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GENETIC ANALYSIS OF LARGE PATCH DISEASE RESISTANCE IN ZOYSIA SPP.

By

NORMA CRISTINA FLOR

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2017

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© 2017 Norma Cristina Flor

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To Isabella and Natalia, the most precious gifts that God and life have given to me. To my parents Carlos and Nelly for their infinite love and support.

To my sister Marta Nelly, my best friend, my other half. To my husband Juan Carlos for his patience and love.

To my family and my friends.

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ACKNOWLEDGMENTS

I thank my advisor Dr. Kevin Kenworthy for his unconditional support during this

pathway. My Ph.D. has been an incredible professional and personal opportunity for

me. Dr. Kenworthy does not know the great impact that this experience has have in my

life and for that, my deepest and sincere acknowledgments are for him. I thank Dr.

Philip Harmon for his support. He has always being available to share knowledge with

me. I thank sincerely to all the members of my committee, Dr. Janping Wang, Dr. Erica

Goss and Dr. Nicolas Dufault. They were always available to help me when I need it. I

especially thank Dr. Kenneth Quesenberry. Dr. “Q” who was member of my committee,

plant breeding professor and office mate. Being close to him means always an

opportunity to learn something.

I thank Dr. Patricio Muñoz, who I met as a student and whose help as a

professor was crucial on the statistical analysis of a chapter. I also thank Ling Xing, a

graduate student and office mate for his support, patience and assertive help in the data

analysis. I thank sincerely to the Turfgrass Breeding Program (University of Florida),

Sod Solutions, Turf Research Florida and Turfgrass Producers of Florida for funding my

research project. I thank to all my classmates, colleagues, professors, from Agronomy

and Plant Pathology Department for their help and support during all these years. It is

difficult to mention all of them. Many have gone, some still around and few are always

present in one way or another. I especially thank to Drs: Jing Yang, Esteban Rios,

Bishow Poudel, Nicole Benda, Yolanda Lopez, Jeffrey Fedenko, Yu-Chien Tseng,

Christian Christensen, Joel Reyes and Pilar Fuentealba, Dolores Cenoz, Christopher

Ryan, and Andrew Schreffler. I thank to all the OPS workers and volunteers: Annai

Santi, Jamie Capps, Samantha Potts, Ethan Spence, Jerome Maleski, Jay Leskowyak,

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Danielle Koushel, Bummi Aina, Kayla Douglas, Kenan Ashouri, Walter Parada, Erick

Motle, Rigen Saltivan, Adam Cook, and Catherine Cellon.

I thank Mark Kann and Paul Reith for their technical assistance and support at

the Plant Science Research & Education (PSRU) and Forage Units, respectively. I

thank Jason Haugh, Natasha Restuccia, Eldon Philman, Herman Brown and Michael

Stilwell for their technical assistance at the greenhouse units and walk-in plant growth

room. I thank Brenda Rutherford, Patricia Hill, and Dr. Liping Wang for their laboratory

assistance. I thank sincerely Dr. James Olmstead and Werner Collante for lending me

the GeneMarker key. This little help allowed keeping working away from the office. I

thank Dr. Patricia Rodriguez, for her technical assistance with the GeneMarker software

and for our dissertation’s chats. I thank Dr. Xiping Yang (Daniel), for his help and

support with the data analysis of the microsatellites markers.

I thank my parents Carlos and Nelly, for their unmeasurable love, invaluable help,

and support during all these years. I would not have made it without their help. I thank

my husband Juan Carlos for his patience, love and support. I know it has been a roll

coaster for him, but he has stand by with me even during the hardest winds. I thank my

sister Marta Nelly, I have missed her every day. I have always have her in my feelings. I

thank my daughters Isabella and Natalia. All the efforts dedicated to this research and

behind the scenes were done for them. I thank to my closest friends Claudia Peñuela,

Catalina Torres, Marcela Oliveros, Socorro Balcazar, Paula Viveros, Carmenza Llanos,

Diana Perez, Claudia Bermudez, and Johana Dossman for their friendship and support

during these years. I Thank God. I trusted him, when I have nothing else to do but keep

going. I asked him to trust myself harder to be able to make it.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES .......................................................................................................... 10

LIST OF FIGURES ........................................................................................................ 13

LIST OF ABBREVIATIONS ........................................................................................... 16

ABSTRACT ................................................................................................................... 17

CHAPTER

1 INTRODUCTION .................................................................................................... 19

Zoysia spp. ............................................................................................................. 19

Taxonomy, Biology and Chromosome Number ................................................ 20 Morphological Variation in Zoysia spp. ............................................................. 20

Uses and Qualities ........................................................................................... 20

Zoysiagrass Breeding in USA ........................................................................... 21 Large Patch Disease............................................................................................... 22

Etiology............................................................................................................. 23 Rhizoctonia solani Anastomosis Group (AG) 2-2 ............................................. 23

Disease Symptoms and Signs .......................................................................... 24

Disease Cycle .................................................................................................. 25 Disease Management ...................................................................................... 25

Genetic Resistance in Zoysiagrass Germplasm ..................................................... 26 Heritability ............................................................................................................... 28 Heritability Estimates in Zoysia spp. ....................................................................... 29

Use of Simple Sequence Repeats (SSR) Markers ................................................. 29 Molecular Markers in Zoysiagrass .................................................................... 32

Genetic Linkage Mapping ................................................................................. 33 Association Mapping Analysis in Zoysia spp. ................................................... 35

2 SCREENING COMMERCIAL ZOYSIAGRASS CULTIVARS FOR RESISTANCE TO LARGE PATCH DISEASE ................................................................................ 37

Introduction ............................................................................................................. 37 Materials and Methods............................................................................................ 39

Walk-in Plant Growth Room ............................................................................. 39 Plant Material ................................................................................................... 40 Rhizoctonia solani Anastomosis Group 2-2 Large Patch Isolate (UF 0714) ..... 40

Inoculum Preparation ....................................................................................... 42 Growth Room Inoculations ............................................................................... 42

Data Analysis .......................................................................................................... 44

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Results .................................................................................................................... 45

Run One and Run Three .................................................................................. 45 Run Two ........................................................................................................... 46

Discussion .............................................................................................................. 47 Conclusions and Future Research .......................................................................... 55

3 HERITABILITY ESTIMATES FOR LARGE PATCH DISEASE RESPONSE IN ZOYSIAGRASS ...................................................................................................... 62

Introduction ............................................................................................................. 62

Materials and Methods............................................................................................ 65 Large Patch Disease Screenings ............................................................................ 68

Germplasm ....................................................................................................... 69 F1 Hybrids ......................................................................................................... 69

F1 Families ....................................................................................................... 70 Statistical Analysis .................................................................................................. 71

Frequency Distribution and Population Segregation ........................................ 71 Heritability Estimates ........................................................................................ 72

Results .................................................................................................................... 73 Large Patch Disease Response of Zoysiagrass Populations ........................... 73 Heritability Estimates for Large Patch Disease ................................................. 77

Correlation and Regression Analysis ............................................................... 77 Discussion .............................................................................................................. 77 Large Patch Disease Response of the Three Zoysia spp. Populations .................. 78

Analysis of Variance of the Large Patch Disease Responses .......................... 81 Observations of the Large Patch Disease in the Field Trial .............................. 83

Frequency Distribution of Large Patch Disease Response in the F1 Segregating Families .................................................................................... 84

Correlation of Large Patch Disease Response between Walk-in Plant Growth Room and Field Plots ....................................................................... 86

Relationship between Leaf Texture and Large Patch Disease Response ........ 86 Broad and Narrow Sense Heritabilities ............................................................. 87

Conclusions and Future Research .......................................................................... 90

4 ASSOCIATION OF SIMPLE SEQUENCE REPEAT PRIMERS WITH LARGE PATCH DISEASE RESPONSE IN ZOYSIAGRASS ............................................. 110

Introduction ........................................................................................................... 110 Materials and Methods.......................................................................................... 113

Development of a Segregating Zoysiagrass F1 Family ................................... 113 DNA Isolation ................................................................................................. 113 Simple Sequence Repeats (SSRs) Survey by Polyacrylamide Gel

Electrophoresis (PAGE) .............................................................................. 115 Polyacrylamide Gel Electrophoresis ............................................................... 117

Large Patch Disease Phenotyping ................................................................. 119 Identification of F1 Hybrids .............................................................................. 120

Selection of Polymorphic SSRs Primers and Segregation Analysis ............... 120

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Data Analysis ........................................................................................................ 122

Fragment Scoring ........................................................................................... 122 Chi-Square X2 Test......................................................................................... 123

Single Marker Analysis and T-test .................................................................. 123 Results .................................................................................................................. 124

Large Patch Disease Response of the F1 Family ........................................... 124 Survey of Polymorphic SSRs on the Zoysiagrass Parental Genotypes .......... 124 Identification of True Hybrids .......................................................................... 125

Segregation Analysis in the Selected 21 Zoysiagrass Genotypes .................. 125 Single Marker Analysis and T-test Results ..................................................... 126 Segregation Distortion .................................................................................... 127

Discussion ............................................................................................................ 128 Polymorphism Assesment .............................................................................. 129

Single Gene Analysis and T-test .................................................................... 130 Segregation Distortion .................................................................................... 131

Duplicated Loci ............................................................................................... 133

Conclusions and Future Research ........................................................................ 134

5 FINAL REMARKS ................................................................................................. 145

APPENDIX

A GENOTYPE × DAY INTERACTION BASED ON INOCULATION RUNS ............. 151

B GENOTYPE × DAY INTERACTION BASED ON RATING DATES ...................... 157

C GENOTYPE × DAY INTERACTION ON EACH INOCULATION RUN .................. 163

D ENVIRONMENTAL CONDITIONS OF THE GREENHOUSE ............................... 165

E BREEDING VALUES OF THE ZOYSIA SPP. GERMPLASM ............................... 166

F BREEDING VALUES OF THE F1 HYBRID POPULATION ................................... 168

G BREEDING VALUES OF THE F1 HYBRID POPULATION IN THE FIELD PLOTS .................................................................................................................. 171

H SUMMARY OF LARGE PATCH DISEASE SEVERITY RATINGS IN THE FIELD PLOTS .................................................................................................................. 174

I ENVIRONMENTAL CONDITIONS IN THE FIELD PLOTS AT THE PLANT SCIENCE RESEARCH & EDUCATION UNIT IN CITRA, FLORIDA. ................... 175

J LINKAGE MAP CONSTRUCTION WITH LOD ≥ 2.0 ............................................ 177

K LINKAGE MAP CONSTRUCTION WITH LOD ≥ 3.0 ............................................ 181

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LIST OF REFERENCES ............................................................................................. 186

BIOGRAPHICAL SKETCH .......................................................................................... 201

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LIST OF TABLES

Table page 2-1 Zoysiagrass commercial cultivars selected for the large patch disease ............ 57

2-2 Analysis of variance of large patch disease severity of twelve zoysiagrass cultivars in three inoculation runs at the walk-in plant growth room. ................... 58

2-3 Analysis of variance of large patch disease severity of twelve zoysiagrass cultivars in three inoculation runs. ...................................................................... 59

2-4 Combined mean separation 7 and 14 days after inoculation (DAI) of large patch disease severity of zoysiagrass commercial cultivars in the first run of inoculation within the walk-in plant growth room. .................................................................. 59

2-5 Combined mean separation 7 and 14 days after inoculation (DAI) of large patch disease severity of zoysiagrass commercial cultivars in the third run of inoculation within the walk-in plant growth room................................................. 60

2-6 Mean separation at 7 and 14 days after the inoculation (DAI) of large patch disease severity of twelve zoysiagrass commercial cultivars in the second run of inoculation within the walk-in plant growth room................................................. 61

3-1 University of Florida zoysiagrass germplasm accessions and cultivars evaluated in the large patch disease screening. ................................................................. 92

3-2 Zoysiagrass coarse-texture F1 hybrids and cultivars evaluated in the large patch disease screening. .............................................................................................. 94

3-3 Zoysiagrass genotypes used to develop the F1 segregating families. ................ 96

3-4 Selected zoysiagrass F1 families for narrow-sense heritability estimation of large patch disease. .................................................................................................... 97

3-5 Statistical model to calculate broad sense heritability of large patch disease in the zoysiagrass germplasm. ..................................................................................... 98

3-6 Statistical model to calculate narrow sense heritability of large patch disease in the zoysiagrass F1 hybrids and F1 segregating families. ..................................... 99

3-7 Genotypic (𝜎𝐺2), genotype × run interaction (𝜎𝐺2x r), genotype × run × replication interaction (𝜎𝐺2x r x rep.) and pooled error (𝜎ɛ2) variance components, broad sense heritability and their associate standard errors (± SE) of the large patch . ....... 100

3-8 Genotypic (𝜎𝐺2), genotype × run interaction (𝜎𝐺2 x r), genotype × run × replication interaction (𝜎𝐺2 x r x rep.) and pooled error (𝜎ɛ2) variance components, narrow sense heritability and their associate standard errors (± SE) of the large ......... 101

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3-9 Genotypic (𝜎𝐺2), genotype × replication interaction (𝜎𝐺2 x rep.) and pooled error (𝜎ɛ2) variance components, narrow sense heritability and their associate standard errors (± SE) of the large patch disease severity of the F1 hybrids .... 101

3-10 Large patch disease severity ranges of the F1 hybrids on selected rating days at the field plots. ................................................................................................... 102

3-11 Genotypic (𝜎𝐺2) and pooled error (σ2ε) variance components, narrow sense

heritability and their associate standard errors (± SE) of the large patch disease severity of the segregating F1 families at 7, 14 and 21 days after the ............. 102

3-12 Large patch disease segregation of the F1 families at 7 days after the ............ 108

3-13 Large patch disease segregation of the F1 families at 14 days after the .......... 109

3-14 Large patch disease segregation of the F1 families at 21 DAI. ......................... 109

4-1 Simple Sequence Repeat (SSRs) primers developed for Zoysia spp. used to screen two zoysiagrass parental accessions with differing large patch disease responses. ........................................................................................................ 136

4-2 Annealing temperature ranges for Zoysia spp. SSRs primer sets in each linkage group. ............................................................................................................... 137

4-3 Selected zoysiagrass accessions with differing large patch disease response for screening with polymorphic SSRs primers. ...................................................... 138

4-4 Simple Sequence Repeat (SSRs) primers developed for Zoysia spp. selected for the segregation analysis of the screening panel of the 5333-53 × 375 F1

segregating family. ........................................................................................... 139

4-5 Minimum, maximum, and average number of fragments observed in the screening panel of a full-sib F1 family after amplification with Simple Sequence Repeat (SSRs) primers. ................................................................................... 140

4-6 Polymorphic SSRs fragments tested for the Mendelian 1:1 segregation ratio. . 140

4-7 Zoysia spp. simple sequence repeat primers with significant association with low large patch disease severity based on the Kruskal-Wallis H test...................... 141

4-8 Zoysia spp. simple sequence repeat (SSRs) primers significantly associated to large patch disease tolerance based on the T-Test. ......................................... 143

D-1 Relative humidity, air temperature and light intensity of the greenhouse prior to transfer of plants to the growth room for inoculation. ........................................ 165

E-1 Breeding values of the zoysiagrass germplasm for large patch disease response at 7 and 14 days after the inoculation (DAI) within the walk-in plant growth .... 166

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F-1 Breeding values of the zoysiagrass F1 hybrid population for large patch disease response at 7 and 14 days after the inoculation (DAI) within the walk-in plant growth room...................................................................................................... 168

G-1 Breeding values of the zoysiagrass F1 hybrid population for large patch disease response at the field plots. ................................................................................ 171

H-1 Large patch disease severity ratings taken in the F1 hybrids at the Plant Science Research & Education Unit. ............................................................................. 174

I-1 Air temperature, soil temperature and relative humidity in the field plots at the Plant Science Research & Education Unit in Citra, Florida. ............................. 176

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LIST OF FIGURES

Figure page 3-1 Frequency distribution of large patch disease severity of the zoysiagrass

germplasm at 7 DAI in the first inoculation run. ................................................ 103

3-2 Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 14 DAI in the first inoculation run. .............................................. 103

3-3 Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 7 DAI in the second inoculation run. .......................................... 104

3-4 Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 14 DAI in the second inoculation run. ........................................ 104

3-5 Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 7 DAI in the first inoculation run. ...................................................... 105

3-6 Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 14 DAI in the first inoculation run. .................................................... 105

3-7 Frequency distribution of large patch disease severity of the zoysiagrass F1 hybrids at 7 DAI in the second inoculation run. ................................................ 106

3-8 Frequency distribution of large patch disease severity of the zoysiagrass F1 hybrids at 14 DAI in the second inoculation run................................................ 106

3-9 Frequency distribution of large patch disease severity of the zoysiagrass F1

segregating families at 7 DAI. ........................................................................... 107

3-10 Frequency distribution of large patch disease severity of the zoysiagrass F1

segregating families at 14 DAI. ......................................................................... 107

3-11 Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 21 DAI. ............................................................................................. 108

A-1 Genotype × DAI interaction of JaMur................................................................ 151

A-2 Genotype × DAI interaction of Empire. ............................................................. 151

A-3 Genotype × DAI interaction of El Toro. ............................................................. 152

A-4 Genotype × DAI interaction of Zeon. ................................................................ 152

A-5 Genotype × DAI interaction of Palisades. ......................................................... 153

A-6 Genotype × DAI interaction of Diamond. .......................................................... 153

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A-7 Genotype × DAI interaction of UltimateFlora. ................................................... 154

A-8 Genotype × DAI interaction of Shadow Turf. .................................................... 154

A-9 Genotype × DAI interaction of Meyer................................................................ 155

A-10 Genotype × DAI interaction of Emerald. ........................................................... 155

A-11 Genotype × DAI interaction of Taccoa Green. .................................................. 156

A-12 Genotype × DAI interaction of Zorro. ................................................................ 156

B-1 Genotype × DAI interaction of JaMur based on rating dates. ........................... 157

B-2 Genotype × DAI interaction of El Toro based on rating dates. .......................... 157

B-3 Genotype × DAI interaction of Empire based on rating dates. .......................... 158

B-4 Genotype × DAI interaction of Zeon based on rating dates. ............................. 158

B-5 Genotype × DAI interaction of Palisades based on rating dates. ...................... 159

B-6 Genotype × DAI interaction of Diamond based on rating dates. ....................... 159

B-7 Genotype × DAI interaction of UltimateFlora based on rating dates. ................ 160

B-8 Genotype × DAI interaction of Shadow Turf based on rating dates. ................. 160

B-9 Genotype × DAI interaction of Meyer based on rating dates. ........................... 161

B-10 Genotype × DAI interaction of Emerald based on rating dates. ........................ 161

B-11 Genotype × DAI interaction of Taccoa Green based on rating dates. .............. 162

B-12 Genotype × DAI interaction of Zorro based on rating dates. ............................. 162

C-1 Genotype × DAI interaction of zoysiagrass cultivars in inoculation run 1.......... 163

C-2 Genotype × DAI interaction of zoysiagrass cultivars in inoculation run 2.......... 163

C-3 Genotype × DAI interaction of zoysiagrass cultivars in inoculation run 3.......... 164

J-1 Linkage map of the markers specific to the Zoysia japonica (5333-53) female parent. Distance (cM) between markers is shown on the left side. ................... 178

J-2 Linkage map of the markers specific to the Zoysia matrella (375) male parent. Distance (cM) between markers is shown on the left side. ............................... 179

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K-1 Linkage map of the markers specific to the Zoysia japonica (5333-53) female parent. Markers linked with LOD = 3.0 were included in the analysis. Distance (cM) between markers is shown on the left side. .............................................. 182

K-2 Linkage map of the markers specific to the Zoysia matrella (375) male parent. Markers linked with LOD = 3.0 were included in the analysis. Distance (cM) between markers is shown on the left side. ...................................................... 184

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LIST OF ABBREVIATIONS

AG

cM

DAI

DDF

F1

LP

PCR

QTL

SDF

Anastomosis group

CentiMorgan

Days after the inoculation

Double Dose Fragment

First filial generation

Large patch disease

Polymerase Chain Reaction

Quantitative trait loci

Single Dose Fragment

SSRs Simple sequence repeats.

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

GENETIC ANALYSIS OF LARGE PATCH DISEASE RESISTANCE IN ZOYSIA SPP.

By

Norma Cristina Flor

May 2017

Chair: Kevin Kenworthy Major: Agronomy

Large patch (LP) caused by Rhizoctonia solani, AG 2-2 LP is the most important

disease in Zoysia spp. The disease is managed through fungicides and cultural

practices. Etiology has been described; but knowledge regarding inheritance, variance

components and heritability is warranted. No cultivar has complete resistance and

genetic resistance could decrease management costs. This study evaluated three

zoysiagrass populations (germplasm collection, F1 hybrids and six F1 families) and 12

cultivars for LP disease response. Screening was done with artificial inoculations and in

field plots (natural infection).

The screening protocol was successful for symptom development. Phenotypic

variation was observed. In the field plots, LP disease developed, but other factors

affected the ratings. Overall, no accession had complete resistance, but different

susceptibility levels were observed. Some accessions performed better than some

cultivars, but these responses need to be confirmed.

Phenotypic segregation patterns and variance components indicated that the

disease is quantitatively inherited and influenced by environmental effects. Broad (H2)

and narrow sense (h2) heritabilities were estimated. The germplasm population had

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moderate H2; indicating that genetic factors affected the LP disease response. The F1

families and F1 hybrids had low and moderate h2, indicating that the phenotypic variation

observed was mainly due to environmental effects rather than additive factors.

This study also evaluated 459 Simple Sequence Repeats (SSRs) primers

previously mapped in Zoysia spp. Some primers (260) were polymorphic between a LP

tolerant (5333-53) and susceptible (375) accessions. Subsequently, 137 of these

primers were evaluated in 21 progeny developed from the F1 family (5333-53 × 375).

Amplified fragments had Mendelian segregation ratios, segregation distortion and

identified duplicated loci.

The single gene marker analysis and T-test identified 20 primers associated with

disease responses. These primers should be evaluated in the entire population by a

Quantitative Trait Loci analysis. If the linkage is confirmed, the primers could be used in

a marker assisted selection. The information from this research will allow for

identification of accessions with improved disease response. These are the first

heritability estimates reported for LP disease in Zoysia spp. and the first report of SSRs

primers to target LP disease resistance.

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CHAPTER 1 INTRODUCTION

Zoysia spp.

Zoysia spp. or zoysiagrasses are perennial C4 warm-season turfgrass native to

the Pacific Rim of East Asia and the South Pacific (Brosnan and Deputy, 2008; Patton,

2012). In the United States, zoysiagrass is grown in the southern and transition zone

regions (Patton, 2012). Zoysia is also grown in Australia (north-eastern coastline),

China, Japan, Korea and Taiwan (Yaneshita et al., 1999; Cai et al., 2004; Weng et al.,

2007; Li et al., 2009).

Zoysia japonica Steudel, was introduced in the USA in 1895; and Zoysia matrella

L. Merril, in 1911 by C.V. Piper (USDA botanist). “Matrella” was the first cultivar

released in 1941 by the Alabama Agricultural Experimental Station (Childers and White,

1947; Grau and Radko, 1951); afterwards more than 30 cultivars have been released

(Patton, 2010). The genus Zoysia was named after Karl von Zois, an austrian botanist,

who introduced the species into USA.

Currently, there are 11 Zoysia spp. reported (Tanaka et al., 2016a), but two are

the most common for turfgrass uses; Zoysia japonica, or Japanese lawngrass and

Zoysia matrella, or Manilagrass (Okeyo et al., 2011). These species primarily differ by

leaf texture; Z. japonica has a coarse texture, and Z. matrella has very fine or fine

texture. They also have different responses to biotic and abiotic factors (Patton, 2010).

Zoysia japonica genotypes are more cold-hardy and have faster establishment than Z.

matrella genotypes (Patton and Reicher, 2007; Brosnan and Deputy, 2008; Okeyo et

al., 2011). In contrast, Z. matrella genotypes are more saline and insect tolerant (Patton,

2010). Inter and intra specific hybridization occurs spontaneously in nature or by

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controlled crossing of desirable genotypes (Forbes, 1952; Yaneshita et al., 1999). Rates

of self fertility are variable: high (0-72%) (Forbes, 1952) and moderate (0-54%)

(Fukuoka, 1989). Zoysiagrass cultivars differ in regional adaptation. In the transition

zone, ‘Meyer’ (Z. japonica) dominates due its adaptation to cold temperatures (Okeyo et

al., 2011); whereas, in Florida ‘Empire’ (Z. japonica) is the most popular cultivar.

Taxonomy, Biology and Chromosome Number

Taxonomically, Zoysia spp. are classified within the subfamily Chloridoideae

(Wang et al., 2015). Zoysia is an allotetraploid species, where 2n = 4x = 40 (Yaneshita

et al., 1999). Zoysiagrass is protogynous, cross pollinated and self-fertile (Forbes, 1952;

Cai et al., 2005); however, it is primarily vegetatively propagated through stolons,

rhizomes (Tsuruta et al., 2005) and sod. Few cultivars are seed propagated.

Morphological Variation in Zoysia spp.

Leaf texture has been the primary morphological trait to classify Zoysia species.

Genotypes with coarse (> 2 mm) and fine leaf textures (< 2 mm) belong to japonica and

matrella types, respectively (Patton, 2010; Tsuruta et al., 2011). Variation for

inflorescence traits has also been reported. Z. matrella genotypes have smaller seed

head, pedicel length and peduncle width compared to Z. japonica genotypes (Tsuruta et

al., 2011; Kimball et al., 2013). However, continuous phenotypic variation and

expression of intermediate categories makes accurate classification difficult for some

genotypes (Yaneshita et al., 1997).

Uses and Qualities

Zoysiagrass is used in sport fields, landscapes, home lawns and golf courses.

Very fine or fine cultivars are suitable for fairways, tee boxes and bunkers faces (Li et

al., 2005; Patton and Reicher, 2007). Sod and turf growers prefer zoysiagrass for its

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uniform growth, density, drought tolerance and low maintenance (Green II et al., 1993;

Patton, 2010; Obasa et al., 2012, 2013; Kimball et al., 2013); and because it has

moderate tolerance to salinity and shade (Patton, 2010). However, zoysiagrass has

freeze sensitivity, excessive thatch production and susceptibility to insects, nematodes

and mites. It also has poor wear tolerance during dormancy, with slow recovery from

damage (Green II et al., 1993; Patton, 2010).

Zoysiagrass Breeding in USA

‘Matrella’ was the first commercial cultivar released in 1941; followed by ‘Meyer’

(1951), ‘Sunburst’ (1952), ‘Midwest’ (1963), ‘El Toro’ (1986), ‘Belair’ (1987), and

‘Cashmere’ (1989) (Patton, 2010). Zoysiagrass has been improved by conventional

breeding; although some cultivars have been produced by mutagenesis (Tsuruta et al.,

2011). Successful cultivars have been developed by controlled crossing or selection of

superior progenies within populations. ‘Emerald’ was the first hybrid (Z. japonica × Z.

pacifica) released in 1955 (Patton, 2010; Tapia, 2015). In the last 25 years, thirty-eight

cultivars have been released in USA with thirty-two commercially available (Patton,

2010). Improved characteristics have included deeper roots with improved drought,

shade and leaf firing responses (Patton, 2010).

In USA six breeding programs lead research on zoysiagrass and other warm

season grasses. Major breeding objectives include turf density, drought, cold, wear,

shade and salinity tolerance; and improved responses (resistance) to insects,

nematodes, mites and fungal diseases (Tsuruta et al., 2011; Genovesi, D., and

Chandra, 2014). Breeding lines are tested in different regional trials to identify

genotypes with superior performance.

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The most damaging insects are hunting billbugs (Sphenophorus venatus vestitus

Chittenden, 1904), mole crickets (Scapteriscus spp.), tropical sod webworms (TSW)

(Herpetogramma phaeopteralis) (Guenée, 1854) and fall armyworms (Spodoptera

frugiperda) (J. E Smith, 1797). Large patch disease is undoubtedly the most significant

and economically damaging disease of zoysiagrass. Recently, breeding for large patch

tolerance has become a major goal for zoysiagrass breeders (Unruh et al., 2013;

Genovesi, D., and Chandra, 2014).

Large Patch Disease

Large patch is the most severe disease of Zoysia spp. in North America (Green II

et al., 1993; Hyakumachi et al., 1998; Toda et al., 2004). The disease was first reported

in Japan as “Rhizoctonia large patch” (Oniki et al., 1986). Other warm-season

turfgrasses such as St. Augustinegrass (Stenotaphrum secundatum [Walter] Kuntze),

hybrid bermudagrass (Cynodon dactylon (L.) Pers. × Cynodon transvaalensis Burtt-

Davy), centipedegrass [Eremochloa ophiuroides (Munro) Hack.], and buffalograss

[Buchloe dactyloides (Nutt.) Engelm.] are susceptible to the disease (Smiley et al.,

2005).

In the USA, the etiology of the disease and the association of a specific

anastomosic group of Rhizoctonia solani were described by Green et al. (1993). Prior to

1993, brown patch was the name used to describe diseases caused by Rhizoctonia

spp. on cool and warm season grasses (Freeman, 1967; Hurd and and Grisham, 1983;

Burpee and and Martin, 1992; Tisserat et al., 1994). Since 1993, both names, brown

and large patch, have been used interchangeably to describe diseases caused by

Rhizoctonia spp. on cool and warm season turfgrasses. This has generated confusion

about the etiology of each disease.

23

Etiology

Large patch is caused by a soil-borne and ubiquitous fungal species complex

Rhizoctonia solani J. G. Kühn, [teleomorph Thanatephorus cucumeris (Frank) Donk];

classified in the sub-division Basidiomycota, class Hymenomycetes (Yang and Li,

2012). Currently, there are fourteen anastomosis groups of Rhizoctonia spp. reported

(Carling et al., 2002a; González Garcia et al., 2006); but only the anastomosis group 2-

2 LP (Large Patch) is associated with the disease on warm season turfgrasses (Green II

et al., 1993). Other anastomosis groups (AG-1, AG 2-2, AG-4 and AG-5) of Rhizoctonia

are also turfgrass pathogens causing foliar blight and sheath rots (Martin and and

Lucas, 1984; Haygood and and Martin, 1990; Burpee and and Martin, 1992).

Rhizoctonia solani Anastomosis Group (AG) 2-2

The anastomosis group 2 is very diverse and it has been sub-divided into three

subgroups: AG 2-1, AG 2-2 and AG 2-3, based on anastomosis frequency, Thiamine

requirement and host specialization (Ogoshi, 1987; Naito and Kanematsu, 1994). The

AG 2-2 has been subsequently divided into four sub-groups: 1, IIIB, IV, and LP based

on host specialization, morphological features on Potato Dextrose Agar (PDA), hyphal

growth rate at 35°C and restriction sites (Eco RI, Ban III, Xba I, Sal I, Msp I and Taq)

(Ogoshi, 1987; Liu and Sinclair, 1991; Stevens and Jones, 1993; Hyakumachi et al.,

1998; Aoyagi et al., 1998; Li et al., 2005). The 2-2 LP subgroup includes isolates

pathogenic to warm-season grasses (Green II et al., 1993; Hyakumachi et al., 1998;

Aoyagi et al., 1998; Toda et al., 2004; Li et al., 2005).

On PDA media at 25°C AG 2-2 LP isolates produce colonies with light brown

mycelia of “buff” appearance. The colonies are aerial and dense with small mycelia

24

aggregates scattered superficially. Sclerotia production has not been observed on

culture media (Hyakumachi et al., 1998; Aoyagi et al., 1998).

Disease Symptoms and Signs

Infections can occur from late fall through mid-spring and when zoysia is dormant

(Green II et al., 1993; Smiley et al., 2005), but in the transition zone symptoms usually

appear during late spring or early summer when zoysia is initiating its period of active

growth (Green II et al., 1993, 1994; Smiley et al., 2005). The most well-known symptom

is a patch of dead tissue that varies in size from 10 cm to 10 m (Aoyagi et al., 1998;

Toda et al., 2004; Smiley et al., 2005). The pathogen infects the crown and base of

leaves sheaths; while roots and stolons remain infection-free (Green II et al., 1993;

Smiley et al., 2005). Leaf sheaths rot after infection and are easily pulled out from the

plant; a common disease symptom (Aoyagi et al., 1998; Smiley et al., 2005). Sheath

dieback can occur as a result of basal infection (Smiley et al., 2005).

On leaf sheaths, infection starts as water-soaked lesions of light brown

with/without yellow coloration; as the infection progresses the tissue turns to brown,

yellow, orange or reddish color. The orange discoloration appears to be associated with

nutrient deficiency caused by the infection process (Tisserat et al., 1994). When the

infected tissue dies, small patches start to develop (Aoyagi et al., 1998; Toda et al.,

2004; Smiley et al., 2005). Patches are usually circular, but as the disease progresses

they become irregular. Patch development is favored by shade, wet soils, and poorly

drained or compacted soils (Green II et al., 1993). Patches can appear recurrently in a

location for many years (Tisserat et al., 1994; Smiley et al., 2005). Zoysiagrass recovers

from non-infected tissue when environmental conditions become favorable for its growth

(Tisserat et al., 1994).

25

Disease Cycle

Rhizoctonia solani AG 2-2 LP is isolated from infected and asymptomatic leaf

sheaths, crowns and thatch debris, but also from soil or rhizosphere samples (Aoyagi et

al., 1998). The fungus can overwinter in mycelia or sclerotia-like bulbis (Tisserat et al.,

1994). When the fungus is present and environmental conditions are favorable, a

susceptible host can become infected. Soil temperatures ranging from 20-25°C (50-

75°F) are optimal for infection (Tisserat et al., 1994). Under high disease pressure with

low levels of control, significant damage can occur.

The fungus slows down growth when temperatures increase above 30°C (86°F)

usually during late spring and early summer (Tisserat et al., 1994). Fungicide

applications during active infection prevent extended damage; but have little effect on

infected tissue.

Disease Management

Best management practices include cultural, chemical and genetic approaches.

Cultural control methods can involve several changes in management during periods of

active infection, including: changing mowing heights, adjusting nitrogen applications,

soil aerification, soil verticutting, adequate irrigation frequency, improvement of soil

drainage and thatch reduction (Tisserat et al., 1994; Smiley et al., 2005; Vann, 2007).

Nitrogen (N) fertilization and higher mowing heights are the most suggested

management practices (Tisserat et al., 1994; Smiley et al., 2005; Obasa et al., 2013).

However, the effect of low N rates, N sources and timing of fertilization on decreasing

large patch disease severity has not been clearly demonstrated (Green II et al., 1994;

Obasa, 2012; Obasa et al., 2013). Core-aerification, verticutting, sand top-dressing and

pre-emergent herbicides did not have a significant effect on decreasing LP severity on

26

Meyer (Green II et al., 1994; Obasa et al., 2013). Obasa et al. (2013) reported

decreases in patch size under higher mowing heights (4.5 cm).

Fungicide programs include both curative and preventative approaches.

Preventive applications are suggested to use before environmental conditions are

optimal for disease onset. Strobilurin-based fungicides such as Heritage®

(Azoxystrobin) (Syngenta® Crop Protection Inc.), Compass® (Trifloxystrobin) (Bayer

CropScience Inc.), Insignia® (Pyraclostrobin) (BASF Corp.) and flutolanil-based

fungicides as Prostar® (Triadimefon + flutolanil) (Bayer CropScience Inc.) are the best

options for disease control (Patton and Latin, 2005). Demethylation-inhibitor (DMI)

fungicides such as BannerMaxx® (Propiconazole) (Syngenta ® Crop Protection Inc.),

Bayleton® (Triadimefon) (Bayer CropScience Inc.), Eagle® (Myclobutanil) (Dow

Chemical Company LLC) and Chipco® 26019 (Iprodione) (Bayer CropScience Inc.),

also provide good control (Tisserat et al., 1994; Patton and Latin, 2005).

Genetic Resistance in Zoysiagrass Germplasm

Breeding for large patch disease resistance is a relatively recent goal for

turfgrass breeders. Therefore, limited information for disease responses of zoysiagrass

cultivars and breeding genotypes is available. Furthermore, cultivar disease response

appears to be specific to regional trials.

Disease field data (1996, 2002 and 2007) reported by the National Turfgrass

Evaluation Program (NTEP) at six locations (Georgia, South Carolina, Texas, Arkansas,

North Carolina, Oklahoma and Florida) (Morris, 2000, 2006, 2012) indicated a possible

cultivar × environment and cultivar × isolate interaction. In these trials, cultivars were

rated for brown patch (BP) (under warm and cool temperatures) and large patch (LP)

diseases. Specific information to characterize the isolates present in these field plots

27

was not included; and it is not known if multiple isolates were affecting the disease

ratings at these locations.

In other research, zoysiagrass cultivars were evaluated to Rhizoctonia foliar

blighting and large (brown) patch diseases in Texas and Florida; respectively. In Texas,

cultivars were evaluated under unfavorable conditions to large patch isolates.

Temperature for incubation was 28°C; higher than the currently recommended

temperature (23°C) for symptom development. Interestingly the same isolate was used

to inoculate Agrostis spp. (L.) (cool season) and Zoysia spp. (warm season) (Metz et al.,

1992). Metz et al. (1992) did mention that their isolate was AG 2-2, but confirmation of

the LP group was not provided. Given that Green et al. (1993) reported host-specificity

between AG 2-2 LP isolates and warm season grasses there is a resulting concern with

regard to the isolate used by Metz et al. (1992) because it led to symptom development

on bentgrass and it caused symptoms at a temperature higher than that recommended

for development of LP. Optimal temperature for symptom development in zoysiagrass

inoculated with AG 2-2 LP isolates has been reported between 15-20°C (Hyakumachi et

al., 1998; Aoyagi et al., 1998) and 20-25°C (Green II et al., 1993; Obasa et al., 2012).

Symptom development was delayed at 30°C (Green II et al., 1993).

In Florida, several cultivars were evaluated. ‘Empire’, ‘JaMur’ and ‘Emerald’

were classified as susceptible; but the level of susceptibility was not described (Unruh et

al., 2013). In summary, no high levels of genetic resistance to large patch disease have

been reported for zoysiagrass cultivars. Data from field trials indicate that cultivars have

different susceptibility levels.

28

Heritability

The first definition of “hereditary” as a function of gene transmission from parents

to offspring was described by Lush, (1940). Heritability is a population specific

parameter that partitions additive, dominance or epistatic variances associated to a

phenotypic trait (Lush, 1940; Visscher et al., 2008). The estimated genetic effects are

only applicable to the individuals measured within a defined population (Visscher et al.,

2008). Heritability estimates indicate the level of progress and the genetic gain that can

be achieved for a particular trait through each selection cycle. Heritability values also

can give insights about the most convenient breeding procedure to improve a trait

(Lush, 1940).

Two types of heritability: broad- (H2) and narrow-sense (h2) can be estimated

(Lush, 1940; Nyquist and Baker, 1991) with related or unrelated individuals. Estimation

can be performed by simple regression (linear mixed) or using the “animal model”

(iterative method) (Visscher et al., 2008). The animal model is extendedly used in

livestock genetic (Quass and Pollak, 1980). Broad-sense heritability (H2) includes all

genetic factors [additive (σ2A), dominant (σ2

D), and epistatic (σ2I)] associated with a trait

(Milton, J., Sleper, 1995). Narrow-sense heritability (h2) estimates additive factors (σ2A)

(Lush, 1940; Nyquist and Baker, 1991; Milton, J., Sleper, 1995) that are more

informative for selection; and thus preferred by plant breeders. Levels to determine if a

trait has low, moderate or high heritability vary between species and differing studies.

Kumar et al. (2015) reported low h2 heritability as < 0.20, moderate between 0.20-0.40

and high heritability > 0.40.

29

Heritability Estimates in Zoysia spp.

Inherent variation exist in zoysiagrass germplasm for turfgrass quality traits,

physiological and diseases responses. However, estimates of broad and narrow-sense

heritabilities are limited. A few reports exist for morphological and physiological traits

(Guo et al., 2009; Schwartz et al., 2009). Broad-sense heritabilities were reported

separately for zoysiagrass germplasm having very fine, fine and coarse leaf textures.

High broad sense heritability estimates (0.62-0.94) were obtained for percent plot

coverage, genetic color, turf density, turf quality and seed-head density; and moderate

to low H2 (0.32-0.58) for fall dormancy, spring green-up and turf quality under

Glufosinate [2-amino4-(hydroxymethylphosphinyl) butanoic acid] application, mole

cricket (Scapteriscus spp.) infestation and Bipolaris spp. Shoemaker (1959) infection

(Schwartz et al., 2009).

High narrow-sense heritabilities (63.22-93.67%) were estimated on F1

populations for density, turf height, leaf length, leaf width, leaf length/width, internode

length, internode diameter and internode length/diameter (Guo et al., 2012a). High h2

(42.72-98.8%) were reported for inflorescence density, reproductive branch height and

length, floret number, length and width; and floret length/width (Guo et al., 2010).

Moderate h2 (0.23-0.66) were estimated for seed head length, leaf width, growth habit,

flowering, floret, stem and leaf color and response to rust [Puccinia spp. Pers. (1801)]

(Flor et al., 2014). Heritability estimates for traits such as winter color retention and

responses to important diseases such as large patch have not been reported.

Use of Simple Sequence Repeats (SSR) Markers

Microsatellites or Simple Sequence Repeats (SSRs) are short DNA sequences

composed of repeated sequences that are widely distributed in the genome of

30

prokaryotes and eukaryotes (Fountain et al., 2011; Moniruzzaman et al., 2015;

Shamjana et al., 2015). The SSRs sequences consist of one to six base pairs repeated

in different numbers (Morgante and Olivieri, 1993; Morgante et al., 2002; Wang et al.,

2003; Moniruzzaman et al., 2015; Shamjana et al., 2015). A microsatellite motif is about

100 base pairs in length (Tautz, 1989). These repeated motifs probably originate due to

slippage during DNA replication or repair events (Tautz et al., 1986; Tautz, 1989).

Microsatellites were first observed on DNA of Drosophila melanogaster Meigen

(1830), whales (Tautz, 1989), human genomic DNA (Weber and May, 1988; Weber,

1996) and the human cardiac muscle actin gene (Litt and Luty, 1989; Tautz, 1989).

Microsatellites distribution is usually non-random with higher frequency to non-coding

regions; although they are found in coding and untranslated regions (UTR) (Tautz et al.,

1986; Tautz, 1989; Wang et al., 1994; Li et al., 2002; Shamjana et al., 2015).

In plants, two types of microsatellites are common: dinucleotide (10 or more

repeated units of two nucleotides) often present in introns or 5’ flanking regions

(Morgante and Olivieri, 1993; Morgante et al., 2002) and trinucleotide (7 or more

repeated units of three nucleotides) often present in genic and transcribed regions

(Morgante and Olivieri, 1993). The frequency of dinucleotide SSRs is higher (Wang et

al., 1994). The AT, AG/TC and TAT and TCT are the most common dinucleotides and

trinucleotides, respectively (Morgante and Olivieri, 1993).

Specific functions of SSRs are debatable. SSRs have functional and structural

significance. Structurally, they play a role in chromatin organization; functionally, SSRs

affect gene transcription, gene translation, DNA replication, recombination and

mismatch repair mechanisms (Li et al., 2002).

31

Several techniques are available for identifying SSR regions. The classical

method is the construction of a genomic-based library with probes (Röder et al., 1998).

Currently, faster methods such as cross-species amplification (Moniruzzaman et al.,

2015), amplification with random amplification of polymorphic DNA (RAPD) and vector

cloning (Lunt et al., 1999), library-based method modification by primer extension

(Ostrander et al., 1992; Paetkau, 1999), hybridization techniques (Karagyozov et al.,

1993; Hamilton et al., 1999), and Fast Isolation by AFLP of Sequences Containing

repeats (FIASCO) are used (Wang et al., 2010). Microsatellites regions are amplified by

Polymerase Chain Reaction (PCR) reaction with specific primers. Polymorphisms are

visualized on high resolution gels such as polyacrylamide or fine agarose. Genotyping is

also performed by genome sequencing or capillary electrophoresis (ABI, Applied

Biosystems, Foster City, CA, USA).

Microsatellites are the predominant molecular markers used in plant sciences

(Morgante and Olivieri, 1993; Pérez-de-Castro et al., 2012; Shamjana et al., 2015).

Advantages include: detection of co-dominance (Mendelian inheritance), locus-

specificity, high conservation within species, ease of implementation and high levels of

polymorphism (Morgante et al., 2002; Tsuruta et al., 2005; Cai et al., 2005; Fountain et

al., 2011; Cubry et al., 2014; Moniruzzaman et al., 2015; Shamjana et al., 2015). These

markers have been widely used for population genetic analysis, conservation genetics,

genome and linkage mapping, gene flow detection, and genetic diversity assessment

within populations (Morgante and Olivieri, 1993; Morgante et al., 2002; Shamjana et al.,

2015). Microsatellites have been essential molecular tools for the development of

linkage maps in plants, fish and humans (Shamjana et al., 2015).

32

Molecular Markers in Zoysiagrass

The use of marker-assisted selection in zoysiagrass has been limited. Molecular

markers have been used to elucidate intra and inter specific genetic relationships and to

conduct linkage analysis (Yaneshita et al., 1997, 1999; Cai et al., 2004, 2005; Tsuruta

et al., 2005; Li et al., 2009, 2010, 2015). DNA-based markers such as RFLP (Restriction

Fragment Analysis Length Polymorphism), RAPD (Random Amplified Polymorphic

DNA), SSRs (Simple Sequence Repeats), SRAP (Sequence Related Amplified

Polymorphism), CISP (Conserved Intron Scanning Primers), and PLUG (PCR-based

Landmark Unique Gene) have been used to study zoysiagrass populations and

cultivars. Genetic diversity and allelic variation (Yaneshita et al., 1997, 1999; Budak et

al., 2004; Tsuruta et al., 2005; Hashiguchi et al., 2007; MA et al., 2007; JiPing et al.,

2008; Chen et al., 2009; La Mantia et al., 2011; Kimball et al., 2013), association

between morphological and physiological traits (salinity tolerance) (Yaneshita et al.,

1997), adaptability to geographical regions (Weng et al., 2007), phylogenetic and

taxonomic relationships (Cai et al., 2005; Kimball et al., 2013) have been associated to

DNA banding profiles. Linkage mapping (Yaneshita et al., 1997, 1999; Cai et al., 2004,

2005; Tsuruta et al., 2005; Li et al., 2009, 2010, 2015) and comparative genetic analysis

(Jessup et al., 2011; Li et al., 2015; Wang et al., 2015) have also been conducted on

zoysiagrass populations. Recently, the first genome assembly of three zoysia species

were published: Zoysia japonica cv. ‘Nagirizaki’, Zoysia matrella cv. ‘Wakaba’ and

Zoysia pacifica cv. ‘Zanpa’ (Tanaka et al., 2016a).

RFLP profiles, RAPD and SSRs fragments have been associated with spikelet,

leaf-blade (Yaneshita et al., 1997), salinity tolerance (Weng et al., 2007) and

relatedness among zoysia germplasm (Yaneshita et al., 1997; Weng et al., 2007;

33

Kimball et al., 2013). Microsatellites and SRAPs loci have been linked to cold tolerance

and green period (Guo et al., 2012a). Quantitative trait loci (QTL) have been associated

to leaf width (Ebina, 2000), cold resistance (ChengLong et al., 2010), and salt tolerance

(Guo et al., 2014). Research projects for identifying QTL associated with large patch

and mite resistance (Chandra et al., 2015) and freezing tolerance (McCamy Pruitt et al.,

2015) are being conducted.

Genetic Linkage Mapping

The first genetic zoysiagrass linkage map with coverage of 1,506.3 centiMorgan

(cM) was constructed with RFLP markers using F2 progeny from Z. japonica x Z.

matrella (Yaneshita et al., 1999). Subsequently, two linkage maps with AFLP markers of

932.5 cM (Cai et al., 2004), and 1,320 cM (Ebina et al., 1999) were constructed. The

first SSR-enriched genomic library was created by Tsuruta et al. (2005) using a Z.

japonica cultivar: ‘Asagake’. In this study 26 SSRs primers were validated on

zoysiagrass cultivars and hybrids (Tsuruta et al., 2005). The first SSRs linkage map

(1,187 cM) was created by Cai et al. (2005). The first integrated SSRs linkage map was

based on Z. japonica and Z. matrella genotypes. This consensus map had 507 loci and

covered 2,066.6 cM (Li et al., 2010).

Currently, three high resolution maps of zoysiagrass constructed with different

markers are available. The first map covers 1,351.2 cM and it was constructed with

SSRs using self-pollinated progeny of ‘Matushima 2’, a Z. matrella genotype as the

mapping population (Li et al., 2010). The second map covers 1,337.2 cM and was

developed with RAD markers using two Z. japonica cultivars (Wang et al., 2015). A third

map with the greatest coverage (2,158.5 cM) was developed with RFLP, CISP and

PLUG markers to evaluate synteny between Z. japonica and rice (Oryza sativa L.) (Li et

34

al., 2015). QTL analysis for salt tolerance and linkage analysis was conducted using a

Z. japonica F1 population. In this study the linkage map covered 1,211 cM (Guo et al.,

2014).

In zoysiagrass, molecular markers have shown Mendelian inheritance (Ebina et

al., 1999; Yaneshita et al., 1999; Cai et al., 2004, 2005; Li et al., 2009; Guo et al., 2014;

Wang et al., 2015), and distorted segregation (Yaneshita et al., 1999; Cai et al., 2004,

2005; Li et al., 2009; Jessup et al., 2011; Guo et al., 2014; Wang et al., 2015). Distorted

segregation is a deviation of the expected Mendelian inheritance (Xu and Hu, 2009). In

zoysiagrass, this phenomenon appears to be the result of genetic effects (Jessup et al.,

2011; Guo et al., 2014) or small population sizes (La Mantia et al., 2011). The presence

of duplicated loci (Yaneshita et al., 1999; Cai et al., 2005) and inversion of markers (Li

et al., 2009, 2010) among linkage groups has also been reported.

A relationship between linkage groups and chromosome number has been

observed in zoysiagrass (Yaneshita et al., 1999; Cai et al., 2004; Li et al., 2009; Wang

et al., 2015). Although, the occurrence of additional linkage groups associated with a

small number of markers has been reported (Cai et al., 2004, 2005; Guo et al., 2014;

Wang et al., 2015). In zoysiagrass mapping populations, 20 (Li et al., 2015), 22

(Yaneshita et al., 1999; Li et al., 2009; Wang et al., 2015), 24 (Cai et al., 2005; Guo et

al., 2014) and 26 (Cai et al., 2004) linkage groups have been reported. These linkage

groups have different marker densities. Averages of 0.6 cM (Wang et al., 2015), 4.1 cM

(Li et al., 2010), 4.2 – 4.8 cM (Li et al., 2015), and 5.0 cM (Guo et al., 2014) have been

estimated. Two type of populations have been utilized for mapping; F1 and selfed (F2)

progenies from Z. japonica × Z. matrella (Yaneshita et al., 1999; Cai et al., 2004, 2005)

35

and from Z. japonica × Z. japonica genotypes (Li et al., 2009; Guo et al., 2014; Wang et

al., 2015).

Association Mapping Analysis in Zoysia spp.

Currently two studies on association mapping have been conducted in

zoysiagrass. In the first study AFLP and gSSRs primers were evaluated in an F1

population of Z. matrella for fall armyworm resistance (FAW). Two loci were identified by

mapping Zfawr1, a FAW resistant locus previously reported in maize (Zea mays L.)

(Jessup et al., 2011). In the second study, 8 SSRs polymorphic markers associated with

salinity tolerance in four Z. matrella cultivars were genetically mapped (La Mantia et al.,

2011).

These findings support the potential use of DNA markers for breeding of

zoysiagrass. However, associations, linkage mapping and QTLs that identify disease

resistance loci have not been reported. Identification of SSRs markers associated with

large patch disease tolerance will improve efficiency and reliability of the selection

process. The objectives of this research were 1) to evaluate the LP disease response of

12 commercial cultivars and 3 zoysiagrass populations by artificial inoculations with a

characterized AG 2-2 LP isolate, 2) Estimate broad and narrow-sense heritability for LP

disease in 3 zoysiagrass populations using artificial inoculations and natural infection

(field plots), 3) Evaluate the correlation of the LP disease response under natural

infection (field plots) and artificial inoculation (walk-in plant growth room), 4) Screen

microsatellite primers for polymorphism in parental zoysiagrass genotypes with differing

LP response, and 5) Perform a segregation analysis of the polymorphic markers in a

subset of segregating F1 progeny. This information will be useful for zoysiagrass

36

breeders and will increase the knowledge relative to the Zoysia spp.-large patch

disease pathosystem.

37

CHAPTER 2 SCREENING COMMERCIAL ZOYSIAGRASS CULTIVARS FOR RESISTANCE TO

LARGE PATCH DISEASE

Introduction

Zoysiagrass (Zoysia spp.) is a perennial warm-season turfgrass native to the

Pacific Rim of East Asia and the South Pacific (Engelke and Anderson, 2002; MA et al.,

2007; Brosnan and Deputy, 2008; Patton, 2012). In the United States zoysiagrass is

used for home lawns, sport fields, golf courses and other landscapes (Li et al., 2005;

Patton, 2009) in the southern and transition zone regions (Yaneshita et al., 1997; Cai et

al., 2004; Weng et al., 2007; Li et al., 2009; Patton, 2012). Among all Zoysia spp., Z.

japonica Steud. and Z. matrella L. Merr., are the most utilized as turfgrass in USA

(Okeyo et al., 2011).

Turfgrass quality of zoysiagrass is affected by shade, drought and cold

temperatures. Susceptibility to biotic factors include damage by insects (crickets,

worms), arthropods (mites) and microorganisms such nematodes and fungi. Large

patch is the most economically important disease of zoysiagrass. The disease is caused

by an specific anastomosis group (AG) of Rhizoctonia solani designated as 2-2 (Green

II et al., 1993). Large patch should be the formal name utilized when referring to the

disease caused by the AG 2-2 (R. solani) only in warm-season grasses, but in some

cases brown patch disease is used to refer to diseases caused by R. solani in warm

season grasses.

Historically, brown patch was the name used to describe diseases caused by

Rhizoctonia solani on cool and warm-season grasses. However, Green et al. (1993)

described the etiology of the large patch disease in zoysiagrass and other warm-season

grasses and reported the specificity of the AG 2-2 LP with large patch disease. This

38

created the formal distinction between brown and large patch diseases. Brown patch is

caused by R. solani biotype AG 2-2 III-B and large patch disease by R. solani biotype

AG 2-2 LP.

Today the terms brown patch and large patch should be used to refer to diseases

of R. solani that occur on cool season and warm-season grasses, respectively.

However, ambiguities remain in the literature regarding usage of the correct name of the

disease on warm season grasses, and in many cases a description of the isolate used

on artificial large patch disease screenings or information of the isolate present in field

plots is not provided. This has resulted in reporting of information that is not clear

relative to brown or large patch diseases, especially because additional anastomosis

groups of Rhizoctonia can induce disease on warm season grasses. Therefore, the use

of a characterized isolate of R. solani of the appropriate anastomosic group is very

important when screening for responses to brown and large patch diseases. The use of

an isolate with an specific characterization (morphological and/or molecular) is also

important because it is unknown if genetic diversity present within a fungal population

affects the disease response; i.e. it is unknown if variants within isolates of the AG 2-2

LP group exist in the same field or in a particular geographical region. The presence of

these variants could create an isolate by environment interaction that could affect the

disease development.

Large patch disease affects zoysiagrass turfgrass quality. However, only limited

information is available detailing the resistance or susceptibility levels of zoysiagrass

cultivars to the disease (Metz et al., 1992; Morris, 2000, 2006, 2012). This information is

important to perform management practices that reduce the disease severity. This study

39

was developed with the following objectives: 1) to develop an efficient screening

protocol using a characterized isolate to identify differences in large patch disease

response in zoysiagrass and 2) to compare the large patch disease responses of 12

commercial zoysiagrass cultivars using a characterized isolate of the AG 2-2 LP. These

results will indicate the levels of tolerance or susceptibility of the cultivars and provide

useful information to turfgrass managers and breeding programs.

Materials and Methods

Twelve commercial cultivars of zoysiagrass (Table 2-1) were evaluated for

development of large patch disease in response to artificial inoculation with a virulent

isolate (UF 0714) of Rhizoctonia solani. The experiment was repeated three times

(runs); where each run was arranged as a completely randomized design with 12

replications of each genotype. For evaluation, six pots were inoculated and six were

non-inoculated controls. Specific details follow.

Walk-in Plant Growth Room

Large patch screenings were conducted in a walk-in plant growth room at the

University of Florida, Gainesville, FL. The room was 2.43 m. by 2.89 m. with three

benches. Each bench measured 0.55 m. by 2.23 m. Illumination on the benches was

achieved by Sun System® 5 400 MH (Sunlight Supply®, Inc., Vancouver, WA) with 400

watt type M59 lamps. Lamps were 1.52 m. above the center of each bench and

provided approximately 32.63 – 49.0 µmol m-2 s-1. The temperature was maintained with

a Friedrich wireless wall thermostat-model KW (ACWholesalers, Inc.), and the

photoperiod was controlled using an electromechanical timer (24 h) (Intermatic, Inc.) set

for 12 hours light and 12 hours night.

40

Plant Material

The 12 cultivars utilized in the inoculation studies were propagated from

vegetative stocks maintained in 10 cm diameter pots (KORD products, Toronto, Ontario,

Canada) under greenhouse conditions. In May 2010, twenty-four, and in April 2012

twelve replications per cultivar were established as vegetative sprigs to perform the 3

inoculation runs separated in time. For preparation of pots, 4 sprigs were planted into

each 7.6 cm diameter pot (KORD products, Toronto, Ontario, Canada) filled with 38 g of

80:20 Metro-Mix® 910 (Sun Gro Horticulture Canada Ltd.) and United States Golf

Association (USGA) specification sand. During establishment and prior to inoculation,

plants were irrigated twice a day for 4 minutes using an automated overhead irrigation

system. Every 2 weeks, plants were hand-trimmed (2.0 cm height) and fertilized at 2.44

kg/ha-1using Miracle-Gro® Water Soluble All Purpose Plant Food 24-8-16 (Scotts

Miracle-Gro Products, Inc., Marysville, OH, USA). The average temperature in the

greenhouse ranged between 25-35°C for run 1 and run 2, and between 21-33°C for run

3. Relative humidity was 72%, 71% and 67% for the three runs, respectively. Inoculation

runs were initiated when plants were fully established. Plants were maintained in the

greenhouse from May to October 2010 for the first two inoculation runs; and from April

to July 2012 for the third run.

Rhizoctonia solani Anastomosis Group 2-2 Large Patch Isolate (UF 0714)

University of Florida R. solani isolate UF 0714 from the Plant Pathology

Department was used to perform the inoculations (Flor et al., 2016). The isolate was

collected on a sod farm in Belle Glade, FL, on April 2007 from ‘Palmetto’ St.

Augustinegrass (Stenotaphrum secundatum) exhibiting large patch-like symptoms. The

fungus was isolated from symptomatic leaf sheaths. Briefly, small pieces of about 0.5

41

cm2 were disinfected in sodium hypochlorite (NaClO) at 10% x 30s and subsequently

rinsed 3 times with sterile water. Pieces were dried with paper towels and plated on

Potato Dextrose Agar (PDA Difco, Becton, Dickinson and Company, Franklin Lakes, NJ)

medium (39 g/L). Plates were incubated at 25°C under dark conditions. Hyphal tip

growth was transferred to a new PDA plate and incubated under the same conditions.

This process was repeated once. Genetic uniformity of the isolate was assumed by

selecting hyphal tip growth.

The isolate was initially classified as Rhizoctonia solani Anastomosis Group 2-2

LP (large patch) based on morphological features associated with this group

(Hyakumachi et al., 1998; Aoyagi et al., 1998) when grown on PDA medium (full

strength). Agar pieces from active growth of the isolate (on PDA) were transferred to

autoclaved wheat seeds and incubated under the same conditions described above.

Colonized seeds with fungal mycelia were transferred to small vials for long term

storage at 4°C to ensure genetic stability of the isolate over time.

Molecular characterization. The isolate amplified (358 base pair amplicon)

using AG 2-2 LP primers (expected 400 base pair amplicon) (Carling et al., 2002b) by

polymerase chain reaction (PCR), and was identified as Rhizoctonia solani or

Thanatephorus cucumeris AG 2-2 LP (99%), AG 2-2 IIIB (97-99%) and web blight (WB)

(98%) using DNA sequence data. The BLAST search was performed in the National

Center for Biotechnology Information, NCBI website (https://www.ncbi.nlm.nih.gov)

using the blast nucleotide option optimized for highly similar sequences (Data accessed

on October 2016). Nucleotide sequencing was performed on the amplicon (700 base

pair) obtained with Internal Transcribed Spacer (ITS) primers 1 and 4 (White et al.,

42

1990) by PCR. These primers amplified the ITS 1 and 2 regions and the 5.8 S RNA

gene (White et al., 1990).

Isolate UF 0714 was an effective virulent isolate previously used to screen St.

Augustinegrass (Flor, 2009). After a preliminary screen on zoysiagrass, it was selected

as the inoculum source for all populations.

Inoculum Preparation

Isolate UF 0714 is maintained on wheat (Triticum aestivum L.) seed for long term

storage at 4°C and activated on PDA medium at 25°C (± 2°C) under dark conditions.

Inoculum preparation and inoculation methodology was similar for all screenings;

where, inoculum was grown on 30 grams of wheat seed using 125 ml Erlenmeyer

flasks. Seed were soaked overnight in 30 ml of deionized water and then autoclaved for

25 minutes at 120°C for three consecutive days. Prior to inoculation, the isolate was

transferred to PDA media and incubated at 25°C under dark conditions. Six mycelia

plugs (5 mm) from a 3 day old colony were transferred to each flask. Flasks were

shaken daily by hand to ensure uniform colonization and were incubated for 1 week at

25°C (dark) until fungal colonization was observed. Flasks with autoclaved seed with no

inoculum were prepared as controls.

Growth Room Inoculations

Inoculations were conducted in the previously described walk-in plant growth

room. Prior to each inoculation of each run, plants were hand-trimmed with scissors to

ensure uniformity.

Three runs of the experiment were conducted in August 2010, October 2010, and

July 2012 using plants at three (run 1 and run 3) and six months (run 2) after planting.

Each inoculation run consisted of 144 plants (12 cultivars × 12 replications). Each pot

43

was an experimental unit. Six plants of each cultivar were inoculated (six replications)

with 15 infected seed placed in the pot center. The remaining six plants were treated

with non-inoculated autoclaved seeds (controls). The experimental design was

completely randomized.

For incubation, individual pots/plants were enclosed in one gallon re-sealable

plastic bags with a paper towel on the bottom. Thirty mL of tap water were dispensed

with a Brinkmann dispensette® to the paper towel to ensure high humidity (100%)

inside the bag. Plants were incubated under optimal environmental conditions for

disease development, as follows: temperature was set at 23/21°C (± 2°C) day/night with

a 12 hour photoperiod. Relative humidity in the walk-in plant growth room oscillated

between 40 and 50%. Three HOBO Pro v2 loggers model U23 (HOBOware® Onset

Computer Corporation, Bourne, MA, USA) were located on the benches (one/bench) to

monitor temperature and relative humidity during incubation. For better severity

assessment, infected tissue was dried by removing the pots from the bags 24 hours

before a rating. Ratings of disease severity were recorded by visually estimating the

percent of the area with symptoms (or lesions) compared to the total leaf sheath area.

Ratings were taken at 7 and 14 days after inoculation (DAI). In previous screenings with

isolate 0714 on St. Augustinegrass genotypes (Flor, 2009) and two Z. japonica cultivars

(Kenworthy, K., personal communication), mycelia and symptoms were observed at 7

DAI, but disease severity was higher at 14 DAI.

For data analysis, the estimated severity percent was converted to a mid-point

value using the modified Horsfall-Barrat rating (Horsfall and Barratt, 1945; Bock et al.,

2009).

44

Data Analysis

Data were analyzed using the generalized linear model procedure (Proc GLM) in

SAS (version 9.4 for Windows, SAS Cary, NC, 1982) at a significance level of P ≤ 0.05.

Disease severity of the 3 runs was analyzed using a one-way analysis of variance

(ANOVA) to detect differences between runs, cultivars, ratings dates and their

associated interactions. Cultivar means were separated using Tukey’s Honestly

Significantly Difference when there was DAI interaction within a run.

No previous report has clearly quantified levels of resistance or susceptibility of

zoysiagrass genotypes to large patch disease; therefore, resistance and susceptibility

responses were defined for this study. Definitions were based on observations of

disease caused by isolate UF 0714 and from other growth chamber and field

assessments of brown patch on cool season turfgrasses. Two studies evaluated

genotypes in a growth chamber estimating the percent of diseased tissue (Han et al.,

2006; Cho et al., 2011); while others evaluated genotypes in field trials estimating the

percentage of diseased area within plots (Yuen et al., 1994; Han et al., 2006) or utilizing

a 0-9 scale to represent disease severity (Bonos et al., 2003, 2004b; Belanger et al.,

2004; Bonos, 2006; Bokmeyer et al., 2009a; b). For example, creeping bentgrass

(Agrostis stolonifera L.) genotypes were considered resistant when brown patch severity

ratings were ≤ 15% (Cho et al., 2011) and tall fescue (Festuca arundinacea Schreb.)

genotypes were labeled as resistant when disease severity was ≤ 23% (Yuen et al.,

1994) and ≤ 30% (Bokmeyer et al., 2009a; b). Creeping bentgrass (Agrostis palustris

Huds.) and colonial bentgrass (Agrostis capillaris L.) genotypes were resistant to dollar

spot (Sclerotinia homoeocarpa F. T. Benn.) when severity was ≤ 22% (Belanger et al.,

2004) and ≤ 25% (Bonos et al., 2003; Bonos, 2006), respectively. Resistance to gray

45

leaf spot [Pyricularia grisea (Cooked) Sacc] was designated for perennial ryegrass

genotypes when severity ratings were ≤ 20% (Bonos et al., 2004b) or ≤ 30% (Han et al.,

2006).

Based on the results from these studies, cultivars with low disease severity (≤

20%) were considered resistant or tolerant. Complete resistance is considered when the

reproduction of the pathogen is totally prevented after initial infection (Parlevliet, 1979;

Horns and Hood, 2012). Tolerance is defined as the host’s ability to reduce disease

severity by decreasing further infections without affecting pathogen reproduction

(Parlevliet, 1979; Horns and Hood, 2012). Conversely, cultivars with disease severity

percentages ≥ 51% and ≥ 71% were classified as susceptible and highly susceptible,

respectively. Cultivars with average severity between ≥ 21% and ≤ 50% were classified

as moderately susceptible.

Results

For the pooled analysis of all runs, cultivars were moderately susceptible,

susceptible and highly susceptible to the isolate. Significant cultivar × run, cultivar × DAI

and run × DAI interactions occurred, although the cultivar × DAI × run interaction was

not significant (Table 2-2). Since a significant cultivar × run interaction occurred, runs

were analyzed and described independently. For run 1 and run 3 there were no cultivar

× DAI interactions, but the interaction was significant for run 2 (Table 2-3). Therefore, 7

and 14 DAI ratings were combined for run 1 and run 3, and separated for run 2.

Run One and Run Three

In run 1 (August 2010), ‘Taccoa Green’, ‘Zorro’ and ‘Shadow Turf’ were

extremely susceptible cultivars (Table 2-4). ‘Emerald’ was not different from Zorro and

Shadow Turf; and ‘Meyer’ was not different from Shadow Turf. Subsequently,

46

‘Palisades’, ‘UltimateFlora’, ‘Diamond’, and ‘Zeon’ were not different from Meyer

indicating that all of these cultivars, with disease severity ranging from 44.7 to 70.7%

were moderately susceptible to susceptible to the isolate, UF 0714. ‘JaMur’ (23.2%), ‘El

Toro’ (24.8%) and Empire (29.5%) were also moderately susceptible, but had

significantly lower severity from other moderately susceptible cultivars in run 1 (Table 2-

4).

In run 3 (July 2012), Zorro had the highest degree of disease severity (79.5%).

Emerald, Zeon and UltimateFlora were also statistically the most susceptible cultivars,

with disease severity >75 % (Table 2-5). Empire (44.1%), was the least susceptible

cultivar, and the only cultivar defined as moderately susceptible in run 3. However,

Empire was not statistically different from Taccoa Green (53.3%), JaMur (54.5%) and El

Toro (58.3%), all of which had susceptible responses. The remaining four cultivars were

also susceptible (Table 2-5).

Run Two

As mentioned, there was a significant cultivar × DAI interaction for run 2 (October

2010); therefore, data are presented separately for 7 and 14 DAI (Table 2-6). At 7 DAI,

Empire (31.1%) had the least amount of disease and a moderately susceptible

response. Empire was not different from JaMur, Meyer, Palisades, El Toro, Zeon, and

Zorro that ranged in classification from moderately susceptible to susceptible (39.5% to

67.8% disease severity). UltimateFlora, Emerald, Taccoa Green, Diamond and Shadow

Turf were not statistically different from most of the above genotypes for disease

severity; however, their severities ranged from 72.5% to 80% classifying them as highly

susceptible.

47

Disease severity increased from 7 to 14 DAI (Table 2-6), and ratings at 14 DAI

did not produce many significant differences due to extremely high disease. With a

rating of 66.2%, Empire had less severity compared to all other cultivars that ranged

from 78.9% (Palisades) to 96% (Emerald). Genotype × DAI interactions are shown in

Appendices A, B and C.

No significant variations in temperature, relative humidity or number of light hours

were registered in the walk-in growth chamber that could explain the differences among

runs and the cultivar × DAI interaction in run 2. However plant age was different

between runs. Plants in the second run were three months older than plants used in the

other two experiments.

Discussion

Rhizoctonia solani AG 2-2 LP, the causal agent of large patch disease, is a soil

inhabiting fungus capable of surviving long periods without a host. The disease can

appear recurrently during seasonal cold periods (fall), making it difficult to completely

eliminate. Genetic resistance has not been reported in zoysiagrass cultivars. As a

result, large patch disease is typically managed through preventative or curative

fungicide applications. Furthermore, levels of resistance or susceptibility for LP disease

in many cultivars are not clearly described.

This current study utilized a screening protocol that induced disease

development under controlled conditions. The feasibility of having a protocol for

screening zoysiagrass genotypes is an important step to breed for disease resistance.

The protocol developed for inoculation of zoysiagrass in the walk-in plant growth room

was successful for symptom development, although in this case, results are specific to

this particular isolate. Isolate 0714 was consistently virulent to zoysiagrass. Conditions

48

within the walk-in plant growth room were appropriate for mycelia and symptoms

development; mycelia were usually abundant at 7 DAI. Symptoms included an orange

coloration and infected leaf blades that were easily removed from leaf sheaths. This

was an indication of active infection and successful reproduction of LP symptoms in the

walk-in plant growth room.

In this study the disease response of 12 zoysiagrass cultivars under controlled

artificial conditions was evaluated. All cultivars had compatible interactions with the

isolate, although severity levels varied.

Empire had the least disease severity in all three runs. During run 1, run 2 (7

DAI), and run 3 it was designated as moderately susceptible, although in run 2 (14 DAI)

it was considered susceptible. JaMur and El Toro had similar disease responses. These

cultivars were moderately susceptible in run 1 and 3; and susceptible in run 2.

Palisades and Meyer were fairly consistent across the runs, but overall had more

disease than Empire, JaMur and El Toro. Palisades and Meyer had disease severity

between 42% - 67.5% among the runs, and would be considered susceptible to the LP

isolate used in this study. The remaining seven genotypes (all Z. matrella except

UltimateFlora) were less consistent, but had at least one or two runs with greater than

71% disease; and therefore, are considered susceptible. Zorro, Emerald and Shadow

Turf were the most consistently highly susceptible cultivars in the study.

Comparisons of these results to other literature can be made for Zorro, Emerald,

and Meyer. Field observations of the other cultivars evaluated in this study are not

currently published. Zorro and Emerald were susceptible to LP in Arkansas and in

Oklahoma (Morris, 2006) and also in the walk-in plant growth chamber (Gainesville, FL).

49

However, the LP disease response was different in the NTEP trial 2002 at Raleigh, NC.;

where both cultivars were rated with little to no disease in the field (Morris, 2006). For

Meyer, the NTEP ratings in Raleigh, NC (Morris, 2006) agree with walk-in plant growth

chamber data; where, in both environments this cultivar showed somewhat less

susceptibility. However, in Arkansas and Florida, NTEP ratings indicate that Meyer was

very susceptible (Morris, 2006, 2012) which is in contrast to the growth room ratings

with our LP isolate UF 0714 . It is expected that the responses of both tolerant and

susceptible cultivars will be better under field conditions where environmental factors

fluctuate and conditions for disease development may not be as favorable. In addition,

high levels of disease pressure in the walk-in plant growth room over 2 weeks may

overcome resistance mechanisms, if they exist.

As noted above, there was a cultivar × run interaction. Zorro, Taccoa Green,

Zeon, UltimateFlora and Shadow Turf were less consistent between the runs and were

the likely causes of the interaction. These inconsistencies in ranking indicate that the

protocol needs to be adjusted to improve factors that may contribute to differences in

the disease response. Using more replications per genotype, performing the

inoculations on plants of the same age and evaluating genotypes grown in bigger pots

are some factors that could improve the protocol and decrease interactions between

runs. Using estimates of percent severity or the utilization of a rating scale with better

defined categories might also increase the consistency between runs and would provide

better descriptions of a genotype’s LP disease response.

It is pertinent to consider that large differences in disease severity ratings

between replications of the same cultivar or between rating days (7 and 14 DAI) could

50

be attributed to the utilization of the “Horsfall-Barrat” (H-B) scale. The H-B is a

logarithmic-based scale with 12 categories that represent severity percentages that

increase and decrease symmetrically from 0 to 50% and from 100 to 50%, respectively

(Horsfall and Cowling, 1978; Bock et al., 2009, 2010; Chiang et al., 2016). For data

analysis the categories are converted to mid-point values (Horsfall and Cowling, 1978;

Bock et al., 2009, 2010; Chiang et al., 2016). The range between some intervals such

as (12-25%), (75-87%) and especially (25-50%) and (50-75%) are wide and decrease

data resolution (Bock et al., 2009; Chiang et al., 2014, 2016). As a result, a rating can

be under or overestimated, affecting the accuracy of the disease assessment (Bock et

al., 2009; Chiang et al., 2014, 2016) and introducing variability into the samples (Bock et

al., 2010). Therefore, the estimation of the proportion (%) of the infected leaf area would

be a better method for assessment of LP disease severity.

The damage caused by the isolate progressed through the incubation period in

all cultivars. At 14 DAI most of the cultivars had high disease severity and this may

indicate that ratings from 7–10 DAI would be better to capture differences between

cultivars. Obasa et al. (2012) reported similar observations under growth chamber

conditions and found significant differences to large patch susceptibility at 5 DAI among

zoysiagrass breeding lines and Meyer. The genotypes evaluated were not significantly

different when rated at 10, 15, 20, and 25 DAI (Obasa et al., 2012). Related to

components of resistance, Parlevliet (1979) proposed that under high inoculum

pressure, polygenic resistance among genotypes can be difficult to identify. Thus, early

ratings and estimation of Area Under Disease Progress Curve (AUDPC), apparent rate

51

of infection (r) and maximum severity (Y max.) would be appropriate when screening for

LP disease resistance.

Information available describing the response of zoysiagrass cultivars to large

patch disease is limited (Metz et al., 1992; Morris, 2000, 2006, 2012; Obasa et al.,

2012). These reports assess the response by qualitative/quantitative ratings of disease

severity or by qualitative classification of resistance or susceptibility. Disease responses

reported by the National Turfgrass Evaluation Program (NTEP) field trials are usually

limited to specific regions where environmental conditions such as soil type,

temperature, rain, and relative humidity may have an effect on disease development.

In field trials cultivar variation (genotype by location) associated with LP

responses have been observed. The NTEP has the most extensive evaluation of

zoysiagrass responses under field conditions to diseases caused by Rhizoctonia spp.

NTEP has conducted three national zoysiagrass trials (1996, 2002 and 2007) with

reports of zoysiagrass to brown patch and large patch (LP) diseases from seven

locations (Georgia, South Carolina, Texas, Arkansas, North Carolina, Florida and

Oklahoma) (Morris, 2000, 2006, 2012). For brown patch disease, Emerald, Zorro,

Meyer, Zenith and Chinese Common produced variable responses between Georgia

(Morris, 2000), Texas, South Carolina (Morris, 2006) and Florida (Morris, 2012).

For Large patch disease, Emerald, Zorro, Meyer, Chinese Common, Zenith,

Himeno and Compadre had variable disease responses in some of the locations

evaluated in the 2002 and 2007 NTEP trials (Morris, 2006, 2012). In Oklahoma the size

of the diseased area was measured, but not associated with the degree of resistance

and susceptibility. Based on the measurements, Meyer appeared to be resistant and

52

Zorro very susceptible. Compadre, Chinese Common, Emerald, Zenith and Himeno

appeared to be moderately susceptible. Disease responses of 6 cultivars were

consistent between the trials in Oklahoma and Arkansas (Morris, 2012). Zorro and

Zenith were moderately susceptible in Oklahoma and Florida (Morris, 2006, 2012).

However, disease ratings in Raleigh were different from disease ratings taken in

Oklahoma and Arkansas (Morris, 2006). In Raleigh, these cultivars were rated with high

resistance (≥ 8.7). Conversely, in Oklahoma and Arkansas the cultivars were moderate

to highly susceptible. Meyer appeared to be moderately resistant in Oklahoma and

Raleigh, but it was susceptible in Arkansas and Florida (Morris, 2006).

Contrasting responses were reported for BP and LP diseases in Arkansas,

South Carolina and Texas for ‘Compadre’, ‘Emerald’, ‘Meyer’, ‘Zenith’, Himeno and

‘Zorro’ (Morris, 2006). Chinese Common had consistent moderately susceptible and

susceptible responses in these locations (Morris, 2006). Metz et al. (1992) classified

Emerald and Meyer differently from the disease ratings obtained in Raleigh (NC) and

Gainesville (FL) (Morris, 2006). For all of the above reports, the variable environmental

conditions of each site and the probable differing isolates make it difficult to clearly

designate a level of resistance or susceptibility of zoysiagrass cultivars under field

conditions. Screening under controlled environments could produce more reliable

disease responses than field trials under natural infection.

However few reports of LP disease assessment in controlled conditions are

reported. Metz et al. (1992) evaluated genotypes of zoysiagrass to Rhizoctonia foliar

blight disease under growth chamber conditions using a virulent isolate of the

Anastomosis Group 2-2 (AG 2-2). This research was conducted prior to the designation

53

of large patch for warm season grasses; and therefore, it is not known if this study was

conducted using a BP (brown patch) or LP (large patch) isolate of AG 2-2. Additionally,

the temperature (28°C) used for incubation by Metz et al. (1992) is not the current

recommended temperature (23°C) that is most conducive for large patch symptom

development. The same isolate was used for inoculating genotypes of Agrostis spp.

(bentgrass). Since bentgrass is a cool season turfgrass, symptom development should

not have been expected if the isolate was from the “LP subgroup”, due to the specificity

between the AG 2-2 LP and warm-season grasses. In their study, the zoysiagrass

disease severity ranged from 10.83% (Meyer) to 100% (‘Omni’), indicating differences in

the disease response among genotypes. ‘Royal’, ‘Cavalier’ and ‘Diamond’ exhibited a

high degree of disease resistance, while ‘Korean Common’, ‘Sunburst’ and ‘Belair’ were

highly susceptible. ‘Emerald’, ‘El Toro’, and ‘Palisades’ were moderately susceptible

and ranked between Diamond and Belair.

Obasa et al. (2012) evaluated the large patch disease response of Meyer and its

progeny obtained from specific crosses with two breeding lines, Diamond, Zorro and

Emerald. Evaluations were conducted under field and growth chamber conditions in

Kansas for two years. In both environments, Meyer exhibited moderate to low

susceptibility to the isolate. In the field, Meyer showed lower Area Under Disease

Progress Curve (AUDPC) values compared to its progeny; however, in the growth

chamber, Meyer had higher and lower AUDPC values than its progeny. In this study no

correlation was found between growth chamber and field data, nor between data taken

in different years (Obasa et al., 2012).

54

In previous screenings for other turfgrass diseases, resistance has been defined

by the amount of severity (≤ 30%) observed on cultivars and genotypes under the

experimental conditions of each study (Yuen et al., 1994; Bonos et al., 2003, 2004b;

Belanger et al., 2004; Bonos, 2006; Han et al., 2006; Bokmeyer et al., 2009a; b; Cho et

al., 2011). In this current study resistance (disease severity ≤ 20%) was not found

among the cultivars evaluated. Thus, the determination of the disease response was

based on levels of susceptibility observed during a specific incubation period. Cultivars

of Z. japonica had the most consistent and positive responses against LP disease. For

discussion purposes, the combined 7 and 14 DAI averages of runs 1 and 3 and 7 DAI

for run 2 will be considered. The disease severity at 14 DAI in run 2 was too high for

consideration of classifying LP response.

The screening methodology was not designed to directly assess the relationship

between leaf texture and disease response, but results indicate that the more winter

hardy japonica cultivars responded similarly with less large patch disease severity.

Coarse textured Z. japonica genotypes are better adapted to low temperatures than Z.

matrella genotypes (Patton and Reicher, 2007; Xuan et al., 2009; Patton, 2009; Tsuruta

et al., 2011). A possible linkage between cold tolerance and disease resistance would

be a good topic to study in the future. Japonica-type cultivars also tend to have a more

open growth (less dense) habit (Wherley et al., 2011), longer and wider (> 2 mm)

(Anderson, 2000; Patton, 2009) leaf blades and wider leaf angles (55°± 1.83) (Choi,

2010); features that may decrease the spread of the mycelia. Conversely, Z. matrella

cultivars have shorter and narrower (< 2 mm) (Anderson, 2000; Patton, 2009) leaf

blades, and narrower leaf angles (60° ± 8.11) (Choi, 2010) with more dense growth

55

(Wherley et al., 2011); features that may facilitate the spread of the mycelia. Turf

canopy density has been associated with increased susceptibility to brown patch

disease in tall fescue (Festuca arundinaceae Schreb) cultivars because of faster

interblade mycelia growth (Giesler et al., 1996a; b; Bonos et al., 2004a; Watkins and

Meyer, 2004), and increased humidity within the canopy (Yuen et al., 1994).

Conclusions and Future Research

Up-to-date response information of commercial cultivars to large patch disease is

important to assist with the development of better management practices to prevent or

cure the disease in known susceptible cultivars. Knowledge of cultivars with resistance

or tolerance will aid in the selection of cultivars for use in areas where large patch is

known to occur and help to reduce maintenance costs and the use of fungicides.

Identification of resistant cultivars would also help to explore the mechanisms

associated with resistance and positively impact the development of future cultivars.

This screening was performed with the purpose of detecting differences for large

patch disease on zoysiagrass cultivars and to identify cultivars with high resistance

levels. Results indicate the variability in susceptibility of commercial zoysiagrass

cultivars to a characterized AG 2-2 LP isolate of R. solani. For some cultivars this data

represents the first documentation of large patch response. Although, among these 12

cultivars none were resistant (≤ 20% disease severity), they showed different degrees of

damage. These results are specific for the isolate used and indicate the disease

response under conditions appropriated for the pathogen to induce infection and

symptoms development (high disease pressure). This data may not reflect the

performance of these cultivars under field conditions. In the field, under natural

56

infection, the response of the cultivars could be different due to fluctuating factors: soil

and air temperature, relative humidity, precipitation, soil type and cultural practices.

This study did show that, in general, commercial cultivars of Z. japonica were

less susceptible to LP infection than Z. matrella. In addition, Empire, JaMur and El Toro

were identified as being the most consistent and least susceptible, while Zorro,

Emerald, and Shadow Turf were the most susceptible to the LP isolate UF 0714.

Further research may help to explore and understand the mechanisms

associated with the disease resistance in zoysiagrass at morphological and molecular

levels in this pathosystem. Since all the cultivars were susceptible to the isolate,

knowledge about of the timing of infection in each cultivar, infection rates during specific

incubation times and lesion expansion rate would be useful to detect components of

resistance among these zoysiagrass cultivars. Also, additional screenings of germplasm

with wider genetic diversity and the utilization of other AG 2-2 LP isolates, along with

field evaluations would be extremely valuable to support the results presented in this

study. These evaluations are important because the response of the cultivars appears

to be specific for each disease and location; indicating genotype interactions (Morris,

2000, 2006, 2012).

57

Table 2-1. Zoysiagrass commercial cultivars selected for the large patch disease screening.

Cultivar Species Developing institution Year of release

Diamond Z. matrella Texas A&M Univ., Dallas, TX 1996 El Toro Z. japonica Univ. of California, Riverside, CA 1986 Emerald Z. japonica × Z. pacifica USDA 1955 Empire Z. japonica Sod Solutions Inc., Mt. Pleasant, SC 1999 JaMur Z. japonica Bladerunner Farms Inc., Poteet, TX 1996 Meyer Z. japonica USDA 1951 Palisades Z. japonica Texas A&M Univ., Dallas, TX 1996 Taccoa Green Z. matrella Univ. of Florida, Belle Glade, FL 2005 Shadow Turf Z. matrella Ivey Gardens Greenhouse, Lubbock, TX 2007 UltimateFlora Z. japonica Univ. of Florida, Belle Glade, FL 2005 Zeon Z. matrella Bladerunner Farms Inc., Poteet, TX 1996 Zorro Z. matrella Texas A&M Univ., Dallas, TX 2001

Source: (Patton, 2009).

58

Table 2-2. Analysis of variance of large patch disease severity of twelve zoysiagrass cultivars in three inoculation runs at the walk-in plant growth room.

Source of variation DF Type III SS Mean Square F value P > F

Runa 2 51360.69 25680.34 98.98 <.0001 DAIb 1 50181.33 50181.33 193.42 <.0001 Rep (run)c 15 3408.14 227.20 0.88 0.5920 Cultivard 11 45053.46 4095.76 15.79 <.0001 Cultivar × run 22 23316.72 1059.85 4.09 <.0001 Cultivar × DAI 11 6053.50 550.31 2.12 0.0184 DAI × run 2 3373.51 1686.75 6.50 0.0017 Cultivar × DAI × run 22 7667.57 348.52 1.34 0.1401 Errore 345 279922.87 259.44

a Inoculation run: 12 zoysiagrass cultivars were inoculated with an LP isolate (0714) and incubate under optimal conditions for disease development. A run consisted of 144 plants (12 cultivars x 12 replications: 6 inoculated and 6 as controls). b DAI: days after the inoculation. c Replications were nested inside an inoculation run. d Twelve zoysiagrass cultivars were evaluated. e Residual error.

59

Table 2-3. Analysis of variance of large patch disease severity of twelve zoysiagrass cultivars in three inoculation runs.

Runa 1 Run 2 Run 3 Source of variation DF Mean Square P > F Mean Square P > F Mean Square P > F

Cultivarb 11 2864.71 <.0001 1724.95 <.0001 1625.80 <.0001 Replicationc 5 117.73 0.8716 174.41 0.6067 389.47 0.1157 DAId 1 10531.89 <.0001 31196.39 <.0001 11826.56 <.0001 Cultivar × DAI 11 386.08 0.2971 605.93 0.0071 255.35 0.3027 Errore 115 322.60 - 240.88 - 214.84 -

a Inoculation run: 12 zoysiagrass cultivars were inoculated with an LP isolate (0714) and incubate under optimal conditions for disease development. A run consisted of 144 plants (12 cultivars x 12 replications: 6 inoculated and 6 as controls). b Twelve zoysiagrass cultivars were evaluated. c Six replications were inoculated for each cultivar. d DAI: days after the inoculation. e Residual error.

Table 2-4. Combined mean separation 7 and 14 days after inoculation (DAI) of large patch disease severity of

zoysiagrass commercial cultivars in the first run of inoculation within the walk-in plant growth room.

Cultivar Disease severitya

Taccoa Green 70.7 ab Zorro 66.8 ab Shadow Turf 60.9 abc Emerald 56.5 bcd Meyer 51.5 cde Palisades 48.3 de UltimateFlora 47.1 de Diamond 45.5 de Zeon 44.7 e Empire 29.5 f El Toro 24.8 f JaMur 23.2 f

a Disease severity mean of six replications of 7 and 14 days (combined) after the inoculation.

b Mean separation obtained with Tukey’s HSD (Honest Significant Difference) at P > 0.05. Minimum Significant Difference: 24.46.

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Table 2-5. Combined mean separation 7 and 14 days after inoculation (DAI) of large patch disease severity of zoysiagrass commercial cultivars in the third run of inoculation within the walk-in plant growth room.

Cultivar Disease severitya

Zorro 79.5 a Emerald 79.1 ab Zeon 79.1 ab UltimateFlora 75.4 abc Meyer 67.5 bcd Palisades 65.4 cde Diamond 62.9 def Shadow Turf 59.1 def El Toro 58.3 efg JaMur 54.5 efg Taccoa Green 53.3 fg Empire 44.1 g

a Disease severity mean of six replications of 7 and 14 days (combined) after the inoculation.

b Mean separation obtained with Tukey’s HSD (Honest Significant Difference) at P > 0.05. Minimum Significant Difference: 19.96.

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Table 2-6. Mean separation at 7 and 14 days after the inoculation (DAI) of large patch disease severity of twelve zoysiagrass commercial cultivars in the second run of inoculation within the walk-in plant growth room.

Cultivar Disease severity at 7 DAIa Disease severity at 14 DAIa

Shadow Turf 80.0 ab 95.5 ac

Diamond 76.7 ab 92.8 a Taccoa Green 75.7 ab 88.7 a Emerald 74.5 ab 96.0 a UltimateFlora 72.5 ab 92.0 a Zorro 67.8 abc 89.4 a Zeon 62.0 abc 87.1 a El Toro 46.8 abc 92.0 a Palisades 42.0 abc 78.9 ab Meyer 39.5 bc 90.0 a JaMur 39.5 bc 92.8 a Empire 31.1 c 66.2 ab

a Disease severity mean of six replications at 7 and 14 days after the inoculation.

b Mean separation obtained with Tukey’s HSD (Honest Significant Difference) at P > 0.05. Minimum Significant Difference: 18.46. c Mean separation obtained with Tukey’s HSD (Honest Significant Difference) at P > 0.05. Minimum Significant Difference: 39.70.

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CHAPTER 3 HERITABILITY ESTIMATES FOR LARGE PATCH DISEASE RESPONSE IN

ZOYSIAGRASS

Introduction

In the United States zoysiagrass (Zoysia spp.) is a popular C4 perennial warm

season turfgrass in Florida and in the transition zone. Qualities of zoysiagrass include

uniform growth and density, reduced inputs and tolerance to drought and saline

conditions (Green II et al., 1993; Patton, 2010; Obasa et al., 2012, 2013; Kimball et al.,

2013). Zoysiagrass is used for home lawns, sport fields, golf courses and other

landscapes (Yaneshita et al., 1999; Li et al., 2005; Patton and Reicher, 2007; Harris-

Shultz et al., 2014).

Zoysia spp. are protogynous, cross pollinated, self-fertile (Forbes, 1952; Cai et

al., 2005) and allotetraploid, where 2n = 4x = 40 (Yaneshita et al., 1999). In general,

Zoysia spp. easily hybridize (Forbes, 1952) increasing the species’ genetic diversity.

Zoysiagrass is primarily propagated vegetatively, although many genotypes are seed

producers.

The genus Zoysia is diverse. Currently, there are eleven Zoysia spp. reported

(Tanaka et al., 2016a), but Z. japonica and Z. matrella are the species most utilized for

turfgrass. In USA Z. japonica was introduced in 1895 and Z. matrella in 1911 (Childers

and White, 1947; Grau and Radko, 1951).

Abiotic factors can affect the quality of zoysiagrass. Problems associated with

zoysiagrass include thatch production, poor wear tolerance during dormancy and slow

recovery from wear; and issues with abiotic stress factors such as low temperatures and

shade (Green II et al., 1993; Patton, 2010). For these reasons there are several

63

zoysiagrass breeding programs emphasizing drought, wear and winter hardiness

responses, color, establishment rate, turf density, shade and salinity tolerance.

Zoysiagrass is also sensitive to several biotic pests in Florida’s sub-tropical

climate. Thus, breeding programs have considered the improvement of responses

(resistance) to insects, nematodes, mites and fungal diseases.

Large patch (LP) caused by the fungus Rhizoctonia solani (Anastomosis Group

2-2 LP) (Green II et al., 1993; Hyakumachi et al., 1998) is the most economically

important disease (Obasa et al., 2012). Large patch most often occurs between late fall

and spring when zoysiagrass is dormant (Green II et al., 1993). The fungus affects the

crown and base of leaf sheaths; thus water and nutrient translocation is limited. Infected

leaf sheaths develop an orange coloration and eventually blast. As a result, small to

large patches of infected-dead tissue form; hence, the name large patch. The fungus

typically becomes inactive when temperatures increase (summer) and zoysiagrass is

actively growing (Green II et al., 1993; Obasa et al., 2012).

When conditions are suitable for symptom development, the disease can be

managed using applications of both preventative and curative fungicides. No

zoysiagrass cultivar exhibits complete resistance. Thus, improvement for large patch

resistance is currently a major breeding goal. Understanding the genetic components of

large patch disease is essential for improvement of zoysiagrass. This knowledge implies

the elucidation of the inheritance mode, genetic variances and heritability estimation.

This information will allow for the determination of an appropriate breeding procedure.

Currently, for the Zoysia spp.- large patch pathosystem this information is not available.

64

Few reports of heritability exist for zoysiagrass traits. Moderate to high broad

sense heritabilities H2 (0.32 - 0.94) were reported for plot coverage, turf density, genetic

color and seed head density (Schwartz et al., 2009). Moderate H2 (0.32 - 0.58) was

reported for spring green-up, fall dormancy and turf quality under Bipolaris spp. infection

and mole cricket (Scapteriscus spp.) damage (Schwartz et al., 2009). Qian et al. (2000)

also reported moderate H2 (0.40 - 0.67) for salinity tolerance, root growth, shoot growth

and relative leaf firing.

Narrow sense heritabilities (h2) have been reported for morphological and

reproductive traits. Moderate to high h2 (63.22 - 93.67%) were reported for density, turf

height, leaf length, leaf width, leaf length/width, internode length, internode diameter

and internode length/diameter (Guo et al., 2012a). For reproductive traits such as

inflorescence density, reproductive branch height, reproductive length, floret number,

length, width and floret length/width, moderate to high h2 (42.72 - 98.8 %) were obtained

(Guo et al., 2010). Also, for seed head length, leaf width, growth habit, flowering, floret,

stem and leaf color and response to rust (Puccinia spp.) low to moderate h2 (0.23-0.66)

were reported (Flor et al., 2014).

For other turfgrasses such as perennial ryegrass (Lolium perenne L.), tall fescue

(Festuca arundinacea Schreb), creeping bentgrass [Agrostis stolonifera L., (1753)], Poa

trivialis L. and seashore paspalum (Paspalum vaginatum Swartz), broad and narrow

sense heritabilities have been reported for stem (Puccinia graminis subsp. graminicola

Z. Urban) and crown rust (Puccinia coronata Corda f. sp. lolii), gray leaf spot

[Magnaporthe oryzae Couch and Kohn, (2002)], brown patch (Rhizoctonia solani) and

dollar spot (Sclerotinia homoeocarpa F. T. Benn) diseases. Over all these studies,

65

broad sense and narrow sense heritabilities were H2 = 0.25-0.92 (Hurley and Funk,

1985; Rose-Fricker et al., 1986; Bonos et al., 2003, 2004b; Bonos, 2006; Bokmeyer et

al., 2009a; b) and h2 = 0.22-0.62 (Reheul and Ghesquiere, 1996; Bonos, 2006; Han et

al., 2006; Bokmeyer et al., 2009b; Flor et al., 2013).

Since no genetic studies and/or heritability estimates have been reported for the

zoysiagrass-large patch pathosystem; this current study was addressed to estimate

these components. At the University of Florida, zoysiagrass populations were evaluated

for their large patch disease response. Objectives of these studies were to describe the

disease responses associated with these zoysiagrass populations and to estimate the

heritability relative to the inheritance of large patch disease response. Results will

provide information of the proportion of genetic factors associated to large patch

disease in the populations. This knowledge can be used to implement an efficient

breeding method for this trait.

Materials and Methods

Three populations were used to screen large patch disease response under

artificial conditions within a walk-in plant growth room. One of these populations was

also evaluated under field conditions (natural infection). The zoysiagrass populations

studied will be referred to as: germplasm accessions, F1 hybrids and segregating F1

families.

The germplasm population was comprised of 50 genotypes and 10 cultivars

(Table 3-1). The genotypes selected from a group of 250 lines evaluated from 2005-

2008 for adaptability to Florida. The F1 hybrids were a group of 78 genotypes (Table 3-

2) selected from a broader group of 800 progeny produced from crossing the previously

selected 50 germplasm lines (Flor et al., 2011). Selection of the hybrids for evaluation

66

was based on their parent’s responses to large patch disease during the germplasm

evaluation. The F1 families were developed by crossing 20 genotypes (Table 3-3)

selected from the germplasm population. These genotypes had variable leaf textures

and large patch disease responses. Commercial zoysiagrass cultivars were included as

checks for screening of the germplasm and F1 hybrids.

Crossing procedure. To produce the F1 hybrids and F1 families, sod of each

parental germplasm line was harvested from the field and brought into the greenhouse.

The F1 hybrids were produced in the fall of 2008 and the F1 families were produced in

early spring 2011. For both populations, crossing was initiated during active flowering

and each genotype was used as female and male. For the F1 hybrid population all

possible cross combinations were attempted among the 50 selected germplasm lines.

However, the F1 family population only utilized 20 germplasm lines and specific crosses

were made that would produce progeny that segregated for leaf texture and LP disease

response. Racemes with mature stigmas at pre-anthesis were used as females.

Stigmas were examined for signs of previous pollination using a triple lens magnifier

(5X, 10X and 15X, RadioShack Corporation, Ft. Worth, TX). Immature stigmas were

covered with borosilicate cylindrical culture tubes of 6 mm x 50 mm (Thermo Fisher

Scientific, NH, USA) or transparent glassine pollination paper bags of 15 cm x 3.5 cm

cut at 10 cm length (Seedburo Equipment Company, Chicago, USA) to avoid unspecific

pollination. Stigmas were pollinated when mature. Racemes with mature anthers were

chosen as males.

Pollination was performed between 8:00 and 9:30 a.m., when stigmas were

receptive and anthers open for pollen release. Pollen was collected on microscope

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cover glass 18 x 18-1 (Fisher Scientific, Co., Pittsburgh, PA) and carefully sprinkled

using a fine brush or by softly placing the stigmas over the pollen. Abundant pollen was

transferred to ensure good coverage of stigmas. Pollinated racemes were labeled and

covered with glassine pollination paper bags for approximately 7 to 10 days. Racemes

of a specific cross were bulk harvested when mature, approximately 30 days after

pollination and stored in 5.7 cm x 8.9 cm coin envelopes (Swinton Avenue Trading Ltd.,

Inc., Boca Raton, FL., USA) at 4°C until use. Open pollinated and controlled self-

pollinated racemes from parental genotypes were also harvested for the F1 family

evaluations.

In 2009 and 2012, seed were germinated for the F1 hybrids and F1 families

respectively. Scarification of seed was performed, prior to germination, with sodium

hypochlorite (10%) for 2 hours followed by three washes with tap water. Subsequently,

seed were planted in 14.3 cm Saucer Plus trays (SP6VUS Gardener’s Blue Ribbon,

Woodstream Corp., Lititz, PA, USA) with approximately 55 gr of 1:1 of Metro-Mix® 910

(Sun Gro Horticulture Canada Ltd.) and United States Golf Association (USGA)

specification sand. Saucers were located in a poly-greenhouse. Individual seedlings of a

cross were re-planted and given a unique genotype or F1 family label.

Propagation, establishment and maintenance of zoysiagrass genotypes for the

three populations used for artificial inoculations were conducted using similar practices

at the University of Florida Turfgrass Envirotron greenhouse. Genotypes were

propagated in 75 mm (7.6 cm) plastic pots (KORD products, Toronto, Ontario, Canada)

with approximately 40 g of 80:20 Metro- Mix® 910 potting mix soil (Sun Gro Horticulture

Canada Ltd.) and USGA sand. Maintenance included automatic irrigation daily (25 mL

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2X); weekly trimming at 5.0 cm and foliar fertilization every 2 weeks. Fertilization was

applied using 24-8-16 Miracle-Gro® Water Soluble All Purpose Plant Food (Scotts

Miracle-Gro Products, Inc., Marysville, OH, USA) at 2.44 Kg/ha. To ensure uniformity,

plants were trimmed the day before or the day of inoculation.

In July 2011, the 78 F1 hybrids, ‘JaMur’ and ‘Empire’ zoysiagrass were planted at

the Plant Science Research and Education Unit (PSREU), Citra, FL. The trial was a

randomized complete block design (CRBD) with 3 replications. Ten, 25 cm2 plugs were

planted in 1.5 m × 1.5 m plots. Plots were separated by 0.30 m alleys. Maintenance of

the plots included mowing at 7.6 cm once a week during the growing season and

automatic irrigation applied as needed, but typically 3 times/week with 16.13 mm3 of

watering/event. Other practices included annual fertilization at 19.52 g N m-2 split in 4

applications (every 3 months), two applications of UFlexx® (46N-0P-0K) at 1.12-2.24

Kg/ha and application of pre and post-emergence herbicides, insecticides and

fungicides. Alleys between plots were maintained using a 2% solution of glyphosate

applied monthly during growing seasons. Environmental conditions of the greenhouse

prior to inoculation of each population are shown in Appendix D.

Large Patch Disease Screenings

Isolate 0714 of Rhizoctonia solani AG 2-2 LP was used to inoculate the 3

populations. Isolate description, inoculation protocol and incubation conditions were

described in Chapter II. Inoculations were conducted in the same walk-in plant growth

room of the UF Plant Pathology Department described in Chapter II. Each pot was

inoculated with 10-12 infected seeds. The mock controls were treated with 10

autoclaved seeds. For the 3 populations evaluated, and due to space limitations in the

walk-in plant growth room, the whole set of accessions was divided in inoculation

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subsets. Attempts were made to have uniform conditions (inoculation procedure and

incubation conditions) between inoculations.

Germplasm

In fall 2009, 50 germplasm accessions and 10 cultivars were evaluated in two

inoculation runs separated in time. Due to limited space within the walk-in plant growth

room, accessions were randomly divided into four subsets for each run; for a total of

eight inoculation subsets. Within a subset of a given run all replications of an included

accession were included. Within a subset, the treatments (pots of accessions) were

arranged as a completely randomized design with nine inoculated and three non-

inoculated (control) pots per genotype. Disease severity was visually estimated based

on the percent of sheath tissue infected and converting the estimated percent to a mid-

point value using the modified Horsfall-Barrat rating (Horsfall and Barratt, 1945; Bock et

al., 2009). Severity ratings were taken at 7 and 14 days after inoculation (DAI).

F1 Hybrids

The F1 hybrids were screened using the walk-in plant growth room and in field

plots. ‘Meyer’, ‘Empire’, ‘Geo’ and ‘Aloysia’ were included in the walk-in plant growth

room as standard checks. Hybrids were evaluated in two runs separated in time; first in

spring and summer (May through August) 2011 and second in spring (Apr. and May)

2015. Again space limitations required that a run of the experiment be divided into four

subsets. A subset of a run included all replications of a given hybrid; and hybrids were

arranged in a completely randomized design with four inoculated and two non-

inoculated pots of each hybrid. Disease severity was visually estimated based on the

percent of infected tissue within a pot. Severity ratings were taken at 7 and 14 days

after inoculation (DAI).

70

In the field, large patch disease severity was visually estimated as the percent of

plot with symptomatic or damaged tissue. Plots were evaluated from February 2012 to

April 2015. A total of 36 ratings were taken in the field. However, the majority of the

ratings were discarded for the analysis due to lack of enough phenotypic variation for

the disease, low disease pressure, overlapping symptoms with Bipolaris and

confounding effects due to winter kill and dormancy. Thus, for the narrow sense

heritability estimation only four ratings were used.

F1 Families

In spring 2013, six F1 families were selected for heritability estimation of large

patch disease response (Table 3-4). Selection was based on disease response and

texture of the parental lines. Morphological segregation and family size were also

considered. Five clones from each genotype/family were produced. In the greenhouse

plants were established in a randomized complete block design. Each block had 275

plants (269 genotypes + 6 parents).

Inoculations were performed in fall 2014 (Oct. and Nov.) and spring 2015 (Feb.

and March) 12 and 14 months after propagation. Because of space limitations in the

walk-in plant growth room, each replication was inoculated separately as a subset of a

run, and the experimental design was a randomized complete block design; where

blocks were the subsets of a given run. Thus, a subset (block) consisted of 275

inoculated genotypes. A total of four runs were performed. An additional “mock run” was

performed Jan. 2015 using non-inoculated, autoclaved seed.

Ratings were taken at 7, 14 and 21 days after inoculation (DAI). Disease severity

was visually estimated based on the percent of infected tissue. Amount of mycelia/pot

was recorded. After 7 and 14 DAI ratings, 20 mL of tap water were added to the paper

71

towel to maintain high humidity inside the bag. Plants were bagged again and incubated

for an additional week. At 14 DAI, plants were trimmed (scissors) to increase symptom

development. After the 21 DAI rating, plants were removed from the walk-in plant

growth room and returned to the greenhouse.

Statistical Analysis

Severity data were analyzed using the animal model with fixed and random

effects as follows: y = Xβ + Za + Z1i + e, where y = disease severity, β = vector of fixed

effects, a = random effect of the genotype ~ N (0, Iσ2a), i = random effect of genotype

interactions ~ N (0, Dσ2i) (Table 3-5 and Table 3-6). I and D were identity and block

diagonal matrices. X, Z and Z1 were incidence matrices. The random residual effect was

e ~ N (0, Iσ2e).

Analysis of variance of disease severity data was obtained using ASREML v3.0

software (Gilmour et al., 2009) with a significance level of P ≤ 0.05. For the walk-in plant

growth room data significance of run, replication, genotype, and their respective

interactions were tested according to each data set. However, because genotypes and

their interactions were considered as random effects the analysis was performed by

combining runs within ratings (7 and 14 DAI) (Table 3-5 and Table 3-6). For the field

trial significance of genotype and genotype × replication were tested.

Frequency Distribution and Population Segregation

Population distribution of the disease response was described at each rating date

and inoculation run using disease severity means. Histograms were elaborated at 10%

severity intervals. The disease response of the genotypes was determined using the

severity percentage estimated as explained in Chapter II. Phenotypic segregation was

described only for the F1 families using disease severity means.

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Heritability Estimates

Broad and narrow sense heritability were estimated using the variance

components obtained from the ASREML v3.0 software (Gilmour et al., 2009). Broad

sense heritability was estimated on a family-mean basis as the ratio of genotypic

variance (σ2G) to the total phenotypic variance (σ2

P) as follows: H2 = σ2G/σ2

P (Nyquist

and Baker, 1991; Falconer, D. S and Mackey, 1996).

Narrow sense heritability h2 (Falconer, D. S and Mackey, 1996) was estimated as

the ratio of additive variance (σ2a) to the total phenotypic variance (σ2

P) as follows: h2 =

σ2a/σ2

P and incorporating the pedigree information. Phenotypic variance (σ2P) was equal

to σ2P = σ2

a + σ2i + σ2

ε; where σ2a is additive genetic variance, σ2

i is epistatic variance and

σ2ε is environmental variance. The expected phenotypic variance for H2 and h2 estimates

was calculated by dividing the variance components by their respective coefficients

(Nyquist and Baker, 1991) (Table 3-5 and Table 3-6). For the field trial the median value

of the disease severity was used to estimate the narrow sense heritability. This value

reflects more accurately the variation observed among replications and ratings. Standard

errors (SE) of the heritability for the germplasm and F1 hybrids were calculated using the

formula (Hallauer and Miranda, 1981) as follows:

SE (heritability) = SE (𝜎𝐺2) / 𝜎𝐺

2 +𝜎𝐺

2 × run

run+

𝜎𝐺2 × run×rep.

run × rep.+

𝜎ɛ2

run × rep. . (3-1)

The standard error of the genotypic variance (SE σ2G) was calculated using the

following formula (Hallauer, 1970):

SE (σ2G) = √

2

𝑐2∑

𝑀𝑖2

𝑑𝑓𝑖+ 2 (3-2)

73

where 𝑐 equals the coefficient of the mean square of the genotype, and Mi and dfi

are the appropriate mean squares and degrees of freedom, respectively, that were

obtained from the components of variance σ2i.

Breeding values were calculated as the sum of BLUP (Best Linear Unbiased

Predictors) + overall mean of the population. BLUP’s of each genotype were obtained

using the ASREML v3.0 software (Gilmour et al., 2009). Breeding values were used to

make correlations of the disease severity among days after the inoculation (DAI) in each

population. For the germplasm and F1 hybrid populations the breeding values were

used for ranking the genotypes based on the disease response at 7 and 14 DAI (walk-in

plant growth room) and in the field trial.

Additionally, for the F1 hybrids the breeding values obtained from the walk-in

plant growth room (7 and 14 DAI) and field trial (one breeding value from the median

estimate) were correlated. Pearson correlation was performed with the CORR

procedure in SAS (SAS, ver. 9.1.3.; SAS Institute, Cary, NC). Also breeding values

were used to observe the relationship between leaf texture and disease severity of the

zoysiagrass germplasm and segregating F1 families. Linear regressions were performed

with the Proc REG in SAS (SAS, ver. 9.1.3.; SAS Institute, Cary, NC).

Results

Large Patch Disease Response of Zoysiagrass Populations

Disease severity varied among populations, inoculation runs and ratings. Within

the walk-in plant growth room and field plots all genotypes had compatible reactions

with the isolate resulting in variable levels of damage. At 7 DAI mycelia were observed

on many genotypes; and it was noticed that the amount of mycelia varied during

74

incubation. However, an association between mycelia abundance and symptoms was

not evaluated. During the incubation period, mock plants (controls) remained symptom-

free, and among inoculated plants the amount of disease increased. In general, fungal

colonization and amount of disease symptoms increased progressively between ratings.

The analysis of variance from the linear mixed model of the germplasm and F1

hybrids indicated that genotype and the genotype × run interactions were significant (P

≤ 0.05) (data not shown). The analysis of variance at 7 and 14 DAI indicated that

genotype and the genotype × run interactions were significant (Table 3-7 and Table 3-

8). For germplasm, the variance components had the same pattern at both ratings. The

genotype × run interaction variance was higher than the genotype variance, indicating

that this interaction contributed significantly to the phenotypic variance. In this

population, the disease severity means ranged from 9.0% to 88.8% at 7 DAI and from

8.0% to 99.5% at 14 DAI, indicating high phenotypic variation between the genotypes.

Histograms illustrate the genotype × run and the genotype × DAI interactions. In

the first run, disease severity had a multimodal distribution at 7 DAI (Figure 3-1) and

was skewed to susceptibility at 14 DAI (Figure 3-2). However in the second run, disease

severity was skewed towards tolerance at 7 DAI (Figure 3-3) and while disease severity

increased with time there was not a clear trend at 14 DAI (Figure 3-4). Overall, within

the germplasm screen, ten accessions: 5458-26, 5458-12, 5459-10, 5458-10, 5458-28,

332, 5332-52, 5458-35, 5333-53, 152, and El Toro, JaMur and Meyer were good

performers (Appendix E). The most susceptible genotypes were 5309-23, 5331-34,

5307-16, 374, and 5309-12. Zorro, Emerald, Palisades, UltimateFlora and Empire,

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were the most susceptible cultivars. The remaining genotypes, Zeon and Taccoa Green

had intermediate disease responses (Appendix E).

For the F1 hybrids, the variance components (Table 3-8) indicated that the

genotype × run interaction variance was higher than the genotype variance at 7 DAI and

extremely higher at 14 DAI; indicating that environmental effects contributed

significantly to the phenotypic variance. Disease severity means ranged from 0% to

92.4% at 7 DAI and from 6.3% to 98.0% at 14 DAI, indicating high phenotypic variability

within the population. Histograms confirmed the run and DAI genotype interactions. For

the first run, disease severity was multimodal with a tendency towards susceptibility at 7

DAI (Figure 3-5) and skewed towards susceptibility at 14 DAI (Figure 3-6). For the

second run, severity was skewed towards tolerance at 7 DAI (Figure 3-7), and although

slightly multimodal at 14 DAI the population was again skewed towards less disease

(Figure 3-8). Genotypes UFZ 154, UFZ 23, 08426, 08022, UFZ 08, UFZ 15, 357, 08754,

Empire, Meyer and ‘Aloysia’ were good performers (Appendix F). The most susceptible

genotypes were UFZ 42, UFZ 158, UFZ 148, 08491, UFZ 43, UFZ 145, UFZ 124,

08798, UFZ 93, and UFZ 144 (Appendix F).

In the field trial, the analysis of variance indicated that genotypes were

statistically significant (Table 3-9). There was not genotype × replication interaction.

Disease severity varied among the four ratings. Overall disease severity ranged from

0% to 95 % (Table 3-10) with average of 15.17% (SE 12.03). The highest average

disease rating (29%) for large patch occurred on January 2013 (Table 3-10). Rating

averages of Feb. 2012, Jan. 2015 and Apr. 2015 were similar. Ranking of the

genotypes indicated that the best performing genotypes were 08854, 08272, 08845,

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Empire, UFZ 155, UFZ 106, 08544, 08173, JaMur and UFZ 98. The most susceptible

genotypes were UFZ 124, UFZ 118, 08548, 08153, UFZ 123, UFZ 08, 08456, BA 182

and UFZ 92 (Appendix G). In general large patch disease varied considerably among

the 36 ratings and within rating days (Appendix H).

Analysis of variance of the F1 families indicated that genotypes were significantly

different at 7, 14 and 21 DAI (Table 3-11). Analysis of variance components indicated

that the genotype variance was low for the three ratings, especially at 7 DAI.

Conversely, the residual variance was consistently higher, indicating that the population

had considerable variation not associated with the genotype variance. Disease severity

means of the genotypes ranged from 3.6 % to 33 % at 7 DAI, from 12 % to 63 % at 14

DAI; and from 27 % to 81 % at 21 DAI, indicating phenotypic variation among

genotypes especially at 14 and 21 DAI.

Histograms indicated that the population was skewed towards resistance at 7

DAI; only a few genotypes with moderately susceptibility were observed (Figure 3-9). At

14 (Figure 3-10) and 21 DAI (Figure 3-11) the population was slightly skewed towards

tolerance and had multimodal distribution. Through time, the number of genotypes with

higher disease severity (≥ 40%) increased.

Segregation of the F1 families. At 7 DAI most accessions exhibited resistant response;

some genotypes were moderately susceptible and none were susceptible (Table 3-12).

At 14 and 21 DAI, the number of moderately susceptible and susceptible genotypes

increased (Table 3-13 and Table 3-14). At 21 DAI some genotypes were highly

susceptible. Only 5 genotypes retained resistance at 21 DAI. They were all from family

5333-53 × 375.

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Heritability Estimates for Large Patch Disease

In the germplasm population broad sense heritability (H2) was moderate. At 7

and 14 DAI H2 estimates were 0.45 (SE 0.18) and 0.54 (SE 0.18), respectively (Table 4-

7). Narrow sense heritability estimates of the F1 hybrids from the walk-in plant growth

room were low. Estimates were h2 = 0.18 (SE 0.14) and h2 = 0.16 (SE 0.12) at 7 and 14

DAI, respectively (Table 4-8). In the field plots, the narrow sense estimate was

moderate to high (0.43 SE 0.15) (Table 4-9). Narrow sense heritability of the F1 families

was also low. Estimates were h2 = 0.09 (SE 0.03), h2 = 0.22 (SE 0.04) and h2 = 0.27

(0.04), at 7, 14 and 21 DAI, respectively (Table 4-11).

Correlation and Regression Analysis

Rating dates of large patch disease were well correlated when comparisons were

made within the three populations evaluated in the walk-in plant growth room:

germplasm (r = 0.76652), F1 hybrids (r = 0.72941) and F1 families (r ranged from 0.8473

to 0.9450). For the F1 hybrids correlations of breeding values between the walk-in plant

growth room (7 and 14 DAI) and field trial (median value) were poor (7 DAI, r = -0.1243;

14 DAI, r = -0.0562).

According to the regression analysis, no relationship was observed between leaf

texture and disease severity of the zoysiagrass germplasm and F1 families at any rating

date. For germplasm, the regression coefficients were R2 = 0.0311 and R2 = 0.1411 at 7

and 14 DAI, respectively. For the F1 families, they were R2 = 0.0067, R2 = 0.0139 and

R2 = 0.0296 at 7, 14 and 21 DAI respectively.

Discussion

Zoysiagrass breeding has been undertaken to improve turfgrass characteristics

such as quality, color, texture, establishment, winter hardiness, and adaptation to

78

different regions. More recently, improvement for large patch disease has become a

targeted trait. However, to date, only the disease etiology and disease management

strategies have been described (Green II et al., 1993; Tisserat et al., 1994; Hyakumachi

et al., 1998; Aoyagi et al., 1998, 1999; Toda et al., 2004; Patton and Latin, 2005). The

identification of resistant genotypes, inheritance mode and estimation of heritability for

the zoysiagrass-large patch disease pathosystem are important factors for breeders to

consider; but little information regarding inheritance of the disease is currently available.

Information regarding disease responses of zoysiagrass cultivars is limited and specific

to field trials (Metz et al., 1992; Morris, 2000, 2006, 2012). Specific conditions in

zoysiagrass growing regions may affect disease onset and intensity; and thus, the

response of the genotypes. Moreover, knowledge of the amount of the genetic diversity

within the AG 2-2 LP group is limited.

Large Patch Disease Response of the Three Zoysia spp. Populations

This study evaluated and described the large patch disease response in three

zoysiagrass populations. The phenotypic variation of the disease severity was utilized to

estimate broad and narrow sense heritabilities. No zoysiagrass genotype or cultivar had

complete resistance (0% disease) under the experimental conditions of this study (walk-

in plant growth room and field plots). However, in each population, tolerant (≤ 20%),

moderately susceptible (≥ 21 ≤ 50%), susceptible (≤ 51 ≤ 70%) and highly susceptible

(≥ 71%) genotypes were identified. Some of these genotypes had superior large patch

disease responses compared to Meyer, Empire and other cultivars. These resistant

genotypes have value for use to improve large patch disease response in zoysiagrass;

although additional evaluations should be conducted to corroborate their disease

responses.

79

The disease response in some cultivars varied among and between screenings.

Other cultivars were consistently susceptible. In the germplasm screen, Meyer, El Toro

and JaMur were the best performers. Conversely, Emerald, Palisades, Empire, Zorro,

and UltimateFlora were the most susceptible cultivars. Zeon and Taccoa Green had

intermediate responses, but overall Taccoa Green was classified as susceptible. Among

the F1 hybrids, Meyer, Aloysia and Empire performed well. Empire’s performance in

both screenings is an example of the variable disease responses that can be observed

for zoysiagrass genotypes. In the field trial, Empire and JaMur performed good with low

disease severity ratings.

Meyer and Empire (both Zoysia japonica) have been the predominant cultivars

within the transition zone and Florida, respectively. Although the National Turf

Evaluation Program (NTEP) reports for Rhizoctonia infections are variable for Meyer, it

is defined as having moderate resistance to large patch disease (Metz et al., 1992;

Morris, 2006), while Empire is considered susceptible (Unruh et al., 2013). Emerald and

Zorro were previously reported as highly susceptible (Metz et al., 1992; Morris, 2006).

The results obtained from the germplasm screen are in agreement with these previous

reports.

However, some results are variable. For example, in the germplasm screen,

Empire and Palisades performed differently from the cultivar screen described in

Chapter II. In the cultivars screen, Palisades was moderately susceptible and Empire

was the least susceptible cultivar. Conversely in the germplasm screen, both cultivars

were highly susceptible with Empire performing slightly better than Palisades. In both

evaluations, the cultivars had compatible reactions with the isolate, and disease severity

80

increased over time. JaMur, El Toro, Meyer and Zeon exhibited moderate susceptibility

in both the germplasm and the cultivar screens described in Chapter II; while Zorro,

Emerald, Taccoa Green and Ultimate were consistently very susceptible. In summary,

results from this current research compared favorably to other reports; although the

variability of some cultivar responses corroborates the effect of environmental

conditions. In summation, Emerald, Zorro and Taccoa Green could be used as standard

susceptible cultivars for artificial inoculations; while JaMur and Empire could be used as

checks to identify genotypes with better tolerance.

In the three zoysiagrass populations evaluated, the number of genotypes with

improved resistance was higher at 7 DAI. However, longer incubation periods (10 DAI)

under high disease pressure may be more suitable to identify potential sources of

resistance, especially if the amount of phenotypic segregation in a population is high.

Among the F1 families, the majority of tolerant genotypes belonged to family 5333-53 ×

375 (largest family: 152 genotypes). This family was developed using a female parent

(5333-53) with an improved response and a susceptible male parent (375) previously

determined by the germplasm screen. Identification of progeny with superior responses

are useful for backward selection of parents to utilize in future crosses. Offspring

between two susceptible or two tolerant parents were not evaluated due to time and

space limitations. But in the future, screening of these progeny would be beneficial to

better understand the inheritance mechanisms to large patch disease in zoysiagrass.

A goal of these screens was to identify zoysiagrass accessions with high levels

of resistance to the selected isolate. However, as mentioned previously, no accession

with complete resistance was identified. Lack of complete resistance to diseases

81

caused by Rhizoctonia solani have been previously reported (Metz et al., 1992; Yuen et

al., 1994; Bokmeyer et al., 2009a; b; YuXiang et al., 2011; Obasa et al., 2012; Hossain

et al., 2014; Wang et al., 2014; Eizenga et al., 2015). Rhizoctonia solani is a

necrotrophic fungus, with a broad host range (Che et al., 2003; Wang et al., 2014) and

with several mechanisms to induce cell death (Mengiste, 2012) and disease; thus, host

species lack specific signaling pathways to activate mechanisms that induce resistant

responses (Wang et al., 2014). Furthermore, no genotype or cultivar with high levels of

resistance has been identified in any turfgrass–pathosystem (Hurley and Funk, 1985;

Rose-Fricker et al., 1986; Giesler et al., 1996b; Green D.E. et al., 1999; Kimbeng, 1999;

Bonos et al., 2003, 2004b; Belanger et al., 2004; Bonos, 2006, 2011; Han et al., 2006;

Cho et al., 2011; Flor et al., 2013).

Analysis of Variance of the Large Patch Disease Responses

The analysis of variance indicated genotype × inoculation run (walk-in plant

growth room) interactions in the germplasm and F1 hybrids. Experimental conditions

between runs and inside the walk-in plant growth room were strictly controlled; however,

there was an intrinsic variation associated with each run. The genotype × run interaction

is indicative of the impact that environmental effects have on the disease response.

Environmental effects could potentially be minimized by increasing the number of

replications and/or pot size. A higher number of replications can increase accuracy of

disease severity estimates. Small pots are useful to screen many genotypes

simultaneously, but disease severity ratings can be more accurate in genotypes that are

grown in bigger pots. Also, plant fitness after long-term maintenance under greenhouse

conditions should be considered.

82

Genotypes were vegetatively propagated in similar time frames, but runs were

spaced through time; which could induce environmental variation. These interactions

agree with low correlations reported by Obasa et al. (2012) for large patch disease

responses between zoysiagrass breeding lines and Meyer, and inoculations under walk-

in plant growth room conditions; i.e. indicating genotype × run interactions. In some

cases, a large environmental effect is unavoidable especially when genotypes are the

treatments and several genotypes are evaluated (Schwartz et al., 2009).

The majority of genotypes showed a steady increase in disease severity at 14

DAI with ratings above 90%; while in other genotypes the amount of tissue infected

decreased. Recovery through faster production of new tissue or delayed development

of disease among differing genotypes could have caused this interaction. The genotype

× DAI interaction could be another indication that large patch disease response was

influenced by environmental effects.

Variable responses of genotypes also indicate the presence of environmental

effects. Genotypes 375 and 5459-10 had differing responses between the germplasm

screen and when subsequently included in the F1 family screen as parents. Genotype

375 was highly susceptible and then tolerant; whereas, 5459-10 responded oppositely.

For accurate assessments of large patch response, given genotypes require multiple

evaluations.

The analysis of variance of the large patch disease severity of the F1 hybrids from

the field trial indicated significant differences for genotypes. Disease severity also varied

among rating days and years. Accurate assessments of large patch from December to

February were difficult due to the confounding effects of dormancy and winter kill. For

83

this reason, many ratings were excluded. Even for those dates that were retained for

analysis many accessions were variable between ratings; and again, support the

importance of genotype by environment effects on disease development.

Observations of the Large Patch Disease in the Field Trial

Environmental conditions such as relative humidity and soil temperature varied

among years; however, air temperatures were similar. Soil temperature may affect

germination of Rhizoctonia’s survival structures. Through the cooler periods of the four

year trial (December through April), the air temperature ranged from -0.5°C to 33°C, soil

temperature between 14°C to 26°C and relative humidity from 66% to 87% (Appendix I).

These environmental factors were conducive to fungal growth and disease onset

(Green II et al., 1993; Tisserat et al., 1994; Obasa et al., 2013). Thus, disease

development appeared to be affected by other factors. Rhizoctonia solani AG 2-2 LP

requires high moisture for several hours (up 3 days) to initiate infection. Mowing height,

soil drainage and fertilization can favor the host or the pathogen and thus, disease

development. Compacted soils with poor drainage increase humidity, water content;

and thus, disease onset (Green II et al., 1993; Tisserat et al., 1994; Obasa et al., 2013).

The sandy soils associated with the field trial were well-drained and not compacted.

Therefore, a lack of sufficient inoculum, effects of cultural practices and sandy soil could

explain the moderate and variable disease severity observed. Additionally, significant

Bipolaris pressure occurred through summer 2013, affecting assessment of large patch.

Several fungicides were applied for control of the Bipolaris from September through

December, and included: PegasusTM HPX [Chlorothalonil (54%)] (Phoenix UPI), Tartan®

[Trifloxystrobin (4.17%) and Triadimefon (20.86%)] (Bayer CropScience LP), Alliette®

WDG [Aluminum tris (O-ethyl phosphonate) (80.0%)] (Bayer CropScience LP), and

84

Dovetail® [Thiophanate Methyl (19.65%) and Iprodione (19.65%)] (Phoenix UPI). These

applications were not targeted for control of large patch, but may have had an effect on

Rhizoctonia inoculum in the field, because subsequent 2014 ratings were low.

The field did not have a known history of large patch disease, although natural

infection occurred. The field isolate was confirmed as “Rhizoctonia AG 2-2 LP” based

on colony morphology (PDA) and positive amplification with AG 2-2 LP primers (Carling

et al., 2002b), but no further characterizations of the isolate were performed. Genetic

differences between UF 0714 and the field isolate could exist. The extent of the genetic

diversity within the AG 2-2 LP has not been clearly determined. Li et al. (2005) did not

detect genetic differences on AFLP patterns of AG 2-2 LP isolates from different warm

season grasses; while Obasa (2012) reported differences in growth rate (PDA medium),

nuclei condition and AFLP fingerprinting patterns between AG 2-2 LP isolates collected

from the same field. However the association between virulence levels and AFLP

profiles was not evaluated in that study. In addition, in the preliminary study used to

select UF 0714, differing isolates produced different amounts of mycelium and levels of

disease when evaluated on El Toro (data not shown). Variation within other

anastomosis groups of Rhizoctonia solani has been reported based on rDNA ITS

regions (Kuninaga et al., 1997).

Frequency Distribution of Large Patch Disease Response in the F1 Segregating Families

Distribution of the disease responses in the germplasm and F1 hybrids was broad

among runs. In the F1 families, diseased tissue was observed on some genotypes at 7

DAI, but in general severity was low. Segregation towards susceptibility with multi-

modal distribution occurred at 14 and 21 DAI. This was not unexpected, as disease

85

should increase for plants under high and constant disease pressure caused by a

virulent isolate. It is possible that resistance (if present) was overcome under these

conditions. At 14 and 21 DAI the population had wide ranges of disease severity (12-

63% and 27-81%, respectively) indicating phenotypic variation; however, the majority of

the accessions were rated as moderately susceptible and susceptible. If resistance is

overcome by 14 DAI, then ratings at 10 DAI could allow for more accurate identification

of superior genotypes. Conversely, genetic differences at early ratings (5 - 7 DAI) may

not be clearly detected due to the need of a longer incubation period (1 week) for

Rhizoctonia solani AG 2-2 LP.

The wide ranges of disease severity observed in the inoculation runs are

indicative of continuous and multimodal distributions associated with quantitative

inheritance (Parlevliet, 1979). Rhizoctonia solani is a necrotrophic fungus, with a wide

host range, thus quantitative inheritance is common. Continuous distribution for

Rhizoctonia spp. associated diseases have been reported in several crops such as rice

(Oryza sativa L.) (Zhao et al., 2008; YanHua et al., 2013; Zeng et al., 2014; Eizenga et

al., 2015), corn (Zea mays L.) (Hooda et al., 2015), tall fescue (Lolium arundinaceum

Schreb) (Bokmeyer et al., 2009ab) and creeping bentgrass (Agrostis stolonifera L.)

(Bonos et al., 2003, 2006; Bonos, 2011). In tall fescue and creeping bentgrass,

research has been conducted on brown patch, a Rhizoctonia solani disease of cool

season grasses caused by AG 1-IA. Similarly, bimodal distribution for sheath blight in

rice (Goita, 1985; Che et al., 2003), sheath blight in corn (Kumar and Singh, 2002;

Hooda et al., 2015) and corn banded diseases (Goita, 1985; Hooda et al., 2015) have

86

also been reported. In summary, results from this current research support previous

reports of quantitative inheritance for some Rhizoctonia diseases.

Correlation of Large Patch Disease Response between Walk-in Plant Growth Room and Field Plots

The disease responses of the F1 hybrids between the walk-in plant growth room

and field trial were not correlated. Disease severity within the growth room was higher

than observed from the field trial (low to moderate). Adequate and constant temperature

(23°C), high humidity (100%) and a virulent isolate favored the infection and disease

development in the walk-in plant growth room. Conversely, as previously mentioned in

the field other factors could have affected the disease intensity. Obasa et al. (2012) also

observed a lack of correlation for large patch disease severity between growth room

and field experiments (r = -0.39, P = 0.16). They reported evidence of strong

environmental effects for the host-pathogen interaction (Obasa et al., 2012). The results

of the current study and Obasa et al. (2012) suggest that genotypes should be screened

under artificial conditions to provide constant and high disease pressure, and that field

conditions should be manipulated to increase disease incidence and minimize the

effects of other abiotic and biotic factors.

Relationship between Leaf Texture and Large Patch Disease Response

Leaf texture did not affect the disease response. This study was not developed to

directly measure this association. However the available data was used to observe if a

trend between these variables existed. Results of linear regressions performed with the

breeding values showed very low coefficients, indicating that a specific (fine or coarse)

texture was not directly associated to a particular disease response.

87

Broad and Narrow Sense Heritabilities

Broad and narrow sense heritabilities for large patch disease were estimated for

the three populations. Heritability estimation is an important parameter to understand

the genetic basis of a trait, and to predict genetic gain and improvement on selection

(Visscher et al., 2008). Broad sense heritability (H2) indicates the level that genetic

factors (additive, dominant and epistatic) are associated with a trait (Milton, J., Sleper,

1995). Narrow sense heritability (h2) (Nyquist and Baker, 1991) indicates the level that

additive factors have on trait expression (Milton, J., Sleper, 1995); and thus, is more

informative for improvement through selection.

In the germplasm population, moderate H2 (0.45-0.54) were obtained at 7 and

14 DAI, respectively. Narrow sense heritabilities associated with the F1 families were

low from the three rating days (7 DAI = 0.09; 14 DAI = 0.22; and 21 DAI = 0.27). Narrow

sense heritability was also low for the F1 hybrids at 7 and 14 DAI (0.18 and 0.16,

respectively) in the walk-in plant growth room. In the field trial, heritability was moderate

to high (h2 = 0.43). Possible causes for the low heritabilities estimates can be the

environmental variation affecting disease development or lack of significant phenotypic

variation in the populations.

Moderate H2 and low h2 values suggest that genetic factors contribute to the

disease response, but additive effects were not significant contributors. It is possible

that the environmental effects have masked the effects of additive genes on the

populations. In the germplasm and F1 hybrids; the genotype × run interaction variance

component was larger than the genotype variance. In the F1 families the genotype

variance was very low at 7 DAI and higher at 14 and 21 DAI. However, the residual

error variance was significantly higher in these ratings. The ratios of variances in these

88

studies, suggest that a high amount of variability in these populations cannot be directly

attributed to the genotypes. The overall phenotypic variation along with higher genotype

× run and residual variances, and low genotypic variances are indicative of

environmental effects and could explain the low narrow sense heritability estimates

obtained.

The results of this current study does not exclude the presence of dominant

effects partially linked with the inheritance of large patch disease responses in

zoysiagrass. A lack of additive effects with more dominant effects implies that

improvement through selection over generations is not possible; however, dominant

effects can be useful in the development of hybrids with improved large patch response

that are then vegetatively propagated. Vegetative production and planting of

zoysiagrass is preferred compared to seeded cultivars.

For other turfgrass diseases, broad and narrow sense heritabilities have been

reported and the associated estimates vary according to the disease, population and

method or statistical model for estimation. In general, the majority of these studies

report H2 and h2 estimates higher than the estimates obtained for zoysiagrass

populations in this current study. Estimated heritability values are subject to the

statistical methods used in their calculation, amounts of genetic variation in the

population, and the use of an experimental design that accounts for environmental

variation such evaluations that include different years and locations.

In zoysiagrass, only broad and narrow sense heritabilities have been reported

for Bipolaris spp. (H2 = 0.40) (Schwartz et al., 2009) and Puccinia spp. (rust) (h2 = 0.36)

under natural infection (Flor et al., 2014). In tall fescue populations, low to high broad

89

sense heritabilities ( 0.25-0.74) and moderate narrow sense heritabilities (0.57-0.62)

have been reported for brown patch disease (Bokmeyer et al., 2009a; b). Broad and

narrow sense heritabilities for perennial ryegrass (Lolium perenne) have been reported

as moderate to high (0.65-0.70) for stem rust (Puccinia graminis subsp. graminicola)

(Rose-Fricker et al., 1986) and high (0.92) for gray leaf spot (Pyricularia grisea) (Bonos

et al., 2004b) diseases. Additionally in perennial ryegrass, the h2 for crown rust

(Puccinia coronata Corda) was moderate (h2 = 0.46) (Reheul and Ghesquiere, 1996)

and gray leaf spot was moderate to high (h2 = 0.57-0.76) (Han et al., 2006).

The narrow sense heritabilities referenced above for crown rust, gray leaf spot,

brown patch and dollar spot were all calculated through the mid-parent progeny

regression. This method has been traditionally used for estimating narrow sense

heritability accurately in simple and balanced designs (Stoskopf et al., 1993a). In this

method, the environmental and non-additive variance are removed from the analysis

(Conner and Hartl, 2004) and the genotype by environment interaction is not included

(Stoskopf et al., 1993a).

For dollar spot disease (Sclerotinia homoeocarpa) moderate to high H2 (0.56-

0.92) were reported on creeping bentgrass (Agrostis stolonifera) (Bonos et al., 2003)

and Poa trivialis (0.57-0.90) (Hurley and Funk, 1985). Low (0.23) and high (0.79) narrow

sense estimates were reported for seashore paspalum genotypes (Paspalum vaginatum

Swartz) (Flor et al., 2013) and for a creeping bentgrass population (Bonos, 2006),

respectively.

Flor et al. (2013) reported h2 estimates (0.23) on seashore paspalum genotypes

to dollar spot disease using the animal model- REML analysis. This estimate was much

90

lower than the narrow sense heritability estimates reported for crown rust, gray leaf

spot, brown patch and dollar spot diseases using the mid-parent progeny regression.

For turfgrass diseases and other traits, the h2 estimate on seashore paspalum is the

only one currently reported using the animal model.

For gray leaf and for dollar spot diseases the highest broad sense heritability

values (0.92) were obtained using small size populations. One study evaluated 11

perennial ryegrass entries (Bonos et al., 2004b) and the other 5 creeping bentgrass

parental lines (Bonos, 2006). Also high H2 (0.56-0.90) for dollar spot disease on

creeping bentgrass clones was estimated in a population of 265 clones, developed from

3 cultivars (Bonos et al., 2003). These studies report high estimates, but data were

generated from few genotypes or from germplasm with narrow diversity. Conversely,

the H2 estimates of the zoysia germplasm were obtained from 60 genotypes with wider

genetic background.

Conclusions and Future Research

This research has presented a framework of large patch disease using three

zoysiagrass populations. A controlled procedure previously used to evaluate

commercial cultivars was useful to efficiently screen large numbers of zoysiagrass

genotypes for their response to a characterized and virulent large patch isolate. Disease

severity data indicate that large patch response is quantitative. Moderate broad-sense

and low narrow-sense heritability estimates from three populations indicate a strong

influence of environmental effects on the disease response. There was no correlation of

the disease severity of the F1 hybrids between data taken in the walk-in plant growth

room and field plots. Artificial inoculations in the growth room always induced higher

disease severity than natural infection in the field. Disease tolerance or susceptibility

91

appears to not be related to leaf texture in the two populations evaluated. Fine and

coarse textured accessions had similar ranges of disease response.

These results constitute the first reports of H2 and h2 for this disease in

zoysiagrass. It is important to consider that because the same characterized isolate was

used for all studies, these results may be specific to isolate UF 0714.

Future research should consider the validation of the gene action and

identification of resistance mechanisms to the AG 2-2 LP in zoysiagrass. This

information will help to develop zoysiagrass cultivars with increased resistance to large

patch disease.

92

Table 3-1. University of Florida zoysiagrass germplasm accessions and cultivars evaluated in the large patch disease screening.

Genotype and cultivar Species Leaf texturea Developing Institution

123 Z. matrella Fine FAESb

152 Z. matrella Fine

182 Z. japonica Medium coarse

coarse

FAES

188 Z. japonica Medium coarse FAES

252 Z. matrella Fine FAES

306 Z. matrella Fine FAES

309 Z. matrella Fine FAES

328 Z. matrella Fine FAES

332 Z. matrella Fine FAES

357 Z. matrella Fine FAES

358 Z. matrella Fine FAES

374 Z. matrella Fine FAES

375 Z. matrella Fine FAES

402 Z. japonica Medium coarse FAES

422 Z. matrella Fine FAES

433 Z. pacifica Very fine FAES

2430 Z. japonica Coarse Texas AgriLIFEc

3363 Z. japonica Coarse Texas AgriLIFE

3588 Z. matrella Fine Texas AgriLIFE

4360 Z. japonica Medium coarse Texas AgriLIFE

4429 Z. matrella Very fine Texas AgriLIFE

5288 Z. japonica Coarse Texas AgriLIFE

5315 Z. matrella Very fine Texas AgriLIFE

8516 Z. japonica Medium coarse Texas AgriLIFE

5256-20 Z. japonica Medium coarse Texas AgriLIFE

5257-8 Z. japonica Medium coarse Texas AgriLIFE

5269-24 Z. japonica Coarse Texas AgriLIFE

5305-48 Z. japonica Medium coarse Texas AgriLIFE

5306-45 Z. japonica Medium coarse Texas AgriLIFE

5307-16 Z. japonica Coarse Texas AgriLIFE

5309-12 Z. japonica Coarse Texas AgriLIFE

5309-23 Z. japonica Coarse Texas AgriLIFE

5309-35 Z. japonica Coarse Texas AgriLIFE

5330-23 Z. japonica Coarse Texas AgriLIFE

5330-38 Z. japonica Coarse Texas AgriLIFE

5331-34 Z. japonica Coarse Texas AgriLIFE

5332-52 Z. japonica Medium coarse Texas AgriLIFE

5332-53 Z. japonica Medium coarse Texas AgriLIFE

5333-53 Z. japonica Coarse Texas AgriLIFE

5335-3 Z. macrantha Z. matrella Coarse Texas AgriLIFE

5337-46 Z. japonica Z. macrantha Medium coarse Texas AgriLIFE

93

Table 3-1. Continued. Genotype and cultivar Species Leaf texturea Developing Institution

5343-52 Z. japonica Z. matrella Medium

coarse

Texas AgriLIFE

5458-10 Z. matrella Z. minima Very fine Texas AgriLIFE

5458-12 Z. matrella Z. minima Very fine Texas AgriLIFE

5458-18 Z. matrella Z. minima Very fine Texas AgriLIFE

5458-28 Z. matrella Z. minima Very fine Texas AgriLIFE

5458-35 Z. matrella Z. minima Very fine Texas AgriLIFE

5458-39 Z. matrella Z. minima Very fine Texas AgriLIFE

5459-10 Z. matrella Z. paciflora Very fine Texas AgriLIFE

5504-6 Z. matrella Z. minima Very fine Texas AgriLIFE

El Toro Z. japonica Coarse University of California

Emerald Z. japonica Z. pacifica Fine USDA and USGAd

Empire Z. japonica Coarse Sod Solutions

JaMur Z. japonica Coarse Bladerunner Farms, Inc.

Meyer Z. japonica Coarse USDA and USGA

Palisades Z. japonica Coarse Texas AgriLIFE

Pristine Flora Z. japonica Z. tenuifolia Fine University of Florida

UltimateFlora Z. japonica Medium

coarse

University of Florida

Zeon Z. matrella Fine Bladerunner Farms, Inc.

Zorro Z. matrella Fine Texas AgriLIFE

a Leaf texture classified based on leaf width as follows: Very fine < 1.0 mm; 1.0 mm < Fine < 2.0 mm; 2.0 mm < Medium coarse < 3.0 mm; 3.0 mm < Coarse.

b Florida Agricultural Experiment Station. c Texas A&M University. d United States Department of Agriculture and United States Golf Association.

94

Table 3-2. Zoysiagrass coarse-texture F1 hybrids and cultivars evaluated in the large patch disease screening.

Genotype Female parent Male parent

08022 5309-12 152 08050 5309-12 123

08054 5309-12 123

08083 5309-12 5256-20

08153 182 2430

08157 182 5333-53

08173 5330-23 5256-20

08184 5330-23 5256-20

08189 5330-23 5269-24

08196 5330-23 5269-24

08203 5332-52 5309-35

08272 5330-23 5330-38

08426 357 5335-3

08456 123 Meyer

08487 252 5309-35

08491 252 5309-35

08544 2430 5335-3

08548 2430 5307-16

08595 2430 5333-53

08597 2430 5333-53

08599 2430 5333-53

08617 UFZ10 358

08631 UFZ10 358

08754 5333-53 357

08778 3363 5309-35

08797 2430 358

08798 2430 358

08820 5309-35 5269-24

08845 5257-8 2430

08854 5330-38 4360

BA182 - -

BA402 - -

BA513 - -

UFZ08

UFZ106

UFZ11

UFZ118

UFZ119

5256-20 5309-12

UFZ106 5333-53 3363

UFZ11 5256-20 5309-35

UFZ118 188 182

UFZ119 188 182

UFZ121 2430 8516

UFZ121B 2430 8516

95

Table 3-2. Continued.

Genotype Female parent Male parent

UFZ123 2430

UFZ1030

UFZ10 UFZ124 2430 UFZ10

UFZ125 2430 5309-35

UFZ126 2430 5333-53

UFZ128 2430 5309-35

UFZ129 2430 5307-16

UFZ132 2430 5333-53

UFZ133 2430 8516

UFZ134 2430 5309-35

UFZ14 5309-12 123

UFZ142 5309-35 3363

UFZ144 5309-35 5257-8

UFZ145 UFZ10 5330-38

UFZ148 5306-45 358

UFZ15 5330-23 5256-20

UFZ151 5330-38 4360

UFZ153 5309-35 5256-20

UFZ154 4360 UFZ10

UFZ155 5330-38 4360

UFZ156 5309-35 5256-20

UFZ158 5309-35 5269-24

UFZ159 5309-35 5269-24

UFZ16 5256-20 182

UFZ160 5309-35 5256-20

UFZ20 182 2430

UFZ23 182 422

UFZ33 5309-12 182

UFZ36 5330-23 5269-24

UFZ37 5330-23 5269-24

UFZ42 5305-48 5256-20

UFZ43 5269-24 3363

UFZ62 5333-53 2430

UFZ84 5333-53 5269-24

UFZ92 5309-35 3257-8

UFZ93 5333-53 5269-24

UFZ94 309 3363

UFZ96 5309-35 5257-8

UFZ98 5333-53 5256-20

Aloysia - -

Empire - -

Geo - -

JaMur - -

96

Table 3-3. Zoysiagrass genotypes used to develop the F1 segregating families. Genotype Large patch disease responsea Leaf textureb Species

374 Susceptible Fine Z. matrella 5335-3 Susceptible Coarse Z. macrantha Z. matrella 309 Susceptible Fine Z. matrella 5331-34 Susceptible Coarse Z. japonica 375 Susceptible Fine Z. matrella 5504-6 Susceptible Very fine Z. matrella Z. minima 5307-16 Susceptible Coarse Z. japonica 5309-23 Susceptible Coarse Z. japonica 433 Susceptible Very fine Z. pacifica 5458-18 Susceptible Very fine Z. matrella Z. minima 4429 Susceptible Very fine Z. matrella 332 Tolerant Fine Z. matrella 2430 Tolerant Coarse Z. japonica 252 Tolerant Fine Z. matrella 422 Tolerant Fine Z. matrella 5333-53 Tolerant Coarse Z. japonica 5332-52 Tolerant Coarse Z. japonica 5459-10 Tolerant Fine Z. matrella Z. paciflora 5458-12 Tolerant Fine Z. matrella Z. minima 5458-26 Tolerant Fine Z. matrella

a Large patch disease response based on the germplasm screening. b Leaf texture classified based on leaf width as follows: Very fine < 1.0 mm; 1.0 mm < Fine < 2.0 mm; 2.0 mm < Medium coarse < 3.0 mm; 3.0 mm < Coarse.

97

Table 3-4. Selected zoysiagrass F1 families for narrow-sense heritability estimation of large patch disease.

Femalea Maleb Family IDc Speciesd Leaf texturee Disease responsef Number of plantsg

5332-52 5307-16 7 Z. japonica × Z. japonica Coarse Tolerant × susceptible 20 5332-52 5331-34 8 Z. japonica × Z. japonica Coarse Tolerant × susceptible 20 5332-52 375 9 Z. japonica × Z. matrella Coarse × fine Tolerant × susceptible 153 5333-53 5307-16 10 Z. japonica × Z. japonica Coarse Tolerant × susceptible 20 5333-53 5331-34 11 Z. japonica × Z. japonica Coarse Tolerant × susceptible 20 5459-10 5331-34 12 Z. pacifica × Z. japonica Fine × coarse Tolerant × susceptible 36

Total 269

a Zoysiagrass accession used as female (pollen receptor). b Zoysiagrass accession used as male (pollen donor). c Family identification given as a quick reference during data analysis in the dissertation.

d First and second species relates to the female and male parent, respectively. e Leaf texture classified based on leaf width as follows: Very fine < 1.0 mm; 1.0 mm < Fine < 2.0 mm; 2.0 mm < Medium coarse < 3.0 mm; 3.0 mm < Coarse. f Large patch disease response based on the germplasm screening. g Number of plants evaluated in the screening.

98

Table 3-5. Statistical model to calculate broad sense heritability of large patch disease in the zoysiagrass germplasm.

a H2: Broad sense heritability, estimated as follows: H2 = 𝜎𝐺

2

𝜎𝑃2

b Days after the inoculation. c Overall population mean. d Inoculation run: 60 zoysiagrass accessions were inoculated with an LP isolate (0714) and incubated under optimal conditions for disease development. In each run 9 inoculated replications and 3 controls of a given accession were evaluated at once. e Replication. f Residual error.

Estimate of H2a

Model Genotypic variance

𝜎𝐺2

Phenotypic variance

𝜎𝑃2

7 and 14 DAIb

Severity = µc + rund + repe. (run) + (genotype +

genotype × run + genotype × run × rep.) + ɛf 𝜎𝐺

2

𝜎𝐺2

+ 𝜎𝐺

2× run

run+

𝜎𝐺2

× run × rep.

run × rep.+

𝜎ɛ2

run × rep.

99

Table 3-6. Statistical model to calculate narrow sense heritability of large patch disease in the zoysiagrass F1 hybrids and F1 segregating families.

Population Estimate of h2a

Model Additive variance

𝜎𝐴2

Phenotypic variance 𝜎𝑃

2

F1 families 7, 14 and

21 DAI

Severity = µc + repd. + (genotype) + ɛe 𝜎𝐺2

𝜎𝐺2

+ 𝜎ɛ2

F1 hybrids 7 and 14 DAI

Severity = µ + runf + rep. (run) + (genotype + genotype × run + genotype × run × rep.) + ɛe

𝜎𝐺2

𝜎𝐺

2+

𝜎𝐺2

× run

run+

𝜎𝐺2

× run × rep.

run × rep.+

𝜎ɛ2

run × rep.

F1 hybrids

Field trial

Overall h2

Severity median = µ + replication + (genotype + genotype ×

replication) + ɛe

𝜎𝐺2

𝜎𝐺2

+ 𝜎𝐺

2× rep.

rep.+

𝜎ɛ2

rep.

a h2: Narrow sense heritability, estimated as follows: h2 = 𝜎𝐴

2

𝜎𝑃2

𝜎𝐴2 = 𝜎

2𝑔𝑒𝑛𝑜𝑡𝑦𝑝𝑒. b Days after the inoculation. c Overall population mean. d Replication.

e Residual error. f Inoculation run: 80 zoysiagrass accessions were inoculated with an LP isolate (0714) and incubated under optimal conditions for disease development. In each run 4 inoculated replications and 2 controls of a given accession were evaluated at once.

100

Table 3-7. Genotypic (𝜎𝐺2), genotype × run interaction (𝜎𝐺

2x r), genotype × run × replication interaction (𝜎𝐺

2x r x rep.) and

pooled error (𝜎ɛ2) variance components, broad sense heritability and their associate standard errors (± SE) of

the large patch disease severity of the zoysiagrass germplasm at 7 and 14 days after the inoculation (DAI) in two inoculation runs.

Variance componenta DF 7 DAIb ± (SE) 14 DAI ± (SE)

𝜎𝐺2 60 115.67 ± 2.13 161.64 ± 2.63

𝜎𝐺2

x run 120 245.02 ± 4.67 249.21 ± 4.81

𝜎𝐺2

x run x rep 1080 0 ± 0 0 ± 0

𝜎ɛ2 1134 344.88 ± 22.25 262.57 ± 22.16

H2 c 0.45 ± 0.18 0.54 ± 0.18 a Variance components were estimated using Restricted Maximum Likelihood Estimation (REML) with the ASReml software (Gilmour et al., 2009). b Days after the inoculation.

c Broad sense heritability was estimated from the variance components as follows: H2 = 𝜎𝐺2

𝜎𝑃2

where 𝜎𝑃2 = 𝜎𝐺

2 + 𝜎𝐺

2 × run

run+

𝜎𝐺2 × run × rep.

run × rep.+

𝜎ɛ2

run × rep.

101

Table 3-8. Genotypic (𝜎𝐺2), genotype × run interaction (𝜎𝐺

2 x r), genotype × run ×

replication interaction (𝜎𝐺2

x r x rep.) and pooled error (𝜎ɛ2) variance components,

narrow sense heritability and their associate standard errors (± SE) of the large patch disease severity of the F1 hybrids at 7 and 14 days after the inoculation (DAI) in two inoculation runs.

Variance componenta

DF 7 DAIb ± (SE) 14 DAI ± (SE)

𝜎𝐺2 111 28.18 ± 0.76 80.31 ± 0.77

𝜎𝐺2 x run

222 199.52 ± 4.12 755.62 ± 5.57

𝜎𝐺2 x run x rep.

888 5.47 ± 0.14 0 ± 0

𝜎ɛ2 704 257.74 ± 6.37 243.57 ± 15.88

h2 c 0.18 ± 0.14 0.16 ± 0.12 a Variance components were estimated using Restricted Maximum Likelihood Estimation (REML) with the ASReml software (Gilmour et al., 2009). b Days after the inoculation.

c Narrow sense heritability was estimated from the variance components as follows:

h2 = 𝜎𝐴2

𝜎𝑃2

where 𝜎𝑃2 =. 𝜎𝐺

2 +𝜎𝐺

2 × run

run+

𝜎𝐺2 × run × rep.

run × rep.+

𝜎ɛ2

run × rep.

𝜎𝐴2 = 𝜎

2𝑔𝑒𝑛𝑜𝑡𝑦𝑝𝑒.

Table 3-9. Genotypic (𝜎𝐺2), genotype × replication interaction (𝜎𝐺

2 x rep.) and pooled error

(𝜎ɛ2) variance components, narrow sense heritability and their associate

standard errors (± SE) of the large patch disease severity of the F1 hybrids at the field plots.

Variance componenta DF Data of four ratingsb ± (SE)

𝜎𝐺2 111 22.77 ± 2.16

𝜎𝐺2

x rep. 333 0 ± 0

𝜎ɛ2 240 85.20 ± 8.92

h2 c 0.43 ± 0.15 a Variance components were estimated using Restricted Maximum Likelihood Estimation (REML) with the ASReml software (Gilmour et al., 2009). b Four large patch disease severity ratings were used for the analysis as follows: Feb. 2012, Jan. 2013, Jan. 2015 and Apr. 2015. c Narrow sense heritability was estimated from the variance components as follows:

h2 = 𝜎𝐴2

𝜎𝑃2

𝜎𝐴2 = 𝜎

2𝑔𝑒𝑛𝑜𝑡𝑦𝑝𝑒.

102

Table 3-10. Large patch disease severity ranges of the F1 hybrids on selected rating

days at the field plots.

Month Year Disease severitya ranges (%)b

Mean Standard error

Standard deviation

February 2012 1 - 50 10.53 0.53 8.34 January 2013 0 - 95 28.70 2.46 31.75 January 2015 0 - 90 11.77 2.50 21.79 April 2015 0 - 40 10.98 0.50 6.91

a Disease severity measured under natural infection. b Disease severity estimated as the percentage of area with symptoms over the total area of the plot.

Table 3-11. Genotypic (𝜎𝐺2) and pooled error (σ2

ε) variance components, narrow sense

heritability and their associate standard errors (± SE) of the large patch disease severity of the segregating F1 families at 7, 14 and 21 days after the inoculation (DAI) in one inoculation run.

Variance componentsa

DF 7 DAIb ± (SE) 14 DAI ± (SE) 21 DAI ± (SE)

𝜎𝐺2 275 16.08 ± 2.35 116.23 ± 4.23 181.24 ± 4.97

𝜎ɛ2 1100 155.86 ± 21.18 400.81 ± 20.40 489.80 ± 20.45

H2 c 0.09 ± 0.03 0.22 ± 0 0.27 ± 0.04 a Variance components were estimated using Restricted Maximum Likelihood Estimation (REML) with the ASReml software (Gilmour et al., 2009). b Days after the inoculation.

c Narrow sense heritability was estimated from the variance components as follows:

h2 = 𝜎𝐴2

𝜎𝑃2 , where 𝜎𝑃

2 = 𝜎𝐴2 + 𝜎ɛ

2

𝜎𝐴2

= 𝜎 2𝑔𝑒𝑛𝑜𝑡𝑦𝑝𝑒.

103

Figure 3-1. Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 7 DAI in the first inoculation run.

Figure 3-2. Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 14 DAI in the first inoculation run.

0

5

10

15

20

25

30

35

40

91 -100 81 -90 71 - 80 61 -70 51 -60 41 -50 31 -40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

0

5

10

15

20

25

30

35

40

91 -100 81 - 90 71 - 80 61 -70 51 - 60 41 -50 31 - 40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

104

Figure 3-3. Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 7 DAI in the second inoculation run.

Figure 3-4. Frequency distribution of large patch disease severity of the zoysiagrass germplasm at 14 DAI in the second inoculation run.

0

5

10

15

20

25

30

35

40

91 -100 81 - 90 71 - 80 61 -70 51 - 60 41 -50 31 - 40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

0

5

10

15

20

25

30

35

40

91 -100 81 - 90 71 - 80 61 -70 51 - 60 41 -50 31 - 40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

105

Figure 3-5. Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 7 DAI in the first inoculation run.

Figure 3-6. Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 14 DAI in the first inoculation run.

0

5

10

15

20

25

91 - 100 81 - 90 71 - 80 61 - 70 51 - 60 41 - 50 31 - 40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

0

5

10

15

20

25

91 - 100 81 - 90 71 - 80 61 - 70 51 - 60 41 - 50 31 - 40 21 - 30 11 - 20 0 - 10

Nu

mb

er

of g

en

oty

pe

s

Disease severity ranges (%)

106

Figure 3-7. Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 7 DAI in the second inoculation run.

Figure 3-8. Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 14 DAI in the second inoculation run.

0

5

10

15

20

25

30

35

40

45

91 - 100 81 - 90 71 - 80 61 - 70 51 - 60 41 - 50 31 - 40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

0

5

10

15

20

25

30

35

40

45

91 - 100 81 - 90 71 - 80 61 - 70 51 - 60 41 - 50 31 - 40 21 - 30 11 - 20 0 - 10

Num

ber

of

genoty

pes

Disease severity ranges (%)

107

Figure 3-9. Frequency distribution of large patch disease severity of the zoysiagrass F1

segregating families at 7 DAI.

Figure 3-10. Frequency distribution of large patch disease severity of the zoysiagrass F1

segregating families at 14 DAI.

0

20

40

60

80

100

120

140

160

91-100 81-90 71-80 61-70 51-60 41-50 31-40 21-30 11-20 0-10

Num

ber

of

genoty

pes

Disease severity ranges (%)

0

20

40

60

80

100

120

140

160

91-100 81-90 71-80 61-70 51-60 41-50 31-40 21-30 11-20 0-10

Num

ber

of

genoty

pes

Disease severity ranges (%)

108

Figure 3-11. Frequency distribution of large patch disease severity of the zoysiagrass F1

hybrids at 21 DAI.

Table 3-12. Large patch disease segregation of the F1 families at 7 days after the

inoculation.

a A given cross was performed using the first and second accessions as female and male parents, respectively. b Family identification given as a quick reference during data analysis in the dissertation.

c Disease response classification based on estimated severity percent: (Resistant ≤ 20%; ≥ 21 ≤ 50% moderate susceptible; ≥ 51% susceptible and ≥ 71% highly susceptible.

0

20

40

60

80

100

120

140

160

91-100 81-90 71-80 61-70 51-60 41-50 31-40 21-30 11-20 0-10

Num

ber

of

genoty

pes

Disease severity ranges (%)

Crossa Family IDb

Resistantc Moderate susceptiblec

Susceptiblec Highly susceptiblec

5332-52 × 5307-16 7 16 4 0 0 5332-52 × 5331-34 8 17 3 0 0 5333-53 × 375 9 140 12 0 0 5333-53 × 5307-16 10 16 4 0 0 5333-53 × 5331-34 11 18 2 0 0 5459-10 × 5331-34 12 22 14 0 0 Total 229 39 0 0

109

Table 3-13. Large patch disease segregation of the F1 families at 14 days after the inoculation.

Crossa Family IDb

Resistantc Moderate susceptiblec

Susceptiblec Highly susceptiblec

5332-52 × 5307-16 7 4 13 3 0 5332-52 × 5331-34 8 5 14 1 0 5333-53 × 375 9 71 78 2 1 5333-53 × 5307-16 10 1 13 6 0 5333-53 × 5331-34 11 2 16 2 0 5459-10 × 5331-34 12 3 22 9 2 Total 86 156 23 3

a A given cross was performed using the first and second accessions as female and male parents, respectively. b Family identification given as a quick reference during data analysis in the dissertation.

c Disease response classification based on estimated severity percent: (Resistant ≤ 20%; ≥ 21 ≤ 50% moderate susceptible; ≥ 51% susceptible and ≥ 71% highly susceptible.

Table 3-14. Large patch disease segregation of the F1 families at 21 DAI.

Crossa Family IDb

Resistantc Moderate susceptiblec

Susceptiblec Highly susceptiblec

5332-52 × 5307-16 7 0 5 11 4 5332-52 × 5331-34 8 0 11 7 2 5333-53 × 375 9 5 110 29 8 5333-53 × 5307-16 10 0 1 13 6 5333-53 × 5331-34 11 0 8 8 4 5459-10 × 5331-34 12 0 3 20 13 Total 5 138 88 37

a A given cross was performed using the first and second accessions as female and male parents, respectively. b Family identification given as a quick reference during data analysis in the dissertation.

c Disease response classification based on estimated severity percent: (Resistant ≤ 20%; ≥ 21 ≤ 50% moderate susceptible; ≥ 51% susceptible and ≥ 71% highly susceptible.

110

CHAPTER 4 ASSOCIATION OF SIMPLE SEQUENCE REPEAT PRIMERS WITH LARGE PATCH

DISEASE RESPONSE IN ZOYSIAGRASS

Introduction

Zoysia spp. (zoysiagrass) are popular turfgrass species in the southern and

transition zone regions of the USA (Patton, 2012). Z. japonica Steudel and Z. matrella

(L.) Merrill (Okeyo et al., 2011) are the most popular for turfgrass uses on sport fields,

parks, landscapes, home lawns, and golf courses (Yaneshita et al., 1999; Li et al., 2005;

Patton and Reicher, 2007; Harris-Shultz et al., 2014). Zoysiagrass is valued for its

aesthetic value, uniform growth, low maintenance (Green II et al., 1993; Patton, 2010;

Obasa et al., 2012, 2013; Kimball et al., 2013; Tanaka et al., 2016a), and tolerance to

shade, cold, drought and saline conditions (Green II et al., 1993; Patton, 2010; Obasa et

al., 2012, 2013; Kimball et al., 2013; Tanaka et al., 2016a). Zoysiagrass species are

segmental allotetraploids (2n = 4x = 40) with protogynous flowers (Forbes, 1952;

Yaneshita et al., 1999) that are both cross- and self-fertile (Forbes, 1952; Yaneshita et

al., 1997, 1999; Cai et al., 2005).

Improvements in zoysiagrass have been made using conventional breeding for

genetic color, establishment rate, seed production and tolerance to cold, wear, drought,

and salinity (Wang et al., 2015). For biotic factors, zoysiagrass has been bred to

improve responses to nematodes and mites (Chandra et al., 2015). Conventional

breeding is a lengthy process that could be improved by the use of molecular markers.

However the use of molecular markers in zoysiagrass for breeding purposes has been

limited.

Molecular genetic studies were initiated with Restriction Fragment Length

Polymorphism (RFLP) markers on self-pollinated progenies of Z. japonica, Z. matrella

111

and a hybrid of these species (Yaneshita et al., 1997). Subsequently, Yaneshita et al.

(1999) published the first linkage map with RFLP markers. Molecular markers have

been developed and utilized to construct genetic linkage maps, explore the genetic

diversity, perform Quantitative Trait Loci (QTL) analysis and comparative genetic

associations (Yaneshita et al., 1993, 1997, 1999; Ebina et al., 1999; M. Yaneshita T.

Sasakuma, 1999; Budak et al., 2004; Tsuruta et al., 2008, 2005; Hashiguchi et al.,

2007; MA et al., 2007; Weng et al., 2007; Hong et al., 2008; Harris-Shultz et al., 2012;

Kimball et al., 2013; Tanaka et al., 2016a).

Currently, genetic linkage maps based on RFLPs (Yaneshita et al., 1997; Li et

al., 2015), Amplified Fragment Length Polymorphisms (AFLP’s) (Ebina et al., 1999; Cai

et al., 2004, 2005), Simple Sequence Repeats (SSRs) (Cai et al., 2005; Li et al., 2009,

2010), Sequence Related Amplified Polymorphisms (SRAPs) (Guo et al., 2014),

Random Amplified Polymorphic DNA (RAPD) (Guo et al., 2014; Wang et al., 2015),

Conserved Intron Scanning Primers (CISP) and PCR Landmark Unique Genes

(PLUGs) (Li et al., 2015) are available for zoysiagrass. Genetic diversity studies have

been performed with RAPDs (Weng et al., 2007), SSRs (Tsuruta et al., 2005, 2008;

Kimball et al., 2013), and AFLPs (Hong et al., 2008).

Comparative genetic (Jessup et al., 2011; Li et al., 2015; Wang et al., 2015) and

association analysis for cold tolerance and green period have also been conducted

(Guo et al., 2012b). For leaf width (Ebina, 2000), cold resistance (ChengLong et al.,

2010), and salt tolerance (Guo et al., 2014) some Quantitative Trait Loci (QTL) have

been detected. Ongoing QTL analysis are underway for mites (Chandra et al., 2015),

large patch resistance (Chandra et al., 2015) and freezing tolerance (McCamy Pruitt et

112

al., 2015). The most recent studies include transcriptome analysis (Tanaka et al.,

2016a), sequence tagged high density genetic maps (Wang et al., 2015), the publication

of the chloroplast genome of Z. matrella (Tanaka et al., 2016ab), and the first genome

assembly project (Tanaka et al., 2016a). Genomes drafts have been validated with

some zoysiagrass linkage maps (Wang et al., 2015; Tanaka et al., 2016a). This

information will be useful to validate molecular markers and linkage groups and also to

search for genes associated to specific responses.

Zoysiagrass has a small genome size [421 Mega base pairs (Mbp], as estimated

by linkage analysis (Arumuganathan and Tallury, 1999). Using cytometry and K-mer

analysis, Tanaka et al. (2016a) estimated the genome size of 3 species. Estimates were

as follows: Z. Japonica, ~ 390 Mb (0.80 pg/2C) and 340 Mb; Z. pacifica, ~ 370 Mb (0.76

pg/2C) and 302 Mb; and Z. matrella, ~ 380 Mb (0.79 pg/2C) and 423 Mb (Tanaka et al.,

2016a).

The number of linkage groups (22 - 44) and the genome size coverage in linkage

mapping analysis have varied from 932.5 centi Morgan (cM) to 3956.1 cM (Yaneshita et

al., 1999; Cai et al., 2004, 2005; Li et al., 2009, 2010; Jessup et al., 2011; Guo et al.,

2014; Wang et al., 2015). The linkage map with the highest density covers 2,158.5 cM

of the zoysiagrass genome (Li et al., 2015). The occurrence of small linkage groups with

unlinked and distorted markers has been reported (Yaneshita et al., 1999; Cai et al.,

2004, 2005; Li et al., 2009, 2010; Jessup et al., 2011; Guo et al., 2014). Large patch

caused by Rhizoctonia solani AG 2-2 LP is the most important disease of zoysiagrass.

No high levels of genetic resistance have been identified, but inherent genetic variation

within germplasm collections appears promising for finding superior genotypes.

113

Currently, LP disease screenings are conducted under field conditions with

natural infections or in walk-in growth rooms using artificial inoculations. The use of

molecular methods can be used to reduce the time to identify resistant/tolerant

accessions, eliminate environmental interactions and potentially hasten cultivar release

(Lammerts van Bueren et al., 2010). To date, molecular markers associated with LP

disease responses have not been identified. Approximately 507 SSRs loci are currently

available for molecular genetic studies in Zoysia spp. (Li et al., 2010). The association

of some SSRs loci with large patch (LP) disease tolerant genotypes would be an

important finding for zoysia breeders. The objectives of this research were 1) evaluate a

set of microsatellite primers for polymorphisms among parental zoysiagrass genotypes

with differing LP responses; 2) perform a segregation analysis of the polymorphic

markers in a subset of a segregating F1 progeny; and 3) identify markers potentially

associated with large patch tolerant genotypes.

Materials and Methods

Development of a Segregating Zoysiagrass F1 Family

In 2012, an F1 family with 153 progeny from crossing a Z. japonica (5333-53) with

a Z. matrella (375) was created. These two parental accessions were previously

identified as large patch disease tolerant and susceptible, respectively (Chapter III). The

F1 progeny was propagated, established and maintained in the University of Florida

Turfgrass Envirotron greenhouse. Propagation of the genotypes and maintenance

practices were performed as previously described (Chapter III).

DNA Isolation

Fresh leaf tissue was collected from parental and progeny lines for genomic DNA

isolation. Leaf tissue was kept on ice until preparation. Tissue was chopped into 0.5 cm

114

pieces and 120 mg from each line was collected in 2.0 mL micro centrifuge Eppendorf

tubes (Seal Rite, USA Scientific Inc.). To facilitate maceration, 2 stainless steel 5 mm

beads (QIAGEN, Silicon Valley, USA) were added to the tubes. Tubes were frozen in

liquid nitrogen for 5 minutes and grinding was done on the Mixer Mill MM 400 (Retsch,

GmbH, Haa, Germany) for 1 minute, 45 seconds at a vibrational frequency of 30 Hz.

Genomic DNA was isolated using the Sodium Dodecyl Sulfate-SDS modified protocol

(Dellaporta et al., 1983). Thus, after maceration, 600 µL of extraction buffer pH = 8.0

[0.04 M Tris-Base, 0.05 M Tris-HCL, 0.5 M NaCL and 0.05 M EDTA

(Ethylenediaminetetraacetic acid)] with Polyvinylpyrrolidone (PVP) a 0.05% (w/v) were

added to tubes and vortexed for 10 seconds. Subsequently 40 µL of sodium dodecyl

sulfate (SDS 20%) were added, tubes were vortexed (20 s) and incubated at 65°C for

30 minutes. For precipitation, 200 µL of 5M Potassium Acetate were added, tubes were

vortexed (10 s), incubated on ice for 20 minutes and centrifuged (5430 R, Eppendorf) at

14000 revolutions per minute (rpm) for 20 minutes. All centrifugation steps were

performed at 14000 rpm. The supernatant (700 µL) was collected in a 2 mL Eppendorf

tube; chloroform was added in a 1:1 ratio (700 µL); tubes were vortexed and spun for 15

minutes. Again, the supernatant was collected in a new tube; 400 µL of isopropanol

were added, tubes were slowly mixed by hand, incubated at -20°C for 1 hour and spun

for 20 minutes. After centrifugation, supernatant was discarded and pellets were dried

by tube inversion on a paper towel for 1 hour or until visually dry. Pellets were re-

suspended in 40 µL of buffer Tris-Base, Tris-HCL and EDTA, pH = 8.0 with 4 µL of

RNAse (10 mg/ml) and incubated at 37°C for 30 minutes. The last precipitation was

performed with 4 µL of 3M of sodium acetate (pH = 5.2) and 100 µL of absolute ethanol.

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Tubes were mixed, incubated at -20°C overnight and spun at 4°C for 15 minutes.

Supernatants were discarded, pellets were washed with 100 µL of 70% ethanol, spun

for 20 minutes and dried for about 1 hour.

DNA was diluted in 200 µL of TE buffer pH = 8.0 (10 mM Tris-Base and 1 mM

EDTA) and stored at -20°C until use. Quality of isolated DNA was verified by agarose

LE general purpose (Apex BioResearch Products, Genesee Scientific Corporation) gel

(0.8%) electrophoresis. Genomic DNA (2.0 µL) was mixed with 7.0 µL of deionized H2O

and 2.0 µL of dye 6X (Apex BioResearch Products, Genesee Scientific Corporation).

Electrophoresis events were ran for 30 minutes at 120 voltage in a Sub-Cell® GT cell

chamber (BIORAD Laboratories, Inc., USA) with 1X Tris-Base EDTA buffer pH = 8.0,

amended with ethidium bromide at 0.625 mg/ml (1 drop per 50 mL of buffer) (Apex

BioResearch Products, Genesee Scientific Corporation). A 100 base pair DNA ladder II

(3.0 µL /lane) (Apex BioResearch Products, Genesee Scientific Corporation) was

loaded as control.

DNA was quantified (ng/µL) at absorbance A260/A230 using a

spectrophotometer (NanoVue plus, GE Healthcare, Bio-Science Corp, NJ, USA) with

automatic path length (0.5 mm), dilution factor 1.0, and factor 50.0. DNA quality was

quantified by absorbance of A260/A280 and visualized in agarose gels of 0.8%.

Simple Sequence Repeats (SSRs) Survey by Polyacrylamide Gel Electrophoresis (PAGE)

A total of 459 SSRs primers (Li et al., 2010) (Table 4-1) were surveyed for

polymorphisms on parental accessions 5333-53 (LP tolerant) and 375 (LP susceptible).

Oligonucleotide primers were synthesized by Invitrogen life technologies (Thermo

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Fisher Scientific Inc.) at a 100 µM concentration. Forward and reverse stocks were

prepared using 10 µM on sterile distilled water (dH2O) and stored at 20°C until use.

DNA amplification. Genomic DNA was amplified by Polymerase Chain Reaction

(PCR). PCR reactions were carried out in 0.2 ml tubes (8-well strips with dome caps,

USA Scientific®) with a total volume of 15 µL. PCR mixtures were prepared with 10X

standard buffer 15 mM MgCl2 or 2X Taq RED Master Mix 1.5 mM MgCl2 (Apex

BioResearch Products, Genesee Scientific Corporation). The Master Mix with standard

buffer included: 9.9 µL of ultra-pure water (Gentrox, Genesee Scientific Corporation),

1.5 µL of 10X standard buffer, 0.3 µL of dNTPs Mix 40 mM (10 mM each), 0.45 µL of 50

mM MgCl2, 1.2 µL of oligonucleotides stock (10 mM), 0.15 µL of Taq DNA polymerase

(5 units/µL) and 1.5 µL of genomic DNA (20-25 ng/µL). The Master Mix with 2X Taq

RED included: 4.8 µL of ultra-pure water, 7.5 µL of Taq RED Master Mix, 1.2 µL of

oligonucleotides stock (10 mM) and 1.5 µL of genomic DNA (20-25 ng/ µL). Reagents

were purchased at Apex BioResearch Products, Genesee Scientific Corporation.

Annealing temperatures for each primer set were based on forward and reverse

sequences (800 nM) (New England Bio Labs Inc. http://tmcalculator.neb.com. Tm

calculator v1.7) (Table 4-2). The touchdown protocol (Li et al., 2010) was eliminated due

to the presence of non-specific bands observed in the agarose gels with several primer

sets. Extension time was also reduced to 30 seconds during cycles and to 3 minutes for

the final extension step. Specific PCR profiles for groups of primers were set based on

the annealing temperature data suggested by the Tm calculator v1.7 (New England Bio

Labs Inc. http://tmcalculator.neb.com. However, for some primer sets it was necessary

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to improve the quality of the PCR amplicon; thus, the annealing temperature was

increased or decreased.

Amplifications were performed in a MultiGene OptiMax thermal cycler (Labnet

International, Inc.) as follows: initial denaturation at 94°C for 5 minutes, followed by 30

cycles of 1 minute at 94°C, annealing for 1 minute using the primer specific

temperatures; and extension for 30 seconds at 72°C. The final extension ran for 3

minutes at 72°C, followed by holding at 4°C. Samples were kept at 4°C until use.

PCR reactions were analyzed in 0.8% or 1.0% agarose general purpose LE gels

(APEX, Bioresearch Products) as previously described. PCR reactions (3.0 to 4.0 µL)

made with 10X standard buffer were mixed with 3 µL of 6X loading dye (Apex

BioResearch Products, Genesee Scientific Corporation). PCR reactions (3.0 to 4.0 µL)

made with 2X Taq RED Master Mix were loaded directly.

Electrophoresis was ran for 30 minutes at 120 volts on 1X Tris-Base EDTA buffer

with a pH = 8.0, as previously described. PCR reactions were adequate when a single

and bright band of expected size was visualized; otherwise PCR amplifications were

adjusted (primer concentration and higher/lower annealing temperature).

Polyacrylamide Gel Electrophoresis

PCR reactions (3.0-5.0 µL) were evaluated in 19:1 Acrylamide/Bis-acrylamide

gels (Alfa Aesar, Johnson Matthey Company, MA, USA) using vertical electrophoresis.

Acrylamide/Bis-acrylamide working stock was at 60% (w/v). Two electrophoresis

systems were used: the OwlTM dual-gel vertical chamber model P10DS (Thermo Fisher

Scientific Inc.) and the JVD-80 – Dual Slab Vertical System (IBI Scientific, IA, USA),

although the majority of the SSRs primers were analyzed using the P10DS model.

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Prior to gel preparation, glass plates were cleaned with 96% Ethanol and dried

with Kimwipes 34721 Kimtech Science® Brand (Kimberly-Clark Worldwide, Inc., GA,

USA). After assembly, plates were leak-proof sealed with 3M Scotch blue 2090 masking

tape ¾” (ABLE Industrial Products, Inc., CA, USA) on both sides and with 2” office

binder clips (Swinton Avenue Trading Ltd., Inc., Boca Raton, Fl., USA) placed at the top

and the bottom of each side. The bottom part of the chamber was sealed with 20 mL of

2% agar (High strength, Research Products International, IL, USA) polymerized for 5

minutes.

Following this process, the non-denaturing gels were made for stacking the

sandwich plates and running electrophoresis. The stacking gel (20 mL) and running gel

(50 mL) were prepared using a 9.0% acrylamide/bis-acrylamide gel solution (60% w/v).

The stacking gel was prepared with 8.43 mL of 0.5X Tris-Base EDTA buffer pH = 8.0,

1.5 mL of acrylamide/Bis-acrylamide solution, 100 µL of fresh 10% of Ammonium

Persulfate APS (Thermo Fisher Scientific Inc.) and 10 µL of ultra-pure TEMED

(N,N,N',N'-Tetramethylethylenediamine, Thermo Fisher Scientific Inc.). This gel was

polymerized for 15 minutes. The running gel was prepared with 32.5 mL of 0.5X Tris-

Base EDTA buffer pH = 8.0, 7.5 mL of Acrylamide/Bis-acrylamide solution (60% w/v),

350 µL of fresh 10% of APS and 35 µL of ultra-pure TEMED. The gel solution was

thoroughly mixed by inverting the tube manually for 6 to10 seconds and poured

immediately inside the glass sandwich plates, followed by the assembly of the combs.

Twenty well combs of 1.5 mm thick (BIO RAD Laboratories, Life Science Group, CA,

USA) were used. Usually 2 gels were made and run at the same time. Running gels

were polymerized for 1 hour.

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After polymerization, the inner side of the chamber was filled with approximately

500 mL 1 X TBE buffer. Once the gels were immersed in the buffer, the combs were

taken out and the samples were loaded. Samples were loaded using 200 µl round 0.6

mm tips (1022-0600) (USA Scientific, Inc.). A volume of 2.0-4.0 µL of a sample was

loaded. As controls, 3.0 µL of Perfect Size 50 base pair ladder (5 PRIME Inc., MD,

USA) and 3.0 µL of 100 base pair DNA ladder II (Genesee Scientific Corporation) were

loaded. Following the loading process, the outside chamber was filled with 1.5 L of 1 X

TBE buffer and the electrophoresis was initiated. Electrophoresis was performed for 3

hours at 120 voltage/100 amperage, on 1X Tris-Base EDTA buffer pH = 8.0.

Following electrophoresis, the top glass plate was carefully removed leaving the

gel attached to the bottom glass plate. Gels (attached to the glass plate) were rinsed

and stained individually on a 2.4 L Rubbermaid freezer container (Blain Supply Inc., WI,

USA). Initially, gels were rinsed with fresh deionized ultrapure water to eliminate buffer

residuals followed by staining with 300 mL of ethidium bromide solution (0.5 µg/ml) for

10-12 minutes with shaking (at 100-120 rpm). Gels were de-stained in 300 mL of fresh

deionized ultra-pure water for 15 minutes (or as it needed) under shaking (at 100 rpm).

After de-staining, a single gel was carefully taken from the glass plate to the UV light

tray of the Molecular Imager® Gel DocTM XR + system (Bio Rad Laboratories Inc., CA,

USA) to be visualized under trans-UV light. The banding pattern was obtained with the

Image LabTM software version 5, using the ethidium bromide protocol and auto exposure

for intense bands. Gel pictures were taken and saved for analysis.

Large Patch Disease Phenotyping

Screening of the selected F1 family was performed in fall 2014 (Oct. and Nov.)

and spring 2015 (Feb. and March) at 12 and 14 months after propagation. The

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inoculations were conducted using a randomized complete block design (RCBD) with 4

inoculated replications and 1 “mock run” (control) (Jan. 2015). Isolate information, and

details of the inoculation procedure and incubation were described in Chapter II. The

rating system was described in Chapter III. Phenotypic segregation and distribution of

the disease response was performed for each rating date using the disease severity

means of the genotypes as described in Chapter III.

Identification of F1 Hybrids

Polymorphic primers, ZB 01N17 (ID 116) and Zj AG 130 (ID 442) (Li et al., 2010),

with specific alleles associated to the female and male parents were selected to identify

F1 hybrids. PCR reactions, agarose and PAGE gels electrophoresis were performed as

described above. Progeny were considered true hybrids when segregation of male

alleles were observed with one or both primer sets. Progeny were questionable when

only segregation of female alleles were observed.

Selection of Polymorphic SSRs Primers and Segregation Analysis

For segregation analysis 137 primers from all linkage groups were selected

(Table 4-4). Primer selection was based on the following criteria: cM distance among

markers, clear polymorphism and differing linkage groups. These criteria were

established after noticing that several polymorphic markers were in close proximity (≤ 5

cM). In these cases, the primer set that best amplified was selected for the segregation

analysis. Some primers were excluded because the polymorphic segments were

ambiguous between parents. In summary, 137 polymorphic primers were selected

based on distance intervals ≥ 20 cM and their segregation was evaluated in a screening

panel of 21 genotypes with differing large patch disease response. To obtain the

segregation data, the “Poor Man fluorescent” with the 3 primers protocol (Schuelke,

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2000) was used. This protocol was selected over the PAGE methodology, because

results and alleles calling process can be accomplished faster.

Oligonucleotides desalt SSRs primers (100 µM) were synthesized by Applied

Biosystems, Foster City, CA, USA). The forward primers were tail labeled at the 5’ end

with the universal M13 sequence (CAC GAC GTT GTA AAA CGA C). In addition, the

primer M13 was fluorescently labeled at the 5’ end with four dyes: 6-carboxy-fluorescine

6-FAM, PET®, NED and VIC) (Applied Biosystems, Foster City, CA, USA) (Schuelke,

2000).

DNA amplification. The PCR mix was prepared in 0.1 mL PCR plates semi skirt

natural (USA Scientific, Inc.) with a final volume of 12 µL reaction consisting of 1.8 µL of

ultra-pure water (Gentrox, Genesee Scientific Corporation); 6.0 µL of 2X Taq RED

Master Mix; 0.3 µL of forward tailed labeled primer (10 µM); 1.2 µL of reverse primer (10

µM); 1.2 µL of M13 fluorescent dye tailed labeled primer (10 µM) and 1.5 µL of genomic

DNA (20-25 ng/ µL).

Amplifications were performed in a MultiGene OptiMax thermal cycler (Labnet

International, Inc.) as follows: initial denaturation at 94°C for 5 minutes, followed by 30

cycles of 30 seconds at 94°C, annealing for 45 seconds at 56°C, and extension for 45

seconds at 72°C. The profile for annealing the M13 fluorescent dye labeled primer was

8 cycles as follows: denaturation at 94°C for 30 seconds, annealing for 45 seconds at

53°C, extension for 45 seconds at 72°C, and final extension for 10 minutes at 72°C,

followed by holding at 4°C. Samples were kept at 4°C until use. PCR products were

analyzed on agarose gel electrophoresis as previously explained.

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Capillary electrophoresis. PCR products were multiplexed for capillary

electrophoresis and genotyping. Reactions were prepared on 0.1 mL PCR plates semi

skirt natural (USA Scientific, Inc.). For each sample (or accession), 1 to 2 µl of PCR

product obtained with 4 dyes were diluted in 80 to 100 µl of ultra-pure molecular water.

Amounts of PCR product for genotyping were determined based on band intensity

observed from the agarose gels.

The analysis of the amplified fragments were performed using an Applied

Biosystems AB 3730 analyzer (96 capillary technology) (Applied Biosystems, Foster

City, CA, USA) with the GeneScanTM 600 LIZ® dye size standard v2.0 (Thermo Fisher

Scientific) as the internal standard. Capillary electrophoresis was performed at the Gene

Expression & Genotyping Core of the Interdisciplinary Center for Biotechnology

Research (ICBR) of the University of Florida, Gainesville, FL. Electropherogram data

(alleles) were assessed using GeneMarker® v.2.4.0 (SoftGenetics®, LLC., PA, USA).

Data Analysis

Fragment Scoring

Fragments from each primer set were obtained for the selected offspring.

Fragment (peak) designation was based on the expected size of the amplicon for each

primer set, although other peaks were called if they were clear and consistently present

in the progeny. Minor and distorted peaks were dropped out of the analysis as

suggested by Rodríguez et al. (2001).

All the fragments associated with the parental genotypes were scored in the

progeny for presence or absence as 1:1 segregating alleles. Several authors have

utilized this approach to overcome polyploid segregation complexities (Wu et al., 1992;

Yaneshita et al., 1999; Ripol et al., 1999; Cai et al., 2004, 2005; Saha et al., 2005;

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Hirata et al., 2006; Li et al., 2009; Jessup et al., 2011; van Dijk et al., 2012; Liu et al.,

2012; Koning-Boucoiran et al., 2012; Honig et al., 2014; Guo et al., 2014; Wang et al.,

2015). This methodology identifies bands or fragments known as single dose restriction

fragments SDRFs (Wu et al., 1992) and double dose fragments DDRFs (Ripol et al.,

1999).

Chi-Square X2 Test

Segregation of the population was tested for expected 1:1 and 3:1 Mendelian

segregation ratios using the Chi-square analysis X2 test at P ≥ 0.05 (Hirata et al., 2006;

Jessup et al., 2011).The 1:1 ratio was tested for goodness of fit for fragments that

associated to either the female or male parents; while the 3:1 ratio was tested for

markers present in both parents. Chi-square values were calculated using the formula:

X2 = ∑ [(O – E)2/ E], where O represents the number of accessions with the observed

value (a given fragment) and E represents the expected value based on the

calculations.

Single Marker Analysis and T-test

In attempts to identify SSRs fragments associated to the tolerant phenotype, the

genotyping and phenotyping data were used to perform the single gene marker analysis

with the Kruskal-Wallis H test (Kruskal and Wallis, 1952) in the MapQTL® software (van

Ooijen, 2009). For this analysis the linkage phase of the markers obtained with JoinMap

4.0 (Stam, 1993) was used. Linkage mapping for the parental genotypes was conducted

with markers that fit the 1:1 segregation ratio (P > 0.05) (Appendices H and I).

Similarly, the binary data set of polymorphic alleles associated to the female and male

parent were tested along with the disease severity data using the T-test. Analysis was

conducted using SAS version 9.4 with Proc TTEST, alpha = 0.05, H0 = 0.

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Results

Large Patch Disease Response of the F1 Family

All 153 genotypes had compatible reactions with the isolate. The F1 family

segregated for large patch disease response with phenotypic variation of the disease

severity. At 7 DAI mycelia growth was observed on many genotypes, but in general the

disease severity was low on many genotypes. Disease severity and mycelia

colonization were higher at 14 and 21 DAI, indicating progress of the disease between

ratings. During the incubation period, mock plants (controls) remained symptom-free,

and among inoculated plants the amount of disease increased.

Variations in tolerance and susceptibility were distinct at 14 and 21 DAI. Average

disease severity of accessions ranged from 0 % to 36.25 % at 7 DAI, from 6.75 % to

71.25 % at 14 DAI; and from 17.50 % to 87.50 % at 21 DAI.

Selection of Accessions for the Screening Panel. Twenty-one accessions

with differing large patch disease response were selected as a screening panel to

analyze the segregation with the polymorphic primers. The accessions were selected

based on their average performance compared to the family average. Accessions with

severity ≤ 20% were considered LP tolerant, while accessions with severity ≥ 30% were

considered LP susceptible. The average disease severity from each rating and overall

average for the 21 selected genotypes are listed in Table 4-3.

Survey of Polymorphic SSRs on the Zoysiagrass Parental Genotypes

A total of 459 primer sets were evaluated in the parental lines for polymorphisms.

From this group, a total of 379 (82.57%) markers were polymorphic between the two

parental accessions, 50 (10.89%) monomorphic, 24 (5.22%) had problematic

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amplifications, 119 (26%) generated ambiguous polymorphisms and 6 (1.30%) did not

amplify in the parental genotypes (Table 4-1).

Identification of True Hybrids

A total 140 out of 153 hybrids were confirmed as true hybrids. Seven progeny

showed only maternal alleles with both primers and six accessions were questionable

because they showed either partial or complete segregation of maternal alleles with

primer ZB 01N17 and Zj AG 130, respectively. These 13 progeny were discarded from

further analysis due to the possibility of self-fertilization.

Segregation Analysis in the Selected 21 Zoysiagrass Genotypes

A total of 137 polymorphic primers (Table 4-4) were selected for genotyping the

screening panel. Selection of the primers from the 379 polymorphic primers was based

on cM distance, clear polymorphism, and quality of the bands after the polyacrylamide

gel electrophoresis (PAGE). Primers were selected acrossing all linkage groups.

All screened primer sets produced polymorphic fragments in the progeny. A total

of 1,163 fragments or markers were generated in the screening panel. The number of

detected fragments for each SSR primer set varied considerably from 1-25

fragments/locus (data not shown). For several sets of primers the observed fragment

size varied considerably from their expected sizes. Among the evaluated primer sets,

the number of female parent-specific fragments (436, 37.4%) was almost equal to the

number of male parent-specific fragments (437, 37.5%). The number of fragments (290,

25%) shared by both parental genotypes was moderate. A total of 22 non-parental

fragments (orphans) were inherited (presence in > 10 accessions) in the progeny.

The minimum number of fragments was 1 to 4 (average = 2.6)/primer; whereas,

the maximum number of fragments was 7 to 25 (average = 13)/primer within the

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screening panel. Several primers from different linkage groups yielded a high number of

fragments (Table 4-6). Higher numbers of fragments (average = 8.6 to 12) was more

common on linkage groups 13, 16, 17, 18, and 19. Conversely, fewer fragments

(average = 3.6 to 4.8) were observed on linkage groups 2, 4, 9, 10 and 22. Three sets

of primers (from LG 10 and LG 12) did not amplify any fragment.

A total of 675 fragments from 22 linkage groups were heritable and scored in the

screening panel. From this group, a total of 536 (79.4%) and 139 (20.5%) fragments

showed Mendelian 1:1 and 3:1 segregation ratios (d.f. = 1.0, P > 0.05), respectively. For

fragments with a 1:1 segregation ratio, a total of 145 (27%) and 132 (24.6%) were

specific to the female and male parents, respectively. Some fragments associated to

one parent or the other did not amplify in the progeny (null fragments). A total of 13

(4.8%) and 16 (6.03%) null fragments were associated to the female and male parents,

respectively (Table 4-7).

Single Marker Analysis and T-test Results

Results from the single marker analysis performed with the Kruskal-Wallis H test

identified 51 fragments (from 35 primers) significantly associated with large patch

disease severity (Table 4-7). From this group, a total of 20 and 31 fragments were

specific to the female and male parents, respectively. Fragments from all linkage groups

were significant, except from the groups 7, 9, 12 and 14. The most significant (P ≤

0.0001) fragment associated with reduced disease amplified from the primer ZB 01G22

(LG 16). Interestingly this marker segregated from the male parent (susceptible).

The T-test identified 34 fragments (26 primers) significantly associated with the

disease response (Table 4-8). From these fragments, 13 and 21 were specific to the

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female and male parents, respectively. Fragments from all the linkage groups were

associated, except from LGs 4, 7, 9, 12 and 14.

Overall 12 primers were associated with reduced LP severity. Three of these

primers produced fragments associated to the female parent. These primers were: ZB

01H23 (P < 0.0256) from LG 15; ZB 01D24 (P < 0.0085) from LG 5; and ZB 01C09 (P <

0.0326) from LG 22. Significant fragments segregating from the male parent were also

identified in the T-test. These fragments were produced after amplification with the

following primers: ZB 09L15 (P <.0001) from LG 19; ZB 02L18 (P< 0.0009) from LG 10;

ZA 03F03 (P< 0.0012) from LG 21; and ZB 02K23 (P<0.0019) from LG 17.

Segregation Distortion

High segregation distortion was observed with single dose fragments (SDF) and

double dose fragments (DDF). A total of 230 SDF (43%) were distorted. Of these, 113

(41.7%) and 117 (44.1%) fragments were associated to the female and male parent,

respectively. For the group of DDF, 79 (56.8%) were distorted.

The number of SDF and DDF varied among linkages groups in both female and

male parents. For SDF, the linkage groups with more fragments (9 to13) specific to the

female parent were LG 1, LG 2, LG 5, LG 11, LG 13, LG 15, LG 17, LG 19 and LG 21.

For the male parent, the linkage groups with more fragments (10 to 14) were LG 7, LG

13, LG 16 and LG 19. For DDF, the linkage groups with fragments (8 to 12) were LG 3,

LG 5, LG 7, LG 12, LG 13, LG 15, LG 16, LG 17, and LG 19. Fragments with distorted

segregation were observed in all linkage groups. However, the linkage groups 17, 1, 2,

15, 16, 3 and 13 had more distorted fragments.

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Discussion

Large patch disease is currently the most economically important disease in

zoysiagrass. Currently, no molecular markers have been associated with large patch

disease which appears to be controlled by quantitative genetic factors (Chapter and III),

and as reported turfgrass diseases of Rhizoctonia solani (Bonos et al., 2003; Bonos,

2006, 2011; Bokmeyer et al., 2009a; b). Traits with quantitative inheritance are more

sensitive to environmental effects (Stoskopf et al., 1993b).

Screenings for large patch disease have been mostly conducted inside walk-in

plant growth rooms under conditions that favor disease development (Green II et al.,

1993; Hyakumachi et al., 1998; Flor et al., 2010; Obasa et al., 2012). These screenings

can be time consuming and not practical for large populations, and effects from

genotype × environment can still occur during different inoculation events (Chapter III).

Field evaluations are adequate when the disease is endemic or when high levels of

disease pressure are purposely maintained for screenings. Therefore, identification of

molecular markers in zoysiagrass linked to LP resistant loci would be a good tool to

speed the selection process and obtain more accurate results. The use of this approach

in zoysiagrass for LP disease has been negligible.

This current study evaluated 459 SSRs primers in two zoysiagrass parental

accessions with differing large patch disease response and identified 379 polymorphic

primers. However, the group of polymorphic primers was reduced using selection

criteria such as cM distance and clear polymorphisms. Thus, a total of 137 polymorphic

primers were selected and evaluated in a screening panel (21 accessions - F1 progeny)

to select informative and heritable markers that segregate with LP tolerance. The

majority of the primers (except 3) amplified in the parental lines and in the panel. These

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primers yielded a high number of fragments (1,163) indicating high heterozygosity

between the parental accessions that manifested in excessive segregation of the

screening panel.

The proportions of polymorphic fragments with 1:1 segregation ratio associated

to each parent were very similar. These fragments indicate heterozygosity in the parent

with the fragment and homozygosity in the other parent. Other primers amplified similar

regions in both parents (3:1 ratio), indicating mutual heterozygosity (Saha et al., 2005).

Polymorphism Assesment

A high number of polymorphic fragments (83.40%) was observed between the

parental lines; conversely the percentage of monomorphic fragments (11.13%) was low.

High polymorphism is useful to detect differences associated to a trait and it is common

in outcrossing species (Saha et al., 2005). In this study, a proportion of the polymorphic

fragments were attributed to the heterozygous state of both parental accessions. In

zoysiagrass, similar findings have been reported with RFLPs (Yaneshita et al.,1999)

and SSRs (Tsuruta et al., 2005) (Cai et al., 2005). High heterozygosity was also

reported by Tanaka et al. (2016a) in sequence data of three cultivars of Z. japonica, Z.

matrella and Z. pacifica. The percentage of monomorphic alleles reported by Tsuruta et

al. (2005) and Cai et al. (2005) ranged from 3.5% to 19.2%. Results from this current

study supports a lower proportion of monomorphic alleles.

High polymorphism was also observed related to the number of amplified

fragments. Zoysia spp. are considered segmental allotetraploid (2n = 4x = 40)

(Yaneshita et al., 1999), with disomic inheritance; thus, no more than 4 alleles per locus

are expected if the locus is not duplicated. The number of alleles/locus should be

associated with the ploidy level of the species (van Dijk et al., 2012). In this study

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several primers amplified more than 4 fragments/locus. Up to 25 fragments were scored

with a few primers. This excessive polymorphism (> 4 alleles) has been observed

previously in zoysiagrass (Cai et al., 2005; MA et al., 2007). In these studies, up to 9

and 10 alleles generated from a single locus were observed. Cai et al. (2005) evaluated

self-pollinated progeny with SSRs and AFLP markers and MA et al. (2007) evaluated 30

Z. japonica accessions with SSRs markers. Conversely, the presence of one or two

alleles per locus for the majority of the SSRs primers evaluated was reported for

zoysiagrass cultivars (Harris-Shultz et al., 2014) and for an F1 mapping population (Li et

al., 2009). The higher number of fragments/locus observed in the screening panel tend

to be more similar to Cai et al. (2005) and MA et al. (2007), and substantially different

from Harris-Shultz et al. (2014) and Li et al. (2009). In all of these studies, SSRs

markers were used; thus, they were not the cause of excessive polymorphism.

The heterozygous nature of the parental lines (5333-53 and 375) combined with

the existence of duplicated loci and possibly additional recombination events are the

likely causes of the excessive polymophism observed with some primer sets. For future

analysis, the high polymoprhism rates can be minimized by using informative primers

that produce the expected number of alleles (up to 4). For linkage mapping, the use of

selfed progeny of heterozygous accessions can minimized excessive polymorphisms.

Single Gene Analysis and T-test

The Chi-square goodness-of-fit results identified a group of fragments that fit the

1:1 segregation ratio. These fragments were further tested with the single gene marker

and T-tests which indicated that fifty-one (single gene) and fifteen fragments (T-test)

were significantly associated with large patch disease severity. These loci were

previously mapped in different linkage groups (Li et al., 2010). The loci with the most

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significant association belong to the linkage groups 5, 10, 15, 16, 17, 19, 21 and 22.

These results should be interpreted with caution and validated using the entire F1

family. This approach will reduce the number of primers to be evaluated and will focus

on screening primers with informative segregation.

Further research should explore these regions (linkage groups) with more SSRs

primers to see if the significant association can be confirmed. Also, the zoysiagrass

genome data can be utilized to locate the SSRs fragments with significance to confirm

their linkage grouping information. Synteny between rice, sorghum and zoysiagrass can

be utilized to support these observations. In rice, QTL linked to resistance to sheath

blight have been mapped in chromosomes 1, 6, 7, 8, and 9 (Yadav et al., 2015).

Segregation Distortion

In this current study several fragments showed deviation (distorted markers) from

the expected Mendelian segregation (Xu and Hu, 2009). High levels (41.7% and 43%)

of fragment distortion were reported within the group of markers associated to the

female and male parents, respectively. The number of distorted fragments was similar,

thus no parental factors were associated with the distortion. Similar results were

reported by Li et al. (2009), with two Z. japonica parental accessions evaluated with

SSRs markers and by Saha et al. (2005) who evaluated tall fescue parental genotypes

and their F1 progeny with SSRs and AFLP markers. These findings are useful to select

primer sets with low distortion. Furthermore, the proportion of fragment distortion should

be confirmed in the complete population.

Different levels of segregation distortion were previously reported in zoysiagrass

(Yaneshita et al., 1999; Cai et al., 2004, 2005; Li et al., 2009, 2010; Jessup et al., 2011;

Guo et al., 2014), and in other turfgrass and forage species (Jones et al., 2002; Saha et

132

al., 2005; Honig et al., 2014). In zoysiagrass the highest level (43.7%) of distorted

segregation was reported by Li et al. (2010) in an F1 population from Z. japonica and Z.

matrella. Guo et al. (2014) suggests that it is normal to observe segregation distortion in

F1 populations and attributes this phenomenon to genetic effects rather than to the

population structure or the nature of the markers used.

Distorted fragments were not included in the preliminary zoysiagrass parental

linkage mapping (Appendices J and K) to avoid including noisy data in the analysis.

However, some authors have safely included these type of fragments in QTL analyses

without detrimental effects on the data resolution (Xu and Hu, 2009). In fact, the

inclusion of these fragments has been considered beneficial for the analysis (Xu and

Hu, 2009) and for identifying regions with high segregation distortion.

Several causes are attributed to segregation distortion. In this study, population

size can have a significant effect. Hirata et al. (2006) mentioned that population size can

affect marker segregation in linkage mapping in terms of marker distance, order and

clustering. The number of zoysiagrass accessions utilized in the screening panel was

low, and it is possible that this population does not accurately reflect some segregation

events. Besides the causes mentioned above, it is important to consider that a

proportion of the segregation distortion may originate from the calling and scoring

process of the fragments.

Distorted segregation has also been attributed to the presence of null alleles,

point mutations, insertions and/or deletions close to the regions amplified by the

markers (Jones et al., 2002), gametic selection, abnormal chromosome pairing (Xu et

133

al., 1995) unequal frequency of alleles due to small population size, gametophytic

genes and hybrid sterility (Wu et al., 2010).

The segregation analysis of the complete F1 family will be important to accurately

determine the extent of the segregation distortion and to determine if distorted

fragments should be included in the linkage mapping analysis.

Duplicated Loci

Results from the screening panel indicated that some SSRs primers amplified

duplicated loci. These primers should be excluded for further analysis because they can

generate inconsistencies in the data. Detection of duplicated loci is important. Causes

can be associated with amplification of similar regions in the same or different

chromosomes or to amplification conditions. The presence of duplicated loci could also

be attributed to the high number of allelic fragments observed in the screening panel. It

is also possible that PCR conditions caused unspecific annealing of the primers due to

the excessive number of fragments in the screening panel. Thus, changes in PCR

conditions could decrease the proportion of duplicated loci (Hirata et al., 2006). In

general, the abundance of polymorphic regions yielded with some primer sets was

extremely high in the population.

Duplicated loci increase the number of the markers that can be expected in

diploid or polyploid crops (Jones et al., 2002); although in polyploid species they appear

to be more common (Helentjaris et al., 1988). In a zoysiagrass mapping study using

RFLPs, Yaneshita et al., (1999) reported the presence of duplicated loci; but this

phenomenon is not common in zoysiagrass mapping populations (Cai et al., 2005).

In other allotetraploid crops such as wheat (Triticum spp.) (Song et al., 2005),

sorghum (Sorghum bicolor L.) (Bhattramakki et al., 2000), oat (Avena nuda L.) (Song et

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al., 2015) zoysiagrass (Cai et al., 2005), tall fescue (Saha et al., 2005), perennial

ryegrass (Lolium perenne L.) (Jones et al., 2002), and creeping bentgrass (Agrostis

stolonifera L.) (Honig et al., 2014) duplicated loci have been detected after amplification

with SSRs primers. These loci can produce inconsistencies in linkage maps; however,

their inclusion (Saha et al., 2005) or exclusion (Jones et al., 2002) is a decision based

on how their presence changes the integrated linkage maps.

Conclusions and Future Research

In this research SSRs primers previously mapped in Zoysia spp. populations (Li

et al., 2010) were evaluated in parental accessions (Z. japonica and Z. matrella) with

differing large patch disease response, and in a screening panel of F1 progeny

(interspecific hybrids) developed from the cross. The amount of polymorphism observed

with these primers in the parental accessions and in the selected F1 hybrids (screening

panel) was high. This initial screening indicated that 111 primers produced clear and

heritable fragments in the progeny. However, many of these primers also had distorted

segregation. From this group (111 primers), a total of 20 and 26 primers with informative

segregation were associated with the female and male parent, respectively. Three

primers segregating from the tolerant parent were significantly associated (T-test) with

less disease severity.

These primers produced fragments that segregated in linkage groups as reported

by Li et al. (2010) and warrant further evaluation in the complete F1 family for QTL

analysis. The identification of QTLs and markers associated to important traits in

zoysiagrass is relatively new (Jessup et al., 2011; Guo et al., 2012b). Markers have

been found to be linked to fall armyworm resistance (Jessup et al., 2011), cold tolerance

135

and green period (Guo et al., 2012b). In general, the majority of these studies report

high levels of polymorphism in the mapping populations.

Candidate screening for genes associated to disease resistance can be an

important step now that the zoysiagrass genome assembly has been completed;

although this approach was not successful to map the Zfawr1 (armyworm resistance

loci) in zoysiagrass (Jessup et al., 2011). Candidate gene analysis has been used for

sheath blight (Rhizoctonia solani AG 1-IA) resistance in rice reporting the β 1–3

glucanase and other genes associated with moderate resistance (Yadav et al., 2015).

Large patch disease caused by Rhizoctonia solani (AG 2-2 LP) is a trait probably

governed by quantitative inheritance with large environmental effects. The identification

of molecular markers associated with resistant phenotypes would allow breeders to

narrow the screening process under artificial conditions to evaluate only those

genotypes with promissory disease response.

The regions with putative SSRs fragments associated to large patch disease

resistance can also be genotyped with other markers such as Single Nucleotide

Polymorphisms (SNP’s) or using next generation sequencing (NGS) techniques. Also

future research can include the evaluation of the polymorphic informative markers in

selfed progeny of accessions with improved resistance levels. With this approach the

level of polymorphism can be reduced (Cai et al., 2004), and the segregation analysis

can be more distinct. These approaches can produce more reliable data to identify

polymorphisms or genome regions associated with the targeted trait.

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Table 4-1. Simple Sequence Repeat (SSRs) primers developed for Zoysia spp. used to screen two zoysiagrass parental accessions with differing large patch disease responses.

a Linkage groups reported by Li et al. (2010). b Number of total SSRs primers mapped by Li et al. (2010) in each linkage group and available for analysis. c Number of polymorphic SSRs primers between two Zoysia spp. parental accessions. d Number of monomorphic SSRs primers between two Zoysia spp. parental accessions. e Number of SSRs primers with ambiguous polymorphism between two Zoysia spp. parental accessions. f Number of SSRs primers that produced fair quality amplicon after amplification with genomic DNA of two Zoysia spp. parental accessions. g Number of SSRs primers that did not produce amplicon (under the PCR conditions used) after

amplification with genomic DNA of two Zoysia spp. parental accessions.

Linkage groupa

Number of

primersb

Number of polymorphic

primersc

Number of monomorphic

primersd

Primers with ambiguous

polymorphisme

Primers with fair

amplificationf

Non-amplificationg

1 36 21 4 6 5 0 2 31 16 4 10 0 1 3 26 16 2 8 0 0 4 14 8 2 4 0 0 5 13 8 1 0 4 0 6 30 18 4 8 0 0 7 25 18 1 4 0 2 8 30 19 5 5 0 1 9 29 16 1 11 1 0

10 26 10 2 10 4 0 11 28 18 3 7 0 0 12 20 7 3 8 2 0 13 22 8 4 8 1 1 14 7 5 0 1 0 1 15 18 11 1 6 0 0 16 22 11 2 8 1 0 17 22 13 3 2 4 0 18 14 7 1 5 1 0 19 13 10 1 2 0 0 20 8 4 3 1 0 0 21 13 7 3 2 1 0 22 12 9 0 3 0 0

Total 459 260 50 119 24 6

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Table 4-2. Annealing temperature ranges for Zoysia spp. SSRs primer sets in each linkage group.

Linkage groupa Annealing temperature ranges (C°)b

1 46 – 62 2 44 – 50 3 44 – 52 4 45 – 51 5 44 – 51 6 43 – 51 7 39 – 52 8 44 – 50 9 44 – 54 10 44 – 52 11 45 – 51 12 45 – 51 13 44 – 51 14 47 – 54 15 46 – 50 16 44 – 51 17 44 – 51 18 43 – 52 19 44 – 51 20 47 – 51 21 43 – 51 22 44 - 49

a Linkage groups reported by Li et al. (2010). b Annealing temperature ranges used to evaluate all the SSRs primers of each linkage group.

138

Table 4-3. Selected zoysiagrass accessions with differing large patch disease response for screening with polymorphic SSRs primers.

Accession identification

LP disease severity at 7

DAIa

LP disease severity at

14 DAIb

LP disease severity at

21 DAIc

Average of the LP disease severityd

Disease response

Genotype 59 3.25 8.75 17.5 9.8 Tolerant Genotype 61 3.75 10.75 18.75 11.0 Tolerant Genotype 115 2.5 6.75 25.0 11.4 Tolerant Genotype 78 4.5 10.5 22.5 12.5 Tolerant Genotype 13 4.5 11.75 22.5 12.9 Tolerant Genotype 118 4.25 10.5 25.0 13.2 Tolerant Genotype 11 6.25 13.25 21.25 13.6 Tolerant Genotype 144 6.25 13.25 23.75 14.4 Tolerant Genotype 29 5.75 17.0 20.75 14.5 Tolerant Genotype 99 8.25 14.5 23.75 15.5 Tolerant Genotype 69 11.0 18.25 25.0 18.1 Tolerant Genotype 150 21.75 38.0 52.0 37.3 Mod. susceptible Genotype 112 16.75 38.75 58.75 38.1 Mod. susceptible Genotype 131 20.5 41.25 52.5 38.1 Mod. susceptible Genotype 4 22.5 35.0 58.75 38.8 Mod. susceptible Genotype 155 22.0 38.75 60.0 40.3 Mod. susceptible Genotype 45 25.0 44.5 58.75 42.8 Mod. susceptible Genotype 129 20.0 47.5 75.0 47.5 Mod. susceptible Genotype 49 12.5 56.25 78.75 49.2 Mod. susceptible Genotype 154 20.0 71.25 77.5 56.3 Susceptible Genotype 158 36.25 62.5 80.0 59.6 Susceptible

a Large patch disease severity at 7 days after the inoculation (DAI) was estimated based on data of four replications. b Large patch disease severity at 14 days after the inoculation (DAI) was estimated based on data of four replications. c Large patch disease severity at 21 days after the inoculation (DAI) was estimated based on data of four replications. d Average of the large patch disease severity was estimated based on average data of the three ratings.

139

Table 4-4. Simple Sequence Repeat (SSRs) primers developed for Zoysia spp. selected for the segregation analysis of the screening panel of the 5333-53 × 375 F1

segregating family.

a Linkage groups reported by Li et al. (2010). b Number of total SSRs primers mapped by Li et al. (2010) available for analysis. c Number of polymorphic SSRs primers between two Zoysia spp. parental accessions. d Number of SSRs primers from each linkage group evaluated in the screening panel.

Linkage groupa

Number of primersb

Number of polymorphic

primersc

Number of primers evaluated in the screening paneld

1 36 21 8 2 31 16 8 3 26 16 7 4 14 8 5 5 13 8 6 6 30 18 7 7 25 18 8 8 30 19 6 9 29 16 7 10 26 10 7 11 28 18 7 12 20 7 7 13 22 8 5 14 7 5 3 15 18 11 7 16 22 11 8 17 22 13 7 18 14 7 5 19 13 10 5 20 8 4 3 21 13 7 5 22 12 9 6

Total 459 260 137

140

Table 4-5. Minimum, maximum, and average number of fragments observed in the screening panel of a full-sib F1 family after amplification with Simple Sequence Repeat (SSRs) primers.

Linkage groupa Minimum number of fragmentsb

Maximum number of fragmentsc

Average number of fragmentsd

1 3 16 6.7 2 2 8 4.6 3 2 10 5.6 4 3 8 4.6 5 4 7 5.7 6 2 9 5.1 7 3 13 7.4 8 2 9 5.3 9 2 7 3.6 10 0 10 4.0 11 3 12 6.1 12 0 16 5.3 13 4 25 12.0 14 3 12 7.7 15 1 24 7.7 16 3 19 8.8 17 2 18 8.7 18 3 14 8.6 19 4 18 10.2 20 4 9 5.7 21 4 12 7.8 22 3 8 4.8

a Linkage groups reported by Li et al. (2010). b Minimum number of fragments scored with a primer set from each linkage group. c Maximum number of fragments scored with a primer set from each linkage group. d Average of number of fragments scored with all the primer sets evaluated from each linkage group.

Table 4-6. Polymorphic SSRs fragments tested for the Mendelian 1:1 segregation ratio. Fit the ratioa Distortedb Nullc

5333-53 (Female)d 145 113 13 374 (Male)e 132 117 16 Total 277 230 29

a Number of fragments that fit the Mendelian 1:1 ratio: P > 0.05. b Number of fragments that did not fit the Mendelian 1:1 ratio: P < 0.05. c Number of fragments scored in the female or male parents but not observed in the 21 accessions of the screening panel. d Zoysiagrass accession used as female parent (pollen receptor). e Zoysiagrass accession used as male parent (pollen donor).

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Table 4-7. Zoysia spp. simple sequence repeat primers with significant association with low large patch disease severity based on the Kruskal-Wallis H test.

Linkage groupa Primer IDb SSR Loci IDc Parentd Significancee Kruskal-Wallis Rankf

19 373 ZB09L15 male **** 8.915 10 155 ZB02L18 male **** 8.753 20 222 ZB04E01 male **** 8.341 16 92 ZB01G22 Female **** 7.92 21 54 ZA03F03 male *** 7.742 16 87 ZB01F13 male *** 7.491 17 153 ZB02K23 male *** 7.432 19 391 ZB10O19 Female *** 6.823 2 411 ZC03C23 Female ** 6.611

19 391 ZB10O19 male ** 6.61 1 225 ZB04H19A Female ** 6.438

17 50 ZA02O09 male ** 6.369 22 266/365 ZB06B08-ZB09I02A male ** 6.369 13 227 ZB04J06 male ** 6.191 22 74 ZB01C09 Female ** 5.541 1 221 ZB04D24 male ** 5.338 1 221 ZB04D24 male ** 5.338

16 290 ZB06N21 male ** 5.338 21 54 ZA03F03 male ** 5.338 8 291 ZB06O03 male ** 5.081 3 18 ZA01C12 male ** 5.075 3 148 ZB02J20 Female ** 5.043

17 153 ZB02K23 Female ** 4.975 6 375 ZB09N07 Female ** 4.685

10 327 ZB08B08 Female ** 4.684 15 356 ZB09C22 male ** 4.67 11 39 ZA01P11 male ** 4.649

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Table 4-7. Continued.

Linkage groupa Primer IDb SSR Loci IDc Parentd Significancee Kruskal-Wallis Rankf

2 187 ZB03G06 male ** 4.548 18 103 ZB01J23 male ** 4.413 19 116 ZB01N17 male ** 4.413 15 96 ZB01H23 Female ** 4.366 13 130 ZB02C08 male ** 4.339 4 395 ZC01C12 Female ** 4.285 18 441 ZjAG125 Female ** 4.173 8 291 ZB06O03 male ** 4.06 5 85 ZB01D24 male ** 3.962

2 411 ZC03C23 male ** 3.866 6 116 ZB01N17 Female * 3.826 6 95 ZB01H17 Female * 3.822 22 266/365 ZB06B08-ZB09I02A Female * 3.752 1 225 ZB04H19A male * 3.686 1 225 ZB04H19A male * 3.686 21 163 ZB02O05 male * 3.686 16 92 ZB01G22 male * 3.574 5 85 ZB01D24 male * 3.548 18 103 ZB01J23 Female * 3.327 5 85 ZB01D24 Female * 3.28 5 66 ZB01A21 Female * 3.181 1 221 ZB04D24 male * 2.978 1 225 ZB04H19A Female * 2.898 13 208 ZB03N18 Female * 2.754

a Linkage groups reported by Li et al. (2010). b Primer identification for dissertation purposes. c Simple Sequence Repeat locus name reported by Li et al. (2010). d Parent makes references to the female (5333-53) and male (375) accessions used as parents to develop the F1 family. e Significance level: *P ≤ 0.05; ** P ≤ 0.01; * * * P ≤ 0.001; * * * * P ≤ 0.0001. f Rank given by the Kruskal-Wallis Test.

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Table 4-8. Zoysia spp. simple sequence repeat (SSRs) primers significantly associated to large patch disease tolerance based on the T-Test.

Parenta SSR loci

IDb Allelec LGd

No. of accessions without the

fragment (0)

Meane Standard

error

No. of accessions

with the fragment (1)

Meanf Standard error

T-value

1 ZB01D24 B 5 13 35.06 17.03 8 17.37 10.58 0.0085 1 ZB01H23 a 15 14 34.04 17.35 7 16.90 9.38 0.0256 1 ZB01C09 b 22 12 35.62 17.43 8 19.31 11.61 0.0326 1 ZB01J23 b 18 11 21.12 13.61 9 35.96 18.56 0.0539 1 ZB01H17 B 6 11 21.42 13.23 10 35.92 18.14 0.0488 1 ZB02P17 B 5 8 37.98 18.43 11 21.75 13.67 0.0410 1 ZB02K23 C 17 8 19.58 13.23 11 37.40 16.21 0.0209 1 ZB01H17 b 6 9 19.13 12.27 12 35.22 17.26 0.0282 1 ZB 02J20 a 3 8 18.97 11.89 13 34.08 17.54 0.0454 1 ZB01G22 a 16 7 16.35 9.63 13 35.90 16.56 0.0106 1 ZB04H19A a 1 7 16.25 9.35 14 34.36 17.0 0.0174 1 ZB08B08 b 10 7 41.75 15.54 14 21.61 13.79 0.0069 1 ZB10O19 D 19 7 16.14 9.49 14 34.42 16.90 0.0162 2 ZA03F03 b 21 9 38.94 16.43 12 20.36 13.07 0.0094 2 ZB02O05 A 21 8 37.85 16.76 13 22.46 14.93 0.0413 2 ZB06O03 C 8 14 34.01 17.23 7 16.95 9.99 0.0265 2 ZB04N13 a 16 11 24.95 15.79 7 40.41 15.36 0.0577 2 ZB01J23 C 18 13 33.64 18.05 7 16.95 9.30 0.0359 2 ZB01D24 D 5 13 22.11 13.85 8 38.42 17.70 0.0292 2 ZB09C22 B 15 12 22.49 13.92 8 38.92 17.56 0.0315 2 ZB 03G06 A 2 12 21.85 12.92 9 36.95 18.72 0.0412 2 ZA01C12 B 3 9 22.34 13.22 9 39.75 16.26 0.0241 2 ZB02C08 b 13 10 37.31 17.95 9 19.11 10.80 0.0173 2 ZB01F13 X8 16 12 20.78 15.64 9 38.38 13.83 0.0149 2 ZB09L15 C 19 12 39.38 14.60 9 13.58 2.45 <.0001 2 ZB01G22 X1 16 10 36.76 18.02 10 21.37 12.82 0.0411 2 ZB04E01 C 20 11 19.51 11.87 10 38.02 17.04 0.0090 2 ZB06O03 D 8 10 19.25 13.46 11 36.58 16.16 0.0156

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Table 4-8. Continued.

Parenta SSR loci IDb

Allelec LGd No. of accessions without the fragment

(0)

Meane Standard error

No. of accessions

with the fragment

(1)

Meanf Standard error

T-value

2 ZB02L18 D 8 10 39.99 15.50 11 17.72 10.28 0.0009 2 ZB02K23 d 17 8 42.93 14.06 11 20.41 12.61 0.0019 2 ZB10O19 c 19 10 38.93 15.49 11 18.69 12.28 0.0035

2 ZA03F03 C 21 10 39.80 15.73 11 17.90 10.37 0.0012

2 ZA01P11 C 11 7 38.81 18.05 12 22.78 14.49 0.0483 a Parent makes references to the female (5333-53) and male (375) accessions used as parents to develop the F1 family. b Simple Sequence Repeat locus name reported by Li et al. (2010). c Designation to identify fragments of different size amplified with the same primer set. d Linkage groups reported by Li et al. (2010). e Disease severity mean of the zoysiagrass accessions without the fragment. f Disease severity mean of the zoysiagrass accessions with the fragment.

145

CHAPTER 5 FINAL REMARKS

This research was focused on the Zoysia spp.- large patch (LP) disease

pathosystem. LP is a disease caused by the fungal pathogen: Rhizoctonia solani.

However only one anastomosis group (AG): 2-2 of the fungus and one biotype (LP)

within the AG caused the disease in warm season grasses. Disease etiology has been

described, but no information related to the genetics of the disease, heritability

estimation, and variance components have been described. Furthermore, no

commercial cultivar has high resistance level. Thus, the search for sources of genetic

resistance within germplasm are very important. However, screening of germplasm and

breeding lines have not been extendedly conducted.

Levels of tolerance or susceptibility of the zoysiagrass cultivars are not clearly

reported. Data from field trials of the National Turfgrass Evaluation Program (NTEP)

indicate genotype by environment interactions. Thus this current study was conducted

to evaluate zoysiagrass cultivars and populations for large patch disease resistance. In

addition, variance components and broad and narrow sense heritabilities were

estimated. One important goal was to identify accessions with high resistance levels. To

accomplish the objectives, two populations of the Turfgrass Breeding Program of the

University of Florida were evaluated. These populations were previously developed by

conventional breeding. A third population was created by crossing accessions with

differing LP response and leaf texture to produce families that segregated for disease

response. Several families were obtained; however only 6 were selected for further

analysis. The remaining families were planted in field plots. In addition, a set of 12

commercial cultivars was evaluated to quantify levels of resistance or susceptibility.

146

The three populations and the set of 12 cultivars were evaluated under artificial

conditions for disease development in a walk-in plant growth room. One population was

also evaluated in a field trial for LP response (natural infection) and for turfgrass

qualities. The screening protocol was adequate for fungal infection and symptoms

development. This screening was conducted with an AG 2-2 LP isolate (UF 0714). The

isolate showed morphological features associated with the AG 2-2 LP group on Potato

Dextrose Agar (PDA). In addition, the isolate was partially characterized at molecular

level with AG 2-2 LP primers (positive amplification) and with sequence data of the

Internal Transcribed Spacer Regions 1 and 2 and with the 5.8 S RNA gene. This

information is useful because the disease responses are specific to this isolate.

Previous reports in the literature lack of specific information of the isolate used for

artificial screenings or the biotype of the isolates collected in the field.

Results from the inoculations indicated that no genotype or cultivar was highly

resistance to the disease. This observation was consistent in all populations evaluated.

Genotypes and cultivars differed by susceptibility levels and probability for infection rate

(r). However, the (r) parameter was not quantified and would be a good research topic.

For some cultivars, the results reported in this study constitute the first documentation

related to LP responses. In general, ‘JaMur’ and ‘Empire’ were good performers and

‘Zorro’, ‘Emerald’ and ‘Taccoa Green’ very susceptible.

Zoysiagrass accessions were evaluated at 7, 14 and 21 days after the

inoculation (DAI). At 7 DAI, fungal mycelia were well developed in the majority of the

accessions. At this rating date, many genotypes in all populations had clearly developed

a compatible reaction with the isolate. Phenotypic variation for the disease response

147

was observed in all populations, especially at earlier ratings, although the tendency was

toward susceptibility, especially at later ratings days (14 and 21 DAI). At 14 and 21 DAI,

the majority of the accessions was susceptible and highly susceptible. This resulted in

enough lack of phenotypic variation to identify accessions with resistance mechanisms.

Thus, for future screenings, earlier ratings at 5 and 10 DAI are suggested to capture

more phenotypic variation.

Although no accession was confirmed as highly resistance, several genotypes

were considered good performers with moderate susceptibility. Some of these

genotypes had better responses than ‘Meyer’, Empire and other zoysiagrass cultivars.

This results are promissory for the Turfgrass Breeding Program. Resistance of these

genotypes need to be confirmed with additional inoculations and field evaluations along

with evaluations for turfgrass performance.

Large Patch disease responses had continuous and multimodal variation in all

the screenings, indicating quantitative inheritance. Also, disease responses appeared to

be highly influenced by environmental factors. Genotypes and cultivars showed

interactions or variable responses in inoculations runs conducted using similar

conditions.

Variance genetic components were described in 3 populations. In many cases

the environmental variance component was higher than the genotypic variance,

indicating a strong effect of the environment. The F1 hybrids population had low genetic

variance, probably indicating low to moderate phenotypic variation for the disease

response.

148

Broad H2 and narrow sense h2 heritabilities were low and moderate. Broad sense

was moderate in the germplasm population. Conversely, narrow sense was low in the

F1 hybrids and F1 segregating families. Moderate H2 can indicate that the disease

response in this population was influenced by some genetic factors. Low h2 can indicate

that the disease response was not influenced by additive factors and thus, the progress

through cycles of crossing between desirable parents can be slow.

In the field, several abiotic and biotic factors affected the disease response.

Several ratings were not included in the analysis for confounding effects of abiotic

factors such dormancy or winter kill and biotic factors such as Bipolaris spp. infection.

No correlation was found between data of artificial inoculations and field plots from the

same population. Levels of disease severity were always higher in the walk-in plant

growth room than in field plots. This indicates that field screenings need to be modified

to favor fungal dispersion, infection and symptoms development. For example,

inoculation of the plots and maintenance of conditions that induce disease, are strongly

suggested.

Characterization of the isolate (s) present in the field will also be important to

confirm a potential genotype by isolate interaction. Currently, little is known about the

genetic diversity within the AG 2-2 LP.

Leaf texture in the germplasm and F1 segregating families was not related with

tolerance or susceptibility. Accessions with fine and coarse leaf texture were both

tolerant and susceptible to the isolate used. Conversely in the cultivars screening, Z.

japonica cultivars were less susceptible than Z. matrella cultivars, but the number of

149

cultivars evaluated in this study was low and the objectives were not directly addressed

to measure the relationship between these variables.

Another objective of this research was to evaluate Simple Sequence Repeat

(SSRs) primers in accessions with differing LP response. The goal was to identify

polymorphic primers and evaluated them in a screening panel for informative

segregation. In this study 459 SSRs primers were evaluated. From this group, 260

primers showed clear polymorphisms and 137 were selected based on centi Morgan

(cM) distance and presence in different linkage groups. These primers were screened in

a panel of 21 accessions of an F1 segregating family (5333-53 x 375), developed from

the same accessions were the primers were initially evaluated for polymorphism.

The screening panel consisted of 21 accessions with tolerance (11 genotypes)

and susceptibility (10 genotypes) responses. Level of tolerance or susceptibility were

determined by the disease severity observed in four different inoculation runs,

conducted under artificial conditions for disease development. Results from the SSRs

survey in this panel, indicated that some primers produced fragments with Mendelian

1:1 and 3:1 segregation ratios. Twenty fragments were associated with the tolerance

phenotype and 26 with susceptibility. Three fragments that segregated from the female

parent (tolerant accession) had high significance levels that indicate evidence of

genotypic-phenotypic association. Fragments associated with the female parent require

further evaluation in the whole population to confirm the association. Data of this study

identified fragments from 8 linkage groups with possible association. This number of

linkage groups still be wide to find a close association. Thus, more SSRs primers from

these linkage groups should be evaluated to identify loci with high significant

150

association. The identification of SSRs primers associated with the tolerant phenotype

could improve the screening process in terms of time and costs.

Future research also should study timing of infection and evaluate if

morphological traits such as leaf thickness are components of resistance. The use of

parametric (apparent rate infection) and non-parametric variables (AUDPC) could help

to elucidate resistance mechanisms in zoysiagrass and identify accessions with

improved LP resistance in a more clear way.

151

APPENDIX A GENOTYPE × DAY INTERACTION BASED ON INOCULATION RUNS

Figure A-1. Genotype × DAI interaction of JaMur.

Figure A-2. Genotype × DAI interaction of Empire.

0

10

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Run 1 Run 2 Run 3

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Inoculation runs

JaMur

7 DAI

14 DAI

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Run 1 Run 2 Run 3

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Inoculation runs

Empire

7 DAI

14 DAI

152

Figure A-3. Genotype × DAI interaction of El Toro.

Figure A-4. Genotype × DAI interaction of Zeon.

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Run 1 Run 2 Run 3

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El Toro

7 DAI

14 DAI

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Run 1 Run 2 Run 3

Dis

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Inoculation runs

Zeon

7 DAI

14 DAI

153

Figure A-5. Genotype × DAI interaction of Palisades.

Figure A-6. Genotype × DAI interaction of Diamond.

0

10

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Figure A-7. Genotype × DAI interaction of UltimateFlora.

Figure A-8. Genotype × DAI interaction of Shadow Turf.

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Figure A-9. Genotype × DAI interaction of Meyer.

Figure A-10. Genotype × DAI interaction of Emerald.

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Figure A-11. Genotype × DAI interaction of Taccoa Green.

Figure A-12. Genotype × DAI interaction of Zorro.

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APPENDIX B GENOTYPE × DAY INTERACTION BASED ON RATING DATES

Figure B-1. Genotype × DAI interaction of JaMur based on rating dates.

Figure B-2. Genotype × DAI interaction of El Toro based on rating dates.

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Figure B-3. Genotype × DAI interaction of Empire based on rating dates.

Figure B-4. Genotype × DAI interaction of Zeon based on rating dates.

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Figure B-5. Genotype × DAI interaction of Palisades based on rating dates.

Figure B-6. Genotype × DAI interaction of Diamond based on rating dates.

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Figure B-7. Genotype × DAI interaction of UltimateFlora based on rating dates.

Figure B-8. Genotype × DAI interaction of Shadow Turf based on rating dates.

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Figure B-9. Genotype × DAI interaction of Meyer based on rating dates.

Figure B-10. Genotype × DAI interaction of Emerald based on rating dates.

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Figure B-11. Genotype × DAI interaction of Taccoa Green based on rating dates.

Figure B-12. Genotype × DAI interaction of Zorro based on rating dates.

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163

APPENDIX C GENOTYPE × DAY INTERACTION ON EACH INOCULATION RUN

Figure C-1. Genotype × DAI interaction of zoysiagrass cultivars in inoculation run 1.

Figure C-2. Genotype × DAI interaction of zoysiagrass cultivars in inoculation run 2.

164

Figure C-3. Genotype × DAI interaction of zoysiagrass cultivars in inoculation run 3.

165

APPENDIX D ENVIRONMENTAL CONDITIONS OF THE GREENHOUSE

Table D-1. Relative humidity, air temperature and light intensity of the greenhouse prior to transfer of plants to the growth room for inoculation.

Population Relative humidity %

Min. air temperature °C

Max. air temperature °C

Light intensity µmol m-2 s-1

Germplasma run 1 66.8 22.2 31.3 NAd Germplasm run 2 65.3 22.4 31.4 NA F1 hybridsb run 1 61.4 24.5 34.3 NA F1 hybrids run 2 67.4 18.5 31.2 132.06 F1 familiesc replication 1 65.1 22.3 34.7 134.94 F1 families replication 2 66.0 21.0 34.0 134.60 F1 families replication 3 68.0 19.0 32.7 121.95 F1 families replication 4 67.8 18.6 32.3 120.23

a Germplasm included 50 accessions and 10 Zoysia spp. cultivars. b F1 hybrids included 78 accessions and 2 Zoysia spp. cultivars. c F1 families included 275 Zoysia spp. accessions from 6 families. d Data not available.

166

APPENDIX E BREEDING VALUES OF THE ZOYSIA SPP. GERMPLASM

Table E-1. Breeding values of the zoysiagrass germplasm for large patch disease response at 7 and 14 days after the inoculation (DAI) within the walk-in plant growth room.

Genotype or cultivar Breeding value at 7

DAI Rank Breeding value at 14

DAI Rank

5458-26 34.63 1 62.59 1 5458-12 35.36 2 64.41 2 5459-10 35.56 3 69.02 3 5458-10 37.02 4 69.98 4 5458-28 38.98 5 72.32 5 332 45.00 28 72.89 6 5332-52 39.36 6 76.32 7 5458-35 42.93 17 78.33 8 5333-53 41.31 10 79.27 9 152 49.73 36 80.62 10 5288 42.95 18 80.68 11 El Toro 42.31 14 80.79 12 5315 42.13 12 81.80 13 252 42.81 15 81.83 14 306 44.11 23 81.99 15 422 41.88 11 82.11 16 5458-18 47.91 33 82.87 17 5330-38 44.16 25 83.65 18 4360 49.62 35 84.00 19 2430 43.43 20 84.48 20 5458-39 42.89 16 84.87 21 JaMur 47.52 31 85.27 22 3363 45.86 29 86.24 23 5309-35 51.38 40 86.30 24 Meyer 40.46 8 86.44 25 357 51.06 39 86.50 26 188 48.70 34 86.60 27

5335-3 58.65 57 86.96 28 5337-46 44.55 26 86.98 29 Zeon 43.13 19 87.30 30 5332-53 54.85 48 87.31 31 358 52.47 42 87.64 32 5504-6 55.62 50 87.77 33 402 43.55 21 88.09 34 5330-23 44.94 27 89.05 35 328 42.19 13 89.12 36 123 50.97 38 89.74 37 5256-20 43.99 22 90.57 38

167

Table E-1. Continued

Genotype or cultivar Breeding value at 7

DAI Rank Breeding value at 14

DAI Rank

4429 47.89 32 91.24 39

5257-8 52.57 43 91.45 40

5306-45 50.43 37 91.56 41

Taccoa Green 56.86 53 92.74 42

182 44.16 24 92.80 43

8516 40.28 7 93.20 44

5343-52 53.34 46 93.21 45

5269-24 46.90 30 94.62 46

375 56.54 52 95.50 47

3588 41.11 9 95.69 48

309 57.72 55 95.74 49

5305-48 53.88 47 97.63 50

5309-12 51.85 41 98.08 51

Empire 58.24 56 98.60 52

Ultimate 55.71 51 98.98 53

Palisades 63.24 59 100.08 54

374 61.92 58 100.71 55

5307-16 53.04 45 101.01 56

5331-34 57.20 54 101.26 57

Emerald 64.43 60 101.30 58

Zorro 55.24 49 101.59 59

5309-23 52.78 44 102.02 60 a Genotypes are sorted based on the ranking obtained at 14 DAI. Ranking is sorted from the genotype with the lowest breeding value to the genotype with the highest breeding value. Lower breeding values indicate better disease response. Higher breeding values indicate susceptibility response.

168

APPENDIX F BREEDING VALUES OF THE F1 HYBRID POPULATION

Table F-1. Breeding values of the zoysiagrass F1 hybrid population for large patch disease response at 7 and 14 days after the inoculation (DAI) within the walk-in plant growth room.

Genotype or cultivar Breeding value-7

DAI Ranka Breeding value-14

DAI Ranka

UFZ154 47.19 6 63.80 1 Meyer 45.84 1 64.14 2 Aloysia 47.15 5 64.24 3 UFZ23 49.24 38 64.30 4 8426 47.39 11 64.30 5 8022 47.04 3 64.41 6 4360 48.31 18 64.53 7 UFZ08 47.25 7 64.59 8 UFZ15 47.26 8 64.63 9 357 47.92 15 64.94 10 Empire 47.32 10 64.97 11 8754 47.30 9 65.09 12 5309-12 47.63 12 65.18 13 UFZ36 48.44 21 65.24 14 8516 49.49 47 65.26 15 UFZ133 49.06 32 65.28 16 UFZ123 48.54 24 65.33 17 UFZ121 49.45 45 65.38 18 UFZ129 48.53 23 65.76 19 UFZ33 47.90 14 65.88 20 422 49.60 48 65.90 21 UFZ20 49.77 52 66.28 22 8083 47.07 4 66.43 23 5330-23 48.74 27 66.44 24 8631 49.28 40 66.55 25 8599 51.02 77 66.80 26 5335-3 49.43 43 66.82 27 UFZ142 50.04 59 66.82 28 UFZ151 49.26 39 66.82 29 152 48.64 25 66.85 30 8854 50.82 75 67.08 31 UFZ62 48.44 22 67.09 32 UFZ98 49.92 56 67.21 33 UFZ14 49.71 50 67.57 34 UFZ106 46.90 2 67.68 35 3257-8 49.44 44 67.71 36 Meyer 49.33 41 67.74 37

169

Table F-1. Continued

Genotype or cultivar Breeding value-7

DAI Ranka Breeding value-14

DAI Ranka

8820 49.15 36 67.79 38 BA402 50.38 67 67.84 39 BA182 49.12 35 67.85 40 5307-16 49.24 37 67.92 41 UFZ10 48.72 26 67.96 42 UFZ92 49.88 55 68.11 43 BA513 50.66 71 68.15 44 8456 50.45 69 68.23 45 8157 48.40 19 68.29 46 GEO 48.77 28 68.43 47 8184 50.36 65 68.44 48 JaMur 49.73 51 68.46 49 UFZ128 50.40 68 68.47 50 8544 51.51 91 68.57 51 UFZ134 51.21 84 68.66 52 UFZ155 49.94 57 68.69 53 8617 48.42 20 68.80 54 8050 51.13 79 68.88 55 8595 47.93 16 68.92 56 8054 52.19 99 68.94 57 UFZ16 50.77 74 68.97 58 8778 51.52 92 68.98 59 UFZ156 50.33 63 69.03 60 8189 51.70 95 69.13 61 5333-53 47.74 13 69.13 62 3363 50.36 66 69.17 63 8272 51.14 80 69.20 64 5256-20 50.27 61 69.27 65 UFZ160 51.50 89 69.28 66 UFZ96 50.28 62 69.41 67 8797 50.56 70 69.45 68 UFZ121B 52.92 106 69.67 69 UFZ37 48.98 31 69.70 70 8196 50.33 64 69.72 71 UFZ159 49.47 46 69.82 72 5309-35 50.91 76 70.00 73 8487 51.25 85 70.12 74 123 52.37 101 70.14 75 UFZ153 51.21 83 70.22 76 UFZ11 50.15 60 70.23 77 UFZ118 48.96 30 70.34 78 2430 51.62 94 70.43 79 309 51.16 81 70.57 80

170

Table F-1. Continued

Genotype or cultivar Breeding value-7

DAI Ranka Breeding value-14

DAI Ranka

358 50.70 72 70.73 81 188 48.92 29 71.09 82 5332-52 49.36 42 71.15 83 UFZ119 48.26 17 71.24 84 UFZ132 49.87 54 71.26 85 252 53.22 108 71.45 86 5257-8 51.36 86 71.45 87 5330-38 51.78 97 71.53 88 8173 49.11 34 71.66 89 UFZ94 52.19 100 71.98 90 5306-45 51.17 82 71.99 91 UFZ84 49.95 58 72.00 92 8548 51.87 98 72.04 93 5269-24 51.03 78 72.14 94 8153 51.43 87 72.33 95 8845 53.52 109 72.44 96 UFZ125 51.44 88 72.48 97 5305-48 52.82 105 72.93 98 8597 49.62 49 73.09 99 8203 49.77 53 73.27 100 UFZ126 49.08 33 73.52 101 UFZ144 51.60 93 73.54 102 UFZ93 50.76 73 73.56 103 8798 53.02 107 73.73 104 UFZ124 52.56 103 73.97 105 UFZ145 51.51 90 74.09 106 UFZ43 51.75 96 74.09 107 8491 56.38 111 74.33 108 UFZ148 52.38 102 74.90 109 UFZ158 52.68 104 74.95 110 UFZ42 54.63 110 75.57 111

a Genotypes are sorted based on the ranking obtained at 14 DAI. Ranking is sorted from the genotype with the lowest breeding value to the genotype with the highest breeding value. Lower breeding values indicate better disease response. Higher breeding values indicate susceptibility response.

171

APPENDIX G BREEDING VALUES OF THE F1 HYBRID POPULATION IN THE FIELD PLOTS

Table G-1. Breeding values of the zoysiagrass F1 hybrid population for large patch disease response at the field plots.

Genotype or cultivar Breeding

value Ranka Population

8854 7.32 1 Progeny 8272 7.63 2 Progeny 5257-8 7.63 3 Parent 8845 8.05 4 Progeny Empire 8.08 5 Progeny UFZ155 8.08 6 Progeny UFZ106 8.20 7 Progeny 4360 8.46 8 Parent 8544 8.52 9 Progeny 5330-38 8.68 10 Parent 8173 8.79 11 Progeny JaMur 8.82 12 Progeny UFZ98 8.98 13 Progeny UFZ151 9.04 14 Progeny 358 9.07 15 Parent UFZ42 9.20 16 Progeny UFZ96 9.28 17 Progeny 8196 9.40 18 Progeny 8798 9.47 19 Progeny UFZ84 9.50 20 Progeny UFZ148 9.60 21 Progeny UFZ144 9.67 22 Progeny UFZ43 9.70 23 Progeny 5330-23 9.72 24 Parent 422 9.76 25 Parent 5335-3 9.76 26 Parent 252 9.84 27 Parent 8487 10.07 28 Progeny 8189 10.07 29 Progeny UFZ36 10.12 30 Progeny 5305-48 10.19 31 Parent 5333-53 10.25 32 Parent UFZ11 10.25 33 Progeny 8184 10.27 34 Progeny UFZ62 10.36 35 Progeny UFZ160 10.34 36 Progeny UFZ94 10.48 37 Progeny 8083 10.55 38 Progeny UFZ132 10.65 39 Progeny

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Table G-1. Continued

Genotype or cultivar Breeding

value Rank Population

3363 10.68 40 Parent UFZ154 10.69 41 Progeny 8491 10.88 42 Progeny 8595 10.89 43 Progeny UFZ23 10.93 44 Progeny 5256-20 11.01 45 Parent 8797 11.09 46 Progeny 5306-45 11.10 47 Parent 309 11.15 48 Parent UFZ37 11.17 49 Progeny 5269-24 11.26 50 Parent 152 11.27 51 Parent 8599 11.36 52 Progeny 8631 11.39 53 Progeny UFZ133 11.49 54 Progeny UFZ93 11.50 55 Progeny UFZ159 11.53 56 Progeny 5332-52 11.54 57 Parent Aloysia 11.59 58 Parent GEO 11.59 59 Parent Meyer 11.59 60 Parent UFZ129 11.63 61 Progeny UFZ128 11.63 62 Progeny UFZ134 11.73 63 Progeny 8022 11.84 64 Progeny BA402 11.86 65 Progeny 8516 11.96 66 Parent 8203 12.16 67 Progeny UFZ156 12.16 68 Progeny UFZ14 12.18 69 Progeny UFZ121B 12.36 70 Progeny UFZ119 12.40 71 Progeny 8820 12.44 72 Progeny 8054 12.52 73 Progeny UFZ145 12.58 74 Progeny UFZ126 12.70 75 Progeny 5309-35 12.87 76 Parent UFZ15 12.90 77 Progeny 8778 12.90 78 Progeny 123 12.92 79 Parent 8597 12.94 80 Progeny

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Table G-1. Continued

Genotype or cultivar Breeding

value Rank Population

5309-12 13.04 81 Parent UFZ16 13.06 82 Progeny 8050 13.19 83 Progeny 2430 13.28 84 Parent UFZ20 13.43 85 Progeny UFZ125 13.64 86 Progeny UFZ142 13.71 87 Progeny UFZ153 13.78 88 Progeny 8426 13.79 89 Progeny 188 13.87 90 Parent 3257-8 13.92 91 Parent Meyer 13.96 92 Parent 8617 14.11 93 Progeny UFZ33 14.11 94 Progeny 5307-16 14.20 95 Parent UFZ121 14.36 96 Progeny BA513 14.39 97 Progeny UFZ158 14.82 98 Progeny 8157 15.02 99 Progeny 357 15.48 100 Parent 8754 15.60 101 Progeny UFZ92 15.73 102 Progeny BA182 15.77 103 Progeny 8456 15.80 104 Progeny UFZ08 16.61 105 Progeny UFZ10 16.66 106 Parent UFZ123 16.94 107 Progeny 8153 18.16 108 Progeny 8548 18.45 109 Progeny UFZ118 19.51 110 Progeny UFZ124 20.24 111 Progeny

a Genotypes are sorted based on the ranking obtained using disease severity data of four measurements. Ranking is sorted from the genotype with the lowest breeding value to the genotype with the highest breeding value. Lower breeding values indicate better disease response. Higher breeding values indicate susceptibility response.

174

APPENDIX H SUMMARY OF LARGE PATCH DISEASE SEVERITY RATINGS IN THE FIELD PLOTS

Table H-1. Large patch disease severity ratings taken in the F1 hybrids at the Plant Science Research & Education Unit.

No. Year Month Severity ranges (%) Average Observations

1 2012 February 1 - 50 10.53 2 2012 March 0 - 19 5.00 3 2012 March 0 - 20 4.33 4 2012 April 0 - 20 6.00 5 2012 May 0 - 10 2.54 6 2012 May 0 - 15 1.62 7 2012 June 0 - 20 3.47 8 2012 June 0 - 25 4.41 9 2012 July 0 - 15 2.64

10 2012 August 0 - 75 10.55 11 2012 September 0 - 40 1.63 Bipolaris 12 2012 September 0 - 50 2.08 Bipolaris 13 2012 October 0 - 35 1.33 Bipolaris 14 2012 November 0 - 40 0.90 Bipolaris 15 2012 November 0 - 25 0.46 Bipolaris 16 2012 December 0 - 20 1.31 Bipolaris 17 2013 January 0 - 95 28.70 Bipolaris 18 2013 February 30 - 100 89.00 Bipolaris 19 2013 March 10 - 100 85.66 Bipolaris 20 2013 April 0 0 Bipolaris 21 2013 May 0 0 Bipolaris 22 2013 June 0 0

23 2013 July 0 - 90 12.30 Bipolaris. 117 plots with * dataa

24 2013 August 0 - 15 7.72 25 2013 September 0 - 20 5.14 26 2013 October 0 - 10 1.59 27 2013 December 0 - 25 2.66 28 2014 January 0 - 15 1.21 29 2014 April 0 - 10 0.48

30 2014 July 0 - 85 14.36 some bipolaris/out of

season 31 2014 September 0 - 5 0.06 32 2014 November 0 - 50 18.53 dormancy/winter kill 33 2014 December 0 - 50 13.00 winter kill

34 2015 January 0 - 90 11.77 WK. LP clear in some

plots 35 2015 April 0 - 40 10.98 36 2015 July 0 - 20 2.17

a Missing data = *

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APPENDIX I ENVIRONMENTAL CONDITIONS IN THE FIELD PLOTS AT THE PLANT SCIENCE

RESEARCH & EDUCATION UNIT IN CITRA, FLORIDA.

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Table I-1. Air temperature, soil temperature and relative humidity in the field plots at the Plant Science Research & Education Unit in Citra, Florida.

Month Year Avg.a Air

temperature °C Min. air temperature

°Ca Max. air temperature

°Ca Avg. b Soil

temperature Avg. relative

humidity

January 2012 13.4 -5.6 28.6 16.2 74 February 2012 16.9 -5.3 28.8 18.9 77 March 2012 20.3 2.3 30.8 23.2 75 April 2012 21.4 6.7 33.3 26.1 69 May 2012 24.4 13.6 35.0 28.9 75 June 2012 25.6 16.8 34.4 29.5 82 July 2012 26.7 19.0 35.0 30.5 85 August 2012 26.1 18.4 34.2 28.4 88 September 2012 25.4 14.3 33.9 27.2 85 October 2012 21.4 4.8 33.4 24.5 82 November 2012 14.8 -1.4 28.9 19.4 82 December 2012 15.0 -2.6 26.8 17.6 83 January 2013 16.3 -0.5 28.5 18.1 82 February 2013 15.1 -5.3 29.8 17.6 76 March 2013 13.6 -3.0 27.8 17.7 66 April 2013 20.8 9.1 31.0 22.8 78 May 2013 22.3 8.0 32.9 25.3 77 June 2013 26.2 19.3 34.7 28.6 85 July 2013 25.8 21.1 34.4 28.6 88 August 2013 26.7 20.0 35.6 30.5 87 September 2013 25.5 18.9 34.8 29.3 85 October 2013 21.7 7.4 32.3 25.8 84 December 2013 16.4 1.9 28.5 18.5 87 January 2014 10.9 -2.3 26.3 13.8 80 April 2014 20.6 0.0 33.4 23.8 77 July 2014 26.4 0.0 34.8 29.9 83 September 2014 25.1 0.0 34.3 28.7 87 November 2014 14.5 -2.4 29.2 18.5 78 December 2014 15.0 -1.1 27.1 17.8 84 January 2015 12.8 -1.1 28.2 16.2 81 April 2015 22.6 14.1 31.1 26.2 81 July 2015 26.5 19.3 35.4 29.8 86

Data was obtained using the Florida Automated Weather Network. a Air temperature data up to 2 m from the soil surface.

b Soil temperature data at -10 cm below the soil surface.

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APPENDIX J LINKAGE MAP CONSTRUCTION WITH LOD ≥ 2.0

Linkage mapping for the parental genotypes was conducted only with fragments that fit

the 1:1 segregation ratio (P > 0.05). The fragments associated to the female and male parent

were coded as <lmxll> and <nnxnp>; respectively, where lm and nn indicate presence of the

fragment, and ll and np indicate absence. Accessions that did not amplify for a particular

fragment (missing data) were treated as --.

The maps were constructed with the following parameters: log-likelihood of the odds

(LOD) > 2.0, maximum recombination (REC) frequency = 0.40, jump threshold for removal of

loci = 5.0, ripple = 1, and with a third round. The cross pollinated (CP) function was used

because parents were heterozygous. The CP function calculates the linkage phase of the

markers. Map distances were calculated using the Kosambi’s mapping function (Kosambi

D.D, 1944). The maximum linkages function was used to assign independent groups.

Identical loci were removed. Linkage maps graphs were obtained with MapChart 2.0

(Voorrips, 2002).

Female and male maps were constructed using 145 and 132 loci with 1:1 segregation

ratios, respectively. The linkage group information of the markers reported by Li et al. (2010)

was used to assign linkage groups. Eighteen (in female) and 26 (in male) fragments were

mapped in seven and ten linkage groups, respectively. The minimum and maximum distances

between two adjacent markers were 4.8 cM, 25.4 cM (male map) and 25.9 cM (female map).

Several fragments were excluded because their linkage grouping did not correspond with the

linkage groups reported by Li et al. (2010), who mapped the SSR loci in Z. matrella and Z.

japonica genotypes. The number of fragments mapped was very low; the majority of

fragments (84.1%) were unlinked.

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ZB 02O05 LG 21 0.0

M3F03 LG 21 4.8

ZB 03J03 LG 15 0.0

ZB 10K23 LG 15 17.3

ZD 01A22 LG 15 0.0

ZD 01A22 LG 15 25.9

5 6 7

Figure J-1. Linkage map of the markers specific to the Zoysia japonica (5333-53) female parent. Distance (cM) between markers is shown on the left side.

ZA 01P11 LG 11 0.0

ZA 01P11 LG 11 5.5

ZB 03N18 LG 13 12.1

ZB 06K09 LG 11 18.1

ZB 03M04 LG 11 22.5

ZC 01021 LG 15 0.0

ZB 08M16 4.8

ZB 02A24 LG 19 0.0

ZB 02A24 LG 19 9.8

ZB 01L11 LG 19 19.6

ZB 07A11 LG 1 0.0

ZB 10O01 LG 1 4.8

1 2 3 4

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ZB 04H19A LG 1 0.0

ZB 04H19 A LG 1 4.8

ZB 01L11 LG 19 0.0

ZB 02A24 LG 19 9.7

ZB 02A24 LG 19 14.5

ZC 01E07 LG 17 0.0

ZA 02009 LG 17 4.8

4 5 6

Figure J-2. Linkage map of the markers specific to the Zoysia matrella (375) male parent. Distance (cM) between markers is shown on the left side.

ZA 03F03 LG 21 0.0

ZA 03F03 LG 21 4.9

M3F03 LG 21 (two alleles) 14.7

ZB 02005 LG 21 19.5

ZB 04D24 LG 1 0.0

ZB 04D24 LG 1 4.8

Zj AG130 LG 7 0.0

Zj AG130 LG 7 7.2

ZB 03B03 LG 7 17.3

ZB 03B03 LG 7 25.4

1 2 3

180

Figure J-2. Continued.

ZB 06O03 LG 8 0.0

ZB 06O03 LG 8 14.7

ZB 03N23 LG 4 0.0

ZB 03N23 LG 4 4.8

ZB 03J15 LG 16 0.0

ZB 04N13 LG 16 11.6

ZB 04N13 LG 16 23.2 ZB 03E06 LG 18 0.0

ZB 03E06 LG 18 4.8

10

7 8 9

181

APPENDIX K LINKAGE MAP CONSTRUCTION WITH LOD ≥ 3.0

An additional map was constructed with the same parameters mentioned in appendix J

but including markers linked with LOD ≥ 3.0. With this approach, fewer markers (69%) were

unlinked. In the female and male parent maps, 50 and 34 markers were mapped in 16 and 13

linkage groups, respectively. The minimum and maximum distances between two markers

were 4.8 cM, 29.6 cM (male map) and 39.9 cM (female map), respectively.

182

Figure K-1. Linkage map of the markers specific to the Zoysia japonica (5333-53) female parent. Markers linked with LOD = 3.0 were included in the analysis. Distance (cM) between markers is shown on the left side.

ZA 01P11 LG 11 0.0

ZA 01P11 LG 11 6.0

ZB 03N18 LG 13 10.8

ZB 06K09 LG 11 16.5

ZB 03M04 LG 11 19.0

ZB 02P17 LG 5 29.3 ZB 06K09 LG 11 30.9

ZB 03J09 LG 11 39.9

ZC 01D15 LG 3 0.0

ZB 06003 LG 8 10.3

ZB 02L18 LG 8 16.8 ZB 02C05 LG 6 18.0

ZB 02L18 LG 8 26.6

ZB 06003 LG 8 31.7

ZB 10K23 LG 15 0.0

ZB 08M16 LG 15 6.1

ZC 01021 LG 15 9.9

ZB 07A11 LG 1 18.6

ZB 02A24 LG 19 0.0

ZB 02A24 LG 19 9.8

ZB 01L11 LG 19 19.6

1 2 3

4

ZC 01C12 LG 4 0.0

ZB 06B08 LG 22 6.2

ZA 02009 LG 17 11.2

ZB 07A16 LG 11 0.0

ZB 10001 LG 1 9.7

ZB 07A11 LG 1 14.5

ZB 06D19 LG 5 0.0

ZB 06D19 LG 5 8.0

ZB 09K11 LG 9 12.3

5 6 7

183

ZB 06D18B LG 5 0.0

ZB 03K17 LG 13 9.6

ZB 01H17 LG 6 0.0

ZB 03N18 LG 13 15.5

ZB 01J23 LG 18 0.0

ZB 03J09 LG 11 15.8

M3F03 LG 21 20.7

ZT-a8 LG 18 0.0

ZB 01J23 LG 18 10.0

ZA 03C15 LG 21 14.9

16

Figure K-1. Continued

ZB 04E01 LG 20 0.0

ZB 02P17 LG 5 10.7

ZB 01H23 LG 15 0.0

ZC 03C23 LG 22 14.7

ZB 03J09 LG 11 0.0

ZB 08B08 LG 10 14.7

ZB 02L18 LG 8 0.0

ZB 08I08 LG 12 9.6

8 9 10 11

12 13 14 15

ZB 02L20 LG 3 0.0

ZB 10019 LG 19 4.8

184

Figure K-2. Linkage map of the markers specific to the Zoysia matrella (375) male parent. Markers linked with LOD = 3.0 were included in the analysis. Distance (cM) between markers is shown on the left side.

ZB 01D24 LG 5

0.0

ZA 02O09 LG 17 14.9

ZB 04D24 LG 1 24.8

ZB 04D24 LG 1 29.6

Zj AG130 LG 7 0.0

Zj AG130 LG 7 7.2

ZB 03B03 LG 7 17.3

ZB 03B03 LG 7 25.4

ZA 03F03 LG 21 0.0

ZA 03F03 LG 21 4.9

M3F03 LG 21 (two alleles) 14.7

ZB 02005 LG 21 19.5

1 2 3

185

ZB 07O09B LG 3 0.0

ZA 01A07 LG 14 9.6

ZB 04BN13 LG 16 0.0

ZB 03J15 LG 16 11.3

ZB 01L03 LG 8 0.0

ZB 01L11 LG 19 6.7

11 12 13

Figure K-2. Continued

ZB 03B01 LG 9 0.0

ZB 03O03 LG 1 6.3

ZB 06B15 LG 1 0.0

ZB 06F22 LG 9 4.8

ZB 02K15 LG 19 0.0

ZB 02K15 LG 19 14.7

ZB 03N23 LG 4 0.0

ZB 03N23 LG 4 4.8

7 8 9 10

186

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BIOGRAPHICAL SKETCH

Norma Cristina Flor-Payan is originally from Colombia. She received her bachelor’s

degree in agronomy of the National University (Palmira) on December 1998. Her bachelor’s

thesis was conducted in the International Center of Tropical Agriculture –CIAT- (Palmira)

under the supervision of Dr. Fernando Correa. She worked with rice blast disease (Pyricularia

grisea Sacc.). At CIAT, she worked as a Research Assistant at the Genetic Resources Unit

(GRU) under the supervision of Dr. Daniel Debouck. Her research was focused on viruses of

quarantine importance of cassava (Manihot sculenta Crantz).

In August 2005, she joined the Plant Pathology Department of the University of Florida

to pursue her master’s degree under the supervision of Dr. Lawrence Datnoff and Dr. Philip

Harmon. Her thesis was focused on screening St. Augustinegrass (Stenotaphrum

secundatum Kuntze) germplasm for brown and large patch diseases (Rhizoctonia solani

Kuhn). After that, she worked for 1 year as a technician at the Turfgrass Breeding Program

(University of Florida) under the supervision of Dr. Kevin Kenworthy, the head breeder of the

warm-season grasses program. She also worked part-time at the Plant Disease Clinic under

supervision of Dr. Carrie Harmon.

In January 2011 she enrolled as a doctor of philosophy student in the Agronomy

Department, with Dr. Kevin Kenworthy as her major advisor. Her dissertation was focused on

genetic studies of large patch disease in Zoysia spp. Norma is the proud mom of Isabella and

Natalia, twins’ girls of 8 years old.