistant to M. trifolia, a previously undocumented species. Pederson and Windham (1995) released MSNR4 after four cycles of recurrent selection from a wide pool of white clover germplasm. This population was shown to be resistant to M. incognita, M. arenaria and M. graminicola. The cultivar UFWC5 was also reported to be tolerant to southern RKN which was developed by recurren t phenotypic selection us ing Osceola as the base population and southern root-knot nematode ( M. incognita ) race 4 as the selective pathogen (Wofford and Ostmark, 2005). An understanding of the inherita nce pattern of resi stance should improve the progress from selection in a breeding program to enhance RKN re sistance. Partitioning the genetic variability to General Combining Ability (GCA) and Speci fic Combining Ability (SCA) would help understand the genetics behind resistance. The information will be very helpful in development of synthetic varieties such as in white clove r (Baker, 1978). The GCA is an indication of the additive genetic variation of the trait while SCA is the measure of the non-additive variation. Griffing (1956) has given a procedure to differe ntiate these combining abilities using diallel crosses. This method has been utilized for seve ral crops to understand the genetics of those crops. The understanding of the relative importance of GCA and SCA would lead to selection of the most efficient plant breeding procedures. Th e recurrent selection program using a set of resistant parents (having higher GCA effect for the resistance) should be the most efficient method to improve the resistance if additive va riation is of primary importance. Conversely, hybridization of specific parential combinations with large SCA ef fects would be more desirable for crops where the non-additive variance comp onent is more important than the additive 77

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component. This procedure has been utilized in many crops to unders tand the inheritance pattern. The objective of this research was to estimate the GCA and SCA effects on expression of hostpathogen interaction responses using a set of white clover dialle l cross progeny inoculated with M. arenaria race 1. Materials and Methods Selection of Parents Seeds of UFWC5 were pl anted in Cone-tainers (Stuewe and Sons, Inc., Tangent, OR) filled with fine commercial building sand. Two week s after germination, the seedling plants were inoculated with ca. 500 eggs of M. incognita race 4. Eight weeks later, these plants were carefully removed from Cone-taine rs. The root systems were rinsed in water to remove the sand. Roots were then immersed in a solution of 0.05% red food color (McCormik & Co., Hunt Valley, MD). The number of egg masses and galls were counted and classi fied in three groups. The plants with 0 to 5 galls or egg masses were cl assified as resistant, plants with 6 to 30 as intermediate and more than 30 galls or egg masses as susceptible (Cal l et al., 1997). Eleven resistant, 11 susceptible and 9 inte rmediate plants were selected. These plants were then planted in 15-cm diameter pot. Three to five clones of ea ch selected plant were produced and planted in additional 15-cm diameter pots. These pots were maintained in a pollinator free greenhouse. Crossing Since white clover is known to be relative ly self incompatible, flowers were not emasculated prior to making crosses. Hand crosses were made with the ai d of a toothpick with emery paper glued to the flat surface of the tooth pick as described by Taylor (1980). Attempts were made to complete all possible crosses within these 31 parents. Since white clover is self incompatible, no selfs were made and attempts at selfing yielded only 6 seeds from about 100 flower heads, each head containing 30 to 40 fl owers (ca. 300 to 4000 florets). White clover is a 78

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long day flowering plant. Under short day conditions, artificial light was used to extend the daylength to 16 hours in the greenhouse to stimulate flowering during winter months. At 20 to 30 days after pollination, the flower heads were harvested and seeds were hand threshed. These seeds were collected in small pa per bags and stored. The seeds of reciprocal crosses were combined. Although we attempted to complete all crosses among the 31 white clover clones, only eight clones were used for this diallel experiments. The availability of enough seeds from every cross for a half diallel design was a major factor dete rmining the number of parents. Rather than attempting a diallel analysis with a larger nu mber of parents, we chose to use only parents for which a high number of proge ny plants per replication were available. Inoculation Eight parents consisting of th ree resistant (R5, R6, R11), tw o intermediate (M3, M4), and three susceptible (S3, S4, S7) were used in this diallel experiment. Ninety eight progeny plants of each cross, a total of 28 crosses, were germinated in Cone-tainers. These plants were arranged in a randomized complete block design with 7 repl ications of 14 plants each. At two weeks after germination, each plant was inoculated with ca. 500 eggs of M. arenaria race 1 with the aid of a continuous flow syringe as described in chapter th ree. An extra tray with 98 plants of Osceola was also inoculated to provide plants for upr ooting to monitor the progression of the disease symptoms on susceptible plants. The source inoc ulum was maintained in a separate greenhouse and eggs were extracted with the same method described in chapter three. Data Collection and Analysis The diallel experiment was terminated when mo st plants of the extra Osceola flat were showing a gall and egg mass score between 3 and 5. The data collection procedure was the same as that described in chapter thr ee. Separate individuals were assi gned by replications to rate egg masses, galls and also to count eggs with the microscope. The variables accessed were 79

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percentage root system galled (PRSG), egg mass score and gall score, eggs g-1 of dry root weight and eggs plant-1. The PRSG variable has been used as a 1 to 5 scale variable by some authors (Pederson and Windham, 1989; Pederson and Windham, 1992; Windham and Pederson, 1989) but we chose to use this variable as absolute percentage. The ot her variables were measured as described in chapter 3 and 4. The data anal ysis was conducted base d on Griffings method 4 model I (Griffings, 1956) using the SAS code as described by Zhang et al. (2005). Results and Discussion Percentage Root System Galled (PRSG) There was a significant difference both due to crosses and replications (Table 5-1). The replication effects may be due to environmental e ffects inside the greenhouse or to differences in how individuals visualized and sc ored PRSG. Any effects due to individuals may also contribute to significant replicati on effects seen for other response variables. Variation among crosses was separated into variation due to GCA effects and variation due to SCA effe cts. Both the GCA and SCA effects were significant ( P < 0.001) (Table 5-1). The GCA:SCA ratio was 0.87. This is an indication that additive e ffects are more important than non-addi tive effects in the expression of white clover tolerance to p eanut RKN based on the PRSG. The individual GCA effects of all three resist ant (R5, R6, R11), one intermediate (M4) and all three susceptible (S3, S4, S7) parents were significant ( P < 0.001) (Table 5-2), but the GCA effect of one intermediate parent (M3) was not. Within the resistant clones, most crosses involving R6 reduced the mean PRSG value in the range of 40% less than ot her resistant parents. The susceptible clones, all increased the PRSG value by almost an equal amount while one intermediate (M4) increased the PRSG value by a bout 10% more than that of other susceptible clones. 80

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Among the twenty eight SCA effects, only eleven were significant ( P < 0.05) and only six produced a favorable response (reduced PRSG). Th e cross between the tw o resistant clones (R5 and R11) increased the PRSG value (SCA = 6.1) in opposite response than expected based on their GCA, while the cross betw een two susceptible clones (S4 and S7) decreased the value (SCA = -5.2), again an opposite response from expected based on their respective GCA effects. The cross of resistant parent (R5) with one su sceptible parent (S3) had a significant negative SCA (-8.3) PRSG value from expected while th e cross of the same parent with another susceptible parent (S4) showed an increased value of PRSG (SCA = 4.8). Both susceptible parents (S3 and S4) had about the same GCA e ffects. Although the above SCA effects were significant, the predominance of GCA effects sugge sts primarily additive effects contributed to reduced PRSG. The differences in such reactions suggest that the inherita nce of resistance to M. arenaria race 1 is not easily explained. This complicat ed inheritance may be due to the fact that the clones used in this diallel study were not selected using M. arenaria race 1 but with M. incognita race 4. If M. arenaria had been used, these GCA effects might have been more consistent with the classification of the parent s. Nevertheless, parental classification based on response to M. incognita race 4 did identify one parent (R6) that showed a highly resistant PRSG response to M. arenaria race 1 as quantified by its very large negative GCA value (-25.6). The crosses M4S3 and M4S4 were the most susceptible based on the PRSG score which was also confirmed by the GCA and SCA values for PRSG added to the population mean of 61.7 (Table 5-3). The crosses R5R 6, R6R11 gave the most resist ance based on the GCA:SCA and their calculated mean PRSG. Wh en the single cross PRSG means were evaluated, no cross combination stands out for reduced PRSG except for all crosses of resistant parent R6. 81

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The higher GCA effect of intermediate (M4) than that of any susceptible parent is further evidence that classification of parental phenotypes using M. incognita race 4 may not be valid for their responses to M. arenaria race 1. This suggests th at there may be different genes involved in the resistance to M. arenaria race 1 than in M. incognita race 4. All other susceptible-resistance classifications were also consistent between M. arenaria race 1 and M. incognita race 4. Egg Mass Score Cross effects were significant for egg mass score ( P < 0.001) (Table 5-1). The crosses effects were then partitioned into variability due to GCA effects and due to SCA effects, both of which were significant (Table 5-1). The GCA:SCA ratio was 0.70. This higher ratio suggests again that additive effects were more important than non-additive effects and selections based on a parents performance should lead to impr oved resistance in the progeny population. The effects due to GCA effects were significant in only one resistant (R6) and two susceptible (S3 and S7) parents. The SCA effects were significant in only 5 of the 28 crosses (Table 5-4). The parent contri buting most to reduced egg mass score was R6 (GCA = -0.2) whereas both S3 and S7 were equal in contribut ing to susceptibility with GCA = 0.3. The most favorable single cross combinations were R5M3, R5R6, R5R11, R6R11 and R6M1 all with egg mass scores below 3.0. Single cross combinations th at increased egg mass scores were S3S7 and S4S7 (Table 5-5). Gall Score There were significant differences in variability both due to replicati ons and due to crosses ( P < 0.001) (Table 5-1). The variabil ity within crosses was partitione d into the variability due to GCA effects and SCA effects, bot h of which were significant (P < 0.001) (Table 5-1). The GCA:SCA ratio was 0.77 indicating th at additive effects are more im portant in the inheritance of white clover gall score in response to M. arenaria race 1 82

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The analysis of individual GCA and SCA effects showed that seven of the eight parents GCA effects were significant with M3 being th e only parent not show ing a significant GCA effect (Table 5-6). The relative magnitudes of these GCA effects were similar to that of the PRSG variable with R6 being the parent contri buting to reduced gall score and M4 being the parent that increased gall scor e. Twelve of the twenty eight combinations had significant SCA effects and six of them reduced the gall score. The most resistant combinations were R5R6 and R6R11 while the most susceptible combinations were M4S3, M4S4, M4S7, S3S4 and S3S7. These combinations suggest that progeny of the most resistance parent (R6) when combined with another resistant parent were resistant and progeny of susceptible parents (M4, S3) were susceptible when combined with other susceptible parents. However, the resistant by susceptible crosses produced progeny ranged from resistant (R5S3) to susceptible (R5S4, R5S7, R6M3) (Table 5-7). Eggs g-1 Dry Root Weight The variable eggs g-1 of dry root weight was tested for normality and then was log transformed to meet the normality assumptions. Crosses were a significant ( P < 0.01) (Table 5-8) source of variation. The effects of the crosses were partitioned in to GCA effects and SCA effects where only the GCA effects were significant ( P < 0.001). The GCA:SCA ratio was 0.97 indicating a high level of importance of additive effects in the expression of eggs g-1 dry root weight when inoculated with M. arenaria race 1. The individual GCA and SCA effect analysis id entified that three of the eight clones have significant GCA effects and only one cross (M4S4) had signifi cant SCA effects and it was toward resistance (reduced eggs g-1 dry root weight SCA effect). Only one resistant clone (R6) had a significant ( P < 0.001) (Table 5-9) GCA effect a nd two susceptible parents (S3 and S7) 83

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had significant GCA effects ( P < 0.01). The best cross combina tion for reducing egg number was R5R6 and the crosses that produced the most eggs were S3S7 and S4S7 (Table 5-10). Eggs Plant-1 An analysis of variance for the variable eggs plant-1 showed similar results as obtained from eggs g-1 dry root weight. Cros ses were significant ( P < 0.001), and when partitioned into GCA and SCA effects, only the GCA effects were significant ( P < 0.001) (Table 5-8). The GCA:SCA ratio was 0.73 indicating that non-additiv e effects are not as important as additive effects in the inheritance of eggs plant-1. The analysis of the individual GCA and SCA effects resulted in significant GCA effects for two resistant (R5, P < 0.05 and R6, P < 0.001) clones and two susceptible (S3 and S7, both P < 0.001) clones (Table 5-11). Only two crosses (R5R6 and R5S4) gave significant SCA effects. The cross of R5 with resistant R6 reduced (SCA = -0.29) the egg number more than expected from GCA effects while the cross of R5 with non-significant GCA effects parent S4 increased (SCA = 0.31) the egg number. The cross combination with the lowest eggs plant-1 was R5R6 and the combination with the highest eggs plant-1 was S3S7 (Table 5-12). Correlation The correlation between PRSG and gall rating was 0.71 ( P < 0.001) (Table 5-13). This very high correlation is as expected because the number of galls and the percentage of galled roots are related variables. The correlation betw een the egg mass score and gall score was low (r = 0.33, P < 0.001). This may indicate that some juvenile M. arenaria race 1 successfully entered the root system and produced galls but could not reach maturity and produce eggs. This correlation suggests a post infect ion mechanism of tolerance in these white clover plants by inhibiting the juvenile maturati on or depressing the number of females that matured to produce egg masses. 84

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The utilization of very resistan t parent (R6) and other resistan t parent (R5 and R11) can be very helpful in a selection program to breed fo r RKN tolerance/resistance in white clover. As most white clover cultivars are developed by population improvement methods and synthetic cultivars are released rather than emphasizing an individual single crosses, findings from this research should be helpful for synthetic cultivar development. The results of this diallel study with M. arenaria race 1 are similar to those of the previous chapters studying combining ability effects of white clover progeny when inoculated with M. incognita race 4. Nevertheless, there were some di fferences in magnitudes of GCA and SCA effects. Among both the resistant and susceptible parents, a wide range in magnitude of GCA effects may suggest the involvement of multiple genes in the inheritanc e of resistance to M. arenaria race 1. Some parents identified as intermediate in response to M. incognita race 4 produced progeny that were as or more suscep tible than progeny from parents identified as susceptible. This result may suggest the involvemen t of different genes in resistance to different RKN populations. These findings are in agre ement with the study by Windham and Pederson (1989) showing that SC-1, develope d by Gibson (1973) as resistant to M. incognita was only moderately tolerant to some RKN populations. The predominance of GCA effects (additive gene tic variation) in the inheritance of all discussed variables in this rese arch is supported by other studies in white clover (Pederson and Windham, 1992) and red clover [ Trifolium pretense (L.); Call et al., 1997]. Furthermore, studies on corn [ Zea mays L., Williams and Windham, 1990], and cotton [ Gossypium hirsutum (L.), McPherson et al., 1995; Zhang et al ., 2007] also identified additive effect to be more important than non-additive effects in inherita nce of resistance to RKN populations. 85

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Table 5-1. Analysis of variance of combining abil ities of the variables pe rcentage root system galled (PRSG) egg mass score, and gall score of selected white clover clones inoculated with M. arenaria race 1. Source DF PRSG Egg Mass Score Gall Score REP 6 7381*** 11.97*** 9.80*** Cross 27 20510*** 5.37*** 12.78*** GCA 7 72832*** 33.61*** 41.78*** SCA 20 2281*** 3.13*** 2.53*** Error 498 0.46 0.54 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively Egg masses and galls were rated on a 1 to 5 sc ale where 0 = 0 galls or egg masses, 1 = 1 to 2 galls or egg masses, 2 = 3 to 10 galls or egg ma sses, 3 = 11 to 30 galls or egg masses, 4 = 31 to 100 galls or egg masses and 5 = more than 100 galls or egg masses 86

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Table 5-2. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects for percentage root system galle d (PRSG) of three resistant, two intermediate and three susceptible white clover clones inoculated with M arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 -3.3*** -2.3 6.1** -6.0*** 2.7 -8.3*** 4.8* 3.0 R6 -25.6*** 0.7 7.8*** 4.9* -3.0 -2.5 -5.5** R11 -4.4*** 0.0 -6.7** 2.8 -3.5 0.7 M3 0.0 -4.9* -2.7 -1.1 7.1*** M4 14.2*** 3.9 3.8 -3.7 S3 6.2*** 3.7 3.7 S4 5.5*** -5.2* S7 7.3*** GCA effects are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the PRSG from the mean and a positive value means it increased. 87

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Table 5-3. Mean percentage root system galle d (PRSG) of roots of three resistant, two intermediate and three susceptibl e white clover inoculated with M. arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 59 31 60 52 75 56 69 69 R6 39 32 44 55 39 39 38 R11 58 57 65 66 59 65 M3 62 71 65 66 76 M4 74 86 85 79 S3 67 77 79 S4 66 69 S7 68 The bold on the diagonal are means of that parent. R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 88

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Table 5-4. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on egg mass score of three resistant, two interm ediate and three susceptible white clover clones inoculated with M arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 -0.1 -0.1 0.1 -0.3*** -0.1 0.1 0.2*** 0.0 R6 -0.2*** 0.0 -0.1 0.2*** 0.0 -0.0 -0.1 R11 -0.1 0.2** -0.1 -0.1 -0.2** 0.1 M3 -0.1 0.0 0.1 -0.1 0.1 M4 -0.1 -0.1 0.1 -0.1 S3 0.3*** -0.0 -0.0 S4 0.0 0.0 S7 0.3*** GCA effects are in bold on the diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the egg mass rating from the mean and th e positive values means it increased. Egg masses were rated on a 1 to 5 scale where 0 = 0 egg masses, 1 = 1 to 2 egg masses, 2 = 3 to 10 egg masses, 3 = 11 to 30 egg masses, 4 = 31 to 100 egg masses and 5 = more than 100 egg masses 89

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Table 5-5. Mean egg mass scores of roots of three resistant, two intermediate and thee susceptible white clover inoculated with M. arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 3.0 2.7 2.9 2.6 2.9 3.3 3.2 3.3 R6 2.9 2.8 2.7 3.1 3.2 2.9 3.1 R11 3.0 3.1 2.9 3.2 2.8 3.3 M3 3.0 3.0 3.4 3.0 3.3 M4 3.1 3.3 3.2 3.3 S3 3.3 3.4 3.7 S4 3.1 3.5 S7 4.2 The bold on the diagonal are means of that parent. R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 Egg masses were rated on a 1 to 5 scale where 0 = 0 egg masses, 1 = 1 to 2 egg masses, 2 = 3 to 10 egg masses, 3 = 11 to 30 egg masses, 4 = 31 to 100 egg masses and 5 = more than 100 egg masses 90

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Table 5-6. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on gall score of three resistant, two intermedia te and thee susceptible white clover clones inoculated with M arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 -0.1*** -0.3*** 0.1 -0.1 0.1 -0.2* 0.2* 0.2* R6 -0.6*** -0.2** 0.2** 0.3*** 0.0 0.1 -0.1 R11 -0.1** 0.0 -0.2** 0.0 -0.1 0.2** M3 0.0 -0.2** -0.1 0.0 0.0 M4 0.3*** 0.0 0.0 0.0 S3 0.2*** 0.2** 0.1 S4 0.1** -0.3*** S7 0.1*** GCA effects are in bold on the diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 the negative value of GCA and/or SCA indicat e that this particular clone and/or cross decreased the gall rating from the mean and the positive value means it increased. Galls were rated on a 1 to 5 scale where 0 = 0 ga lls, 1 = 1 to 2 galls, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls and 5 = more than 100 galls 91

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Table 5-7. Means of gall score of roots of three resistant, two in termediate and thee susceptible white clover inoculated with M. arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 4.0 3.1 4.0 4.0 4.4 4.0 4.2 4.3 R6 3.6 3.3 3.8 4.1 3.7 3.7 3.5 R11 4.0 4.1 4.2 4.2 4.1 4.4 M3 4.2 4.3 4.3 4.2 4.3 M4 4.4 4.7 4.5 4.6 S3 4.3 4.6 4.6 S4 4.2 4.0 S7 4.2 The bold values on the diagonal are means of that parent. R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 Galls were rated on a 1 to 5 scale where 0 = 0 ga lls, 1 = 1 to 2 galls, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls and 5 = more than 100 galls 92

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Table 5-8. Analysis of variance of comb ining abilities for the variables eggs g-1 of dry root weight and egg splant-1 of selected white clover clones inoculated with M. arenaria race 1. Source DF Egg per gram Egg per plant REP 6 1.05*** 2.22*** Cross 27 0.49** 0.72*** GCA 7 1.23*** 2.15*** SCA 20 0.23 0.23 Error 0.24 0.12 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively 93

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Table 5-9. General combining ab ility (GCA) and specific combin ing ability (SCA) effects on eggs g-1 of dry root weight of three resistant, two intermediate and thee susceptible white clover clones inoculated with M arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 -0.09 -0.22 -0.03 -0.07 0.11 -0.10 0.22 0.09 R6 -0.28*** -0.03 -0.04 0.25 0.10 0.01 -0.07 R11 -0.05 0.21 0.08 0.07 0.00 -0.30 M3 0.00 0.10 -0.19 -0.18 0.16 M4 -0.06 -0.03 -0.36* -0.15 S3 0.23** 0.10 0.06 S4 0.01 0.22 S7 0.25** GCA values are in bold on the diagonal. The or iginal data was log transformed to meet the requirements of analysis. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the eggs g-1 from the mean and the positive value means it increased. 94

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Table 5-10. Means of eggs g-1 of dry root weight of three resistant, two intermediate and three susceptible white clover inoculated with M. arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 130,000 74,300 116,100 114,800 131,500 136,400 151,500 184,500 R6 116,500 97,600 115,900 125,600 156,700 108,900 136,500 R11 132,300 157,300 131,100 171,700 125,100 127,600 M3 141,000 141,100 140,400 111,300 202,500 M4 139,700 157,000 151,600 140,300 S3 169,100 191,400 224,300 S4 150,700 221,300 S7 176,700 The values in bold on the diagona l are means of that parent. R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 95

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Table 5-11. General combining ability (GCA) a nd specific combining ab ility (SCA) effects on eggs plant-1 of three resistant, two intermediate and thee susceptible white clover clones inoculated with M arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 -0.14* -0.29* 0.09 -0.23 0.01 -0.13 0.31* 0.24 R6 -0.44*** 0.16 0.09 0.21 -0.01 -0.03 -0.13 R11 -0.07 0.01 0.08 -0.10 -0.06 -0.18 M3 0.02 0.08 0.04 -0.15 0.16 M4 0.05 -0.20 -0.10 -0.07 S3 0.27*** 0.23 0.16 S4 0.05 -0.18 S7 0.27*** GCA effects are in bold on the diagonal. The or iginal data was log transformed to meet the requirements of analysis. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the eggs plant-1 from the mean and positive values means it increased. 96

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Table 5-12. Means of eggs plant-1 of three resistant, two intermediate and three susceptible white clover clones inoculated with M. arenaria race 1. R5 R6 R11 M3 M4 S3 S4 S7 R5 25,700 11,700 24,300 19,500 25,300 25,800 33,100 39,200 R6 20,200 19,800 22,300 22,500 25,500 19,100 20,600 R11 26,300 26,100 29,500 31,400 25,000 27,800 M3 29,400 31,100 38,100 24,800 43,400 M4 29,400 33,500 28,700 35,000 S3 36,700 47,100 55,200 S4 29,600 31,900 S7 36,100 The bold values on the diagonal are means of that parent. R5, R6 and R11 resistant; M3 and M4 intermediate and S 3, S4 and S7 susceptible to M. incognita race 4 97

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Table 5-13. Correlations among egg mass score, gall score and PRSG of eight clones of white clover inoculated with M. arenaria race 1. Gall Score PRSG Egg Score 0.34*** 0.33*** Gall Score 0.71*** *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively 98

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CHAPTER 6 QUANTITATIVE GENETIC BASIS OF INHERITANCE OF RESISTANCE IN WHITE CLOVER TO JAVANESE ROOT-KNOT NEMATODE Abstract White clover (T rifolium repens L.) is an important forage legume of the southeastern USA including Florida. Root-knot nematodes (Meloidogyne spp.) can be one of the major limiting factors in white clover production and persistenc e in this region. This study was conducted to determine the relative importance of additive and non-additive variance in the inheritance of resistance to M. javanica in a selected group of white clover clones. Eight parents including three resistant, two intermediate and three susceptibl e clones were crossed in a partial diallel design and the progeny from these 28 crosses were eval uated for egg mass score, gall score, eggs per gram dry root weight and eggs per plant. Th e parent plants resist ance reaction was based on prior response to M. incognita race 4. Progeny of the 28 crosses were arranged in a randomized complete block design with 5 replications and 14 plants in each replicat ion in a greenhouse. Two week old progeny seedlings were in oculated with ca. 500 eggs of M. javanica Eight weeks after inoculation, the plant roots were washed and eval uated for the above variables. Analysis of the variables gall score, egg mass score and eggs per plant showed that both General Combining Ability (GCA) and Specific Combining Ability (SCA ) effects were significant for these root response variables. The variable eggs per gram dry root weight showed that only GCA effects were significant. A high GCA:SCA ratio for every variable indicat ed that additive effects were more important than non-additive effects. The GCA effects of both resistant and susceptible clones varied in magnitude from other clones of the same resistance class. Only a small number of the SCA effects were significan t. The clone R7 which was classified as a resistant parent based on its response to M. incognita race 4 was susceptible to M. javanica This indicated the 99

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involvement of different genes contro lling the resistance response between M. javanica and M. incognita Introduction White clover ( Trifolium repens L.) is one of the major legume forage crops worldwide and also in the southeastern USA. Although it is a cool season perenni al legume, it generally behaves as a reseeding annual in Florida. With higher crud e protein and digestibility than grasses, it can be an important component of Florida pastures. It is suitable for hay, silage, green chop and importantly for grazed pastures. Several pathological problems exist that may limit the production and persistence of white clover. Root-knot nematodes ( Meloidogyne spp.) can be a factor, espe cially on light textured soils which are common in Florid a. There are four predominant species of root-knot nematodes (RKN) that account for more than 95% of the wo rld distribution (Sasser et al., 1983). They are M. incognita (Kofoid and White) Chitwood, M. arenaria (Neal) Chitwood, M. javanica (Treub) Chitwood and M. hapla Chitwood. M. javanica is best adapted in the areas with dist inct dry and wet seas ons (Sasser et al., 1983). M. javanica does not have pathological ra ces based on host specificity. M. javanica is found in warm regions of the world and often predom inant in higher altitudes of warm climate. It is the most serious nematode pest in central Africa (Ferris, 1999). Since RKN invade and damage fine roots, the RKN infected plants wilt easily, become stunted and eventually may die. Symptoms of chlorosis may also be seen. The RKN damage in infected fields often is manifested as patches of dead plants indicati ng localized areas of high infection. A clear sign of root-knot nematodes is that the roots are swollen due to galling and have a knot like appearance (Thorne, 1961). 100

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Control of RKN disease is very difficult a nd the most effective control will be the combination of all available cont rol measures including resistant cu ltivars, chemical and cultural practices. The first reported selection for tolerance and /or resistance to RKN in white clover dates back to Bain (1959). Bain evaluated lines of white clover seedlings and selected for RKN tolerance. Gibson (1973) developed SC-1 white clover which was reported to be resistant to RKN. Mercer et al., (2000) gained some success in selecting white clover strains resistant to M. trifolia (previously identified as M. hapla ). Pederson and Windham (1995) released MSNR4 after four cycles of recurrent se lection from a wide genetic base of white clover germplasm. This population was shown to be resistant to M. incognita, M. arenaria and M. garaminicola. The cultivar UFWC5 was also developed by recurren t phenotypic selection us ing Osceola as the base population and southern root-knot nematode ( M. incognita ) as the selective pathogen (Wofford and Ostmark, 2005). The progress from the selection in a breed ing program should be improved with the understanding of the inheritance pa ttern of any trait such as RKN resistance. The information on the relative importance of additive and non-additi ve variations which gives the total genetic variation would help in unders tanding the genetics c onditioning the resistance. Those variations can be related to General Combining Ability (GCA) and Specific Combining Ability (SCA) effects. Such information should be helpful in development of synthe tic varieties that are common in white clover (Baker, 1978). The GCA provides a measure of the additive variation and SCA provides a measure of the non-additiv e variations. Griffi ng (1956) has given a procedure to differentiate these co mbining abilities using diallel cr osses. This proc edure has been utilized in many crops to understa nd the inheritance pattern. The objective of this research was 101

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to estimate the GCA and SCA effects on expression of host-pathogen inter action responses using a set of white clover diallel cr oss progeny inoculated with M. javanica Pederson and Windham (1992) found that selected resistant parents prod uced progeny with the least M. incognita reproduction in a diallel study of th ree resistant and three susceptible plants. Their study found that additive gene action was of much greater importance in inheritance of RKN resistance in white clove r than non-additive gene action. A different diallel analysis by Call et al. (1997) using four re sistant, three intermediate and two susceptible red clover ( Trifolium repens L.) parents also showed predominantly significant GCA effects and nonsignificant SCA effects. Some othe r diallel studies have also id entified GCA effects as more important than SCA effects in resistance to RKN (Williams and Windham, 1990; Mcpherson et al., 1995; Zhang et al., 2007). Materials and Methods Selection of Parents Seeds of UFWC5 were pl anted in Cone-tainers (Stuewe and Sons, Inc., Tangent, OR) filled with fine sand. Two weeks af ter germination, the seedling plan ts were inoculated with ca. 500 eggs of M. incognita race 4. Eight weeks later, these plants were carefully taken out from each container. The root systems were rinsed in water to remove the sand. Roots were then immersed in a solution of 0.05% red food color (McCormik & Co., Hunt Valley, MD) to stain and highlight the egg masses. Other researchers had used Phloxine-B to stain the egg masses (Holbrook et al., 1983), but we found the red food color to be equally e ffective with a reduced level of toxicity than that of Phloxine-B. The number of egg masses and galls were counted and the plants were classified. The plants with 0 to 5 galls or egg masses were classified as resistant, plants with 6 to 30 as intermediate and thos e with more than 30 galls or egg masses as susceptible (Call et al., 1997). Elev en resistant, eleven susceptibl e and nine intermediate plants 102

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were selected. These plants were then plante d in 15-cm diameter pots. Two to five clonal cuttings of each plant were produced and plante d to other 15cm diameter pots. These pots were maintained in a pollinator free greenhouse. Crossing Since white clover is known to be relative ly self incompatible, flowers were not emasculated prior to making crosses. Hand crosse s were made with the aid of a toothpick and emery paper glued to the flat su rface of tooth pick as described by Taylor (1980). Attempts were made to complete all possible crosses within these 31 parents. As white clover is self incompatible, no selfs were made and attempts at selfing yielded only 6 seeds from about 100 flower heads, each head containing 30 to 40 flowers (ca. 3000 to 4000 total florets). White clover is a long day flowering plant. Under short day co nditions, artificial light was used to extend the daylength to 16 hours in the greenhouse. At 20-30 days after pollinati on, the flower heads were harvested and seeds were hand threshed. These seeds were collected in small pa per bags, labeled by crosses and replications and stored. The seeds of recipro cal crosses were combined. Although we attempted to complete all cro sses among the 31 white clover clones, only progeny from eight clones were used for this diallel experiment. The availability of enough seeds from every cross for a half diallel design wa s the major factor determining the number of parents. Rather than attempting analysis with a large number of plants, we chose to use a larger number of progeny of each cr oss in each replication. Inoculation Eight parents consisting three resistant (R 1, R4, R7), two medium (M1, M3) and three susceptible (S1, S3, S7) were used in this diallel experiment. Seventy plants of each cross, from a total of 28 crosses, were planted in the cone-taine rs. Prior to inoculation, pl ants were arranged in 103

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a randomized complete block design with 5 repl ications of 14 plants each for inoculation. We also included cuttings of the eight parents to compare their resistance reaction with the GCA given by Griffings analysis. The cuttings were made at the same time as seeding of progeny. Two weeks after planting, 14 clones of each parent were selected and arranged in a randomized block design. After two weeks of seedling grow th, both the progeny plan ts and parent clones were inoculated with ca. 500 eggs of M. javanica with the aid of a continuous flow syringe as described in chapter three. An ex tra tray with 98 plants of Osceola was also inoculated to provide plants for uprooting to monitor the progression of the disease sy mptoms on susceptible plants. The source inoculum was maintained in a separa te greenhouse and eggs were extracted with the same method described in chapter three. Data Collection and Analysis The diallel experiment was terminated when most plants of Osceola were showing a gall and egg mass score between 3 and 5. Variables eval uated were egg mass score, gall score, eggs g-1 dry root weight and eggs plant-1. Individual plants were scored for egg masses and galls. The scores used were 0 = 0 galls or egg masses, 1 = 1 to 2 galls or egg masses, 2 = 3 to 10 galls or egg masses, 3 = 11 to 30 galls or egg masses, 4 = 31 to 100 galls or egg masses and 5 = more than 100 galls or egg masses (Taylor and Sasser, 1978). All plants in a replication were pooled for egg extraction and the eggs were counted on a replication basi s with the aid of a hemocytometer slide. Four grids on the hemocy tometer slide were counted, and 3 sub-samples from each replication were counted and averaged to calculate to tal egg numbers extracted from each replication of each progeny. The egg counts we re then divided by the dry root weight to obtain eggs g-1 of dry root weight. Although the experiment was initiated with 14 plants in each replication, all did not survive. Thus at the time of termination and we divided the egg count by the number of surviving plants to obtain the eggs plant-1 variable. The data collection procedure 104

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was the same as that of chapter three. Individuals were associated with replications for counting egg masses and gall numbers and for counting egg numbers with the microscope. The data analysis was conducted based usi ng Griffings method 4 model I (Griffing, 1956) using the SAS code as described by Zhang et al., (2005). Results and Discussions Egg Mass Score The analysis of variance for egg mass score show ed that both replicat ions and crosses were significant ( P < 0.001) sources of variability (Table 6-1). The replication effects may be due to environmental effects inside the greenhouse or to differences in how indi viduals visualized and scored egg mass score. Any effects due to in dividuals may also contribute to significant replication effects seen for other response variab les. The variation within crosses was partitioned into GCA and SCA effects. Both GC A and SCA effects were significant ( P < 0.001) (Table 6-1). The GCA:SCA ratio was 0.51. Although this ratio is not as high as found for most variables in the previous two chapters, it still suggests that additive genetic variances were as important as non-additive genetic variances. Analysis of the individual GCA effects of all parents showed five significant GCA effects including three resistant (R1, R4 and R7) and tw o susceptible (S3 and S7 ; Table 6-2). The GCA effects of both the intermediate parents and the susceptible parent S1 were not significant. The positive direction of the GCA effect of the resistant parent R7 showed that this parent actually increased the number of egg masses in the roots of white clover which is c ontrary to its expected reaction. Its magnitude (0.2) was the same as one susceptible S7 (0.2) and was similar to another susceptible (0.3) parent. The remaining two resi stant clones conferred a negative GCA effect suggesting they had additive genetic effects for reducing the number of egg masses. 105

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This aberrant positive GCA value of the resist ant clone R7 may suggest that plants which are resistant to M. incognita may not be resistant to M. javanica and that there could be different genes conferring resistance to di fferent populations of RKN. Seven SCA effects out of twenty eight were si gnificant (Table 6-2) and three were in the desirable direction (lower egg mass score). The combinations R1 R7 and R1M1 were the most resistant crosses and R7S3 was the most susceptible cross (Table 6-3). From the overall means, it would seem that parent R1 was the most resi stant parent which produced progeny with more resistance in each cross combination. We also analyzed the egg mass scores of individual parent means obtained from inoculated rooted vegetative cuttings of each parent. The co rrelation between GCA effect and the mean of rooted cuttings of the pare nts themselves was r = 0.62 ( P < 0.05). The mean of the egg mass score from Osceola was 4.67 which was higher than any of the 28 crosses. But two parents S1 and S7 which we re susceptible gave a higher score (5.0) than Osceola (Table 6-3). Gall Score The analysis of variance for gall score showed the significant cross effect ( P < 0.001; Table 6-1). The GCA and SCA effects within the cross variation were also significant ( P < 0.001 and P < 0.01 respectively). The GCA:SCA ratio wa s 0.57 indicating not so strong effect of additive variation in the inherita nce of gall score in response to M. javanica in white clover. The GCA effects of all the thre e resistant (R1, R4 and R7), one intermediate (M3) and one susceptible (S3) were significan t (Table 6-4). The gall score also showed similar GCA effects as for egg mass score in the case of the resistant clones. The resistant clone (R7) had a positive GCA effect indicating its inclination towards su sceptibility. The remaining resistant clones (R1 and R4) had negative GCA effects as expected from their original parental resistance reaction 106

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classification. The significant GCA effects of the intermediate parent M3 and the susceptible parent S3 both were in a positive direction indi cating they increased the number of galls in infected roots. Only six out of twenty eight SCA effects were significant (Table 6-4) and three decreased the number of galls. The resistant parent R1 when crossed with resistant parent R4 gave a group of progeny that were more susceptible (SCA effect = 0.3) than expected from GCA effects. However, when it was crossed with susceptible (R 7; although classified as resistant, the GCA value suggested it to be a susceptible parent), the progeny gall score was less (SCA = -0.4) than expected from GCA (Table 6-4) Another resistant clone (R4) when crossed with two clones having non-significant GCA effects (M1 and S1) showed SCA effect s in contrasting directions; the cross R4M1 increased the gall score (SCA = 0.3) more than expected while the cross R4S1 decreased the gall score (S CA = 0.3). This type of resistance reaction indicates the complexity of M. javanica resistance in white clover. The comple xity may have originated due to the selection of parents based on response to M. incognita race 4 rather than M. javanica There could be a higher GCA:SCA ratio as found with variables discussed in previous chapters if we have used the same pathogen ( M. javanica ) both for selection of parents and for this diallel study. The GCA effects of those parents could also be consistent with th e resistance/susceptible classification of those parents. Still, by using a different RKN population in selection of parents and in the diallel study allowed us to identify the involvement of different genes in the resistance responses to different RKN population in white clover. The most resistant cross was R1R7 and the mo st susceptible were R7S3 and M3S3 (Table 6-5). These results were in accordance with the results found from egg mass score. The 107

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correlation between GCA effects and actual mean gall score of rooted cuttings of the parents was not significant. The mean of the gall score from the Osceola was 4.2 which was higher than the mean of any of the 28 crosses. But inoculated rooted cutt ings of the three parents M1, S1 and S7 which were susceptible (based on GCA effects of prog eny) had higher mean gall score than Osceola (Table 6-5). Eggs g-1 Dry Root Weight The analysis of variance for eggs g-1 dry root weight showed crosses were significant ( P < 0.001; Table 6-6). The within cross variance was further partitioned into GCA and SCA effects in which only GCA effects were significant ( P < 0.001). The GCA:SCA ratio was 0.84 indicating the greater importance of additive variance than non-additive variance. Only three of the eight clones had significant GCA effects (Table 6-7). Only one resistant clone (R1) showed a negative significant GCA eff ect and two susceptible clones (S3 and S7) had significant positive GCA effects. The only significant SCA effect was of resistant (R1) by resistance (R4) cross, but this SCA effect was in an undesirable direction (increased the number of eggs, SCA = 0.36) more than expected from the GCA of these parents. The combinations with the lowest eggs g-1 of dry root weight were R1R7 and R1M3 while the combinations with the highest eggs g-1 of dry root weight were M1S7 and S3S7 (Table 6-8). As the additive effects appear to be more impor tant, the best parent for use in production of a synthetic cultivar would be R1 which gave th e least number of eggs and also had the most negative GCA effects (-0.69). The correlation between the GCA effects and mean of the parents from crosses was 0.99 ( P < 0.001) while the correlation between the GCA effects and actual means from parent clones was 0.65 ( P < 0.05). 108

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The mean of the eggs g-1 dry root from the Osceola was 252,200 which was higher than any of the 28 crosses. But one susceptible pa rent (S7) also gave egg numbers of over 250,000 (Table 6-8). Eggs Plant-1 The analysis of variance for the RKN eggs plant-1 of white clover showed crosses were significant sources of vari ation (Table 6-6). The variation du e to crosses was partitioned into GCA and SCA effects. The GCA ef fect was highly significant (P < 0.001) while SCA effect was significant ( P < 0.05). The GCA:SCA ratio was 0.71. This higher ratio signifies that additive variation is more important than non-addi tive in the inherita nce of eggs plant-1 in white clover. Analysis of the individual GC A and SCA effects only showed significant GCA effects for two resistant parents (R1 and R7) and two susc eptible parents (S3 and S7) (Table 6-9). The resistant parent R1 had negative GCA effects (-0.69) indicating a reduction in egg numbers while another resistant parent R7 had positive GCA effects (0.17). Th e findings were similar to egg mass score and gall score and support our previous statement that although R7 was identified as a parent resistant to M. incognita, it was not resistant for M. javanica Four out of twenty eight SCA effects were significant and only one of these SCA effects (R1R7, -0.44) was negative indicating a reduction in egg number more than expected from GCA. The cross of resistant parent R1 with another resistant parent, R4, (having non-significant GCA effects) had a significantly more positive SCA effect (SCA = 0.43) for number of eggs than would have been estimated from GCA effects, This same resist ant parent R1 yielded a lower egg number when crossed with another suscep tible R7 (SCA = -0.44). The cross combination with lowest egg number plant-1 was R1R7 and the cross with the highest egg number plant-1 was R7S3 (Table 6-10). The parent that resulted in the most overall reduction in egg number plant-1 was R1 both in terms of estimat ed GCA (-0.69) and mean eggs 109

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plant-1(14,300). The correlation between the GCA eff ects and actual mean number of eggs plant-1 from inoculation of rooted parent clones was 0.56 ( P < 0.05). The mean number of eggs plant-1 from inoculation of Osceola was 81,500 which was higher than the mean of any of the 28 crosses. Howe ver, rooted cuttings of one susceptible parent (S7) gave higher eggs plant-1 (128,700) than Osceola (Table 6-10). Correlation The correlation coefficient of egg mass score and gall score was 0.72 ( P < 0.01) indicating that most nematodes that induced the formation of a gall also resulted in the production of an egg mass. Gall scores are indicative of the plants response to the presence of invading RKN. If fewer egg masses were produced than galls, this would be an indicator that the plant is reducing fecundity of the RKN by reducing number of juveniles that matu re to reproductive females and produce egg masses. Our results do not indicate that such a reduction occurred with these progeny. The findings of this diallel study with M. javanica were similar to our findings with M. incognita race 4 and M. arenaria race 1. The higher GCA:SCA ra tio of egg count variables (egg plant-1, egg g-1 dry root weight) implies the higher im portance of additive genetic effects which are also found by Pederson and Windham (1992). The egg mass score and gall score variable, however, gave the lower GCA:SCA ratio which implies non-additive genetic effects are also as much important as additive genetic effects. This finding was different from our previous findings for these variable with M. incognita race 4 and M. arenaria race 1. The reason behind this could be that the parents we re selected utilizing M. incognita race 4 rather than M. javanica This selection process even resulted in on e parent (R7) to be susceptible to M. javanica which was classified as resistance to M. incognita race 4. This study identified the differences in genes that confer resistance to different populations of RKN. But all the parents resistant to M. incognita 110

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race 4 were not susceptible to M. javanica only one out of three resistant parents was susceptible. 111

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Table 6-1. Analysis of variance of egg mass scor es and gall scores combining ability of progeny from crosses of selected white clover parents inoculated with M. javanica Source DF Egg mass score Gall score REP 4 26.51*** 99.51*** Cross 27 7.20*** 5.64*** GCA 7 18.42*** 15.12*** SCA 20 3.52*** 2.70** Error 1.14 1.17 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively Egg masses and galls were rated on a 1 to 5 sc ale where 0 = 0 galls or egg masses, 1 = 1 to 2 galls or egg masses, 2 = 3 to 10 galls or egg ma sses, 3 = 11 to 30 galls or egg masses, 4 = 31 to 100 galls or egg masses and 5 = more than 100 galls or egg masses 112

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Table 6-2. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on egg mass score of three resistant, two inte rmediate and thee susceptible white clover clones inoculated with M javanica R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.5*** 0.4** -0.3* -0.2 0.1 0.3* -0.1 -0.1 R4 -0.2*** -0.1 0.2 0.0 -0.3* -0.2 0.0 R7 0.2*** -0.2 0.0 0.1 0.5*** 0.0 M1 0.1 0.2 0.1 -0.4 0.3* M3 0.0 0.0 0.2 -0.3* S1 -0.1 -0.1 0.0 S3 0.3*** 0.1 S7 0.2** GCA are in bold on diagonal. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 the negative value of GCA and/or SCA indicat e that this particular clone and/or cross decreased the egg mass score from the mean and the positive value means it increased. Egg masses were rated on a 1 to 5 scale where 0 = 0 egg masses, 1 = 1 to 2 egg masses, 2 = 3 to 10 egg masses, 3 = 11 to 30 egg masses, 4 = 31 to 100 egg masses and 5 = more than 100 egg masses 113

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Table 6-3. Mean egg mass scores of roots of three resistant, two intermediate and thee susceptible white clover inoculated with M. javanica R1 R4 R7 M1 M3 S1 S3 S7 Parent mean R1 2.8 3.0 2.6 2.6 2.8 2.9 3.0 2.8 2.0 R4 3.1 3.2 3.4 3.0 2.6 3.2 3.2 3.9 R7 3.4 3.3 3.4 3.4 4.2 3.6 3.4 M1 3.3 3.5 3.4 3.2 3.8 4.4 M3 3.3 3.1 3.7 3.1 3.8 S1 3.1 3.3 3.3 5.0 S3 3.5 3.8 4.2 S7 3.4 5.0 The bold values on the diagonal are means of that pa rent crossed with others. The last column is the mean obtained from inoculation of r ooted cuttings of the parent clones. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Egg masses were rated on a 1 to 5 scale where 0 = 0 egg masses, 1 = 1 to 2 egg masses, 2 = 3 to 10 egg masses, 3 = 11 to 30 egg masses, 4 = 31 to 100 egg masses and 5 = more than 100 egg masses 114

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Table 6-4. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on gall score of three resistant, two intermedia te and thee susceptible white clover clones inoculated with M javanica R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.4*** 0.3* -0.4* 0.0 -0.3 0.4* 0.0 0.0 R4 -0.2** 0.1 0.3* 0.1 -0.3* -0.3 -0.2 R7 0.2* -0.1 0.1 0.2 0.2 0.0 M1 0.0 0.1 -0.1 -0.3* 0.1 M3 0.2** -0.1 0.2 -0.1 S1 -0.1 -0.1 0.0 S3 0.3*** 0.2 S7 0.1 GCA values are in bold on the diagonal *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the gall score from the mean and the positive value means it increased. Galls were rated on a 1 to 5 scale where 0 = 0 ga lls, 1 = 1 to 2 galls, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls and 5 = more than 100 galls 115

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Table 6-5. Means of gall score of roots of three resistant, two in termediate and thee susceptible white clover inoculated with M. javanica R1 R4 R7 M1 M3 S1 S3 S7 Parent Mean R1 2.9 2.9 2.6 2.8 2.8 3.1 3.2 2.9 3.0 R4 3.1 3.3 3.3 3.3 2.6 3.1 3.0 3.8 R7 3.4 3.2 3.6 3.5 3.9 3.4 3.4 M1 3.2 3.5 3.0 3.2 3.4 4.4 M3 3.4 3.3 3.9 3.4 3.8 S1 3.1 3.3 3.2 4.5 S3 3.5 3.8 3.6 S7 3.3 4.5 The bold values on the diagonal are means of that pa rent crossed with others. The last column is the mean obtained from inoculation of r ooted cuttings of the parent clones. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Galls were rated on a 1 to 5 scal e where 0 = 0 galls, 1 = 1 to 2 galls, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls and 5 = more than 100 galls 116

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Table 6-6. Analysis of variance of eggs g-1 of dry root weig ht and eggs plant-1 combining abilities of selected white clover parents inoculated with M. javanica. Source DF Eggs g-1 Eggs plant-1 REP 6 0.84*** 0.64** Cross 27 0.92*** 1.04*** GCA 7 2.99*** 2.95*** SCA 20 0.20 0.37* Error 0.16 0.19 *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively 117

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Table 6-7. General combining ab ility (GCA) and specific combin ing ability (SCA) effects on eggs g-1 of dry root weight of three resistant, two intermediate and thee susceptible white clover clones inoculated with M. javanica R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.69*** 0.36* -0.17 -0.21 -0.21 0.11 0.08 0.04 R4 -0.01 0.03 -0.18 -0.01 -0.17 -0.01 -0.01 R7 0.03 -0.16 0.11 -0.10 0.19 0.09 M1 0.07 0.23 0.30 -0.26 0.28 M3 -0.05 0.07 0.05 -0.24 S1 -0.01 -0.05 -0.16 S3 0.33*** 0.00 S7 0.32*** GCA effects are in bold on the diagonal. The or iginal data was log transformed to meet the normality requirements of analysis. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative values of GCA and /or SCA indicate that this particular clone and/or cross decreased the eggs g-1 of dry root wei ght from the mean and the positive value means it increased. 118

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Table 6-8. Means of eggs g-1 of dry root weight of three resistant, two intermediate and thee susceptible white clover inoculated with M. javanica R1 R4 R7 M1 M3 S1 S3 S7 Parent Mean R1 64,900 75,500 48,900 56,800 49,000 58,000 88,100 78,100 30,700 R4 110,600 110,800 92,800 102,500 107,000 140,700 144,900 74,700 R7 117,200 96,900 118,900 97,600 181,900 165,700 221,500 M1 125,600 134,100 166,000 126,900 205,700 141,200 M3 111,600 106,100 156,500 114,400 120,300 S1 114,700 143,900 124,200 208,600 S3 148,900 204,300 120,700 S7 148,200 273,900 The bold values on the diagonal are means of that pa rent crossed with others. The last column is the mean obtained from inoculation of r ooted cuttings of the parent clones. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 119

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Table 6-9. General combining ab ility (GCA) and Specific combin ing ability (SCA) effects on eggs plant-1of three resistant, two intermediate and thee susceptible white clover clones inoculated with M. javanica. R1 R4 R7 M1 M3 S1 S3 S7 R1 -0.69*** 0.43* -0.44* 0.11 -0.16 0.22 -0.17 0.00 R4 0.01 -0.03 -0.25 0.12 -0.33 0.10 -0.03 R7 0.17* -0.25 0.11 -0.01 0.47** 0.14 M1 0.00 0.14 0.35* -0.32 0.21 M3 0.03 0.09 0.03 -0.33 S1 -0.10 -0.22 -0.09 S3 0.29*** 0.11 S7 0.29*** GCA values are in bold on the diagonal. The or iginal data was log transformed to meet the normality requirements of analysis. *, **, *** significant at 0.05, 0.01 and 0.001 pr obability levels, respectively R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 Negative values of GCA and/or SCA indicate that this particular clone and/or cross decreased the eggs plant-1 from the mean and the positive value means it increased. 120

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Table 6-10. Means of eggs plant-1of roots of three resistant, two intermediate and thee susceptible white clover inoculated with M. javanica R1 R4 R7 M1 M3 S1 S3 S7 Parent Mean R1 14,300 18,400 9,800 15,000 13,200 13,400 14,000 16,600 8,800 R4 25,300 27,600 19,000 28,200 17,300 34,800 32,000 28,300 R7 32,000 21,500 32,900 25,200 60,800 46,000 52,400 M1 27,300 28,800 41,100 25,500 40,400 36,000 M3 26,800 25,000 34,600 24,600 74,600 S1 25,100 24,100 29,400 78,700 S3 34,300 46,700 38,800 S7 33,700 128,700 The bold values on the diagonal are means of that pa rent crossed with others. The last column is the mean obtained from inoculation of r ooted cuttings of the parent clones. R1, R4 and R7 resistant; M1 and M3 interm ediate and S1, S3 and S7 susceptible to M. incognita race 4 121

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CHAPTER 7 CONCLUSIONS Root-knot nematodes (RKN) can be one of the major problems limiting production and persistence of forage legumes in cluding white clover in light textured soils. Four major RKN species limiting the economic production of white clovers are M. incognita, M. arenaria, M. javanica and M. hapla. Southern RKN ( M. incognita) has four physiological races all of which may attack white clover but race four is th e less aggressive one (Windham and Pederson, 1989). Meloidogyne hapla is generally found in cooler regions and is not a signif icant problem for Florida. There have been attemp ts at various locations over a nu mber of years to breed a white clover variety for RKN resistance (Bain, 1959; Gibson, 1973). The cultivar UFWC5, developed by five cycles recurrent selection from Osceola using race 4 of M. incognita was recently released from the University of Florida as having an improved level of RKN resistance (Wofford and Ostmark, 2005). When this cultivar was evaluated for respons e to various RKN populations using the four races of M. incognita M. arenaria race 1, and M. javanica the resistance re action of UFWC5 white clover to these different RKN populations was variable. UFWC5 produced significantly lower numbers of egg masses and galls when inoculated with the four races of M. incognita Mean root egg mass and gall scores of UFWC5 plants inoculated with the M. incognita races were all below 2.0 signifying resistance to th ese populations. The roots of UFWC5 plants inoculated with M. javanica and M. arenaria race 1 also had reduced galling and egg mass production as compared to Osceola but were above the level (2.0) where they could be classified resistant (Call et al., 1997). This study pointed out the differences in the virulence of different RKN populations. This may suggest the involvemen t of different genes for resistance to the 122

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different populations of RKN. There will likely be a need for multiple cycles of selection using the same RKN population for which resistance is desired. There were no significant differe nces in both the root and sh oot weights of non-inoculated Osceola and UFWC5. This leads us to the conclu sion that selecting for RKN resistance did not hamper the yield potential of this selected whit e clover cultivar. The root weight of inoculated plants was higher than those of non-inoculated plan ts, likely because of the large galls instead of small fibrous roots. Based on three different diallel analysis studie s, additive genetic variance appeared to be the principal type of gene action involved in se lection for RKN resistance in UFWC5. All three RKN populations used for genetic study showed th at additive variance was more important than non-additive variance in the inherita nce of resistance to RKN. The pl ants which were resistant to M. incognita race 4 were not necessarily re sistant in the same degree to M. arenaria race 1 or M. javanica and the degree of susceptibility was also different in these three populations. One parent that showed resistance to M. incognita race 4 was susceptible to M. javanica This observation suggests that there are differences in the genes th at confer resistance to different populations of RKN. The importance of additive vari ance suggests that selection of a few superior parents for development of a synthetic variety would be th e most appropriate bree ding strategy. Based on our research, the clones R1, R4 and M3 would be best parent s for breeding resistance to M. incognita For resistance to M. arenaria race 1 and M. javanica, only one parent in each case (R6 and R1 respectively) was outstanding. In the future, it will be important to screen for response to specif ic RKN populations that are of interest if the target is resistance to those populat ions because of the differences in 123

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virulence of different populations. In any such screening and breeding pr ogram, plant breeders have to focus on additive variance rather than non -additive. Thus, a search for superior sets of parents should be a major goal rather than iden tifying one or two superior hybrid combinations. 124

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REFERENCES Abd-El-Samie, and Y. Taha.1993. Nematode in teractions with root-nodule bacteria. In Khan, M.W. (ed.) Nematode Interac tions. Chapman & Hall. London. Bain, D.C. 1959. Selection for re sistance to root-knot of white and red clover. Plant Dis. Rep. 43:318-322. Baker, R.J. 1978. Issues in dia llel analysis. Crop Sci. 18:533-536. Baltensperger, D.D., C.E. Dean, E.S. Horner. 1984. Registration of Osceola white clover. Crop Sci. 24:1211. Barrett, B., C. Mercer, and D. Woodfield. 2005. Genetic mappi ng of a root-knot nematode resistance locus in Trifolium Euphytica 143:85-92. Baxter, L.W., and P.B. Gibson. 1959. Effect of root-knot nematodes on persistence of white clover. Agron. J. 10:603-604. Bird, A.F. 1979. Histopathology and physiology of synctia. P. 155-172. In Lamberti, F. and C.E.Taylor (ed.). Root-knot nematodes (Meloidogyne Species) systematics, biology and control. Academic Press Inc. London. Brink, G.E., and G.L. Windham. 1990. White clover response to nematode infestation and plant density. Crop Sci. 30:1295-1298. Bunte', R., J. Muller' and W. Friedt. 1997. Genetic variation and response to selection for resistance to root-knot nematodes in oil ra dish (Raphanus sativus ssp. oleiferus). Plant Breed. 116:263-266. Burow, M.D. and J.G. Coors, 1994. DIALLEL. A microcomputer program for the simulation and analysis of diallel crosses. Agron. J. 86:154-158. Call, N.M., K.H. Quesenberry, D.S. Wofford, a nd R.A. Dunn. 1997. Combining ability analysis of resistance of southern root-knot nematode in red clover. Crop Sci. 37:121-124. Chahal., G.S., and S.S. Gosal. 2002. Principles a nd procedures of plant breeding. Alpha Science Int'l Ltd, Oxford, UK. Chambliss, C.G., and D.S. Wofford. 2006. Wh ite clover [Online]. Agronomy Department, Florida Cooperative Extension Service, Inst itute of Food and Agricultural Sciences, University of Florida. Available at http://edis.ifas.ufl.edu/AA198 (verified 10/01/2008). Chitwood, B.G. 1949. Root-knot nematodes. I. A revision of the genus Meloidogyne Goeldi 1887. Proc. Helminthol. Soc. Wash. 16:90-104. 125

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Griffing, B. 1956a. Concept of general and specif ic combining ability in relation to diallel crossing systems. Aust. J. Biol. Sci. 9:463-493. Griffing, B. 1956b. A generalized treatment of th e use of diallel crosses in quantitative inheritance. Heredity. 23:31-50. Hartman, K.M., and J.N. Sasser. 1985. Identification of Meloidogyne species on the basis of differential host test and peri neal pattern morphology. p. 69-77. In Barker K.R., C.C. Carter, and J.N. Sasser (eds.). An advanced treatise on Meloidogyne. Vol. 2. Methodology. North Carolina State University Graphics, Raleigh, NC. Holbrook, C.C., D.A. Knauft, and D.W. Dicks on. 1983. A technique for screening peanut for resistance to Meloidogyne arenaria Plant Dis. 67:957-958. Hussey, R.S., and G.J.W. Janssen. 2002. Root-knot nematodes: Meloidogyne Species .p. 43-70. In Starr, J.L., R.J. Cook, and J. Bridge (ed.). Pl ant resistance to parasitic nematodes. CABI Publishing, Egham, UK Kouam, C., K.H. Quesenberry, D.S. Wofford, and R. Dunn. 1998. Genetic diversity for rootknot nematode resistance in white clover and related species. Genetic Resources and Crop Evolution 45:1-8. Magari, R., and M.S. Kang. 1994. Interactive BASIC program for Griffings diallel analyses. J. Hered. 85:336. McLeish, L.J., G.N. Berg, J.M. Hinch, L.V. Nambiar, and M.R. Norton. 1997. Plant parasitic nematodes in white clover and soil from white clover pastures in Australia. Aust. J. Exp. Agric. 37:75-82. McPherson, G.R., J.N. Jenkins, J.C. McCa rty, and C.E. Watson. 1995. Combining ability analysis of root-knot nematode resi stance in cotton. Crop Sci. 35:373-375. Melakeberhan, H. and J.M. Webster. 1993. The phenology of plant-nematode interaction and yield loss. p. 26-41. In Khan, M.W. (ed.). Nematode interactions. Chapman & Hall. London. Mercer, C.F., J. Van Den Bosch, K.J. Miller. 2000. Progress in recurr ent selection and in crossing cultivars with white clover resi stant to the clover root-knot nematode Meloidogyne trifoliophila N.Z. J. Agric. Res. 43:1:41. Pederson, G. A., and G.L. Windham. 1989. Resistance to Meloidogyne incognita in Trifolium interspecific hybrids and species related to white clover. Plant Dis. 73:567-569. Pederson, G.A., G.L. Windham, M.M. Ellsbury, M. R. Mcloughlin, R.G. Pratt, and G.E. Brink. 1991. White clover yield and persistence as influenced by cypermethrin, benomyl, and root-knot nematode. Crop Sci. 31:1297-1302. 127

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131 BIOGRAPHICAL SKETCH I was born in Chitwan, Nepal, 80 miles from the capital, Kathmandu. I was born to Mr. Hari Prasad Acharya and Mrs. Radha Devi Acharya as their youngest child. I completed my high school, always the first in my class. I received my Bachelor of Science degree in agriculture in 2005 from Institute of Agriculture and Animal Sc iences (Tribhuvan University, Nepal) with a major in plant breeding. I joined The University of Florida in Spring 2007 for an M.S. in agronomy (genetics). I completed my M.S. in Fa ll 2008. I will join The University of Georgia for a Ph.D. degree from The Institute of Plant Breed ing, Genetics and Genomics beginning in Spring 2009.