University of Florida Thesis Or Dissertation

NEMATODES AFFECTING BANANA AND PLANTAIN AND MIRACLE FRUIT PRODUCTION IN SOUTH FLORIDA

LYNHE DEMESYEUX

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2019

To my loving mother for her endless love, sacrifices and for teaching me many valuable lessons that have guided me throughout my life

ACKNOWLEDGMENTS

I would like to thank the chair of my committee, Dr. Alan H. Chambers for being so patient and understanding towards me and helping me to achieve this goal.

I also thank my committee members Dr. William T. Crow, Dr. Jonathan Crane and Dr. Randy Ploetz for their guidance and wisdom all along my time as a graduate student at UF.

My friends Frantz Marc Penson Deroy, Cassandre Feuillé and Carina Theodore for their supports and the laughter shared.

I also acknowledge Maria de Lourde Mendes PhD, Maria Brym and Sarah

Brewer for their help in data collection, proof reading and support.

Finally, I thank the AREA/Feed the future project for funding my research and Dr.

Rosalie Koenig for her diligent administrative guidance.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 10

ABSTRACT ...... 11

CHAPTER

1 LITERATURE REVIEW ...... 13

Introduction to Banana and Plantain ...... 13 Introduction to Nematodes ...... 14 Pathogenicity, Biology and Life Cycle of Plant Parasititc Nematodes on Banana and Plantain ...... 16 Radopholus similis ...... 16 Generalities ...... 16 Symptoms and economic importance ...... 17 Biology and life cycle ...... 17 Helicotylenchus spp...... 18 Generalities ...... 18 Symptoms ...... 18 Biology and life cycle ...... 19 Generalities ...... 19 Symptoms and economic importance ...... 20 Biology and life cycle ...... 20 Meloidogyne spp...... 20 Generalities ...... 20 Symptoms and economic importance ...... 21 Biology and life cycle ...... 21 Rotylenchulus reniformis ...... 21 Control Methods ...... 21 Introduction to Miracle Fruit ...... 22 Introduction ...... 22 Botany ...... 23 Utilization ...... 24 Miraculin Extraction ...... 24 Miraculin Purification ...... 25 Potential Uses of Miraculin ...... 26 Taste-Altering Mechanism ...... 27 Current Medical Applications ...... 27

2 PLANT-PARASITIC NEMATODES ASSOCIATED WITH EIGHT BANANA CULTIVARS IN SOUTHERN FLORIDA...... 28

Summary ...... 28 Introduction ...... 29 Material and Methods ...... 31 Site Selection ...... 31 Musa Cultivars ...... 32 Root Sampling ...... 32 Nematode Extraction ...... 32 Nematode Identification and Quantification ...... 33 Preliminary Screening for Nematode Resistance ...... 33 Results ...... 34 Plant Parasitic Nematodes Nematodes from Soil Samples ...... 34 Plant Parasitic Nematodes Associated with Musa Roots ...... 35 Nematodes Associated with Musa Cultivars in Histosol and Limestone Soils .. 36 Preliminary Screening of Nematode Reproduction on Diverse Banana Accessions ...... 37 Discussion ...... 44 Recognition ...... 47

3 YIELD PERFORMANCE AND MIRACULIN CONTENT OF A NOVEL FRUIT (SYNSEPALUM DULCIFICUM) IN HOMESTEAD, FLORIDA ...... 48

Summary ...... 48 Introduction ...... 48 Material and Methods ...... 50 Plant Material ...... 50 Yield Evaluation ...... 51 Miraculin Crude Extract ...... 51 Miraculin IMAC Purification and Protein Sequencing ...... 52 SDS-PAGE (Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis) ..... 52 Miraculin Quantification ...... 53 Statistical Analysis ...... 53 Results ...... 53 Miracle Fruit Total Yield by Accession ...... 53 Miracle Fruit Seasonal Yield ...... 54 Miracle Fruit Total Yield per Tree ...... 54 Miracle Fruit Average Fruit Weight ...... 55 Protein Sequencing ...... 55 Protein Extraction ...... 55 Discussion ...... 59 Seasonal Yield ...... 59 Yield per Tree ...... 59 Average Fruit Weight ...... 60 Tree Clustering ...... 60 Protein Quantification ...... 60

Conclusion ...... 60

LIST OF REFERENCES ...... 61

BIOGRAPHICAL SKETCH ...... 70

LIST OF TABLES

Table page 2-1 Banana farm management; soil type description; color of the soil, pH range and age of the plantations for each surveyed location...... 37

2-2 A list of the eight banana and plantain cultivars used in this study...... 38

2-3 Plant parasitic nematode counts per 100 g of roots from different banana accessions from two different farms in Southern Florida with differing soil types...... 38

2-4 A list of the 26 accessions used in the screening experiment...... 39

3-1 Average yield for each accession in kg/tree/year for fruit fresh weight. Averages connected by the same letter are not significantly different using Tukey’s HSD (alpha=<0.05)...... 56

3-2 Average miracle fruit yield among the six harvest windows. Data represents the sum and average yield of all trees by harvest window...... 56

LIST OF FIGURES

Figure page

1-1 Purification procedure of protein with Ni-NTA. (Image credit: ThermoFisher Scientific website)...... 26

2-1 Percentage of the total number of plant parasitic nematodes by genus in soil samples from histosol...... 40

2-2 Percentage of the total number of plant parasitic nematodes by genus in soil samples from limestone...... 40

2-3 Percentage of the total number of plant parasitic nematodes by genus in soil samples from an additional independent limestone site collection...... 40

2-4 Representative microscope images of the most common nematode species isolated from banana roots in southern Florida...... 41

2-5 Absolute frequency (presence/absence) of nematode genus in roots and soil samples in the histosol site and limestone site...... 41

2-6 Percentage of the total number of plant parasitic nematode genus extracted from 100 g of banana roots from the histosol site in August...... 42

2-7 Percentage of the total number of plant parasitic nematode genus extracted from 100 g of banana roots from the histosol site in May...... 42

2-8 Percentage of the total number of plant parasitic nematode genus extracted from 100 g banana roots from the limestone site in August...... 42

2-9 Percentage of the total number of plant parasitic nematode genus extracted from 100 g of banana roots...... 43

2-10 Nematode counts from an inoculation study from diverse Musa accessions...... 43

3-1 Average fruit yield for all 66 accessions in grams per tree over 52 weeks. Error bars represent standard error for all accession yield data...... 56

3-2 Total yield per tree over 52 weeks color coded by morphotype...... 57

3-3 Total yield per tree over 52 weeks color coded by clusters...... 57

3-4 Miracle fruit crude extract. Figure shows the total protein content of the sodium chloride solution. Y axis is for the absorbance while the X axis is the retention time...... 58

3-5 Single peak of miraculin after purification with IMAC. Y axis is for the absorbance while the X axis is the retention time...... 58

LIST OF ABBREVIATIONS

HPLC High Performance Liquid Chromatography

IMAC Immobilized-metal Affinity Chromatography

Ni-NITA Nickel Nitriloacetic Acid

SDS-PAGE Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

NEMATODES AFFECTING BANANA AND PLANTAIN AND MIRACLE FRUIT PRODUCTION IN SOUTH FLORIDA

Lynhe Demesyeux

August 2019

Chair: Alan H. Chambers Major: Horticultural Sciences

Banana and plantain are large perennial herbs from the Musa genus which bear a delicious fruit that can be eaten fresh or cooked, depending on the plant type. It plays an important role in human nutrition, especifically in developing countries as it provides food and cash income for millions of farmers. However, its production is threatened by plant parasitic nematodes that feed on their roots and corms. As the proper management of these pests depends on the type of nematode present in the field, a nematode survey was conducted on eight cultivars in two contrasting soil types in South

Florida. Results revealded Helicotylenchus and Meloidogyne as the most prevalent and abundant phytonematodes genera for these locations. Of the eight surveyed cultivars, the lowest number of phytonematodes genera was consistently found in ‘Wiliams’,

‘Pisang Rajah’ and ‘Giant plantain’ in both locations.

Moreover, new information on the production pattern of a high value crop, miracle fruit (Synsepalum dulcificum), was generated by taking yield data from nine different morphological types for 52 weeks. The highest yielding type was ‘Typical’ (~2.8 kg/tree/year) while ‘Holly’ was the lowest yielding type (~0.5 kg/tree/year). Miracle fruit is primarily valued for the protein miraculin, a natural, non-caloric sweeter that

accumulates in the tissue of the berries. This protein stimulates sweet taste receptors in response to acids. Miraculin was successfully extracted from fruit tissue and purified using affinity chromatography.

CHAPTER 1 LITERATURE REVIEW

Introduction to Banana and Plantain

Banana and plantain are are ranked among the most important crops in the world. Their combined annual production is above 150 million tonnes of fruit produced on approximately 10 million hectares worldwide (FAOSTAT, 2017). Banana and plantain originated in Southeast Asia, but they are currently produced and consumed in nearly 140 countries in the tropics and subtropics. As the major exported and consumed fruit globally, banana plays an important role in human nutrition and in the economy since it generates income and employment for millions of households (Tripathi et al.,

2015). Recent statistics about banana export in 2016 indicate that this industry generates approximately US $9 billion annually although 85% of world production is consumed in the producer’s household or sold in local markets (FAOSTAT, 2017).

This perennial crop stands as one of the most important syapple food after rice, maize, wheat, cassava and potato (Tripathi et al., 2015). In East Africa, for example, bananas are one of the most important staple food crops, particularly in Uganda, where the annual per capita consumption the highest in the world between 220-460 kg/person.

About 70% of this production is consumed in the household while 20% is sold in local, urban or international markets. The rest is processed into alcoholic beverages and secondary foods. Similarly, in Tanzania, 60% of the production is consumed by the producer’s family. Green banana comprises 30% of the total consumed carbohydrates and it is the principal staple food for 75% to 95% of the Tanzanian population (Kilimo

Trust, 2012; Perrier et al., 2011).

Banana is also highly consumed in the Western hemisphere including the

Caribbean region. In Haiti for example, the annual consumption of plantain alone is around 22 kg per year per person. This amount is three times higher if the person lives in a rural banana production region (Cé spedes, 2012). Plantain consumption provides a significant amount of carbohydrates, fiber, potassium, and vitamin B6 and is low in sodium (Seenivasan, 2017).

While banana is not an important crop in the USA, the per capita consumption of banana in this country is around 11 kg and it is consumed mainly as a dessert fruit instead of a major source of dietary calories. Banana is cultivated only on 1,600 acres distributed between Florida and Hawaii on 500 acres and 1100 acres, respectively, which represent 0.01% of the world production annually. The United States is the largest single country of fresh banana (Evans et al., 2015).

Introduction to Nematodes

Nematodes (phylum Nematoda) are roundworms, with an unsegmented body unlike non-parasitic earthworms. They are the most abundant multicellular organisms on earth and feed on wide range of organisms such as bacteria, fungi, animals and plants (Poinar et al., 1994; Manum et al., 1994). The length of the plant feeders varies from 250 µm to 12 mm, with an average of 1 mm, and their width is between 15 to 35

µm (Lambert and Bekal, 2002).

The plant parasitic nematodes are generally less than 1,000 cells, transparent and have a long and slender tubular body, except for the females of some sedentary species that become swollen and rounded in their adult stage (Lambert and Bekal,

2002). Plant parasitic nematodes are a huge problem in agriculture. They reduce the

global food prodcutiom up tp 12% annually with an economic impact of US $157 billion

(Singh et al., 2015).

Plant parasitic nematodes feed on all parts of plants by using a special feature of their mouth part called a stylet, which is used for penetration, suction of plant juice and for secretion. The stylet varies in size and shape depending of the feeding mode of the species. Some nematodes kill the cells of the plants by withdrawing all their content, resulting in large lesions in the plant tissue, whereas others modify the cell of the plant by creating a large and long-term feeding site. This can be accomplished by the fusion of adjacent plant cells by breaking down their cell walls, or through the continuous nuclear division in the absence of cell division of a single cell (Lambert and Bekal,

2002).

Virulent plant parasitic nematodes feeding on results in a reduction of root mass and an alteration of the root structure. This morphological modification reduces the ability of roots to absorb water and nutrients from the soil causing yellowing of the leaves, fewer and smaller leaves, nutrient deficiency, wilting, stunting, yield reduction, and in the most drastic stage, the death of the plant (Lambert and Bekal, 2002).

In contrast, foliar nematodes damage leaves by feeding directly on the foliage and destroying their aesthetic value. For many ornamental plants, the damage is often more esthetic than lethal. However, in severe infections the pathogen can cause severe injury, inducing defoliation and inhibiting or limiting flowering. The symptoms caused by foliar nematodes differ depending on the stage of plant development. They can cause the young leaves to curl, twist and stunt, but the leaves remain on the plant. Yet, in more mature plants, they can induce blotches that are small, angular, and vein limited.

In advanced stages, the blotches turn brown and dry and may eventually fall leaving a hole on the leaf (Meyers and Hudelson, 2014; Southey, 1993).

Plant parasitic nematodes are a huge problem in agriculture. They reduce the global food production up to 12% annually with an economic impact of US $157 billion

(Singh et al., 2015).

Pathogenicity, Biology and Life Cycle of Plant Parasititc Nematodes on Banana and Plantain

Several nematodes are important pathogens of Musa spp. These include

Radopholus similis, some Pratylenchus spp., Helicotylenchus multicintus, Meloidogyne incognita, Hoplolaimus pararobustus, Heterodera oryzae and Heterodera oryzicola

(Commonwealth Agricultural Bureaux International (CABI), 2017). The first three in the above list are the most important and widespread (Gowen et al., 2005). These have been documented as banana damaging nematodes in West African banana producing countries, South and Central America, Florida and in other Caribbean countries including the Dominican Republic (CABI, 2017). Some destroy the primary roots of host plants reducing their anchorage, which leads to toppling (Gowen et al., 2005).

Radopholus similis

Generalities

R. similis, also known as the burrowing nematode, is the most damaging nematodes pest of bananas in the tropics. Discovered in banana in Fiji by Cobb at the end of the 19th century, the parasite is now widespread in most tropical regions that grow bananas. It is suggested that its worldwide occurrence was facilitated by the international trade of infected propagative plant materials between banana-producing countries (Gowen et al., 2005; O’Bannon, 1977).

Symptoms and economic importance

The persistent damage caused by R. similis can cause up to 75% of yield loss in banana (Hölscher et al., 2013). Fortunately, this nematode rarely affects commercial production areas in Florida (Mc Sorley, 1986). Where it occurs, it causes “black head toppling disease” (Gowen et al., 2005). The nematode affects the root cortex but can also feed on the rhizome or corm and produce lesions similar to those it causes in the roots. One diagnostic feature for the presence of the burrowing nematode in a plant is a reddish lesion in the larger roots that can be revealed by a longitudinal section of the root (McSorley, 1986). Furthermore, the injuries caused by this nematode usually provide an entry that favors secondary infections by fungi and/or bacteria, inducing the development of other diseases (O’Bannon, 1977).

Yield reduction caused by R. similis is due to the inability of affected plants to absorb water and nutrients which results in a reduction of the bunch weight and flower production, as well as the stunting and the toppling of the plants which root systems are destroyed by the parasite (Speijer, 1999).

Biology and life cycle

As a migratory endoparasitic nematode, all the infective stages of R. similis can complete their life cycle within the root tissue. From the second-stage juvenile to the adult stage (except the adult males that are not infective due to a degenerate stylet), the nematodes use their stylet to penetrate the root system. The penetration usually occurs at the tip of the root although it can occur anywhere along the root length (Kaplan and

Opperman, 2000; Loos, 1962). The invasion is made via the epidermis of the roots from where they migrate to occupy an intracellular position in the cortex. By extracting the cytoplasmic content of the nearby cells, the cell walls collapse to form lesions and

cavities that will be eventually occupied by fungi. R. similis has never been reported to feed on the stele, but the secondary infection of the stele by other parasites hasten the root damage process and leads to their breakage. Short root stubs at the base of the corm are often observed (Loos, 1962).

The life cycle (from egg to egg) of the parasite is completed within 20 to 25 days at 24-32°C and females lay 4-5 eggs daily for 2 weeks which take 8-10 days to hatch.

(Loos, 1962).

Helicotylenchus spp.

Generalities

Helicotylenchus spp. and R. similis frequently occur together in banana plantation even under optimal conditions and are of greatest concern (Wang and Hooks,

2009). H. multicintus, also called spiral nematode, may be considered as the most damaging nematode of bananas after the burrowing nematode in subtropical regions where the conditions are suboptimal for banana production and for R. similis

(Quénéhervé, 2009; McSorley and Parrado, 1986).

Symptoms

Spiral nematodes feed on the outer cells of the roots and cause small and shallow necrotic lesions that take longer time to develop than those caused by lesion nematodes. Nevertheless, in heavy infestation, the small lesions merge to form extensive necrotic lesions in the outer cortex of the roots (Quénéhervé and Cadet,

1985). Other typical plant parasitic nematode symptoms such as stunted plants, lengthening of vegetative phase, reduction of plant and bunch size as well as toppling can be observed under high infestation of H. multicintus (Gowen et al., 2005).

Biology and life cycle

Helicotylenchus species are often classified as endoparasitic nematodes as the eggs, both sexes, and all juvenile stages can be found in the cortex of the plant roots

(Zuckerman and Strich-Harari, 1964). A few days after entry Helicotylenchus can be found in the cortex usually to a depth of 4 to 6 cells. They feed on the surrounding cytoplasm cells close to the epidermis (Blake, 1966), and cause different kind of cellular damage such as rupture of cell wall, nucleus enlargement, contracted cytoplasm and other impairments. Females lay eggs in roots, which hatch within 5 to 6 days. Under suitable conditions in the field, the life cycle is reported to be completed in about 32 days. Helicotylenchus multicintus is bisexual and reproduces by cross-fertilization or amphimixis (Southey, 1973) but many other Helicotylenchus spp. reproduce by parthenogenesis.

Pratylenchus spp.

Generalities

Pratylenchus spp. are another migratory endoparasitic nematode that affect banana is. Only 2 of the 8 reported Pratylenchus species attacking Musa spp. worldwide are documented as the most common and damaging pathogens for this crop

(P. coffeae and P. goodeyi). P. coffeae was observed for the first time in plantains roots in Grenada by Cobb in 1919 and was described by the author as Tylenchus musicola

(Quénéhervé, 2009; Gowen et al., 2005). This species is considered as a pan-tropical species and an important pest of tuber crops, ornamentals coffee, banana and other fruit trees (Gowen et al., 2005).

Symptoms and economic importance

Plants infested with Pratylenchus present the same symptoms as those attacked by R. similis (plant stunting, extended vegetative phase, reduced size of the bunch and number of leaves and toppling). Epidermal and cortical tissue present extensive black or purple necrotic areas that are often subjected to secondary infections (Bridge and Page,

1984). P. goodeyi can eventually penetrate and destroy the cortical parenchyma of the roots and the corm which impair root functions (De Guiran and Vilardebó, 1962).

Biology and life cycle

As migratory endoparasites, P. coffeae and P. goodeyi complete their life cycle within the cortex of the roots and corm of banana. Adults and juvenile stages of both sexes are invasive (Gowen et al., 2005).

Once in the roots, the migration is done between and within cells to reach and occupy a position parallel to the stele from where they feed on the cytoplasm of surrounding cells. Their feeding habit creates cavities that collapse as described for R. similis. The cortical parenchyma is also destroyed but no cell enlargement is observed as described for R. similis (Pinochet, 1978; Blake, 1961).

The life cycle form egg to egg for this pathogen is typically completed within 27 days on average at 25-30C (Gotoh, 1964).

Meloidogyne spp.

Generalities

Many Meloidogyne species are found associated with banana and plantains.

Among them, the most common are M. incognita, M. arenaria, M. javanica and M. hapla. More than one species can be found in a single gall (Pinochet, 1977). They are widely distributed throughout the tropics and affect bananas as well as a wide range of

crops (Gowen et al., 2005). As for Helicotylenchus species, they are found in greater numbers in the absence of R. similis due to competition (Quénéhervé, 1990). This species was the most widely found in American Samoa (Brooks, 2004) and was the only species feeding on banana in Taiwan in the 1980’s (Lin and Tsay, 1985).

Symptoms and economic importance

The typical galling on primary and secondary roots is the major symptom caused by Meloidogyne (Gowen et al., 2005). Stunted growth can be observed too as well as root rot when associated with Fusarium solani or Rhizoctonia spp. (Sudha and Prabhoo,

1983; Lin and Tsay, 1985).

Biology and life cycle

Life cycle of Meloidogyne spp. from egg to egg is usually completed between 25 to 30 days at 21-28C. The infective second juvenile stage enters the root, starts feeding and become stationary. Females lay 200-400 eggs in an egg sac inside of the roots (Narasimhamurthy et al., 2018; Osunlola and Fawole, 2014).

Rotylenchulus reniformis

Also called reniform nematode, Rotylenchulus spp. are another genus of plant parasitic nematode that have been reported in multiple banana producing areas. Its life cycle and histopathology on banana is similar to those on other hosts (Sivakumar and

Seshadri, 1972). They are mostly extracted from soil samples and from secondary roots as their permanent feeding location mostly occur on these roots (Ayala, 1962;

Edmunds, 1968).

Control Methods

There are several strategies to control nematodes in banana plantations. These can be classified into three main types of nematode management. First, methods such

as crop rotation, soil fumigation, solarization and, use of clean planting material are used as prevention method. Second, mulching and regular fertilization application are used as cultural controls. Third, plant-based genetic resistance.

While the utilization of resistant plant material to plant parasitic nematodes appear to be the most economic and environmentally friendly method, farmers choose their cultivars primarily based on yield, market value and resistance to other major key abiotic and biotic threats rather than their resistance to phytonematodes. Thus, an integrated pest management strategy for nematode is presented as the most suitable method to control this pest as previously suggested by Quénéhervé (2009) and Wang and Hooks (2009).

Based on the importance of this crop for human consumption and the severity of infection in certain banana production areas, several studies have been conducted in order to find cultivars that are resistant to nematodes affecting the yield of the crop.

The objectives in this project are to identify the most important plant-parasitic nematodes to banana plants under South Florida conditions and to identify cultivars of bananas and plantains that are resistant to them.

Introduction to Miracle Fruit

Introduction

Synsepalum dulcificum commony called miracle fruit or miracle berry, is a perennial, evergreen shrub originating from the hot, humid lowlands of West Africa. It grows best in partial shade and in well drained soils with a low pH. Although it can reach up to 6 m tall in its native habitat, it is usually no more than 1.5 m in pots (Oumorou et al., 2010; CRFG, 1996; Bartoshuk et al., 1974; Brouwer et al., 1968).

Botany

The plant belongs to the Sapotaceae which is composed of around 40 genera.

The genus Synsepalum is restricted to the tropical regions of Africa and comprises 30 or so species. The Synsepalum dulcificum species is one of the eight most useful species for West African communities and the most widely distributed species within this genus (Akoègninou et al., 2006; Burkill, 2000).

The miracle fruit bush has relatively small leaves pointed at the apex. The leaves are entire, symmetrical and obovate–lanceolate to broadly lanceolate. They measure in general 4-7.5 cm long over 3-4 cm wide for a length/width ratio of 2:1. A small petiole attached them to the branch around which they are distributed in small clusters.

The flowers are white, small and can be in solitary or in small clusters. Miracle fruit flowers are composed by 4 to 5 sepals and 4 to 5 petals. The reproductive parts are composed of 5 stamens and a single ovary with a simple style and an inconspicuous stigma. They are hermaphroditic and autogamous (Ayensu, 1972; Lim, 2013). Although cross pollination is possible, a more in-depth understandings of the pollination mechanisms is still needed.

Multiple times a year, the plant flowers and produces a small ellipsoid berry of approximately 2 cm long and 1 cm wide that turns from green to red at maturity. Some types reportedely bear yellow fruits (Ayensu, 1972; Lim, 2013). The fruit contains a seed that is surrounded by a thin layer of white pulp that has a mild cherry-like taste (Inglett and Chen, 2011). This pulp contains a non-caloric, natural sweetener glycoprotein, called miraculin and this is the primary purpose for miracle fruit cultivation.

Utilization

In its center of origin, the plant has been exploited by the indigenous people for centuries in their diet as well as for its medicinal properties (Burkill, 2000; Most et al.,

1979). The leaves, bark and roots are used in the treatment of diabetes, enuresis, kidney diseases and other afflictions. The fruit is a rich source of vitamin C, leucine, flavonols and anthocyanin (Du et al., 2014). The fruits also render sour foods and drinks more palatable changing a sour taste into sweet taste (Burkill, 2000; Most et al., 1979).

The sensory impact is more similar to sugar than other natural, non-caloric sweeteners.

This change in taste is triggered by a glycoprotein called miraculin, which is not sweet itself (Brouwer et al., 1968). The production of miraculin in the fruit starts at the color break, around 6 weeks after pollination (Sun et al., 2007). The native state of miraculin is a dimer (40-48 kD) while its denatured state a monomer. The taste- modifying activity of the protein occurs at an acidic pH, with a maximum activity at pH

3.0 and lowest at neutral pH. The monomeric form of miraculin itself has no activity at all pHs, and the dimerization of the monomeric form is required in order to trigger the taste altering property of the miraculin (Ito et al., 2007).

The sweetening impact of miraculin can last more than 1 hour after being held in the mouth for approximately 3 minutes. It can trigger sweetness from a large range of acids such as HCl, lactic acid, formic acid, oxalic acid, acetic acid and citric acid

(Kurihara and Beidler, 1969).

Miraculin Extraction

The first attempts to isolate isolate miraculin from the berries was made by Inglett et al., (1965). They discovered that it was neither soluble in water nor in organic solvents. In 1968, it was extracted simultaneously by two separate groups of

investigators using different extraction methods. Kurihara and Beiddler used ion- exchange chromatography to extract miraculin from berries and described it as a glycoprotein. Simultaneously, Brouwer et al. (1968) extracted the active constituent by gel filtration on a Sephadex column and was named it “miraculin.” Other investigators have also attempted to extract this protein with modified extraction parameters (Giroux and Henkin, 1974; Kurihara and Terasaki 1982).

None of these extraction procedures resulted in pure miraculin and the solvents that were used were suspected to reduce the sweet-inducing activities. Theerasilp and

Kurihara (1988) determined that miraculin was composed of a polypeptide chain of 191 amino acids with two sugar molecules linked to two Asn residues (Asn-42 and Asn-

186).

In 2015, an optimized method to extract and purify miraculin using affinity chromatography was developed by He et al. (2015).

Miraculin Purification

Theerasilp and Kurihara (1988) affirmed that miraculin can be purified by ammonium sulfate fractionation followed by two chromatographic steps. An additional ultrafiltration after the ammonium sulfate fractionation and after each of chromatographic steps is required for this method. Although relatively high purity of miraculin was obtained, this method was tedious and costly (He et al., 2015).

The taste modifying property of the miraculin is generated by two histidine residues (His-30 and His-60) located at the interface of each of the two monomer subunits of the miraculin (Ito et al., 2007; Paladino et al., 2008). These four histidine residues can complex with immobilized-metal affinity chromatography (IMAC), thus the possibility of exploiting them for miraculin purification. Duhita et al. (2009) purified

miraculin using an IMAC column. He et al. (2015) purified miraculin to more than 97% by using a crude extract at pH 7, Tris–HCl as binding buffer (pH 7) and 300 mM imidazole as elution buffer. IMAC was loaded with nickel-NTA and was successfully used as a single step process for the purification of miraculin from the crude extract of miracle fruit. Figure 1-1. Explains the purification procedure with Ni-NTA

Figure 1-1. Purification procedure of protein with Ni-NTA. (Image credit: ThermoFisher Scientific website).

Potential Uses of Miraculin

Over the past four decades, the consumption of sugar has increased considerably among children and adults in the US. This is correlated with a growing number of people affected by chronic diseases such as obesity, diabetes type II, cardiovascular disorders and fatty liver disease (Ogden et al., 2015). This has led to the popular adoption of alternative sweeteners, many of them artificial. Unfortunately, some of these alternative sweetener compounds have negative sensory impacts or are now suspected to have negative impacts on human health (Suez et al., 2014). Thus, the

demand for natural, non-caloric sweeteners such as miraculin is predicted to increase over the coming years.

Taste-Altering Mechanism

The mechanism of the taste altering property of the miraculin has been studied by several investigators. According to Kurihara and Beidler (1969) sweetness is induced when the miraculin molecule binds to the taste receptors and activates the sweet receptors at low pH. Koizumi et al. (2011) confirmed that miraculin activates hT1R2- hT1R3 which is the human sweet taste receptor at low pH, suggesting the same mechanism previously described by the authors mentioned above.

Current Medical Applications

This crop has also been studied in the medical application. For example, its utilization has shown positive results in helping to treat diabetic patients with insulin resistance (Chen et al., 2006). Moreover, testimonies of both medical doctors and cancer patients suggest that the ingestion of miraculin improves chemotherapy- associated taste changes, thus, improving nutrition and quality of life of these patients. It has been approved by Baptist Health South Florida Hospital as the first and only dietary supplement to be distributed in their hospital network. (Wilken and Satiroff; 2012;

Miracle Fruit Farm, 2019).

CHAPTER 2 PLANT-PARASITIC NEMATODES ASSOCIATED WITH EIGHT BANANA CULTIVARS IN SOUTHERN FLORIDA

Summary

Niche and specialty banana cultivation provides growers in southern Florida with economic opportunities that do not directly compete with imports. These specialty bananas include dessert, cooking, and plantain types. Specific cultivars often lack foundational agronomic information that could guide the adoption of higher quality types. One major concern for banana growers in southern Florida is damage caused by plant parasitic nematodes. Plant parasitic nematodes limit the long-term economic return of some cultivars by reducing plant health until plants become unproductive.

While some cultural and chemical methods exist for reducing pathogenic nematode populations, these can be expensive, ineffective, or cause environmental concerns.

Understanding the prevalence and identity of nematodes in southern Florida and their impact on specific banana cultivars can help establish grower recommendations when establishing new plantings. Seven phytonematode genera (Helicotylenchus,

Meloidogyne, Pratylenchus, Rotylenchulus, Trichodorus, Tylenchorhynchus, and

Xiphinema) were identified in histosol and limestone soil types in long-term banana plantings in southern Florida. Of these, only Helicotylenchus (0-4,704 100 g-1 roots),

Meloidogyne (0-365 100 g-1 roots), and Pratylenchus (0-604 100 g-1 roots) were consistently abundant over two sampling dates and for both soil types. Plant parasitic nematodes were isolated from the roots of all eight banana cultivars with ‘Giant

Plantain’ AAB, ‘Pisang Raja’ AAB, and ‘Williams’ AAA showing a trend towards fewer total isolated nematodes. This is the first report of nematodes associated with a diverse banana accessions in both histosol and limestone sites in southern Florida. On the

other hand, a preliminary screening of 26 Musa cultivar with plant parasitic nematodes found in South Florida was realized in a greenhouse at the Tropical Research and

Education Center in Homestead, Florida and results showed that the accession

ITC1483 ( Monthan) suppressed nematode growth and reproduction while the highest number of nematode was found ITC1403 (Pisang Mas) roots.

Introduction

Globally, banana (Musa spp.) is ranked as the fourth most important source of dietary calories after rice, wheat, and maize (FAOSTAT, 2017; Sagi et al., 1998).

Bananas are grown and consumed in more than 130 countries in the tropics and the subtropics and include dessert, cooking, and brewing types (Quénéhervé, 2009).

Around 85% of global banana production is consumed locally with most banana growers maintaining small plantings (Quénéhervé, 2009; Tripathi et al., 2015). In the

United States, dessert quality bananas are the most prevalent with a per capita consumption of 12kg of fruit per annuum (Statista, 2019). While most bananas in the

United States are imported, many domestic growers seek specialty types for niche markets (Evans and Ballen, 2012). Specialty bananas include types not available through common retail supply chains with some types being specifically grown for their superior qualities including flavor (Schupska, 2008).

Conventionally, banana cultivation relies on clonally propagated, seedless cultivars that are maintained as monocultures for extended periods of time. These factors increase the risk of global disease epidemics. Historically, this was demonstrated with the demise of the ‘Gros Michel’ banana when destroyed by Panama

Disease (Fusarium oxysporum f. sp. cubense race 1) that required the entire industry to adapt to the modern ‘Cavendish’ type bananas (Jones, 2000; Ploetz, 2015; Laliberté,

2016). Similarly, common banana cultivation practices facilitate the increase of plant parasitic nematodes and limits the effectiveness of chemical or cultural control methods.

Under constant cultivation, banana yield losses of 30-60% can be common under high nematode pressure (Davide,1996).

Unfortunately, the most common types of banana are similarly susceptible to a few species of nematode and represent a major concern in all banana growing regions

(Stover, 1972; Gowen and Quénéhervé, 1990; Gowen et al., 2005). The three most important banana nematodes globally are R. similis (burrowing nematode),

Pratylenchus spp. (lesion nematode), and H. multicinctus (spiral nematode) with others like Meloidogyne spp, and Rotylenchulus reniformis also creating significant negative impacts (Ploetz, 1999; Gowen, 1990). Nematode control relies on preventing the spread of nematodes or application of nematicides that can negatively impact the environment and applicators (Haegeman, 2010). Banana cultivar-specific differences in nematode abundance have been reported previously, and this suggests that banana diversity may play a role in reducing the overall economic risks from plant parasitic nematode damage

(Brooks, 2004).

Growers in southern Florida often rely on specialty and niche crops to increase profitability while facing higher production costs than many fruit exporting countries.

Banana production in southern Florida comprises around 500 acres and is worth ~$2M per year (Evans and Ballen, 2012). There are thousands of banana cultivars available for commercial production and selecting specific cultivars for specialty banana commercial production can be challenging due to limited cultivar-specific information.

Agronomic performance among cultivars can vary for yield, fruit quality, and important traits like disease resistance (Heslop-Harrison and Schwarzacher, 2007).

Some growers in southern Florida cultivate diverse banana types in order to meet the growing demand from various domestic ethnic populations (Crane and Balerdi, 2016).

The diverse types of banana being grown in southern Florida could be used as a method to identify those that naturally support lower rates of nematode infection.

Generating cultivar specific information on yield, quality, and disease resistance for specific growing regions helps to reduce grower risk and support locally grown produce. Screening long-term banana cultivation sites for nematode response could also help identify prevalent nematode species and potentially susceptible banana cultivars. This information can help reduce grower risks when selecting cultivars for new plantings. The purpose of this study was, therefore, to identify the most abundant plant parasitic nematodes in soil and in banana roots at two selected sites in southern Florida under natural conditions, and to screen a diverse collection of banana accessions for nematode reproduction. The outcomes of this research provide preliminary data facilitating future research investigating the genetic basis for nematode resistance in diverse banana accessions.

Material and Methods

Site Selection

Two sites were selected for the nematode screening based on contrasting soil types and the availability of diverse Musa species under commercial cultivation. Both sites had been under commercial banana or plantain cultivation for at least 10 years, and both sites have favorable climatic conditions for Musa agricultural production.

Average annual temperature of 28.9°C and annual precipitation between 1200 and

1400 mm (FAWN, 2017). Neither site had applied nematicides for at least the previous

5 years (Table 2-1).

Musa Cultivars

A total of eight accessions were selected for the nematode survey (Table 2-2).

The accessions were selected to capture some genetic diversity and represent both dessert and cooking banana types. The eight accessions represent all cultivars that were common between both long-term banana growing sites.

Root Sampling

Samples were collected in May and again in August 2018.Two to four plants were sampled for each accession at each site preferentially from recently flowering plants. Roots and soil were collected from an approximately 30 cm x 30 cm area on two sides of each plant. The root and soil samples from each mat for a given genotype were bulked to form composite samples and placed into a plastic bag properly identified with the date, location, and cultivar name. All samples were transported in insulated boxes to protect samples from direct sunlight and temperature fluctuations. Samples were stored at 4°C until processing for nematode extraction.

Nematode Extraction

Soil and root samples and were processed separately. Nematodes were extracted from plant roots and soil following the modified Baermann tray/Funnel technique/pie-pan method (Whitehead and Hemming, 1965). Prior to the extraction of nematodes, all soil samples were sieved using a #30 (595 µm) mesh to remove debris and stones that might be in the sample. Two subsamples of 50 g of soil were taken from each bulk sample for nematode extraction. Each subsample was placed into a 500 mL beaker and thoroughly mixed with 200 mL of water. The mixture was then poured onto a

coffee filter supported by a screen placed inside of a plastic bowl. The water level inside the plastic recipient was kept even with the soil in the coffee filter throughout a room temperature incubation for 72 hours (Mekete et al., 2012; Coyne and Claudius-Cole,

2007). After incubation, the filtered water from subsamples was pooled. Nematodes were collected and concentrated using a 38 µm sieve. Nematodes were then identified and quantified as the number of nematodes per 100 g of soil.

For root samples, the same method was used with some modifications

(Whitehead and Hemming, 1965). Roots from each accession and location were independently processed. Roots were cleaned with tap water and left to be air-dried for two hours before processing. Roots were then cut into small portions of 1 or 2 cm and mixed thoroughly. Twenty-five grams of cut roots were then macerated in a kitchen blender with ~200 mL of water for ~20 seconds. The blended root samples were then poured onto a coffee filter placed inside of a mesh sieve in a plastic bowl as described for the soil samples (Mekete et al., 2012; Coyne and Claudius-Cole, 2007). Nematode quantification is reported as nematodes per 100 g of roots.

Nematode Identification and Quantification

An inverted microscope was used to identify the nematodes at the genus level and to enumerate the number of individuals per genus contained in each sample. A petri dish with scored lines was used to prevent recounting of the same individuals.

Preliminary Screening for Nematode Resistance

A preliminary evaluation for resistance to Florida endemic nematodes was conducted with 26 Musa accessions from September 2018 to January 2019 under greenhouse conditions at the Tropical Research and Education Center in Homestead,

Florida. Accessions were received as in vitro plants from the International Transit

Center (ITC, http://www.promusa.org/ITC) and the group was composed of AAA, ABB,

AAB, and AA genomes (Table 2-4). Plants were hardened for two weeks at 23°C under artificial lighting in 3.5-inch-deep well cell trays filled with soilless mix and slow release fertilizer. Plants were transferred to the greenhouse for one month before transplanting into 1-gallon pots with soilless mix and ~10 grams of slow release fertilizer per pot.

Plants were allowed to grow for two months prior to inoculation.

A bulk nematode extraction from infected banana roots was used as to inoculate each pot with ~28 nematodes at the base of the plant. Plants were watered every two days throughout the experiment. The entire root system of each plant was collected and prepared for nematode extraction 21 weeks post inoculation. The roots were washed, cut, and then macerated in a kitchen blender for 20 seconds following the previously described methods with minor modifications (Coolen et al., 1971; Seinhorst, 1988). The blended mixture was decanted through a set of sieves with a 53 µm aperture over a 38

µm sieve. Nematodes were collected in the 38 µm sieve and were washed into a modified Baermann tray. The nematodes were collected 72 hours after incubation and were used to calculate the reproductive fitness of the bulk nematode population.

Results

Plant Parasitic Nematodes Nematodes from Soil Samples

Nematode counts from soil samples were taken from the histosol and limestone sites to identify the percentages of plant-parasitic nematodes present by genus. The percentage of isolated genera are shown in Figure 2-1; Figure 2-2; Figure 2-3 and representative microscope images are shown in Figure 2-4. Helicotylenchus,

Meloidogyne, Rotylenchulus, and Xiphinema were isolated at both sites. Pratylenchus,

Trichodorus and Tylenchorhynchus were only isolated in soil samples from the histosol

site (Figure 2-1). The May soil sampling for the limestone site was qualitatively similar to the August sampling, but with increased Helicotylenchus and decreased Meloidogyne percentages (Figure 2-2 and 2-3). Helicotylenchus and Meloidogyne nematodes were the most abundant genera and represented 2/3 to 3/4 of total plant parasitic nematodes isolated from soil samples. Pratylenchus nematodes were much less abundant in histosol soils (0-0.2%) compared to limestone soils (1.9 to 17.3%).

In term of absolute frequency, the plant parasitic nematode population of the soil samples of both locations was more diverse than that of the root samples. Trichodorus,

Rotylenchulus, Tylenchorhynchus and Xiphinema were detected with low frequency rating in the histosol site of 25% for Trichodorus and 13% for the three other phytonematodes mentioned above. Helicotylenchus and Meloidogyne had similar distribution, present in 75% of the soil samples of the histosol site (Figure 2-5).

Contrariwise, Helicotylenchus was found in the totality of the soil samples of the limestone site. Meloidogyne, Pratylenchus and Rotylenchulus also were frequent, being present in 62.3% and 37.5 % respectively of the soil samples collected in this location

(Figure 2-5).

Plant Parasitic Nematodes Associated with Musa Roots

Roots from banana and plantain were sampled in May and August from eight cultivars growing at both sites (Table 2-1). Helicotylenchus was the predominant nematode genus and accounted for 63.9 to 96.3% of all plant-parasitic nematodes associated with banana roots (Figure 2-6; Figure 2-7; Figure 2-8; Figure 2-9).

Meloidogyne was found at each sampling time and site and ranged from 3.7 to 17.3% of all nematodes sampled. Hoplolaimus was only identified once from histosol root samples in May (0.8% of all nematodes) (Figure 2-7).

In roots from histosol soils, Meloidogyne was the most frequent plant-parasitic nematode (absolute frequency) with a frequency rating of 87.5% of the root samples followed by Helicotylenchus that was found in 75% of samples. Hoplolaimus and

Pratylenchus were also identified, but, with lower frequency rating of 12.5% and 6.3% respectively. In roots from limestone soils, Helicotylenchus was extracted in 81.3% of the samples, while Meloidogyne was found in 56%. The absolute frequency of

Pratylenchus in banana root samples was 3 times higher in the limestone than in the histosol location (Figure 2-5). Helicotylenchus, Meloidogyne, and Pratylenchus were in general the most recurrent and abundant genera.

Nematodes Associated with Musa Cultivars in Histosol and Limestone Soils

Number of plant parasitic nematodes associated with each of the eight banana cultivars grown at the histosol site are shown in Table 2-3. Meloidogyne,

Helicotylenchus, Hoplolaimus, and Pratylenchus were isolated from root samples from histosol soils. Meloidogyne was isolated from every cultivar during one or both sampling dates and ranged from 0 to 365 nematodes. Helicotylenchus was the most abundant nematodes associated with banana roots and ranged from 0 to 4,704 nematodes per

100 g of roots. Hoplolaimus was only isolated from two cultivars during a single timepoint, and Pratylenchus was only isolated from a single cultivar at one timepoint.

Plant parasitic nematodes were also isolated from banana roots growing in limestone soil (Table 2-3). Helicotylenchus, Meloidogyne, and Pratylenchus were isolated from banana roots growing in limestone soil. Helicotylenchus was the most abundant nematode in limestone root samples and ranged from 0 to 1344 per sample.

Meloidogyne were isolated from each cultivar and ranged from 0 to 204 nematodes per

100 g of roots. Pratylenchus was only isolated from two cultivars (‘African Rhino’ and

‘Williams’) growing in limestone soil and ranged from 0 to 604 per 100 g of roots.

Preliminary Screening of Nematode Reproduction on Diverse Banana Accessions

A diverse collection of 26 Musa accessions was inoculated with Musa parasitic nematodes and their reproduction was quantified (Figure 2-10). Accessions ranged from an average of two nematodes (ITC1483: Monthan) to 251 nematodes (ITC1403: Pisang

Mas). Overall, only accessions ITC1177 (Zebrina), ITC1403 (Pisang Mas), ITC0277

(Leite), and ITC0450 (Pisang Palembang) showed a higher average nematode count 21 weeks after inoculation.

Table 2-1. Banana farm management; soil type description; color of the soil, pH range and age of the plantations for each surveyed location. Site Farm Soil Type Color pH Age of Management the plantation Pahokee/ Occasional weed Mostly Histosol 0 – 25 cm: Slightly ~10 years Palm beach control mostly by characterized by a thick Black (N acidic County mowing and black muck soil layer 2/) muck; herbicide over limestone bedrock. application. Zero Their structure ranges 25 – 71 input of fertilizer from moderate coarse cm, color drawing upon the blocky to fine and is black moderate fertility medium granular (5YR 2/1) of the soil. structure. They muck originate from organic matter. These soils are poorly drained, covered with water for 6 to 12 months, except for very dry years.

Homestead/ Low fertilizer Calcareous Entisol of 7 – 22 cm Midly to > 20 Miami-Dade input with recent limestone origin deep: dark moderately years County additional water that have been brown alkaline supply through rockplowed to facilitate (10YR micro-aspersion. agricultural production. 3/3) or Herbicides and They have poor water brown (10 mowing were retention and nutrient YR 5/3 used as means content of weed control.

Table 2-2. A list of the eight banana and plantain cultivars used in this study. Cultivar is the name of the accession; Genome Group is the subgenome composition and Description is the primary use of the banana. Cultivar Genome Group Description African Rhino AAB Cooking banana DwarfCavendish AAA Dessert FHIA 1 AAAB Dessert FHIA 18 AAAB Dessert Giant Plantain AAB Cooking Hua Moa AAB Cooking/dessert Pisang Rajah AAB Dessert Williams AAA Dessert

Table 2-3. Plant parasitic nematode counts per 100 g of roots from different banana accessions from two different farms in Southern Florida with differing soil types. Data from the three consistently isolated genera (Helicotylenchus, Meloidogyne and Pratylenchus) are shown for histosol and limestone during both May and August extractions. Helicotylenchus Meloidogyne Pratylenchus

H L H L H L H L H L H L

Banana Cultivar M M A A M M A A M M A A

African Rhino 75 300 0 108 150 0 4 68 10 10 0 604

Dwarf Cavendish 0 10 100 1344 365 0 40 36 0 0 0 0

FHIA 1 1495 0 132 176 55 0 4 4 0 0 0 0

FHIA 18 3135 5 504 0 215 0 0 204 0 0 0 0

Giant Plantain 10 125 4 108 180 35 48 128 0 0 0 0

Hua Moa 15 0 4704 328 80 0 88 32 0 0 0 0

Pisang Rajah 335 10 0 20 5 0 12 4 0 0 0 0

Williams 0 40 136 172 0 0 20 184 0 0 0 8

H = histosol; L = limestone; M = May extraction; A = August extraction

Table 2-4. A list of the 26 accessions used in the screening experiment. ITC code is the accession ID, Accession name is name for the accession; Genome Group is the subgenome composition and Description is the primary use of the banana. ITC code Accession name Genome Origin Description group ITC0081 Igitsiri (Intuntu) AAA Burundi _ ITC0084 Mbwazirume AAA Burundi Cooking

ITC0123 Simili Radjah ABB _ Cooking

ITC0127 Kamaramasenge AAB _ Dessert

ITC0249 Calcutta 4 Acuminata India _

ITC0277 Leite AAA _ _

ITC0283 Long Tavoy Acuminata _ _

ITC361 Blue Java ABB _ _

ITC0450 Pisang Palembang AAB Malaysia Dessert

ITC0472 Pelipita ABB Philipines Cooking

ITC0484 Gros Michel AAA _ Dessert

ITC0643 Cachaco ABB _ Cooking

ITC649 Foconah AAB _ _

ITC0654 Petite Naine AAA _ Dessert

ITC659 Namwa Khom ABB Thailand _

ITC662 Khai Thong Ruang AAA Thailand _

ITC0685 Pisang Pipit AA Indonesia _ ITC0767 Dole ABB _ _

ITC0769 Figue Pomme Géante AAB _ _ ITC0825 Uzakan AAB Papua New _ Guinea

ITC1121 Pisang Lilin AA _ _ ITC1177 Zebrina Acuminata Indonesia _

ITC1403 Pisang Mas AA _ _

ITC1418 FHIA-25 AAB Honduras _

ITC1441 Pisang Ceylan AAB _ _

ITC1483 Monthan ABB _ Cooking

Figure 2-1. Percentage of the total number of plant parasitic nematodes by genus in soil samples from histosol.

Figure 2-2. Percentage of the total number of plant parasitic nematodes by genus in soil samples from limestone.

Figure 2-3. Percentage of the total number of plant parasitic nematodes by genus in soil samples from an additional independent limestone site collection. Data represent nematode percentage from eight independent soil samples for figure 2-1; 2-2 and 2-3.

Figure 2-4. Representative microscope images of the most common nematode species isolated from banana roots in southern Florida. Helicotylenchus (A), Meloidogyne (B), and Pratylenchus (C) are shown.

Figure 2-5. Absolute frequency (presence/absence) of nematode genus in roots and soil samples in the histosol site and limestone site.

Figure 2-6. Percentage of the total number of plant parasitic nematode genus extracted from 100 g of banana roots from the histosol site in August.

Figure 2-7. Percentage of the total number of plant parasitic nematode genus extracted from 100 g of banana roots from the histosol site in May.

Figure 2-8. Percentage of the total number of plant parasitic nematode genus extracted from 100 g banana roots from the limestone site in August.

Figure 2-9. Percentage of the total number of plant parasitic nematode genus extracted from 100 g of banana roots from the limestone site in May. Samplings data represent bulked root samples across all banana cultivars at each site for each sampling date.

Figure 2-10. Nematode counts from an inoculation study from diverse Musa accessions. Accessions are labeled by their ITC number. Data represents three independent plants inoculated with banana root-associated nematodes.

Discussion

This study identified the most frequent and abundant nematode genera isolated from soil and banana roots from two long-term banana plantings in southern Florida.

Three of them (Helicotylenchus, Meloidogyne and Pratylenchus) are known to cause serious economic damage to banana plantations (Quénéhervé,2009). Helicotylenchus and Meloidogyne were in general the most abundant phytonematodes recovered from soil and root samples. Similar cases have been found in South Africa and in Hawaii where complex of Meloidogyne and Helicotylenchus represented more than 2/3 of the plant parasitic nematode population recovered in these locations (De Jager et al., 1999;

Wang and Hooks, 2004). Nematodes were isolated from all banana cultivars, but trends suggest that ‘Giant Plantain’, ‘Pisang Raja’, and ‘Williams’ may support lower levels of nematode populations. These preliminary results will need to be confirmed in future studies using increased replication throughout the growing season. Unfortunately, this was not possible for the current study due to the limited plant materials and limited planting designs available in the long-term banana cultivation areas.

In general, the results of this study agree with previous studies that isolated

Helicotylenchus and Meloidogyne from roots of a commercial planting of ‘Burro’ banana in southern Florida (McSorley and Parrado, 1981). However, R. similis was not isolated from any soil or root samples even though this has been reported as a problem in southern Florida (McSorley et al., 1982). These results agree with conclusions by other authors that Helicotylenchus is the most important nematode in the subtropics where conditions are suboptimal for banana production and R. similis survival (Ploetz, 1997;

Gowen and Quénéhervé, 1990). We also isolated Hoplolaimus from two cultivar root

sample in histosol soils (Nguyen et al., 2015; Quénéhervé, 2009; Adiko, 1988; Marteille et al.,1988b).

The presence of Trichodorus, Tylenchorhynchus and Xiphinema in soil samples might be explained by the presence of surrounding weed species in the surveyed areas.

These nematode species were not a focus in the present study because they were not associated with banana root samples, though each has been associated with banana production areas previously (Chau et al., 1997; Khan and Hasan, 2010; Pathan et al.,

2004). The differences between the sites might explain some of the more subtle differences in this study. For example, the increased abundance of total nematodes at the histosol site might be due to the rich organic soil or due to farm management. The histosol site relied primarily on mowing to control weeds and prevent soil erosion, whereas the limestone site leveraged leaf mulch to suppress weeds around banana stems. Soil influence as well as climatic influence on the dynamic of banana parasitic nematode have been investigated in the past by several researchers and results reveal that species of Helicotylenchus thrive better than Radopholus spp. in wet organic soils

(Quénéhervé, 1988). The diversity of non-banana plants at the histosol site might therefore support a more favorable environment for nematodes. The increased abundance of Pratylenchus nematodes in the limestone site could also be related to this soil type, because the same cultivars were under long-term cultivation at both sites and the histosol site had fewer of these nematodes.

Previous studies have found large variability in nematode abundance among different banana cultivars (Brooks, 2004). Our preliminary screening of nematode growth on a diverse collection of banana accessions presented some interesting results.

The rapid growth of nematodes on accessions ITC1177 (Zebrina), ITC 1403 (Pisang

Mas), ITC 0277 (Leite), and ITC 0450 (Pisang Palembang) and unsupported growth on accessions like ITC 0484 (Gros Michel), ITC 1483 (Monthan), ITC 0361 (Blue Java), and ITC 0081 (Igitsiri) suggests that there could be some interesting genetics to investigate for future work. A focused study with increased replication and inoculation with reared nematode populations could help in the elucidation of nematode resistance if the preliminary findings are validated.

The results of this study while useful, should be cautiously interpreted as it relates to generalizations about cultivar vigor and quality. Some banana accessions can support relatively high nematode populations without showing symptoms of stress.

Therefore, nematode abundance alone cannot be used to predict banana yield. Also, only limited replication was available for this study because of the minimal availability of plants for each cultivar as is common in commercial plantings involving many diverse accessions. Regardless, these results provide novel information for future work that could combine the most promising cultivars in terms of yield and fruit quality with multi- year trials in order to validate these results and enable confident grower recommendations.

In conclusion, the purpose of this study was to identify the most common nematode species associated with soil and banana roots in southern Florida. We identified Helicotylenchus, Meloidogyne, and Pratylenchus as the most common nematodes associated with banana roots. Also, Pratylenchus nematodes seem to be less abundant in histosol than limestone soils. ‘Giant Plantain’, ‘Pisang Raja’, and

‘Williams’ all seemed to consistently yield fewer nematodes from root extractions. These

accessions might be favorable for growers that are currently looking for accessions that could be more resistant to nematode colonization.

Recognition

The authors acknowledge Nicholas Larsen of Lago’s Farm and Don and Katie

Chafin of Going Bananas for providing access to banana materials for this study.

CHAPTER 3 YIELD PERFORMANCE AND MIRACULIN CONTENT OF A NOVEL FRUIT (SYNSEPALUM DULCIFICUM) IN HOMESTEAD, FLORIDA

Summary

Miracle fruit (Synsepalum dulcificum) is the botanical source of miraculin and an understudied species with a promising future. This crop has been used in the Africa for its medicinal and sweetening properties for the past 100 years. Due to the growing number of people affected by chronic diseases associated with high sugar consumption, the demand for natural, non-caloric sweeteners such as miraculin is increasing.

However, there is a lack of information regarding agronomic differences and variation in miraculin content among cultivars for this crop. Our research focused on investigating the yield performance and the miraculin content across nine miracle fruit morphotypes growing in Homestead, Florida. Data of mature fruit weight/tree and average weight/fruit were collected in order to obtain insights on the production pattern of this perennial crop which flowers and bears fruits multiple times per year. In parallel to the yield trial, miraculin was extracted and quantified using high performance liquid chromatography.

Data reveal differences in performance among cultivars. The highest production of ~2.8 kg/tree/year (p<0.05) was obtained for ‘Typical’ while the lowest was obtained for ‘Holly’

(~0.7kg/tree/year, p<0.05). No significant difference was observed among the remaining cultivars. This will be the first report on yield performance of miracle fruit.

Introduction

Non-caloric, natural sweeteners are increasing in popularity as a mean to reduce sugar content in foods and beverages (Rodrigues, et al., 2016; Philippe et al., 2014;

Kant, 2005). These natural sweeteners come from various botanical sources and each has distinct flavor characteristics, strengths and weaknesses (Gibbs et al., 1996; Inglett

and May,1968). Miraculin extracted from miracle fruit (Synsepalum dulcificum) is a non- caloric protein, natural sweetener. The protein interacts with sweet taste receptors and increases the perception of sweetness in the presence of an acid (Yamamoto et al.,

2006; Kurihara, 1992; Kurihara and Beidler, 1969). For example, the perception of sweetness for foods like lemons and strawberries are greatly increased (Wong and

Kern, 2011; Kurihara and Beidler, 1968; Inglett and May,1968). The major benefit of the miraculin protein as a non-caloric sweetener is that its flavor profile is closer to sugar compared to other natural sweeteners that can impart metallic or other off-flavors

(Rodrigues, et al., 2016; Hellekant et al., 1998). The miraculin protein itself is not heat stable in its native form and must be carefully prepared in order to preserve its taste- modifying properties (Kurihara and Beidler, 1968). Nonetheless its natural properties remain intact for more than 6 months at pH 4 and stored at 5°C (Gibbs et al., 1996).

The miracle fruit plant is an evergreen bush native to African countries from

Ghana to the Congo. The plant belongs to the Sapoteceae and reportedly grows best in partial shade in well-drained soils with slightly acidic pH (Tchokponhoue et al., 2019;

Milhet and Costes, 1984). The flowers are small, white, and bisexual that usually form in small clusters. Miracle fruit flowers are hermaphroditic and self-compatible. The fruit is attractive with a bright red peel, ellipsoid, and approximately 2 cm long by 1 cm wide.

The fruit itself is not especially flavorful. A large seed comprises most of the fruit volume and is surrounded by a white pulp that contains the miraculin protein (Du et al., 2013;

Lim, 2013; Ayensu, 1972). The viability of seeds rapidly declines after harvesting the pulp (Tchokponhoue et al., 2018).

Miraculin has been well-studied for its sweet-inducing properties. The overall sweetening sensory impact is ~400 times that of sugar on a molecular basis when stimulated by foods or beverages with acidic pH (Gibbs et al., 1996; Kurihara and

Beidler, 1968). Miraculin and miracle fruit are available through various online vendors in various formats from plants to freeze dried fruit powder (Wilken and Satiroff, 2012).

The miracle fruit tablets are often used in a recreational setting to change the flavor perception of common foods like cheese, wine, and acidic fruits. Miraculin has also been trialed in clinical settings as it reportedly reduces the metallic taste of foodsfor patients undergoing chemotherapy treatments (Wilken and Satiroff, 2012.; Shi et al.,

2016). While miraculin is been actively studied, the plant has only benefited from limited horticultural research (Xingwei et al., 2016).

The purpose of this study was to fill critical knowledge gaps for yield and miraculin content of miracle fruit berries. Currently, there are no available reports on this information in the scientific literature. This study was conducted in southern Florida where the environment is favorable for miracle fruit cultivation. We anticipate that the results from this study will further our understanding of this horticulturally interesting species and provide valuable information for those considering its cultivation.

Material and Methods

Plant Material

The study was carried out from May 2018 to May 2019 on a private farm in

Homestead, Florida, USA. Plants were propagated vegetatively and were maintained in

~80 L containers in a shade house with supplemental irrigation, and a slow release fertilizer was applied once a year. The plants received the same amount of fertilizer and water. Supplemental irrigation was reduced in summer due to regular rainfall. All plants

were five years old at the time of sampling. Plants were separated into nine different types based on morphological characteristics (Table 3-4). Six types had ten plants available for evaluation and comprised the bulk of the study materials. Three additional types including ‘Christmas’, ‘Weeping’, and ‘Holly’ were also selected for sampling but only had fruits from 1, 2 and 3 plants available for analysis, respectively. All plants were pruned to manage total bush size in February of each year.

Yield Evaluation

Fruit from each plant were harvested each week in the morning over 52 weeks.

This harvest interval captured almost all the fruit with only minor loss to premature fruit dropping. Fruit data was collected by individual tree. Total weekly fruit weight was collected throughout the study. Fruit number data was collected over multiple harvest windows.

Miraculin Crude Extract

Fruit for miraculin quantification were processed within four hours of harvesting.

Fruits were juiced manually in plastic zip closure bags. Juice samples of ~20 mL

(without seeds or peel) were then frozen at -80°C until miraculin extraction.

Miraculin was extracted as previously described (He et al., 2015) with some modifications. One gram of finely ground tissues was vortexed with 10 mL of distilled water for 2 mins, centrifuged at 12,000 rcf for 30 min, and the supernatant was carefully discarded. The water extraction was repeated twice. The pellet was resuspended in two mL of 0.5 M NaCl buffer, vortexed for 2 mins, mixed on a Belly Button® shaker for 60 mins at 4°C, and centrifuged at 12000 rcf for 20 mins. The supernatant was collected as the miraculin crude extract and stored at -20°C until further analysis.

Miraculin IMAC Purification and Protein Sequencing

IMAC purification was previously described (He et al., 2015). All IMAC steps were performed at 4°C. The crude extract was pH adjusted to 7.0 using NaOH. A six mL chromatography column was fixed to a vertical support and washed with five mL binding buffer (20 mM Tris-HCl, 300 mM NaCl, pH 7.0). The flow through was discarded and

500 µL Qiagen Ni-NTA Agarose (50% Ni-NTA slurry, catalog #30210) was added to the column, allowed to settle, and washed with five mL binding buffer. Two mL of the pH adjusted crude extract was then added to the column, and the flow through was applied to the column twice more. The column was then washed twice with five mL of binding buffer each time and the flow through discarded. Bound proteins were then eluted with

500 µL aliquots of elution buffer (300 mM imidazole, 200 mM Na2HPO4, 300 mM NaCl, pH 8), and fractions were collected for quantification.

Ni-NTA purified miraculin was submitted to the University of Florida Proteomics

Core for sequencing. The sequencing results were analyzed using Scaffold Viewer

4.8.3.

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis)

The crude extract and purified miraculin were analyzed by SDS-PAGE gel electrophoresis. Ten µl samples were mixed with 2x SDS-PAGE Laemmli Buffer (4%

SDS, 20% glycerol, 120 mM Tris-HCl, and 0.02% w/v bromophenol blue) and 0.5 ul 2- mercaptoethanol, heated to 95°C for 5 minutes, and centrifuged at 21,000 x g for 1 min.

Then, 10 ul of each sample or Precision Plus Protein™ protein standard (Bio Rad, Cat

No 1610363) was subjected to electrophoresis using a 12% polyacrylamide Mini-

PROTEAN® TGX™ precast gel (Bio Rad, Cat No 456-1045). Gels were run at 200 V for

30-40 minutes until the indicator dye reached the bottom of the gel. Gels were rinsed

once using distilled water, stained with filtered Coomassie R250 buffer (50:10:40 methanol: acetic acid: water, 0.2% w/v Coomassie R250), rinsed with distilled water, and destained (50:10:40 methanol: acetic acid: water) until clear bands were apparent.

Miraculin Quantification

Miraculin content was measured using high-performance liquid chromatography

(HPLC, Infinity II, Agilent Biosystems) for the crude extract and elution samples.

Samples were syringe filtered using a 4.5 µm PES syringe filter. Filtered samples were then added to amber vials and run on the HPLC using an Agilent Poroshell 300SB-C18,

2.1x75 mm, 5 µm column. HPLC grade water with 0.1%TFA and acetonitrile were used for the mobile phases. The method included 1 minute 100% water, then applied an increasing acetonitrile gradient up to 70% over nine minutes. The HPLC needle was rinsed in between samples. The flow rate was held constant at 1 ml/min, and absorbance was measured using a UV-vis detector at 280 nm.

Statistical Analysis

Data for yield evaluation was submitted to the software JMP Pro 14 for statistical analysis and means were separated by Tukey’s HSD test (alpha=<0.05).

Results

Miracle Fruit Total Yield by Accession

The total yield of miracle fruit has not been previously reported, and this information is vital for calculating the economics of growing miracle fruit in southern

Florida. The total yield (fresh weight) from the 66 trees over one year was 135.9 kg with an average of 2.1 kg per tree per year. The average yield was 0.4 kg per tree per week.

The highest yielding tree type was ‘Typical’ with 2.76 kg/tree/year and was significantly different than ‘Curly leaf’ (1.86 kg/tree/year), ‘Combo’ (1.68 kg/tree/year), and ‘Holly’

(0.73 kg/tree/year), the lowest yield value recorded. Yield value for ‘Christmas’, ‘Plates’,

‘Wavy leaf’, ‘Columnar’ and ‘Weeping’ with an average of 2.1kg/tree/year of fruit were not statistically different with the yield of the other studied types (Table 3-1).

Miracle Fruit Seasonal Yield

Six peaks in miracle fruit production were identified during the 52-week study

(Figure 3-1). A production peak was observed approximately every two months. These peaks were used to identify harvest windows between peak production times. Total yield for all accessions was significantly different among the six harvest windows (Table

3-2). Harvest window 1 from May 21, 2018 to July 12, 2018 had the highest average yield with 706.25 g/tree. The average production for harvest window 2 (July 16 to

September 4, 2018) and 3 (September 10 to November 13, 2018) were not significantly different but were twice lower than the production obtained in harvest window 1.

Another major decline in the production was observed from November 19 to May 8,

2019, for harvest windows 4 to 6 specifically. The lowest averaged production was recorded for harvest window 6 (March 20 to May 8, 2019, 177g) and was not significantly different with the production for harvest window 4 (19 Nov to 23 Jan 2019,

266g) and 5 (23 Jan to 13 March 2019, 241g).

Miracle Fruit Total Yield per Tree

The classification of miracle fruit trees by morphological types could introduce a subjective assignment of genetic relatedness. Therefore, individual trees were analyzed for yield performance over 52 weeks to investigate if superior types were present

(Figure 3-2). Production patterns were mostly homogeneous among trees whithing the

‘Typical’ group as well as for the ‘Plates’ type. However, nonuniform production trends were observed for trees across the other surveyed groups. For this reason, trees were

also analyzed based on their production pattern and 5 different clusters were identified

(figure 3-3).

Miracle Fruit Average Fruit Weight

Average fruit weight was measured for the first 25 weeks of the study during harvest windows 1-3 (Table3-1). The average fruit weight for the nine miracle fruit types ranged from 1.53 to 1.22 g/fruit. ‘Curly Leaf’, ‘Typical’, ‘Wavy leaf’ and ‘Plates’ were not significantly different and were bigger in size (average 1.5 g/fruit) than Columnar,

‘Combo’, ‘Holly’, ‘Weeping’ and ‘Christmas’ (~1.3 g/fruit, < 0.05).

Protein Sequencing

The Ni-NTA purified protein was sequenced to confirm the protein’s identity. A perfect hit was identified as the miraculin precurser (Synsepalum dulcificum) corresponding to Genebank BAA07603 (gi 1109652) with 100% probability, 40.5% coverage (89/220 amino acids), six exclusive unique peptides, and eight exclusive unique spectra.

Protein Extraction

Miraculin was extracted from miracle fruit freeze dried tissue following the method described by He et al. (2015). Figure 3-4 shows different absorbance peaks at different retention time for all the proteins of miracle fruit that are soluble in sodium the chloride buffer. After the purification of the miraculin using affinity chromatography, a single peak was consistently observed at 7.0 minutes of retention time displaying the miraculin (Figure 3-5).

Table 3-1. Average yield for each accession in kg/tree/year for fruit fresh weight. Averages connected by the same letter are not significantly different using Tukey’s HSD (alpha=<0.05). Accessions kgtree/year/tree/year g/fruit ‘Typical’ 2.76 (A) 1.23 (A) ‘Christmas’ 2.41 (ABC) 1.22 (B) ‘Plates’ 2.25 (AB) 1.27 (B) ‘Wavy leaf’ 2.21 (AB) 1.48 (A) ‘Columnar’ 2.01 (AB) 1.50 (A) ‘Curly leaf’ 1.86 (BC) 1.40 (B) ‘Weeping’ 1.80 (ABC) 1.54 (A) ‘Combo’ 1.68 (BC) 1.35 (B) ‘Holly’ 0.73 (C) 1.27 (B)

Table 3-2. Average miracle fruit yield among the six harvest windows. Data represents the sum and average yield of all trees by harvest window. Harvest Window Harvest Weeks Sum Yield (g) Ave Yield (g) 1 May-July 2018 46612.59 706.25 (A) 2 Jul-Sept 2018 15899.72 357.51 (B) 3 Sept-Nov 2018 11669.16 311.23 (B) 4 Nov 2018-Jan 2019 23595.36 266.42 (BC) 5 Jan-March 2019 20541.14 240.90 (BC) 6 March-May 2019 17583.50 176.81 (C)

Figure 3-1. Average fruit yield for all 66 accessions in grams per tree over 52 weeks. Error bars represent standard error for all accession yield data.

Figure 3-2. Total yield per tree over 52 weeks color coded by morphotype.

Figure 3-3. Total yield per tree over 52 weeks color coded by clusters.

Figure 3-4. Miracle fruit crude extract. Figure shows the total protein content of the sodium chloride solution. Y axis is for the absorbance while the X axis is the retention time.

Figure 3-5. Single peak of miraculin after purification with IMAC. Y axis is for the absorbance while the X axis is the retention time.

Discussion

Seasonal Yield

Results from this study revealed the major harvest periods for this crop which were identified in June, then from late November to mid-March (Table 3-2, Figure 3-1).

Although the yield performance for this species has not being previously published,

Irvine, (1961) reported that the harvest period for this crop in Ghana is from December to June. More recently, Oumorou et al. (2010) suggested a harvest window for the crop that differs from our findings. According to theses authors, the harvest period in Benin is from December to February. Fruits are also available in May, August and in October. All of these reports are only qualitative or based on preliminary observations and require further investigation (Achigan-Dako et al., 2015). The difference in harvest time might be explained by differences in environment, but it is impossible to directly compare unless yield is quantified and reported in these other areas.

Yield per Tree

As for the yield performance of the crop, 2 kg /tree/year of mature fruits can be harvested on average. This is the first report on miracle fruit yield that is based on a structured investigation that captured the total fruit production of the crop for a yearlong period. The highest yield per tree found during the course of this study was 2.7 kg/tree/year which is about ~ 6 times lower than the yield/year reported by Achigan-

Dako et al., 2015 (12-15kg per tree). The same authors stated that variation in yield for this species might be influenced by the branching, habitat and the age of the plants

(Achigan-Dako et al., 2015). Since information regarding the plant materials and the data collection methods used for this research were not provided, comparison with our findings is not possible.

Average Fruit Weight

The average weight per fruit can be a good indicator for plant breeders working on the improvement of the crop in terms of yield components, as well as for farmers when selecting a variety to grow. Fruit weighted an average of 1.3 to 1.5 gindividually.

Preliminary studies on this parameter have not been published yet. Hence, this data can be used as reference for farmers interested in growing the crop as well as in future investigations on the species.

Tree Clustering

Clustering the shrubs based on production behavior helped us understand the overall production pattern for this crop. Since trees from same morphological types presented different production trends, investigations at the molecular level might help obtain better insights on this behavior if there is a genetic component involved.

Protein Quantification

Miraculin is was successfully extracted, purified, and identified during this study.

Confirmation was provided by SDS-PAGE analysis, affinity chromatography, protein sequencing, and results that matched expectations from other literature sources.

Conclusion

Interest for miracle fruit is increasing due to its multiple applications in pharmaceutical and food industries. Research to answer fundamental questions on miracle fruit such as yield performance, are essential to make productive use of this species as a crop. Results from our research has made a substantial contribution to the scientific community especially for potential growers in southern Florida.

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BIOGRAPHICAL SKETCH

Lynhe Demesyeux was born in Port-au-Prince, Haiti in 1990 in a small family of two children. In October 2009, she was awarded a full scholarship to study in Costa

Rica. In 2012, she realized a full 4 months internship in Jamaica where she oversaw the designing and execution of a carbon neutral project for Pantrepant farm, under Gustavo

Diaz Flores supervision and advised by German Obando MSc.

In December 2013 she successfully obtained her bachelor’s degree in agronomy from EARTH University.

Driven by the desire to contribute to improvement of agricultural practices in

Haiti, she went back to her country the same year and works in extention for two different institutions for 3 years.

She also has a strong passion for teaching and volunteered in an agriculture vocational school teaching sorghum production for several months to young student.

She received her Master of Science from the University of Florida in 2019. Her goal is to continue her education in agriculture and life science, have her own farm in a in Haiti and to continue exercicing her passion for teaching and research.