Ethan Nielsen Dissertation

Home , Melogold, New Zealand grapefruit, Robert Soost

EXPLOITATION OF PUMMELO (CITRUS MAXIMA (BURM.) MERRILL) THROUGH BREEDING, PLOIDY MANIPULATION, AND INTERSTOCKS FOR IMPROVEMENT OF CULTIVATED CITRUS

ETHAN RIRIE NIELSEN

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

To my loving wife, Harmonie

ACKNOWLEDGMENTS

I would like to thank my supervisory committee chair Dr. Jude Grosser for his insights and guidance throughout this long journey towards the attainment of the highest degree in education. I admire his dedication to developing better Citrus and improving the livelihood of the growers that look to him for solutions, which he freely gives.

I also want to thank my other committee members, Dr. Jose X. Chaparro who taught me many insights in perennial plant breeding, Dr. Fred Gmitter, Jr., Dr. Arnold

Schumann, and Dr. Kevin Folta whose excitement for discovery through the scientific method is infectious.

I want to thank my fellow Lab members: Dr. Manjul Dutt, Gary Barthe, Dr. Ahmad

Omar, Dr. Miliça Ćalović, and Elaine Moreira. Thanks to my fellow graduate students

Aditi Satpute and Flavia Zambon, and the other undergraduates and lab assistants who helped make this possible.

Thanks to Dr. James Graham for his instructions on working with citrus canker and Kayla Gerberich who assisted with the canker inoculum preparation. Thanks to Dr.

Larry Duncan and Dr. Fahiem EL-Borai Kora for the assistance with nematodes. Thanks to Dr. Timothy Ebert for assistance with statistical analyses.

Thanks to my wife and children who let me pursue my dream of becoming a plant breeder. Thanks to my parents for instilling in me the values and work ethic that have helped me to succeed.

TABLE OF CONTENTS

page ACKNOWLEDGMENTS ...... 4 LIST OF TABLES ...... 8 LIST OF FIGURES ...... 10 LIST OF ABBREVIATIONS ...... 12 ABSTRACT ...... 13 CHAPTER 1 INTRODUCTION ...... 15 2 REVIEW OF LITERATURE ...... 19 History of Cultivation in Florida ...... 19 Taxonomy ...... 22 Genetic Variation in Citrus ...... 23 Ancestral and Contemporary Cultivars ...... 24 Pummelo (Citrus maxima (Burm.) Merrill) ...... 24 Grapefruit (Citrus x paradisi Macfadyen)...... 27 Oranges ...... 30 Citron, Lemon, and Lime ...... 32 Mandarin and Mandarin Hybrids ...... 32 Kumquats ...... 33 Trifoliate Orange (Poncirus trifoliata [L.] Raf.) ...... 34 Papedas ...... 35 Near Citrus Relatives ...... 36 Rootstocks ...... 36 Interstocks ...... 39 Protein and RNA Signals ...... 40 Citrus Breeding ...... 40 Mutation Breeding ...... 41 Polyploidy ...... 41 Protoplast Culture and Somatic Hybridization ...... 43 Seedless Citrus ...... 44 Interploid Hybridization ...... 44 Cybrids in Citrus ...... 46 Diseases of Citrus ...... 47 Citrus Canker ...... 48 Citrus Canker Inoculation ...... 49 Huanglongbing ...... 50 CLas Detection ...... 52 Nematode Caused Diseases ...... 53 Objectives ...... 54

3 EVALUATION OF CITRUS CANKER RESISTANCE IN PUMMELO PARENT SELECTIONS ...... 56 Background and Objective ...... 56 Materials and Methods ...... 59 Material Background ...... 59 Pummelo Candidate Selection ...... 60 Bacterial culture and inoculum preparation ...... 61 Leaf Inoculation ...... 62 Candidatus Liberibacter asiaticus Testing ...... 63 Results and Discussion ...... 64 4 INTERPLOID HYBRIDIZATION TO PRODUCE SEEDLESS TRIPLOID HYBRIDS OF PUMMELO AND GRAPEFRUIT ...... 85 Background and Objective ...... 85 Materials and Methods ...... 87 Easy Peel Grapefruit ...... 90 Embryo Rescue for Interploid Crosses ...... 91 Flow Cytometry for Ploidy Determination ...... 91 Seedling Grafting ...... 92 Citrus Canker Inoculations ...... 92 Results and Discussion ...... 93 Citrus Canker Inoculation ...... 95 Preliminary Evaluation of Field trees ...... 98 5 TESTING OF SELECTED PUMMELO AND HYBRID INTERSTOCKS TO MITIGATE HLB SYMPTOMS IN COMMERCIAL SWEET ORANGE/SWINGLE TREES ...... 119 Background and Objectives ...... 119 Materials and Methods ...... 120 Stick Grafting ...... 121 Control Trees ...... 123 Rootstocks ...... 123 HLB Development ...... 123 CLas Detection ...... 124 Growth Data ...... 125 Results and Discussion ...... 125 6 SUMMARY AND CONCLUSIONS ...... 144 Evaluation of Citrus Canker Resistance in Pummelo Parent Selections ...... 146 Interploid Hybridization to Produce Seedless Triploid Hybrids of Pummelo and Grapefruit ...... 148 Testing of Selected Pummelo and Hybrid Interstocks to Mitigate HLB Symptoms in Commercial Sweet Orange/Swingle Trees ...... 151 Further Breeding Efforts ...... 153 Conclusions ...... 155

APPENDIX A CITROPSIS GILLETIANA (SWINGLE) FOR CITRUS GERMPLASM ENHANCEMENT AND CULTIVAR DEVELOPMENT ...... 160 Introduction ...... 160 Citropsis in Citriculture ...... 161 Materials and Methods ...... 163 Somatic Fusion ...... 163 Sexual Hybrid ...... 165 Pollen Viability ...... 165 Molecular Markers ...... 165 Nematode Assay ...... 166 Results and Discussion ...... 166 Summary and Conclusion ...... 167 B MICROCITRUS HYBRIDS: POTENTIAL NEW CROPS FOR CITRUS GROWERS ...... 177 Introduction ...... 177 Finger Limes and HLB ...... 178 New Hybrids ...... 179 Tetraploid Sydney Hybrid ...... 181 Summary and Conclusion ...... 182 C MEDIA COMPOSITION ...... 192 LIST OF REFERENCES ...... 194 BIOGRAPHICAL SKETCH ...... 218

LIST OF TABLES

Table page

3-1 Parental Cultivars of Pummelo (C. maxima) ...... 72 3-2 Pummelo selections for citrus canker study...... 73 3-3 Citrus Canker Lesion Rating Scale ...... 74 3-4 F values and significant levels from ANOVA for number of lesions and lesion size rating for selections...... 74 3-5 Mean comparison of number of lesions between pummelos, grapefruit controls, and kumquat controls...... 75 3-6 Mean comparison of lesion rating between pummelos, grapefruit controls, and kumquat controls...... 75 3-7 P-values and squared distances (Mahalanobis distances) from canonical discrimination procedure for pummelos and controls comparing number of lesions and lesion ratings for selections...... 76 3-8 PCR results for CLas detection in pummelo samples ...... 77 4-1 2014 Crosses to generate seedless, triploid progeny ...... 100 4-2 Spring 2015 Crosses to generate seedless, triploid progeny...... 101 4-3 F values and significant levels from ANOVA for number of lesions and lesion size rating for all seedlings and controls...... 102 4-4 Tukey’s HSD pairwise analysis for number of lesions between triploid seedlings and controls...... 103 4-5 Tukey’s HSD pairwise analysis for rating of lesions between hybrid seedlings and controls ...... 105 4-6 Canonical Multivariate analysis for both rating of lesions and number of lesions between hybrid seedlings and controls...... 107 4-7 Triploid offspring significantly different (α=0.05) from the grapefruit controls but not significantly different from the Meiwa kumquat control...... 110 4-8 Triploid hybrids prone to dropping leaves following inoculation with citrus canker...... 110 4-9 Triploid hybrid family citrus canker tolerance comparison...... 111 4-10 Differences in parent families using both lesion size and rating using Mahalanobis distance (P-values) and squared distances compared to grapefruit, pummelo, and Meiwa kumquat controls...... 112 4-11 Field tree citrus canker response...... 113 5-1 Summary of Selected Interstock Tree Combinations, with Swingle citrumelo rootstock and HLB-infected scion, initial number of trees...... 131

5-2 HLB Disease Severity Rating Scale ...... 131 5-3 ANOVA for measured interstock attributes...... 132 5-4 Mean values and standard deviation (SD) for plant height, weight, rootstock diameter, interstock diameter, scion diameter, interstock length, and Ct value ...... 133 5-5 Correlation matrix for mean values of measured attributes...... 134 5-6 P-values and squared distances (Mahalanobis distances) from canonical discrimination procedure for all measured attributes by interstock ...... 135 B-1 Microcitrus species and hybrids available for hybridizing...... 185 B-2 Parents crossed and resulting seedlings...... 186

LIST OF FIGURES

Figure page

2-1 Florida Citrus Production, 1915-2015 (in 1000s of boxes)...... 55 2-2 Pigmented grapefruit descended from Duncan grapefruit...... 55 3-1 Grapefruit with its genetic progenitors ...... 78 3-2 Pummelo tree 5-4-99-7 in research grove...... 78 3-3 Pummelo fruits and tree habit ...... 79 3-4 Citrus canker inoculation pattern...... 82 3-5 Control leaves four weeks post inoculation...... 82 3-6 Sampling of pummelo leaves showing citrus canker lesions four weeks post inoculation ...... 83 3-7 Multivariate canonical scatterplot comparing sum of lesions and lesion ratings for selections...... 84 4-1 Tetraploid pummelo-orange hybrids...... 114 4-2 Embryo rescue of all seeds from a single fruit of 5-1-99-2 S5 x Hudson 4x. .... 114 4-3 Potentially triploid seedlings germinating in the greenhouse ...... 115 4-4 Flow cytometry read out on Partec PA machine...... 115 4-5 Triploid offspring: Citrus canker lesion size vs. number of lesions...... 116 4-6 5-1-99-2 S5 x Huds4x-14-10 tree in field, exhibiting no citrus canker...... 117 4-7 5-1-99-2 S5 x Huds4x-14-9 trees in field, exhibiting low citrus canker ...... 117 5-1 C2-4-1 (left) and C2-4-8 (right)...... 136 5-2 Nova+Citropsis gilletiana (N+C) growing in research grove, Spring 2017 ...... 136 5-3 Process of stick grafting...... 137 5-4 Grafted interstocks on Swingle rootsocks, prior to grafting of Valencia scions . 137 5-5 Grafted interstocks on Swingle rootsocks, beginning to leaf out ...... 138 5-6 Valencia on interstock trees prepared for field planting ...... 138 5-7 Plot of Canonical1*Canonical2 for measured attributes of interstocks ...... 139 5-8 Valencia scion on Swingle rootstock with interstock trees planted in November 2016 near Avon Park, FL, photo taken in January 2017 ...... 140 5-9 CLAS-infected interstock trees planted in November 2016 near Avon Park, FL, photo taken in October 2017 ...... 141 5-10 CLAS-infected interstock trees planted in November 2016 near Avon Park, FL, photo taken in October 2017 ...... 142

5-11 CLAS-infected interstock trees planted in November 2016 near Avon Park, FL, photo taken in October 2017 ...... 143 A-1 Nova+Citropsis gilletiana growing in research grove, Spring 2017 ...... 170 A-2 Protoplasts suspended in a density gradient ...... 171 A-3 Readout from Partec PA flow cytometry machine ...... 171 A-4 First flower of Nova+Citropsis gilletiana ...... 172 A-5 Pollen germinating on 10% sucrose + 1% agar ...... 172 A-6 Sole fruit obtained from first flowering...... 173 A-7 Developing embryo obtained from Nova+Citropsis gilletiana fruit ...... 173 A-8 Tetraploid Nova+Citropsis hybrid roots compared to diploid W. Murcott roots . 174 A-9 W.Murcott+Citropsis gilletiana leaves and spines ...... 174 A-10 SSRs of Citropsis gilletiana, W.Murcott+Citropsis gilletiana, and W.Murcott .... 175 A-11 EST-SSR markers for what appears to be a cybrid...... 175 B-1 Cross section of two finger limes showing the spherical juice vesicles ...... 187 B-2 Microcitrus at the Florida DPI Citrus Arboretum in Winter haven, FL ...... 187 B-3 5-1-99-2 S5 x Microcitrus australis ...... 188 B-4 Seedling of Microcitrus australasica DPI 50-36 x Poncirus trifoliata DPI 50-7 .. 188 B-5 Open pollinated seedlings of Sydney Hybrid treated with oryzalin ...... 189 B-6 PartecPA flow cytometry histogram, showing 2x peak for key lime and 4x peak for tetraploid Sydney Hybrid seedling...... 189 B-7 Comparison of leaves between parents and offspring ...... 190

LIST OF ABBREVIATIONS

ACP Asian citrus psyllid CLas Candidatus Liberibacter asiaticus CREC University of Florida Citrus Research and Education Center in Lake Alfred, Florida. CTV Citrus Tristeza Virus CFU Colony Forming Units Ct Threshold Cycle dpi Days post inoculation EBN Endosperm Balance Number HLB Huanglongbing, formerly known as Citrus Greening Disease N+C Nova+Citropsis gilletiana. Somatic fusion of Nova mandarin hybrid with Citropsis gilletiana tetrazyg Zygotic offspring from sexual crosses of somatic fusion parents. Xcc Xanthomonas citri sub. citri. Causal agent of Asiatic citrus canker.

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

EXPLOITATION OF PUMMELO (CITRUS MAXIMA (BURM.) MERRILL) THROUGH BREEDING, PLOIDY MANIPULATION, AND INTERSTOCKS FOR IMPROVEMENT OF CULTIVATED CITRUS

Ethan Ririe Nielsen

December 2017

Chair: Jude Grosser Major: Horticultural Sciences

Citrus in Florida is in desperate need of new and improved cultivars in light of recent disease epidemics which have ravaged the citrus industry and threaten all citrus producing regions of the world. The primary diseases at present are Huanglongbing

(HLB) and citrus canker. Most cultivated varieties of citrus are hybrids derived from a few ancestral species. Disease tolerance found in the ancestors may be used to develop synthetic new hybrids that approximate the genetic make-up of modern cultivated forms. This study focuses on selecting and utilizing pummelo (Citrus maxima

Burm. Merrill) cutivars and accessions that are exhibiting tolerance to citrus canker

(Xanthomonas citri var. citri), with additional consideration of tolerance to

Huanglongbing (presumed causal agent Candidatus Liberibactor asiaticus).

These pummelo selections will be used to breed new synthetic grapefruit hybrids to replace current susceptible varieties while maintaining the appearance and flavor of consumer-accepted forms. New hybrids will take advantage of parthenocarpy and triploidy to produce seedless fruits.

One of the symptoms of HLB is the loss of feeder roots contributing to the decreased nutrient uptake and decline of the tree’s health. Discovery of CLas infected trees exhibiting healthy and expansive root systems on standard Swingle rootstocks after being infected for several years led to the question of whether the scion or an interstock could remediate the effects of HLB. To explore this idea, HLB infected

Valencia sweet oranges were grafted onto putative HLB tolerant or resistant pummelo

and hybrid interstocks, which were grafted onto standard rootstocks in order to

determine if their putative resistance to HLB could be conferred to the scion and

rootstock. Hybrids using Citropsis gilletiana and Microcitrus species are also discussed

as sources of disease resistance or tolerance.

CHAPTER 1 INTRODUCTION

Citrus fruits are consumed globally as fresh fruit or as juice, with Florida historically having been the largest producer of citrus in the world from 1945 until 1985

when Brazil’s orange production surpassed the US (Neves et al., 2011; Reuther et al.,

1967; Schuler and Scarborough, 1965). Current global production of citrus is estimated

to be 91.3 million metric tons (49.6 million metric tons of oranges, 28.4 million metric

tons of tangerines and mandarins, 7.3 million metric tons of lemons and limes, and 6.0

million metric tons of grapefruit) (USDA Foreign Agricultural Service, 2017). Citrus fruits

and juices are excellent sources of Vitamin C and contribute other key nutrients such as

potassium, folate, magnesium, and vitamin A (Rampersaud and Valim, 2015).

Domestication of citrus began several thousands of years ago in Southeast Asia,

spreading globally by traders following ancient land and sea routes (Reuther et al.,

1967; Tolkowsky, 1938). The specific lineages that eventually led to modern citrus

cultivars were undocumented and lost, and the ancestry has been controversial (Barrett

and Rhodes, 1976; Nicolosi, 2007; Scora, 1975; Scora et al., 1982; Wu et al., 2014).

Some of the traits of citrus biology and cultivation makes it difficult to decipher this ancestry. Trees are propagated asexually through either grafting or apomictic seed (via nucellar polyembryony) to continue lines with desirable traits (Castle et al., 1993;

Williamson and Jackson, 1994). Many types or classifications of citrus arose through

interspecific hybridization events and introgressize hybridization with wild ancestral

species (Wu et al., 2014). These events occurred anciently before being globally

dispersed, with no written record of the domestication process. Diversity within these

clonally propagated groups has occurred through somatic mutations, usually without

sexual recombination, either arising from bud sports on mature trees or variation in apomictic nucellar propagated seed (Wu et al., 2014).

Nucellar embryos are advantageous for fixing traits in a seedling population,

especially when it is desirable to create a homogenous population from seed, which is

the industry standard for rootstock production (Castle et al., 1993; Grosser et al., 1995).

This is problematic when diseases arise due to the narrow genetic base, especially in

light of the recent Huanglongbing (HLB) epidemic, devastating citrus groves in Florida

and other parts of the world (Bové, 2014; Halbert, 2005). No commercial cultivar is

immune to its devastating effects, which is exacerbated by the lack of genetic diversity

in groves (Burrow et al., 2015; Ramadugu et al., 2016). If one tree of a particular cultivar

is susceptible, its clonally derived siblings are as well (Grosser and Gmitter, 2012).

Monocultures of Citrus allow for uniformity in ripening times, fruit size, fruit flavors

and colors, consistent levels of soluble solids and acidity, and other desirable traits

pertaining to habit or growth. The disadvantages of growing crops in a monoculture

results from both a lack of biodiversity and narrow genetics, as varied plants in

polyculture will host various insect and animal populations, whose absence in a

monoculture may allow certain pest species to become more numerous and become

economically damaging (Altieri, 1999; Zeller et al., 2012).

Due to the long generation times of citrus, it would be risky to attempt to found a

grove from zygotic seed despite its merits. Growers want to reduce risk as much as

possible, as the life of a grower already involves a high level of risk due to events

beyond their control: adequate rain in the right season, extreme temperatures, disease

outbreaks, and whether fruit prices will stay high enough to make a crop worth harvesting.

Citrus with a varied genetic background has shown to be more adaptive to environmental pressures (Grosser, 2012; Grosser et al., 2016). There is abundant genetic variation in ancestral species and near relatives (Herrero et al., 1996; Uzun and

Yesiloglu, 2012). Using this genetic diversity in a breeding program could potentially provide the genes or alleles to overcome many of the current issues, and perhaps prepare the industry for the next disease or problem lying in wait beyond the horizon.

We now know genetically what the ancestors are of modern citrus cultivars

(Barrett and Rhodes, 1976; Curk et al., 2015; Nicolosi, 2007; Scora, 1975; Wu et al.,

2014). The cultivars selected in ancient times often occurred by chance and were selected based on flavor, not necessarily disease resistance. By selecting parents with increased disease resistance or tolerance, we can recreate cultivars with built in disease tolerances that have fruit approximating accepted cultivars. This has been attempted with some success in citrus (Beach, 2012; Gmitter, 2015; Grosser et al.,

2004; Mohamed, 2009).

Pummelos (C. maxima (Burm.) Merrill) have contributed their genetics to many modern cultivars, from more recent hybrids such as the grapefruit and tangelo, to admixtures of more ancient origin such as sweet oranges sour oranges (Curk et al.,

2015; Wu et al., 2014). The University of Florida Citrus breeding program contains a diverse collection of pummelos, with attributes ranging various fruit shapes, sizes, and flavors, to trees that appear to have shown varied disease response to some of the more economically devastating diseases currently affecting the citrus industry

(Ananthakrishnan et al., 2006; Grosser et al., 2004). Few citrus breeding programs have taken advantage of directly breeding elite pummelo selections to either recreate citrus

types or develop new disease resistant cultivars.

CHAPTER 2 REVIEW OF LITERATURE

History of Cultivation in Florida

The center of origin of cultivated citrus is in Southeast Asia, somewhere between

India and China (Scora, 1975). The oldest recorded mention of citrus fruits come from ancient Chinese literature, with the earliest known mention in the Shu-king, a collection of old documents thought to have been collected and edited by Confucius himself around 500 B.C., which mention kü (oranges) and yu (pummelos) (Tolkowsky, 1938).

From Southeast Asia, it is believed, that citrus began to spread by land and sea trade routes to the surrounding regions (Swingle and Reece, 1967). Trade routes from India then spread citrus further west, first the citron reaching the middle east, followed by the sour orange (Tolkowsky, 1938). When the Spanish came west to the New World, they brought citrus with them, and first planted sour oranges in Cuba and throughout the colonies as they settled in the new world (Díaz del Castillo, 1908). They discovered that the citrus they brought with them from Spain grew well in the new world, introducing plants and seeds of sour orange, sweet orange, lemons, limes, and citron (Laszlo,

2007). The exact date of the introduction of citrus into Florida is unknown, with oranges

arriving in St. Augustine sometime after it was settled in 1565 (Connor, 1930, p. 227).

Many wild groves became established in the New World, being further spread by

Indians and pioneers who settled the frontier (Morton, 2013). There was limited commercial cultivation of these wild plantings until better transportation allowed for quicker paths to northern markets (Reuther et al., 1967, p. 21). The first budded trees were introduced around 1830, and were used to develop the first commercial groves

(Castle et al., 1993). Growers began topworking improved scions onto wild groves of

sour oranges to increase production because seedling sweet oranges often died from foot rot (Castle et al., 1993). Sour orange became the favored rootstock in Florida due to its resistance to foot rot, as well as its innate cold tolerance and ability to yield fruit of excellent quality (Bitters, 1986).

Budding allowed propagators to easily increase numbers and multiply superior

cultivars in sufficient quantities to growers because the trees came into production

sooner (Castle et al., 1993). The success of sour orange as a rootstock so popularized the use of propagation by budding that the technique soon became the sole method of propagation by many nurseries, phasing out seedling propagation entirely (Castle et al.,

1993). This also increased interest in citrus, resulting in a wider diversity of scions and rootstocks available (Castle et al., 1993). The budding system allowed propagators and growers to evaluate grafted material quicker than could be achieved by seedling trees

(Castle et al., 1993).

Growers learned that Florida’s climate and soil were ideal for producing profitable orange and other Citrus crops, and began shipping them up the east coast, first by boat

and then by rail (Webber, 1967). After the civil war, citrus production in Florida reached

around one million boxes, gradually increasing to five million boxes by 1893. The

following year, 1894, a major freeze nearly destroyed all citrus in the state. Production

in 1895 was a meager 147 thousand boxes (Reuther et al., 1967, p. 23). Learning from

the disaster, growers shifted production to areas further south in the state, eventually

reaching pre-freeze production levels by 1910 (Ziegler and Wolfe, 1961).

In 1915, growers produced ten million boxes, and continued increasing

production, reaching 100 million boxes for the first time in 1950. Production continued to

increase through the next several decades, with occasional freezes which forced production further south, centralizing in Polk county as the primary production area for much of the state’s annual citrus crop (Ziegler and Wolfe, 1961)

In 2004, several disasters converged to wreak havoc on the citrus industry.

Citrus Canker had been detected in Florida in 1995, and government officials,

encouraged by previously successful eradication programs, had initiated a program to

eliminate it from the state (Gottwald et al., 2001a). To contain the citrus canker

epidemic, the Florida Supreme Court ruled the state had the right to destroy healthy

citrus trees growing within 1,900 feet or less from an infected tree. Thousands of acres

and millions of trees were burned in an attempt to eliminate the pathogen, to the

devastation of both growers and homeowners (Dahlburg, 2005).

Between 2004 and 2005, several hurricanes swept through the state, spreading

citrus canker far and wide, infecting thousands of acres, eventually forcing authorities to

realize it would no longer be feasible to try to control the spread of the pathogen (“5B-

58.001 : Citrus Canker Eradication (Repealed) - Florida Administrative Rules, Law,

Code, Register - FAC, FAR, eRulemaking” 2017). In 2005, Huanglongbing was first

detected in Dade County, near Homestead, in the southern tip of Florida (Halbert,

2005). Citrus psyllids (Diaphorina citri Kuwayama), the vector of Candidatus

Liberibactor asiaticus, had first been detected in Florida in 1998 (Halbert and

Manjunath, 2004). From the time of introduction until detection of Huanglongbing seven

years later, citrus psyllids had been reproducing and spreading as the population grew.

This infestation was likely helped in the 2004 Hurricane season, further scattering

psyllids across the state. By 2008, citrus psyllids as well as Huanglongbing were found in every citrus production area of the state, (Dixon et al., 2008).

Citrus production forecast for the 2017 season estimates production at

80,560,000 boxes of all citrus for Florida (189,310,000 for all US production areas), down from peak production in 1997-98 of 304,450,000 boxes (“NASS - Florida Citrus

Production Forecasts,” 2017). Current area of citrus in production is 480,121 acres in

Florida (USDA NASS Citrus Acreage, 2016).

Taxonomy

The genus Citrus L. belongs to the subtribe Citrineae, of the tribe Citreae within the subfamily Aurantioideae of the Rutaceae family. They are dicotyledonous angiosperms, evergreen, rarely deciduous; fruit with leathery (or rarely soft) exocarp and spongy mesocarp; seeds embedded in pulp vesicles; leaves 1-foliolate, simple, or rarely digitately 3-foliolate (Swingle and Reece, 1967; Zhang and Mabberly, 2008).

Most of the genus Citrus evolved in the tropical and subtropical monsoon regions of Southeast Asia and the Malay archipelago, from whence they spread into other sections of the world (Swingle and Reece, 1967). True citrus fruits belong to genus

Citrus, formerly one of six genera although recently some taxonomists have lumped them all together into genus Citrus: Clymenia, Eremocitrus, Microcitrus, Poncirus,

Fortunella and Citrus, (Mabberley, 1997; Uzun and Yesiloglu, 2012; Zhang and

Mabberly, 2008). Citrus taxonomy and phylogeny are confusing due to their long history of cultivation, wide distribution, nucellar polyembryony, and sexual compatibility between species (Nicolosi, 2007). Linnaeus first classified all Citrus as three different species: Citrus aurantium, C. medica, and C. decumana (Linnaeus, 1758, 1753). Later taxonomists had varied, wider interpretations. There are two widely used schemes of

classification, the classification of Tanaka (1954) who recognized 162 separate species,

or the classification of Swingle (1967), who suggested lumping the same number

together into 16 species, subdivided into various horticultural forms and hybrids.

Scora (1975) first suggested cultivated citrus originated from only three ‘basic’

true species within the subgenus Citrus as follows: citron (C. medica L.), mandarin (C.

reticulata Blanco), and pummelo (C. maxima (Burm.) Merrill). Early taxonomists were

limited in their classification to using data gathered from morphology, anatomy, and

geographical distribution. Barrett and Rhodes (1976) investigated Citrus relationships by

studying many differentiating morphological characteristics, and by comparing species

to known hybrids was able to construct a system of relatedness. Without using modern

genetic tools he drew a similar conclusion: there were three basic ancestral species,

along with other wild species that played a smaller part in developing cultivated citrus.

The other important types (orange, grapefruit, lemon, and lime) are believed to have

originated from one or more generations of hybridization between the three ancestral

species with additional hybridization among Subgenus Papeda, validated by recent

genetic studies (Curk et al., 2015; Garcia-Lor et al., 2013; Liang, 1990; Liang et al.,

2007; Uzun et al., 2010; Wu et al., 2014).

Genetic Variation in Citrus

Many citrus cultivars are closely related, having arisen through various bud

mutations or sports which have altered specific horticultural traits, and have been

maintained through grafting onto rootstocks (Uzun et al., 2010; Webber, 1967).

Additionally, many citrus cultivars produce apomictic seed through nucellar

polyembryony, their progeny selected for mutations that may be subtle differences from

the parent or selected as disease-free cultivars (Uzun et al., 2010; Webber, 1967).

Because of the slight differences it can be difficult to distinguish cultivars using morphological characteristics alone (Fang and Roose, 1997; Uzun et al., 2010).

Studies of genetic diversity in different citrus collections have found that there is

a good amount of genetic diversity in citrus, just not within certain classes of citrus

(Barkley et al., 2006; Herrero et al., 1996; Roose et al., 1992; Uzun and Yesiloglu,

2012). Sweet oranges, sour oranges, and grapefruit, have been shown to highly

heterozygous, but have low diversity between cultivars (Herrero et al., 1996; Novelli et

al., 2004; Uzun et al., 2010). Within the progenitor species exists much higher levels of

diversity (Curk et al., 2015; Herrero et al., 1996).

Ancestral and Contemporary Cultivars

Citrus cultivars can be classified into eight basic categories: citron, pummelo,

mandarin, sweet orange, sour orange, grapefruit, kumquat, lemon, and lime. These

distinctions can be difficult to make as many of these groups intercross, making new

hybrid forms, such as the tangor (tangerine x orange), tangelo (tangerine x pomelo

(grapefruit)) or limequat (kumquat x lime). Many of the modern cultivars are admixtures

of multiple species, often with the cross having naturally occurred anciently (Scora,

1975; Wu et al., 2014).

Pummelo (Citrus maxima (Burm.) Merrill)

Pummelo, also known as shaddock, (Citrus maxima (Burm.) Merrill, C. grandis

(L.) Osbeck, or C. decumana L.) is native to the Malayan and East Indian archipelagos,

where it spread to India and China (Swingle and Reece, 1967). In the mid-17th century,

an English sea captain, Phillip Chaddock, brought pummelo seeds from the East Indies

to Barbados, where settlers started growing the fruits, corrupting his name and calling

the fruits “shaddocks” (Webber, 1967; Karp, 2000). Pummelos is one of the largest

citrus fruits (only a few citron cultivars attain larger size (Ramadugu et al., 2015)), with a

diameter of 15-25 cm and weighing between 1-2kg, but many cultivars produce smaller fruit similar to a large grapefruit in size (Saunt, 2000). Fruit shape is also variable, from

spherical to oblate to pyriform. Pummelo fruit frequently have between 12 and 25

segments, often asymmetrical in size and arrangement, with many ‘blind’ segments that

don’t reach the peel (Swingle and Reece, 1967).

Peels have a smooth but pebbly texture created by prominent oil glands (Swingle

and Reece, 1967). Ripe fruit are pleasantly fragrant, and are often used to scent a

room. Fruit are yellow to light green when ripe, occasionally with a pink blush; flesh

color may be white (yellow), pink, or red, which can be as dark in hue as a red fleshed

grapefruit (Roose et al., 1992). The thickness of the peel varies greatly, with some

varieties having an albedo several centimeters thick (Saunt, 2000). Fruit are often very

seedy-with well over one hundred seeds per fruit—although some varieties may be

seedless in the absence of cross pollination (Hodgson, 1967). Pummelo produces

monoembryonic seeds with embryos of zygotic origin, with each seedling a new genetic

combination of its parents (Uzun and Yesiloglu, 2012).

Pummelos are a prized fruit in many Asian countries where they are presented

as gifts and considered lucky (Chan, 1999; Karp, 2000). For example, during the

Chinese Moon Festival, pummelos are often given as gifts, used in holiday cooking, and

the peels made into children’s hats for the moon goddess to answer their prayers

(Chan, 1999; Lin, 2010).

Pummelo remains largely unknown within the U.S., partly because the name

causes confusion since pomelo is an alternative name for grapefruit, especially in

foreign countries. The name appears to be derived from the Dutch “pompelmoes” meaning “big lemon”, given to it by Dutch settlers in Indonesia (Hodgson, 1967; Karp,

2000). There is some confusion as “pomelo” is used as a synonym for grapefruit, but

“pummelo” is the preferred spelling (Hodgson, 1967).

Pummelo has played an impressively important role as the progenitor of most commercially important citrus fruits in the world today: sweet orange, sour orange, lemon, and grapefruit (Uzun and Yesiloglu, 2012; Wu et al., 2014). These crosses occurred anciently in some of these types, with grapefruit the most recently derived commercially important type (Gmitter, 1995). Of the Citrus species, Citrus maxima

(Burm.) Merrill, is perhaps the most important, offering a great resource in attributes and genetic diversity (Uzun and Yesiloglu, 2012).

Pummelos comprise a highly variable group, with some trees extremely vigorous and reaching large sizes, and others much less vigorous and smaller (Hodgson, 1967).

Pummelos have good heat tolerance, although there is range of heat requirements for fruit to ripen with some ripening before grapefruits and others only obtaining the best fruit quality in the hottest environments (Hodgson, 1967) Cold tolerance also varies, with some varieties showing a similar cold tolerance to grapefruits and others with a higher sensitivity to cold (Hodgson, 1967).

Flesh of the pummelo generally has a firmer texture than other citrus, usually eaten fresh out of hand as opposed to spooning the flesh out as in grapefruit, and is difficult to juice (Hodgson, 1967). After peeling the fruit and pulling the segments apart, the carpellary membranes may be easily peeled off leaving the juice vesicles intact and adhering to each other. The juice vesicles are typically solid, and this process may be

done with only a small amount of juice released. The segments are then served either with or without sugar, and typically do not have the bitterness found in grapefruit, although there are bitter pummelos (Hodgson, 1967; Saunt, 2000)

Sweetness of the flesh varies considerably, as does flavor, acidity, and bitterness; the best cultivars have fruity flavors, low acidity and low bitterness (Saunt,

2000). Bitterness from the citrus compound naringin is often found in the segment walls

(Park et al., 1983). In the U.S., the major factor regarding the pummelos’ public relations

problem with the American consumer is probably their large size. Packing equipment

are typically only sized for a fruit with the maximum size of a large grapefruit, which is

the equivalent of a small pummelo (Gmitter, 2012). Larger fruit typically command

higher prices per individual fruit making the resulting cost of fruit unaffordable to the

average uninformed buyer, and purchasers are further put off by large fruit consisting

mostly of thick, spongy peel (Lange, 2016). Most of the pummelos sold in the US are

grown in California, largely due to a larger Asian base who are familiar with the fruit

(Karp, 2000).

Grapefruit (Citrus x paradisi Macfadyen)

The grapefruit, (C. x paradisi Macfadyen) was a chance hybrid between

pummelo and sweet orange, originating in the Caribbean in the 1700’s (Gmitter, 1995).

It was first brought to America by Count Odette Phillippe, who settled in Safety Harbor

near Tampa, FL, in 1823 (Saunt, 2000). While grapefruit accounts for 11% of citrus

production in Florida, the state historically has been the largest producer and exporter of

grapefruit, producing over 2.3 million tons, with 45% of it shipped as fresh fruit (“Citrus

Budwood Annual Report 2014-2015,” 2015). Production in Florida declined steadily

since the introduction of citrus canker in 1996 and Huanglongbing (HLB) in 2005, with

production for 2017 projected to be 688,000 tons (USDA, 2017). Grapefruit is highly

susceptible to Huanglongbing —which reduces production and kills the tree—as well as

citrus canker, causing unsightly, unmarketable fruit (Hodges et al., 2014). New,

disease-resistant grapefruit cultivars are essential to continue production in Florida

where HLB and citrus canker are now firmly established (Grosser and Gmitter, 2012).

Grapefruit is naturally difficult to use as a female due to its high rate of nucellar

polyembryony, with nearly every seedling producing an identical clone of the mother

tree (Ferguson and Grafton-Cardwell, 2014; Reuther et al., 1967). Breeding is not

impossible, as many tangelos have grapefruit as the female parent, including

‘Minneola’, ‘Orlando’, ‘Sampson’, and ‘Seminole’, as well as the Swingle citrumelo

(Reuther et al., 1967). Grapefruit genetic improvement in the past has focused mainly

on mutation selection, mostly by finding natural mutations such as decreased seediness

or improved pigmentation, which occur naturally and randomly (Gmitter, 1995).

Additionally, attempts have been made to generate new mutations by exposure to

chemicals or irradiation to hopefully cause mutations with reduced seed or improved

color. However, these methods are random in effect and will also cause deleterious

mutations at the same frequency. ‘Star Ruby’ is an example of a radiation induced

mutation, produced by irradiating seeds of ‘Hudson’ grapefruit (Hensz, 1971). The

resulting cultivar has deep-red fleshed, low seeded fruits and a smooth yellow rind with

a red blush. However, some negative attributes accompany the improved traits.

According to Ed M. Nauer of the University of California-Riverside, Citrus Variety

Collection, “It exhibits greater susceptibility to Phytophthora, nutrient deficiencies, cold

temperatures, and pest problems. It does not appear to grow as vigorously as other

grapefruit varieties, and the fruit is often smaller.“ (Nauer, 1988).

While the exact parents of grapefruit are unknown, genetic research has shown

that grapefruit is descended from a hybrid between a pummelo (C. maxima (Burm.)

Merrill) and sweet orange (C. sinensis L.), with sweet orange itself an ancient admixture

between pummelo and mandarin (Garcia-Lor et al., 2013; Wu et al., 2014). The original

cross of the grapefruit was by chance, between two random cultivars of pummelo and

sweet orange. Improvement attempts will focus on using improved, disease resistant

cultivars to create a synthetic grapefruit or grapefruit-like hybrid.

While it is possible to create a grapefruit-like hybrid by remaking the cross

(pummelo x sweet orange), this would be difficult to accomplish due to the time until

bearing (typically five or more years), the eventual large size of the trees, and the high

heterozygosity of Citrus would require a grow-out of large numbers of seedlings

(Gmitter, 1995). Focused back-crosses using elite grapefruit selections as one of the

parents and a pummelo as the other parent is more likely to ease the process of finding

a fruit that is closer to a grapefruit.

Precedence for triploid, grapefruit-like pummelo hybrids can be found from the

University of California breeding program with ‘Oroblanco’ and ‘Melogold’ cultivars, both seedless, triploid grapefruit-like hybrids developed in California as crosses from

‘Siamese Sweet’ acidless pummelo with a tetraploid ‘Marsh’ grapefruit (Soost and

Cameron, 1980, 1985). These are most often marketed as grapefruit, and generally taste and appear similar to white-fleshed grapefruit, although their peels are thicker.

University of Florida has also released a triploid pummelo-grapefruit hybrid called ‘914’

which is red fleshed and seedless, and notable for its low furanocoumarin levels; furanocoumarins in grapefruit interfere with the body’s ability to metabolize certain medications (Beach, 2012; Gmitter, 2015).

One of the oldest known named grapefruit cultivars, the Duncan likely originated

as a seedling from the first grapefruit trees grown in Florida. It is the paragon of

grapefruit in terms of fruit flavor and quality, representing the ideal standard for what a

grapefruit should taste like. It is white fleshed but excessively seedy, causing it to fall

out of popular demand for seedless, pigmented flesh cultivars (Gmitter, 1995). It is also

the ancestor of most cultivated grapefruit, as various mutations or sports have arisen with

reduced seed numbers or improved flesh color (Gmitter, 1995). This is illustrated in

Figure 2-2, showing the series of mutations leading to popular modern pigmented

cultivars, all descended from ‘Duncan’ grapefruit.

Oranges

The sweet orange (Citrus x sinensis (L.) Osb.) is the fruit of most commercial

importance in the world, widely cultivated for its fruit and juice (FAO, 2016). Its origin is

unknown and first theorized as a hybrid of ancient origin between C. maxima (Burm.)

Merrill (pummelo) and C. reticulata (L.) Osb. (mandarin orange) (Barrett and Rhodes,

1976; Scora, 1975). Xu et al. (2013) proposed that the cross was more complex: (C.

maxima × C. reticulata) × C. maxima as the female parent and C. reticulata as the male

parent. This was later proved incorrect by Wu et al. (2014) by the presence of

homozygous pummelo alleles on the second chromosome, showing that both parents

must have had some pummelo ancestry. The sweet orange appears to be an admixture

of mandarin and pummelo, with the chloroplast genome of pummelo, and shared alleles

of mandarin (found in Ponkan, Willow-leaf, and Huanglingmiao mandarins) across

three-quarters of its genome (Wu et al., 2014). Clementine (C. x clementina) was also shown in the same study to be a hybrid between sweet orange and mandarin.

Sour orange, (Citrus x aurantium (L.) Osb.) has also been theorized to be

another ancient hybrid between C. maxima (pummelo) and C. reticulata (L.) Osb.

(mandarin orange) (Barrett and Rhodes, 1976; Scora, 1975). Wu et al. (2014)

determined that the sour orange is an F1 hybrid between a female pummelo and male

mandarin, perpetuated through the production of nucellar seeds. No recent relationship

was detected between sweet orange and sour orange.

Outside of the United States, sour orange has many uses, as a condiment, used

in marmalade, as a cleaning agent and using the blossoms for perfume extraction

(Morton, 2013, pp. 130–133; Wu et al., 2014). In the U.S., it has been used primarily as

a rootstock. Sour orange was historically a very popular rootstock in Florida, giving good

yields of high quality fruit. It has lost popularity because when it is grafted with sensitive

scion cultivars, it is susceptible to Citrus Tristeza Virus (CTV) “quick decline”,

accounting for only 10.6% of the rootstocks in 2015-2016. However, 47% of all

grapefruit during that time were budded onto sour orange. Sour orange was frequently

the most used rootstock in Florida prior to 1974 (“Citrus Budwood Annual Report 2014-

2015,” 2015). Sour orange performs well on calcareous and wet soils, yielding less on

sandy soils compared to some other rootstocks (Saunt, 2000). It is susceptible to citrus

and burrowing nematodes, as well as Diaprepes breviatus root weevils (Bitters, 1986;

Castle et al., 1993).

Although there is wide amount of genetic diversity within citrus, there is not a lot genetic diversity within sweet orange or sour orange cultivars, due to clonal propagation

and variation arising from somaclonal mutations (Herrero et al., 1996; Wu et al., 2014).

Citron, Lemon, and Lime

Citron (Citrus medica L.) is the largest of true citrus, documented weighing up to

15 kg (33 lbs.) with the greatest diversity of citrons found in south-east Asia (Ramadugu et al., 2015). They generally fall into two categories—acid or sweet (Swingle and Reece,

1967). Flavor is typically lemon-like. In Asia they are valued for their edible albedo which is used in cooking, and in the Middle-east region esteemed with religious significance (Arias and Ramón-Laca, 2005). Citrons are the progenitors of lemons and limes; lemons have been found to be hybrids between citron and the sour orange

(pummelo x mandarin), and limes were found to be hybrids between citron and the small-flowered papeda (Citrus micrantha Wester) (Curk et al., 2016).

Mandarin and Mandarin Hybrids

One of the ‘true’ species of citrus, Citrus reticulata (L.) Osb.) is native to

Southeast Asia found in warm temperate to subtropical regions. The origin of the

mandarin name is theorized to be from when it was introduced into England in 1805.

Mandarin was the name of the language spoken by Chinese officials, who wore orange

colored robes at the time, so this Chinese import was dubbed a mandarin. The fruit is

also known as a tangerine, from a similar story of import origin when these citrus fruits

were imported from Tangiers, Morocco, in the late 19th century. (Hodgson, 1967)

The mandarin has probably been cultivated in China for several thousand years, and the earliest mention is from 12th century BCE (Saunt, 2000). It is grown primarily for

its fruit, but is occasionally used as a rootstock. The term tangor was one of a number of

terms developed by Webber and Swingle in the early 1900s to classify hybrids of mandarin and orange. Most of these resemble mandarins and include cultivars such as

‘Temple’, ‘Ellendale’ and ‘Ortanique’. The presence of orange in the ancestry of complex hybrids makes it difficult to determine how to classify cultivars that resemble

mandarins. Since there are a number of cultivars that have a small proportion of their

heritage that is not mandarin, a number of scientists now commonly refer to all cultivars

that produced fruits that resemble mandarins be called mandarins or alternatively as

mandarins and mandarin hybrids (Kahn, 2008). This was confirmed in recent genetic

studies, which have shown even those cultivars of mandarin thought to be pure C.

reticulata had alleles from C. maxima (Curk et al., 2015; Wu et al., 2014)

Mandarins have richly colored orange peels, with a smooth, pebbly texture. Peels

are typically thin and tear easily, with a white colored albedo. Between the peel and the

segments is a layer of reticulated tissue, from which the scientific name derives.

Kumquats

A close relative of Citrus, kumquats were originally put in their own genus,

Fortunella, named after the plant explorer Robert Fortune. Recently some taxonomists have moved them to genus Citrus (Zhang et al., 2008), but will be referred to herein as

Fortunella. Kumquats are native to Southeast Asia, from tropical to temperate subtropical regions. Kumquats are typically small fruits, with edible rinds, and minor commercial value. Some of the cultivars exhibit cold tolerance and winter dormancy not found in other citrus cultivars, with the exception of P. trifoliata and C. ichangensis

(Swingle and Reece, 1967). Genetic investigation has revealed that the various described cultivars separate into two main species, Fortunella hindsii Swingle and the F. margarita Lour. species complex, which includes F. margarita, F. japonica, and F.

crassifolia (Yasuda et al., 2016). Fortunella polyandra (Ridl.) Tan. (Malayan kumquat)

and F. obovata hort. ex Tan. (Changshou kumquat) were both shown to have

chromosomes from Citrus, and are not valid species (Yasuda et al., 2016). Calamondin

(Citrus madurensis Lour.) has long been thought to be a an ancient hybrid between

Citrus and Fortunella (Handa and Oogaki, 1985; Swingle, 1943). Other hybrids with

citrus include limequats, lemonquats, orangequats, and citrangequats (Hodgson, 1967).

Meiwa kumquat (F. margarita ‘Meiwa’) has shown exceptional canker tolerance

and is widely regarded as immune, while Nagami kumquat (F. margarita ‘Nagami’) has

less citrus canker tolerance (Chen et al., 2012; Khalaf et al., 2007). Nagami kumquats

produce zygotic seed, while Meiwa produce polyembryonic, nucellar seed. They are

graft compatible and sexually compatible with citrus; however, some crosses with Citrus

exhibit delayed graft incompatibilities (J. Chaparro, pers. comm.).

Trifoliate Orange (Poncirus trifoliata [L.] Raf.)

The trifoliate orange, Poncirus trifoliata, is sexually and graft compatible with

citrus. Some taxonomists classify it as genus Citrus (Zhang et al., 2008), although it

exhibits distinct traits not found in other true citrus. It is a unique member of the citrus

family, owing to its trifoliate leaves and deciduous habit. It has long been used as a

rootstock, as it produces primarily nucellar seeds, and the cultivar ‘Flying Dragon’ also

valued for providing tree size control (Bitters, 1986). Its fruit are essentially inedible

although the fruit is used in Chinese medicine (Swingle and Reece, 1967). It shows

great winter hardiness, and can impart this cold tolerance onto the scions grafted to it

(Castle et al., 1993; Swingle and Reece, 1967). It can be crossed with other citrus with

hybrids being used for rootstocks as well. It has gained wider use since some cultivars

are immune to CTV and xyloporosis, has shown some tolerance to HLB, and is a non-

preferred host for Asian citrus psyllids (Ramadugu et al., 2016; Yoshida, 1996).

Trifoliate oranges are cross compatible with citrus and have been useful in the creation

of many rootstocks (Swingle and Reece, 1967). These rootstocks often extend good

fruiting characteristics to the scion, confer cold hardiness, disease resistance, and the

trifoliate leaves help to spot shoots that may arise from below the graft (Bitters, 1986;

Castle et al., 1993).

Papedas

Papedas are a truly wild branch of the citrus family, many of them still to be found

growing in primeval forests of the Monsoon region in south-east Asia and India (Arora,

2000). Most have fruits that are inedible or barely edible, and characteristically have

acrid oils in their juice vesicles (Swingle and Reece, 1967). Leaves typically are the

same size as the petiole, so that leaves appear to be split in half (Swingle and Reece,

1967). Citrus micrantha Wester, the small-flowered papeda or Samuyao of the

Philippines, has been discovered to be one of the progenitors of the modern lime (Curk

et al., 2016, 2015; Gmitter et al., 2012). The Khasi papeda, Citrus latipes, is a wild

papeda from north-eastern India, and has shown to be tolerant to HLB (Ramadugu et

al., 2016). Giuseppe Reforgiato of the CRA-ACM (Research Centre for Citriculture and

Mediterranean Crops) in Acireale, Sicily, has developed some Citrus latipes hybrids:

Citrus latipes x trifoliate orange, and C. latipes x sour orange hybrids that may prove to

be HLB tolerant. The Ichang papeda, C. ichangensis, is a wild species that may be the

most cold tolerant citrus species, but fruits are inedible and the interior comprised

mostly of large seeds (Malik et al., 2015).

Near Citrus Relatives

Many of the near citrus relatives are sexually compatible and graft compatible, such as species in the genera Clymenia, Fortunella, Oxanthera, Poncirus, Microcitrus,

and Eremocitrus, which have been reclassified as genus Citrus (Mabberley, 1997).

Some near relatives are sexually incompatible but graft compatible, such as species in

the genera Atalantia, Citropsis, Feronia, Feroniella, Severinia, and Swinglea (Yoshida,

1996). Severinia buxifolia is graft compatible with Citrus, showing resistance to boron toxicity, and some cultivars exhibiting CTV immunity (Swingle and Reece, 1967;

Yoshida, 1996).

These near relatives, although frequently graft compatible, are difficult to hybridize with true Citrus. Very few sexual hybrids exist and documented hybrids that have resulted have not fruited or have been sterile (Medina-Filho et al., 1998); Smith was able to obtain fruit from Citropsis hybrids, which have been seedless (Smith et al.,

2013). Somatic hybrids have been made using protoplast fusion, but these have also been sterile, using Severinia, Citropsis, Atlantia, and Feronia (Grosser et al., 1995,

1996b; Grosser and Gmitter, 1990a; Ling et al., 1990; Mourão Fo et al., 1994). Many of these intergeneric hybrids have been created for the purpose of developing disease resistant rootstocks (Grosser, 1996), but have been unable to be used in standard rootstock production due to lack of seeds, which relies on the use of nucellar seed as an inexpensive source of uniform germplasm.

Rootstocks

Grafting is an ancient technology, likely originating in China as a way to propagate citrus, allowing people to propagate mature plants that were difficult to propagate by cuttings (Pollan, 2002). It is widely used in fruit trees and increasingly with

vegetable crops (Lee et al., 2010). The strong influence the rootstock has on the scion directly affects where and how successfully citrus grow. The rootstock will alter many different horticultural and pathological traits of the scion. Rootstocks serve three general purposes:

Reduction of juvenility: Trees grown from seed are slow to bear, often very vigorous and upright in growth habit, and can be very thorny. This can be avoided by budding a scion from a mature tree, resulting in trees more uniform, quicker to bear, and thornless (Wareing, 1959).

Environmental adaptation: Rootstocks vary in their ability to cope with soil quality, soil pH, environmental stresses, pests such as nematodes, soil-borne diseases, and mineral nutrition problems. They will also modify plant growth, biomass accumulation and repartition, and phenology, affecting whole plant performance (Ollat et al., 2016).

Using known attributes, a grower will select the scion and rootstock combination that will perform best for a specific location.

Horticultural performance: The rootstock interacts with the scion and environment affecting nutrition, growth, productivity, water usage, and several aspects of fruit quality.

(Castle et al., 1993) The effect of the rootstock on whole plant performance is associated with the structural (root morphology) and physiological properties (Ollat et al., 2016).

Any species in the genus Citrus can theoretically be used as a rootstock, and different factors determine what rootstock to use, such as climate, soil characteristics, and desired tree size. Popular rootstocks vary by region, but in Florida eleven rootstocks account for 95% of all citrus rootstocks used in 2015-2016.

Top Rootstocks in Florida 2015-2016:

1. ‘Kuharske’ citrange (P. trifoliata x C. x sinensis) 2. ‘X639’ (C. reticulata Cleopatra x P. trifoliata) 3. Sour orange (C. x aurantium) 4. US-897 (C. reticulata x P. trifoliata) 5. ‘Swingle’ citrumelo (C. x paradisi x P. trifoliata) 6. US-942 (Sunki mandarin C. reticulata x Flying Dragon P. trifoliata) 7. US-802 (Siamese pummelo C. maxima x P. trifoliata) 8. US-812 (C. reticulata Blanco x P. trifoliata) 9. ‘Cleopatra’ mandarin (C. reticulata) 10. UFR-4 (‘Nova’+Hirado Buntan seedling x Cleopatra+Argentine trifoliate) 11. Volkamer lemon (C. x volkameriana) (“Citrus Budwood Annual Report 2015-2016,” 2016).

The ideal rootstock confers disease resistance, tolerance of environmental stresses, enhanced nutritional uptake, soil adaptation, and improves yield. Additionally, rootstocks can influence fruit quality, affecting the acidity and amount of soluble solids in the fruit, or affecting the texture of the rind in some cases (Castle et al., 1993). Sour orange has long been a popular rootstock in Florida, but is susceptible to diseases like

CTV and HLB. Results of the St. Helena rootstock trials have shown that some of the most popular commercial rootstocks are highly susceptible to HLB (Grosser, 2012). The worst offenders are, in order of decreasing susceptibility: Kuharske, Carrizo, Volkamer,

Swingle, and rough lemon. The trials have also shown that along with some diploid hybrids, tetraploid zygotic cultivars that combine genetics from several progenitors, i.e. the “tetrazygs”, such as UFR-4, have shown ability to impart increased HLB tolerance to grafted sweet orange trees (Grosser, 2012, 2015a).

Other cultivars and species have disease tolerance and other positive characteristics that would be useful in rootstocks, but in themselves are not necessarily good rootstocks. These species could be combined with rootstocks which lack the desired trait using sexual or somatic hybridization (Grosser et al., 1995).

Interstocks

Interstocks are a grafting solution largely used to mediate interactions between the scion and rootstock. They are more frequently used in apple for a dwarfing effect, which could be caused by the additional graft union, or an imperfect graft union, which may slow translocation, regulating growth between a more vigorous scion or rootstock

(Seleznyova et al., 2008; Zhao et al., 2016). The interstock may influence the quantities

of auxin passing down to the root and thereby affects tree development and cytokinin

synthesis (Lochard and Schneider, 1981). The interstock may also affect nutrient

translocation or introduce endogenous growth factors which regulate growth (Hartmann

and Kester, 1975). Castle et al. (2011) used a sweet orange interstock in attempt to

help ‘Pera’ sweet orange scion incompatibility with Swingle citrumelo rootstock. Bitters

et al. used Citrus relatives as interstocks for tree size control (Bitters et al., 1978).

Some research has been done to determine whether interstocks will impart some

disease tolerance in citrus. Researchers in Brazil experimented to see if an interstock of

trifoliate or Cleopatra mandarin between Rangpur lime and the Valencia sweet orange

scion could stop the transmission of Citrus sudden death, finding it did not help avoid

the disease (Pompeu Jr. and Blumer, 2008). Researchers in Malaysia conducted an

experiment to see if an interstock could help combat HLB symptoms for HLB sensitive

mandarin scions, which they found to be highly susceptible to HLB. They used a

combination of a local variety of pummelo (listed as C. grandis (L.) ‘Limau Bali’), as well

as Citrus hystrix Chakr., both found to be HLB tolerant, and C. aurantium L. and C.

aurantifolia S. as tolerant species in a previous experiment (Shokrollah, 2009), using

these as both rootstocks and interstocks, with C. reticulata ‘Limau Madu’ as scion, and

side grafted with HLB positive material (Shokrollah et al., 2011). It was determined that

the rootstock and interstock combination of C. grandis and C. hystrix, in either position, resulted in a healthy C. reticulata scion.

Protein and RNA Signals

Research has shown that some molecules are able to pass through the graft union,

such as proteins and RNAs, in addition to photoassimilates, amino acids and other

small molecules found in the phloem sap, and move in either direction from the scion to

the rootstock and vice versa (Ruiz-Medrano et al., 1999; Spiegelman et al., 2013).

Intracellular trafficking will move products generated from one graft partner into distant

plant organs, affecting growth and regulation of the other.

Citrus Breeding

Although there are many diverse hybrids in citrus, and cultivars and species

generally will intercross readily, citrus breeding is complicated due to a variety of

reasons. Many cultivars are apomictic, producing polyembryonic, nucellar seeds,

resulting in each seed producing one or more clones of the mother plant derived from

the nucellus of the seed. Breeding is additionally hampered by high heterozygosity and

long juvenility, with seedling trees developing a relatively large size before flowering.

This time period may be from five to twenty years before flowering, and makes it difficult

to maintain a large seedling population necessary to observe segregating traits

(Cameron and Frost, 1968; Gradziel et al., 2009; Lebowitz, 1985). Time between when

a cultivar is selected and when it is released has typically been long: Kara, an Owari

Satsuma x King mandarin hybrid was produced in 1915, and not released until 1935,

and Flame grapefruit originated from seed collected in 1973, and not released until

1987 (Saunt, 2000). Many of the recent releases by the University of Florida originated

over 20 years ago (Grosser et al., 2015).

Interfertility is high between species and cultivars, allowing for diverse choice

when choosing breeding parents. In addition to sexually derived progeny, spontaneous

mutations have contributed to many of the variations in cultivated citrus (Cameron and

Frost, 1968; Gmitter, 1995; Salomon and Weinbaum, 1984; Uzun and Yesiloglu, 2012).

Mutation Breeding

Grapefruit and sweet orange genetic improvement in the past has focused mainly on mutation selection, mostly by finding natural mutations such as decreased seediness or improved pigmentation, which occur naturally and randomly (Cameron and Frost,

1968; Graça et al., 2004; Grosser et al., 2015; Salomon and Weinbaum, 1984).

Additionally, attempts have been made to generate new mutations by exposure to chemicals or irradiation to disrupt the DNA and hopefully cause the same types of mutations. However, these methods are also random in effect and will also cause deleterious mutations at the same frequency. ‘Star Ruby’ is an example of a radiation induced mutation, produced by irradiating seeds of ‘Hudson’ grapefruit (Hensz, 1971).

Polyploidy

Most animals and plants existing in a diploid state containing two sets of chromosomes. An organism typically receives one set from each of its parents.

Polyploidy, a state with one or more sets of chromosomes than the average for an organism, is a common biological phenomenon in plants and plays an important role in the evolutionary history of plants (Otto and Whitton, 2000). Citrus are generally diploid

(2n=2x=18), but polyploid forms have been either discovered or induced (Swingle and

Reece, 1967). These polyploid forms have proven useful in breeding and studying genetics, resulting in plants with overall larger features, and frequently used horticulturally to increase fruit and flower size. Polyploidy in citrus results in thicker

leaves, usually broader than their diploid counterparts. The petiole wings are often wider, sometimes fused with the leaf blade (Cameron and Frost, 1968, p. 353).

Tetraploid trees will generally have stouter thorns, which are retained into maturity longer than diploids (Frost, 1925). There is wide variation of vigor of tetraploids, with some showing similar or greater vigor than diploids, while many of the autotetraploids

(duplication of a diploid set of chromosomes) have reduced vigor, reluctance to flower, and reduced fruit set(Frost, 1925). Allotetraploid (tetraploid containing two sets of chromosomes from different species) hybrids have shown increased vigor, and are being used successfully as rootstocks (Ananthakrishnan et al., 2006; Grosser and

Gmitter, 2011; Grosser, 2015a, 2015b). Tetraploid rootstocks often have a dwarfing effect on a diploid scion, which can be an attractive trait for tree size control (Grosser,

2012).

Longley (1925) was the first to report a natural tetraploid of the wild Hong Kong kumquat (Fortunella hindsii Swingle). Frost later identified nucellar progeny with “thick leaves’ in Citrus and Poncirus seedlings, and determined cytologically that they were

tetraploids (1926, 1925). Cameron and Frost (1968) also reported that out of 3,600

nucellar progeny, approximately 2.5% were tetraploid. Most spontaneous tetraploids

arose from nucellar seedlings; Lapin (1937) studied the oldest tetraploids in the

Riverside collection and determined that they were nucellar by absence of pollen parent

characteristics and uniformity within a variety. Cameron and Frost (1968) concluded that

doubling of the chromosome often occurs in the parent tree, in the tissue of the ovule or

ovary. These spontaneous tetraploids from nucellar seedlings are otherwise identical to

their diploid siblings, other than some morphological differences due to the polyploidy,

and can be considered autotetraploids. Polyploidy has also been induced using chemical agents such as colchicine or oryzalin which prevent microtubule formation during mitosis, interrupting the separation of the chromosomes following duplication and resulting in cells with doubled ploidy (Bajer and Molè-Bajer, 1986; Pickett-Heaps, 1967).

Protoplast Culture and Somatic Hybridization

Protoplasts are ‘naked cells’ stripped of their cell wall without damaging the cell membrane or nucleus, and are produced from either plants or fungi. The cell wall is typically dissolved using enzymes extracted from certain fungi, i.e. cellulase, macerase and pectinase. Protoplasts can be isolated from any part of the plant, but in citrus the totipotent protoplasts, capable of regenerating an entire plant, are recovered using embryogenic callus or suspension cultures, usually regenerated from immature citrus ovules. Protoplasts are also useful in transformation, as the cells can take up plasmids, and since they are single cells, every single cell in the recovered plant will be transformed (Grosser and Gmitter, 2011, 1990b; Grosser and Omar, 2011).

Somatic hybridization through protoplast fusion is a technique with various applications, especially useful with concern to ploidy manipulation. It can be used to make crosses that are difficult or otherwise impossible, due to parents being too distantly related, sterile, having differing ploidy levels, and in Citrus it is especially useful to create new combinations from cultivars with nucellar seeds (Grosser and Gmitter,

2005, 1990b).

Protoplasts are isolated from two donors, typically one source from an embryogenic callus line and the other isolated from leaf mesophyll, as the leaf mesophyll protoplasts will not continue to develop unless fused, reducing the number of potential unfused plants in the offspring (Guo and Grosser, 2005). Protoplasts can be

fused either using polyethylene glycol (PEG) or by electroporation (Grosser et al.,

2010). The process creates allotetraploids and autotetraploids, which can be used in breeding programs to create seedless, triploid cultivars, or in breeding of tetraploid rootstocks (Grosser et al., 2010; Grosser and Omar, 2011).

Seedless Citrus

Citrus fruits are naturally seedy, with some cultivars excessively so. Consumer

preference has shifted to seedless cultivars, and current breeding now prioritizes this as a desired trait in new cultivars (Asins et al. 2015; Gmitter et al. 2012). Seeds may also

contribute to bitter off-flavors in juice, leading juice producers to also favor seedless fruit

(Ollitrault et al., 2006). Many cultivars of citrus will set fruit through parthenocarpy (i.e.,

fruit formation without fertilization or embryo abortion), without any external stimulation

and produce seedless fruit. Some cultivars have facultative parthenocarpy, producing

seedless fruit only if grown in isolated stands; if allowed to cross-pollinate with other

citrus, these cultivars will be seedy (Gambetta et al., 2013; Mesejo et al., 2013). Best

results can be attained when combining parthenocarpy with self-incompatibilty or male

sterility, as this increases the amount of seedless fruit (Vardi et al., 2008). The Makaku

Kishu mandarin is genetically seedless, which some breeding programs are using to

generate new, seedless cultivars (Chavez and Chaparro, 2011; Yamasaki et al., 2008).

A third method is to combine parthenocarpy with triploidy, which has been successfully

used in other horticultural crops to produce seedless fruit, such as watermelon (Thayyil

et al., 2016) and commercial bananas.

Interploid Hybridization

Triploids’ three sets of chromosomes make it very unlikely for meiosis to produce

fertile gametes, which causes pollen and ovule abortion resulting in seedless fruit (Frost

and Soost, 1968). Triploids can exhibit some fertility, producing low numbers of haploid,

diploid, and triploid gametes (Otto and Whitton, 2000). Some diploid varieties naturally

produce some 2n gametes, which is probably how the triploid Persian and Bearss limes

arose (Bacchi, 1940). Triploid citrus can also be produced by hybridizing natural or

induced tetraploids with diploids (Aleza et al., 2009).

Differences in ploidy will cause abnormalities in seed development, especially

when the seed parent is diploid, resulting in underdeveloped or aborted seeds (Navarro

et al., 2003). The endosperm balance number hypothesis offers an explanation of

making successful crosses with mismatched ploidy; it suggests that there must be a two

to one maternal to paternal ratio in the endosperm for normal development (4x × 2x

(pentaploid endosperm) as opposed to 2x × 4x (4x endosperm)) (Carputo et al., 1999;

Ollitrault et al., 2006).

Researchers have discovered that most angiosperms have double fertilization: a pollen cell will fertilize both the haploid egg cell and the diploid endosperm, resulting in triploid endosperm (Russell, 1992). If there is a ploidy mismatch that does not meet that

ratio, then endosperm does not develop normally resulting in underdeveloped or small

seeds, if they do not abort (Carputo et al., 1999). These seeds can be salvaged using

embryo rescue and culturing them in vitro (Aleza et al., 2010; Viloria et al., 2005).

Normal seeds containing triploid embryos have been shown to have pentaploid

endosperm, which seems to confirm the theory of the two to one endosperm balance

number applying to Citrus (Esen et al., 1979; Esen and Soost, 1977; Wakana et al.,

1982).

Tetraploid breeding parents in the University of Florida breeding program have been developed either from selecting naturally occurring autopolyploids (screening seedlings for tetraploid characteristics or from seeds from fruit with gigas chimeric

sectors), somatic fusions, sexual crosses of somatic fusions (tetrazygs), or artificially

doubling ploidy using colchicine (Bowman et al., 1991; Gmitter et al., 1991; Grosser et

al., 2014, 2007).

Cybrids in Citrus

Cytoplasmic hybrids, or cybrids, are an unequal fusion of two cells, wherein the nucleus

of one parent is lost following somatic fusion. The resulting cybrid will have a nucleus

from one parent, with cytoplasm (including the organelles) from one or both parents.

The process of creating somatic fusions will occasionally produce cybrids as a

byproduct (Grosser et al., 1996a; Satpute et al., 2015). It has been discovered that the

inheritance of organelles in such cybrids is usually random for plastids, but that the

mitochondria are inherited from the embryogenic line used in the fusion (Cabasson et

al., 2001; Grosser et al., 1996a; Moreira et al., 2000). Cybrids typically display

characteristics of the leaf parent used in fusions, and can sometimes be identified

visually. Cybrids will also be diploid or aneuploid and can be separated from somatic

fusions by flow cytometry. Molecular markers have been developed for mitochondrial

DNA, useful for differentiating hybrid from non-hybrid individuals using SSR markers

(Froelicher et al., 2010; Moreira et al., 2002, 2000). If genes are known to be located in

the cytoplasm, as in cytoplasmic male sterility, these could then be transferred without

changing fruiting characteristics and be useful in scion improvement (Guo et al., 2004).

The ‘Summer Gold’ grapefruit is a cybrid with cytoplasm from Dancy mandarin,

and the nucleus of a Ruby grapefruit. This resulted from an effort to generate triploids

directly by fusing protoplasts isolated from mandarin embryogenic callus with protoplasts isolated from grapefruit haploid pollen tetrads. Plants regenerated exhibited grapefruit morphology. These plants produce delicious grapefruit with an exceptionally

long harvest window.

Diseases of Citrus

While there are many diseases that affect citrus production today, the two with the most severe impact currently are citrus canker and Huanglongbing. After citrus canker was detected in Florida in 1910, and declared eradicated in 1933, reappeared in the late 1980’s, and again declared eradicated by 1994 (Gottwald et al., 2001a). It appeared again in Miami in 1995 and in Manatee county in 1996 (Gottwald et al.,

2001a, 2002). The state began a citrus canker eradication program in 1996, eliminating

trees from commercial and private plantings, angering both growers and home-owners

over the loss of their trees. The citrus canker eradication program was eventually

determined to be infeasible and the program terminated in 2008; it did not eradicate

citrus canker, which is now declared endemic to Florida (“USDA APHIS | Citrus

Canker,” 2016). Huanglongbing is the more destructive of the two diseases. While

citrus canker results in blemished, unsightly fruit which drop prematurely, it does not

negatively impact the health of the tree except in the most severe infections (Gottwald

et al., 2002). Fruits detected with citrus canker are restricted from being imported to

foreign countries, such as the EU and Japan, with one affected fruit causing an entire

shipment to be destroyed (Graham and Dewdney, 2010). Huanglongbing, however, not

only ruins the fruit, it also kills the tree. Solving these problems at a breeding level will

allow citrus growers to reduce costs of controlling these diseases and remain

competitive in a global market.

Citrus tristeza virus (CTV) became a problem after its vector, the brown citrus aphid (Toxoptera citricida Kirkaldy), was discovered in Florida in 1995 (Halbert and

Brown, 1996). Sour orange had been the most popular rootstock in Florida, and its susceptibility caused growers to choose alternative rootstocks while breeders worked at producing sour orange replacements (Ananthakrishnan et al., 2006; Grosser et al.,

2004; Voosen, 2014). CTV has become less of a problem as growers have planted other rootstocks (“Citrus Budwood Annual Report 2015-2016,” 2016), as well as growers spraying more for psyllid control, resulting in better control of the brown citrus aphid.

Citrus Canker

Citrus canker disease is caused by a gram-negative bacterium, Xanthomonas citri subsp. citri (Xcc)(Brunings and Gabriel, 2003), previously known as Xanthomonas

axonopodis pv. citri. Severe infection may cause defoliation, shoot dieback, and

premature fruit drop (Graham and Dewdney, 2010). Symptoms of citrus canker are

necrotic lesions found on the leaves, stems, and fruit. Infected areas appear as tan-

colored, raised, corky lesions with water-soaked margins surrounded by a yellow halo

(“2003 Florida Citrus Invasive Pest and Disease Identification Handbook,” 2003). Citrus

canker is spread primarily by wind and rain, and to a lesser extent birds and mammals,

as well as equipment and people. Citrus canker affects a wide variety of citrus species

and cultivars, and symptom severity is related to Xcc pathotype, host genotype, and

environmental conditions (Gottwald et al., 1993).

Screening for canker resistant cultivars of citrus began in the early twentieth

century when citrus canker made its first appearance on U.S. soil. The USDA then

began a study to screen citrus and citrus relatives to identify resistant varieties, begun in

1917 by George L. Peltier, which lasted for several years. In the first published report from 1918, he noted that growers regarded the oval, or Nagami, kumquat (F. margarita

‘Nagami’) as being resistant. His conclusion was similar; he was able to infect the oval kumquat, but the disease did not spread and the tree was not adversely affected. They found some related species to have what is regarded to be immunity, but were not closely related enough to be useful in a traditional breeding program, such as species in the genus Xanthophyllum and Glycosmis (Peltier, 1918).

Gottwald and Graham had similar results to the earlier study of Peltier: Citrus

from their study with high resistance included Nagami kumquat (F. margarita ‘Nagami’, only kumquat variety tested), calamondin (kumquat/mandarin hybrid), and etrog (Citrus medica L.). Trifoliate orange (Poncirus trifoliata) and its hybrids are particularly susceptible when wounded, followed by grapefruit. Specimens with high resistance or immunity also included citrus relatives Murraya paniculata Jack, and Severinia buxifolia

Jack (Gottwald et al., 1993).

A study screening for molecular markers associated with canker resistance identified the most resistant citrus cultivars as kumquat (cultivar not specified), ichang

papeda, yuzu, and mandarin (cultivar not specified) (Peng et al., 2010).

Citrus Canker Inoculation

Canker screening for determining susceptibility consists of inoculating the leaves

or fruit with liquid cultures of Xcc. Early inoculations consisted of pricking the leaves with

a pin and applying the inoculum to the surface of the leaf (Peltier, 1918; Peltier and

Frederich, 1924, 1920). Later it was discovered that direct inoculation with a syringe into

the leaf mesophyll gave more consistent disease infection (Stall et al., 1982, 1980). The

discovery of using a needless syringe pressed directly against the abaxial surface of the

leaf allowed for direct penetration into the mesophyll through the open stomates without causing damage to the surface of the leaf or fruit (Francis et al., 2010; Graham et al.,

2016a). Typically this is manually performed, with the user attempting to inject a similar sized area into the mesophyll, although an automated system using pressurized gas to precisely deliver consistent amounts of inoculum with a needless injector has been developed (Francis et al., 2011a). Leaves may be used in either attached or detached assays, although attached assays have shown higher levels of infection response compared to detached leaves (Francis et al., 2010).

Degree of infection has primarily consisted of counting lesions following inoculation (Bock et al., 2011; Francis et al., 2010; Graham et al., 2016a). Bock (2010) suggests using digital photography and hyperspectral imaging using software to analyze leaf area to accurately assess disease severity, if the means and budget allow for using high tech methods.

Huanglongbing

Huanglongbing (HLB), also known as citrus greening disease, is currently considered the most serious disease affecting citrus worldwide (Hall et al., 2013). It is thought to be caused by Candidatus Liberibacter asiaticus, characterized as a phloem- inhabiting α-proteobacteria, and is transmitted by two kinds of phloem-feeding citrus psyllids, Diaphorina citri and Trioza erytreae (Mead, 1977). In the U.S. it is vectored by the Asian citrus psyllid (ACP), Diaphorina citri, which was first detected in Florida at

Delray Beach, Palm County, FL, in 1998. It was likely spread to other areas of the state via the nursery trade, hitchhiking on citrus relatives in the ornamental trade, such as orange jessamine (Murraya paniculata) and insect migration possibly being assisted by hurricanes (Hall et al., 2013). Huanglongbing was not detected in Florida until 2005

(Halbert, 2005). By the time HLB was detected in Florida, the ACP was found in many areas of the state, and in a relatively short time the disease had spread to most citrus

production areas. Currently, Huanglongbing has been detected in 37 counties in Florida,

and is found in all major citrus production areas (Albritton, 2011).

Although new to the US, HLB has been known in China for over one hundred

years (Husain and Nath, 1927). It was described in South Africa in 1947, where it was

named Citrus Greening disease. When it was realized that Citrus greening was the

same disease as Huanglongbing in China, the earlier described name took precedence.

It was discovered in Brazil in 2004, and is now found throughout the Caribbean, having

spread to Cuba, Mexico, and several other islands, and threatens all commercial citrus

growing regions (Bové, 2014).

While there is variation of symptoms between citrus genotypes, common

symptoms of HLB resemble nutrient deficiencies, with leaves exhibiting yellowing of the

veins and surrounding tissues, followed by yellowing or mottling of the entire leaf, and

veins occasionally becoming corky (Halbert, 2005). This is followed by premature

defoliation and twig dieback. Affected fruit is frequently small, lopsided and bitter, fail to

ripen normally remaining green with a thick peel, and often abscise prematurely (Burrow

et al., 2015). Plants will show stunted growth, as well as frequent off-season flowering,

with most flowers falling off. As the disease progresses, trees become less productive,

and eventually the leaves dehisce and the tree dies (Su, 2001). Current control

strategies focus on eliminating psyllids through chemical means, but these treatments

are costly and generally considered less effective (Hall et al., 2013). Plant resistance to

HLB is one method that is being pursued, either by traditional breeding (Miles et al.,

2017; Ramadugu et al., 2016) or transgenic methods (Dutt et al., 2016; Zou et al.,

2017), but neither can offer immediate relief to the growing epidemic. Other experiments

and alternatives have been suggested and are in trial, such as spraying with

brassinosteroids (Canales et al., 2016), Kaolin clay to discourage psyllid feeding

(McKenzie et al., 2002), and using antibiotics either directly injected into the trunk or as

a foliar spray (Hu and Wang, 2016; Zhang et al., 2014).

Generally, sweet orange and grapefruit are very susceptible; some lemons and

limes display tolerance, evidenced by less severe symptoms and much slower or no

decline in both greenhouse and field plantings; and ungrafted Poncirus trifoliata appears

to be most tolerant to HLB (Fan et al., 2012). Rough lemon appears to have anatomical

differences that allow it to be less affected to CLas than other citrus, infected plants

showing fewer symptoms such as less phloem collapse and sieve cell blockage, less

starch accumulation, and greater survival of feeder roots (Fan et al., 2013, 2012).

Recent studies of citrus has shown that while all commercial cultivars of citrus are

susceptible to HLB, there are some differences in severity of infection, especially with

some relatives or species showing tolerance or immunity (Folimonova et al., 2009;

Ramadugu et al., 2016; Shokrollah, 2009).

CLas Detection

HLB symptoms are similar to other diseases and nutrient deficiency, and with a

long incubation time until trees develop symptoms, detection can be difficult (Burrow et

al., 2015). The most accepted method to confirm HLB is by using PCR with molecular

markers developed for detection of Candidatus Liberibacter asiaticus (Hocquellet et al.,

1999; Hung et al., 2004; Li et al., 2006; Teixeira et al., 2005).

Detection of CLas by real time PCR (or qPCR) is explained by the results of Ct

counts, which are Threshold Cycles, or the number of replication cycles required before

a sufficient quantity of DNA is produced so that the qPCR machine can detect the

fluorescent molecular tags and register a positive result (“Real-Time PCR,” n.d.). Ct values are inversely proportional to the amount DNA in the sample, with low Ct counts equivalent to higher amounts of nucleic acid. qPCR machines typically use forty cycles to amplify samples, with Ct values above thirty-six falling within the range of error and are considered negative (Caraguel et al., 2011). This could mean that a very small amount of bacterial DNA was detected or that there was some environmental contamination. Using thirty-six for the cut-off is an arbitrarily assigned number, and different numbers have been used as low as 32, ideally setting the value high enough to eliminate false positives and false negatives (Ladero et al., 2010). More recent advances have led not only to the detection of the bacteria, but also quantification of the bacteria to determine the extent of the bacterial population (Ladero et al., 2010; Li et al.,

2008; Wang et al., 2006).

Nematode Caused Diseases

There are a few species of plant pathogenic nematodes affecting the Citrus industry in Florida. The two most economically important are the citrus nematode

(Tylenchulus semipenetrans Cobb), which causes slow decline disease, and the burrowing nematode (Radopholus similis Cobb), which causes spreading decline disease (Duncan, 2005). A few other nematodes will attack the roots of citrus, including the sting nematode (Belonolaimus longicaudatus, Belonolaimus sp.) and the root knot nematode (Meloidogyne sp.), but both of these pests are not exclusive to citrus.

Feeding on the roots does not kill the citrus tree, but reduces the tree’s ability to uptake

nutrients and water, as well as reducing yield. If left untreated the tree will die over

time. Feeding also allows for the entry of other secondary diseases (Duncan, 2005).

Objectives

The overall objective of this research project is to evaluate pummelo genetic

diversity to develop grapefruit/pummelo triploid hybrids with disease resistance to

replace current disease-susceptible, seedy grapefruit, as well as investigate the

usefulness of the pummelos as interstocks to increase the disease resistance of

commercial sweet orange trees.

This research project aims to test three hypotheses:

H1: Pummelos displaying resistance to citrus in canker in the field will show

resistance when inoculated under greenhouse conditions, as necessary to employ rapid

greenhouse screening in the breeding program.

H2: Pummelos displaying resistance to citrus canker will pass on this trait to their

progeny when used as parents in interploid crosses to generate improved seedless

triploid progeny

H3: Prospective HLB tolerant pummelos and hybrids will improve the growth of

CLas infected scion and rootstock when used as an interstock in commercial sweet

orange/Swingle trees.

Florida Citrus Production 1915-2015 (in 1000s of boxes) 300,000 250,000 200,000 150,000 100,000 50,000 0 1915-1916 1918-1919 1921-1922 1924-1925 1927-1928 1930-1931 1933-1934 1936-1937 1939-1940 1942-1943 1945-1946 1948-1949 1951-1952 1954-1955 1957-1958 1960-1961 1963-1964 1966-1967 1969-1970 1972-1973 1975-1976 1978-1979 1981-1982 1984-1985 1987-1988 1990-1991 1993-1994 1996-1997 1999-2000 2002-2003 2005-2006 2008-2009 2011-2012 2014-2015

Oranges Grapefruit Other

Figure 2-1. Florida Citrus Production, 1915-2015 (in 1000s of boxes). Source: Florida Citrus Statistics, USDA-NASS (2016)

Figure 2-2. Pigmented grapefruit descended from Duncan grapefruit. (Adapted from Saunt, 2000)

CHAPTER 3 EVALUATION OF CITRUS CANKER RESISTANCE IN PUMMELO PARENT SELECTIONS

Background and Objective

Citrus canker is one of the most economically damaging diseases affecting citrus growers today, and is present in most citrus producing areas of the world (Graham et al., 2004; Pruvost et al., 2014). The bacteria enter through stomata or mechanical injury, affecting the leaves, fruits and stems forming raised pustules (Gottwald et al., 2002).

The bacteria is spread by multiple means, primarily from wind driven rain over short distances, or longer distances with severe storms (Dewdney and Graham, 2016).

Workers can spread the bacteria on hands, clothes and equipment, which can spread the bacteria to other groves. Citrus leafminer (Phyllocnistis citrella Stainton) also assists dispersal, allowing for entry and colonization of the bacteria in affected leaves. Once established in the grove citrus canker will cause defoliation, blemished fruit, premature fruit drop, and general tree decline (Gottwald et al., 2002; Graham et al., 2004). In an attempt to restrict the spread of the pathogen, quarantine areas have been established in Florida and elsewhere (Gottwald et al., 2002). In Florida, citrus plants and fruits require inspection and certification by the USDA before they can travel out of state

(Graham and Dewdney, 2010).

Citrus canker is also costly to control. In affected areas, people, vehicles and equipment must be decontaminated before entering groves, and infected material must be promptly disposed of when detected to further prevent the spread of the disease

(Gottwald et al., 2001b; Graham et al., 2004). Windbreaks also help to prevent the spread, as well spraying copper bactericidal sprays to protect young leaves and fruit,

pesticides for control of leaf miner, and planting of citrus canker tolerant varieties (Das and Singh, 2003; Graham and Dewdney, 2010).

The preferable way to control or prevent the spread of citrus canker is through

using cultivars with natural resistance or tolerance to the disease (de Carvalho et al.,

2015; Deng et al., 2010). While most commercial citrus is susceptible to citrus canker to

various degrees, grapefruits are highly susceptible to citrus canker and several disease

management strategies are required to successfully produce grapefruit in endemic

citrus canker areas (Graham et al., 2016a; Grosser and Gmitter, 2012; Peltier and

Frederich, 1920).

Conventional breeding approaches can use citrus germplasm as a source of resistance to citrus canker. Some non-commercial Citrus and citrus relatives have what

is referred to as ‘field resistance’ to citrus canker, where under typical field conditions

plants do not show any canker lesions. Calamondin (Citrofortunella microcarpa (Bunge)

Wijnands) and Meiwa and Nagami kumquats (Fortunella sp.) are regarded as highly

resistant (Deng et al., 2010; Khalaf et al., 2007; Peltier, 1918). Resistance of varying

degrees has been found in other sexually compatible species and selections, including

C. medica, C. maxima, and C. reticulata ( Ponkan, Satsuma, Tankan, Satsuma,

Cleopatra, Sunki, and Sun Chu Sha cultivars) (Deng et al., 2010; Gottwald et al., 2002).

The lack of genetic knowledge for the inheritance of important horticultural traits makes

it difficult to identify promising crosses (Navarro et al., 2004).

Plants suspected to be resistant to citrus canker must be screened to determine

their susceptibility. Several methods have been developed for disease screening of

citrus canker which can be divided into two categories depending on the type of assay

and the inoculation method. Disease screening may be done in vivo (attached leaf

assay) (Deng et al., 2010; Viloria et al., 2004) or in vitro (detached leaf assay) . Three

methods have been widely used for bacteria inoculation of citrus canker: pressure

infiltration (Francis et al., 2011b; Viloria et al., 2004; Zhang et al., 2010), pin puncture

(Deng et al., 2010; Gonçalves-Zuliani et al., 2016; Yang et al., 2011), and spray inoculation (Deng et al., 2010; Graham et al., 1992; Yang et al., 2011). Method of choice will depend on the material available, number of genotypes to test, replicates, and time available for the experiment. The in vivo method using pressure infiltration has proven to be an efficient method for citrus canker screening.

Most commercial citrus is susceptible to citrus canker, and it would be more

effective to cultivate selections that have been bred to have innate resistance to

infections of citrus canker. Naturally resistant cultivars would reduce economic losses

by reducing the requirements of control methods.

Pummelos are ancestors and genetic contributors to many modern cultivated

citrus, including oranges, grapefruit, tangelos, and other hybrid fruits. Identifying

pummelo varieties with increased resistance to citrus canker will be useful in developing

new citrus varieties. This study may lead to the development of important commercial

grapefruit-like varieties with resistance to citrus canker.

Grapefruit production has rapidly decreased with the introduction of both citrus

canker and Huanglongbing in the major areas of production within Florida, facing an

81% decrease in production since 2004 (Hudson, 2016; “NASS - Florida Citrus

Production Forecasts,” 2017). The new status quo, where citrus canker and

Huanglongbing are endemic, requires new resistant varieties to be developed (Grosser

and Gmitter, 2012).

It is necessary to identify genetic contributors with improved horticultural traits to

breed better grapefruit, from fruit attributes including flavor, fruit shape, peel color, flesh

color, and peel thickness, ºBrix, and titratable acidity, to physical traits of the tree,

especially resistance or tolerance to disease. Grapefruit is a chance hybrid between

pummelo and sweet orange, with sweet orange being an ancient admixture between

pummelo and mandarin (Wu et al., 2014). The University of Florida breeding program

has a genetically diverse collection of pummelos. These are further evaluated for

disease resistance or tolerance to identify parents for further breeding to create

grapefruit-like hybrids.

Materials and Methods

Material Background

In 1999 the Grosser lab initiated a project to select superior types of pummelo

seedlings to use in further experiments, with the main goal of producing disease

resistant rootstocks, specifically to create a synthetic sour orange (Ananthakrishnan et

al., 2006; Grosser et al., 2004). Seeds were collected from fruits of C. maxima (Burm.)

Merrill trees planted at the Department of Plant Industry Florida Citrus Arboretum in

Winter Haven, Florida, and from the home of a private individual (Table 3-1). In total,

200 seed each of eleven standard pummelos were planted directly into flats of a high

pH (8 to 8.5) calcareous Winder depressional soil collected directly from the Indian

River citrus production area in Fort Pierce, FL. (with higher clay content and pH than

typical Winder soil because it was from a double-bedded area), that was inoculated with

both Phytophthora nicotianae and P. palmivora. After three months, superior seedlings

were selected (approximately 250) and propagated for further study and use in breeding and fusion experiments (Ananthakrishnan et al., 2006; Grosser et al., 2004) Further

assays were conducted to determine the resistance to CTV of seedlings (Mohamed,

2009; Mohamed et al., 2008). Propagated trees were grafted with Valencia scion and planted at a challenging field site at the IRREC in Fort Pierce in cooperation with Bob

Pelosi in 2001. The purpose of this planting was to assess the rootstock ability of the selected pummelo germplasm. Immediately after planting, the site became infested with

Diaprepes root weevils and inadvertently became a screen for resistance to the

Diaprepes/Phytophthora complex. After three years in the field, the healthiest trees

were identified and twenty-five pummelo seedlings were selected for further study

(including an assay for CTV resistance) and use in somatic hybridization experiments

with selected mandarins to produce sour orange-like hybrids at the tetraploid level; one

of the standout performers in that trial was 5-1-99-2 S5, which was selected for this trial

(unpublished data).

These surviving pummelos were grafted to Swingle citrumelo rootstock and

planted in a University of Florida grove in Lake Alfred, FL. Of these surviving offspring

many interesting traits have been observed: various fruit shapes from oblate to pyriform,

flesh from white to red, many with delicious fruits, peels ranging from thick to thin, and

most interesting of all: trees with little to no symptoms of citrus canker or HLB in a grove

where both diseases are prevalent.

Pummelo Candidate Selection

Selections were made with priority given to trees that were free of citrus canker

and HLB symptoms, with other traits being secondary. Trees were rated on a scale from

0-5 for citrus canker and HLB symptoms, with 0 representing no visible symptoms, and

5 representing a heavily diseased tree. Fruit flesh color varied from white to pink to red

(Figure 3-3), and fruit size varied from 12-20+cm in diameter (Table 3-2). Peel color was generally yellow, with some varieties exhibiting a red blush (see 7-2-99-5, Figure 3-3).

Psyllid and pest control followed commercial recommendations, managed using

industry accepted practices. Citrus canker was not managed.

Beginning in the summer of 2013, field trees were rated for these relevant traits

and disease incidence. The CREC HLB scout (Luba Polonik) had previously flagged

trees not showing HLB symptoms. Trees observed to be asymptomatic of both citrus canker and HLB were selected for the initial canker challenge assay and later narrowed down to the top eleven, using ‘Marsh’, ‘Ruby Red’ and ‘Flame’ grapefruits as positive controls and ‘Nagami’ and ‘Meiwa’ kumquats as negative controls.

Bacterial culture and inoculum preparation

All trees were budded onto white grapefruit+trifoliate orange 50-7 somatic hybrid experimental rootstock in July/August 2013. Plants were cultivated in soilless medium

(The Scotts Co., Marysville, OH, USA) contained in 3.8 L pots and maintained in the greenhouse between 20 to 30°C temperature. Plants were fertilized twice a year with

NPK plus minor elements 12-3-9 controlled release fertilizer (Harrell’s LCC, Lakeland,

FL, USA).

When budded trees were over two feet tall in November 2013, all trees were placed in a citrus canker quarantine greenhouse and foliage trimmed to induce a flush of new growth. When new leaves had expanded to 75% full size, the leaves were inoculated with Xanthomonas citri subsp.citri (Xcc), at a concentration of 104 cfu ml-1

according to methods described by Francis, Pena, and Graham (2010).

The Xcc strain 2004-00054 was used for all inoculation assays. This strain was isolated in 2004 from sweet orange (C. sinensis) in Dade County, FL. The strain was stored in glycerol at −80°C. For inoculation assay, the Xcc culture stored in glycerol was

thawed and streaked on DifcoTM Nutrient Agar (0.3% beef extract, 0.5% peptone, 1.5%

agar). The plate was kept at 28°C for 48 to 72 hours. A single bacterial colony was

seeded into 25 ml of sterile DifcoTM Nutrient Broth (0.3% beef extract, 0.5% peptone)

and grown at 28°C for 24 hours at 150 rpm. This condition is required for Xcc to reach

the log phase, a stage at which the bacterium is actively growing. The bacterial

suspension was centrifuged at 5,000 g for 10 minutes at 4°C and re-suspended in

sterile saline phosphate buffer (PBS; 40 mM Na2HPO4+25 mM KH2PO4) and kept on

ice. The bacterial suspension was adjusted to 0.3 OD at 600 nm, equivalent to 108

colony-forming units (cfu) per ml. For attached leaf assay, the bacterial suspension was

diluted and adjusted to 104 colony-forming units (cfu) per ml. To confirm viability of the

bacterium, the suspension was serially diluted and 50 μl of the final dilution was spread

over two nutrient agar plates.(Graham et al., 2016b).

Leaf Inoculation

The plants were pruned to stimulate the growth of new flushes. The most

susceptible leaf stage was reached two to three weeks after pruning when immature

leaves were 75% expanded.

Bacterial suspension was pressure infiltrated in young leaves using a 1 cm3

needle-less tuberculin syringe. The syringe tip was pressed against the abaxial surface

of the leaf using a latex gloved hand as support. Bacterial suspension (approximately 2

μl) was infiltrated into the leaf until the water-soaked area reached about 6 mm in diameter

(Francis et al., 2010). Three injections were performed on each side of the mid-vein, five

leaves per plant (See Figure 3-4), and three plants each per pummelo variety, and two

plants each for control cultivars. Excess of inoculum was wiped from the leaf surface with

KIMTECH delicate task wipes (Kimberly-Clark, Koblenz, Germany). Five leaves per plant

and three plants per genotype were inoculated. The inoculated flushes were covered with

clear plastic bags for 24 hours to maintain high humidity. The plants were completely

randomized and kept in the greenhouse with temperature range from 20°C to 30°C.

Development of symptoms on leaves was observed weekly up to 28 dpi. The number of

lesions per leaf was counted at 14 dpi.

While number of lesions have typically been used in previous attached or detached

leaf assays (Francis et al., 2011a, 2010; Graham et al., 2016b), lesion size was also used

in this analysis. Citrus canker lesions have a raised irregular surface, growing vertically

as well as laterally, making it difficult to gauge infection based solely on a two-dimensional

analysis such as measuring lesion area. A five-point scale was developed to rate lesion

size based on width and height, by comparing lesions to a printed size scale, shown in

Table 3-3, rating the largest lesion per leaf. Lesion rating was performed four weeks post

inoculation Trees were inoculated three times over five months, in November 2014,

January 2015, and March 2015.

The number of lesions and lesion ratings were analyzed using using SAS JMP®

Pro 13 statistical software, using Tukey’s HSD. Multivariate canonical discrimination

method (CANDISC procedure) was performed using SAS 9.

Candidatus Liberibacter asiaticus Testing

Parent trees which appeared symptom-free were tested to determine if they were

infected with CLas. Three leaves were selected from each tree, which exhibited some

signs of nutrient deficiency, and sent to the University of Florida Plant Pathology HLB

Detection Lab to be analyzed using PCR in November 2014. Results are shown in

Table 3-3. Ct values of 36 or higher were considered negative. Testing was repeated in

December 2016, DNA was isolated from the petioles of three to four leaves gathered from each variety, weighed into 100µg portions, ground using liquid nitrogen and a

BeadBug Microtube Homogenizer. DNA was extracted and purified with GeneJET Plant

Genomic DNA Purification Kit (#K0722-Thermo Scientific), PCR was performed using

Applied Biosystems StepOnePlus™ Real-Time PCR system and quantitative TaqMan™

PCR using 16S rDNA-based TaqMan™ primer-probe sets specific to Ca. Liberobacter

asiaticus developed by Li et.al. (2006). Results were analyzed using Life Technologies

StepOne™ Software V2.3.

Results and Discussion

The main hypothesis in this study is that pummelos displaying resistance to citrus

in canker in the field will show resistance when inoculated under greenhouse conditions,

as necessary to employ rapid greenhouse screening in the breeding program. As

previously stated, grapefruits are highly susceptible to citrus canker, and losses

attributable to citrus canker can be quite severe and control is challenging (Bock et al.,

2011; Gottwald et al., 2002; Graham et al., 1992). Control of citrus canker for fresh

market grapefruit requires windbreaks to reduce wind penetration into the canopy and

copper sprays every three weeks to protect the growing fruit from infection (Dewdney

and Graham, 2016). Inoculation with Xcc has confirmed that ‘Marsh’, ‘Flame’, and ‘Ruby

Red’ grapefruit are highly susceptible to citrus canker. These selections produced large

lesions and pustules typical of citrus canker infection (Figure 3-4).

Lesion size on the pummelo selections varied from large lesions similar to those

found on grapefruit, to lesions that remained small and dark, and exhibited yellow halos

around the lesions (Figure 3-6). Citrus canker symptoms started to appear around 7 dpi

with Xcc at 104 cfu ml−1. The lesions are characterized by raised, light brown, canker-

like pustules typical of the compatible host reaction (Figure 3-5). Pummelos showed the

same typical canker lesions however the size of lesions varied (Figure 3-6). ‘Nagami’

kumquat showed few symptoms, more than ‘Meiwa’ and lower than all other candidates.

Most of ‘Meiwa’ kumquat leaves did not show any symptoms, but a few inoculated sites

showed slightly raised, dark brown pustules. No hypersensitive response was observed.

‘Marsh’, ‘Flame’ and ‘Ruby’ grapefruit varieties had consistently larger lesions in

all experiments (Rating >3 per leaf), although numbers of lesions were not as high as

some of the pummelo selections. C2-5-12 pummelo had the highest number of lesions

(Lesions >150 lesions per leaf). Numbers of lesions varied highly between pummelo

selections and series of inoculations (Figure 3-6), as did values for grapefruit selections.

‘Meiwa’ kumquat had the lowest number of lesions in all inoculations (1 to 20 lesions

per leaf) (Figure 3-4).

Some cultivars, while appearing to be resistant to citrus canker in the field, seem

to be susceptible from inoculations, showing increased severity compared to grapefruit

controls. This could be due to the method of infection, as trees in the field do not

typically have inoculum injected directly into the leaf mesophyll in high concentrations,

or perhaps from using a biotype of Xcc that is more virulent than the endemic variety in

our research grove. Efforts were made to inoculate leaves at the same stage in

development, although there may have been some variation as all plants did not grow at

the same rate. The first two inoculations took place during the winter months and while

trees were in a temperature controlled greenhouse they were not given supplemental

lighting and subject to reduced daylength; lesion severity may not have been as severe

compared to active growth in spring, however a correlation test between inoculations

showed that the there was little difference between inoculations (number of lesions:

r2=0.235467, rating of lesions: r2=0.069269) allowing the data to be pooled (Tables 3-4,

3-5). The ANOVA showed that there were significant differences for number of lesions

(L) and lesion rating (R) (P ≤ 0.0001), but slightly less for L x R interaction (P = 0.0117)

(Table 3-4).

The kumquat controls proved to be difficult to infect under lab conditions, showing few slow-growing lesions which remained small. Meiwa kumquat is classified

as highly resistant to citrus canker under field conditions, Nagami kumquat as resistant,

and both showed some infection but still less than the other citrus selections examined

(Francis et al., 2009; Kumar et al., 2013) (Figure 3-5, Table 3-5). Although kumquats

have been classified as genus Fortunella, researchers have used kumquats to study

inheritance of canker resistance due to their canker resistance and sexual compatibility

with citrus (Grosser et al., 2008; Kumar et al., 2013). The kumquat resistance to citrus

canker is characterized as a hypersensitive response (HR) reaction to Xcc infection that

blocks the bacterial proliferation in the plant (Francis et al., 2009; Trivedi and Wang,

2014).

Following leaf inoculation of ‘Meiwa’ kumquat with an Xcc suspension of 104 cfu ml-1, no canker lesions were observed on most of the inoculated leaves and just a few

leaves showed slightly raised dark brown lesions at the inoculation site (Figure 3-5). No

hypersensitive response was observed. The lack of HR reaction in this trial is explained

by the low bacterial concentration used for inoculation. Most of the studies that show

HR reaction in kumquat leaves used Xcc inoculum at 106 or 108 cfu ml-1, a 1000-fold concentration higher than used this in this experiment (Fu et al., 2012; Khalaf et al.,

2011, 2007). Even though HR reaction was not observed, kumquat is considered highly resistant to citrus canker based on the present study.

There was varied response of the grapefruit controls, although all could be infected and developed lesions typical of citrus canker. The grapefruits were not significantly different (95% confidence) from each other for both number of lesions and lesion rating. ‘Ruby Red’ had consistently larger lesions. C2-5-12 consistently had higher numbers of lesions that were intermediate in size, and was significantly different from both control groups (Tables 3-5, 3-6).

The hypothesis that trees that showed field resistance would show resistance to citrus canker inoculations was not clearly demonstrated, as all pummelos showed

increased infection than what was observed under field conditions. In comparison to

citrus canker resistant cultivars ‘Meiwa’ and ‘Nagami’ kumquats, all pummelos were

able to be infected. Number of lesions had less statistical differences at 95%, with little

separation between the pummelos and both control groups (Tables 3-5, 3-6).

Lesion size rating showed more separation, having a clear separation between

the two control groups, with all pummelos falling between the two control groups (Table

3-5). There was a wide range of response among the pummelos. 5-1-99-3 was the

closest to the kumquat controls for both reduced number of lesions and reduced size of

lesions; 5-1-99-3, 8-1-99-1A, and 5-1-99-2 S5 were the only pummelo selections that

were not significantly different (95% confidence) from Meiwa kumquat, and all were

significantly different from grapefruit controls (Tables 3-5, 3-6).

Combining the data showed less separation, the only clear separation was ‘Ruby

Red’ grapefruit from all of the other selections (Figure 3-7, Table 3-7). Lesion rating was the more useful for determining differences between selections.

While all trees showed increased infection over the kumquat controls, with little significant differences between lesion number for all selections, there were differences in lesion sizes. Many pummelos appear to be more tolerant to citrus canker, producing less and smaller lesions than grapefruit. No HR reactions were observed in any of the pummelo selections.

It is possible that the reduced appearance of citrus canker lesions on field trees may be a combination of circumstances, including leaves that are more resistant to bacterial penetration and growth flushes occurring when the bacteria is less active.

Trees showing field resistance to citrus canker is likely due in part to the physical structure of the leaves themselves as opposed to plant cellular defenses as in ‘Meiwa’ kumquat. Pummelos tend to have larger, thicker, more rigid leaves than grapefruit with a thick, waxy cuticle. Size of the stomatal pores may also contribute to the plants ability to exclude bacterial entry, as grapefruit have larger stomatal pores than other citrus varieties (Turrell, 1947) Leaves in citrus tend to last multiple seasons and are usually only susceptible to bacterial infiltration during a brief period in early development. The few citrus canker lesions observed on field trees of the pummelo selections appear to primarily originate from mechanical damage as a result of hedging.

There may be other genetic attributes which suppress the growth of citrus canker, as lesions in pummelo were generally smaller than those of the grapefruit controls (Table 3-5). There appears to be some degree of antibiosis to suppress the

growth of lesions. Figure 3-6 shows the size comparison of some of the typical lesions

found in the pummelo selections; the lesions of the best selections (5-1-99-3, 8-1-99-

1A) have smaller lesions that grew at a reduced rate, resulting lesions that smaller than

the grapefruit controls in the same time period (Figure 3-5).

The inoculation of citrus canker into the leaves of may not accurately reflect field performance of trees, but is useful for gauging resistance to Xcc infection when

compared to highly susceptible genotypes. The data appears to support the conclusion

that while the pummelos could be infected with Xcc, the infections were less severe

than grapefruits. Direct infiltration of Xcc into the leaf mesophyll is useful for determining

plant response to infection, however does not accurately reflect the physical properties

of the leaves which may help prevent entry of the bacteria. A screening method of

spraying inoculum onto leaf surfaces may be useful for determining the ability of the leaf

to resist infection, however this method is not preferred for most assays. This may be

due to the difficulty in controlling affected areas, maintaining consistent concentrations

and other variables affecting penetration and ability to gauge effectiveness and difficulty

in reproducibility. Attached leaf assay is an effective way to determine citrus canker

response, however size of lesions appear to be a better metric for evaluation.

While the greenhouse inoculations may not be directly correlated with observed

field resistance, there does appear to be value in performing the greenhouse screening

exercise to assist in determining value of individual pummelo selections for further use

as breeding parents. Trees in the grove largely all appeared to be relatively equivalent

in their responses to citrus canker, which could mean that they could be escapes or

there may have been a lack of ideal environmental conditions for infection during the

most susceptible leaf growth period (i.e. lack of rainfall during flush). Mature trees were observed as single replicates resulting in too small of a sample size to make adequate

conclusions of field resistance to citrus canker. Inoculations of Xcc to trees in the

greenhouse revealed more differences in response to infections of citrus canker,

showing variation in number and size of citrus canker lesions (Table 3-5), which appear

to show variation of the trees ability to prevent the bacteria from replicating within its

tissues. Under field conditions this could be replicated by entry points resulting from

mechanical or insect damage, allowing the bacteria to bypass the physical properties of

the leaf that may prevent infection. A combination of abilities to both prevent bacterial

infiltration and retard or inhibit bacterial replication within the tree would be ideal traits to

pass onto future generations.

Multivariate analysis gave a combined score for both lesion size and rating

(Table 3-7). All pummelos were significantly different (α=0.05) from Ruby Red

grapefruit, but not from the other grapefruit controls, and both Flame and Marsh were

also significantly different from Ruby Red. Most of the pummelos were also significantly

different from Meiwa kumquat except for 5-1-99-3. Pummelo 5-1-99-3 had the lowest

mean values of the pummelos for both numbers of lesions and lesion size rating (Tables

3-5, 3-6). It was not significantly different from Nagami kumquat, but was significantly different from all three grapefruit controls (Table 3-7). This analysis suggests the best pummelo for canker resistance is 5-1-99-3. Pummelo 5-4-99-7 had the next highest P- value of 0.0461 compared to Meiwa, followed pummelo 4-4-99-6 with a P-value 0.0262.

Both of these selections were significantly different from Ruby Red but not Flame or

Marsh grapefruit.

Under field conditions some of the selected pummelos initially appeared to be unaffected by HLB; trees in the grove had been previously marked as free of HLB symptoms by CREC HLB scout Luba Polonik. Parent trees which remained symptom free were tested to determine if they were infected with CLas. (Table 3-7) In 2013, no

CLas bacteria were detected in two pummelo selections, 5-4-99-3 and 8-1-99-1B, while two had low levels to be considered HLB negative (5-4-99-7 and 5-1-99-2 S5) using a

Ct value threshold of 36. The cause behind this absence of bacterium or low level of infection is unknown but surrounding trees show symptoms and decline, so it’s not likely an escape scenario. One possibility is that some trees are less attractive (or more resistant) to the psyllid vector, or perhaps there is an interaction with the rootstock, as all of the field pummelos are grafted, however psyllid feeding preference tests were not performed. Some of the initial selections have since shown HLB symptoms (i.e. 5-4-99-

3, 8-1-99-1B), and it may just be a matter of time before all of the promising pummelo selections become infected. Only 5-4-99-7 may still be considered negative, with a Ct value of 36.5617 in December 2016. Bacteria is still being detected, but at a very low level, and similar to its previous result in 2014 (36.5024). The tree continues to appear healthy.

Table 3-1. Parental Cultivars of Pummelo (C. maxima) Tree I.D. Parent Pummelo Variety (Origin) % Seedling Survival* DPI 8-1 Liang Ping Yau sdlg. (China) 69% DPI 7-2 Large Pink Pummelo (SE Asia) 67% HBJL Hirado Buntan sdlg. (Japan) 56% DPI 5-4 Red Shaddock Pummelo (SE Asia) 47% DPI 4-3 Sha Tian You sdlg. (China) 30% DPI 7-3 Siamese Sweet (Thailand) 27% DPI 4-4 Siamese Pummelo (Thailand) 18% DPI 5-1 Hirado Buntan sdlg. (Japan) 17% DPI 8-2 Pummelo NW (Florida) 12% DPI 7-1 Chinese Pummelo (China) 4% DPI 6-1 Kao Phuang (Thailand) 0% *Survival data from Grosser (2014).

Table 3-2. Pummelo selections for citrus canker study. Citrus canker ratings and HLB flagged if symptomatic (0-5, 0=no lesions, 1=1-5 lesions found on tree, 2=5-15 lesions, 5=Many Lesions) made in summer 2013 and summer 2014. Fruit data averaged from 6 fruits of each variety in February 2013. Fruit Fruit Flesh Titratable 2013 2014 2013 2014 Selection Peel ºBrix Parent diameter Shape Color Acidity Canker Canker HLB HLB

necked- 10- Sha Tian 4-3-99-1 12-14 cm white 8.2 1.1 0 0 N N pyriform 12mm You sdlg. light 10- Siamese 4-4-99-6 15 cm obovoid 9.1 1.3 0 1 N N pink 12mm Pummelo Chandler 48-OP-01-3 15 cm obovoid pink 10mm 9.5 1 0 2 N N Pummelo Hirado 8- 5-1-99-2 S5 16-18 cm globose red 10.5 1.45 0 1 N N Buntan 10mm sdlg. Hirado 7- 5-1-99-3 15 cm obovoid pink 11 1.15 0 0 N N Buntan 10mm sdlg. 10- Red 5-4-99-3 15-20 cm obovoid pink 9 1 0 0 N N 18mm Shaddock obovoid- 12- Red 5-4-99-7 13-15 cm white 11 0.8 0 1 N N necked 20mm Shaddock Large 16- 7-2-99-5 15 cm obovoid pink 7.8 1.45 0 1 N Y Pink 20mm Pummelo globose- 15- Liang 8-1-99-1A 16-17 cm white 9 1.1 0 1 N N obovoid 20mm Ping Yau 10- Liang 8-1-99-1B 15-16 cm obovoid pink 10.1 0.7 0 0 N N 20mm Ping Yau Pink- 8- Liang C2-5-12 15-16 cm obovoid 10.1 1.24 0 0 N N red 18mm Ping Yau Hirado 12- HBJL-1 16-17 cm globose red 8.7 0.55 0 1 N N Buntan 15mm sdlg.

Table 3-3. Citrus Canker Lesion Rating Scale Rating Description 0 Lesions absent or very small and dark, to 0.5mm

1 0.5-1.0mm

2 1.0-1.5mm

3 1.5-2.0mm

4 Large, raised pustules. 2.0-2.5+mm

Table 3-4. F values and significant levels from ANOVA for number of lesions and lesion size rating for selections.z F Value and significance df F Value Prob>F Model 45 8.9499 P ≤ 0.0001 Lesions (L) 15 4.4251 P ≤ 0.0001 Lesion Size Rating (R) 15 16.3218 P ≤ 0.0001 Lesions x Rating (L x R) 15 2.0033 P = 0.0117 ZData from 11 pummelo selections, 3 grapefruit selections, and 2 kumquat selections.

Table 3-5. Mean comparison of number of lesions between pummelos, grapefruit controls, and kumquat controls. Grapefruit Controls Kumquat Controls Name Meanz ± SDy Ruby Red Flame Marsh Nagami Meiwa Ruby Red 39.50 ± 16.63 bc 0x 10.86 22.5 3.66 32.05 Flame 50.36 ± 54.02 bc 10.86 0 11.64 14.52 42.91 Marsh 62.00 ± 39.5 abc 22.5 11.64 0 26.16 54.55 C2-5-12 110.0 ± 114.1 a 70.47***w 59.61 47.97* 74.13*** 102.52*** 4-3-99-1 73.78 ± 79.26 ab 34.28 23.42 11.78 37.94 66.33** 48-OP-01-3 73.11 ± 65.87ab 33.61 22.75 11.11 37.27 65.66** 5-1-99-2 S5 62.86 ± 71.6 abc 23.36 12.5 0.86 27.02 55.41 5-4-99-3 56.86 ± 58.45 bc 17.36 6.5 5.14 21.02 49.41 8-1-99-1B 56.13 ± 54.6 bc 16.63 5.77 5.87 20.29 48.68 7-2-99-5 51.78 ± 71.5 bc 12.28 1.42 10.22 15.94 44.33 Pummelos 8-1-99-1A 49.50 ± 69.2 bc 10 0.86 12.5 13.66 42.05 4-4-99-6 42.30 ± 40.4 bc 2.8 8.06 19.7 6.46 34.85 5-4-99-7 40.73 ± 46.0 bc 1.23 9.63 21.27 4.89 33.28 5-1-99-3 21.83 ± 23.63 c 17.68 28.54 40.18 14.02 14.38 Nagami 35.84 ± 38.32 bc 3.66 14.52 26.16 0 28.39 Meiwa 7.450 ± 11.10 c 32.05 42.91 54.55 28.39 0 ZMean separation by column not connected by same letter are significantly different. Least squares mean analysis using Tukey’s HSD (α=0.05). YStandard deviation. XDif=Mean(i)–Mean(j). q*=3.44143, α=0.05 W*, **, and *** indicates significance at P ≤ 0.05, 0.01, and 0.001, respectively.

Table 3-6. Mean comparison of lesion rating between pummelos, grapefruit controls, and kumquat controls. Grapefruit Controls Kumquat Controls Name Meanz ± SDy Ruby Red Flame Marsh Nagami Meiwa Ruby Red 2.93±0.94 a 0x 0.0533 0.2762 2.2933*** 2.6333*** Flame 2.88±0.97 ab 0.0533 0 0.2229 2.24*** 2.58*** Marsh 2.66±0.91 abc 0.2762 0.2229 0 2.0171*** 2.3571*** 4-4-99-6 2.16±0.83 bcd 0.7742* 0.7209 0.4981 1.5191*** 1.8591*** 4-3-99-1 2.11±0.83 cd 0.8222** 0.7689* 0.546 1.4711*** 1.8111*** 7-2-99-5 2.09±1.13 cde 0.8444** 0.7911* 0.5683 1.4489 1.7889 5-4-99-7 1.89±0.89 def 1.0444*** 0.9911*** 0.7683 1.2489 1.5889 C2-5-12 1.86±1.09 def 1.0762* 1.0229*** 0.8 1.2171*** 1.5571*** 48-OP-01-3 1.69±0.73 defg 1.2444*** 1.1911*** 0.968*** 1.0489*** 1.3889*** 8-1-99-1B 1.44±1.41 fgh 1.4889*** 1.4356*** 1.2127 0.8044** 1.1444*** Pummelos 5-4-99-3 1.43±0.87 efgh 1.5009*** 1.4476*** 1.2247 0.7924* 1.1324*** 5-1-99-2 S5 1.09±0.56 ghi 1.8476*** 1.7943*** 1.571*** 0.4457 0.7857 8-1-99-1A 1.05±0.39 ghi 1.8833*** 1.83*** 1.607*** 0.41 0.75 5-1-99-3 0.98±0.48 hi 1.9583*** 1.905*** 1.6821*** 0.335 0.675 Nagami 0.64±0.49 i 2.2933*** 2.24*** 2.0171*** 0 0.34 Meiwa 0.3±0.47 i 2.6333*** 2.58*** 2.3571*** 0.34 0 ZMean separation by column not connected by same letter are significantly different. Least squares mean analysis using Tukey’s HSD (α=0.05). YStandard deviation. XDif=Mean(i)–Mean(j). q*=3.44143, α=0.05 W*, **, and *** indicates significance at P ≤ 0.05, 0.01, and 0.001, respectively.

Table 3-7. P-values and squared distances (Mahalanobis distances) from canonical discrimination procedure for pummelos and controls comparing number of lesions and lesion ratings for selections.

Meiwa Nagami Flame Marsh Ruby Red

P-value Sq. Dist. P-value Sq. Dist. P-value Sq. Dist. P-value Sq. Dist. P-value Sq. Dist. Meiwa 1 0 0.290 0 0.007 1 0.0009 1 <0.001 105 Nagami 0.289 0 1 0 0.093 0 0.0423 0 <0.001 104 4-3-99-1 <0.001 1 0.017 1 0.200 0 0.5433 0 <0.001 98 4-4-99-6 0.026 1 0.272 0 0.671 0 0.3172 0 <0.001 97 48-OP-01 <0.001 1 0.031 1 0.131 0 0.3601 0 <0.001 100 5-1-99-2 0.004 1 0.222 0 0.135 0 0.2277 0 <0.001 103 5-1-99-3 0.577 0 0.610 0 0.034 0 0.004 1 <0.001 102 5-4-99-3 0.009 1 0.316 0 0.304 0 0.3856 0 <0.001 101 5-4-99-7 0.046 0 0.415 0 0.497 0 0.2176 0 <0.001 98 7-2-99-5 0.008 1 0.201 0 0.703 0 0.6268 0 <0.001 97 8-1-99-1 0.008 1 0.376 0 0.177 0 0.1653 0 <0.001 102 C2-5-12 <0.001 3 <0.001 2 <0.001 1 0.0024 1 <0.001 102 Flame 0.007 1 0.093 0 1 0 0.7 0 <0.001 93 Marsh <0.001 1 0.042 0 1 0 1 0 <0.001 95 Ruby Red <0.001 105 <0.001 104 <0.001 93 <0.001 94.8749 1 0

Table 3-8. PCR results for CLas detection in pummelo samples ID Ct Value 2013 HLB Result Ct Value 2016 HLB Result 5-4-99-4 20.7865 POS 25.3551 POS 5-1-99-3 20.8685 POS 22.8782 POS UKP-1 21.7696 POS 22.5284 POS 8-1-99-1A 23.1326 POS 24.2651 POS HBJL-1 33.8471 POS 28.5942 POS HBJL-4 34.9564 POS 23.3691 POS 7-2-99-11 35.5183 POS 34.8945 POS 5-1-99-2 S5 36.1627 NEG 35.1045 POS 5-4-99-7 36.5024 NEG 36.5617 NEG 8-1-99-1B Undetected NEG 24.6965 POS 5-4-99-3 Undetected NEG 30.584 POS

Figure 3-1. Grapefruit with its genetic progenitors. From left: mandarin (Citrus reticulata), sweet orange (C. x sinensis), grapefruit (C. x paradisi), and pummelo (C. maxima). Photo by author.

Figure 3-2. Pummelo tree 5-4-99-7 in research grove, photo taken March 2015. Surrounding trees showing HLB deterioration while 5-4-99-7 remains asymptomatic and healthy. Photo by author.

Fruit Cross Section Fruit exterior Tree in situ

4-3-99-1

4-4-99-6

48-OP-01-3

5-1-99-2 S5 Figure 3-3. Pummelo fruits and tree habit. Photos by author.

5-4-99-3

5-4-99-7

7-2-99-5

8-1-99-1A Figure 3-3 (continued). Pummelo fruits and tree habit

8-1-99-1B

C2-5-12

HBJL-1 Figure 3-3 (continued). Pummelo fruits and tree habit

Figure 3-4. Citrus canker inoculation pattern.

Figure 3-5. Control leaves four weeks post inoculation. From left to right: Meiwa kumquat, Flame, Marsh, and Ruby Red grapefruits. Photos by author.

48-OP-01-3 4-3-99-1 4-4-99-6 5-1-99-2 S5

C2-5-12 5-4-99-3 (‘Monster’) 5-1-99-3 8-1-99-1A

Figure 3-6. Sampling of pummelo leaves showing citrus canker lesions four weeks post inoculation. Photos by author.

Figure 3-7. Multivariate canonical scatterplot comparing sum of lesions and lesion ratings for selections.

CHAPTER 4 INTERPLOID HYBRIDIZATION TO PRODUCE SEEDLESS TRIPLOID HYBRIDS OF PUMMELO AND GRAPEFRUIT

Background and Objective

Originating in the Caribbean, grapefruit (Citrus paradisi Macfadyen) is first

documented in the early 18th century. Genetic testing has shown that C. paradisi

Macfayden is not a true species, resulting from a chance hybridization of C. maxima

(Burm.) Merrill (pummelo) with C. sinensis (L.) Osb. (sweet orange). Grapefruit

improvement schemes typically have relied on mutations, as grapefruit is highly nucellar

and each seed is apomictic and polyembryonic. Grapefruit also has a narrow flavor

profile that consumers will accept as grapefruit; similar crosses between a pummelo and a

sweet orange may not necessarily produce a fruit that will have the same flavor profile,

favoring one of the parents more than the other.

Due to the increased cost of controlling bacterial diseases, natural or genetic

resistance to disease is the preferred method to combat diseases such as citrus canker

and Huanglongbing. Biotechnological methods have been used to overcome some of the

natural breeding barriers imposed by Citrus (Grosser et al., 2000; Grosser and Gmitter,

2012). Genetic transformation is the subject of many current Citrus research efforts

(Barbosa-Mendes et al., 2009; Dutt et al., 2016; Mendes et al., 2010; Zou et al., 2017),

however there are many obstacles facing the production of genetically modified organisms

(GMO) including public perception and deregulation to approve products for human

consumption (Maghari and Ardekani, 2011). For these reasons, it is preferred to produce new varieties through accepted traditional sexual breeding methods.

Most grapefruit development has been through naturally occurring or induced somatic mutations that have been recognized and subsequently propagated asexually,

such as the Star Ruby, Flame, Rio Red, and Ray Ruby grapefruits (Gmitter, 1995;

Graça et al., 2004; Saunt, 2000). Crosses of grapefruit with other Citrus have led to the

creation of new, useful cultivars such as the tangelo and orangelo (Gmitter, 1995;

Hodgson, 1967).

There is some precedence for producing fruit similar to grapefruit. The first named

grapefruit in Florida was the Triumph grapefruit named in 1884, but it is thought to be an

orangelo, a cross between orange and grapefruit (Hodgson, 1967). Fruit of similar

heritage to the grapefruit include the Attani of India, the Natsudaidai and Asahikun of

Japan, the Wheeny grapefruit, and Poorman Orange from Australia which has been

marketed in New Zealand as the ‘New Zealand grapefruit’. All have a flavor somewhat

similar to grapefruit, but all in general have an appearance that looks more like an orange

(Hodgson, 1967; Saunt, 2000).

Although it would be possible to recreate a grapefruit-like hybrid by using elite

cultivars of pummelo and sweet orange, this is difficult as citrus is highly heterozygous

and would result in highly variable offspring genotypes, most that would be quite seedy.

It would be time consuming and costly to screen the numbers of progeny required to

develop something that consumers may recognize as a grapefruit, but could be a viable

approach if interploid crosses can be designed to produce seedless triploid offspring. As

grapefruit is genetically a cross between pummelo and sweet orange, sweet orange

itself is an admixture of mandarin and pummelo, with the chloroplast genome of

pummelo, and shared alleles of mandarin (found in Ponkan, Willow-leaf, and

Huanglingmiao mandarins) across three-quarters of its genome (Wu et al., 2014). The

creation of a fruit that contains a higher proportion of pummelo may result in something

similar to grapefruit. Similar approaches have been used to create fruits that are similar

in appearance and flavor profile to grapefruit such as the grapefruit-pummelo hybrids

Oroblanco, Melogold, and UF 914 (Gmitter, 2015; Soost and Cameron, 1980, 1985).

These triploid hybrids are seedless and similar in flavor to grapefruit, and Oroblanco

and Melogold are marketed as grapefruit, although they have thicker peels and lack the

rich flesh color of pigmented grapefruit. UF 914 has good flavor and an attractive red

flesh color, but it has only recently been released by University of Florida, so there is not

yet data on how it will be regarded by the public. Initial impressions are good, with fruits

that are less bitter, sweet, and additionally have low furanocoumarins that interfere with

certain medications (Beach, 2012; Gmitter, 2015).

Focus on interploid crosses to improve disease resistance required in new

cultivars, with primary emphasis on improving canker tolerance, is the major objective of

this chapter, combined with focus on fruit quality. Availability of flowering parents was

influenced by the ongoing HLB endemic in Florida, with some correlation of healthy tree

flowering with natural HLB tolerance.

Materials and Methods

Interploid crosses were designed to combine complementary fruit attributes

observed in the parents. Initial crosses were conducted in 2014 (Table 4.1). Pollen

donors included tetraploid Hudson grapefruit (Grosser et al., 2014), using pollen

obtained from a tree growing in the Rapid Evaluation System (RES) at the Lake Alfred

CREC, and challenged by HLB under field conditions (Grosser and Gmitter, 2012). The

original tree was infected with HLB and died in 2016, but buds of the infected tree were

grafted to a vigorous ‘tetrazyg’ tetraploid rootstock: a+volk x Orange19 ((Citrus

amblycarpa + Volkamer lemon) x ((Nova mandarin + Hirado Buntan pummelo seedling)

x (Cleopatra mandarin + Argentine trifoliate orange)). Tetrazygs are zygotic offspring

from sexual crosses of somatic fusion parents that have the combined genetics of at

least four citrus cultivars. Subsequent growth of the grafted Hudson tetraploid appears

to have recovered with reduced or nonexistent HLB symptoms.

Tetraploid somatic hybrids used in interploid crosses targeting a fruit that was

grapefruit-like but possibly easy peeling were: ‘Nova’ mandarin hybrid + ‘Osceola’

mandarin hybrid and ‘Murcott’ tangor + ‘Dancy’ mandarin. Diploid pummelo females

utilized were selected from previous evaluations for increased disease resistance to

HLB and citrus canker (see chapter 3), using females that were blooming at the time of

crossing.

Azza Mohamed previously identified pummelos that were resistant to CTV from

the UF citrus collection (Mohamed, 2009; Mohamed et al., 2008). The only pummelo

that was used from her experiments was the red pummelo 5-1-99-2-S5, which is

showing excellent tolerance to HLB and still exhibiting few symptoms, although it has

been confirmed to be infected with CLas (see Table 3-7).

2015 crosses were significantly expanded, taking advantage of many selections

that had finally come through juvenility and were flowering (Table 4.2). Three

autotetraploid pummelo selections created with colchicine by Divia Kainth (Grosser et

al., 2014) began to flower in spring 2015: DV2, DV5, and 5-1-99-2 DV13. These were

created from open pollinated seed of red-fleshed pummelos and are likely

autotetraploids produced from zygotic diploids derived from Hirado Buntan pink

pummelo. All appeared to be HLB negative and exhibited few citrus canker lesions,

even though there was obvious high HLB and canker pressure in the surrounding grove.

Diploid grapefruit pollen of dark red grapefruit somaclones N11-3, N11-7, and

N11-15 were used for pollinating tetraploids to create seedless grapefruit-like hybrids.

Somaclone N11-11 was regenerated from ‘Ruby’ embryogenic callus. It has shown higher than average ˚Brix in the last few growing seasons, and the original tree is showing better tolerance to HLB as compared to other grapefruit somaclones (J.W.

Grosser, personal communication). N11-7 and N11-15 were regenerated from embryogenic callus derived from commercially purchased ‘Red Cooper’ dark red grapefruit from Texas (Red Cooper Grapefruit Co., Alamo, TX), and these two clones are also showing elevated ˚Brix. (J.W. Grosser, personal communication). Tetraploid pollen from the DV selections above was also applied to some diploid cultivars, on both elite pummelos and mandarin-like hybrids, in efforts to produce a synthetic grapefruit.

The Liang Ping Yau pummelo growing in the Florida Department of Plant

Industry Citrus Arboretum has shown very little HLB symptoms in a highly affected

grove, and is a parent of the 8-1 series of pummelo offspring which have appeared to

be HLB and citrus canker resistant. The tree was pollinated with several cultivars using

both tetraploid and diploid pollen but all attempts were unsuccessful in producing a

single fruit (Table 4-2).

Two additional pummelo-orange tetraploids were also used, both of which have shown good tolerance to HLB and produce zygotic seeds (Figure 4.1):

• C2-4-1 is a tetraploid resulting from an interploid cross of a somatic hybrid Succari

sweet orange + Hirado Buntan pummelo seedling, with Hirado Buntan pink

pummelo. Fruit are orange-like in appearance and tending to be orange-like in flavor

but very acidic, although acidity does decrease through the season. This tree was

severely infected with HLB sometime prior to 2010 but after applying controlled

release fertilizer with micro-elements the tree has rebounded with stable health,

showing very few symptoms. This selection is not tolerant to citrus canker.

• Murcott+Chandler80 is a somatic hybrid that resulted from a fusion of Murcott

mandarin with a Chandler pummelo open-pollinated seedling #80. Trees at two

locations in groves with heavy HLB pressure continue to exhibit good health and

very few HLB symptoms. Fruit also appear more orange-like, but more grapefruit-like

in flavor, exhibiting a bitter flavor. Trees are quite tolerant of citrus canker, showing a

low number of small lesions.

Easy Peel Grapefruit

In order to create an easy-peel grapefruit-like hybrid, crosses were made using

several good tasting, Ambersweet orange seedlings derived from open-pollination,

showing good HLB tolerance and no citrus canker lesions (produced by W. S. Castle).

Ambersweet is a hybrid of Clementine tangerine by Orlando tangelo crossed with a

seedling mid-season sweet orange (Hearn, 1989). Stored pollen from the 4x pummelos

was applied to several trees, and seeds recovered using embryo rescue techniques.

Reciprocal crosses to the tetraploid pummelos were not able to be accomplished due to

asynchronous flowering of the parents.

Crosses were also attempted using tetraploid Hudson pollen on mandarin

selections held at the Florida Department of Plant Industry Citrus Arboretum using

Monreal, Marisol, Nules, and Fina Sodea Clementines, all of which were unsuccessful.

Crosses were attempted a using an Okitsu somaclone that had been labeled as

tetraploid but later testing revealed that it was diploid; regardless, all seeds recovered

produced diploid polyembryonic, nucellar seedlings. Temple Tangor was also used, but

did not produce any fruits.

Embryo Rescue for Interploid Crosses

Diploid pummelos as parents in interploid crosses requires the use of embryo

rescue to obtain seedlings, due to imbalance of the endosperm balance number (EBN)

causing insufficient or absent endosperm (Shen et al., 2011). Fruit from diploid

pummelos were allowed to grow and develop until August when fruit were harvested

and surface sterilized using a solution of 20% bleach and four drops of Tween® 20

(Sigma Aldrich) per liter, submerging fruit for thirty minutes. Fruit were then cut open

inside of a laminar flow hood and seeds extracted. The seed coat was removed and

embryos placed on 13 mm cellulose acetate discs (Fisher Scientific) and allowed to

germinate on EME-Maltose medium (Figure 4-2). Germinating seeds were then transferred to Magenta boxes containing 100mL of rooting medium. When seedlings had grown to the tops of the magenta boxes and had a sufficient quantity of roots, they

were transferred to soil and covered in plastic bags and allowed to acclimate indoors

under fluorescent lights for one week. During this period the plastic covering was

opened and the humidity reduced until the plant adjusted and the plastic was completely

removed. When plants were hardened off they were transferred to the greenhouse.

Flow Cytometry for Ploidy Determination

Flow cytometry is a simplified way to ascertain nuclear DNA content, measuring

the reflectance of a nuclear stain to determine ploidy level, and well established in

Citrus ploidy determination (Arumuganathan and Earle, 1991; Lucretti et al., 1990;

Omar et al., 2016). Seedlings were analyzed for ploidy using a Partec PA tabletop flow

cytometer (Sysmex Partec GmbH, Görlitz, Germany). Approximately 100µg of leaf

tissue from a fully expanded leaf was finely chopped using a straight edge razor blade with .5mL of Sysmex CyStain UV Precise P Nuclei Extraction Buffer (Sysmex Partec

GmbH, Görlitz, Germany). The finely chopped leaf and solution were then passed

through a CellTrics 50µm strainer (Sysmex America) and then added 0.5mL Sysmex

CyStain UV Precise P “automate” staining buffer. Key lime (2x), Persian lime (3x), and

Giant Key lime (4x) were used as internal standards to calibrate the machine and

determine ploidy level of test subjects.

Fruits from remaining tetraploid x diploid crosses were harvested between

September and October and germinated in soilless medium (The Scotts Co., Marysville,

OH, USA) and maintained in the greenhouse between 20 to 30°C temperature (Figure

4-3). When seedlings had two to three fully expanded leaves, flow cytometry was performed to determine ploidy status.

Seedling Grafting

Triploid seedlings were grafted while still small, when the seedlings had at least four mature leaves. The tip of each plant was severed and divided into three nodes which were then wedge grafted into the tip of Orange 4 and Orange 14 experimental

‘tetrazyg’ rootstocks and wrapped with parafilm. Grafted trees were grown in 6.25 cm

(2.5 in) diameter deepots (Stuewe and Sons, Tangent, Oregon). Trees from 2014 were transferred to 10 cm (4 in) deepots (Stuewe and Sons, Tangent, Oregon).

Citrus Canker Inoculations

When grafted seedlings had five to six newly expanded leaves they were moved

to a citrus canker approved greenhouse and subjected to citrus canker inoculations, testing seedlings from 2014 and 2015. The most susceptible leaf stage was reached two to three weeks after pruning when immature leaves were 75% expanded. The

leaves were then inoculated with Xanthomonas citri subsp.citri (Xcc), Xcc strain 2004-

00054 was used for all inoculation assays, at a concentration of 104 cfu ml-1 according

to methods described by Francis, Pena, and Graham (2010) and described in the

previous chapter. Inoculations were performed at three time points: May 26, June 28,

and August 16, 2016, in an air-conditioned citrus canker quarantine greenhouse

maintained at 29 C (85˚F).

Infection was rated after 14 days by counting number of lesions per inoculation

site, and rating lesion size of largest lesion on each leaf from 0-4 using a printed scale

for comparison (Table 3-3). Following counting of lesions inoculated leaves were

removed from the plant.

Statistical analysis was performed using SAS® 9.4 and JMP® PRO 13 using

Tukey’s HSD and canonical multivariate discrimination methods and an alpha of 0.05.

Results and Discussion

A population of seedlings were confirmed to be triploid through flow cytometry.

Seedlings have displayed good vigor and appear to demonstrate polyploid features,

such as thicker leaves and roots. Some of the seedlings with DV5 as a parent have also

shown reduced thorniness, an unusual trait for juvenile trees. Many of the attempted

crosses did not result in any fruits, and many of the seedlings that were obtained did not

appear to be hybrid based on their morphology, even though flowers were emasculated

before controlled pollinations (Tables 4-1, 4-2). It had been assumed that flowers devoid

of petals would be unattractive to pollinators as this technique has worked successfully

with other controlled pollinations in Citrus, however pummelo flowers are large and

produce abundant nectar and it appears that bees or other insects may still have visited

the flowers. Thrips could also be involved, as high populations of thrips were observed

on the pummelo flowers. It is also possible the flowers are cleistogamous, self-

pollinating before flowers opened. It is known that there are self-compatible varieties of

pummelos (Yamamoto et al., 2006), and cleistogamy has been reported in citron (Curk

et al., 2016), however cleistogamy in pummelo has not been previously documented or

little reported. Hybrid status was confirmed using flow cytometry (Figure 4-3). Seedlings

with the same ploidy level as their female parent were discarded.

Both the Ambersweet seedling-derived trees and tetraploid pummelo DV13

produced apomictic nucellar seeds when used as females. DV13 is the result of a

colchicine doubled, open-pollinated Hirado Buntan pink pummelo seedling and appears to have crossed with an orange, producing large, orange colored fruit and nucellar seed.

Interestingly, 5-1-99-2 S5 also produced seedlings that were more than 3x obtained from embryo rescue of seeds, producing two 4x seedlings (5-1-99-2S5 x Hudson4x-14-

15, 5-1-99-2S5 x Hudson4x-14-18) and one that was 5x or more (peak was to the right of 4x). Unfortunately, the 5x seedling was not vigorous and died before it was to be grafted. It would appear that 5-1-99-2 S5 has the ability to produce 2n unreduced gametes, which could be useful to develop triploid seedlings using pollen from diploid cultivars, or for further use in tetraploid parent breeding.

Interploid crosses that produced many large seeds (specifically 5-1-99-2 S5 x

Huds 4x, HBJL-1 x Valencia 4x, DV5 x N11-3, Murc+Chan #80 4x x 5-4-99-3, C2-4-1 4x x 5-1-99-2 S5) were planted in the greenhouse in seedling trays using standard potting soil. Over the course of one weekend rodents had unexpectedly eaten or damaged large numbers of the seeds before precautions were taken to stop further predation, such as covering the seeds with tray covers and placement of poison bait.

Citrus Canker Inoculation

Seedlings displayed a wide variety of response to citrus canker inoculations.

Many of the plants appeared to perform nearly as well as the negative control and the pummelo parent, while most had less or smaller lesions than the Ruby Red and Flame

grapefruit controls. ANOVA testing confirmed the presence of significant variation in the

attributes observed (Table 4-3). The triploid hybrids in this test were from pummelo

females 5-1-99-2 S5, 5-4-99-3, 5-4-99-7, and 8-1-99-2B. However, due to uneven growth flushes, only pummelos 5-1-99-2 S5 and 5-4-99-3 were included in repeated inoculations. Triploid seedlings with single inoculations (only one replicate per assay), were removed from statistical analysis due to too few data points; seedlings and controls used for the statistical analysis are shown in Tables 4-4, 4-5, 4-6, and in Figure

4-7.

While many triploid seedlings were inoculated with citrus canker, there were a

number that were not able to be tested, either due to failure of being successfully

grafted, or due to poor growth following grafting. Some that were tested once were

unable to be tested further due to lack of subsequent growth. This was probably mostly

due to genetic weaknesses in these hybrids.

Size and number of lesion response resulting from each inoculation did vary per

clone, although many showed consistency between tests. There was little correlation

between number of lesions and size of lesions, only a few seedlings with large lesions

also had high numbers of lesions (Ratings over 3.4 and lesions greater than 200 per

leaf: DV5xN11-3-15-06, 8-1-99-2BxHuds4x-14-03, 5-1-99-2xHuds4x-14-18,

M+Chan80xN11-3-15-04, DV2xN11-7-15-03).

Number of lesions showed little significant variation (α=0.05, 0.1, and 0.15; Table

4-4) between controls and seedlings, with only 5-1-99-2xHuds4x-14-18 showing a significant difference from the Meiwa kumquat control. Lesion ratings showed more variation (Table 4-5) with Meiwa being significantly different from the majority of seedlings. Only DV2xN11-3-15-01 was significantly different (α=0.05) from Flame grapefruit.

While many seedlings were not significantly different from the controls for number of lesions, more significant differences (a=0.05) were found when both number of lesions and lesion rating were analyzed using a multivariate canonical discrimination analysis (Table 4-6). Both grapefruit controls and Meiwa kumquat were significantly different from many of the seedlings. Ideal candidates that should have improved canker lesion response will be significantly different from the grapefruit controls but not from the meiwa kumquat control. Table 4-7 lists the seedlings that display these criteria.

These seedlings will need to be further evaluated under field conditions to determine if the greenhouse inoculations are indicative of field resistance to citrus canker.

Some seedlings were prone to dropping their leaves after being inoculated. The triploid seedling M+Chan80xN11-3-15-02 dropped the most leaves (Table 4-7), and had a 50% rate of dropped leaves; it had high numbers of lesions and an average rating for lesion size. The selections that dropped 100% of their leaves, also only had one inoculation and consisted of single replicate. These selections were poor growers and were unable to be inoculated more than once as a result. It is difficult to equivocate leaf dropping with resistance to citrus canker, as more inoculations would be needed to

show that these selections consistently drop leaves when inoculated, but some may

prove to have a reaction similar to a hypersensitive reaction.

Seedling data may not have been in sufficient quantity to make good

determinations of a seedlings ability to resist infection of citrus canker. Some seedlings

only had one seedling for each assay. However, the seedlings could be evaluated as

groups to determine if the groups of certain parental crosses were significantly different

from other parental combinations.

Testing for inheritance of canker tolerance in families showed that some parents

passed on their canker resistance to their offspring better than others (Tables 4-8, 4-9).

When examining number of lesions and lesion rating separately, some families

appeared to perform better than others, shown in Table 4-8. Using these criteria

separately it seems the best parental combinations were M+Chan80 x N11-7,

M+Chan80 x 7-2-99-11, and M+Chan80 x 5-4-99-3, all showing reduced number of

lesions and smaller lesions that were significantly different from Ruby Red and Flame

grapefruits. M+Chan80 has good resistance to citrus canker, and the offspring

collectively appear to retain this ability, especially when combined with a canker

resistant parent like 5-4-99-3. Unfortunately, M+Chan80 fruit has a light colored flesh,

so it should be crossed with dark red selections in future crosses.

Table 4-9 uses a multivariate canonical discrimination to compare groups, using

both number of lesions and lesion rating. M+Chan80x7-2-99-11 was the best parental

combination, being significantly different from Flame and Ruby Red grapefruit controls,

and not significantly different from the pummelo 5-4-99-3. This combination appears to

be improved over the grapefruit controls, but not as improved as the Meiwa kumquat. All

parental combinations had less lesions than the grapefruit controls, although most were not significantly different at the 95% confidence level, with the exception of M+Chan80 x

7-2-99-11. The combination of M+Chan80 x N11-3 was significantly different from Ruby

Red, pummelo 5-4-99-3, and Meiwa kumquat, but not Flame.

Grafted plants will be planted in the field for further evaluation under high canker

pressure field conditions. Greenhouse inoculations may not accurately represent field

performance, as shown by the parent pummelo trees 5-4-99-3, 5-1-99-2, and 5-4-99-7,

which have continued to appear blemish free from citrus canker in a high canker

pressure field site. Further testing is warranted to determine triploid seedling tolerance

or resistance to CLas infection given that HLB is now endemic to Florida and continued

success of citrus will be dependent on introducing new cultivars with improved

resistance or tolerance to this disease.

Preliminary Evaluation of Field trees

Nine of the triploid hybrids produced from 2014 crosses reached sufficient size

for field planting and were planted into a research grove in Lake Alfred in Summer 2016,

prior to the completion of the citrus canker screening (Table 4-11, highlighted in Figure

4-8). Trees are located on an end row with no wind screens and and no citrus canker

control, under heavy canker pressure. Trees are planted in rows that include heavily

symptomatic Rio-Red grapefruit, and are also next to a block of grapefruit trees where

citrus canker is abundant, creating ample opportunity for infection. These trees are able

to offer some preliminary field data (Figure 4-10) to either confirm or refute lab data.

The field trees of 4-4-99-7 x Huds4x-14-11 and 4-4-99-7 x Huds4x-14-13 both

had low numbers of inoculations and are found in Figure 4-8, which shows all tested

offspring. 5-1-99-2 S5 x Huds4x-14-9 had two clones field planted with one showing

some lesions and the other very few. The only field planted tree to show no canker infection is 5-1-99-2 S5 x Huds4x-14-10, which is better than grapefruit controls for number of lesions and better than all of its full siblings that were also field planted, with

5-1-99-2 S5 x Huds4x-14-9 being the next closest (Figure 4-8); one clone had few

lesions while a second clone had many lesions This data shows some correlation with

the greenhouse data. However, sample size is not large enough to draw definite

conclusions. More robust field trials of the hybrids are planned for planting in 2018.

Table 4-1. 2014 Crosses to generate seedless, triploid progeny. Number of seedlings and associated ploidy in right columns. Large Small No. of Seedlings Female Male Fruit Seeds Seeds 2x 3x 4x 5x 5-4-99-3 Hudson 4x 0 0 0 0 0 0 0 5-1-99-2 S5 Hudson 4x 1 10 30 10 23 2 1 5-1-99-2 S5 Nova + Osceola 0 0 0 0 0 0 0 5-1-99-2 S5 Murcott + Dancy 0 0 0 0 0 0 0 8-1-99-1B Hudson 4x 1 17 3 17 3 0 0 8-1-99-2A Nova + Osceola 1 1 0 0 0 0 0 8-1-99-2A Murcott + Dancy 0 0 0 0 0 0 0 8-1-99-2A Hudson 4x 1 35 0 35 0 0 0 4-4-99-7 Hudson 4x 1 5 25 5 17 0 0 4-4-99-7 Nova + Osceola 0 0 0 0 0 0 0 4-4-99-7 Murcott + Dancy 0 0 0 0 0 0 0 Total 5 67 58 67 43 2 1

100

Table 4-2. Spring 2015 Crosses to generate seedless, triploid progeny. Number of seedlings and associated ploidy in right columns. No. of Seedlings Large Small 2x 3x 4x Female Male Fruit Seeds Seeds 4-4-99-7 Hudson 4x 0 - - - - - 5-1-99-2 S5 Hudson 4x 6 258Z 20 119 4 0 5-4-99-7 DV5 2 0 0 0 0 0 7-2-99-11 DV5 0 - - - - - 7-3-99-2 Hudson 4x 1 0 0 0 0 0 Ambersweet Sdlg 2 DV5 1 3 0 4 0 0 Ambersweet Sdlg 2 DV13 1 3 0 3 0 0 Ambersweet Sdlg 6 DV5 2 14 0 12 0 0 Ambersweet Sdlg 6 DV13 1 3 0 3 0 0 Ambersweet Sdlg 11 DV5 1 5 0 6 0 0 Ambersweet Sdlg 11 DV13 2 2 0 2 0 0 C2-4-1 4x 5-4-99-3 2 18 0 0 0 17 C2-4-1 4x 5-1-99-2 S5 5 48 0 0 0 36 C2-4-1 4x N11-3 2 22 0 0 11 10 DV2 N11-3 0 - 0 - - - DV2 N11-7 1 23 0 0 21 0 DV5 N11-3 3 130Z 0 0 11 20 DV5 N11-7 0 - 0 - - - DV13 N11-3 0 - 0 - - - DV13 N11-7 2 21 0 0 0 20 Fina Sodea DV5 0 - - - - - HBJL-1 DV5 3 45 2 45 2 0 HBJL-1 Valencia 4x 2 140Z 0 36 0 0 HBJL-4 N11-7 0 - - - - - HBJL-4 DV5 1 1 0 1 0 0 Liang Ping Yau Furr 4x 0 - - - - - Liang Ping Yau Hudson 4x 0 - - - - - Liang Ping Yau DV5 0 - - - - - Liang Ping Yau N11-7 0 - - - - - 5-4-99-3 Hudson 4x 0 - - - - - 5-4-99-3 DV5 1 2 4 2 4 0 Marisol DV5 0 - - - - - Monreal DV5 0 - - - - - Murc+Chan #80 4x 5-4-99-3 4 115Z 30 1 23 0 Murc+Chan #80 4x 5-1-99-2 S5 0 0 0 - - - Murc+Chan #80 4x 5-4-99-7 2 32 0 20 12 0 Murc+Chan #80 4x 7-2-99-11 1 10 0 0 10 0 Murc+Chan #80 4x N11-3 4 26 0 0 9 17 Murc+Chan #80 4x N11-7 8 48 0 1 21 26 Murc+Chan #80 4x N11-15 1 3 0 0 1 2 Nules DV5 0 - - - - - Okitsu mandarin somaclone 5-1-99-2 S5 0 - - - - - Okitsu mandarin somaclone 5-4-99-3 1 10 - 32 - - Okitsu mandarin somaclone N11-3 0 - - - - - Okitsu mandarin somaclone N11-7 0 - - - - - Temple tangor DV5 0 - - - - - UKP-1 Hudson 4x 1 0 - - - - UKP-1 DV5 1 0 24 - 6 - UKP-1 N11-7 0 - - - - - Total 62 982 80 287 135 148 ZSeeds eaten or damaged by rodents after planting in seedling trays in the greenhouse, resulting in discrepancy between numbers of planted seeds and tested seedlings.

101

Table 4-3. F values and significant levels from ANOVA for number of lesions and lesion size rating for all seedlings and controls.z Number of Lesions Lesion Size Rating Sum of Mean Sum of Source DF Squares Square F Ratio Squares Mean Square F Ratio Model 163 3112713.6 19096.4 1.5326 349.09916 2.14171 2.1959 Error 319 3974875.3 12460.4 Prob > F 311.12308 0.97531 Prob > F C. Total 482 7087588.8 0.0007* 660.22224 <.0001* ZData from 168 hybrid selections, 2 grapefruit selections, 2 pummelo selections, and 1 kumquat selection.

102

Table 4-4. Tukey’s HSD pairwise analysis for number of lesions between triploid seedlings and controls (Flame grapefruit, 5-4-99-3 pummelo, and Meiwa kumquat) (α=0.05, significant P-values marked with *). Mean total number of citrus canker lesions and lesion ratings on attached leaves over three assays on all triploid grapefruit-pummelo selections. α† Grapefruit-Flame Pummelo-5-4-99-3 Kumquat-Meiwa Y Level Least Sq Mean 0.05 0.1 0.15 Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t|

Grapefruit-Flame 191.25 ab ab ab - - - - 181 81.9 2.21 1 69.6 2.7 0.9846 -115.115 Grapefruit-RubyRed 150.67 ab ab ab 40.58 88.5 0.46 1 140.42 88.5 1.59 1 77.21 1.9 1 -188.838

DV2xN11-3-15-07 291.33 ab ab ab 100.08 88.5 1.13 1 281.08 88.5 3.18 1 287.89 77.2 3.73 0.3214

M+Chan80xN11-3-15-04 283.25 ab ab ab -92 81.9 -1.12 1 273 81.9 3.33 1 -279.81 69.6 -4.02 0.1482

5-1-99-2xHuds4x-14-18 279.17 a a a 87.92 74.8 1.18 1 268.92 74.8 3.6 0 275.72 61 4.52 0.0272*

M+Chan80x5-4-99-7-15-06 271.75 ab ab ab -80.5 81.9 -0.98 1 261.5 81.9 3.19 1 -268.31 69.6 -3.86 0.2351

M+Chan80xN11-3-15-01 270.20 ab ab ab -78.95 77.7 -1.02 1 259.95 77.7 3.35 1 -266.76 64.6 -4.13 0.1061

M+Chan80xN11-3-15-02 265.00 ab ab ab -73.75 88.5 -0.83 1 254.75 88.5 2.88 1 -261.56 77.2 -3.39 0.6148

DV2xN11-3-15-30 251.00 ab ab ab 59.75 81.9 0.73 1 240.75 81.9 2.94 1 247.56 69.6 3.56 0.4621

DV2xN11-3-15-23 250.33 ab ab ab 59.08 88.5 0.67 1 240.08 88.5 2.71 1 246.89 77.2 3.2 0.7752

DV2xN11-7-15-17 250.00 ab ab ab 58.75 88.5 0.66 1 239.75 88.5 2.71 1 246.56 77.2 3.19 0.7785

DV5xN11-3-15-07 232.33 ab ab ab 41.08 88.5 0.46 1 222.08 88.5 2.51 1 228.89 77.2 2.96 0.9156

C2-4-1xN11-3-15-05 223.50 ab ab ab -32.25 100.3 -0.32 1 -213.25 100.3 -2.13 1 -220.06 90.5 -2.43 0.9988

DV2xN11-7-15-03 220.50 ab ab ab 29.25 100.3 0.29 1 210.25 100.3 2.1 1 217.06 90.5 2.4 0.9992

M+Chan80x5-4-99-7-15-04 218.00 ab ab ab -26.75 88.5 -0.3 1 207.75 88.5 2.35 1 -214.56 77.2 -2.78 0.9722

M+Chan80xN11-7-15-05 215.33 ab ab ab -24.08 88.5 -0.27 1 205.08 88.5 2.32 1 -211.89 77.2 -2.74 0.9782

M+Chan80x5-4-99-7-15-05 213.67 ab ab ab -22.42 88.5 -0.25 1 203.42 88.5 2.3 1 -210.22 77.2 -2.72 0.9815

5-1-99-2xHuds4x-14-02 210.00 ab ab ab 18.75 88.5 0.21 1 199.75 88.5 2.26 1 206.56 77.2 2.68 0.9872

DV2xN11-7-15-01 208.00 ab ab ab 16.75 81.9 0.2 1 197.75 81.9 2.41 1 204.56 69.6 2.94 0.926

M+Chan80xN11-15-15-1 205.75 ab ab ab -14.5 81.9 -0.18 1 195.5 81.9 2.39 1 -202.31 69.6 -2.91 0.938

DV5xN11-3-15-06 204.00 ab ab ab 12.75 81.9 0.16 1 193.75 81.9 2.37 1 200.56 69.6 2.88 0.9464

M+Chan80xN11-7-15-07 204.00 ab ab ab -12.75 88.5 -0.14 1 193.75 88.5 2.19 1 -200.56 77.2 -2.6 0.9935

DV2xN11-3-15-21 202.75 ab ab ab 11.5 81.9 0.14 1 192.5 81.9 2.35 1 199.31 69.6 2.86 0.9518

M+Chan80x5-4-99-3-15-07 201.75 ab ab ab -10.5 81.9 -0.13 1 191.5 81.9 2.34 1 -198.31 69.6 -2.85 0.9559

C2-4-1xN11-3-15-03 198.00 ab ab ab -6.75 88.5 -0.08 1 -187.75 88.5 -2.12 1 -194.56 77.2 -2.52 0.9969

8-1-99-2BxHuds4x-14-03 196.67 ab ab ab 5.42 74.8 0.07 1 186.42 74.8 2.49 1 193.22 61 3.17 0.799

C2-4-1xN11-3-15-07 196.00 ab ab ab -4.75 88.5 -0.05 1 -185.75 88.5 -2.1 1 -192.56 77.2 -2.49 0.9977

DV2xN11-7-15-14 187.67 ab ab ab -3.58 88.5 -0.04 1 177.42 88.5 2.01 1 184.22 77.2 2.39 0.9993

5-1-99-2xHuds4x-14-01 187.57 ab ab ab -3.68 72.6 -0.05 1 177.32 72.6 2.44 1 184.13 58.4 3.15 0.8067

4-4-99-7xHuds4x-14-13 185.50 ab ab ab -5.75 100.3 -0.06 1 175.25 100.3 1.75 1 182.06 90.5 2.01 1

5-1-99-2xHuds4x-14-17 178.00 ab ab ab -13.25 81.9 -0.16 1 167.75 81.9 2.05 1 174.56 69.6 2.51 0.9973

M+Chan80xN11-3-15-07 177.60 ab ab ab 13.65 77.7 0.18 1 167.35 77.7 2.15 1 -174.16 64.6 -2.7 0.9849

5-1-99-2xHuds4x-14-13 177.29 ab ab ab -13.96 72.6 -0.19 1 167.04 72.6 2.3 1 173.84 58.4 2.98 0.9094

DV2xN11-7-15-16 174.20 ab ab ab -17.05 77.7 -0.22 1 163.95 77.7 2.11 1 170.76 64.6 2.64 0.9902

M+Chan80x5-4-99-3-15-09 168.00 ab ab ab 23.25 88.5 0.26 1 157.75 88.5 1.78 1 -164.56 77.2 -2.13 1

5-1-99-2xHuds4x-14-08 167.83 ab ab ab -23.42 74.8 -0.31 1 157.58 74.8 2.11 1 164.39 61 2.69 0.9853

DV2xN11-7-15-06 167.67 ab ab ab -23.58 88.5 -0.27 1 157.42 88.5 1.78 1 164.22 77.2 2.13 1

DV5xN11-3-15-01 165.50 ab ab ab -25.75 81.9 -0.31 1 155.25 81.9 1.9 1 162.06 69.6 2.33 0.9997

5-1-99-2xHuds4x-14-09 164.50 ab ab ab -26.75 81.9 -0.33 1 154.25 81.9 1.88 1 161.06 69.6 2.31 0.9997

DV2xN11-7-15-08 161.75 ab ab ab -29.5 81.9 -0.36 1 151.5 81.9 1.85 1 158.31 69.6 2.27 0.9998

4-4-99-7xHuds4x-14-04 154.25 ab ab ab -37 81.9 -0.45 1 144 81.9 1.76 1 150.81 69.6 2.17 1

M+Chan80x5-4-99-3-15-02 153.33 ab ab ab 37.92 88.5 0.43 1 143.08 88.5 1.62 1 -149.89 77.2 -1.94 1

5-1-99-2xHuds4x-14-23 151.40 ab ab ab -39.85 77.7 -0.51 1 141.15 77.7 1.82 1 147.96 64.6 2.29 0.9998

C2-4-1xN11-3-15-11 150.33 ab ab ab 40.92 88.5 0.46 1 -140.08 88.5 -1.58 1 -146.89 77.2 -1.9 1

M+Chan80x7-2-99-11-15-05 150.25 ab ab ab 41 81.9 0.5 1 140 81.9 1.71 1 -146.81 69.6 -2.11 1

5-1-99-2xHuds4x-14-16 144.20 ab ab ab -47.05 77.7 -0.61 1 133.95 77.7 1.72 1 140.76 64.6 2.18 1

5-1-99-2xHuds4x-14-10 142.50 ab ab ab -48.75 81.9 -0.6 1 132.25 81.9 1.61 1 139.06 69.6 2 1

C2-4-1xN11-3-15-08 139.25 ab ab ab 52 81.9 0.63 1 -129 81.9 -1.58 1 -135.81 69.6 -1.95 1

M+Chan80xN11-3-15-03 139.20 ab ab ab 52.05 77.7 0.67 1 128.95 77.7 1.66 1 -135.76 64.6 -2.1 1

M+Chan80x5-4-99-7-15-01 138.14 ab ab ab 53.11 72.6 0.73 1 127.89 72.6 1.76 1 -134.7 58.4 -2.31 0.9998

DV2xN11-3-15-01 134.00 ab ab ab -57.25 100.3 -0.57 1 123.75 100.3 1.23 1 130.56 90.5 1.44 1

5-1-99-2xHuds4x-14-19 133.17 ab ab ab -58.08 74.8 -0.78 1 122.92 74.8 1.64 1 129.72 61 2.13 1

DV2xN11-7-15-13 129.33 ab ab ab -61.92 88.5 -0.7 1 119.08 88.5 1.35 1 125.89 77.2 1.63 1

C2-4-1xN11-3-15-01 128.75 ab ab ab 62.5 81.9 0.76 1 -118.5 81.9 -1.45 1 -125.31 69.6 -1.8 1

M+Chan80xN11-7-15-15 126.50 ab ab ab 64.75 81.9 0.79 1 116.25 81.9 1.42 1 -123.06 69.6 -1.77 1

5-4-99-3xDV5-15-1 124.75 ab ab ab 66.5 81.9 0.81 1 114.5 81.9 1.4 1 -121.31 69.6 -1.74 1

8-1-99-2BxHuds4x-14-01 124.71 ab ab ab -66.54 72.6 -0.92 1 114.46 72.6 1.58 1 121.27 58.4 2.08 1

5-1-99-2xHuds4x-14-22 123.75 ab ab ab -67.5 70.9 -0.95 1 113.5 70.9 1.6 1 120.31 56.3 2.14 1

DV2xN11-7-15-11 122.00 ab ab ab -69.25 88.5 -0.78 1 111.75 88.5 1.26 1 118.56 77.2 1.54 1

4-4-99-7xHuds4x-14-06 121.20 ab ab ab -70.05 77.7 -0.9 1 110.95 77.7 1.43 1 117.76 64.6 1.82 1

DV2xN11-7-15-09 119.25 ab ab ab -72 81.9 -0.88 1 109 81.9 1.33 1 115.81 69.6 1.66 1

M+Chan80x5-4-99-3-15-21 117.67 ab ab ab 73.58 88.5 0.83 1 107.42 88.5 1.21 1 -114.22 77.2 -1.48 1

M+Chan80x5-4-99-3-15-11 117.40 ab ab ab 73.85 77.7 0.95 1 107.15 77.7 1.38 1 -113.96 64.6 -1.76 1

8-1-99-2BxHuds4x-14-02 114.20 ab ab ab -77.05 77.7 -0.99 1 103.95 77.7 1.34 1 110.76 64.6 1.71 1

M+Chan80x5-4-99-3-15-01 113.67 ab ab ab 77.58 88.5 0.88 1 103.42 88.5 1.17 1 -110.22 77.2 -1.43 1

103

Table 4-4 Continued. α† Grapefruit-Flame Pummelo-5-4-99-3 Kumquat-Meiwa Level Least Sq Mean 0.05 0.1 0.15 DifferenceY Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t|

DV2xN11-7-15-15 113.40 ab ab ab -77.85 77.7 -1 1 103.15 77.7 1.33 1 109.96 64.6 1.7 1

M+Chan80x5-4-99-3-15-03 110.75 ab ab ab 80.5 81.9 0.98 1 100.5 81.9 1.23 1 -107.31 69.6 -1.54 1

M+Chan80x5-4-99-3-15-10 108.00 ab ab ab 83.25 100.3 0.83 1 97.75 100.3 0.97 1 -104.56 90.5 -1.15 1

DV2xN11-3-15-02 102.00 ab ab ab -89.25 129.5 -0.69 1 91.75 129.5 0.71 1 98.56 122.1 0.81 1

M+Chan80x7-2-99-11-15-06 100.20 ab ab ab 91.05 77.7 1.17 1 89.95 77.7 1.16 1 -96.76 64.6 -1.5 1

M+Chan80x5-4-99-3-15-13 100.00 ab ab ab 91.25 81.9 1.11 1 89.75 81.9 1.1 1 -96.56 69.6 -1.39 1

M+Chan80xN11-7-15-12 99.00 ab ab ab 92.25 81.9 1.13 1 88.75 81.9 1.08 1 -95.56 69.6 -1.37 1

4-4-99-7xHuds4x-14-01 97.25 ab ab ab -94 81.9 -1.15 1 87 81.9 1.06 1 93.81 69.6 1.35 1

M+Chan80x7-2-99-11-15-03 97.20 ab ab ab 94.05 77.7 1.21 1 86.95 77.7 1.12 1 -93.76 64.6 -1.45 1

M+Chan80x5-4-99-7-15-03 96.67 ab ab ab 94.58 88.5 1.07 1 86.42 88.5 0.98 1 -93.22 77.2 -1.21 1

M+Chan80x5-4-99-3-15-12 89.00 ab ab ab 102.25 88.5 1.16 1 78.75 88.5 0.89 1 -85.56 77.2 -1.11 1

DV2xN11-7-15-07 87.25 ab ab ab -104 81.9 -1.27 1 77 81.9 0.94 1 83.81 69.6 1.2 1

M+Chan80x5-4-99-7-15-08 86.25 ab ab ab 105 81.9 1.28 1 76 81.9 0.93 1 -82.81 69.6 -1.19 1

5-1-99-2xHuds4x-14-15 86.00 ab ab ab -105.25 72.6 -1.45 1 75.75 72.6 1.04 1 82.56 58.4 1.41 1

M+Chan80xN11-3-15-05 85.80 ab ab ab 105.45 77.7 1.36 1 75.55 77.7 0.97 1 -82.36 64.6 -1.27 1

M+Chan80x5-4-99-3-15-14 83.00 ab ab ab 108.25 88.5 1.22 1 72.75 88.5 0.82 1 -79.56 77.2 -1.03 1

5-1-99-2xHuds4x-14-07 79.33 ab ab ab -111.92 88.5 -1.27 1 69.08 88.5 0.78 1 75.89 77.2 0.98 1

5-1-99-2xHuds4x-14-05 73.75 ab ab ab -117.5 81.9 -1.43 1 63.5 81.9 0.78 1 70.31 69.6 1.01 1

M+Chan80x5-4-99-7-15-02 73.25 ab ab ab 118 81.9 1.44 1 63 81.9 0.77 1 -69.81 69.6 -1 1

4-4-99-7xHuds4x-14-11 69.67 ab ab ab -121.58 88.5 -1.37 1 59.42 88.5 0.67 1 66.22 77.2 0.86 1

5-1-99-2xHuds4x-14-20 65.00 ab ab ab -126.25 74.8 -1.69 1 54.75 74.8 0.73 1 61.56 61 1.01 1

M+Chan80x7-2-99-11-15-02 54.33 ab ab ab 136.92 88.5 1.55 1 44.08 88.5 0.5 1 -50.89 77.2 -0.66 1

M+Chan80x7-2-99-11-15-07 51.00 ab ab ab 140.25 88.5 1.59 1 40.75 88.5 0.46 1 -47.56 77.2 -0.62 1

4-4-99-7xHuds4x-14-02 49.33 ab ab ab -141.92 88.5 -1.6 1 39.08 88.5 0.44 1 45.89 77.2 0.59 1

DV2xN11-7-15-10 46.33 ab ab ab -144.92 88.5 -1.64 1 36.08 88.5 0.41 1 42.89 77.2 0.56 1

M+Chan80x5-4-99-3-15-15 43.25 ab ab ab 148 81.9 1.81 1 33 81.9 0.4 1 -39.81 69.6 -0.57 1

M+Chan80x5-4-99-3-15-08 43.00 ab ab ab 148.25 77.7 1.91 1 32.75 77.7 0.42 1 -39.56 64.6 -0.61 1

M+Chan80x5-4-99-3-15-19 31.33 ab ab ab 159.92 88.5 1.81 1 21.08 88.5 0.24 1 -27.89 77.2 -0.36 1

4-4-99-7xHuds4x-14-12 30.33 ab ab ab -160.92 88.5 -1.82 1 20.08 88.5 0.23 1 26.89 77.2 0.35 1

M+Chan80xN11-3-15-06 17.50 ab ab ab 173.75 81.9 2.12 1 7.25 81.9 0.09 1 -14.06 69.6 -0.2 1 M+Chan80x5-4-99-3-15-17 16.00 ab ab ab 175.25 88.5 1.98 1 5.75 88.5 0.07 1 -12.56 77.2 -0.16 1

Pummelo-5-1-99-2 270.25 ab ab ab -79 81.9 -0.96 1 260 81.9 3.17 1 -266.81 69.6 -3.83 0.2486

Pummelo-5-4-99-3 10.25 ab ab ab 181 81.9 2.21 0.9999 - - - - -6.81 69.6 -0.1 1

Kumquat-Meiwa 3.44 b b b 187.81 69.6 2.7 0.9846 -6.81 69.6 -0.1 1 - - - - ZMean separation not connected by same letter are significantly different. Least squares mean analysis using Tukey’s HSD (α=0.05). YDifference=Mean(i)–Mean(j). †α=0.05, Quantile = 4.35253; α=0.1, Quantile = 4.14869; α=0.15, Quantile = 4.01701; Adjusted DF = 293.0, Adjustment = Tukey-Kramer

104

Table 4-5. Tukey’s HSD† pairwise analysis for rating of lesions between hybrid seedlings and controls (Flame grapefruit, and Meiwa kumquat) showing seedlings with significant differences from controls at different alpha levels, α=0.05, 0.1, and 0.15. Mean of total number of citrus canker lesions on attached leaves over three assays on all triploid grapefruit- pummelo selections. α Grapefruit-Flame Pummelo-5-4-99-3 Kumquat-Meiwa

Name Least Sq Mean 0.05 0.1 0.15 Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t| Grapefruit-RubyRed 4 ab ad ad - - - - 1.7 0.68 2.48 1 3.75 0.58 6.44 <0.001* Grapefruit-Flame 3.93 ab ad ad -0.18 0.74 -0.25 1 1.88 0.74 2.55 1 3.93 0.65 6.09 <0.001* 5-1-99-2xHuds4x-14-10 3.92 a a a -0.4 0.68 -0.58 1 1.3 0.68 1.9 1 3.35 0.58 5.76 <0.001* 4-4-99-7xHuds4x-14-06 3.83 a a a -0.62 0.74 -0.83 1 1.08 0.74 1.46 1 3.13 0.65 4.85 0.007* DV5xN11-3-15-06 3.75 a ad ad -0.25 0.68 -0.37 1 1.45 0.68 2.12 1 3.5 0.58 6.02 <0.001* 8-1-99-2BxHuds4x-14-02 3.7 ab ad ad 0.17 0.65 0.26 1 1.87 0.65 2.88 1 3.92 0.54 7.26 <0.001* 5-1-99-2xHuds4x-14-05 3.55 ab abd ad -0.82 0.74 -1.1 1 0.88 0.74 1.19 1 2.93 0.65 4.54 0.025* DV5xN11-3-15-01 3.55 ab abd ad -2.38 0.74 -3.22 1 -0.68 0.74 -0.92 1 1.37 0.65 2.12 1 4-4-99-7xHuds4x-14-04 3.5 ab abd abd -0.85 0.84 -1.01 1 0.85 0.84 1.01 1 2.9 0.76 3.83 0.25 5-1-99-2xHuds4x-14-08 3.5 ab ad ad -0.2 0.68 -0.29 1 1.5 0.68 2.19 1 3.55 0.58 6.1 <0.001* 5-1-99-2xHuds4x-14-09 3.5 ab abd abd -0.64 0.61 -1.05 1 1.06 0.61 1.75 1 3.11 0.49 6.38 <0.001* 8-1-99-2BxHuds4x-14-03 3.5 ab ad ad -0.48 0.74 -0.65 1 1.22 0.74 1.65 1 3.27 0.65 5.06 0.003* 5-1-99-2xHuds4x-14-18 3.47 ab ad ad -0.62 0.74 -0.83 1 1.08 0.74 1.46 1 3.13 0.65 4.85 0.007* 5-1-99-2xHuds4x-14-13 3.46 a ad ad -0.25 0.63 -0.4 1 1.45 0.63 2.32 1 3.5 0.51 6.86 <0.001* M+Chan80xN11-3-15-04 3.43 ab abd abd -0.25 0.68 -0.37 1 1.45 0.68 2.12 1 3.5 0.58 6.02 <0.001* 5-1-99-2xHuds4x-14-23 3.4 ab abd ad -0.29 0.61 -0.47 1 1.41 0.61 2.33 1 3.46 0.49 7.1 <0.001* DV2xN11-7-15-03 3.4 abc abd abd -0.51 0.65 -0.79 1 1.19 0.65 1.83 1 3.24 0.54 6 <0.001* DV2xN11-7-15-15 3.4 ab abd ad 0.25 0.68 0.37 1 1.95 0.68 2.85 1 4 0.58 6.87 <0.001* M+Chan80x5-4-99-3-15-09 3.4 ab abd abd -0.86 0.61 -1.41 1 0.84 0.61 1.39 1 2.89 0.49 5.93 <0.001* 5-1-99-2xHuds4x-14-17 3.37 ab abd abd -0.38 0.68 -0.56 1 1.32 0.68 1.92 1 3.37 0.58 5.79 <0.001* M+Chan80x7-2-99-11-15-6 3.36 ab abd abd -0.28 0.63 -0.45 1 1.42 0.63 2.27 1 3.47 0.51 6.79 <0.001* 4-4-99-7xHuds4x-14-01 3.35 ab abd abd -0.6 0.63 -0.96 1 1.1 0.63 1.76 1 3.15 0.51 6.17 <0.001* DV2xN11-7-15-14 3.33 ab abd abd -0.82 0.63 -1.31 1 0.88 0.63 1.41 1 2.93 0.51 5.75 <0.001* M+Chan80x5-4-99-3-15-01 3.33 ab abd abd -0.65 0.59 -1.1 1 1.05 0.59 1.77 1 3.1 0.47 6.59 <0.001* M+Chan80x5-4-99-7-15-04 3.33 ab abd abd -0.35 0.65 -0.54 1 1.35 0.65 2.08 1 3.4 0.54 6.3 <0.001* M+Chan80xN11-3-15-03 3.28 ab abd abd -0.6 0.68 -0.88 1 1.1 0.68 1.61 1 3.15 0.58 5.41 <0.001* 5-1-99-2xHuds4x-14-02 3.27 ab abd abd -0.73 0.61 -1.2 1 0.97 0.61 1.6 1 3.02 0.49 6.19 <0.001* DV2xN11-3-15-07 3.27 ab abd abd -0.05 0.68 -0.07 1 1.65 0.68 2.41 1 3.7 0.58 6.36 <0.001* DV2xN11-3-15-30 3.25 ab abd abd -0.25 0.63 -0.4 1 1.45 0.63 2.32 1 3.5 0.51 6.86 <0.001* DV2xN11-7-15-01 3.25 ab abd abd -0.75 0.68 -1.1 1 0.95 0.68 1.39 1 3 0.58 5.16 0.002* 5-1-99-2xHuds4x-14-16 3.24 ab abd abd -1.55 0.84 -1.85 1 0.15 0.84 0.18 1 2.2 0.76 2.91 0.94 M+Chan80xN11-3-15-07 3.21 ab abd abd -1.35 0.84 -1.61 1 0.35 0.84 0.42 1 2.4 0.76 3.17 0.80 5-1-99-2xHuds4x-14-19 3.15 ab abd abd -1.04 0.68 -1.52 1 0.66 0.68 0.96 1 2.71 0.58 4.65 0.02* 5-4-99-3xDV5-15-1 3.15 ab abd abd -0.68 0.74 -0.92 1 1.02 0.74 1.37 1 3.07 0.65 4.75 0.01* 4-4-99-7xHuds4x-14-02 3.13 ab abd abd -1.42 0.74 -1.92 1 0.28 0.74 0.38 1 2.33 0.65 3.61 0.41 5-1-99-2xHuds4x-14-07 3.13 ab abd abd -0.75 0.84 -0.89 1 0.95 0.84 1.13 1 3 0.76 3.96 0.18 DV2xN11-7-15-17 3.13 ab abd abd -1.35 1.08 -1.25 1 0.35 1.08 0.32 1 2.4 1.02 2.35 1 M+Chan80x5-4-99-7-15-05 3.13 ab abd abd -0.48 0.74 -0.65 1 1.22 0.74 1.65 1 3.27 0.65 5.06 0.003*

5-1-99-2xHuds4x-14-01 3.11 ab abd abd -2.15 0.68 -3.14 1 -0.45 0.68 -0.66 1 1.6 0.58 2.75 1 5-1-99-2xHuds4x-14-22 3.1 ab abd abd -1.02 0.74 -1.37 1 0.68 0.74 0.92 1 2.73 0.65 4.23 0.08 M+Chan80x5-4-99-7-15-02 3.1 ab abd abd -0.5 0.68 -0.73 1 1.2 0.68 1.75 1 3.25 0.58 5.59 <0.001* DV5xN11-3-15-07 3.08 ab abd abd -0.5 0.68 -0.73 1 1.2 0.68 1.75 1 3.25 0.58 5.59 <0.001* C2-4-1xN11-3-15-07 3.07 ab abd abd -0.35 0.84 -0.42 1 1.35 0.84 1.61 1 3.4 0.76 4.49 0.03* M+Chan80x5-4-99-3-15-02 3.07 ab abd abd -1.35 0.74 -1.83 1 0.35 0.74 0.47 1 2.4 0.65 3.72 0.3296 8-1-99-2BxHuds4x-14-01 3.02 ab abd abd -1.45 0.74 -1.96 1 0.25 0.74 0.34 1 2.3 0.65 3.56 0.46 M+Chan80x5-4-99-3-15-21 3.02 ab abd abd -0.95 0.68 -1.39 1 0.75 0.68 1.1 1 2.8 0.58 4.81 0.008* C2-4-1xN11-3-15-01 3 ab abd abd -1.45 0.68 -2.12 1 0.25 0.68 0.37 1 2.3 0.58 3.95 0.18 DV2xN11-3-15-01 3 abc abcd abcd -3.42 0.74 -4.62 0.02 -1.72 0.74 -2.32 1 0.33 0.65 0.52 1 M+Chan80xN11-7-15-12 3 ab abd abd -2.75 0.74 -3.72 0.33 -1.05 0.74 -1.42 1 1 0.65 1.55 1 M+Chan80x5-4-99-3-15-17 2.96 abc abd abd -1.88 0.74 -2.55 1 -0.18 0.74 -0.25 1 1.87 0.65 2.89 0.94 M+Chan80x7-2-99-11-15-3 2.95 ab abd abd -0.42 0.74 -0.56 1 1.28 0.74 1.74 1 3.33 0.65 5.16 0.002* 4-4-99-7xHuds4x-14-11 2.93 ab abd abd -0.87 0.65 -1.34 1 0.83 0.65 1.28 1 2.88 0.54 5.33 0.001* 5-1-99-2xHuds4x-14-20 2.93 ab abd abd -0.62 0.74 -0.83 1 1.08 0.74 1.46 1 3.13 0.65 4.85 0.007* M+Chan80xN11-7-15-05 2.93 abc abd abd -0.35 0.65 -0.54 1 1.35 0.65 2.08 1 3.4 0.54 6.3 <.0001* 4-4-99-7xHuds4x-14-13 2.9 abc abcd abcd -0.2 0.68 -0.29 1 1.5 0.68 2.19 1 3.55 0.58 6.1 <.0001* 5-1-99-2xHuds4x-14-15 2.89 ab abd abd 0.08 0.68 0.12 1 1.78 0.68 2.6 1 3.83 0.58 6.59 <.0001* DV2xN11-7-15-16 2.88 ab abd abd -0.67 0.74 -0.9 1 1.03 0.74 1.4 1 3.08 0.65 4.78 0* M+Chan80x5-4-99-3-15-15 2.88 ab abd abd 0.42 0.74 0.56 1 1.28 0.74 1.74 1 -3.33 0.65 -5.16 0* M+Chan80xN11-7-15-07 2.87 abc abcd abd 0.68 0.74 0.92 1 1.02 0.74 1.37 1 -3.07 0.65 -4.75 0* DV2xN11-7-15-08 2.8 ab abd abd 1.43 0.68 2.09 1 0.27 0.68 0.39 1 -2.32 0.58 -3.98 0 M+Chan80x5-4-99-3-15-07 2.8 ab abd abd 1 0.68 1.46 1 0.7 0.68 1.02 1 -2.75 0.58 -4.73 0 DV2xN11-3-15-23 2.75 abc abcd abcd 0.35 0.74 0.47 1 1.35 0.74 1.83 1 -3.4 0.65 -5.27 0.0011*

105

Table 4-5 Continued. α Grapefruit-Flame Pummelo-5-4-99-3 Kumquat-Meiwa Name Least Sq Mean 0.05 0.1 0.15 Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t| Difference Std Error t Ratio Prob>|t| M+Chan80x7-2-99-11-15-5 2.75 ab abd abd 1.95 0.65 3 0.8981 -0.25 0.65 -0.38 1 -1.8 0.54 -3.33 0.6635 C2-4-1xN11-3-15-08 2.73 abc abd abd 1.25 0.84 1.49 1 0.45 0.84 0.54 1 -2.5 0.76 -3.3 0.6896 M+Chan80x5-4-99-7-15-01 2.71 ab abd abd 1.51 0.65 2.32 0.9997 0.19 0.65 0.29 1 -2.24 0.54 -4.15 0.1004 M+Chan80x5-4-99-7-15-06 2.69 abc abd abd 1.42 0.74 1.92 1 0.28 0.74 0.38 1 -2.33 0.65 -3.61 0.4125 M+Chan80x5-4-99-7-15-03 2.6 abc abcd abcd 1.3 0.68 1.9 1 0.4 0.68 0.58 1 -2.45 0.58 -4.21 0.0817 M+Chan80xN11-3-15-05 2.6 ab abd abd 1.48 0.74 2.01 1 0.22 0.74 0.29 1 -2.27 0.65 -3.51 0.5028 M+Chan80x5-4-99-3-15-10 2.59 abc abcd abcd 0.88 0.68 1.28 1 0.83 0.68 1.2 1 -2.88 0.58 -4.94 0.0048* M+Chan80x5-4-99-3-15-13 2.5 abc abcd abcd 0.79 0.74 1.07 1 0.91 0.74 1.22 1 -2.96 0.65 -4.58 0.0216* DV2xN11-3-15-02 2.45 abc abcd abcd 2.48 0.74 3.36 0.6413 -0.78 0.74 -1.06 1 -1.27 0.65 -1.96 1 C2-4-1xN11-3-15-05 2.4 abc abcd abcd 1.06 0.61 1.74 1 0.64 0.61 1.06 1 -2.69 0.49 -5.52 <0.001* C2-4-1xN11-3-15-11 2.4 abc abcd abcd 0.65 0.68 0.95 1 1.05 0.68 1.53 1 -3.1 0.58 -5.33 <0.001* DV2xN11-7-15-06 2.4 abc abcd abcd 0.73 0.74 0.99 1 0.97 0.74 1.31 1 -3.02 0.65 -4.67 0.0148* M+Chan80x5-4-99-3-15-03 2.33 abc abcd abcd 0.62 0.74 0.83 1 1.08 0.74 1.46 1 -3.13 0.65 -4.85 0.007* M+Chan80x5-4-99-3-15-12 2.33 abc abcd abcd 0.42 0.74 0.56 1 1.28 0.74 1.74 1 -3.33 0.65 -5.16 0.0018* M+Chan80xN11-3-15-02 2.33 abc abcd abcd 1.15 0.74 1.56 1 0.55 0.74 0.74 1 -2.6 0.65 -4.03 0.1453 DV2xN11-7-15-07 2.32 abc abcd abcd 1.15 0.68 1.68 1 0.55 0.68 0.8 1 -2.6 0.58 -4.47 0.0329* DV2xN11-7-15-09 2.3 abc abcd abcd 2 0.68 2.92 0.9328 -0.3 0.68 -0.44 1 -1.75 0.58 -3.01 0.8954 M+Chan80x5-4-99-3-15-14 2.3 abc abcd abcd 1.95 0.74 2.64 0.9907 -0.25 0.74 -0.34 1 -1.8 0.65 -2.79 0.9702 M+Chan80xN11-3-15-01 2.27 abc abcd abcd 0.8 0.68 1.17 1 0.9 0.68 1.31 1 -2.95 0.58 -5.07 0.0027* M+Chan80x5-4-99-3-15-11 2.25 abc abcd abcd 1 0.68 1.46 1 0.7 0.68 1.02 1 -2.75 0.58 -4.73 0.0119* C2-4-1xN11-3-15-03 2.24 abc abcd abcd 0.39 0.65 0.6 1 1.31 0.65 2.02 1 -3.36 0.54 -6.22 <0.001* M+Chan80xN11-15-15-1 2.2 abc abcd abcd 2.22 0.74 3 0.9004 -0.52 0.74 -0.7 1 -1.53 0.65 -2.38 0.9994 DV2xN11-7-15-13 2.05 abc abcd abcd 1.7 0.68 2.48 0.9979 0 0.68 0 1 -2.05 0.58 -3.52 0.4922 M+Chan80x5-4-99-3-15-08 2.05 abc abcd abcd 1.5 0.68 2.19 1 0.2 0.68 0.29 1 -2.25 0.58 -3.87 0.2282 M+Chan80x7-2-99-11-15-2 1.87 abc abcd abcd 1.42 0.74 1.92 1 0.28 0.74 0.38 1 -2.33 0.65 -3.61 0.4125 M+Chan80x5-4-99-7-15-08 1.8 abc abcd abcd 0.33 0.68 0.47 1 1.38 0.68 2.01 1 -3.43 0.58 -5.89 <0.001* M+Chan80xN11-7-15-15 1.8 abc abcd abcd 0.47 0.65 0.72 1 1.23 0.65 1.89 1 -3.28 0.54 -6.07 <0.001* DV2xN11-3-15-21 1.76 abc abcd abcd 1.16 0.65 1.79 1 0.54 0.65 0.83 1 -2.59 0.54 -4.8 0.009* M+Chan80xN11-3-15-06 1.75 abc abcd abcd 2.18 0.68 3.18 0.7907 -0.48 0.68 -0.69 1 -1.58 0.58 -2.71 0.9835 M+Chan80x7-2-99-11-15-7 1.6 abc abcd abcd 0.54 0.65 0.83 1 1.16 0.65 1.79 1 -3.21 0.54 -5.94 <0.001* 4-4-99-7xHuds4x-14-12 1.58 abc abcd abcd 0.82 0.74 1.1 1 0.88 0.74 1.19 1 -2.93 0.65 -4.54 0.0247* M+Chan80x5-4-99-3-15-19 1.53 abc abcd abcd 0.88 0.74 1.19 1 0.82 0.74 1.1 1 -2.87 0.65 -4.44 0.0364* DV2xN11-7-15-11 1.37 abc abcd abcd 0.75 0.68 1.1 1 0.95 0.68 1.39 1 -3 0.58 -5.16 0.0018* DV2xN11-7-15-10 1.27 bc bc bc 1.99 0.68 2.9 0.9393 -0.29 0.68 -0.42 1 -1.76 0.58 -3.03 0.8844 Pummelo-5-1-99-2 1 ab abd abd 0.95 0.68 1.39 1 0.75 0.68 1.1 1 -2.8 0.58 -4.81 0.0084* Pummelo-5-4-99-3 0.33 abc abcd abcd 1.7 0.68 2.48 0.9979 - 0.58 - - -2.05 0.58 -3.52 0.4922

Kumquat-Meiwa <0.001 c c c 3.75 0.58 6.44 <0.001* -2.05 -3.52 0.4922 - - - -

†α=0.05, Quantile = 4.35253; α=0.1, Quantile = 4.14869; α=0.15, Quantile = 4.01701; Adjusted DF = 293.0, Adjustment = Tukey-Kramer

106

Table 4-6. Canonical Multivariate analysis for both rating of lesions and number of lesions between hybrid seedlings and controls (Flame and Ruby red grapefruit, 5-1-99-2 and 5-4-99-3 pummelos, and Meiwa kumquat) showing seedlings with significant differences from controls (α=0.05, significant values marked with *). Mean of total number of citrus canker lesions on attached leaves over three assays on all triploid grapefruit-pummelo selections.

Name Grapefruit-Flame Grapefruit-RubyRed Pummelo-5-1-99-2 Pummelo-5-4-99-3 Kumquat-Meiwa P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist.

Grapefruit-Flame 1 0 0.818 0.20108 0.0159* 4.20277 0.1001 2.31973 <0.001* 20.45079 Grapefruit-RubyRed 0.818 0.20108 1 0 0.031* 3.51613 0.0989 2.33209 <0.001* 18.04846 4-4-99-7xHuds4x-14-01 0.4049 0.90707 0.3893 0.94662 0.5728 0.55831 0.773 0.25767 <0.001* 6.18587 4-4-99-7xHuds4x-14-02 0.3885 0.94849 0.5104 0.67412 0.5249 0.64597 0.5577 0.58507 <0.001* 7.82655 4-4-99-7xHuds4x-14-03 0.9744 0.02593 0.9254 0.07758 0.1291 2.06209 0.2282 1.48496 <0.001* 7.55171 4-4-99-7xHuds4x-14-04 0.8764 0.13197 0.8212 0.19712 0.0575 2.88414 0.2012 1.61233 <0.001* 17.63042 4-4-99-7xHuds4x-14-06 0.545 0.60827 0.9326 0.06976 0.0161* 4.18848 0.0656 2.75064 <0.001* 25.74019 4-4-99-7xHuds4x-14-07 0.8106 0.21015 0.9955 0.00448 0.0585 2.86689 0.1274 2.07521 <0.001* 13.5784 4-4-99-7xHuds4x-14-08 0.0589 2.85935 0.0263* 3.68622 0.023* 3.82355 0.1174 2.15805 <0.001* 4.51609 4-4-99-7xHuds4x-14-09 0.2197 1.52346 0.1903 1.66889 0.6427 0.44275 0.9301 0.07249 <0.001* 4.49792 4-4-99-7xHuds4x-14-10 0.2661 1.33011 0.2756 1.2945 0.9626 0.03812 0.972 0.0284 <0.001* 2.20462 4-4-99-7xHuds4x-14-11 0.2524 1.38323 0.2333 1.46297 0.6827 0.38222 0.9137 0.0903 <0.001* 4.88691 4-4-99-7xHuds4x-14-12 0.0049* 5.41108 0.0064* 5.1411 0.5674 0.56787 0.7759 0.254 <0.001* 2.25606 4-4-99-7xHuds4x-14-13 0.8194 0.19938 0.6048 0.50379 0.0783 2.56963 0.1948 1.64529 <0.001* 7.32043 5-1-99-2xHuds4x-14-01 0.5606 0.57999 0.3155 1.15826 0.0282* 3.61146 0.2409 1.43042 <0.001* 20.56861 5-1-99-2xHuds4x-14-02 0.7325 0.31157 0.4415 0.81983 0.0461* 3.10884 0.2285 1.48369 <0.001* 13.13587 5-1-99-2xHuds4x-14-05 0.3307 1.11094 0.6534 0.42618 0.0984 2.33725 0.1645 1.8163 <0.001* 18.59431 5-1-99-2xHuds4x-14-07 0.3944 0.93346 0.5441 0.60982 0.3332 1.10317 0.4298 0.84694 <0.001* 11.63333 5-1-99-2xHuds4x-14-08 0.9062 0.09849 0.7453 0.29428 0.025* 3.73784 0.1541 1.8821 <0.001* 23.02905 5-1-99-2xHuds4x-14-09 0.9128 0.09129 0.7898 0.23612 0.0486* 3.05522 0.1927 1.65608 <0.001* 17.6898 5-1-99-2xHuds4x-14-10 0.9128 0.09126 0.9535 0.04768 0.0182* 4.06488 0.079 2.56101 <0.001* 18.72436 5-1-99-2xHuds4x-14-13 0.8969 0.10886 0.656 0.42226 0.0177* 4.09256 0.1455 1.94082 <0.001* 24.82139 5-1-99-2xHuds4x-14-15 0.2104 1.56732 0.2914 1.23839 0.3167 1.15453 0.5192 0.65692 <0.001* 17.2096 5-1-99-2xHuds4x-14-16 0.6873 0.37552 0.6073 0.49964 0.0916 2.40984 0.303 1.19896 <0.001* 17.54227 5-1-99-2xHuds4x-14-17 0.743 0.29742 0.6542 0.42504 0.159 1.8509 0.3739 0.98713 <0.001* 12.15381 5-1-99-2xHuds4x-14-18 0.3236 1.13268 0.1224 2.11545 0.0007* 7.42236 0.0378* 3.31352 <0.001* 25.76595 5-1-99-2xHuds4x-14-19 0.56 0.58106 0.5232 0.64926 0.1118 2.20816 0.3463 1.06425 <0.001* 18.5387 5-1-99-2xHuds4x-14-20 0.1678 1.7959 0.291 1.23972 0.3621 1.0194 0.4652 0.76723 <0.001* 16.44771 5-1-99-2xHuds4x-14-22 0.4496 0.8016 0.4559 0.7876 0.1164 2.16711 0.3615 1.02117 <0.001* 21.10455 5-1-99-2xHuds4x-14-23 0.81 0.21081 0.7317 0.31273 0.0567 2.89838 0.2212 1.5164 <0.001* 19.31898 8-1-99-2BxHuds4x-14-1 0.4177 0.87562 0.4009 0.91703 0.1482 1.92161 0.4225 0.86424 <0.001* 18.61659 8-1-99-2BxHuds4x-14-2 0.5655 0.57114 0.8346 0.1809 0.1115 2.21064 0.1976 1.63045 <0.001* 15.27163 8-1-99-2BxHuds4x-14-3 0.6689 0.40269 0.3373 1.09102 0.0082* 4.88534 0.1106 2.21904 <0.001* 20.62111 8-1-99-2BxHuds4x-14-7 0.0162* 4.18521 0.0185* 4.04538 0.6866 0.37644 0.8148 0.20492 0.1772 1.74107 8-1-99-2BxHuds4x-14-8 0.3924 0.93851 0.736 0.30688 0.0737 2.63114 0.1063 2.2585 <0.001* 14.00539 DV2xN11-3-15-01 0.6457 0.43814 0.5716 0.56037 0.3413 1.07908 0.5732 0.55751 <0.001* 7.65712 DV2xN11-3-15-03 0.8001 0.22316 0.5826 0.54126 0.2113 1.56279 0.4234 0.86208 0.0062* 5.16545 DV2xN11-3-15-05 0.8637 0.14664 0.984 0.01609 0.2562 1.36822 0.3274 1.12082 0.0014* 6.75116 DV2xN11-3-15-06 0.0188* 4.02976 0.0093* 4.75903 0.0595 2.84869 0.3116 1.17064 0.0366* 3.34536 DV2xN11-3-15-07 0.2824 1.26994 0.1159 2.17129 0.0045* 5.50748 0.0716 2.66066 <0.001* 15.13969 DV2xN11-3-15-08 0.9004 0.10494 0.8611 0.14959 0.3893 0.94638 0.5292 0.63779 0.006* 5.19444 DV2xN11-3-15-09 0.0008* 7.30334 0.0007* 7.4304 0.0927 2.39861 0.2757 1.29413 0.7158 0.33473 DV2xN11-3-15-10 0.0862 2.47167 0.078 2.57378 0.7105 0.34224 0.9832 0.01691 0.0482* 3.06381 DV2xN11-3-15-13 0.837 0.17804 0.635 0.45481 0.2478 1.40197 0.4612 0.77607 0.0069* 5.06332 DV2xN11-3-15-14 0.4224 0.86428 0.2471 1.40465 0.1151 2.17793 0.322 1.13754 0.0096* 4.72571 DV2xN11-3-15-21 0.0433* 3.17472 0.0229* 3.82761 0.1035 2.28612 0.4949 0.7051 0.005* 5.40585 DV2xN11-3-15-22 0.0429 3.18346 0.0364* 3.35008 0.5043 0.68629 0.6978 0.36031 0.6252 0.47042 DV2xN11-3-15-23 0.1958 1.6398 0.0826 2.51557 0.0214* 3.89653 0.198 1.62847 <.0001* 10.76766 DV2xN11-3-15-27 0.003* 5.91966 0.0028* 6.01622 0.151 1.90311 0.252 1.38505 0.9995 0.000472 DV2xN11-3-15-29 0.0025* 6.09937 0.0009* 7.2112 0.0004* 7.9844 0.0065* 5.11932 0.0004* 7.99518 DV2xN11-3-15-30 0.4474 0.80654 0.1993 1.62215 0.0083* 4.87132 0.1136 2.19141 <.0001* 17.07111 DV2xN11-7-15-01 0.6899 0.37168 0.392 0.93952 0.03* 3.5481 0.2028 1.6042 <.0001* 15.98361 DV2xN11-7-15-02 0.0002* 8.59447 <.0001* 9.77553 <.0001* 9.64248 0.002* 6.35256 0.0004* 8.13165

107

Table 4-6 Continued. Grapefruit- Kumquat- Name Grapefruit-Flame Pummelo-5-1-99-2 Pummelo-5-4-99-3 RubyRed Meiwa P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. DV2xN11-7-15-03 0.8261 0.19112 0.5457 0.60691 0.0653 2.75538 0.232 1.46828 <.0001* 10.37536 DV2xN11-7-15-05 0.2612 1.34881 0.2355 1.45348 0.8415 0.17268 0.9961 0.0039 0.1565 1.86657 DV2xN11-7-15-06 0.3849 0.95794 0.2476 1.40255 0.2406 1.43163 0.5995 0.5125 0.0025* 6.12412 DV2xN11-7-15-07 0.1515 1.89942 0.1213 2.1246 0.4668 0.76376 0.8741 0.13466 0.0022* 6.23827 DV2xN11-7-15-08 0.6389 0.44876 0.5331 0.63049 0.2011 1.61266 0.4582 0.78262 <.0001* 11.03313 DV2xN11-7-15-09 0.112 2.20637 0.0889 2.44089 0.3912 0.94169 0.8494 0.16337 0.0006* 7.69746 DV2xN11-7-15-10 <.0001* 10.39464 <.0001* 10.14606 0.035* 3.39245 0.163 1.82564 0.8019 0.22095 DV2xN11-7-15-11 0.0013* 6.81298 0.0009* 7.14025 0.0676 2.71917 0.3395 1.08427 0.1646 1.81579 DV2xN11-7-15-12 0.136 2.009 0.1254 2.09127 0.7457 0.29375 0.9883 0.01179 0.0249* 3.74154 DV2xN11-7-15-13 0.0439* 3.15911 0.0304* 3.53566 0.2926 1.23416 0.7746 0.25562 0.0135* 4.36967 DV2xN11-7-15-14 0.8475 0.16553 0.5973 0.51632 0.0673 2.72417 0.2552 1.37199 <.0001* 13.29954 DV2xN11-7-15-15 0.5766 0.55168 0.7405 0.3008 0.0956 2.36695 0.2353 1.45403 <.0001* 19.31414 DV2xN11-7-15-16 0.409 0.89673 0.2455 1.41146 0.0821 2.52178 0.3982 0.92379 <.0001* 14.36784 DV2xN11-7-15-17 0.429 0.84867 0.2013 1.61183 0.0186* 4.04058 0.1592 1.84958 <.0001* 13.08732 DV2xN11-7-15-18 0.0438* 3.16201 0.0306* 3.52861 0.3071 1.18546 0.5717 0.56023 0.5087 0.67741 DV5xN11-3-15-01 0.9968 0.00324 0.866 0.14389 0.0333* 3.44393 0.1352 2.01461 <.0001* 16.41704 DV5xN11-3-15-02 0.5987 0.51395 0.9 0.10538 0.0923 2.40235 0.1484 1.92081 <.0001* 13.05666 DV5xN11-3-15-03 0.9076 0.097 0.9815 0.01868 0.0479* 3.07053 0.1231 2.10962 <.0001* 13.59296 DV5xN11-3-15-04 0.1099 2.22559 0.0553 2.92485 0.0021* 6.31944 0.0163* 4.1788 <.0001* 10.93981 DV5xN11-3-15-05 0.0905 2.42258 0.0691 2.69769 0.5075 0.67978 0.7802 0.24843 0.4048 0.90721 DV5xN11-3-15-06 0.9318 0.07062 0.6566 0.42131 0.0145* 4.29468 0.0937 2.38664 <.0001* 17.2305 DV5xN11-3-15-07 0.4837 0.72821 0.2431 1.4212 0.0318* 3.48964 0.2123 1.5582 <.0001* 12.34705 DV5xN11-3-15-08 0.0456* 3.12001 0.0205* 3.94185 0.0008* 7.32484 0.0085* 4.84662 <.0001* 11.42058 DV5xN11-3-15-09 0.6437 0.44117 0.8024 0.22034 0.4682 0.76081 0.4775 0.74102 0.0044* 5.53263 DV5xN11-3-15-11 0.003* 5.91966 0.0028* 6.01622 0.151 1.90311 0.252 1.38505 0.9995 0.000472 M+Chan80x5-4-99-7-15-01 0.2261 1.49438 0.1696 1.78513 0.1749 1.75396 0.5895 0.52936 <.0001* 14.99274 M+Chan80x5-4-99-7-15-02 0.2995 1.21052 0.4607 0.77716 0.3053 1.19134 0.4045 0.90802 <.0001* 14.07275 M+Chan80x5-4-99-7-15-03 0.2548 1.37364 0.2524 1.38347 0.5519 0.59552 0.7971 0.22696 0.0005* 7.8886 M+Chan80x5-4-99-7-15-04 0.7475 0.29129 0.4425 0.81763 0.0352 3.38707 0.1941 1.64877 <.0001* 13.7428 M+Chan80x5-4-99-7-15-05 0.6091 0.49656 0.3428 1.07462 0.0491 3.04507 0.254 1.37701 <.0001* 12.29194 M+Chan80x5-4-99-7-15-06 0.0668 2.7311 0.0233* 3.81021 0.0048 5.44045 0.111 2.21506 <.0001* 13.24798 M+Chan80x5-4-99-7-15-07 0.0141* 4.32581 0.0072* 5.01254 0.0502 3.02216 0.1868 1.6876 0.1826 1.71068 M+Chan80x5-4-99-7-15-08 0.0025* 6.10199 0.0033* 5.83119 0.4312 0.84366 0.6704 0.4005 0.1816 1.71631 M+Chan80x5-4-99-7-15-09 0.9907 0.00934 0.9019 0.10326 0.0808 2.5372 0.2043 1.59716 <.0001* 11.70209 M+Chan80x5-4-99-7-15-10 0.9324 0.07005 0.7369 0.30563 0.1542 1.8819 0.3088 1.17996 0.002* 6.35231 M+Chan80x7-2-99-11-15-1 0.2907 1.24064 0.3539 1.04252 0.7163 0.33407 0.7427 0.29783 0.002* 6.3251 M+Chan80x7-2-99-11-15-2 0.0263* 3.6828 0.0298* 3.55584 0.7614 0.27289 0.9743 0.02601 0.0233* 3.80897 M+Chan80x7-2-99-11-15-3 0.4495 0.8018 0.4224 0.86429 0.2563 1.36772 0.5252 0.64541 <.0001* 12.50021 M+Chan80x7-2-99-11-15-4 0.3644 1.01301 0.3836 0.96144 0.9176 0.08598 0.9266 0.0762 0.0657 2.74884 M+Chan80x7-2-99-11-15-5 0.3555 1.03787 0.2499 1.39349 0.1941 1.64879 0.5662 0.56992 <.0001* 11.08326 M+Chan80x7-2-99-11-15-6 0.4744 0.74773 0.6785 0.38834 0.1198 2.138 0.2508 1.38978 <.0001* 18.94461 M+Chan80x7-2-99-11-15-7 0.0107* 4.60915 0.0121* 4.48498 0.5993 0.51283 0.8731 0.13574 0.0654 2.75352 M+Chan80x5-4-99-3-15-01 0.6417 0.44429 0.7458 0.29359 0.1859 1.69252 0.3321 1.10664 <.0001* 12.98672 M+Chan80x5-4-99-3-15-02 0.6535 0.42611 0.5138 0.66742 0.1757 1.74938 0.4402 0.82277 <.0001* 11.08595 M+Chan80x5-4-99-3-15-03 0.1136 2.19169 0.0965 2.35708 0.4516 0.79715 0.8775 0.13079 0.0005* 7.75355 M+Chan80x5-4-99-3-15-04 0.5337 0.62936 0.5017 0.69131 0.7457 0.29379 0.88 0.12789 0.0441* 3.15454 M+Chan80x5-4-99-3-15-06 0.0424* 3.19431 0.0365* 3.34764 0.5155 0.66408 0.7011 0.35553 0.6268 0.46795 M+Chan80x5-4-99-3-15-07 0.29 1.24334 0.1455 1.94076 0.0556 2.91824 0.3468 1.06298 <.0001* 11.9491 M+Chan80x5-4-99-3-15-08 0.0079* 4.92749 0.0121* 4.48498 0.7812 0.24708 0.9553 0.04569 0.0045* 5.51491 M+Chan80x5-4-99-3-15-09 0.8901 0.1165 0.7304 0.31449 0.0852 2.48372 0.2622 1.34478 <.0001* 13.61499 M+Chan80x5-4-99-3-15-10 0.4489 0.80328 0.5788 0.54787 0.6856 0.37799 0.6173 0.48322 0.0124* 4.45984 M+Chan80x5-4-99-3-15-11 0.072 2.65475 0.0586 2.86588 0.3604 1.0241 0.858 0.15324 0.0003* 8.48404 M+Chan80x5-4-99-3-15-12 0.2231 1.50815 0.1891 1.67529 0.6177 0.48253 0.9215 0.08175 0.0118* 4.50582 M+Chan80x5-4-99-3-15-13 0.15 1.90939 0.143 1.95786 0.5183 0.65874 0.8556 0.15603 0.0002* 8.62023 M+Chan80x5-4-99-3-15-14 0.1169 2.16219 0.117 2.16178 0.6995 0.35784 0.9546 0.04643 0.0028* 5.99734 M+Chan80x5-4-99-3-15-15 0.1424 1.96262 0.2634 1.34023 0.4944 0.70607 0.4894 0.7164 <.0001* 12.44858 M+Chan80x5-4-99-3-15-16 0.9266 0.07622 0.9951 0.00492 0.1674 1.79836 0.2494 1.3955 0.0007* 7.46889 M+Chan80x5-4-99-3-15-17 0.1171 2.16043 0.254 1.3771 0.4547 0.79036 0.3817 0.96639 <.0001* 11.16905 M+Chan80x5-4-99-3-15-18 0.0871 2.46091 0.0343* 3.41256 0.0127* 4.43192 0.123 2.1105 0.0004* 8.17134 M+Chan80x5-4-99-3-15-19 0.0034* 5.81223 0.0043* 5.55326 0.4779 0.74026 0.7138 0.33754 0.1497 1.91147

108

Table 4-6 Continued. Grapefruit- Kumquat- Name Grapefruit-Flame Pummelo-5-1-99-2 Pummelo-5-4-99-3 RubyRed Meiwa P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. M+Chan80x5-4-99-3-15-20 0.9903 0.00975 0.8788 0.12928 0.2232 1.50757 0.3698 0.99834 0.0024* 6.14506 M+Chan80x5-4-99-3-15-21 0.9123 0.09185 0.7808 0.24767 0.1486 1.91925 0.3289 1.11639 <.0001* 9.89666 M+Chan80x5-4-99-3-15-22 0.2086 1.57568 0.1234 2.10758 0.2126 1.55649 0.509 0.67694 0.0734 2.6354 M+Chan80x5-4-99-3-15-23 0.0436* 3.1672 0.0362* 3.35691 0.4838 0.72788 0.6904 0.37097 0.6206 0.47781 M+Chan80xN11-15-15-1 0.0301* 3.54558 0.0136* 4.36174 0.0353* 3.38175 0.3294 1.11469 0.0004* 7.94273 M+Chan80xN11-3-15-01 <.0001* 9.85296 <.0001* 11.16434 <.0001* 12.13834 0.0023* 6.19768 <.0001* 13.89916 M+Chan80xN11-3-15-02 0.0507 3.01273 0.019* 4.01878 0.0109* 4.58811 0.1509 1.90383 0.0001* 9.34308 M+Chan80xN11-3-15-03 0.691 0.37008 0.6513 0.42947 0.0915 2.41162 0.2878 1.25092 <.0001* 17.94905 M+Chan80xN11-3-15-04 0.3438 1.07177 0.1403 1.9772 0.0021* 6.28316 0.0529 2.9689 <.0001* 19.6122 M+Chan80xN11-3-15-05 0.0861 2.47349 0.0783 2.57031 0.5256 0.64472 0.9276 0.0752 0.0009* 7.20648 M+Chan80xN11-3-15-06 0.0039* 5.64694 0.0067* 5.09042 0.7484 0.29017 0.828 0.18886 0.0232* 3.81499 M+Chan80xN11-3-15-07 0.7117 0.34047 0.4719 0.75297 0.0489* 3.05 0.2597 1.35448 <.0001* 17.56907 M+Chan80xN11-7-15-01 0.7461 0.29322 0.5269 0.64222 0.0627 2.7957 0.1707 1.77881 0.0007* 7.4847 M+Chan80xN11-7-15-05 0.7095 0.34366 0.4539 0.79207 0.0116 4.52527 0.0719 2.65648 <.0001* 13.95826 M+Chan80xN11-7-15-06 0.4743 0.74791 0.2719 1.30824 0.0039* 5.64277 0.0375* 3.32118 <.0001* 15.32229 M+Chan80xN11-7-15-07 0.4264 0.85496 0.2324 1.46687 0.0756 2.60491 0.3514 1.04976 <.0001* 10.43297 M+Chan80xN11-7-15-08 0.1857 1.69339 0.2161 1.54032 0.8682 0.14145 0.891 0.11549 0.0073* 5.00074 M+Chan80xN11-7-15-10 0.1587 1.85242 0.2446 1.41518 0.7891 0.23703 0.6574 0.42007 0.002* 6.34078 M+Chan80xN11-7-15-11 0.0898 2.43009 0.1107 2.21801 0.9742 0.02618 0.934 0.06833 0.0208* 3.92482 M+Chan80xN11-7-15-12 0.39 0.94461 0.4527 0.79464 0.3079 1.18293 0.5009 0.69296 <.0001* 12.96106 M+Chan80xN11-7-15-15 0.0166* 4.15823 0.0162* 4.17922 0.5334 0.62984 0.8935 0.11264 0.0487* 3.05451 M+Chan80xN11-7-15-16 0.2772 1.28882 0.2001 1.61795 0.5018 0.69116 0.8222 0.1959 0.1298 2.05589 M+Chan80xN11-7-15-18 0.5645 0.57287 0.4193 0.87167 0.429 0.84882 0.7144 0.33664 0.036* 3.36247 M+Chan80xN11-7-15-19 0.018* 4.07377 0.0166* 4.15534 0.4057 0.90486 0.5415 0.61462 0.8059 0.21593 5-4-99-3xDV5-15-1 0.5815 0.54322 0.5796 0.54642 0.18 1.72525 0.3969 0.92714 <.0001* 14.24723 5-4-99-3xDV5-15-2 0.3722 0.99163 0.6377 0.45058 0.2341 1.45943 0.2552 1.37232 <.0001* 10.86498 5-4-99-3xDV5-15-3 0.3572 1.03317 0.6137 0.48902 0.1895 1.67274 0.1773 1.74008 0.0004* 8.06486 5-4-99-3xDV5-15-4 0.9266 0.07622 0.9951 0.00492 0.1674 1.79836 0.2494 1.3955 0.0007* 7.46889 HBJL-1xDV5-15-1 0.2801 1.27835 0.2275 1.48831 0.6934 0.36666 0.9457 0.05581 0.1507 1.9047 C2-4-1xN11-3-15-1 0.5042 0.68649 0.4558 0.7878 0.2163 1.53944 0.7626 0.72658 <.0001* 12.93949 C2-4-1xN11-3-15-2 0.1754 1.75132 0.1164 2.16691 0.343 1.07397 0.0305* 0.27129 0.0148* 4.27627 C2-4-1xN11-3-15-3 0.0278* 3.62614 0.011* 4.57785 0.0035 5.75673 0.3858 3.53291 0.001* 7.04601 C2-4-1xN11-3-15-4 0.856 0.15555 0.74 0.30148 0.1889 1.676 0.2938 0.95563 0.0001* 9.3037 C2-4-1xN11-3-15-5 0.1005 2.31573 0.0517 2.99246 0.0889 2.44056 0.1273 1.22994 0.0535 2.95716 C2-4-1xN11-3-15-6 0.7427 0.29782 0.9799 0.02032 0.0635 2.7824 0.3244 2.07614 <.0001* 13.60262 C2-4-1xN11-3-15-7 0.6156 0.48594 0.3733 0.98882 0.0787 2.56476 0.6245 1.13015 <.0001* 11.55579 C2-4-1xN11-3-15-8 0.3215 1.13928 0.2419 1.42608 0.2462 1.40859 0.194 0.47155 <.0001* 10.66861 C2-4-1xN11-3-15-10 0.4234 0.86201 0.687 0.37585 0.1986 1.62554 0.8614 1.64931 0.0004* 7.92734 C2-4-1xN11-3-15-11 0.314 1.16314 0.2786 1.28354 0.6008 0.51038 0.8179 0.14932 0.0054* 5.30794 Pummelo-5-1-99-2 0.0159* 4.20277 0.031* 3.51613 1 0 0.8179 0.20112 0.0015* 6.64587 Pummelo-5-4-99-3 0.1001 2.31973 0.0989 2.33209 0.8179 0.20112 1 0 0.0345* 3.40506 Kumquat-Meiwa <.0001* 20.45079 <.0001* 18.04846 0.0015* 6.64587 0.0345* 3.40506 1 0

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Table 4-7. Triploid offspring significantly different (α=0.05) from the grapefruit controls but not significantly different from the Meiwa kumquat control. Grapefruit- Grapefruit- Grapefruit- Grapefruit- Kumquat- Kumquat- Name Flame Flame RubyRed RubyRed Meiwa Meiwa Grapefruit-Flame 1 0 0.818 0.20108 <.0001* 20.45079 Grapefruit-RubyRed 0.818 0.20108 1 0 <.0001* 18.04846 DV2xN11-7-15-18 0.0438* 3.16201 0.0306* 3.52861 0.5087 0.67741 M+Chan80x5-4-99-3-15-23 0.0436* 3.1672 0.0362* 3.35691 0.6206 0.47781 M+Chan80x5-4-99-3-15-06 0.0424* 3.19431 0.0365* 3.34764 0.6268 0.46795 M+Chan80xN11-7-15-19 0.018* 4.07377 0.0166* 4.15534 0.8059 0.21593 8-1-99-2BxHuds4x-14-7 0.0162* 4.18521 0.0185* 4.04538 0.1772 1.74107 M+Chan80x5-4-99-7-15-07 0.0141* 4.32581 0.0072* 5.01254 0.1826 1.71068 M+Chan80x7-2-99-11-15-7 0.0107* 4.60915 0.0121* 4.48498 0.0654 2.75352 4-4-99-7xHuds4x-14-12 0.0049* 5.41108 0.0064* 5.1411 0.1066 2.25606 M+Chan80x5-4-99-3-15-19 0.0034* 5.81223 0.0043* 5.55326 0.1497 1.91147 DV2xN11-3-15-27 0.003* 5.91966 0.0028* 6.01622 0.9995 0.000472 DV5xN11-3-15-11 0.003* 5.91966 0.0028* 6.01622 0.9995 0.000472 M+Chan80x5-4-99-7-15-08 0.0025* 6.10199 0.0033* 5.83119 0.1816 1.71631 DV2xN11-7-15-11 0.0013* 6.81298 0.0009* 7.14025 0.1646 1.81579 DV2xN11-3-15-09 0.0008* 7.30334 0.0007* 7.4304 0.7158 0.33473 DV2xN11-7-15-10 <.0001* 10.39464 <.0001* 10.14606 0.8019 0.22095 Kumquat-Meiwa <.0001* 20.45079 <.0001* 18.04846 1 0

Table 4-8. Triploid hybrids prone to dropping leaves following inoculation with citrus canker. Leaves Leaves Percent Average Name Sum of Lesions Inoculated Dropped Dropped Rating DV2xN11-3-15-15 5 5 100% - - DV2xN11-3-15-20 5 5 100% - - DV2xN11-3-15-24 5 5 100% - - DV5xN11-3-15-10 5 5 100% - - 4-4-99-7xHuds4x-14-11 10 8 80% 69.67 2.93 C2-4-1xN11-3-15-6 10 6 60% 180 4 DV2xN11-3-15-19 10 6 60% 63 3.5 DV2xN11-7-15-18 10 6 60% 114 1 M+Chan80x5-4-99-3-15-10 10 6 60% 108 2.59 M+Chan80xN11-3-15-02 30 15 50% 265 2.33 4-4-99-7xHuds4x-14-13 20 10 50% 185.5 2.9 DV2xN11-3-15-01 10 5 50% 134 3 DV2xN11-3-15-17 10 5 50% 250 3.13 DV2xN11-7-15-03 10 5 50% 220.5 3.4 M+Chan80x5-4-99-3-15-20 10 5 50% 19 2.25 M+Chan80x5-4-99-7-15-07 10 5 50% 222 0.8 M+Chan80xN11-7-15-01 10 5 50% 284 3.8 M+Chan80xN11-7-15-18 10 5 50% 174 2.6 DV2xN11-7-15-07 15 6 40% 87.25 2.317 M+Chan80x5-4-99-7-15-04 15 6 40% 218 3.33 M+Chan80x5-4-99-3-15-15 25 9 36% 43.25 2.875 M+Chan80x5-4-99-3-15-11 25 8 32% 117.4 2.25 4-4-99-7xHuds4x-14-02 20 5 25% 49.3 3.13 M+Chan80xN11-3-15-01 20 5 25% 270.2 2.27 M+Chan80xN11-7-15-12 20 5 25% 99 3 8-1-99-2BxHuds4x-14-03 40 8 20% 196.7 3.5 5-1-99-2xHuds4x-14-16 25 5 20% 144.2 3.24 M+Chan80x7-2-99-11-15-3 25 5 20% 97.2 2.95

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Table 4-9. Triploid hybrid family citrus canker tolerance comparison. LS Means differences for number of lesions per leaf and lesion rating per leaf using Tukey’s LSD test for Parental Combinations and Controls. Name Number of Hybrids Number of Lesions SD Lesion Rating SD Grapefruit (Flame and Ruby Red) - 45.07 ab 30.63 3.78 a 0.42 5-4-99-3xDV5 4 26.80 bcdef 22.23 3.57 a 1.04 5-1-99-2xHuds4x 16 32.47 bc 25.65 3.27 a 0.92 8-1-99-2BxHuds4x 3 29.13 bcd 24.38 3.22 abc 1.00 4-4-99-7xHuds4x 13 22.29 def 17.88 3.20 ab 1.08 DV5xN11-3 11 39.91 ab 37.40 3.16 abcd 1.36 Pummelo 5-1-99-2 S5 - 54.05 a 38.00 2.80 abcde 1.40 M+Chan80x5-4-99-7 10 31.08 bcd 26.38 2.74 bcde 1.34 M+Chan80xN11-3 8 35.91 ab 29.17 2.69 cde 1.36 M+Chan80xN11-7 12 25.02 cde 24.23 2.66 de 1.13 C2-4-1xN11-3 10 35.86 abc 22.89 2.66 cde 1.16 M+Chan80x7-2-99-11 7 15.06 efgh 13.23 2.62 de 1.22 DV2xN11-7 18 31.31 bcd 24.56 2.43 e 1.28 M+Chan80x5-4-99-3 10 14.86 fg 13.07 2.42 e 1.21 DV2xN11-3 8 39.34 ab 30.91 2.37 e 1.35 Pummelo 5-4-99-3 - 2.050 gh 1.99 2.05 e 1.36 Meiwa - 0.690 h 1.93 0.00 f 0 ZMean separation by column not connected by same letter are significantly different. Least squares mean analysis using Tukey’s HSD (α=0.05).

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Table 4-10. Differences in parent families using both lesion size and rating using Mahalanobis distance (P-values) and squared distances compared to grapefruit, pummelo, and Meiwa kumquat controls. Grapefruit Pummelo Kumquat Flame Ruby Red 5-1-99-2 S5 5-4-99-3 Meiwa

P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist. P-Value Sq. Dist.

Flame 1 0 0.822 2.4448 0.7060 2.7317 0.0043* 12.432 0.0001* 13.791

Ruby Red 0.8220 2.4448 1 0 0.7178 2.6737 0.0255* 9.552 <0.0001* 17.162

4-4-99-7 x Huds4x 0.6458 1.9017 0.3082 3.0906 0.1378 3.1863 0.0010* 7.116 <0.0001* 10.435

5-1-99-2 S5 x Huds4x 0.9053 0.9687 0.5764 1.9952 0.2794 2.3076 0.0003* 7.474 <0.0001* 11.761

5-4-99-3 x DV5 0.7460 1.9908 0.4655 3.0983 0.1454 4.1506 0.0046* 7.935 <0.0001* 13.580

8-1-99-2B x Huds4x 0.8807 1.1812 0.5485 2.3047 0.2037 2.9781 0.0001* 9.247 <0.0001* 10.764

C2-4-1 x N11-3 0.7905 1.4830 0.2213 3.6140 0.8225 1.0682 0.0001* 8.923 <0.0001* 10.690

DV2 x N11-3 0.6168 1.9695 0.0756 4.7709 0.4894 1.8271 <0.0001* 9.644 <0.0001* 8.9176

DV2 x N11-7 0.5815 2.0216 0.0897 4.4538 0.4308 1.9122 0.0002* 7.821 <0.0001* 7.8864

DV5 x N11-3 0.6511 1.9712 0.8819 1.1759 0.8840 0.9136 0.0010* 7.542 <0.0001* 13.343

M+Chan80 x 5-4-99-7 0.1928 3.6989 0.0609 5.0496 0.5820 1.6158 0.0025* 6.424 <0.0001* 13.800

M+Chan80 x 7-2-99-11 0.0426* 5.6371 0.0317* 5.9657 0.0944 3.6811 0.1418 3.295 <0.0001* 14.347

M+Chan80 x 5-4-99-3 0.2926 3.0185 0.1300 4.0029 0.3836 2.0192 0.0128* 4.891 <0.0001* 9.8515

M+Chan80 x N11-3 0.2987 3.1436 0.0495* 5.3094 0.6518 1.4612 0.0015* 6.847 <0.0001* 12.826

M+Chan80 x N11-7 0.4253 2.6375 0.2120 3.6127 0.5812 1.6374 0.0078* 5.631 <0.0001* 10.372

Pummelo 5-1-99-2 S5 0.7060 2.7317 0.7178 2.6737 1 0 0.0233* 8.318 <0.0001* 12.878

Pummelo 5-4-99-3 0.0043* 12.432 0.0255* 9.5517 0.0233* 8.3175 1 0 <0.0001* 15.623

Meiwa 0.0001* 13.791 <0.0001* 17.162 <0.0001* 12.878 <0.0001* 15.623 1 0

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Table 4-11. Field tree citrus canker response. Name Field Rating 4-4-99-7 x Huds4x-14-1 Many citrus canker lesions 4-4-99-7 x Huds4x-14-11 Many citrus canker lesions 4-4-99-7 x Huds4x-14-13 Many citrus canker lesions 5-1-99-2 S5 x Huds4x-14-8 Many citrus canker lesions 5-1-99-2 S5 x Huds4x-14-9 Many citrus canker lesions 5-1-99-2 S5 x Huds4x-14-9 Few citrus canker lesions 5-1-99-2 S5 x Huds4x-14-10 No citrus canker lesions 5-1-99-2 S5 x Huds4x-14-13 Many citrus canker lesions 8-1-99-2B x Huds4x-14-1 Many citrus canker lesions

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Figure 4-1. Tetraploid pummelo-orange hybrids. Trees in groves on top, with respective fruit below. Murcott+Chandler80 (left), C2-4-1 (right). Photos by author.

Figure 4-2. Embryo rescue of all seeds from a single fruit of 5-1-99-2 S5 x Hudson 4x. Immature seeds extracted from their seed coat were placed on cellulose acetate discs on EME-Maltose media. Large seeds were likely selfs resulting from pollen contamination. Photo by author.

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Figure 4-3. Potentially triploid seedlings germinating in the greenhouse. Photo by author.

Figure 4-4. Flow cytometry read out on Partec PA machine. Three peaks on the histogram represent a 2x key lime on left, 3x pummelo x grapefruit hybrid in center, and 4x giant key lime on right. Photo by author.

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Figure 4-5. Triploid offspring: Citrus canker lesion size vs. number of lesions. Color and size of dot represents lesion size rating (single replicate hybrids removed).

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Figure 4-6. 5-1-99-2 S5 x Huds4x-14-10 tree in field, exhibiting no citrus canker. Photos by author.

Figure 4-7. 5-1-99-2 S5 x Huds4x-14-9 trees in field, exhibiting low citrus canker. Photos by author.

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Figure 4-8. Triploid offspring sorted by number of citrus canker lesions, excluding hybrids with only one replicate per assay. Field planted trees highlighted in red text with darker red and blue bars; grapefruit, pummelo, and kumquat controls in blue text.

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CHAPTER 5 TESTING OF SELECTED PUMMELO AND HYBRID INTERSTOCKS TO MITIGATE HLB SYMPTOMS IN COMMERCIAL SWEET ORANGE/SWINGLE TREES

Background and Objectives

The inspiration for this project came from a research grove where trees of different genetic combinations were grown on Swingle rootstock and were being culled(for fruit characteristics) to make room for new trees. It was noticed that some of the trees even though they were probably CLas infected had extensive root systems,

although Swingle is not regarded as being tolerant. The typical loss of root mass

expected from Swingle rootstock under an HLB-affected scion was not observed.

Grafting is a common method for overcoming problems that the scion alone

cannot address. These may be abiotic in nature, for example, when the soil is alkaline

or prone to flooding. Grafting is also used to overcome biotic threats, such as soil-borne

diseases, or when the tree is attacked by pests such as nematodes or Diaprepes root

weevils. The resistance or tolerance of the rootstock may confer this defense to the

scion.

It is known that certain products, such as hormones and RNAs will pass through

the graft union, in addition to photoassimilates, amino acids and other small molecules

found in the phloem sap, and travel in either direction from the scion to the rootstock

and vice versa (Harada, 2010; Ruiz-Medrano et al., 1999; Spiegelman et al., 2013).

Some of these products may move in response to the tree’s natural defenses and may

improve its chances of fighting HLB.

Interstocks are most commonly used to compensate for scion/rootstock

incompatibility, preventing decline of the tree or to regulate the growth when one grafted

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element is more vigorous than the other (Zhao et al., 2016). The interstock may prevent a bulge that can slow sap and stunt the growth of the tree (Castle, 1992). Additionally, interstocks may increase the lifespan of the tree and increase production and fruit quality (Gil-Izquierdo et al., 2004).

Interstocks in other genera, specifically Malus and Prunus, are more common but are an uncommon practice in citrus cultivation (Castle et al., 1993; Seleznyova et al., 2008). However, there has been limited use of interstocks to challenge diseases such as CTV and citrus decline (Castle, 1992).

Using interstocks that are tolerant to HLB may be able to confer this tolerance to the scion and the rootstock. Some rootstocks appear to be conferring this ability to the scion in early rootstock trials (Grosser, 2012).

Materials and Methods

Interstock candidates were selected based upon their field performance as scions on Swingle citrumelo rootstock, showing few or no HLB symptoms, and have shown exceptional tree vigor. Some pummelo selections have been described previously while others were different than those from previous experiments, selected due to better graft survival, described in Table 5-1.

Two of the interstocks, C2-4-1 and C2-4-8 (Figure 5-1), both resulted from a tetraploid somatic fusion of Succari sweet orange with Hirado Buntan pummelo seedling back-crossed to Succari, presumed to be the result of unreduced gametes of Succari.

Both have shown exceptional vigor even though the trees are CLas infected and are grafted onto Swingle citrumelo rootstock, which is not known for being HLB resistant.

They have shown exceptional ability to recover from a CLas infection when treated with

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controlled release fertilizer containing increased micronutrients (J. Grosser, personal

communication).

Also used in this experiment was a somatic hybrid obtained from protoplast

fusion of Nova mandarin hybrid + Citropsis gilletiana (N+C), created by the Grosser lab

(Mourão Fo et al., 1994), chosen for its unique genetic background and because it was

used in another experiment (described in Appendix A). It had performed well as a

rootstock in a previous rootstock trial before the trial was destroyed by the state of

Florida’s canker eradication program (Grosser, 2012). It is susceptible to HLB but

appears to be tolerant and has continued to grow in spite of CLas infection (Figure 5-2).

Stick Grafting

Stick grafting was chosen as a method for grafting due to the use of CLas infected materials. This technique is routinely used in the CREC citrus breeding program to efficiently produce grafted trees infected with CLas. Infection rates higher than 95% are routinely obtained with this technique, whereas standard inverted-T budding with infected scion results are highly variable with usually less than 50% infection (J.W. Grosser, personal communication).

A schematic depicting the process of creating the interstock trees is provided in

Figure 5-3. Swingle rootstocks were nursery propagated liners of uniform size and age, selected with diameters of 8-10mm. Interstock bud sticks were taken from field trees,

selected from mature flushes with diameters of 8-10mm that were semi-hard and still

green, trimmed to approximately 26cm in length. Most of the interstock source trees in

the field on Swingle citumelo rootstock appeared to be vigorous and healthy, but did

show mild HLB symptoms, suggesting CLas infection. Leaves were removed and sticks

washed and scrubbed with a plastic brush using soap and water, and then surface

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sterilized using 20% bleach solution with one drop Tween® 20 per L, soaking the material for twenty minutes and then rinsing thoroughly with tap water

Rootstocks were severed approximately 30cm above the soil line and prepared for cleft grafting by making an incision through the center of the tip, splitting it down the middle for about 2-3cm. Interstock sections were cut at the base into a V-shape using a sharp grafting knife (Figure 5-3, A) and inserted into the cleft of the rootstock, selected of a similar diameter and matching cambium to cambium. The graft union was tightly wrapped with polyethylene Plastrip budding strips (John F. Malaney Co., Sacramento,

CA). The entire budstick was then wrapped with Parafilm M (Bemis Co. Inc., Neenah,

WS) to seal in moisture while the graft healed. Parafilm was not removed as new shoots were able to grow through the film which eventually deteriorated. Plastrip budding tape was removed when the interstock appeared to have healed and had sprouted new leaves. The interstock was prepared for cleft grafting of the scion when the new flush had grown several cm and had hardened off with fully expanded leaves. The new shoot was cut back and, by making an incision through the center of the tip of the main shoot, split down the middle for about two to three centimeters. Interstock grafting is shown in

Figures 5-4 & 5-5, and completed interstock trees with Valencia scion are shown in

Figure 5-6.

Scion budwood came from Valencia trees less than fifteen years old, growing in the CREC grove in Lake Alfred, FL, where HLB is present and most trees exhibit severe infection. Scion budwood source trees exhibited strong HLB symptoms and were assumed to be HLB-infected at the time of grafting and later confirmed by PCR.

Budwood sticks selected were less than a year old (woody yet still green and pliable)

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and at least eight to ten millimeters in diameter. Leaves were removed and sticks were

trimmed to 26cm and surface sterilized as described for the interstock. Budsticks were

then immediately used for cleft grafting, using the method described previously for the

interstock. Completed interstock trees are shown in Figure 5-6.

Control Trees

Swingle citrumelo was used as self-grafted control by severing the tree at thirty

centimeters above the soil line, splitting the tip with a budding knife and cleft grafting a

stick with leaves removed back onto itself, as described for the interstock. Experimental

OLL-15 sweet orange was used as a non-pummelo scion control.

Rootstocks

The total number of trees in the study was 150. The rootstock used was Swingle citrumelo ('Duncan' grapefruit x Poncirus trifoliata), selected because it is a common industry standard rootstock and has been the most propagated and planted rootstock in

Florida over the past decade. However, throughout the Florida industry, trees on this rootstock have been heavily impacted by HLB, and trees have been shown to exhibit severe feeder root loss immediately after infection (Johnson et al., 2014).

HLB Development

Trees were allowed to heal and flush in a warm greenhouse. When new growth was over 0.25m, trees were removed to an air-conditioned greenhouse maintained at

27 C (80˚F) to create a more ideal growing environment for the CLas bacterium.

Following successful scion grafting, trees were then allowed to grow for six months in the HLB greenhouse when they were rated for HLB severity using a scale of

0-4, as shown in Table 5-2.

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CLas Detection

Quantitative real-time PCR (qRT-PCR) based CaLas detection was conducted to test the cycle threshold (Ct) values and bacterial titer of the ‘Valencia’ scions. DNA was isolated from the petioles of three to four leaves gathered from each ‘Valencia’ scion, weighed into 100µg portions, ground using liquid nitrogen and a BeadBug Microtube

Homogenizer (Benchmark Scientific, Edison, NJ). DNA was extracted and purified with

GeneJET Plant Genomic DNA Purification Kit (#K0722-Thermo Scientific). The

extracted DNA was quantified in a NanoDrop ND-100 spectrophotometer (Thermo

Scientific, Wilmington, DE). The TaqMan® Gene Expression Master Mix Kit (Applied

Biosystems, Foster City, CA) was used to perform qPCR assay. CaLas specific

CQULA04F and CQULA04R primer pair and CQULAP10 TaqMan-probe (FAM

fluorophore dye) were used for the amplification of CaLas 16sRNA (Wang et al., 2006).

All reactions were carried out in 25 µl reaction volume containing 50 ng DNA and 0.3

µM probe and primer concentrations. Amplification was conducted over 40 cycles of

qPCR in StepOnePlus™ PCR system (Applied Biosystems). Results were analyzed

using Life Technologies StepOne™ Software V2.3.

A more accurate representation of the amount of DNA present in leaf tissue was

calculated using a series of dilutions of plasmids containing cloned targets of the HLB

tags, where the amount of bacterial genomes could be quantified in a measured sample

(Li et al., 2008, 2006). Each sample was then diluted in a series of solutions, from

1,000,000 to ten copies per µL, which was then used to quantify the amount of HLB

bacterial DNA in samples isolated from 100mg of leaf petiole.

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Growth Data

Measurements of the scion/interstock/rootstock combination were performed

using a digital caliper (Digimatic 500-321 CD-6”, Mitutoyo Corporation, Japan.) measuring at three positions on each tree: on the rootstock 2.5 cm below the interstock;

in the center of the interstock; and on the scion 2.5 cm above the interstock. The length

of the interstock was measured from the center of each graft union. Height was

measured from the soil line to the tip of the longest branch.

The day before field planting, roots were washed of all soil and air dried, and the

entire plant was weighed. Following weighing, roots were wrapped in damp paper

towels and trees bundled into five-gallon buckets for transport to the field site.

Statistical analysis was performed on all measured growth and disease

parameters using JMP® Pro 13 using Tukey’s HSD. Multivariate analysis performed

with JMP® Pro 13 and SAS 9.4 using the CANDISC procedure.

Results and Discussion

Many of the trees growing in the greenhouse appeared healthy, with many

showing no HLB symptoms, although a few of the scions and interstocks died by the

end of the experiment (‘n’ column in Table 5-4). All trees have tested positive for the

presence of the Candidatus Liberibactor asiaticus bacterium (Figure 5-7). This further

validates the efficiency of the ‘stick’ grafting method for obtaining CLas infected trees.

Ct values were used to approximate the amount of CLas bacteria in the scion.

The Ct, or “threshold cycle”, values are a measure of the number of cycles of

amplification performed by the PCR machine until a measurable quantity of sample is

detected (Porterfield, 2015). Ct values are inverse to the amount of nucleic acid in the

sample, with lower Ct values representing higher CLas titers, and higher numbers

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representing lower amounts and possibly none at all. The lower the value of the Ct, then the higher amount of DNA in the sample, reflecting a lower number of cycles to detect

DNA above the threshold. Ct value above 36 is considered to be HLB negative, as this would reflect a very small amount of DNA, perhaps one copy, and falls within the range of error making it questionable if there actually is any target DNA in the sample or if there was some environmental contamination.

Pummelo HBJL-1 was the most standout interstock performer for most traits evaluated, although it wasn’t statistically different (α=0.05) from pummelo 5-4-99-7 except for rootstock diameter. It had the highest Ct values, and consistently had the heaviest and taller plants, and had thicker scion, interstock, and rootstock diameters

(Table 5-4). The parent tree in the field has shown some HLB symptoms but perhaps the addition of the scion and rootstock allows for a new combination of genetics that contribute to better plant defense, where the sum of the whole is greater than the individual parts.

As expected, the rootstock control Swingle interstock was the worst performer in nearly all categories (Table 5-4), as trees were both the shortest and lightest, with the smallest rootstock and interstock diameters, the smallest scion diameter and the lowest

Ct values indicating higher levels of CLas detected in the scion. The sweet orange OLL-

15 control exhibited the second highest levels of bacterial titer, but was intermediate for height and weight.

Pummelo 8-1-99-1B, also performed well high Ct values (low CLas bacterial populations) and producing more robust plants when used as interstock (Table 5-4).

The mother tree has performed well in the field regarding HLB tolerance, and continues

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to show good health with only mild HLB symptoms. The mother tree initially tested CLas negative in 2013, but tested positive in 2016 (Table 3-8).

Pummelo hybrids C2-4-1 and C2-4-8 were both top performers for measured attributes; neither were significantly different from each other for all measured attributes, both having some of the tallest and heaviest trees as well as having high Ct values.

Both trees have appeared to show good resistance to HLB under field conditions and it would appear it transfers some this ability to the scion, growing vigorously and appearing healthy and able to keep HLB symptoms to a minimum.

Nova+Citropsis gilletiana also appeared to perform well as an interstock. These trees had weights and heights above the 90% quantile of all trees, with no significant differences among height and weight for the top five interstock selections, and had lower Ct values and bacterial titer than the worst affected, but not statistically better than the controls (Table 5-4). The Nova+Citropsis gilletiana interstocks had a tendency to produce flowers and shoots, and will likely require covering to prevent shoots and flowers as an interstock. Prior to field planting interstocks were wrapped with aluminum foil for this purpose.

It is surprising that pummelo 5-4-99-3 didn’t perform better, as it has shown under field conditions to have exceptionally low levels of HLB symptom development.

As an interstock it appeared to perform poorly. It was difficult to keep alive, with only eight interstocks surviving the duration of the testing and some of the lowest Ct values

(Table 5-4). It had two siblings in the test: 5-4-99-4 and 5-4-99-7. The former performed nearly as poorly as 5-4-99-3, however 5-4-99-7 was one of the best interstocks for most

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attributes and had a high Ct value and trees that were among the tallest and heaviest

(Table 5-4).

A correlation matrix produced from JMP was used to determine if there were

significant relationships between variables (Table 5-5). The strongest correlation was

between plant height and weight (0.6722) which would be logical, as weight would

increase with increased mass corresponding to height. Scion diameter also correlated

strongly with plant height (0.478496) and weight (0.623078). HLB rating had the strongest correlation to plant weight (0.35092), followed by Ct average (0.29302) which is not very strong but logically should be associated with CLas infection.

Interstock length did not have any strong correlations to any of the measured attributes. The strongest correlation was with interstock diameter (0.188793), which is not a strong correlation. Interstock length had weak negative correlations to HLB rating

(-0.13772), scion diameter (-0.00455), and Ct Average (-0.1771). C2-4-1 had the longest interstocks (359.44 ± 53.76 mm, Table 5-4) and had some of the highest Ct values. Swingle had some of the shortest interstocks, but not significantly different from

HBJL-1 interstock lengths, which had the highest Ct values. It would appear that interstock length does not have as much importance as the physical attributes of the interstock being used.

It’s difficult to say what interstock is best based on plant height and weight, as there were few significant differences between selections. The most variation was within

Ct values, with HBJL-1 and 5-4-99-7 having the highest values. These two selections were at the top of the list for the attributes measured, suggesting that these are two of

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the most robust interstock selections. C2-4-1 and C2-4-8 were also very similar with good performance, and had the heaviest and tallest trees with high Ct values.

A multivariate approach was used to help determine the best interstocks, using a combination of the measured attributes. The canonical plot (Figure 5-7) shows that most of the interstocks overlapped with no clear separation of interstocks. Interstocks that had higher Ct values and thicker rootstock diameters, both which had the most variation in levels of separation (Table 5-4), appear on the upper right, show HBJL-1 and C2-4-8, while swingle is on the lower left indicating it had values on the other end of the spectrum. C2-4-1, HBJL-4, and UKP-1 appear on the lower right, all of which do not show an overlap of the 95% confidence interval ellipses with Swingle.

The Mahalanobis values (Table 5-6) from the canonical discrimination analysis showed that Swingle was significantly different from every other interstock except 5-4-

99-3, confirming previous observations that Swingle had the smallest plants and the lowest Ct values. C2-4-1, C2-4-8, HBJL-1, HBJL-4 had the most significant values from other interstocks and confirm that these plants were among the best of the interstock selections, with C2-4-1 significantly different from all other interstocks. This would imply that C2-4-1 was the most successful interstock as much as Swingle was the least successful.

The combined data from this study strongly suggests that some interstocks have the ability to impact the disease resistance/tolerance response of an otherwise HLB susceptible scion/rootstock combination. Since the interstock is only a small portion of the total tree, this positive response is likely due to some signaling mechanism moving across the graft union into the scion and rootstock. If the response continues, more

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research is warranted to ascertain the responsible mechanism. Trees from this study have been planted in a commercial grove near Avon Park, FL (Figures 5-8 to 5-11) and will continue to be observed to determine if this interstock effect will continue to confer resistance over time. A few of the trees have died, and a small number of trees that did not get well established were removed. Many of the trees remain in the field trial, and after 11 months, most were showing good health and vigorous growth, in spite of their

CLas infection (Figures 5-8 to 5-11). The CREC citrus improvement team will continue to monitor these trees to determine the long-term effects of the interstocks on tree growth and subsequent productivity. It will be interesting to determine the long-term impact of the interstock on these trees. Positive results so far suggest that other highly tolerant or resistant genotypes should be tested as interstocks, as if a reliable interstock is identified, it could restore the use of popular scion/rootstock combinations in the

Florida industry and elsewhere.

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Table 5-1. Summary of Selected Interstock Tree Combinations, with Swingle citrumelo rootstock and HLB-infected scion, initial number of trees. Number Interstock of Trees Description C2-4-1 10 Tetraploid (Succari sweet orange + Hirado Buntan pummelo seedling) x Succari sweet orange C2-4-8 10 Tetraploid (Succari sweet orange + Hirado Buntan pummelo seedling) x Succari sweet orange Nova+Citropsis 10 Somatic fusion of Nova mandarin hybrid + Citropsis gilletiana (N+C) gilletiana OLL-15 10 Sweet orange selection used as a control interstock 5-1-99-3 10 Hirado Buntan pummelo seedling 7-2-99-11 10 Large Pink pummelo seedling 5-1-99-2 S5 10 Hirado Buntan pummelo seedling 5-4-99-4 10 Red Shaddock pummelo seedling 5-4-99-3 10 Red Shaddock pummelo seedling 5-4-99-7 10 Red Shaddock pummelo seedling 8-1-99-1B 10 Liang Ping Yau pummelo seedling HBJL-1 10 Hirado Buntan pummelo seedling HBJL-4 10 Hirado Buntan pummelo seedling UKP-1 10 Red fleshed pummelo seedling of unknown parentage Swingle Citrumelo 10 Control interstock, Duncan grapefruit x Poncirus trifoliata

Table 5-2. HLB Disease Severity Rating Scale Rating Description 0 Healthy, NO HLB Symptoms 1 Healthy new growth, 1/4 leaves yellow 2 Half of the leaves show yellowing, mottle 3 ½-¾ of the leaves show yellow mottling, some twig dieback 4 Tree deceased

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Table 5-3. ANOVA for measured interstock attributes. Source Plant Height DF Sum of Squares Mean Square F Ratio Prob > F Interstock 14 1,855,614.2 132544 3.6163 <0.0001* Error 111 4,068,374.2 36652 C. Total 125 5,923,988.4

Plant Weight Interstock 14 139948.47 9996.32 2.4322 0.0051* Error 111 456201.84 4109.93 C. Total 125 596150.31

HLB Visual Rating Interstock 14 25.91277 1.85091 2.1314 0.0147* Error 117 101.60238 0.86840 C. Total 131 127.51515

Ct Value Interstock 14 1,494.832 106.774 9.8023 <0.0001* Error 301 3,278.717 10.893 C. Total 315 4,773.548

Rootstock Diameter Interstock 14 80.43509 5.74536 5.0521 <0.0001* Error 111 126.23075 1.13721 C. Total 125 206.66584

Interstock Diameter Interstock 14 60.08937 4.29210 4.6231 <0.0001* Error 111 103.05371 0.92841 C. Total 125 163.14308

Scion Diameter Interstock 14 52.70673 3.76477 4.4390 <0.0001* Error 110 93.29279 0.84812 C. Total 124 145.99952

Interstock Length Interstock 14 281,927.94 20137.7 3.7516 <0.0001* Error 110 590,453.26 5367.8 C. Total 124 872,381.20 * Significant value

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Table 5-4. Mean values and standard deviation (SD) for plant height, weight, rootstock diameter, interstock diameter, scion diameter, interstock length, and Ct value. Raw means and standard deviations are reported, but mean separation on LSMeans was done using Tukey’s test, α=0.05. Rootstock Interstock Scion Interstock Height Weight Diameter Diameter Diameter Length Rating Interstock n (cm) SD (g) SD (mm) SD (mm) SD (mm) SD (mm) SD (0-4) SD Ct Value SD

HBJL-1 10 142.56 abcz 135.57 248.7 a 66.92 12.02 ab 1.16 8.58 abcd 0.6 7.20 ab 0.56 246.5 bc 47.85 0.22 b 0.44 33.2531 a 3.65

5-4-99-7 8 130.02 abc 226.09 176.3 ab 48.88 10.19 cde 0.89 8.37 abcd 0.53 6.32 bcd 0.88 246.25 bc 67.18 0.86 ab 1.07 29.6398 abcd 3.65

C2-4-1 9 152.26 ab 95.92 188.0 ab 45.26 11.97 abc 0.74 9.22 ab 1 8.08 a 0.61 359.44 a 53.76 0.63 ab 0.52 29.4732 b 2.59

C2-4-8 9 156.07 a 123.69 245.2 a 45.02 11.38 abcd 1.18 9.67 a 1.27 7.10 abcd 0.77 270.0 ab 77.78 0.89 ab 0.6 29.4721 bc 2.74

5-1-99-3 10 121.16 bc 216.89 194.3 ab 79.94 11.21 abcd 0.92 9.05 ab 0.73 6.53 abcd 0.86 209.5 bc 37.38 0.9 ab 0.32 28.2957 bcde 3.71

8-1-99-1B 9 139.35 abc 215.27 231.5 ab 94.27 11.13 abcd 0.72 8.90 abc 0.66 7.23 ab 1.19 252.22 b 57.67 1.13 ab 1.25 27.6208 bcde 3.41

HBJL-4 10 134.79 abc 207.89 203.6 ab 78.06 12.20 a 1.2 9.25 ab 0.97 7.28 ab 0.86 236.5 bc 54.47 1.07 ab 0.92 26.851 bcde 4.12

5-1-99-2 S5 10 126.79 abc 319.18 166.7 ab 69.62 10.92 abcde 1.14 9.08 ab 1.22 7.12 abc 0.83 265.0 b 48.99 1 ab 1.31 26.1782 cde 3.08

Nova+ 9 143.16 abc 188.56 208.0 ab 77.11 10.24 cde 1.09 7.94 bcd 0.81 7.32 ab 0.96 275.0 ab 51.78 1.56 ab 1.01 26.1767 bcde 1.73 Citropsis

UKP-1 9 127.85 abc 172.27 168.6 ab 62.82 11.33 abcd 0.72 9.07 ab 0.69 6.91 abcd 1.39 246.67 bc 65.38 1.2 ab 1.14 25.8388 de 4.21

7-2-99-11 10 120.71 bc 276.86 199.8 ab 114.95 11.07 abcd 0.97 9.11 ab 0.99 6.86 abcd 1.13 197.5 bc 42.18 1.2 ab 0.63 25.7278 e 1.91

5-4-99-3 8 122.40 abc 230.8 177.3 ab 73.84 10.83 abcde 1.44 8.45 abcd 1.04 6.14 bcd 1.28 200.0 bc 61.3 2 a 1.22 25.7139 de 4.67

OLL-15 10 134.56 abc 173.19 170.3 ab 47.67 10.63 bcde 0.85 7.35 cd 0.93 6.59 abcd 0.81 271.5 ab 66.46 0.8 ab 0.84 25.5907 e 3.28

5-4-99-4 10 129.48 abc 286.04 148.5 ab 56.54 10.25 de 1.13 8.07 bcd 1.27 5.55 d 1.49 242.0 bc 66.38 0.5 ab 0.53 25.3789 e 3.19

Swingle 8 112.40 c 207.61 121.3 b 53.72 9.29 e 1.19 7.19 d 1.54 5.48 cd 0.85 155.0 c 33.81 1.75 ab 1.39 24.8944 e 2.08

ZMean separation by column not connected by same letter are significantly different.

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Table 5-5. Correlation matrix for mean values of measured attributes. Plant Plant HLB Rootstock Interstock Scion Interstock Ct Height Weight Rating Diameter Diameter Diameter Length Average

Plant Height 1 0.66069 0.19947 0.264119 0.320053 0.478496 0.130652 0.20762

Plant Weight 0.66069 1 0.35092 0.407815 0.505002 0.623078 0.127107 0.172826 HLB Rating 0.19947 0.35092 1 0.15628 0.27897 0.2276 -0.13772 0.29302 Rootstock 0.264119 0.407815 0.15628 1 0.568454 0.456185 0.146871 0.154046 Diameter Interstock 0.320053 0.505002 0.27897 0.568454 1 0.504717 0.188793 0.188443 Diameter Scion 0.478496 0.623078 0.2276 0.456185 0.504717 1 -0.00455 0.253158 Diameter Interstock 0.130652 0.127107 -0.1377 0.146871 0.188793 -0.00455 1 -0.1771 Length Ct Average 0.20762 0.172826 0.29302 0.154046 0.188443 0.253158 -0.1771 1

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Table 5-6. P-values and squared distances (Mahalanobis distances) from canonical discrimination procedure for all measured attributes by interstock. (α=0.05)

Interstock 5-1-99-2 S5 5-1-99-3 5-4-99-3 5-4-99-4 5-4-99-7 7-2-99-1 8-1-99-1 C2-4-1 C2-4-8 HBJL-1 HBJL-4 Nova+Cit UKP-1 OLL-15 Swingle

5-1-99-2 S5 P-Value 1 0.0142* 0.0051* 0.0304* 0.2486 0.0077* 0.4963 0.0033* 0.009* <.0001* 0.001* 0.0064 0.4097 0.0048* 0.0007* Distancez 0 4.86544 6.27377 5.08738 2.77771 5.33384 1.86614 6.26325 5.47245 12.7621 6.86956 5.74108 2.21694 5.67945 8.48504

5-1-99-3 P-Value 0.0142* 1 0.8543 0.059 0.1865 0.107 0.1165 <.0001* 0.0151* 0.0006* 0.0234* <.0001* 0.0395* <.0001* 0.0058* Distance 4.86544 0 0.95669 4.04179 2.76614 2.89009 2.98297 15.4974 4.51679 6.79673 3.9802 10.2202 4.06644 8.46425 5.98529

5-4-99-3 P-Value 0.0051* 0.8543 1 0.1502 0.2626 0.2269 0.1226 <.0001* 0.0134* 0.0001* 0.0126* 0.0002* 0.0425* 0.0012* 0.1106 Distance 6.27377 0.95669 0 3.52249 2.7194 2.58562 3.29042 17.0835 5.1531 8.92925 4.95791 8.32957 4.45353 6.69946 3.83707

5-4-99-4 P-Value 0.0304* 0.059 0.1502 1 0.3868 0.0006* 0.0526 <.0001* 0.009* 0.0002* 0.0024* <.0001* 0.0079* 0.0117* 0.0397* Distance 5.08738 4.04179 3.52249 0 2.44888 7.8459 4.33155 14.7958 5.88296 9.34352 6.68548 10.3673 6.32546 5.41172 5.15754

5-4-99-7 P-Value 0.2486 0.1865 0.2626 0.3868 1 0.0032* 0.3592 <.0001* 0.1619 0.0002* 0.0005* 0.0189* 0.0128* 0.0209* 0.0355* Distance 2.77771 2.76614 2.7194 2.44888 0 5.98064 2.24054 10.6557 3.03547 8.60246 7.43621 4.87255 5.49011 4.56409 4.94192

7-2-99-1 P-Value 0.0077* 0.107 0.2269 0.0006* 0.0032* 1 0.1019 <.0001* <.0001* <.0001* 0.0002* <.0001* 0.3404 <.0001* 0.0084* Distance 5.33384 2.89009 2.58562 7.8459 5.98064 0 3.08987 17.4739 8.92751 15.4998 7.20419 8.56699 2.19051 8.90253 5.68104

8-1-99-1 P-Value 0.4963 0.1165 0.1226 0.0526 0.3592 0.1019* 1 <.0001* 0.0271* <.0001* 0.006* 0.0551 0.3768 0.075 0.0014* Distance 1.86614 2.98297 3.29042 4.33155 2.24054 3.08987 0 8.65866 4.30858 8.6447 5.17559 3.75305 2.18778 3.32941 7.46358

C2-4-1 P-Value 0.0033* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* 1 <.0001* <.0001* <.0001* 0.0001* <.0001* <.0001* <.0001* Distance 6.26325 15.4974 17.0835 14.7958 10.6557 17.4739 8.65866 0 10.4711 16.3717 10.2441 8.46874 9.12536 8.39847 24.3587

C2-4-8 P-Value 0.009* 0.0151* 0.0134* 0.009* 0.1619 <.0001* 0.0271* <.0001* 1 <.0001* 0.0003* <.0001* 0.0004* <.0001* <.0001* Distance 5.47245 4.51679 5.1531 5.88296 3.03547 8.92751 4.30858 10.4711 0 8.70451 7.22407 8.55253 7.83905 9.73804 12.7472

HBJL-1 P-Value <.0001* 0.0006* 0.0001* 0.0002* 0.0002* <.0001* <.0001* <.0001* <.0001* 1 0.0021* <.0001* <.0001* <.0001* <.0001* Distance 12.7621 6.79673 8.92925 9.34352 8.60246 15.4998 8.6447 16.3717 8.70451 0 5.90165 16.0403 13.6101 10.7998 18.4924

HBJL-4 P-Value 0.001* 0.0234* 0.0126* 0.0024* 0.0005* 0.0002* 0.006* <.0001* 0.0003* 0.0021* 1 <.0001* 0.0555 <.0001* <.0001* Distance 6.86956 3.9802 4.95791 6.68548 7.43621 7.20419 5.17559 10.2441 7.22407 5.90165 0 12.494 3.79356 7.64725 13.374

Nova+ P-Value 0.0064* <.0001* 0.0002* <.0001* 0.0189* <.0001* 0.0551 0.0001* <.0001* <.0001* <.0001* 1 0.001* 0.1119 <.0001* Citropsis Distance 5.74108 10.2202 8.32957 10.3673 4.87255 8.56699 3.75305 8.46874 8.55253 16.0403 12.494 0 7.23845 3.01477 10.3045

UKP-1 P-Value 0.4097 0.0395* 0.0425* 0.0079* 0.0128* 0.3404 0.3768 <.0001* 0.0004* <.0001* 0.0555 0.001* 1 0.0038* 0.001* Distance 2.21694 4.06644 4.45353 6.32546 5.49011 2.19051 2.18778 9.12536 7.83905 13.6101 3.79356 7.23845 0 5.85718 8.14171

OLL-15 P-Value 0.0048* <.0001* 0.0012* 0.0117* 0.0209* <.0001* 0.075 <.0001* <.0001* <.0001* <.0001* 0.1119 0.0038* 1 0.0002* Distance 5.67945 8.46425 6.69946 5.41172 4.56409 8.90253 3.32941 8.39847 9.73804 10.7998 7.64725 3.01477 5.85718 0 8.8373

Swingle P-Value 0.0007* 0.0058* 0.1106 0.0397* 0.0355* 0.0084* 0.0014* <.0001* <.0001* <.0001* <.0001* <.0001* 0.001* 0.0002* 1 Distance 8.48504 5.98529 3.83707 5.15754 4.94192 5.68104 7.46358 24.3587 12.7472 18.4924 13.374 10.3045 8.14171 8.8373 0 zSquared distance.

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Figure 5-1. C2-4-1 (left) and C2-4-8 (right). Both are (Succari sweet orange + Hirado Buntan pummelo seedling) x Succari sweet orange and have shown good tolerance to HLB. Pictures taken Spring 2017, photos by J. Grosser.

Figure 5-2. Nova+Citropsis gilletiana (N+C) growing in research grove, Spring 2017. Photo by author.

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Figure 5-3. Process of stick grafting. Rootstock is cleft grafted with interstock (A). Interstock leafs out and pushes out a new shoot (B). New shoot is trimmed and cleft grafted with scion (C). Interstock leaves help to sustain the HLB infected scion (D), which are removed when scion is well established (E).

Figure 5-4. Grafted interstocks on Swingle rootsocks, prior to grafting of Valencia scions. Photo by author.

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Figure 5-5. Grafted interstocks on Swingle rootsocks, beginning to leaf out. Photo by author.

Figure 5-6. Valencia on interstock trees prepared for field planting. All are HLB infected in an air-conditioned greenhouse and most show few symptoms. Photo by author.

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Figure 5-7. Plot of Canonical1*Canonical2 for measured attributes of interstocks (excluding visual rating and interstock length). Ellipses represent 95% confidence interval. This is a graphical representation of Table 5-6.

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Figure 5-8. Valencia scion on Swingle rootstock with interstock trees planted in November 2016 near Avon Park, FL, photo taken in January 2017. Interstocks were wrapped with aluminum foil to prevent sprouting from the interstock. Photo by author.

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Figure 5-9. CLAS-infected interstock trees planted in November 2016 near Avon Park, FL, photo taken in October 2017. From left to right: HBJL-1 #1, HBJL-4 #2, and 8-1-99-1B #2. Photos by Maria Quirico Bautista.

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Figure 5-10. CLAS-infected interstock trees planted in November 2016 near Avon Park, FL, photo taken in October 2017. From left to right: 7-2-99-11 #8, 7-2-99-11 #9, 7-2-99-11 #10. Photos by Maria Quirico Bautista.

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Figure 5-11. CLAS-infected interstock trees planted in November 2016 near Avon Park, FL, photo taken in October 2017. From left to right: UKP-1 #1, UKP-1 #5, and Swingle #11. Photos by Maria Quirico Bautista.

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CHAPTER 6 SUMMARY AND CONCLUSIONS

Citrus fruit such as oranges, mandarins, lemons, and limes are some of the most valuable crops in the world from international trade in terms of fresh fruit production and orange juice consumption. Citrus is the most produced category of fruit in the entire world, with world fruit production in 2014 at 139,796,997 tonnes (FAO, 2016). Orange juice production is important to the economy of Florida generating billions of dollars of revenue in Florida, and has been the worlds second largest producer of orange juice after Brazil (USDA, 2017). However, several bacterial diseases threaten the industry in

Florida, decreasing productivity and reducing quality for the fresh fruit and processed juice markets.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc) is serious quarantine disease that infects all commercial citrus varieties, reducing production and impairing the exportation of fruit and transporting of citrus plants. This disease directly affects production as many commercial citrus cultivars are highly susceptible (Gottwald et al., 2001b). Additionally, all citrus is currently being threatened with Huanglongbing, presumably caused by Candidatus Liberibacter asiaticus (CLas), which is more damaging overall, reducing fruit quality and production and causing the decline of the tree (Halbert, 2005; Miles et al., 2017). Both diseases have been declared endemic to

Florida, and all future cultivars released for production will need to have some form of tolerance if the citrus industry in Florida is to survive (“5B-58.001 : Citrus Canker

Eradication (Repealed) - Florida Administrative Rules, Law, Code, Register - FAC, FAR, eRulemaking,” n.d.; Canales et al., 2016).

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Natural plant resistance is the most economical and effective method of control,

with less environmental impact and is a more sustainable crop protection strategy.

Recent research, as well this project, have concentrated on this method of defense,

aimed at harnessing innate plant defense against these disease (Deng et al., 2010;

Miles et al., 2017).

The primary research goal of this dissertation was to create a short-term mini- breeding program utilizing available pummelo genetic resources to facilitate the

contributions of pummelo to the overall UF-CREC citrus breeding effort. Pummelo (C.

maxima) is a major genetic contributor in the ancestry of many modern citrus cultivars,

including oranges, tangors, tangelos, grapefruit and lemon as well as rootstocks such

as sour orange, citranges, and citrumelos (Curk et al., 2016, 2015; Garcia-Lor et al.,

2013). Pummelo (C. maxima (Burm.) is a good resource with wide genetic diversity

which could be used for further breeding to improve existing cultivars or develop novel

types of citrus (Barkley et al., 2006; Roose et al., 1992; Yu et al., 2017).

Pummelos have been successfully used to recreate other citrus types, such as

sour orange replacement candidates for rootstock improvement (Grosser, 2014;

Grosser et al., 2004), and alternative grapefruit-like hybrids (Siebert et al., 2010; Soost

and Cameron, 1980, 1985).

This work capitalizes on other projects prior to this one, from screening of the

seedlings in 1999 (Ananthakrishnan et al., 2006), to work by Azza Mohamed to select

citrus tristeza virus (CTV) tolerant clones (Mohamed et al., 2008), and by Divya Kainth

to produce allotetraploid pummelos (Kainth, 2010).

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Pummelo selections have been identified and reported herein that have

displayed positive disease responses to both citrus canker and HLB. All future cultivar

releases will be required to show improved resistance or tolerance to these diseases for

citrus cultivation to remain commercially viable in citrus production areas where these diseases have been introduced. The progress made from identifying disease resistant selections and introgressing disease resistance into triploid hybrids moves the UF

breeding program closer to this goal. More long-term data collection is required for a

more definitive conclusion on the triploid population studied in this dissertation, as fruit

quality and bearing habits will need to be observed.

Evaluation of Citrus Canker Resistance in Pummelo Parent Selections

Field grown pummelos in Lake Alfred, FL, in a grove with extensively present

citrus canker and HLB for many years allowed ample opportunity for infection. Seedling-

derived pummelo trees planted here have been shown to be a good resource for

genetic improvement, as the trees have been evaluated in situ for disease response.

The small remaining population of pummelos resulted from initial trials growing the

seeds in Phytophthora inoculated, calcareous soil that screened out the weaker

seedlings and left behind pummelo selections that may have higher disease resistance

and better soil adaptation, as the original work was to identify pummelos that may have

value in rootstock breeding (J.W. Grosser, personal communication). The population

was also screened for CTV tolerance/resistance (Mohamed et al., 2008). The remaining

trees have continued to grow, producing fruit with various flesh colors and flavors, and

some have shown strong resistance to citrus canker and continue to be either HLB

negative or have very low levels of HLB bacteria detected. Some selections have

continued to show only minor HLB symptoms, such as 5-4-99-3 which was HLB

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negative in 2013 (Table 3-6) and recent testing confirms that bacterial populations are still low. 5-4-99-7 has also continued to have low infection, negative in 2013 (Table 3-6) and very low in 2016.

The canker screen of pummelos revealed that while some of the pummelos had fewer lesions than the grapefruit controls, examining only numbers of lesions showed little significant differences between the pummelos, at the 95% confidence level. The addition of using lesion size was helpful to show that analyzing numbers of lesions is useful, but size of lesions and rate of growth are also helpful in determination canker tolerance. The grapefruit controls frequently had fewer numbers of lesions, but the size of the lesions was bigger than the pummelo lesions. Smaller lesions that do not develop may be more useful to the grower, depending on how such lesions are expressed on fruit, which was not possible to study here due to space and time constraints. Growers will be more interested in a saleable fruit that may have no visible lesions or lesions that stay small, as opposed to the heavy infections that currently affect grapefruit resulting in severely affected, unsaleable fruit with many large lesions.

The time of year that testing is done is also important for more accurate results.

Canker screens should be done when plants are actively growing; two of the three initial screens were performed in fall and winter months. Even though they were performed in a temperature controlled greenhouse, day length was natural. This led to marked differences in responses comparing the separate series of inoculations (data not shown).

While there was little statistical difference looking at only numbers of lesions (at the 95% confidence level) for all three assays (Table 3-5), only 5-1-99-3 was statistically

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better than ‘Marsh’ and ‘Ruby Red’ grapefruit controls and not significantly different from

‘Meiwa’ and ‘Nagami’ kumquat controls (Table 3-5). This is a pink fleshed cultivar with the potential to create better cultivars.

5-1-99-3 which was the best for canker resistance was not able to be used as a female the following spring to make crosses as it flowered very early before other potential pollen sources were available, and was done flowering by the time sources of tetraploid pollen were available. This selection should be used in further crosses to determine how its canker resistance will be passed on to its progeny.

Later testing of 5-4-99-3 as a control with the triploid seedlings showed much better response, with smaller lesions and lower numbers of lesions, second only to the

‘Meiwa’ kumquat control (Table 4-5).

Interploid Hybridization to Produce Seedless Triploid Hybrids of Pummelo and Grapefruit

Crosses of the elite pummelos with tetraploid grapefruit, and tetraploid pummelos and grapefruit like hybrids with grapefruit resulted in a population of healthy, vigorous seedlings. The seedlings were tested for ploidy level via flow cytometry, identifying many triploids. These triploid hybrids were grafted and inoculated with citrus canker to identify potentially tolerant or resistant seedlings.

Inoculations with citrus canker gave a varied response in the seedling population.

There is a quantitative response to infection, and given the right set of circumstances

(or wrong) it is possible to infect even Meiwa kumquat, which is regarded as being resistant, as discussed in chapter 3. Time of year and growth activity of the tree affect successful infection; Meiwa would show more lesions during the winter months but was

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immune during periods of active growth, as observed during the seedling inoculations, which took place in spring and summer.

No single seedling was statistically better than the grapefruit controls at the 95% confidence level for number of lesions. While there are differences, and Meiwa kumquats showed very little infection, all were able to be infected to some degree.

Heavily infected leaves had a tendency to abscise before lesions could be counted and rated. Seedlings that were more inclined to drop their leaves were compiled in Table 4-7. This immune response to infection is something that could prove useful; if a tree has the ability to self-prune infected leaves or fruit, it could in theory reduce the amount of inoculum in the canopy. Unfortunately, the seedlings that dropped all of their leaves were also usually poor growers and may be related to other genetic weaknesses. Many that did drop leaves only dropped a few or dropped them only during one inoculation, which would not suggest these selections have a hypersensitive response to Xcc.

Many of the triploid seedlings do appear to be better than grapefruit regarding citrus canker response, with fewer and smaller sized lesions, which also appear to grow slower. If this study were to be repeated it might have be helpful to use a camera to measure actual area of the canker lesions, especially with measurements of the same leaf over several time points.

Statistical analysis of seedling families, offspring from the same parental combination, gave a more detailed response due to the additional data points. The overall comparison showed that certain parents were better able to pass on their citrus canker resistance. The best family was DV2xN11-3, followed by M+Chan80x5-4-99-3.

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The 5-4-99-3 control had fewer lesions than during the initial canker screen, with

no statistical difference at the 95% confidence level between Meiwa for lesion numbers.

This more accurately reflects the field data of 5-4-99-3, which has no visible lesions

under field conditions. Again, this selection is still showing only mild HLB symptoms in

the field. As a parent the seedlings M+Chan80x5-4-99-3 showed a very good response to citrus canker, however seedlings of 5-4-99-3xDV5 had some of the poorest canker response having had larger lesions and more of them.

Testing of trees under actual field conditions would further elucidate actual

response to citrus canker. Injecting Xcc inoculum directly into the leaf mesophyll may

not be an accurate representation of actual plant defense. Pummelos tend to have very

tough and waxy leaves, which may explain why field trees tend to have very low levels

of citrus canker. Some of the resistance to citrus canker may be due to physical

properties as opposed to immune response.

HLB testing of the new triploid hybrids will need to be performed to verify any

HLB tolerance/resistance. These seedlings will need to be subjected to HLB-positive psyllid screening to determine their attractiveness to psyllids and infection susceptibility.

Trees are being prepared for field planting which will expose them to unprotected

conditions. Trees already planted in the field have not shown typical blotchy mottle

symptoms, but they have only been in the field for a short time, and may have not had

sufficient time to develop symptoms.

Fruit evaluation will have to take place in the future, as pummelo and grapefruit

typically take several years to fruit. This time may be reduced with grafted trees, or if

grown in the Rapid Evaluation System. Alternatively, these hybrids could be grafted to

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the newly developed transgenic Carrizo expressing the early flowering gene FT3 (M.

Dutt, personal communication). This could significantly speed up flowering and fruiting, however, USDA approval would be required for field evaluation. Continued evaluation of the triploid population herein should result in the identification of a few new hybrids with both canker and HLB tolerance, and that produce seedless fruit with adequate commercial potential.

Testing of Selected Pummelo and Hybrid Interstocks to Mitigate HLB Symptoms in Commercial Sweet Orange/Swingle Trees

The idea to use the pummelo selections as interstocks came after observing experimental trees grafted onto Swingle rootstock that were being removed from existing field trials due to poor fruit quality. These trees were CLas infected, which typically destroys the feeder roots of affected trees on Swingle, yet these trees exhibited healthy and extensive root systems. Swingle is not known to be HLB tolerant/resistant as a rootstock, leaving the alternate explanation that in the case of these pummelos, there was something from the scion possibly crossing the graft union that could somehow protect the roots.

While it is unknown what is occurring through the use of the pummelo and pummelo hybrid interstocks, the data generated herein shows there is a definite interaction occurring. The interstock appears to be conferring an amount of resistance to the interstock when compared to the OLL-15 and Swingle control interstocks for all the interstocks tested. However, some of the Swingle interstocks have continued to appear healthy even under field conditions (figure 5-12, far right). Trees with the pummelo and pummelo hybrid interstocks showed better growth, thicker calipers, and

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lower Liberibacter populations in the grafted Valencia scion. What is happening or how

this occurs is still under investigation.

Growth of the trees under climate controlled greenhouse conditions, maintained

trees at lower temperatures to encourage HLB bacteria growth in the trees. Many of the

trees had healthy flushes of growth, while the Swingle controls showed typical HLB

symptoms of blotchy mottle and off-season flowering. Field planted trees will be

observed to determine if this protection extends beyond the climate controlled

greenhouse and continue to show healthy growth. To date many trees still appear to be

healthy after 11 months in the grove.

It is known that messenger RNAs will travel throughout the plant and across graft

unions, carried by the phloem (Froelicher et al., 2010; Spiegelman et al., 2013). Other molecules move about as well, such as amino acids and small molecules including plant hormones. Intracellular trafficking will transfer products generated from one graft partner into distant plant organs, affecting growth and regulation of the other (Ruiz-Medrano et al., 1999). It is therefore reasonable to believe that the interstocks are adding their own mix of products which appear to be boosting the immune system of the plant and adding to the plants ability to grow in spite of HLB.

Stress response in plants can elicit a complex cascade of signaling and transcriptions that are not yet well understood (Hasegawa et al., 2000). These signals will elicit production of secondary metabolites such as avirulence determinants and corresponding plant R proteins; heterotrimeric and small GTP binding proteins; ion fluxes, especially Ca2+ influx, and Ca2+ signaling; medium alkalinization and cytoplasmic

acidification; oxidative burst and reactive oxygen species; inositol trisphosphates and

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cyclic nucleotides (cAMP and cGMP); salicylic acid and nitric oxide; jasmonate, ethylene, and abscisic acid signaling; oxylipin signals such as allene oxide synthase- dependent jasmonate and hydroperoxide lyase-dependent C12 and C6 volatiles; as well as other lipid messengers such as lysophosphatidylcholine, phosphatidic acid, and diacylglycerol (Zhao et al., 2005).

It is known that plant defense in response to microbial infection is regulated in a complex network of pathways involving three signaling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (Kunkel and Brooks, 2002). The interstock may be altering the transcriptome and the metabolome of the grafted organism, providing resources that the individual parts may not have and interacting in the phloem in new and different ways, improving plant defense.

There is the possibility of systemic acquired resistance (SAR) coming from the interstocks that came from trees already exposed to the HLB pathogen. The interstocks were obtained from HLB-infected field grown trees, possibly providing an immune boost to the grafted plant (Vallad and Goodman, 2004). While some compounds have been used to elicit induced systemic resistance in citrus, little work has been done to investigate SARs specific to citrus (Graham and Leite, 2004).

Further Breeding Efforts

The analysis of parental groups gives some insights at which parents may be more useful to pass on their canker resistance to their progeny. Murcott+Chandler80 was one of the better parents in examination of parental group crosses for citrus canker resistance, it was also more amenable to crossing, producing more offspring from pollination attempts. The tree is vigorous and healthy in spite of surrounding HLB pressure. Fruits are small and orange-like, but may be improved by crossing with a red

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fleshed pummelo or grapefruit; resulting seedlings from these crosses may provide that improved characteristic.

5-4-99-3 was also one of the better pummelos for citrus canker response, exhibiting small lesions in low numbers comparable to Meiwa, reflecting the field observations canker response. This did not seem to reflect well on its triploid progeny when used as the female, but seedlings using it’s pollen performed better. The better canker response may just be due to the other parent having better canker response and not because 5-4-99-3 isn’t a good parent selection. 5-4-99-3 has been difficult to use as female as it frequently aborts flowers in controlled pollinations.

C2-4-1 and C2-4-8 were two of the best interstocks, and have also produced some seedlings which are being used in other experiments and have shown to be good breeding parents. Only the seedling C2-4-1xN11-3-15-3 from the triploid seedling citrus canker inoculations has shown to be better than controls (Table 4-6).

M+Chan80 x 7-2-99-11 family showed an interesting level of citrus canker response (Table 4-4), being significantly different at the 95% confidence level from

Meiwa and Ruby Red grapefruit, but not significantly different from 5-4-99-3 controls. 7-

2-99-11 is a diploid pummelo that requires more investigation for future crosses.

5-1-99-2S5 is interesting as it is red-fleshed and shows good HLB tolerance in the field, but in general its offspring have tended to show a higher level of citrus canker response comparable to the grapefruit controls. It is also the only pummelo that has produced tetraploid offspring, and may be useful for further breeding at the tetraploid level or to produce triploids from diploid crossing partners.

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Many of these pummelos tested also have potential for rootstock breeding. The

Divya tetraploid pummelos are from open pollinated seed of some of these parental cultivars, and could be crossed to a tetraploid C. reticulata to possibly create a synthetic sour orange, which could offer some tree size control due to it being tetraploid. The field trees have shown excellent disease resistance to Phytophthora and good HLB tolerance and may be able to extend that resistance to the scion. 5-4-99-3 and 5-4-99-7 are good candidates for further rootstock breeding, having shown low levels of HLB symptoms after several years of infection, however whole plant resistance doesn’t always transfer to good rootstock performance.

Conclusions

This core idea of this project was to establish a breeding program using elite C. maxima selections to improve grapefruit. It was necessary to establish that the apparently citrus canker resistant trees, which in the field exhibit no citrus canker lesions, could be verified using available greenhouse inoculation techniques. Identified potentially canker tolerant parents were then used in interploid crosses, back crosses with grapefruit in some instances, to create triploid hybrids, which were also repeatedly challenged with citrus canker to determine if they had any level of tolerance or resistance inherited from a resistant parent. This was be a difficult task given the usual time frame of a PhD program, and there was not adequate time to validate the results under real world conditions in the field. Future studies should determine the correlation comparing results from greenhouse leaf assays with that of natural field exposure.

The selected pummelos identified and used as parents in this study have shown tremendous potential, having been refined through various selections, starting with phytophthora infected challenging calcareous soil, and then grown in the field for 16

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years on HLB-susceptible Swingle rootstock, exposed to citrus canker and HLB for the past 8-10 years. It is necessary for growers to have new cultivars of grapefruit or grapefruit-like hybrids, as traditional grapefruit has seen significant reductions in production following the introduction and establishment of both citrus canker and HLB in

Florida. HLB is also now spreading in Texas, threatening another key area of US grapefruit production. The research herein has developed a framework from which future grapefruit/pummelo breeding can be built and expanded.

Comments on the evolution of this dissertation research project are relevant.

Initially, the primary goal of the project was to build superior allotetraploid breeding parents by the fusion of red grapefruit with elite pummelo selections. Somatic fusions were attempted for 1.5 years at the start of this breeding project between advanced pummelo selections and two red grapefruit embryogenic callus lines, but were unsuccessful. The established system of using embryogenic callus obtained from immature seeds of nucellar seeded cultivars meant that grapefruit would be the source of the embryogenic suspension cultures, using callus derived from Ruby Red and Red

Cooper grapefruits. Pummelo (C. maxima) produces zygotic seed and is not amenable to embryogenic callus production, and thus was used as the source of leaf mesophyll protoplasts. The embryogenic callus lines used in fusion experiments turned out to be recalcitrant for normal embryo production, producing only callus and numerous abnormal embryos when fused with pummelos. Despite months of effort and many media tested, none of the obtained abnormal embryos could be germinated or made to product adventitious shoots (Only four plants were recovered from somatic fusions

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using grapefruit callus when fused with Citropsis gilletiana, discussed in Appendix A.).

Due to this problem, other uses of the available pummelo germplasm were explored.

Following the unsuccessful somatic hybridization research, the amount of progress that could be achieved in the amount of time remaining for a PhD project was limiting, but significant progress was made. Excellent selections of HLB tolerant and citrus canker resistant cultivars of pummelo were identified including 5-4-99-3, 8-1-99-

1B, 5-1-99-2 S5, 8-1-99-1A, and 5-1-99-3. Data from the interploid crosses suggests that some of these are better parents for scion improvement than others, such as 5-4-

99-3 and 5-1-99-2 S5.

No triploid seedling by itself appeared to be completely resistant to citrus canker, nor appeared to be as tolerant as the 5-4-99-3 parent control. Citrus canker inoculations, when performed at the right time of year, showed good correlation with the limited field data. There was a varied response comparing different repeats of the experiment, which could be due to various factors, such as inoculation method, pathovar potency, or temperature fluctuations. Further study is warranted for the derived triploid seedling population, especially in relation to fruit quality; it should be possible to identify canker and HLB tolerant hybrids that produce seedless fruit of commercial quality. If successful, such a new cultivar could replace or augment current grapefruit cultivars that are suffering from disease pressure.

The interstock experiment showed that pummelo-derived interstocks do have an effect on grafted commercial sweet orange trees in response to HLB, especially compared to the Swingle interstock controls. HLB titer in the trees correlated well with phenotypic ratings of disease response, as highly symptomatic trees consistently had

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high titers of bacteria. Overall field response is yet to be determined, as the interstock trees have only been in the field for one season. Some trees are doing poorly while others (mostly those that did well in the greenhouse study) have grown well.

The method used to create the interstock trees in this study may prove too costly for nursery production. An alternative method of using graft bridges or inarching may be viable to rescue mature, affected trees in the grove. Similar methods have been used to topwork trees or save trees with failing rootstocks (Webber and Batchelor, 1943, pp.

44–59, 152–161).

Using trees as interstocks may be good way to evaluate trees for further rootstock breeding. Grafted trees are comprised of multiple components and the resulting combinations may have different responses to environmental pressures that will be different than any of the parts as individuals. For example US897 has shown better resistance to HLB by itself as opposed to when being used as a rootstock

(Albrecht and Bowman, 2011).

Pummelos represent a large and relatively untapped reservoir of genetic diversity, have a lot of potential to offer to both citrus scion and rootstock breeding. As the progenitors of many modern citrus cultivars, most produced through chance hybridizations, pummelos have the potential to recreate and improve upon accepted forms. Grapefruit has a narrowly accepted flavor profile, and may be difficult to recapture in offspring. These new triploid seedlings offer some hope if fruiting quality is comparable to accepted grapefruit flavor.

If the disease resistance of pummelo can be utilized to improve grapefruit, it has the potential to revolutionize a failing crop (the current crop of Florida grapefruit

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produced was the smallest grapefruit crop in decades). Other technologies and cultural practices are being developed to assist growers, and the techniques described herein may be useful to develop commercial quality trees that guarantee long-term sustainability and profitability.

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APPENDIX A CITROPSIS GILLETIANA (SWINGLE) FOR CITRUS GERMPLASM ENHANCEMENT AND CULTIVAR DEVELOPMENT

Introduction

Citropsis (Swingle and Kellerman) is a genus of citrus relatives native to tropical

Africa that are graft compatible and only slightly sexually compatible with true Citrus, with known sexual hybrids having been sterile (Smith et al., 2013; Yasuda et al., 2010).

Harry Ford first began investigating Citropsis as a source of genes for resistance to tropical diseases present in this gene pool, among which Citropsis gilletiana Swingle &

M. Kell. has been documented as exhibiting outstanding resistance to the burrowing nematode (Radopholus similis Cobb) and Phytophthora complex (Ford and Feder,

1960; Grosser et al., 1990). Milam rough lemon was released as a burrowing nematode resistant rootstock in 1964. This rootstock was highly regarded until the 1970’s when a second race of burrowing nematode was discovered in Florida that infested trees previously thought to be resistant (Kaplan and O’Bannon, 1985). It was found that widely planting resistant rootstocks led to the development of new biotypes that could feed on resistant rootstocks, such as Carrizo and Milam (Kaplan, 1986). Tolerant rootstocks are Carrizo citrange, ‘Ridge Pineapple’ sweet orange, ‘Milam’ rough lemon and Kuharske citrange, P. trifoliata, and 1584 (P. trifoliata x ‘Milam’ rough lemon)

(Castle et al., 2016, 1993). While these rootstock selections are reported to be resistant, they are not truly resistant but are tolerant and able to grow and produce in spite of infestation, with minimal loss of production compared to susceptible rootstocks (H. Ford, personal communication).

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Citropsis in Citriculture

African cherry-oranges (Citropsis species) are near citrus relatives that are a source of novel traits that could be used for improvement of citrus (Grosser et al., 1990).

Citropsis sp. are graft compatible with Citrus, but are only marginally sexually compatible, and until recently were regarded as incompatible. Walter Swingle recognized that Citropsis was closely related to citrus, first attempting to cross Citropsis with Citrus in 1913 (Swingle, 1913). He noted in grafting experiments that Ugandan cherry-orange (Citropsis schweinfurthii Engler) grafted readily with Citrus and grew rapidly and vigorously on sweet orange, sour orange, grapefruit and lemon stocks, and successfully budded grapefruit and orange onto C. schweinfurthii. He noted also that C. schweinfurthii was also well adapted to growing on poor, sandy soils, and may be a source of resistance to diseases and adaptations to growing conditions and climates not found in other citrus types (Swingle and Kellerman, 1914). He was able to procure fruit using C. schweinfurthii pollen on Citrus aurantifolia (Christm.) Swing., but the few seeds generated failed to produce hybrids. After decades of creating other citrus hybrids, he concluded that Citrus and Citropsis were sexually incompatible (Swingle, 1945).

Belgian expert horticulturists and pathologists performed experiments in the

Belgian Congo using C. gilletiana Swingle and Kellerm. as a rootstock, and discovered that it was resistant to root rot (Phytophthora) and some other local diseases that Citrus rootstocks were unable to mitigate (Pynaert, 1935).

Harry Ford discovered that C. gilletiana was a vigorous rootstock, with trees performing admirably in greenhouse conditions. This high-level of congruity between

Citrus and Citropsis led him to conduct more experiments with it, and he discovered that it was resistant to the burrowing nematode (Radopholus similis Cobb). He attempted to

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make hybrids using it, stating in 1960: “The immediate use of C. gilletiana will be as the female parent in a breeding program since the seeds are monoembryonic. A successful cross has already been made by using pollen of the nematode tolerant Clone-X hybrid

(Milam rough lemon) and Rough lemon-B.”(Ford and Feder, 1960) No further mention of successful hybrids are discussed in later publications, and we can only assume that he was unsuccessful, as his successor did not inherit any Citropsis hybrids (Bill Castle, personal communication). Dr. Ford stated that the main reason Citropsis was abandoned as a rootstock was due to its tropical origin, having a complete lack of cold tolerance (Harry Ford, personal communication).

Conventional breeding methods have been unable to incorporate Citropsis due to hybrid sterility. Smith was able to cross Citropsis with Citrus wakonai, resulting in hybrids that have flowered and fruited but have been pollen sterile and seedless (Smith et al., 2013). In Japan, researchers seeking to understand the genetic relationship between Citrus and Citropsis created hybrids using Citropsis schweinfurthii and two citrus cultivars (Yahata et al., 2006; Yasuda et al., 2010). Dr. Hisato Kunitake reported in 2015: “Unfortunately, the Citrus and Citropsis hybrids in our study team had still no flowers and fruits.” (H. Kunitake, via email)

Somatic hybridization is useful to incorporate genetically similar materials with obstacles to sexual hybridization. The first somatic hybrid between Citrus and Citropsis was reported in 1990 by Jude Grosser, between Citrus reticulata and Citropsis gilletiana, using polyethylene glycol to fuse protoplasts (Grosser et al., 1990). A few years later, a Japanese researcher reported somatic fusion of Citrus reticulata cv.

Ponkan with Citropsis gabunensis using electrofusion (Ling and Iwamasa, 1994). The

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first hybrids of the Grosser Lab are no longer maintained (C. sinensis cv. Hamlin + C. gilletiana and C. reticulata Blanco cv. Cleopatra + C. gilletiana), but Citrus reticulata

Blanco cv. ‘Nova’ + Citropsis gilletiana Swing. & M.Kell. has been used successfully as a rootstock in an experimental trial (Grosser and Chandler, 2003), before it was burned during citrus canker eradication (J.W. Grosser, personal communication).

Current theory suggests Africa as the source of the Liberobacter family responsible for Huanglongbing (HLB) disease (Bové, 2014). It may be possible that genes for resistance exists somewhere in the Citropsis genepool, which may have evolved in the same general vicinity.

The objective is to produce new hybrids using Citropsis gilletiana, either sexually or using somatic fusion, to be used as burrowing nematode resistant rootstocks.

Materials and Methods

Grosser lab created the first Citropsis gilletiana somatic hybrid in 1990 (Grosser and Gmitter, 1990a), as well as creating a few more in subsequent years (Mourão Fo et al., 1994). While some of these did not perform well as rootstocks, one selection performed well in a rootstock trial that was destroyed before its conclusion during citrus canker eradication: ‘Nova’ mandarin hybrid + Citropsis gilletiana (Grosser and Chandler

2003, J. Grosser personal communication). This is the only somatic hybrid of Citropsis gilletiana still maintained by the University of Florida Citrus breeding program.

Somatic Fusion

Somatic fusion was performed according to protocol developed by J.W. Grosser

(Grosser and Omar, 2011), obtaining protoplasts from young leaves of Citropsis gilletiana Swingle & M. Kell. (clone obtained from the Florida Department of Agriculture,

Department of Plant Industry Citrus Arboretum in Winter Haven, FL). C. gilletiana

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Swingle & M. Kell. was grown in a greenhouse under shade and leaves were harvested

from trees when nearly full to fully expanded but still tender and pliable. Leaves were

dipped in 1N hydrochloric acid for one second, then soaked in 0.825% sodium

hypochlorite solution for eight minutes. Leaves were then rinsed three times with sterile

water, feathered with a razor blade and inserted into a beaker with 8mL enzymatic solution (active agents 2% Cellulase Onozuka RS and 2% Macerozyme® R-10 (Yakult

Honsha, Co.)) and shaken at 2rpm.

Embryogenic callus from ‘W. Murcott’ mandarin and Red Cooper grapefruit were obtained from liquid cultures, separated from the nutrient mixture, and placed in a 4cm petri dish with 5mL of enzymatic solution. Protoplast preparation was passed through a sterile 45 um nylon mesh to remove undigested cell clumps and debris. Protoplast containing filtrate was transferred to the screw-cap centrifuge tube and centrifuged at

700-1000 rpm for 5-10 min.

Supernatant was removed with a Pasteur pipette. Pellet was resuspended by adding 25% sucrose + CPW up to the five mL line and mixed. Two mL of 13% mannitol

+ CPW was carefully injected directly on top of the sucrose layer, taking care to avoid mixing layers, and centrifuged for 10 min at 700 rpm. Viable protoplasts (ring at the interface between two layers) was collected and transfered into another screw-cap centrifuge tube.

Protoplasts were fused using polyethylene glycol 8000mw (Sigma-Aldrich).

Following fusion, protoplasts were cultured in-vitro until plantlets were formed; Hybrids were identified phenotypically, exhibiting thicker, dark green, and often trifoliate leaves,

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and orange colored roots, which were then grafted onto ‘Changsha’+Flying Dragon trifoliate rootstock.

Ploidy of fusion products was confirmed using Partec PA flow cytometer, measured against diploid, triploid, and tetraploid Citrus aurantifolia internal standards.

Sexual Hybrid

Citrus reticulata Blanco ‘Nova’ mandarin + C. gilletiana Swing & M. Kell., produced in the Grosser lab around 1993, flowered for the first time in December 2014, over 20 years since this hybrid was created (Mourão Fo et al., 1994). Because flowering was off-season from most Citrus, flowers were pollinated using pollen stored from the previous season using various tetraploid rootstock cultivars. Fruit failed to develop naturally past 30 days. However, a single embryo was obtained from an abscised ovary, pollinated using citrus rootstock variety UFR-4 and cultured in vitro until it could be grafted onto Changsha + Flying Dragon trifoliate rootstock.

Flowers in subsequent seasons have also failed to produce fruit when hand pollinated. Fruit were discovered on a field tree in December 2016, presumably produced from off-season blooms during the summer months.

Pollen Viability

Pollen viability was determined by smearing five anthers of ‘Nova’+Citropsis gilletiana pollen on 10% sucrose, 1% agar media, stored in the dark at 20C for 10 hours, then grains counted in ten fields of view.

Molecular Markers

DNA was isolated using GeneJET Plant Genomic DNA Purification Kit (Thermo

Scientific) and PCR was performed using Bio Rad T100 Thermal Cycler, with 8 EST-

SSR primers developed by Chunxian Chen et al. (2008). PCR products were then

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analyzed. PCR products were then analysed using Applied Biosystems 3130 Genetic

Analyzer and results analyzed using SoftGenetics GeneMarker V2.6.3.

Nematode Assay

Cuttings were rooted in a mist bed, using ten cuttings of each Citropsis hybrid, as well as the Nova+Citropsis hybrid. Soil pH was adjusted to 7.5 using 15mL of water with

10% calcium carbonate. Tylenchulus semipenetrans (Cobb) nematodes were isolated from roots gathered from a nematode infested grove in Aurburndale, FL. Inoculation of the citrus nematode, T. semipenetrans was performed on one date, 10 mL per plant at a concentration of 100 nematodes per mL: and burrowing nematode, Radopholus similis

(Cobb) was inoculated at three dates.

Results and Discussion

Citropsis gilletiana was used successfully to make new hybrids with W. Murcott mandarin. These can be confirmed phenotypically, as hybrids have thicker leaves, leaflet number intermediate between parents, and paired spines similar to Citropsis gilletiana. Crushing leaves also releases a pungent ‘Citropsis’ odor, dissimilar from W.

Murcott leaves. SSR markers further confirm hybrid status, as hybrids displayed intermediate histograms (Figure A-10).

Pollen viability of ‘Nova’ + C. gilletiana hybrid was 1.05% on the 10% sucrose media. While this is not optimal fertility, hybrids may be possible in the future using its pollen, and crosses were made using tetraploid pummelo and tetraploid pummelo/orange hybrid C2-4-1. This resulted in a single fruit from C2-4-1, with a single seed. The seed produced three embryos, which were lost due to contamination.

EST-SSR molecular markers confirmed the hybrid status of the ‘W. Murcott’ + C. gilletiana fusions. The seedling obtained from the pollination attempt of (Nova+C.

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gilletiana) x UFR-4 was determined to be of nucellar origin, and was identical at all marker locations to the ‘Nova+C. gilletiana’ parent. The Red Grapefruit+C. gilletiana fusions have largely lacked vigor, and the single plant large enough to be tested appears to be a cybrid (Figure A-11,12).

Fruit obtained from the Nova+Citropsis hybrid in 2017 (Figure A-13) all turned out to be completely seedless. Fruit were juicy, with an odd, unpleasant ‘Citropsis’ flavor, highly acid at 3.36 titratable acidity, but with 18.6-21.6 ºBrix.

Further testing will be required to test for burrowing nematode resistance; initial inoculations of burrowing nematodes have not been successful with further inoculations planned when new burrowing nematode cultures are available.

Summary and Conclusion

The ‘Nova’+Citropsis gilletiana has performed well in rootstock trials (Grosser,

2012), ‘Murcott’ has performed well as a rootstock (Personal communication, W.

Castle), and ‘W. Murcott’ may behave similarly as a rootstock, although it typically has not been used as a rootstock. The ‘W.Murcott’+ C. gilletiana fusions are extremely vigorous and easy to graft. If they prove to be burrowing nematode resistant, they will offer a much-needed source of burrowing nematode resistance, as there are few cultivars with burrowing nematode resistance. ‘Nova’+Citropsis gilletiana has not been promoted as a rootstock due to a lack of fruiting, as nucellar seeds are the traditional method of propagating rootstocks. Plants were easily propagated via cuttings, which may be a viable alternative, although cutting propagated plants do not make tap roots that seedlings will typically make. Current interest in producing rootstocks via tissue culture may make the Citropsis hybrid rootstocks more feasible (Bowman et al., 1997).

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W. Murcott is known to be vigorous and produce precocious seedlings, perhaps making it possible for one of the hybrids to produce seeds in less time than

Nova+Citropsis (J.W. Grosser, personal communication). The W.Murcott+C.gilletiana hybrids are vigorous and should prove to be good rootstocks, judging by the example of

Nova+C.gilletiana. Further testing will be required to prove rootstock capability. The fertility of the W.Murcott+C.gilletiana hybrids is unknown, and is likely to be unknown for many years, based on past performance of Nova+Citropsis gilletiana.

Burrowing nematode resistance would be very beneficial for a new rootstock, as there are few burrowing nematode resistant rootstocks available: Carrizo citrange,

Milam, 1584, and Ridge Pineapple. Milam and 1584 both have the negative trait of being low-seeded, which makes it difficult for current seed grown operations.

Propagation by seed may not be a limiting factor for industry adoption of these new hybrids, as tissue culture for rootstock production is being developed by some major rootstock producers (Chaires, 2015a, 2015b). None of these rootstocks have resistance to the second race of burrowing nematode (Ford, 1979).

The Red Cooper grapefruit+C. gilletiana cybrids lack vigor, and do not appear to be useful for further investigations, serving only as curiosities. These will likely be discarded.

It is unknown why the Nova+Citropsis hybrid took over twenty years to flower and fruit. It may have flowered as a stress response to HLB infection, or perhaps due to environmental needs matching up with plant maturity. The fruit to date have been seedless and it is unknown if eventually any seeds will form; the single ovule rescued from the aborted fruit appears to be a clone of the mother. It is unknown if this is

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because the resulting plant was from maternal tissue or derived from nucellar tissue. If it

is from nucellar, it would likely not be very useful to use as a female parent in further

crosses. The pollen does appear to have some viability, if obtained from greenhouse

grown flowers (pollen from field trees didn’t release, but pollen obtained from a tree in

an air-conditioned greenhouse did have flowers that released pollen, possibly from

growing in cooler temperatures, increased humidity, or increased shade). Future

crosses are planned to attempt to produce further complex hybrids, especially using a parent that will increase seediness.

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Figure A-1. Nova+Citropsis gilletiana growing in research grove, Spring 2017. Photo by author.

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Figure A-2. Protoplasts suspended in a density gradient. Green leaf protoplasts on the left and white callus derived protoplasts on the right. Photo by author.

Figure A-3. Readout from Partec PA flow cytometry machine. Tetraploid peak of somatic hybrid on right, with Persian lime as triploid standard for comparison. Photo by author.

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Figure A-4. First flower of Nova+Citropsis gilletiana. Photo by author.

Figure A-5. Pollen germinating on 10% sucrose + 1% agar. Photo by author.

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Figure A-6. Sole fruit obtained from first flowering. Ovule rescue recovered a single embryo. Photo by author.

Figure A-7. Developing embryo obtained from Nova+Citropsis gilletiana fruit. Photo by author.

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Figure A-8. Tetraploid Nova+Citropsis hybrid roots on left, compared to diploid W. Murcott roots on right, showing the dark orange color of Citropsis roots compared to Citrus roots. Photo by author.

Figure A-9. W.Murcott+Citropsis gilletiana. Leaves vary from unifoliate to trifoliate, with spines frequently paired or occasionally single. Photo by author.

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Figure A-10. SSRs of Citropsis gilletiana (top left), W.Murcott+Citropsis gilletiana (12 middle boxes), and W.Murcott (lower right).

Figure A-11. EST-SSR markers for what appears to be a cybrid.

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Figure A-12. Red Cooper grapefruit + Citropsis gilletiana cybrid. Photo by author.

Figure A-13. Open pollinated Nova+Citropsis fruits. Photo by author.

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APPENDIX B MICROCITRUS HYBRIDS: POTENTIAL NEW CROPS FOR CITRUS GROWERS

Introduction

The genus Microcitrus is a group of Citrus relatives from Australia and the

surrounding region. They are characterized by tiny leaves and flowers, and some have

fruit unlike other cultivars of true Citrus (Figure C-1). Recently, taxonomists have

lumped these species, as well as Eremocitrus and Clymenia, together with genus Citrus

(Mabberley, 1998; Zhang et al., 2008). While they are different in some physiological

traits, they are sexually and graft compatible with other citrus cultivars.

The most notable is the finger lime, Microcitrus australasica F. Muell., native to

Australia and so named for their long, cylindrical fruits. There are eight other species

originating from Australia and New Guinea, including the Round Lime (M. australis

Planch.), Mount White lime (M. garrowayi F.M. Bail.), Kakadu Lime (M. gracilis), Russell

River Lime (M. inodora F.M. Bail., M. maideniana (Domin) Swing.), M. papuana, New

Guinea wild lime (M. warburgiana F.M. Bail.) and Desert lime (Eremocitrus glauca

Lindl.). In Australia, they have become popular as ‘bush tucker’, a category of foods that come from the wild, where they command high prices as a specialty crop. In addition to domestic consumption, a significant amount is also exported to Asia and

Europe. Recent interest has encouraged selection of new cultivars with a range of peel

colors (from black, green, red, purple, and yellow) and flesh colors (shades of green,

pink, and red) with improved flavors, now available to Australian growers. In the United

States they have remained rare, only occasionally found as a citrus curiosity and

cultivated to a limited extent in California and Florida (Peter Chaires, personal

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communication). Improved selections are being imported into the U.S, which will be

available in the near future for US growers.

Finger lime juice vesicles are spherical and separate easily. These have been

marketed as ‘citrus caviar’, used in restaurants to make mixed drinks and as a plate

garnish, and are said to accompany fish and seafood very well (Karp, 2009). Like limes

and lemons, the finger lime is an acid fruit, as are most Microcitrus. Flavor is variable,

with most having a prominent lime flavor, but some have a ‘resinous’ flavor.

Microcitrus are also characteristically very spiny; they usually have shorter

internodes with thorns as long or longer than the leaves at every node. Although

Microcitrus is known to be sexually compatible with Citrus, little selection or

improvement of Citrus using Microcitrus has taken place.

Finger Limes and HLB

Finger limes and several of its close Australian relatives are also unique in their

tolerance to HLB. Experiments by the USDA have shown that Asian citrus psyllids avoid

feeding on Microcitrus (Westbrook et al., 2011) and a recently published study also by

the USDA have shown that in the field they have a low infection rate, showing a low

presence of the HLB bacterium in its tissues and few symptoms of the disease. Recent

studies conducted by the USDA in Ft. Pierce, FL, used 96 seed-source families representing a broad cross-section of genetic diversity from the Riverside citrus gene

bank were planted in a replicated study and evaluated for six years. The recently

published paper (Ramadugu et al., 2016) indicates significant resistance almost

exclusively in genera other than Citrus. Ed Stover, USDA, Ft. Pierce, explains, “Within

the citrus genepool, only accessions of Poncirus, Eremocitrus, and Microcitrus showed

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considerable resistance to HLB, with most PCR tests showing Ct>35 (negative for

HLB). Even after six years finger limes had a Ct≥35 in 4 of 5 surviving plants.”

This matches colloquial observations by those who have grown Microcitrus,

which do not appear to show symptoms of HLB when grown in affected areas. The DPI

Citrus Arboretum in Winter Haven is a good example of this; there are many infected

citrus trees in the arboretum but the finger limes remain healthy. Microcitrus inodora is

the exception, as the tree has succumbed to HLB. Microcitrus australis may have

shown some symptoms, with a few branches with leaves that appeared to have the

blotchy mottle symptoms, and this tree has since been removed. The remaining

Microcitrus in the arboretum (several finger limes, giant finger lime, Sydney hybrid, M.

papuana, and Eremocitrus glauca) all appeared to be symptom free.

New Hybrids

Parents were selected based on different attributes, mainly influenced by apparent HLB resistance, with goal of producing HLB tolerant offspring combining the best features of both parents. Using both conventional and laboratory techniques, a population of diploid, triploid and tetraploid offspring were created and their hybrid

status confirmed phenotypically and using flow cytometry for interploid crosses. Diploid

and triploid progeny were created using conventional breeding methods between

selected monoembryonic citrus with pollen obtained from Microcitrus accessions, or

using available Microcitrus to cross with other Citrus cultivars. Selections came from the

USDA-ARS National Germplasm Repository for Citrus and Dates in Riverside, CA,

Florida Department of Plant Industry (DPI) finger lime accession 50-36 and Giant Finger

Lime, and University of Florida clones grown from seed obtained from the Citrus and

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Date Repository (two Microcitrus australasica var. sanguinea trees that began to flower for the first time in spring 2015).

Tetraploid parents were chosen with the intended goal of making seedless offspring: lemons (C4-10-19, C4-6-48), giant key lime, and lemon-lime hybrids

(Lapithiotiki lemon+Lakeland limequat, Lakeland limequat x lime), and a grapefruit-like hybrid (Murc+Chandler#80).

Eight HLB tolerant pummelo selections were also attempted to cross with, including 5-1-99-2 S5, 5-4-99-7, 4-4-99-7, 7-2-99-11, 8-1-99-1A, HBJL-1, and UKP-1.

Several mandarin hybrids were selected including Nules and Marisol clementines, and an Ambersweet orange seedling tree (a complex mandarin/orange hybrid), which appear to be doing well in spite of HLB pressure. Two diploid acid cultivars were used:

Bearss lemon and a key lime hybrid (50x5011-00-6) that had been labeled tetraploid, but was actually diploid. Crosses were made using finger lime 50-36, giant finger lime and EN-2 as females, using pollen from Sinton citrangequat (Fortunella margarita

'Nagami' x Citroncirus 'Rusk'), Poncirus trifoliata (DPI 50-7), Miaray sour orange, 1584 hybrid (Poncirus trifoliata x Milam rough lemon), Nagami kumquat, and Meiwa kumquat.

Results of crosses are shown in Table B-2.

Microcitrus hybrids have shown intermediate leaf characteristics between their parents, but tend to have the thorniness and short internodes of Microcitrus. Leaf size varies, but are larger than the Microcitrus parent and smaller than the other Citrus parent. Citrus seedlings are hypogeous, having cotyledons that remain in the seed, with a shoot that emerges with the first two leaves arranged opposite each other (Zhang and

Mabberly, 2008). Microcitrus seedlings germinate similar to Poncirus seedlings, having

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the first postcotyledonary leaves in the form of alternate cataphylls (Figure B-3). This

character appears to be dominant in Microcitrus, helping to identify possible hybrids as

seedlings emerge, when using Citrus as the female parent.

Tetraploid Sydney Hybrid

Seeds of open pollinated Sydney Hybrid (Microcitrus x virgata, a hybrid between

M. australis with M. australasica), were treated to create a tetraploid that could be used as breeding parent. 50 seeds obtained from fruits grown at the Florida Department of

Plant Industry Citrus Arboretum in Winter Haven, FL. Seeds were surface sterilized by soaking in 20% bleach (8.25% sodium hypochlorite, Great Value, Wal-Mart) with 2 drops/L TweenTM 20 (Fisher Scientific) for 20 minutes. Seeds were rinsed three times

using sterile water inside a laminar flow hood. Seeds were then placed on petri dishes

with Murashige and Skoog media (Fisher Scientific) with 20g/L sucrose and 8g/L agar,

pH 5.6. When seeds began to germinate and the radicle began emerging from many

seeds, all seeds were placed in a solution of 1 mL/L SurflanAS (40.4% oryzalin (3,5-

dinitro-N4N4-dipropylsulfanilamide), Southern Agricultural Insecticides, Inc.), with 1 mL/L

SilEnergy non-ionic surfactant (Brewer International), 25g/L trehalose (Swanson Health

Products), and 2 mL/L Plant Preservative Mixture (Plant Cell Technology). This protocol

is similar to one developed by Contreras et al.(2010) with the addition of trehalose as

recommended by Denny Gerondis ([email protected]) used to double Vitis ploidy,

and used successfully in previous unpublished experiments to double ploidy of

germinating Citrus seeds. Seeds were immersed in 40mL solution inside a 50mL

conical centrifuge tube (Falcon 50mL tube, Thermo Fisher), placed in a Mini Labroller

(Labnet Intl., Inc) and agitated for 24 hours. After 24 hours, seeds were rinsed three

times with sterile water and transferred to magenta boxes containing 80mL media: MS

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Basal media with vitamins (Phytotechlab.com), 20g/L sucrose, 0.5g/L activated charcoal powder, 8g/L agar, and pH 5.6. Seeds were grown in the magenta box for two months, and then transferred to soil and moved to the greenhouse for further observation.

Ploidy of seedlings was determined using a PartecPA flow cytometer, using diploid ‘Key’ lime, triploid ‘Persian’ lime, and tetraploid ‘Giant Key’ lime as internal standards.

Of the 50 seeds, 21 seedlings survived, and of the 21 tested, two are tetraploid and the remainder diploid. Sydney Hybrid produces zygotic seeds, however all seedlings appear to have been pollinated with Microcitrus, either self-pollinated or pollinated by nearby Microcitrus.

Summary and Conclusion

Microcitrus are an untapped resource, with few studies currently examining its breeding potential. Microcitrus, especially M. australis, the finger lime, has shown to be resistant to HLB (Ramadugu et al., 2016) and may offer a genetic resource to be incorporated into other commercial quality citrus. It is not well understood if the resistance to the bacterium is due to antibiosis, which causes the bacteria to not survive well in Microcitrus tissues, or if it’s due to non-host preference by psyllids. The leaves are phenotypically very different from other true Citrus and it’s possible the chemical constituents of Microcitrus change the psyllids ability to recognize it as a potential food source. In the long term field evaluation of Microcitrus, at the end of the study trees were found to be HLB positive, but with low levels of infection in spite of severe infection from the surrounding grove in an area of high HLB pressure (Ramadugu et al., 2016).

This implies that there is more likely a case of antibiosis.

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It is unknown whether or not this protection will extend to the hybrid offspring. It

will be interesting to see if offspring of the crosses using HLB tolerant selections, such

as Pummelo 5-1-99-2 S5 and P. trifoliata 50-7, will exhibit enhanced disease tolerance.

It will be necessary to perform HLB inoculations on all seedlings using infected material

as well psyllid feeding preference tests to verify the cause of tolerance.

Some of the hybrids have potential use as rootstocks. 5-1-99-2S5 x round lime may have good potential as a rootstock; seedling trees are very vigorous, much more vigorous than M. australis seedlings, with leaf size in between the parents. The downside of that cross is that both parents produce zygotic seed, which would make it a less desirable candidate for rootstock production. It may be more valuable for further breeding, crossing with a parent that produces nucellar seeds. The Giant Finger Lime x

Miaray sour orange and finger lime 50-36 x P. trifoliata 50-7 hybrids also have potential

use as rootstocks, showing good vigor as well as having the potential to produce

nucellar seeds.

Giant finger lime x Sinton citrangequat is unique in that it combines four genera:

Citrus, Fortunella, Poncirus, and Microcitrus. These seedlings show good vigor and may

be useful as a scion, possibly as an ornamental Citrus, as Sinton fruits tend to hang on

the tree for long periods. Sinton fruit are edible although sour, useful for making a drink

similar to lemonade. The offspring may be decorative and tasty.

The triploid offspring offer good potential for seedless, acid fruits. There may be

some valuable hybrids in the acid-fruit triploids if they prove to be HLB resistant and

seedless, with fruit quality acceptable as lime or lemon replacements. Finger limes may

have more potential in the development of limes and lemons, especially as the finger

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lime has a more identifiable lime flavor. Round lime has a peculiar flavor, and it is

unknown if this flavor will dominate in the offsprings’ fruit. The trait for spherical juice

vesicles may be dominant, but it’s unknown how the 1/3 ratio will affect this expression.

There is good potential for increased size and good flavor from the acid fruit parents.

Hybrid offspring exhibit the increased thorniness and short internodes of

Microcitrus. In many Citrus cultivars thorniness is a juvenile trait, with mature trees exhibiting fewer or are absent of thorns, ideal for production and harvesting. It is also

unknown if this will be expressed as a juvenile trait in the hybrids. Fruit qualities are yet unknown although some Microcitrus tend to be precocious bearing, fruiting sometimes

in three years or less from seed (Figure B-8). Grafting may also reduce time until bearing; all field plants have been grafted to Swingle citrumelo rootstocks.

Further analysis is required to observe fruiting potential and other traits, such as use as rootstocks, disease resistance/tolerance, and environmental adaptation. Little breeding work has been done with species in Microcitrus, probably due to being relatively unknown in US markets or regarded as a curiosity. In the ongoing battle with

HLB, Microcitrus may be a resource that will allow growers to be regain lost productivity.

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Table B-1. Microcitrus species and hybrids available for hybridizing. Name Species HLB Fruit Color Fruit Shape Finger Lime 50-36 C. australasica Tolerant Red Long, narrow Giant Finger Lime C. australasica x ? Tolerant Green Long and wide Sydney Hybrid C. x virgata Tolerant Green Long and wide Round lime, Dooja C. australis Tolerant Green Spherical Russel River Lime C. inodora Susceptible Yellow Bell shaped Fingerlime EN-1 C. australasica Tolerant Yellow Long, narrow Fingerlime EN-2 C. australasica Tolerant Yellow Long, narrow Red Fingerlime CA C. australasica Tolerant Red Long, narrow

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Table B-2. Parents crossed and resulting seedlings. Resulting Hybrid Female Male Fruit Seedlings Giant Finger Lime Poncirus trifoliata 50-7 4 3 Giant Finger Lime 1584 (Milam x P. trifoliata) 1 0 Giant Finger Lime Miaray sour orange 4 14 Giant Finger Lime Sinton citrangequat 3 20 Bearss Lemon Round Lime 0 Bearss Lemon Sydney Hybrid 0 Bearss Lemon Giant Finger Lime 0 50x5011-00-6 2x lime Finger Lime EN2 1 1 (2x) 50x5011-00-6 2x lime Round Lime 1 3 (2x) (Lakeland Limequat x Lime) 4x Round Lime 0 (Lakeland Limequat x Lime) 4x Finger Lime EN1 0 (Lakeland Limequat x Lime) 4x Finger Lime EN2 2 6 (3x) Giant Key Lime 4x Round Lime 0 Giant Key Lime 4x Finger Lime EN1 1 3 (3x) Giant Key Lime 4x Finger Lime EN2 2 11 (3x) C4-10-19 4x Lemon Round Lime 0 C4-10-19 4x Lemon Finger Lime EN1 0 C4-10-19 4x Lemon Finger Lime EN2 0 C4-6-48 4x Lemon Round Lime 0 C4-6-48 4x Lemon Finger Lime EN1 0 C4-6-48 4x Lemon Finger Lime EN2 0 4-4-99-7 Pummelo Round Lime 0 5-1-99-2 S5 Pummelo Round Lime 1 3 5-1-99-2 S5 Pummelo Red Finger Lime California 1 2 5-4-99-7 Round Lime 1 3 5-4-99-7 Finger Lime EN2 0 7-2-99-11 Round Lime 0 7-2-99-11 Finger Lime EN2 0 8-1-99-1A Round Lime 0 HBJL-1 Finger Lime EN2 0 Murc+Chan#80 4x Finger Lime EN2 0 UKP-1 Pummelo Round Lime 0 UKP-1 Pummelo Finger Lime EN2 0 Ambersweet Orange Seedling Round Lime 0 Ambersweet Orange Seedling Finger Lime EN2 0 Marisol mandarin Round Lime 3 14 Nules mandarin Round Lime 2 6 Nagami kumquat Giant Finger Lime 2 0 Lapithiotiki Lemon x Lakeland Limequat Finger Lime EN2 2 5 Finger Lime 50-36 Poncirus trifoliata 50-7 1 6 Finger Lime EN2 Nagami kumquat 1 5 Finger Lime 50-36 Meiwa kumquat 1 3 Total 108

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Figure B-1. Cross section of two finger limes (left) showing the spherical juice vesicles. Finger lime 50-36 above finger lime EN-1. Flower of Microcitrus australasica (10 mm diameter), also illustrating spiny stems and small leaves. Photo by author.

Figure B-2. Microcitrus at the Florida DPI Citrus Arboretum in Winter haven, FL (summer 2015). Giant finger lime is just visible behind Microcitrus australis (left), Sydney Hybrid (near center), and finger lime DPI-50-36 (right). Photo by author.

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Figure B-3. 5-1-99-2 S5 x Microcitrus australis. Many seedlings were not hybrid, but the seedling in the lower center shows the alternating leaves in contrast to the paired leaves of the non-hybrid seedlings. Photo by author.

Figure B-4. Seedling of Microcitrus australasica DPI 50-36 x Poncirus trifoliata DPI 50-7. Photo by author.

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Figure B-5. Open pollinated seedlings of Sydney Hybrid treated with oryzalin. (Top row of tray are Microcitrus australasica DPI-50-36 x Poncirus trifoliata DPI-50-7, and second row are Microcitrus australasica DPI-50-36 x Fortunella japonica ‘Meiwa’ kumquat). Photo by author.

Figure B-6. PartecPA flow cytometry histogram, showing 2x peak for key lime (left) and 4x peak for tetraploid Sydney Hybrid seedling. Photo by author.

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Figure B-7. Comparison of leaves between parents and offspring. Photos by author.

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Figure B-8. Grafted Microcitrus hybrids in field planting at the University of Florida Citrus Research and Education Center in Lake Alfred, FL. Photo by author.

Figure B-9. Three-year-old seedling tree of giant finger lime x OP, flowering and fruiting in greenhouse. Photo by author.

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APPENDIX C MEDIA COMPOSITION

CPW Medium BH3 Medium EME+ Maltose Components (g/L) (g/L) (g/L) Macronutrient Salts Ammonium Nitrate NH4NO3 1.65 Calcium Chloride Anhydrous CaCl2 0.15 0.44 0.44 Magnesium Sulfate MgSO4*5H2O 0.25 anhydrous 0.37 0.37

Potassium Nitrate KNO3 0.1 1.9 Potassium Phosphate monobasic KH2PO4 0.0272 Potassium Phosphate dibasic KH2PO4 0.17 0.17 Potassium Chloride KCl 1.5 Micronutrient Salts Boric Acid H3BO3 0.0062 0.0062 Cobalt Chloride Anhydrous CoCl2 0.000025 0.000025 Cupric Sulfate Anhydrous CuSO4 0.00000025 0.000025 0.000025 Ferrous Sulfate-H20 FeSO4*7H2O 0.0025 0.0278 0.0278 Manganese Sulfate-H20 MnSO4.H2O 0.0223 0.0223

Na2-EDTA-2H2O 0.0373 0.0373 Potassium Iodide KI 0.00016 0.00083 0.00083 Zinc Sulfate-7H20 ZnSO4 0.0086 0.0086

Sodium Molybdate Na2MoO4*2H2O 0.00025 0.00025

Vitamins etc. myo-Inositol 0.1 0.1 Nicotinamide (Niacinamide) 0.001 0.001 Pyridoxine-HCl 0.001 0.001 Thiamine-HCl 0.001 0.001 Citric Acid (free acid) anhydrous 0.04 0.04 DL-Malic Acid 0.04 0.04 Fumaric Acid 0.04 0.04 Vitamin B12 (Cyanocobalamin) 0.00002 0.00002 D-Calcium Pantothenate 0.001 0.001 L-Ascorbic Acid 0.002 0.002 Choline Chloride 0.001 0.001 p-aminobezoic Acid 0.00002 0.00002 Pyruvic Acid Sodium Salt 0.02 0.02 Folic Acid 0.0004 0.0004 Riboflavin 0.0002 0.0002 D-Biotin 0.00001 0.00001 Vitamin A (retinol) 0.00001 0.00001 Vitamin D3 (cholecalciferol) 0.00001 0.00001 Glutamine 3.1 Malt Extract 0.5 0.5 Casein Hydrolysate 0.25 Coconut Water 20 mL

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CPW Medium BH3 Medium EME+ Maltose Components (g/L) (g/L) (g/L) Sugars Sucrose 51.35 0.2054 Maltose 50 Mannitol 81.9 Fructose 0.25 0.25 Ribose 0.25 0.25 D-xylose 0.25 0.25 Mannose 0.25 0.25 Rhamnose 0.25 0.25 Cellobiose 0.25 0.25 Galactose 0.25 0.25 Glucose 0.25 0.25 Hormones 3-Naphthalene Acetic Acid NAA Kinetin

pH 5.8 5.7 5.78 Agar 8 8 8

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LIST OF REFERENCES

5B-58.001 : Citrus Canker Eradication (Repealed) - Florida Administrative Rules, Law, Code, Register - FAC, FAR, eRulemaking [WWW Document], n.d. URL https://www.flrules.org/gateway/RuleNo.asp?title=CITRUS%20CANKER&ID=5B- 58.001 (accessed 3.7.17).

2003 Florida Citrus Invasive Pest and Disease Identification Handbook, 2003.

Albrecht, U., Bowman, K.D., 2011. Tolerance of the Trifoliate Citrus Hybrid US-897 (Citrus reticulata Blanco × Poncirus trifoliata L. Raf.) to Huanglongbing. HortScience 46, 16–22.

Albritton, M., 2011. Sections (TRS) Positive for Huanglongbing (HLB, Citrus Greening) in Florida.

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BIOGRAPHICAL SKETCH

Ethan Ririe Nielsen was born in 1978, in Bozeman, Montana, USA. He obtained his GED in Kansas City, Missouri in 1995. He graduated with an associate’s in art from

Maple Woods Community College in 1997, then graduated from Brigham Young

University in 2001 with a bachelor’s degree in visual arts. He worked as a graphic designer and illustrator until 2008 when he returned to school at Utah State University where he received a second bachelor’s degree in horticultural science in 2011. He then joined University of Florida to pursue doctoral studies as a graduate research assistant in the Horticulture Sciences Department under the supervision of Jude Grosser. He received his Ph.D. from the University of Florida in the fall of 2017.

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