Fusarium Oxysporum FORMAE SPECIALES ASSOCIATED with ORNAMENTAL PALMS

DIAGNOSTICS, GENOMICS AND POPULATION STUDIES ON Fusarium oxysporum FORMAE SPECIALES ASSOCIATED WITH ORNAMENTAL PALMS

SUSHMA V. PONUKUMATI

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 family for their unconditional love and support

ACKNOWLEDGMENTS

I would like to express my gratitude to Dr. Monica Elliott, my advisor, for supporting me in my research in all possible ways. She always encouraged me to be a passionate, independent researcher and gave me freedom to explore new research fields. She helped me to develop the habits of logical thinking and proper planning. Dr.Elliott selfless time and care were sometimes all that kept me going during the writing of this dissertation.

I would like to thank Dr. Jeffrey Rollins for inducing passion towards bioinfomatics and helping me understand the importance of critical thinking. I am also appreciative to Dr. Rollins for his constant guidance and dedication in his role as co-advisor.

Profound gratitiude goes to my other Ph.D committee members, Dr. Mathew Smith and

Dr. Robin Giblin Davis, for serving on my committee and for providing their time and expertise.

Their advice and recommendations were valuable resources for my dissertation. I will always cherish Dr. Smith for his inspirational chats. Dr.Smith has been a good role model in teaching, who encouraged and expected students to think independently with a purpose regarding any experiment or project. I am also thankful to Dr. Robin Giblin Davis for his constant support and motivation.

My profound gratitude goes to Dr. Jose C. Huguet Tapia who took extreme care and time to teach me bioinformatics. He was always available to trouble shoot and advise me regarding any bioinformatics related questions during the dissertation.

A special mention goes to Ms. Beth Des Jardin for providing me a good support system in Dr. Elliott’s lab. She encouraged me and gave fantastic plant pathology skills training.

I would like to extend thanks to many people (friends, Dr. Elliott’s interns and co- graduate students) both at Fort Lauderdale Research and Extension Center and Plant Pathology

Department in Gainesville who have supported me by their friendship and their advice.

I would like to thank my husband Rajasekhar Ponukumati for his unconditional love and support; without it, this dissertation would not be possible. Finally, I express my gratitude to my parents and in-laws, my brother and sister for their belief in me and for their unending love and encouragement.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 11

ABSTRACT ...... 13

CHAPTER

1 LITERATURE REVIEW ...... 15

Introduction on Palms ...... 15 Fusarium oxysporum ...... 16 Fusarium Wilt Pathogens in Palms ...... 18 Fusarium oxysporum Pathogenesis ...... 23 Cell wall degrading enzymes ...... 24 Signaling proteins ...... 24 Transcriptional factors ...... 25 Blocking host defenses ...... 25 Fusarium oxysporum Genomic Studies ...... 26 Secreted in Xylem Gene Family ...... 28 Objectives ...... 30

2 DIAGNOSTIC PRIMER PAIR TO IDENTIFY FUSARIUM WILT PATHOGENS ON ORNAMENTAL PALMS IN FLORIDA ...... 32

Introduction ...... 32 Material and Methods ...... 35 Isolates ...... 35 Genomic DNA and EF-1α Sequencing ...... 36 Primer Design ...... 36 Genomic DNA Amplification with PFW Primer Pair ...... 37

DNA Amplification with Agar Plug and PFW Primer Pair ...... 38 DNA Amplification with the FOA Primer Pair ...... 39 Phylogenetic Analysis ...... 39 Results...... 40 Genomic DNA and EF-1α Sequencing ...... 40 Primer Design ...... 40 Genomic DNA Amplification with PFW Primer Pair ...... 40 DNA Amplification with Agar Plug and PFW Primer Pair ...... 41 DNA Amplification with FOA Primer Pair ...... 41 Phylogenetic Analysis ...... 41 Discussion ...... 43

3 GENOME CHARACTERIZATION OF Fusarium oxysporum f. sp. palmarum ...... 61

Introduction ...... 61 Material and Methods ...... 66 Isolates ...... 66 DNA Extraction and Whole Genome Sequencing ...... 66 Genome Sequencing ...... 67 Benchmarking Universal Single Copy Orthologs ...... 68 Comparative Genomics to Identify Orthologous Gene Families ...... 68 Identification of Secretory Proteins ...... 69 Detection of Carbohydrate Active Enzymes ...... 69 Detection of Secondary Metabolite Clusters ...... 70 Secreted in Xylem (SIX) Genes in FOC and FOP ...... 70 Detection of Virulence Genes in FOP and FOC ...... 71 Results...... 71 Genome Sequencing ...... 71 Benchmarking Universal Single Copy Orthologs ...... 72 Proteome Analysis of FOC and FOP ...... 72 Identifying Orthologous Families among Fusarium Genomes ...... 73

Secretome Analysis ...... 74 Carbohydrate Active Enzymes of FOC and FOP ...... 75 Detection of Secondary Metabolite Clusters ...... 76 Secreted in Xylem (SIX) Gene Family ...... 78 Virulence Genes ...... 78 Discussion ...... 79

4 SCREENING FOR SECRETED IN XYLEM (SIX) GENES IN FUSARIUM WILT PATHOGENS OF ORNAMENTAL PALMS ...... 108

Introduction ...... 108 Material and Methods ...... 112 Isolates ...... 112 DNA Extraction and Quality Assessment ...... 112 Molecular Characterization by EF-1α Gene ...... 113 Screening of SIX Genes from Assembled Genomes ...... 113 Detection of SIX Genes by PCR ...... 113 Phylogenetic Analysis ...... 114 Results...... 115 Molecular Characterization by EF-1α Gene ...... 115 Screening of SIX Genes from Assembled Genomes ...... 116 Detection of SIX Genes by PCR ...... 116 SIX1 ...... 116 SIX7 ...... 117 SIX10 ...... 117 SIX12 ...... 118 SIX8 and SIX9 ...... 119 Discussion ...... 119

5 RESEARCH SUMMARY ...... 145

LIST OF REFERENCES ...... 148

BIOGRAPHICAL SKETCH ...... 163

LIST OF TABLES

Table page

2-1 Fusarium isolates used in the study, indicating isolate, palm host, geographic location and tissue sampled. Genomic DNA or a fungal colonized agar plug was used as template for PCR using PFW primers and three different polymerases...... 48

3-1 List of the Fusarium genomes used in the study...... 90

3-2 Genomic statistics of F. oxysporum f. sp. canariensis and F. oxysporum f. sp. palmarum with selected Fusarium oxysporum formae speciales...... 91

3-3 Comparison of functional annotations among Fusarium oxysporum f. sp. canariensis and Fusarium oxysporum f. sp. palmarum...... 92

3-4 Important proteins grouped by Pfam domain/families based on Fusarium oxysporum f.sp. canariensis (FOC) and Fusarium oxysporum f. sp. palmarum (FOP) predicted proteomes...... 93

3-5 Comparison of important PFAM domains/families between Fusarium oxysporum f. sp. canariensis (FOC) and Fusarium oxysporum f. sp.palmarum (FOP)...... 94

3-6 The common Pfam domains that are present in the secretome of Fusarium oxysporum f. sp. palmarum...... 95

3-7 Fusarium oxysporum f. sp. palmarum expanded gene clusters based on orthoMCL comparison with 15 Fusarium genomes...... 96

3-8 Expanded gene families of Fusarium oxysporum f. sp. palmarum with genes and their Pfam domain description...... 97

3-9 Carbohydrate active enzymes (CAZymes) families involved in cellulose, hemicellulose and pectin degradation in Fusarium genomes...... 99

3-10 Description of Fusarium oxysporum f. sp. palmarum secondary metabolite gene clusters and its orthologous genes present Fusarium oxysporum genomes that were studied by ORTHOMCL...... 100

3-11 Secreted in Xylem (SIX) genes recovered from Fusarium. oxysporum f. sp. canariensis and F. oxysporum f. sp. palmarum genome assemblies using multiple bioinformatics approaches. . 102

3-12 Putative virulence gene distribution in Fusarium oxysporum f. sp. canariensis and F. oxysporum f. sp palmarum predicted by PHI-Base...... 102

3-13 Putative virulence associated genes orthologs identified in predicted proteome of Fusarium oxysporum f. sp. palmarum by PHI-Base, classified based on role in infection process...... 103

4-1 Previous studies on prominent Fusarium oxysporum formae speciales plus F. foetens with their SIX gene profiles elucidated by various approaches...... 124

4-2 Isolates used in the study and PCR based presence or absence of Secreted in Xylem (SIX) genes...... 125

4-3 PCR primer pairs that were used in this study, with their annealing temperature and amplicon size...... 128

4-4 Secreted in Xylem (SIX) genes recovered from Fusarium oxysporum f. sp. canariensis and Fusarium oxysporum f. sp. palmarum draft genomes using multiple bioinformatics approaches...... 129

LIST OF FIGURES

Figure page

2-1 Comparison of genomic DNA amplification using three different polymerases with the PFW primer pair as viewed on a 1.5% agarose gel...... 54

2-2 Comparison of DNA amplification using mycelial agar plug as template for PCR with three different polymerases and PFW primer pair as viewed on a 1.5% agarose gel...... 55

2-3 Unrooted consensus tree inferred from 10 most parsimonious trees using the Maximum Parsimony method based on a portion of the translation elongation factor (EF-1α) gene...... 56

2-4 Unrooted consensus tree inferred from 10 most parsimonious trees using the Maximum Parsimony method based on the PFW primer...... 58

2-5 Flowchart of the proposed diagnostic method for identification of Fusarium wilt pathogens on ornamental palms...... 60

3-1 Distribution of Fusarium oxysporum f. sp.canariensis (FOC) and F.oxysporum f. sp. palmarum (FOP) proteomes into sub categories of Eukaryotic Orthologous Groups (KOG) gene families...... 105

3-2 The carbohydrate active enzymes (CAZymes) class distribution in Fusarium genomes...... 106

3-3 SMURF secondary metabolite (SM) backbone gene prediction of Fusarium proteomes used in this study...... 107

4-1 EF-1α based phylogenetic tree of Fusarium oxysporum f. sp. canariensis (FOC) and F. oxysporum f. sp. palmarum (FOP) including various formae speciales inferred by Maximum Likelihood...... 130

4-2 SIX1 based phylogenetic tree of Fusarium oxysporum f. sp. canariensis (FOC) by Maximum Likelihood based on the Kimura 2-parameter model...... 132

4-3 SIX7 based phylogenetic tree of Fusarium oxysporum f. sp. canariensis (FOC) by Maximum Likelihood method based on the Kimura 2-parameter model...... 134

4-4 SIX10 based phylogenetic tree of Fusarium oxysporum f. sp. canariensis (FOC) along with F.oxysporum f. sp.palmarum (FOP) using the Maximum Likelihood method based on the Jukes-Cantor model...... 135

4-5 SIX12 based phylogenetic tree of Fusarium oxysporum f. sp. canariensis (FOC) inferred by Maximum Likelihood based on the Jukes-Cantor model...... 136

4-6 SIX8 based phylogenetic tree of Fusarium oxysporum f. sp. palmarum (FOP) by Maximum Likelihood method based on the Kimura 2-parameter model...... 137

4-7 SIX9 based phylogenetic tree of Fusarium oxysporum f. sp. palmarum (FOP) inferred by Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (-968.1485) is shown...... 138

4-8 Genomic DNA amplification of Fusarium oxysporum f. sp. canariensis (FOC) isolates using SIX1 primer pair as viewed on 1.5 % agarose gel stained with GelRed...... 139

4-9 Genomic DNA amplification of Fusarium oxysporum f. sp. canariensis (FOC) and F. oxysporum f.sp.palmarum (FOP) isolates using SIX7 primer pair as viewed on 1.5 % agarose gel stained with GelRed...... 140

4-10 Genomic DNA amplification of Fusarium oxysporum f. sp. palmarum (FOP) isolates using SIX8 primer pair as viewed on 1.5 % agarose gel stained with GelRed...... 141

4-11 Genomic DNA amplification of Fusarium oxysporum f. sp. palmarum (FOP) and F. oxysporum f.sp. canariensis (FOC) isolates using SIX9 primer pair as viewed on 1.5 % agarose gel stained with GelRed...... 142

4-12 Genomic DNA amplification of Fusarium oxysporum f. sp. canariensis (FOC) and F. oxysporum f. sp. palmarum (FOP) isolates using SIX10 primer pair as viewed on 1.5 % agarose gel stained with GelRed...... 143

4-13 Genomic DNA amplification of Fusarium oxysporum f. sp. canariensis (FOC) isolates using SIX12 primer pair as viewed on 1.5 % agarose gel stained with GelRed...... 144

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

DIAGNOSTICS, GENOMICS AND POPULATION STUDIES ON Fusarium oxysporum FORMAE SPECIALES ASSOCIATED WITH ORNAMENTAL PALMS

Sushma V.Ponukumati

August 2017

Chair: Monica L. Elliott Cochair: Jeffrey A. Rollins Major: Plant Pathology

Fusarium wilt on ornamental palms in Florida is a lethal disease caused by Fusarium oxysporum f. sp. canariensis (FOC) and F. oxysporum f. sp. palmarum (FOP). FOP has a wider palm host range, causes an aggressive disease and is primarily localized to Florida, whereas FOC is found worldwide and restricted primarily to one palm species. The first objective was to design an efficient diagnostic method to differentiate palm Fusarium wilt pathogens from other

Fusarium species often associated with palms. A translation elongation factor based primer pair

(PFW) was designed. All FOP and FOC isolates, which were obtained from symptomatic palm petiole or rachis tissue, were amplified. No other Fusarium species were amplified with PFW primers. Results indicate that symptomatic petiole or rachis is the only tissue to use for isolation of palm Fusarium wilt pathogens. For the second objective, the FOP and FOC genomes were sequenced, assembled and annotated using multiple bioinformatic approaches to compare FOC and FOP proteomes to identify common and unique gene families. Results indicate FOP and

FOC share similar copies of conserved pathogenicity factors. FOP has more secondary

metabolite potential than FOC. At least three nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) clusters appear novel to FOP compared to FOC and other Fusarium genomes, suggesting the possible role of secondary metabolites in FOP pathogenicity. The third objective was screening for Secreted in Xylem (SIX) genes that produce small secretory proteins, which may play a role in virulence and in the infection process of Fusarium oxysporum formae speciales. Whole genome sequencing of FOC and FOP indicates the presence of SIX7, SIX10 and

SIX12 in FOC and SIX8 and SIX9 in FOP. A PCR based approach was used to screen 47 isolates of FOC and FOP, obtained from different palm hosts and geographical locations, to detect the presence of SIX genes with published primers. PCR results confirmed the genomic results but

SIX1 was also detected in FOC isolates. Insights about these formae speciales of F. oxysporum will enhance our understanding of host specific interactions and may lead to effective management strategies.

CHAPTER 1 LITERATURE REVIEW

Introduction on Palms

Carl Linnaeus described palms as “Principes–the princes of the plant kingdom” (Plotkin and Balick 1984). The family Arecaceae consists of approximately 2,600 palm species placed in more than 200 genera (Christenhusz and Byng 2016). Palms are grown in tropical, subtropical and Mediterranean climates with an extended life span averaging greater than 60 years (Meerow

1994; Tomlinson and Huggett 2012). Across the world, palms are grown for food, oil, fruits, wine and economic products such as rope, mats, tannin and roofing material (Elliott et al. 2004).

Within the continental USA, palms are primarily used for aesthetic purposes in commercial and residential landscapes.

Palms are monocots with adventitious roots and scattered vascular bundles in the stem that survive the complete life span of the palm. Palms have no vascular cambium, which means there is no secondary growth. Moreover, with a single apical meristem per stem, palms are vulnerable to any damage to the meristem, which can result in death. Palm leaves are some of the largest in the plant kingdom and can be either fan shape (palmate, costa-palmate), feather

(pinnate) or entire. All palm leaves are divided into blade, petiole and leaf base. There are four stages of palm development: seedling, juvenile (leaves but no above-ground stem), adult vegetative (above-ground stem but not mature enough to flower) and adult mature phase

(reproductive). Even though the palm flowers are tiny, they produce some of the largest fruits and seeds in the plant kingdom (Elliott et al. 2004).

In the USA, palms are extensively planted in Hawaii and southern states as ornamentals.

As of 2010, palms rank fourth in the list of nursery sales in Florida accounting for a $404 million

dollar industry (Khachatryan and Hodges 2012). Common palms that are grown in Florida include Bismarckia nobilis (Bismarckia palm), Butia odorato (pindo palm), x Butyagrus nabonnandi (mule palm), Cocos nucifera (coconut), Dypsis lutescens (areca palm), Phoenix canariensis (Canary Island date palm), Phoenix dactylifera (date palm), Phoenix reclinata

(Senegal date palm), Phoenix sylvestris (wild date palm), Ravenea rivularus (majesty palm),

Roystonea regia (royal palm), Sabal palmetto (cabbage palm), Syagrus romanzoffiana (queen palm), and Washingtonia robusta (Mexican fan palm) (Broschat and Black 2016). The average cost of palms in Florida ranges from $100 (areca palm) - $3800 (date palm)

(http://realpalmtrees.com). With few exceptions, the majority of these palms are not native to the

USA.

Similar to other plants, palms are subjected to various diseases, insects, nutritional, and physiological disorders. Palms are susceptible to diseases caused by a wide variety of organisms that include algae, bacteria, fungi, oomycetes, nematodes, phytoplasmas and viruses. Among these, phytoplasmas and fungi are more common in Florida. Common fungal diseases of palms in Florida include bud rot, Fusarium wilt of Canary Island date palm, Fusarium wilt of queen palm and Mexican fan palm, Ganoderma butt rot, Graphiola leaf spot, petiole blight and

Thielaviopsis trunk rot (Elliott et al. 2004).

Fusarium oxysporum

The genus Fusarium belongs to Domain Eukaryota, Kingdom Fungi, Phylum

Ascomycota, Subphylum Pezizimycotina, Class Sordariomycetes, Order Hypocreales, and

Family Nectriceae. H. F. Link first described the Fusarium genus in 1809 as Fusisporium based on banana shaped conidia (Gordon and Martyn 1997; Leslie and Summerell 2006). In 1935,

Wollenweber and Reinking grouped at least 1000 proposed Fusarium like species into 16 16

sections, comprising 65 species, 55 varieties and 22 forms based on culture characteristics in media and spore morphology (macroconidia, microconidia and chlamydospores) (Nelson et al.

1983). They published the first atlas, Die Fusarien, describing these 142 Fusarium-like species.

In 1940, Synder and Hansen consolidated 16 sections into nine species using single spore isolates. They further proposed 25 formae speciales based on the hosts they infect (Gordon and

Martyn 1997). Fusarium oxysporum, one of the nine species, was first described by D. F. L. von

Schlechtendahl in 1824, and later amended by Synder and Hansen in 1940 (Booth 1971; Nelson et al. 1983).

Fusarium oxysporum Schlechtend: Fr. is an asexual species complex comprising a wide variety of biological forms including biocontrol, endophytic, saprophytic and pathogenic isolates. The pathogenic forms affect animals, humans, insects and plants. The plant pathogenic strains are necrotrophs and affect a wide variety of economically important plants, causing either vascular wilt, bulb rot, crown rot or root rot (Michielse and Rep 2009). Fusarium oxysporum is ranked fifth in the list of most notorious plant pathogens (Dean et al. 2012). Plant pathogenic F. oxysporum strains are divided into formae speciales (f. sp singular; ff. spp. plural) based on host plants that they infect. Currently more than 120 formae speciales have been classified, and a few are further classified into races based on host lines or cultivars that they infect (Armstrong and

Armstrong 1981; Baayen et al. 2000). For example, the forma specialis that infects Canary Island date palm is named F. oxysporum f. sp. canariensis, the forma specialis that infects banana is named F. oxysporum f. sp. cubense and so forth.

Fusarium oxysporum has no known teleomorph and is considered to reproduce clonally.

It produces three types of asexual spores: microconidia, macroconidia and chlamydospores

(Nelson et al. 1983). Microconidia are usually one-celled and oval to elliptical in shape. 17

Macroconidia are produced abundantly on infected plants and consist of three to five-celled spores. Chlamydospores are thick-walled, survival structures that can survive in soil for at least

25 years (Downer et al. 2013). They can be produced terminally or intercalary. Morphological characterization of F. oxysporum include shape of macroconidia, microconidia and chlamydospores (Leslie and Summerell 2006).

Fusarium oxysporum vascular wilts are initated primarily as root infections, in which the chlamydospores present on plant debris or in soil germinate and mycelia penetrate into plant roots through natural openings or wounds. The mycelium advances into root cortex reaching xylem vessels through the pits. The mycelium is confined to xylem vessels, but continues to grow and produce microconidia. These microconidia are carried upward by xylem sap, where they germinate and invade more xylem vessels. By this time, plants are in severe water stress, resulting in wilt symptoms as the xylem vessels are clogged by mycelium, conidia, gels, gum and tyloses that are produced by plant defenses. Finally, the fungus invades neighboring tissues of the plant and becomes exposed to the external environment. Newly produce micro and macrocondia may be dispersed to new plants by wind, water or insects (Agrios 2005).

Fusarium Wilt Pathogens in Palms

Currently, there are four Fusarium wilt diseases on palms, each with lethal consequences.

F. oxysporum f. sp. albedinis (FOA) causes date palm wilt on Phoenix dactylifera and is observed in Algeria, Morocco and Mauritania (EPPO 2003). FOA appears to be monophyletic with all isolates belonging to one vegetative compatibility group (Fernandez et al. 1998). Disease progression is slow, and it is usually spread by transport of infected plant material (EPPO 2003).

F. oxysporum f. sp. eleidis (FOE) causes oil palm wilt on Elaeis guineensis (African oil palm) and is reported from countries in Africa, and localized areas in Brazil and Ecuador (Flood

2006; Rusli 2012). FOE may be spread by contaminated seeds. FOE attacks oil palms at various growth stages and has two disease types: acute wilt, which is a fast-paced disease where palms die within 2-3 months, and chronic wilt, which is a slow, progressive decline of trees over years

(Flood 2006).

Canary Island date palm wilt, caused by F. oxysporum f. sp. canariensis (FOC), is present worldwide on Phoenix canariensis. It was first reported in France in 1973 (Mercier and

Louvet 1973), followed by the USA in 1976 (Feather et al. 1989), Japan in 1977 (Arai and

Yamamoto 1977), Spain in 1987 (Fernandez et al. 1997), Australia in 1990 (Priest and Letham

1996), Greece in 2002 (Elena 2005), Italy (Sardinia) in 2004 (Migheli et al. 2005) and Argentina in 2005 (Palmucci 2005). In the USA, FOC was first reported on Phoenix canariensis in

California in 1976 (Feather et al. 1989) and then spread to Florida in 1994 (Simone 2004). It has been reported in Nevada (Wang and McKie 2007), Louisiana (Singh et al. 2011) and South

Carolina and Texas (Elliott et al. 2011). FOC has also been documented on Phoenix reclinata in

Florida (Elliott 2015) and California (personal communication, California Department of Food and Agriculture).

Fusarium wilt of queen (Syagrus romanzoffiana) and Mexican fan (Washingtonia robusta) palms is caused by F. oxysporum f. sp. palmarum (FOP) and was first observed in

Florida (Elliott et al. 2010). Except for one report from a single location in Texas (Giesbrecht et al. 2013), this pathogen is not known anywhere else in the world. FOP has a wider host range than most F. oxysporum formae speciales, as it not only causes wilt diseases of queen and

Mexican fan palms, but also on Phoenix canariensis, and x Butyagrus nabonnandii (Elliott 2011; 19

Elliott et al. 2017). It has also been detected in wilt symptomatic specimens of Bismarckia nobilis and Phoenix reclinata in Florida (Elliott, personal communication).

Fusarium wilts caused by FOC and FOP share similarities, including symptoms and disease cycle, with an overlapping host range, and yet they are distinct diseases and pathogens.

With FOC and FOP, initial symptoms are usually observed in lower fronds and include typical one-sided wilt and necrosis of the leaflets. A brownish streak on the petiole and rachis is always associated with necrotic dead leaflets. Cross sections of the petiole show an internal discoloration that corresponds to the necrotic dead leaflets. Eventually the whole leaf dies and the process is repeated on the next oldest leaf. The primary difference between these two Fusarium wilt pathogens is disease progression. From the onset of symptoms, the disease progression caused by

FOC is slow, taking up to a year to two for palms to die. In the disease caused by FOP, progression is fast, killing the palms within weeks to months (Elliott et al. 2010). This suggests

FOP is a more aggressive pathogen than FOC. Compared to other formae speciales, FOC and

FOP are special cases in a few regards. Typically, a F. oxysporum forma specialis is host specific with one or a few hosts. However, FOC can infect at least two species within the genus Phoenix, and FOP can infect multiple genera within the Arecaceae family including Phoenix, Syagrus and

Washingtonia. FOA and FOE primarily infect palms via root infections, whereas, FOC and FOP primarily infect leaves, but can also infect via the roots.

Fusarium wilt of Canary Island date palm is spread by contaminated pruning tools, infested soil and dead infected plant material. FOP is assumed to be spread primarily by windblown spores (Elliott et al. 2010). There are no cures or treatment for either of the Fusarium wilts caused by FOP or FOC. Prevention is the only way to limit the spread of the disease, and early detection is the key to implement management strategies. Management strategies for Fusarium 20

wilt caused by both pathogens include use of clean and disinfected tools when pruning healthy or diseased palm trees, and removal of the diseased tree as quickly as possible. It is strongly suggested not to replant a susceptible palm species in the infested soils (Elliott et al. 2004).

Phylogenetic studies have revealed that isolates of different formae speciales are often more closely related than isolates within the same forma specialis (Kistler 1997; Lievens et al.

2008). Many formae speciales are polyphyletic in nature where isolates may have adapted to the same host by convergent evolution as seen in F. oxysporum f. sp. cubense and F. oxysporum f. sp. lycopersici (O’Donnell et al. 1998; Ma et al. 2013). A few formae specialis, such as F. oxysporum f. sp. loti and F. oxysporum f. sp. ciceris, are monophyletic with isolates of the same forma specialis all resolved within a clonal lineage (Jimenez-Gasco et al. 2002; Wunsch et al.

2009).

Vegetative compatibility grouping (VCG), polymerase chain reaction assays and DNA fingerprinting are routinely used to understand genetic diversity in the Fusarium oxysporum species complex (Baayen et al. 2000; Gunn and Summerell 2002). A few studies have explored genetic diversity among FOC, but only one study examined FOP isolates. Most FOC isolates

(71%) group into a single VCG group (0240), and mitochondrial DNA markers revealed FOC isolates belonged to a few haplotypes, implying the presence of low to moderate diversity and that FOC is monophyletic (Plyler et al. 2000). Translation elongation factor gene (EF-1α) phylogenetic analysis based on Australian and non-Australian isolates, including USA FOC isolates, indicated that Australian FOC isolates are more diverse than non-Australian isolates with at least three different lineages present, suggesting the Australian FOC are polyphyletic

(Gunn and Summerell 2002; Laurence et al. 2015). A study conducted by O’Donnell et al.

(2009), using two-locus DNA sequence typing, revealed the presence of 256 sequence types (ST) 21

among 850 F. oxysporum species complex isolates. The two loci were EF-1α and nuclear ribosomal intergenic spacer region (IGS). This robust study, which included five isolates of FOP obtained in Florida, revealed the presence of three sequence types in FOP (ST250, ST251 and

ST284) and two sequence types in FOC (ST41 and ST200). None of the FOC isolates were from

Florida. No correlation among sequence types, palm host or disease severity has been observed among FOP isolates.

The most reliable method to identify F. oxysporum formae speciales are through pathogenicity assay in which host plants are inoculated with the pathogens to observe disease development. Additionally, morphological and cultural characteristics and symptoms on infected host tissues are used to identify F. oxysporum (Baayen et al. 2000). However, F. oxysporum formae speciales are morphologically similar and pathogenicity assays are time consuming and laborious. Molecular methods provide an alternative to identify F.oxysporum formae speciales

(Lievens et al. 2008). Designing a diagnostic tool to identify F. oxysporum formae speciales is often challenging and time consuming due to their polyphyletic lineages (Plyler et al. 2000; Suga et al. 2013).

Diagnosis of Fusarium wilt caused by FOC and FOP include isolation of the pathogen from the infected palm tissue, observation of colony and spore morphology (microconidia and macroconidia) and molecular assays. For the latter, DNA is extracted from single-spored isolates. Molecular identification of FOC includes a positive PCR amplification using the

HK66/HK67 primer pair (Plyler et al. 1999). However, this primer pair routinely detects F. proliferatum, which is sometimes isolated from symptomatic palms (Viteroli 2008). Presently,

FOC and FOP are molecularly identified by PCR amplification using the primer pair ef1 and ef2, which amplifies a portion of the EF-1α (Geiser et al. 2004). The amplicon is sequenced and a 22

similarity match is identified using the NCBI database and BLASTn (O’Donnell et al. 1998;

Elliott et al. 2010; Geiser et al. 2004).

Fusarium oxysporum Pathogenesis

To establish a successful infection, fungal plant pathogens have to invade the plant host and overcome host defenses, either by suppressing or counteracting them. Pathogenesis describes

“the process of disease development in the host from initial infection to onset of symptom”

(Lucas 1998). The fungal genes that are required for pathogenesis are defined in multiple ways.

Idnurm and Howlett (2001) broadly defined pathogenicity genes as “genes necessary for disease development, but not essential for its life cycle”. A subclass of pathogenic genes called effectors are defined as small-secreted proteins that function within the plant host to alter host cell structure, promote infections and suppress plant immunity responses. These proteins are generally cysteine rich and lack homologs in closely-related species (Van de Wouw and Howlett

2011). Some of these effectors, “defeated effectors”, can act as avirulence genes that can be recognized by the plant host and contribute to plant resistance. Casadevall and Pirofski (1999) defined virulence as “relative capacity to cause damage in a host”. Virulence factors are determinants of pathogenicity.

Several studies using forward and reverse genetic approaches along with comparative genomic studies have explored pathogenicity genes of F. oxysporum. In this study, pathogenic genes are defined as those that are essential for disease development. A mini-review of some well-known pathogenicity related genes of Fusarium oxysporum are discussed below.

Cell wall degrading enzymes

Plants cell walls are complex, made up of cellulose, hemicellulose, lignin, and pectin. Along with the cuticle, they act as the first barriers against pathogens. Fusarium oxysporum, being a nectrotroph produces an array of cell wall degrading enzymes, such as cutinases (CUT), cellulases, polygalacturnases (PG1, PG5, PGX), pectate lyases (PL1), xylanases (XYL3, XYL4,

XYL5) and proteases like subtilase (PRT1), which assist in host penetration and colonization

(Beckman 1987). However, target mutagenesis studies failed to implicitate them in virulence (Di

Pietro et al. 2003). In F. oxysporum f. sp. lycopersici, xylanases were studied for their involvement in pathogenicity by inactivating transcription factor XlnR that regulates xylanases and celluloses. Although it resulted in low expression of xylanase, no effect on pathogenicity was observed (Calero-Nieto et al. 2007). CTF1, a transcriptional activator for cutinase (CUT1) and lipase (LIP1), was studied in axenic conditions (Rocha et al. 2008), and it was thought to be involved in esterase and lipase activities. Yet, in planta studies suggested that CTF1 does not regulate LIP1 and CUT1, as it was expressed in low levels during root infections. Cell wall degrading enzymes also influence carbohydrate metabolism by carbon catabolite repression.

Disruption of protein kinases like SNF1 and FRP1 (F-box protein) resulted in poor root colonization due to impaired carbohydrate metabolism (Duyvesteijin et al. 2005; Jonkers et al.

2009; Ospina-Giraldo et al. 2003). It is difficult to prove the role of cell wall degrading enzymes in pathogenicity due to their functional redundancy.

Signaling proteins

For successful infection, a pathogen has to recognize the host and adapt to their environment by triggering signaling pathways. In F. oxysporum cAMP-PKA (cyclic adenosine monophosphate-protein kinase) and MAPK (mitogen-activated protein kinase) cascades, activate

transcriptional factors which regulate pathogenicity genes. Studies have shown that MAPK

(FMK1), G protein subunits α (FGA1) and β (FGB1) are required for pathogenicity (review by

Di Pietro et al., 2003, Michielse and Rep 2009a). FGA2 disrupted mutants of F. oxysporum f. sp. cucumerinum resulted in altered colony morphology, reduced conidiation and reduced pathogenicity (Jain et al. 2005). This suggests that kinases proteins have pleiotropic effects on housekeeping and pathogenicity related genes.

Transcriptional factors

Recent studies have indicated that transcriptional factors play a role in virulence as they might regulate pathogenicity genes and are gaining importance in understanding pathogenicity processes. Mutations of FOW2 in F. oxysporum f. sp. melonis and SGE1 in F. oxysporum f. sp. lycopersici have resulted in loss of pathogenicity (Imazaki et al. 2007; Michielse et al. 2009b). In

F. oxysporum f. sp. phaseoli, FTF, a multi-copy transcriptional factor gene, is present only in virulent strains and located on a lineage specific chromosome. The genes that are regulated by

FTF1 are currently unknown (Ramos et al. 2007).

Blocking host defenses

In order to survive in hosts, F. oxysporum has to overcome physical barriers and antifungal compounds that are secreted by plant hosts (Beckman 1987; VanEtten et al. 1994). In a well studied example, α-tomatine is produced by tomato plants and attacks fungal membranes resulting in their lysis (Roddick 1977). F. oxysporum f. sp. lycopersici secretes tomatinases which neutralizes α-tomatine (Larini et al. 1996). Disruption of one of the tomatinases, TOM1, in

F. oxysporum f. sp. lycopersici resulted in mutants with decreased progression of symptoms, suggesting their involvement in virulence (Pareja-Jaime et al. 2008). In response to pathogens, host plants deposit cell wall bound phenolic compounds, which are sensed by F. oxysporum and 25

trigger the production of mycotoxins and enzymes like pectinase, cellulose and amylase. Studies involving insertional mutagenesis of carboxy-cis, cis–muconate cylase (CMLE1) resulted in a decreased degradation of phenolic compounds suggesting their role in pathogenesis (Michielse and Rep 2009; Michielse et al. 2009a).

Fusarium oxysporum Genomic Studies

Reverse genetic approaches have focused on selective genes and model organisms, but comparative genomic studies offer the potential for greater insights into profiles of pathogenic genes in non-model organisms and helps to understand the origins and clues regarding the evolution of these genes. An advantage of comparing genomes comes with the ability to identify the presence of orthologous genes (Okagaki et al. 2016). Orthologs are homologs that are derived from a common ancestor and often retain similar functions. Moreover, comparative genomics can also reveal unique genes that are specific or novel to each genome. Comparative genomic studies are widely being used to address different aspects of fungal plant pathogen biology.

Krijger et al. (2014) conducted a comparative study involving 33 fungal pathogen secretomes, revealing that pathogens with similar lifestyles share certain gene families, but the expansion and contraction of gene families are strongly lineage specific and not confined by fungal lifestyle. In another study Guyon et al. (2014) identified 486 candidate virulence genes in Sclerotinia sclerotiorum using bioinformatic tools and evolution analysis, of which 78 were effector candidates. This research will facilitate future studies to understand host-pathgoen interactions by necrotrophic pathogens. For clinical purposes, a comparative genomic study was used to design a single step PCR based diagnostic assay for F. oxysporum f. sp. conglutians (Ling et al.

2016).

There are multiple platforms with annotated genomes that are publicly available and can be used as reference, and numerous bioinformatics tools are available to study phylogenic, structural and functional aspects of identified gene families. The two main platforms include

Mycocosm, maintained by the Department of Energy Joint Genome Institute, which provides information on 1000 fungal genomes (Grigoriev et al. 2012), and Fusarium Comparative

Genomics, a platform designed for Fusarium (http://genomics.fusariumdb.org/) (Park et al.

2011).

Three significant contributions using comparative genomics that enhanced our understanding about Fusarium oxysporum are described below. A landmark comparative genomic study was conducted by Ma et al. (2010) on F. verticillioides, F. graminearum and F. oxysporum f. sp. lycopersici (FOL) at the Broad Institute. Their study revealed that the FOL genome is partitioned into core and conditionally dispensable or lineage-specific chromosomes

(LS). LS regions are rich in transposons, pathogenicity genes and repetitive elements. The genes present on LS had different phylogenetic profiles (ancestry) than Fusarium housekeeping genes.

This was the first study to suggest genome level horizontal gene transfer as a mode of transfer for pathogenicity genes between F. oxysporum isolates resulting in new pathogenic strains.

Van Dam et al. (2016) conducted a large scale whole genome analysis on 59 isolates belonging to five F. oxysporum formae speciales (ff. spp. cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici) of which the first four infect members of the Curcurbita genus. They identified a total of 104 effectors and indicated that effector profiles of different formae speciales infecting the same hosts tend to be similar when compared to other formae speciales. Strains belonging to different lineages within the same forma specialis tend to share identical effector profiles. 27

A similar study conducted by Williams et al. (2016) on three legume infecting F. oxysporum formae speciales (ff. spp. medicaginis, ciceris and pisi) revealed the presence of genomes segregated into core and lineage specific (LS) regions. The research concluded that effectors are usually located in LS regions. Each forma specialis had unique effectors, indicating convergent evolution and an absence of a common ancestor. Yet, a small set of effectors were conserved among all the legume-infecting formae speciales implying their role in host specificity

Secreted in Xylem Gene Family

Secreted in Xylem (SIX) gene family were the first effectors identified in Fusarium oxysporum f. sp. lycoperisci (FOL) using a combination of proteomic and in-silico approaches, along with gene knock-out and Agrobacterium-based assays (Rep et al. 2004; Schmidt et al.

2013). The SIX1 protein was first identified by proteome analysis of xylem sap of a tomato plant infected by F. oxysporum f. sp. lycopersici (Rep et al. 2004). So far, fourteen proteins have been identified in FOL, and the majority of SIX proteins are located on lineage specific chromosome

14 (Ma et al. 2010; Schmidt et al. 2013). SIX proteins are generally cysteine rich with 300 amino acids, an N-terminal signal and non-autonomous transposable elements (MITEs) present at the promoter sites for the majority of these SIX genes (Houterman et al. 2007; Lievens et al. 2009;

Ma et al. 2010; Rep et al. 2004; Schmidt et al. 2013). Besides FOL, homologs of SIX genes are present in other formae speciales, including Fusarium oxysporum ff. spp. cepae, canariensis, cubense, pisi and vasinfectum, with SIX gene profiles distinct from one another (Chakrabarti et al. 2011; Fraser-Smith et al. 2014; Laurence et al. 2015; Lievens et al. 2009; Meldrum et al.

2012; Taylor et al. 2016).

Although SIX1 through SIX6 genes have been characterized, their biological functional roles and the plant host proteins they interact with are still unknown. In the tomato and FOL 28

pathosystem, there are three resistant tomato cultivars, I-1, I-2, I-3, and subsequently, three races of FOL (1, 2, and 3) have been identified. SIX1 (AVR3) was the first SIX gene to be identified in xylem sap of I-3 resistant tomato cultivar (Rep et al. 2004). On susceptible plants, SIX1 is required for pathogenicity, is expressed during initial colonization and requires the presence of living cells (van der Does et al. 2008). A transcriptional factor for SIX gene expression (SGE1) is required for its regulation and contains eight cysteine residues (Houterman et al. 2007; Michielse et al. 2009b; Rep et al. 2004). The FOL SIX4 is an avirulence gene recognized by the I-1 tomato cultivar. It is present only in race 1 isolates of FOL. The protein is secreted during colonization

(Houterman et al. 2007). SIX3, also called AVR2, has a signal peptide with two cysteine residues. AVR2 is recognized by the I-2 tomato cultivar, and on resistant cultivars, it is required for pathogenicity and expressed in xylem vessels during the early stages of root colonization

(Houterman et al. 2007). SIX5, which has six cysteine residues, and SIX3 share a 1365bp upstream region. Additionally, a shared transcriptional regulator SGE1 controls SIX3 and SIX5.

Both genes physically interact with each other and are required for I-2 mediated resistance.

Deletion of SIX5 leads to loss of pathogenicity implying its role as an effector (Ma et al. 2015).

The combination of SIX3 and SIX5 is extensively present in FOL isolates (Lievens et al. 2009).

SIX6 is an exception to the general rule that SIX genes are specific to Fusarium.

Homologs of SIX6 are present in Colletotrichum orbiculare and C. higginsiantum (Gan et al.

2013; Kleemann et al. 2012). SIX6 consists of eight cysteine residues, requires the presence of living cells and its protein is needed for full virulence in FOL. It suppresses I-2 mediated cell death in Nictoniana benthamiana (Gawehns et al. 2014). SIX1 (AVR3), SIX3 (AVR2), and SIX4

(AVR1) act as avirulence genes and are recognized by R proteins in resistant tomato cultivars.

SIX1, SIX3, SIX5 and SIX6 gene knock out and transformation experiments have proven their

role in virulence and are classified as effectors on susceptible tomato lines (Gawehns et al, 2014;

Lievens et al. 2009; Ma et al. 2015; Rep et al. 2004).

The majority of SIX genes are single copy and located on chromosome 14 with the exception of SIX8. In the FOL 4287 genome, two variants of SIX8 were observed, SIX8a and

SIX8b, with nine identical copies of SIX8a and four copies of SIX8b present (Schmidt et al.

2013). While the majority of SIX8 copies are present on chromosome 14, a few are present on chromosomes 3 and 6 (Schmidt et al. 2013). Using a proteomic approach, SIX8a was identified in xylem sap of infected tomato plant, whereas SIX8b was absent (Schmidt et al. 2013). There are three SIX8 variants observed in F. oxysporum f. sp. cubense, and their presence varies among the different races (1, 2 and 4 (ST4 and TR4)) (Fraser-Smith et al. 2014).The function of SIX8 remains unknown.

Objectives

In the USA, palms are extensively planted as ornamentals. In Florida, Fusarium wilt caused by FOC and FOP has severe consequences because the disease is always lethal. Florida is the only geographical location where both palm-infecting formae speciales have been reported and coexist. The present research focuses on diagnostics, genomics and population studies of these two Fusarium wilt pathogens. The dissertation is divided into three main objectives, which comprise individual chapters. Chapter 5 summarizes the results from all three objectives and suggests further direction for research.

1. The first objective (Chapter 2) is to design a diagnostic primer pair for Fusarium wilt pathogens (FOC and FOP) isolated from ornamental palms in Florida.

2. The second objective (Chapter 3) is to identify candidate pathogenicity factors, by employing comparative genomics, which are unique or conserved across ornamental

palm-infecting formae speciales and can explain the aggressiveness of FOP compared with FOC.

3. The third objective (Chapter 4) is to identify the SIX gene profiles of FOP and FOC based on the 14 SIX genes identified previously from F. oxysporum f. sp. lycopersici.

CHAPTER 2 DIAGNOSTIC PRIMER PAIR TO IDENTIFY FUSARIUM WILT PATHOGENS ON ORNAMENTAL PALMS IN FLORIDA

Introduction

Palms are economically important ornamentals in Florida. In 2010, the retail sale value of palms was equal to all other trees (deciduous, evergreen, flowering and fruit) sold in Florida

(Hodges et al. 2011). However, as with any plant, they are prone to various nutritional deficiencies and diseases. Fusarium wilt caused by Fusarium oxysporum is one of the important fungal diseases threatening ornamental palms in Florida (Elliott et al. 2010; Plyler et al. 2000).

Fusarium oxysporum is classified into more than 120 formae speciales based on the specific host plants infected (Michielse and Rep 2009). Currently, there are four Fusarium wilt diseases on palms. F. oxysporum f. sp. albedinis (FOA) causes date palm wilt on Phoenix dactylifera and is observed in Algeria, Morocco and Mauritania (EPPO 2003). F. oxysporum f. sp. eleidis (FOE) causes oil palm wilt on Elaeis guineensis and is reported from countries in Africa and localized areas in Brazil and Ecuador (Flood 2006). Canary Island date palm wilt, caused by F. oxysporum f. sp. canariensis (FOC), is present worldwide on Phoenix canariensis including Australia (Priest and Letham 1996), Argentina (Palmucci 2005), the Canary Island Islands (Hernandez-Hernandez et al. 2010), France (Mercier and Louvet 1973), Greece (Elena 2005), Japan (Arai and

Yamamoto1977), Spain (Fernandez et al. 1997) and USA (Feather et al. 1989). In the USA, FOC has been reported on Phoenix canariensis in California (Feather et al. 1989), Florida (Simone

2004), Louisiana (Singh et al. 2011), Nevada (Wang and McKie 2007), South Carolina and

Texas (Elliott et al. 2011). FOC has also been documented on Phoenix reclinata in Florida

(Elliott 2015) and California (California Department of Agriculture). Fusarium wilt of queen

(Syagrus romanzoffiana) and Mexican fan (Washingtonia robusta) palms is caused by F.

oxysporum f. sp. palmarum (FOP), which is widespread in Florida (Elliott et al. 2010). Except for one report from one location in Texas (Giesbrecht et al. 2013), this pathogen is not known anywhere else in the world. FOP has a wider host range than most Fusarium oxysporum formae speciales, as it not only causes wilt diseases on queen and Mexican fan palms, but also on

Phoenix canariensis and x Butyagrus nabonnandii ( Elliott 2011; Elliott et al. 2010; 2017). It has also been detected in wilt symptomatic specimens of Bismarckia nobilis and Phoenix reclinata in

Florida (Elliott, personal observation).

Unlike the rest of the world where palms are grown, Florida has two F. oxysporum formae speciales affecting palms, FOC and FOP, with Phoenix canariensis and possibly P. reclinata afflicted by both formae speciales. These two Fusarium wilt pathogens primarily cause a canopy infection with similar symptoms. A one-sided necrosis of the leaf blade is first observed on older leaves with a brown stripe on the rachis or petiole associated with the necrotic leaflet tissue. There is corresponding internal discoloration of the petiole or rachis, and it is this tissue that is used to isolate the pathogen. The symptoms progress upwards through the palm canopy until the palm is killed. Once symptoms are expressed, FOP can kill susceptible palm hosts within a few weeks to several months, as compared to FOC, which usually takes a year to kill the palm (Elliott et al. 2010; Plyler et al. 2000). If the pathogen becomes incorporated into the soil, then infection can occur via the root system, with similar symptoms observed in the canopy. Currently, there are no treatments available to cure these Fusarium wilt diseases (Elliott et al. 2010; Plyler et al. 2000). Disease management relies heavily on prevention and reducing the spread of inoculum (Elliott et al. 2010; Plyler et al. 2000).

It is crucial to identify palm Fusarium wilt pathogens rapidly in order to implement management strategies to prevent the spread of disease. Traditional methods to identify palm 33

Fusarium wilt pathogens are based on symptomology, isolation of the pathogen from the infected palm tissue, and growing isolates on specific media in order to observe colony and spore morphology, both microconidia and macroconidia (Leslie and Summerell 2006). These methods need expertise and are laborious. Additionally, nonpathogenic Fusarium oxysporum and other

Fusarium species are occasionally isolated from symptomatic palm tissue, at times, from the same symptomatic palm tissue (Elliott et al. 2010). These traditional methods are limiting as these Fusarium species might share similar colony and spore morphology, leading to misidentification (Zhang et al. 2005).

PCR-based assays are being used to detect and identify pathogens, as they are fast, reliable and specific, allowing one to differentiate closely related species or even races within a species (Edel et al. 1995; Glass and Donaldson 1995; Li and Hartman 2003; Nayaka et al. 2011).

Designing a diagnostic tool to identify F. oxysporum formae speciales is challenging due to the presence of polyphyletic lineages and possibly races within some formae speciales (Plyler et al.

2000; Suga et al. 2013). There is a PCR-based assay for detecting FOC that uses the primer pair

HK66 and HK67, which is based on the beta-glucosidase gene (Plyler et al. 1999). However, frequent false positives are obtained because this primer pair can amplify Fusarium proliferatum, a common saprophyte associated with ornamental palm tissue (Elliot et al. 2010; Vitoreli 2008).

This problem was solved for detecting FOC by developing a duplex PCR based assay (Vitoreli et al. 2009)

Molecular markers that are routinely used in identifying Fusarium are the partial translation elongation factor 1–alpha (EF-1α), β-tubulin, and calmodulin genes (Baayen et al.

2000; O’Donnell et al. 1998; 2009; Wulff et al. 2010). EF-1α gene is an ideal choice to differentiate Fusarium because it is a single locus gene and contains adequate polymorphism to 34

distinguish Fusarium species and formae speciales of F. oxysporum (Geiser et al. 2004; Nayaka et al. 2011; Wulff et al. 2010).

The first objective of this study was to design a primer pair for a PCR-based assay for rapid, accurate identification of Fusarium wilt pathogens on ornamental palms. The initial goal was for identification of FOP only, but as the study progressed, it became clear that the primer pair could be used for FOC and possibly for FOA and FOE also. The second objective was to evaluate the newly designed primer pair using three different polymerases and associated PCR buffers and two types of template DNA with a collection of 62 pathogenic and nonpathogenic

Fusarium isolates that had been recovered from symptomatic palm tissue in the past ten years in

Florida. Additional template DNA was obtained of FOC, FOA and FOE and were evaluated accordingly.

Material and Methods

Isolates

Table 2-1 is a list of all isolates used in this study. Sixty-two isolates comprised of FOP (18),

FOC (12), F. proliferatum (5), Fusarium incarnatum-equiseti species complex (FIESC) (4), F. solani (3), F. sacchari (1), F. concentricum (3), and nonpathogenic F. oxysporum (16) had been obtained from palm tissues over a ten-year period, primarily from Florida, but also California,

South Carolina and Texas. Most, but not all of these isolates, were obtained from the petiole of palms with Fusarium wilt symptoms. Half of the nonpathogenic F. oxysporum isolates were obtained from roots, leaflets or trunk tissue. All isolates were single-spored and stored at room temperature as mycelial agar plugs in cryogenic vials containing sterile deionized water at the

University of Florida-IFAS, Fort Lauderdale Research and Education Center. Additionally, genomic DNA of 12 isolates was acquired from USDA Agricultural Research Service Culture 35

Collection (NRRL), Peoria, IL and California Department of Agriculture (Table 2-1). These included F. oxysporum ff. spp. albedinis (3), canariensis (4) and elaeidis (5).

Genomic DNA and EF-1α Sequencing

Each isolate from the Florida collection was revived from storage by placing a single agar plug on 1/5 strength potato dextrose agar (1/5 PDA) (Difco Laboratories, Detroit, MI) and grown for 5 days at 28°C. Five agar plugs from 1/5 PDA were transferred to a conical flask with 20 ml of 1/5 strength potato dextrose broth (1/5 PDB) and incubated for 5 days at 28°C without shaking. DNA was extracted from fungal mycelial tissue using Gentra Puregene Tissue Kit

(Qiagen, Valencia, CA) according to the manufacturer instructions. The resulting DNA pellet was suspended in 50 µl of molecular grade water (HyCloneTM, Fisher Scientific, Pittsburg, PA) and quantified using a QubitTM fluorometer (Thermo Fisher Scientific, Pittsburg, PA). The DNA was stored at 4°C for further use. All DNA, either from the Florida collection or provided by other agencies, was assessed for quality and identity by amplifying with ef1 and ef2 primers

(Geiser et al. 2004), which amplify a portion of the EF-1α gene, sequencing the amplicons and comparing sequences with those deposited in GenBank using BLASTn. Purification of PCR products was performed on spin columns using the Wizard® PCR Preps DNA Purification

System (Promega Corp. Madison, WI). Sanger sequencing in both directions was conducted at

University of Florida’s Interdisciplinary Center for Biotechnology (UF-ICBR), (Gainesville,

Florida) with the same primers.

Primer Design

A multiple sequence alignment of 12 EF-1α sequences of Fusarium oxysporum f. sp. palmarum from GenBank (GQ154454, GQ154455, GQ154458, GQ154459, GQ154460,

GQ154462, GQ154463, GQ154464, GQ154465, GQ154468, GQ154469, and GQ154457) was 36

conducted to generate a consensus sequence (Clustal W, Lasergene, DNASTAR, Madison, WI).

Primer-BLAST created five primer pairs based on this consensus sequence (Ye et al. 2012).

Primer Stat (Sequence Manipulation Suite, 2000) (Stothard 2000) analyzed each primer pair based on GC clamp, hairpin formation, melting temperature and self-annealing criteria. A primer set (PFW-F and PFW-R) satisfied all the parameters and was selected for further study. Primers were synthesized by Sigma-Aldrich (St. Louis, MO). The melting temperature for the PFW primer pair was determined by a gradient PCR.

Genomic DNA Amplification with PFW Primer Pair

Genomic DNA of all isolates listed in Table 2-1 was used for the PCR assay using the

PFW primer pair. PCR was performed using three different sources of polymerases: Clontech

(Takara Bio USA, Mountain View, CA), New England Biolabs (NEB) (Ipswich, MA) and

Sigma-Aldrich (Sigma) (St. Louis, MO). These three PCR kits were selected as they were commonly used in plant diagnostic settings. Each PCR was carried out in a 50 µl reaction mixture brought to volume with sterile deionized water. The NEB PCR assay was set up with a final concentration of 1X PCR standard Taq Reaction Buffer (10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2), 200 µM of each dNTP, 1 µM of each primer and 1U of NEB Taq DNA polymerase. The Clontech PCR final concentration consisted of 25 µl of 2X TerraTM PCR Direct

TM Buffer (400 µM of each dNTP; 2 mM MgCl2), 1 µM of each primer and 1.25U of Terra PCR

Direct Polymerase. The Sigma PCR final concentration consisted of 25 µl of 2X ReadyMixTM

Taq Reaction Mix (1.5U Taq polymerase, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2,

0.001% gelatin, and 200 µM of each dNTP) with 1 µM of each primer. Approximately, 10 ng of genomic DNA served as the template for each reaction. Each PCR assay included a negative control consisting of sterile deionized water.

PCR parameters for all the three polymerase sources were: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 90 sec, and extension at 72°C for 2 min, with a final extension step at 72°C for 10 min. An Eppendorf

Thermocycler (Eppendorf AG, Foster City, CA) was used for all PCR assays. PCR products were examined by 1.5% agarose gel electrophoresis in Tris borate EDTA (TBE) buffer with ethidium bromide staining. Purification of selected PCR products was performed on spin columns using Wizard® PCR Preps DNA Purification System (Promega Corp. Madison, WI).

Sanger sequencing in both directions was conducted at UF-ICBR (Gainesville, Florida) with the same primers.

DNA Amplification with Agar Plug and PFW Primer Pair

The PFW primer pair was tested in a PCR assay using a single colonized agar plug (1 mm in width and 3-5 mm in length) as DNA template instead of genomic DNA to eliminate the need to grow the fungus in liquid culture and extract DNA. A total of 26 isolates were grown on

1/5 PDA for 5 days at 28°C: F. concentricum (PLM57D, PLM62D), Fusarium oxysporum ff. spp canariensis (PLM696A, PLM706A, PLM724A, PLM754A) and palmarum (PLM153B,

PLM249A, PLM510A, PLM541D, PLM619A, PLM632A, PLM814A, PLM853A), F. proliferatum (PLM196B, PLM782A), F. sacchari (PLM 700D), F. solani (PLM280D,

PLM332C), F. incarnatum–equiseti species complex (PLM187D, PLM 261D) and Fusarium oxysporum (PLM64A, PLM182C, PLM 428A, PLM 818A, PLM851A). PCR assays were performed using Clontech, NEB and Sigma polymerases and PCR parameters as described above. Each PCR assay included a negative control consisting of sterile deionized water.

DNA Amplification with the FOA Primer Pair

A PCR-based screening was performed on selected representative isolates to test the specificity of F. oxysporum f. sp. albedinis primer pair TL3/FOA28. Genomic DNA of 21 isolates were used in this assay including three isolates of Fusarium oxysporum (PLM208A,

PLM818A, PLM820A), five isolates each of F. oxysporum ff. spp canariensis (PLM221B,

PLM387B, PLM511A, PLM588A, PLM908A), eleidis (NRRL22543, NRRL36358,

NRRL36359, NRRL38313, NRRL38314) palmarum (PLM153B, PLM249A, PLM344E,

PLM632A, PLM710A) and albedinis (NRRL26622, NRRL38288, NRRL38298). The latter were used as positive controls. PCR assays were set up with a total 50 µl volume with final concentration of 1X PCR standard Taq Reaction Buffer (10 mM Tris –HCl, 50 mM KCl, 1.5 mM MgCl2), 200 µM of each dNTP, 1 µM of each primer and 1U of NEB Taq DNA polymerase. Approximately 10 ng of genomic DNA served as the template for each reaction.

Each PCR assay included a negative control consisting of sterile deionized water. PCR was performed using cycling conditions as described by Fernandez et al. (1998) with initial denaturation of 4 mins at 95°C followed by 30 cycles of denaturation at 30 sec at 92°C, annealing at 62°C for 30 sec and extension for 45 sec at 72°C with a final extension step of 15 min at 72°C. All the PCR products were analyzed for the presence or absence of PCR product using 1.5% agarose gel electrophoresis in TBE buffer with GelRedTM staining (Biotium,

Hayward, CA).

Phylogenetic Analysis

Sequences derived for EF-1α from PCR assays using ef1 and ef2 primers (Geiser et al

2004) and sequences from the amplicons derived from the PFW primer pair were queried against known sequences in GenBank using BLASTn to confirm their identity. A phylogenetic analysis

with MEGA version 7 (Kumar et al. 2016) was conducted independently for the two data sets: 35 sequences obtained with EF-1α primers and 35 sequences obtained with PFW primers. A multiple sequence alignment was performed with MUSCLE using the default parameters (Edgar

2004). The phylogenetic tree was constructed by the Maximum Parsimony method using the heuristic search option Tree-Bisection-Reconnection (TBR) branch swapping algorithm with

1000 random addition sequences with 1000 bootstrap replications. The consistency index (CI) and the retention index (RI) were also calculated. All the gaps and missing data were eliminated from the analysis. Unrooted EF-1α and PFW based phylogenetic trees were constructed to infer evolutionary relationships between isolates (Kinene et al. 2016).

Results

Genomic DNA and EF-1α Sequencing

Using genomic DNA for all 74 isolates, a portion of the EF-1α gene was amplified for all

74 isolates using ef1 and ef2 primers. Resulting amplicons were sequenced. Sequences were queried against GenBank using BLASTn to confirm their identity (Table 2-1).

Primer Design

The newly evaluated primer pair was designated PFW-F (5`-

CCATCGTCAATCCCGACCA-3’) and PFW-R (5`-GGAGCGTCTGAGTGATATGTTAG-3’).

The resulting amplicon size is 569 bp. The melting temperature for the PFW primer is 65°C as determined by a gradient PCR.

Genomic DNA Amplification with PFW Primer Pair

Using genomic DNA, the PFW primer pair amplified all isolates of F. oxysporum ff. spp albedinis (3), canarienis (16), elaedis (5), and palmarum (18) across all three polymerases and

PCR buffer mixes evaluated (Table 2-1 and Figure 2-1). Of the 16 nonpathogenic Fusarium oxysporum isolates tested, Clontech TerraTM PCR Direct polymerase amplified all of them

(Table 2-1). However, only 12 and 8 isolates were amplified by the NEB and Sigma polymerases, respectively (Table 2-1). Genomic DNA of other Fusarium species in the study group were not amplified with the PFW primer pair with any of the polymerases tested (Table 2-

1 and Figure 2-1).

DNA Amplification with Agar Plug and PFW Primer Pair

Using a colonized agar plug, Clontech TerraTM Direct polymerase, but not the NEB or

Sigma polymerases, amplified all Fusarium oxysporum (PLM64A, PLM182C, PLM 428A,

PLM818A, PLM851A), f. sp. canariensis (PLM696A, PLM706A, PLM724A, PLM754A) and f. sp. palmarum (PLM153B, PLM249A, PLM510A, PLM541D, PLM619A, PLM632A,

PLM814A, PLM853A) (Table 2-1 and Figure 2-2). As with genomic DNA, no amplification was observed for F. proliferatum, F. solani, F. incarnatum–equiseti species complex, F. concentricum and F. sacchari with any of the evaluated polymerases (Table 2-1 and Figure 2-2).

DNA Amplification with FOA Primer Pair

The Fot1 transposable element based primer pair TL3/FOA28 amplified only the genomic DNA of F. oxysporum f. sp. albedinis (NRRL26622, NRRL38288, NRRL38298), resulting in a 400 bp PCR product.

Phylogenetic Analysis

An alignment of the 35 EF-1α data set resulted in 600 characters in which 21 were parsimony informative sites. An unrooted consensus tree generated by Maximum Parsimony analysis indicated there are four well-established clades (Figure 2-3). Clade 1 is divided into

FOP and FOA subgroups. All FOP sequences, irrespective of palm host and geographical location, clustered within the same clade, but two single nucleotide polymorphism within FOP are reflected in two isolates (PLM320B and PLM676A), which were isolated from different palm hosts. A single point mutation, distinct from other FOP sequences, was observed in PLM632A.

No variation was observed in the three FOA sequences, and they clustered in a single subgroup.

Variation was observed within nonpathogenic Fusarium oxysporum sequences. The majority of F. oxysporum sequences clustered in clade 2. F. oxysporum sequences PLM51A

(leaflets), PLM208A (petiole), PLM476C (petiole) and PLM851A (roots) had identical EF-1α sequences, even though they were recovered from different palm hosts and sampling tissues.

Clade 3 is represented by FOE and two nonpathogenic F. oxysporum isolates. The F. oxysporum isolates PLM568A (roots) and PLM271A (petiole) were more similar to FOE than other F. oxysporum. All FOC isolates clustered together in clade 4 with 100% sequence similarity, irrespective of the location or palm host. No polymorphism was observed among the FOC isolates suggesting a monophyletic lineage. Among the formae speciales that infect palm species,

FOC appeared more distinct with at least ten polymorphic sites compared with FOA, FOE and

FOP.

The phylogenetic tree based on the PFW primer pair sequenced amplicons resulted in 431 characters with 13 parsimony informative sites. The PFW phylogenetic tree yielded similar topology to the EF-1α phylogenetic tree, with the exception of clade 1 (Figure 2-4). In clade 1, subgrouping of FOA and FOP was not observed. The unique patterns of PLM320B, PLM676A and PLM632A observed in the EF-1α tree were also displayed in the PWF tree. The same clade groupings observed with EF- 1α were observed for FOE, FOC and nonpathogenic F. oxysporum isolates (Figure 2-4). 42

Discussion

Fusarium oxysporum consists of both nonpathogenic and pathogenic isolates with the latter classified into formae speciales based on the host plant pathogenicity. Each forma specialis is confined to a limited host range, normally comprised of only one or two host plant species.

FOP is an exception, as it has a wide host range, infecting palms belonging to Phoenix,

Washingtonia and Syagrus genera within the family Arecaceae. Diseases caused by both FOP and FOC are lethal and result in pathogen-infested soils that are unsuited for planting susceptible palm hosts. Canary Island date palm wilt caused by FOC is spread mainly by pruning tools, whereas windblown spores and pruning tools are thought to spread Fusarium wilt on queen and

Mexican fan palms caused by FOP (Elliott et al. 2010). With no fungicide treatments available, timely diagnosis can prevent the spread of the diseases by implementation of phytosanitary measures, primarily palm removal. Furthermore, both FOP and FOC occur in Florida, and their host ranges continue to expand, with P. canariensis and possibly P. reclinata being affected by both pathogens.

Currently, while there is a primer pair (HK66/HK67) used for detection of FOC (Plyler et al. 1999), it does not detect FOP (Elliott, unpublished results), and it also often yields false positives if the fungus isolate is F. proliferatum (Vitoreli 2008). To solve this latter problem, a duplex PCR-based assay is used that requires the FOC primer pair and a primer pair for F. proliferatum (Vitoreli et al. 2009). Another forma specialis specific primer pair TL3/FOA28, based on Fot1 transposon, detects only FOA (Fernandez et al. 1998; results reported herein).

There are no published FOE-specific primers, although they were reported to be in development

(Rusli 2012).

The present study objective was to design primers for a PCR-based assay, which would be fast, cost-effective and reliable for identifying Fusarium wilt pathogens of palm occurring in

Florida. Because the diseases caused by FOC and FOP are lethal, for initial management purposes (i.e. removal), it is not important to know which pathogen, FOC or FOP, is present. It was also desirable to have a PCR based assay that could use mycelial agar plugs as DNA template to eliminate the DNA extraction process. To evaluate the primers developed from multiple sequence alignment, a collection of Fusarium species isolated in Florida from symptomatic Fusarium wilt palms, but not necessarily from petiole or rachis tissue (leaflets, roots and trunk) was used, with the main intent to include comprehensively all the Fusarium species that are expected to be encountered in Florida plant diagnostic labs.

Genomic DNA of all the evaluated isolates of FOC and FOP were amplified using the three polymerases tested and the PFW primer pair developed. Along with FOC and FOP, nonpathogenic F. oxysporum were also amplified but not consistently with every polymerase. F. proliferatum, F. solani, F. concentricum, F. sacchari and F. incarnatum-equiseti species complex failed to amplify with any of the polymerases evaluated, indicating that the PFW primers are specific for F. oxysporum. This is not surprising because the primers are based on the

EF-1α gene. Nonpathogenic F. oxysporum isolates may be recovered from symptomatic palm tissues. However, for greater accuracy with the PCR based assay, the type of tissue sampled is important, and perhaps, the polymerase used for the PCR reaction. As both FOC and FOP primarily cause a canopy infection, the petiole is the ideal tissue to sample (Elliott et al. 2010).

Only 8 of 16 nonpathogenic F. oxysporum isolates from Florida were recovered from petiole tissue, whereas all but one of 25 FOC and FOP isolates from Florida were obtained from petiole tissue. Because the PFW primers amplify both FOP and FOC, if it is necessary to identify the

forma specialis, sequencing of the PCR amplicon derived from the PFW primer, and subsequent

BLAST analysis will distinguish between FOC and FOP (Figure 2-5).

The three PCR buffers and polymerase kits used in this study, Clontech, NewEngland

Biolabs and Sigma-Aldrich, were choosen as they are premade ready-to-use buffers. Moreover, at the time of this research, SigmaReady mix was routinely used for diagnositic purposes at the

Plant Diagnostic Clinic (University of Florida, Gainesville, Florida). Clontech was selected based on its ability to amplify DNA from crude samples, such as colonized agar plugs, fungal spores or other fungal tissue. New England Biolab PCR kit was included in the study as it was the normal PCR kit used in the Elliott laboratory, where this study was conducted. While the type of polymerase and/or associated buffer mix did not affect PCR results for FOC or FOP when using genomic DNA, it did affect PCR results of the 16 nonpathogenic F. oxysporum isolates. All were amplified by the Clontech polymerase, but not all were amplified by NEB and

Sigma polymerases. The disparity among polymerases may be due to more than the polymerase itself, as there is a difference in the buffer mixes. Magnesium (Mg2+) is a vital cofactor needed for DNA polymerase, and lower concentration leads to higher specificity (Lorenz 2012). The

Mg2+ concentration in the buffer mixes used with the NEB and Sigma polymerases (1.5 mM

MgCl2) was lower compared to the Clontech polymerase buffer mix (2.0 mM MgCl2) leading to more positive PCR results with the Clontech polymerase. However, the Clontech polymerase has properties that can be exploited if one wants to use mycelial agar plugs as the DNA template for the PCR assay. The Clontech TerraTM PCR Direct Polymerase, according to the manufacturer,

“is a novel enzyme developed for direct amplification from tissue samples, crude extracts, and dirty templates.” This probably explains why DNA of F. oxysporum, FOP and FOC could be

amplified using agar plugs with the Clontech polymerase but not with the NEB and Sigma polymerases.

PFW primers also amplified FOA and FOE, the formae speciales that cause Fusarium wilts on date and oil palms, respectively. However, these two pathogens are not present in the

USA, and thus far have limited geographic ranges. Nonetheless, the PFW primers could be useful for detection of FOA and FOE in those countries where those diseases occur. While sequencing the PCR amplicon using the PFW primer will identify FOE, the sequences of FOP and FOA are the same (Figure 2-4). This is not surprising considering the previously shown close relationship (Elliott et al. 2010) and results in this study (Figure 2-3).

This study points out that there is a need for additional markers that can separate F. oxysporum formae speciales, especially those associated with palm species. Host specific or pathogenicity genes, like the family of secreted in xylem (SIX) genes, are currently being explored to help solve this problem (Suga et al. 2013). In Florida, confirming the presence of

Fusarium wilt pathogens is necessary for monitoring the continued expansion of the host range of FOC and FOP and to aid in decisions regarding palm removal and nursery quarantines. As both FOP and FOC continue to expand their geographic range, the PFW primer pair may be useful for other states where palms are part of the landscape or grown in nurseries.

While the PFW primers are not specific for FOC and FOP, they are specific for F. oxysporum, and this eliminates false positives if other Fusarium species are isolated. False positives can also be reduced by using only symptomatic petiole tissue for isolation purposes. If necessary, sequencing the amplicons resulting from the PCR-based assay using the PFW primers will separate FOP from FOC. Previously, a second PCR assay using the EF-1α primers (ef1 and

ef2) would have had to be completed for their identification. Thus, best diagnostic practices for identifying palm Fusarium wilt pathogens in Florida include: 1) only isolate from Fusarium wilt symptomatic petiole tissue; 2) use the PFW primer pair in a PCR assay; and 3) if necessary, to distinguish between FOP and FOC, sequence amplicons derived from the PCR assay (Figure 2-

5).

Table 2-1. Fusarium isolates used in the study, indicating isolate, palm host, geographic location and tissue sampled. Genomic DNA or a fungal colonized agar plug was used as template for PCR using PFW primers and three different polymerases. Isolatea Palm host Geographic Sampled Genomic DNA Agar plug amplification locationb tissue amplificationc

Clontech NEB Sigma Clontech NEB Sigma Fusarium oxysporum f. sp. albedinis (FOA) NRRL 26622 Phoenix sp. Morocco Unknown + + + NTd NT NT NRRL 38288 Phoenix dactylifera - Unknown + + + NT NT NT NRRL 38298 Phoenix dactylifera Algeria Unknown + + + NT NT NT F. oxysporum f. sp. canariensis (FOC) PLM221B Phoenix canariensis Florida Petiole + + + NT NT NT PLM224A Phoenix sylvestris Florida Petiole + + + NT NT NT PLM385B Phoenix canariensis Texas Petiole + + + NT NT NT PLM387B Phoenix reclinata Florida Petiole + + + NT NT NT PLM511A Phoenix canariensis South Petiole + + + NT NT NT Carolina PLM588A Phoenix canariensis Florida Petiole + + + NT NT NT PLM696A Phoenix reclinata Florida Petiole + + + + - - PLM706A Phoenix canariensis California Petiole + + - + - - PLM724A Phoenix canariensis Florida Petiole + + + + - - PLM754A Phoenix canariensis Florida Petiole + + + + - -