Citation
Initial Population Densities and Effects of Temperature and Cultivars on the Pathogenic Potential of Meloidogyne Haplanaria, an Emerging Threat to Tomato Production in Florida

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Title:
Initial Population Densities and Effects of Temperature and Cultivars on the Pathogenic Potential of Meloidogyne Haplanaria, an Emerging Threat to Tomato Production in Florida
Creator:
Espinoza Lozano, Lisbeth Delrocio
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (71 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
MENGISTU,TESFAMARIAM M
Committee Co-Chair:
CROW,WILLIAM T
Committee Members:
DUNCAN,LARRY WAYNE
NOLING,JOSEPH W

Subjects

Subjects / Keywords:
haplanaria -- meloidogyne -- mi-gene -- temperature -- tomato
Entomology and Nematology -- Dissertations, Academic -- UF
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Entomology and Nematology thesis, M.S.

Notes

Abstract:
Root-knot nematodes are today a major group of plant-parasitic nematodes that cause important economic losses on a global scale in a wide range of crops. The use of resistant cultivars is one of the key tools to manage these nematodes; however, the emergence of resistance breaking populations of root-knot nematodes has been reported. Meloidogyne haplanaria a root-knot nematode species that was first reported in 2003 in Texas causing significant damage in peanut fields. In 2015 this nematode was also identified from tomato fields in Naples, Florida. This Florida population of root-knot nematode was reported breaking the resistance conferred by the Mi-gene. The Mi-gene is widely used in different tomato cultivars to provide resistance to few species of root-knot nematodes such as M. javanica, M. incognita, M. hapla and M. arenaria. Tomato cultivars with the Mi-gene are widely used in fields in some states. However, many factors affect its performance; factors such as high temperatures, high initial population and gene dosage can interfere with the expression of this gene and limit its use in Florida. The objectives of this project were to determine the damage threshold of M. haplanaria and to analyze the impact of air temperature and genetic background of tomato plants on resistance breaking of M. haplanaria in tomato cultivars. Results from this study show a damage threshold of 3 eggs and second-stage juveniles/ per cm3 of soil. Additionally, it was found that at high temperatures, the life cycle of M. haplanaria is shorter than the virulent M. enterolobii, and that M. haplanaria it can infect and cause severe damage on homozygous or heterozygous resistant tomato plants. This research also confirmed this species to be highly virulent. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
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Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2017.
Local:
Adviser: MENGISTU,TESFAMARIAM M.
Local:
Co-adviser: CROW,WILLIAM T.
Statement of Responsibility:
by Lisbeth Delrocio Espinoza Lozano.

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Applicable rights reserved.
Classification:
LD1780 2017 ( lcc )

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INITIAL POPULATION DENSITIES AND EFFECTS OF TEMPERATURE AND CULTIVARS ON THE PATHOGENIC POTENTIAL OF MELOIDOGYNE HAPLANARIA AN EMERGING THREAT TO TOMATO PRODUCTION IN FLORIDA By LISBETH ESPINOZA LOZANO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017

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2017 Lisbeth Espinoza Lozano

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To everyone who had faith in me

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4 ACKNOWLEDGMENTS I give thank s to my mom Rocio, my dad Rodrigo and my grandparents Rosa Emilia and Adalberto for support ing me all the time, and inspiring me to go further ; t o my brothers Fernando, Diego, and my sisters Mirian, and Katty, for being my emotional support; to Daniel, for being my friend and p artner from the first day of this journey To Dr. Mengistu for his guidanc e support and patience through all this process and for being a good advisor; t o all my committee members for enlightening me and made me a better professional. T o my lab mates Gideon, Anil, Alexandros, and R uhi yyih and lab staff, A lex, Marice, Laban and Matt for being such a nice group of friends and for helping me during my research, without your valuable support this thesis would not be possible. A special acknowledgement to Esther Lilia Peralta m y former advisor, for her advising and for teaching me to give my best in everything I do.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTERS 1 TOMATO INDUSTRY AND NEMATODES ................................ ............................. 12 Tomato Industry Overview ................................ ................................ ...................... 12 Pests and Diseases of Tomato ................................ ................................ ............... 12 Plant Parasitic Nematodes ................................ ................................ ..................... 13 Root Knot Nematode Biology and Life Cycle ................................ .......................... 13 Economic Importance of Root Knot Nematodes ................................ ..................... 15 General Nematode Management Practices ................................ ............................ 16 Cultural Control ................................ ................................ ................................ ....... 17 Chemical Control ................................ ................................ ................................ .... 18 Biological C ontrol ................................ ................................ ................................ .... 19 Plant Resistance ................................ ................................ ................................ ..... 20 Resistance Management and Mi Gene ................................ ................................ ... 21 2 DAMAGE POTENTIAL OF M. HAPLANARIA ON THE MI GENE CONTAINING ................................ ...................... 25 Introduction ................................ ................................ ................................ ............. 25 Materials and Methods ................................ ................................ ............................ 27 Plant Material ................................ ................................ ................................ ... 27 Inoculum Preparation ................................ ................................ ....................... 27 Pot Prep aration and Inoculation of M. haplanaria ................................ ............. 28 Data Collection ................................ ................................ ................................ 28 Results ................................ ................................ ................................ .................... 29 Discussion ................................ ................................ ................................ .............. 30 3 IMPACT OF TEMPER ATURE ON THE STABILITY OF THE MI GENE, AND COMPARISON OF THE DIFFERENT DEVELOPMENTAL PROCESSES OF M. HAPLANARIA M. INCOGNITA AND M. ENTEROLOBII ................................ ....... 3 6 Introduction ................................ ................................ ................................ ............. 36 Materials and Methods ................................ ................................ ............................ 38 Inoculum Preparation ................................ ................................ ....................... 38 Plant Material ................................ ................................ ................................ ... 38 Inoculum Preparation ................................ ................................ ....................... 39 Data collection ................................ ................................ ................................ .. 39

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6 Results ................................ ................................ ................................ .................... 40 Discussion ................................ ................................ ................................ .............. 43 4 THE RESPONSE OF MI GENE RESISTANT TOMATO CULTIVARS AND ROOTSTOCKS TO ROOT INFECTION BY MELOIDOGYNE HAPLANARIA MELOIDOGYNE INCOGNITA AND MELOIDOGYNE ENTEROLOBII ................. 54 Introduction ................................ ................................ ................................ ............. 54 Materials and Methods ................................ ................................ ............................ 55 Plant Material ................................ ................................ ................................ ... 55 Inoculum Preparation ................................ ................................ ....................... 56 Pot Preparation and Inoculation ................................ ................................ ....... 56 Data Collection ................................ ................................ ................................ 56 Results ................................ ................................ ................................ .................... 57 Discussion ................................ ................................ ................................ .............. 59 5 CONCLUSIONS ................................ ................................ ................................ ...... 63 LIST OF REFERENCES ................................ ................................ ............................... 64 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 71

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7 LIST OF FIGURES Figure page 2 1 Effect of initial population densities on total egg masses of M. haplanaria on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16, 32 and 64 eggs and J2/ g of soil in greenhouse conditions. ............. 32 2 2 Effect of initial population densities on total production of M. haplanaria eggs on tomato cultivars Rutgers and Sanibel, 60 days after inoc ulation of 0, 0.25, 1, 2, 4, 8, 16, 32 and 64 eggs and J2/ g of soil in greenhouse conditions. ......... 32 2 3 Effect of initial popula tion densities on root gall index of M. haplanaria on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16, 32 and 64 eggs and J2/ g of soil in greenhouse conditions. ............. 33 2 4 Effect of initial population densities on the production of M. haplanaria eggs / g of root on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16, 32 and 64 eggs and J2/ g of soil in greenhouse. ......... 33 2 5 Effect of initial population densities on the reproductive factor of M. haplanaria / g of root on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16, 32 and 64 eggs and J2/ g of soil in ............ 34 2 6 Relationship between initial population density (Pi) of M. haplanaria and relative shoot height (cm) on tomato cultivars Rutgers and Sanibel. Plants were harvested after 60 days and each point in the graph represents a mean .. 34 2 7 Relationship between initial population density (Pi) of M. haplanaria and relative shoot fresh weight (gm) on tomato cultivars Rutgers and Sanibel. Plants were harvested after 60 days and each point in the graph represents a 35 2 8 Relationship between initial population density (Pi) of M. haplanaria and relative root length (cm) on tomato cultivars Rutgers and Sanibel. Plant s were harvested after 60 days and each point in the graph represents a mean .. 35 3 1 Impact of temperature on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria, and M. incognita at A) 24 C, B) 28 C and C) 32 C. Photographs by Lisbeth Espinoza. ................................ ....................... 46 3 2 Effect of temperatures on total egg production (log x + 1) on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita 40 days after inoculation in growth chamber maintained at 24 28 .. 46 3 3 Effect of temperature on the total egg mass production (log x + 1) on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita 40 days after inoculation in growth chamber maintained ........ 47

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8 3 4 Effect of temperature on RGI on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, M. haplanaria and M. incognita, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ........................ 47 3 5 Effect of temperature on eggs / g of roo t (log x + 1) on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, M. haplanaria and M. incognita, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C.. ................................ ................................ ................................ ........... 48 3 6 Linear regression analysis of temperature on eggs on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ................................ .......... 48 3 7 Linear regression analysis of temperature on eggs/ g of root on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ........................ 49 3 8 Linear regression analysis of temperature on RGI on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ................................ .......................... 49 3 9 Linear regression analysis of temperature on egg masses on tomato cult ivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ................................ .......... 50 3 10 Effect of temperature on eggs / g of root (log x + 1) on tomato cultivars Rutgers and Sanibel inoculated with M. incognita, 40 days after inoculation i n growth chamber maintained at 24 28 and 32 C. ................................ .............. 50 3 11 Effect of temperature on eggs / g of root (log x + 1) on tomato culti vars Rutgers and Sanibel inoculated with M. incognita, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ................................ .............. 51 3 12 Effect of temperature on eggs / g of root (log x + 1) on tomato cultivars Rutgers and Sanibel inoculated with M. incognita, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. ................................ .............. 51 3 13 Effect of temperature on eggs / g of root (log x + 1) on tomato cultivars Rutgers and Sanibel inoculated with M. haplanaria, 40 days afte r inoculation in growth chamber maintained at 24 28 and 32 C. ................................ .......... 52 3 14 Effect of temperature on the total number of J2s/ g of root on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C.. ................................ ......... 52 3 15 Effect of temperature on the number of total J2s/ g of root on tomato cultivars Rutgers and Sanibel inoculated with M. haplanaria 40 days after inoculation in grow th chamber maintained at 24 28 and 32 C.. ................................ ......... 53

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9 3 16 Effect of temperature on the number of total J2s/ g of root on tomato culti vars Rutgers and Sanibel inoculated with M. incognita, 40 days after inoculation in growth chamber maintained at 24 28 and 32 C. Letters represent significant 53 4 1 Effect of M. enterolobii, M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions.. ........ 61 4 2 Effect of M. enterolobii, M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions.. ........ 61 4 3 Effect of M. enterolobii, M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions.. ........ 62 4 4 Effect of M. enterolobii, M. haplanaria and M. incognita on the total egg mass p roduction of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions.. ........ 62

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requir ements for the Degree of Master of Science INITIAL POPULATION DENSITIES AND EFFECTS OF TEMPERATURE AND CULTIVARS ON THE PATHOGENIC POTENTIAL OF MELOIDOGYNE HAPLANARIA, AN EMERGING THREAT TO TOMATO PRODUCTION IN FLORIDA By Lisbeth Espinoza Lozano December 2017 Chair: Tesfamariam Mengistu Major: Entomology and Nematology Root knot nematodes are a major group of plant parasitic nematodes that cause important economic losses on a global scale in a wide range of crops. The use of resistant cultivars is one of the key tool s to manage these nematodes; however, the emergence of resistance breaking populations of root knot nematodes has been reported Meloidogyne haplanaria is a root knot nematode species that was first reported in 2003 in Texas causing significant damage in peanut fields. In 2015 this nematode was also identified from tomato fields in Naples, Florida. This Florida population of root knot nematode was reported breaking the resistance conferred by the Mi gene. Th e Mi gene is widely used in different tomato cultivars to provide resistance to a few spe cies of root knot nema todes such as M. javanica M. incognita M. hapla and M. arenaria Tomato cultivars with the Mi gene are widely used in fields in some states However, many factors affect its performance such as high ; temperatures, initial population density and gene dosa ge can interfere with the expression of this gene and limit its use in Florida

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11 Th e objectives of this project were to determine the damage threshold of M. haplanaria and to a nalyze the impact of air temperature and genetic background of tomato plants on resistance breaking of M. haplanaria in tomato cultivars. Result s from this study show a damage threshold of 3 eggs and second stage juveniles / per cm 3 of soil A dditionally it was found that at high temperatures the life cycle of M. haplanaria is sho rter than the virulent M. enterolobii and that M. haplanaria it can infect and cause severe damage on homozygous or heterozygous resistant tomato plant s. This research also confirmed this species to be highly virulent on tomato

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12 CHAPTER 1 TOMATO INDUSTRY AND NEMATODES Tomato Industry Overview Tomato ( Solanum lycopersicum ) belongs to the Solanaceae family, which contains many important food crops such as potato, pepper and eggplant. The United States is recognized as one of the major tomato producers and ranks second after China in overall production In the U S ., f resh tomatoes are grown on over 39,456 ha, and it was valued at more than $1 243 million in 2015 (USDA NASS, 2016) Historically, California and Florida were the leading producers of tomato yielding almost 65% of the total production in the US However, over the past decades Florida has become the highest value produce r of tomato in the US contributing approximately 36% of the total production of fresh market tomatoes in the U S (USDA NASS, 2016) The majority of the Florid a market occurs in the months of November to April with a total production of $500 million dollars. The total cost of production is approximately $7,000/ a, with 20% allocated for soil and foliar pest and disease control (USDA NASS, 2016) Pests and Diseases o f Tomato Tomato is susceptible to different pest and diseases. Several species of pathogens from different etiologies such as oomycetes, fungi, bacteria, viruses and phytoplasmas infect tomatoes and produce symptoms like damping off, late blight, cankers, speck, leaf spots, root rot stunting, and wilting, among others ( Jones, 1991) Insects such as aphids, whiteflies, thrips, and stink bugs affect tomatoes by sucki ng their juices and weakening the plant s In addition, some insect pests serve as vector s for several viruses and pathogens. Other insects like caterpillars can cause severe

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13 defoliation of the plants, this compromises the photosynthetic capacity of the pla nt, reducing total yield (Webb et al., 2013) The environment has a strong influence on the severity and incidence of pests and diseases. Temp erature, light and humidity are related to the development of di seases by influencing spore germination, infection and propagation of pathogens (Huber and Gillespie, 1992) F or insects, temperature and humidity have a direct effect reducing or extending its life cycle and the reproductive rate (Mattson and Haack, 1987) Plant Parasitic Nematodes Plant parasitic nematodes are known to be a major problem in many crops throughout the world. Worldwide crop losses due to plant parasitic nematodes have been estimated at $118 billion annually, with root knot nematodes, Meloidogyne sp p ranking first in terms of economic losses (Sasser and Carter, 1983) Root Knot Nematode Biology and Life Cycle Root knot nematodes ( Meloidogyne spp. ) are sedentary endoparasites that invade plant roots and establish a prolonged and intimate relationship with their host. Root knot nematodes are distributed worldwide ( Jones et al., 2013) This cosmopolitan group contains more th an 100 species and have thousands of hosts attacking a diverse group of plants including vegetables, fruits, grasses, trees and weeds (Mitkowski and Abawi, 2003) They display sexual dimorphism with females becoming sedentary and maturing into a rounded apple shape as they reach maturity, whereas male s maintain their vermiform form though all their life cycle. M ature females lay eggs into gelatinous masse s composed of a glycoprotein matrix produced from rectal glands; this matrix keep s the eggs together and protected from extreme environmental conditions and

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14 predators (Moens et al., 2009) The egg masses can be found on the surface of galled roots or embedded within the gall tissue and can contain up to 1000 eggs per mass However, the presence of galls is not always necessa ry ( Jones et al., 2013) Within the egg, embryogenesis proceeds to the first stage juvenile (J1), which molts to the infective second stage juvenile (J2). J2s hatch from the egg and, in general, hatching is dependent solely on suitable temperature and moisture conditions, with no stimulus from host plants being required (Moens et al., 2009; Jones et al., 2013) J2s then move through the root to initiate and develop a permanent feeding site called giant cells within or near the vascular system of the plant This feeding s ite serves as a nutrient s ink for the developing J2. N ematode growth and reproduction entirely depend on the development of giant cells Under favo rable conditions, the J2 molts to the third stage juvenile (J3) after about 14 days, then to the fourth stage juvenile (J4), and finally to the adult stage (Moens et al., 2009) The J3 and J4 do not feed; a dult females continue to feed and enlarg e to become round to pear shaped. Root knot nematodes exhibit var i ations in rep roductive strategies that range from amphimixis to obligatory mitotic parthenogenesis. Most species are parthenogenetic and males are only formed under adverse conditions. Root knot nematodes have unbalanced sex ratios ( Jones et al., 2013) In general t he life cycle of root knot nematodes takes three to six weeks to complete, depending on the species, the host plant and environmental conditions (Castagnone Sereno et al., 2013) Root knot nematodes can have several g enerations in one cropping season Many Meloidogyne species have a broad ho st range. The overall host range of Meloidogyne species

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15 encompasses from 3 000 to 5 500 plant species (Abad et al., 2003; Mitkowski and Abawi, 2003) Symptoms are expressions of root dysfunction, t he above ground symptoms of roo t knot nematodes are characterized as a patchy distribution of chlorotic, stunted, necrotic, and wilted plants. The below ground symptoms typically include root galling, however other nematode species such as Nacobbus sp p or the false root knot nematode are able to cause extensive root galling Crop yield reduction is commonly associated with high initial population den sitie s of nematodes in the soil, and of the loss of root function resulting from population increase and reinfection P opulation growth is favored by favorable environmental condition s that promote the early appearance of symptoms and increas e the damage severity (Noling, 1999; Ploeg, 2002) Eco nomic Importance o f Root Knot Nematodes Root knot nematodes are economically important pests on a wide range of vegetables throughout the wo rld (Castagnone Sereno et al., 2013) A recent survey globally ranked root knot nematodes first in the list of the plant parasitic nematodes based on their scientific and econo mic importance. They are considered to be the most destructive and difficult nematode pest to control in tropical and subtropical countries (Simpson and Starr, 2001) Moreover, their involvement in many disease complexes together with their a bility to break most plant resistance contribute significantly to their importance as global pest of vegetables (Luc et al., 2005) As a group, Meloidogyne spp are estimated to cause global losses of US $157 billion (Abad et al., 2008) Important crops around the world such as corn, wheat, plantains, rice, cassava, and potato can be infected by single or multiple species of root knot nem atodes (Manzanilla Lopez and Starr, 2009)

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16 Meloidogyne incognita M. javanica M. arenaria and M. hapla are considered the mo st important root knot nematode species worldwide, given their wide host range and their global distribution (Moens et al., 2009) Some of these species are espec ially important to tomato production. Yield reductions i n tomato from root knot nematodes have been reported glo bally to be higher than 40% (Reddy, 1985) Several species within this genus are reported in Florida infecting tomato including: M incognita M. arenaria M. javanica M. enterolobii and recently reported M. haplanaria Meloidogyne haplanaria the Texas peanut root knot nematode was first described from Texas in 2003 It reduc ed yields on peanut but it was less aggressive and less widely distributed compared to crop impacts induced by the more widely distributed M. javanica and M. arenaria In addition to peanut, the M. haplanaria host range includes tomato, pepper, some legumes, and radish. It was also reported that the re sistance conferred by the Mi gene in tomato was not effective against this nematode (Bendezu et al., 2004) General Nematode Management Practices A number of nematode management strategies have been consid ered including use of; cultural practices, resistant cultivars, chemicals, soil solarization, fumigation, trap crops, organic amendments, and biological control agents. The integration of different management approaches is generally considered a requireme nt to maintain nematode population densitie s under the economic threshold and to minimize the potentia l damage on the crop (Barker and Olthof, 1976) Cropping history and information about common nematode species in the field and previous crops planted is t he first thi ng to do before establishing a cro p in order to determine the best management pr actices that fit

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17 in the agricultural system, followed by a continuous monitoring of the soil and root s to evaluate the efficacy of the management plan (Roberts, 1993) Cultural Control Several cultural practices are useful in suppressing populations of plant parasitic nematodes. Some of these are : early crop destruction following harvest, crop rotation, fallowing, and use of cover crops, flooding, soil amendments and infected plant removal. Crop rotation is one of the oldest and most important cultural nematode management strategies. This method involves seasonally alternating a poor or non host crop with a host crop (Talavera et al., 2009) The success of crop rotation in reducing nematode populations below damaging levels depends on several factors such as accurate identification of the nematode species, h ost range of the given species the presence of alternate hosts in the area such as weeds, and the ability of the nematode to survive in the absence of the host (Widmer et al., 2002) An optimal rotation will also prevent buildup of other parasitic nematode species, and other pests or pathogens that may damage future crops with the rotation sequence By rotating a susceptible host crop with a non host c rop, nematode populations usually are maintained below damaging levels. Other techniques such as fl ooding and solarizat ion of fields have been used to manage nematodes, however they are not widely used as they are labor and resource expensive and are typically considered unsuitable for large scale implementation (Heald and Stapelton, 1990) Although cultural control methods are extremely valuable management tools, they require extensive consideration planning and economic investme nt before successfu l implemented within the field

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18 Chemical Control The management of plant parasitic n ematodes in the soil by chemical means is dependent upon bringing the nematicide into contact with the nematode in concentrations high enough to affect the m The use of ne maticides is generally recommended in situations such as ; high initial population den sity using a highly susceptible plant when the given crop is highly valuable, and when immediate and quick results are needed. For many years chemical treatments have been used to battle the harmful effects of plant parasitic nematodes, but due to increa sing concerns about adverse effects and environmenta l impacts, many have been removed from the market. F or example, methyl bromide was the predominant product for the control of nematodes and many other soil borne pests and pathogens in high value horticul tural c rops. In 2001, production of methyl bromide was halted due to its adverse effects to the environment (Ristaino and Thomas, 1996) Chemical control of nem atodes mainly relies on fumigant and non fumigant nematicides the differences between these types of nematicides are based on the grade of dispersion on the soil. Fumigant nematicides are applied as liquid in the soil Upon contact with air the fumigant v aporizes and moves through the soil in a gaseous phase The use of broad spectrum fumigants helps to significantly reduce the nematode abundance in the soil before planting Some of the fumigant molecules have been designed to exclusively target nematode s whereas other compounds primarily target other organism such as soil borne pathogens and weed seeds (Noli ng, 199 9). Non fumigants can have contact or systemic activity, and are applied as liquid or granular formulations and are typically incorporated in t he soil or applied through chemigation

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19 Generally, non fumigant nematicides have a reduced efficacy compared to fumigants ( Giannakou, et al. 2005 ). Some of the fumigant nematicides that are currently registered for use in tomato are : 1,3 d ichloropropene m etam p otassium d imethyl d isulfide and c hloropicrin but none are as effective as methyl bromide (Zasada et al., 2010) M ost studies conducted for evaluating non fumigant nematicides have shown that they are less consist ent for controlling nematodes and obtaining consistent economic returns to the grower (Noling, 1999) Biological Control Concerns about the environmental hazards of using chemical nematicides and limited alternative crops for rotation have led to the development of biological control agents as a component of crop protection. Biological control is another strategy used for controlling pests worldwide. De Batch ( 1964) defined biological control as the action of parasites, predators or pathogens i n maintaining another organism density at a lower average th There are several organisms such as viruses, fungi, predatory nematodes, and mites that attack nematodes in the soil. Mechanisms of biocontrol of nematodes inclu de predation, pathogenicity and toxicity (Sharon et al., 2001) Some of the most desirable characteristics for a biological control agent are ; host specificity, capability for mass production easy application using standard equipment, not harmful to the environment, provide control for an extended period of time, and have potential for establishment and recycling, etc. The more desirable chara cteristics that an organism has, the better candidate it is as a biological control.

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2 0 Certain b acteria and fungi are major parasites of plant parasitic nematodes. For example, the parasitic fungi Nematophthora gynophila and Verticillium chlamidosporium attack the developing female of wheat cyst nematode, Heterodera avenae (Kerry, 1982) There are two major types of bacteria that are antagonistic to nematodes. The first type includes bacte ria that are pathogenic to nematodes such as Pasteuria penetrans and the second includes those that produce compounds that are toxic to nematodes, for example Bacillus subtilis Pasteuria includes several species that have shown potential for management o f plant parasitic nematodes that attack many agricultural crops. These gram positive, mycelial, endospore formi ng bacteria are mainly obligate parasites of nematodes in that they cannot survive without their host. Pasteuria species are ubiquitous in most e nvironments and are found in different parts of the world. Pasteuria penetrans i s probably the most studied species of Pasteuria within laboratory and field experiments Pasteuria penetrans is an obligate parasite of root knot nematodes (Lamovsek et al., 2013) Plant Resistance Plant resistance is the ability of a plant to limit the grown or development of any detrimental organism. Plant resistance is considered the foundation of integrate nematode management For several decades natural sources of resistance have been found and bred into commercial cultivars. However, the presence of resistance breaking populations as result of a complex interaction between the plant, nematode and environment is bec oming more common (Davies and Elling, 2015) With the availability of germplasm containing nematode resistance genes and high tech molecular transfer techniques, resistant cultivars should become increasingly a primary management tactic of nematodes within crop production Resistant crops provide an effective and

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21 economical method for managing nematodes in both hi gh and low cash value cropping systems. Several resistant cultivars are commercially available for management of root knot nematodes in tomato R esistant cultivars containing the Mi gene have been extensively used for more than 45 years and provide protection against several species of root knot nematodes. The Mi gene was initially found in Lycopersicon peruvianum a wild type of tomato, and incorporated into commercial cultivars (Roberts, 1995) However, there are some concerns about the use of plant resistance, and its long term effectiveness. A major problem with the Mi gene is the lack of horticultural characters and of resistance t o other key fungal and bacterial pathogens (Noling, personal communication). The over reliance on a single resistant cultivar will almost certainly select for virulent races of Meloidogyne capable of overcoming the resistance. Therefore, integration of multiple management practices will help to efficiently manage nematodes and avoid outbreak of potential virulent populations (Roberts and Thomason, 1989; Haroon et al., 1993; Verdejo Lucas et al., 2009) Resistance Management and Mi Gene The use of grafted tomato plants ha s increased during recent years. This technique allows growers to use nematode resistant rootstocks and introduce scions with desirable fruit characters. Some of the se rootstocks carry the Mi gene in their genome providing resistance to certain s pecies of root knot nematodes. The Mi gene is a single dominant gene that confers resistant to M. javanica M. incognita and M. arenaria which are the most destructive root knot nematode species on tomato ( Milligan et al., 1998) This constitutive gene produces a hypersensitive response in the

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22 plant initiating a programed cell death around the area of nematode feeding (Dropkin et al., 1969) The Mi gene confers resistance and not immunity to nematodes, a few juveniles can penetrate the roots and slowly develop with little to no reproduction (Talavera et al., 2009) However, this reproduction level may be influenced by the gene dosage of the cultivar Some research has sho wn that M. javanica presented higher levels of reproduction on heterozygous cultivars compared to homozygous resistant plants (Tzortzakakis et al., 1998) As consequence of the continuous use of Mi to mato cultivars, resistance breaking populations have emerged and are becoming a common problem for many growers In addition, the heat sensitivity of the Mi gene has also been demonstrated in field and laboratory studies at temperatures above 28 o C (Araujo et al., 1982; Ornat and Sorribas, 2008; Devran et al., 2010; Verdejo Lucas et al., 2013) Other sources of nematode resistance such as Mi 2 through Mi 8 genes, Me and N genes were originally found in Lycopersicon species and pepper. However, these genes also become unstable at temperatures higher than 25 o C and completely lose their resistance at 32 o C. New thermostable genes are under study and evaluatio n but they are not yet commercially available (Jablonska et al., 2007) Another limitation to the use of Mi gene cultivars involves root knot nematode species such as M. enterolobii to which Mi resistant cultivars are not effective W ith the continuous spread and esta blishment of nematode species into new geographical areas other root knot nematode species that affect tomato and are not a ffected by the

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23 Mi gene are bec oming increasingly important (Liu and Williamson, 2006; Kiewnick et al., 2009) In August 2015, the UF/IFAS Nema tode Assay Lab reported a virulent root knot nematode species infecting a resistant cultivar of tomato from Naples, Collier County, Florida. The identity of this new resistance breaking population was confirmed as M. haplanaria based on molecular technique s and morphological characters (Joseph et al., 2016) Meloidogyne haplanaria was reported for the first time in Florida and it is important to study this new nematode because of the potential implications that coul d and agronomic crops. Information on crop nematode relationship s is vital for growers to decide on economically viable managemen t strategies within their own crop production systems. Such information is a prerequisite to design effective nematode management strategies and advisory programs. Hence, this research will provide basic information to understand biological aspects of this newly discovered root knot nematode species The impact of different nematode i nitial population densities and temperatures on the response of different Mi gene resistant tomato cultivars towards M. haplanaria will be studies. We will establish a basel ine threshold to be used in further research of management practices to reduce damage associated with this nematode T he objectives of this study are : 1. To determine the damage thresholds of M. haplanaria on the Mi gene containing resistant tomato cultivar 2. To determine the impact of temperature on stability of the Mi gen e and compare the developmental rates of M. haplanaria M. incognita and M. enterolobii

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24 3. To compare the respons e of Mi gene resistant cultivars and rootstocks to infection by M. haplanaria M. incognita and M. enterolobii

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25 CHAPTER 2 DAMAGE POTENTIAL OF M. HAPLANARIA ON THE MI GENE CONTAINING RESISTANT TOMATO CULTIVAR Introduction Root knot nematodes ( Meloidogyne spp. ) a re economically important pests worldwide with over 5,500 plant hosts (Trudgill and Blok, 2001) Solanaceous plants such as tomato, potato eggplant, and pepper are among the main hosts for root knot nematodes. In 2015, the Texas root knot nematode, Meloidogyne haplanaria was reported for the first time in Florida attacking a Mi resistant tomato rootstock grown in Naples, FL. The current kn own distribution of M. haplanaria is limited to Texas, Arkansas, and Florida (Eisenback et al., 2003; Churamani et al., 2015; Joseph et al., 2016) Host range studies revealed that M. haplanaria can parasitize several legume and crucifer crops (Eisenback et al., 2003) and has also been s hown to infect M. arenaria susceptible cul tivars of peanut, garden pea, and radish (Bendezu et al., 2004) The degree of damage caused by a pa rticular nematode species is largely determined by relating pre plant initial soil population densities to growth and yield. The minimal population density that causes measurable reduction in plant growth or yield varies with nematode species, host plant, cultivar and environment (Barker and Olthof, 1976) D amage threshold is the population density in which measurable plant damage or yield loss occurs Thes e thresholds depend on nematode species, crop, soil type and environmental conditions (Ferris, 1978; Trudgill and Phillips, 1997) For e xample, the Nematode Assay Lab at the University of Florida considers the damage threshold for ro ot knot nematodes on tomato under field condit i ons at 1/100 cm 3 of soil whereas for corn it is 80/100 c m 3 (Unpublished data). This variability on the damage threshold is applicable for each plant parasitic nematode attacking any particular crop.

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26 Damage functions are generally defined as mathematical, expressions relating initial populations and yield; however, more complex and specific models that consider economics have also been developed (Seinhorst, 1965; Ferris, 1978) It i s important t o understand how initial population densities can affect the normal cou rse of plant growth, development and yield; in tomato for example, only a few root knot nematodes in the soil can induce plant symptoms such as chlorosis, wilting, and stunting that ca n be confused with nutr ient deficiencies, and which ultimately, compromise tomato yield. These symptoms are the physiological respons e of the plant to the limited uptake of nutrients and water caused by the presence of nematodes inside the root syst e m In contrast to this situation, when root knot nematodes are present the response of the plant can be different than expected ; at low numbers and under certain condition s they can enhance plant growth and yield, however this effect is followed by a dramatically reduction in root growth as numbers increase (Barker and Olthof, 1976) High initial population densities not only affect crop yield and fruit quality, but interfere with the level of resistance conferred by the Mi gene Results presented by Maleit a et al. (2012) sugge st t hat the Mi gene only provides partial resistance ; in the scena rio of high population densities the plant and root growth is reduced and reproductive paramet ers for the nematodes increase. In order to develop and predict potential damage from new species of nematode s it is important to consider the relation between initial population densities and plant growth and yield Meloidogyne haplanaria is a recently discovered root k not nematode species and so far the impact of M. haplanaria ini tial popula tion density on yield reduction of tomato is unknown The objective of this research was to identify an initial

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27 damage threshold level of M. haplanaria on both resistant and susceptible tomato cultivars under greenhouse conditions. This research will provide preliminary damage threshold values and baseline information for future micro plot and field experiments. Materials and Methods Plant Material Tomato cultivars Sanibel (Reimer S eeds ; Saint Leonard, Maryland ), and Rutgers ( Burpee & Co Warminster Pennsylvania ) were used in this study. Seedlings were grown on Miracle G ro P otting mix ( The Scotts Company, Marysville, Ohio) and were maintained inside a growth chamber at 26 C and watered daily. Inoculum Preparation Meloidogyne haplanaria inoculum was obtained from a pure population multiplied on susceptible Rutgers tomato This population was identified using the protocol described by Joseph, et al. ( 2016 ) Heavily infest ed tomato roots were used to extr act eggs using the NaOCl method described by Hussey and Barker ( 1973) C lean roots were choppe d and blended with tap water for one minute. The egg suspension was then transferred into a glass bottle with 0.5% NaOCl, and mixed thoroughly for 2 minutes. The suspension was poured onto nested 75 m and 25 m sieves respectively and rinsed with tap water to remove all the residu al NaOCl. The suspension was centrifuged at 3500 rpm for 3 minutes, and the supernatant was discharged and refilled with sucrose solution (454 g/ L) The new sucrose solution was centrifuged at 3500 rpm for 3 minutes and the supernatant with the eggs was poured on a small 25 m me sh, rinsed with tap and collected in a clean tube for counting.

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28 Pot Preparation and Inoculation o f M. haplanaria Four week old tomato see dlings were transplanted into 15.24 cm diameter pots filled with 1000 cm 3 autoc laved sandy loam soil. Nine initial population densities of M. haplanaria were inoculated (0, 0.25, 1, 2, 4, 8, 16, 32, 64 eggs and J2 / cm 3 of soil ) on to resistant (Sanibel) and a susceptible (Rutgers) tomato by pipetting inoculum solution in to four 3 cm deep holes in the soil of eac h pot After 48 hours, the seedlings were transplanted in to the pots and maintained in a greenhouse with temperature 28C 2. Pots were arranged on greenhouse tables using a randomized block design with eight replicates per treatment. Plants were wate red daily and fertilized 20 days after transplanting with 3 g Osmocote Plus Smart R elease Plant F ood (15 9 12, N P K ) (The Scotts Company, Marysville, Ohio ) per pot. Data Collection After 60 days, plants w ere harvested, and r oot gall index was assessed using the rating scale from 0 to 10 des cribed by Zeck ( 1971 ) Total numbers of egg s masses were ascertained after staining the whole root with 0.0015% p hloxine B f or 20 min at room temperature. After recording total egg mass count, eggs were extracted using the NaOCl method as described above Eggs were counted from 1 ml aliquot of egg suspension under an inverted microscope ( Olympus CK30 ; Center Valley, Pennsylvania ) The reproductive factor was obtai ned from the division of the final population by the initial population. This experiment was repeated one time Collected data was analyzed according to the general lineal model and if using SAS 9.1.3 software (SAS Institute Inc. ; Cary, North Carolina ) a nd R studio (RStudio, Inc. ; Boston, Massachusetts ) R egression analysis was performed on reproductive

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29 parameters Seinhorst's model y = m + (1 m) z P T (Seinhorst, 1965) was fitted to the data in R In this model y is the relative yield (the ratio between the yield at a given Pi and the average yield at Pi T ) with y = 1 at P i T) m is the minimum relative yield (the value of y at very large Pi), P (= Pi) is the initial nematode p opulation density at the time of transplanting, and z is a constant < 1 with z T = 1.05. Results The data sets from the repetitions were consistent on all the evaluated parameters so the data from the repetitions was combined for analysis Meloidogyne haplanaria was able to reproduce at all initial population densities on both Sanibel and Rutgers tomato Tomato cultivars and initial population densities ( Pi ) had a n effect ( P ) on reproductive parameters. Signifi cant differences across the were obtained in the following nematode reproductive parameters for Rutgers and Sanibel respectively: egg masses ( P < 0.0001 ; P < 0.0001 ), total eggs ( P < 0.0001 ; P < 0.0001 ) root gall index ( P < 0.0001 ; P < 0.0001 ) and eggs / g of root ( P < 0.0001 ; P < 0.0001 ) and reproductive factor ( P < 0.0001; P < 0.0001) The regression analysis of the reproductive parameters are presented on figure s 2 1 2 2 2 3, and 2 4 the data fitted on a logarithmic model and it is possible to observe a separation on the curves of Rutgers and Sanibel; in addition the curves start flattening around initial populations of 32 eggs J2/ cm 3 of soil. T he regression analysis on reproducti ve factor displayed on figure 2 5 showed that Rutgers was significantly high compared to San ibel at the lowest initial population density ( 0.25 eggs J2/ cm 3 ) ( P < 0.0001). The regression analysis presented a negative slope for Rutgers and Sanibel ( 0.5246; 0.2129) The Seinhorst model fitted for shoot height (Figure 2 6) and root length (Figur e 2 8) for both Rutgers and Sanibel but shoot

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30 weight fitt ed only for Rutgers (Figure 2 7 ). The tolerance limit (T) was determine at 1 and 3 eggs J2/ cm 3 soil for root length and plant height parameters for both cultivars Rutgers and Sanibel; however, the model did not fit well for root weight of either cultivar Discussion Reproduction of M. haplanaria was observed to increase in both tomato c ultivars Rutgers and Sanibel indicating that both tomato cultivars are host s fo r M. haplanaria and suggesting that plant damage was correlated with high initial populations and reproductive output Root galling severity, number of egg masses, and total number of eggs were observed to increa se with the Pi inoculum level of M. haplana ria indicating their virulence on the tested tomato cultivars This is in agreement with several other studies (Ei senback et al., 2003; Bendezu et al., 2004; Joseph et al., 2016) This study did not find shoot weight and length, and root length and weight to be well correlated with Pi Plant growth parameters were not very informative or consistent through the different initial population densities. The Seinhorst model was fitted for plant height and root length against Pi for both Rutgers and Sanibel but shoot weight fitted only for Rutgers (Figure 2 9) The tolerance limit ( T ) was 1 a nd 3 nematodes/ cm 3 soil for root length and plant height parameters for both Rutgers and Sanibel indicating that these cultivars are not suitable to plant in Meloidogyne haplanaria infested areas. Nematode growth parameters were more informative and consist ent across the initial population densities and cultivars. Rutgers presented higher reproduction than Sanibel as expected. As the population increases, there was also an increase of the egg masses, total eggs, RGI and eggs / g of root. These results could represent the breaking of the Mi resistance on Sanibel plants. It wa s also observed that the carrying capacity for the nematode infection was around 32

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31 eggs J2/ cm 3 of soil. Studies performed by Inserra et al, ( 1983) demonstrated the different tolerance levels for susceptible and resistant cultivars of alfalfa to M. hapla ; they determine the tolerance limit in 1.6 and 7 eggs/ cm 3 of soil for the susceptible and resistant cultivars respectively. Additionally, studies performed from Di Vito (Di Vito and Ekanayake, 1984; Di Vito et al., 1991) determined different tolerance levels depending of the nematode species and the type of experiment (pot, greenhouse, field, etc). The refore, the data collected on this study provided a preliminary tolerance value to be used for future micro plots or field experiment s Additionally, the reproductive factor (Rf) for both cultivars showed a negative slope across the different population de nsities, meaning that the Rf decreased as the initial populations densities increased. Rutgers presented an Rf of 27.9, whereas Sanibel had 13.82 under greenhouse conditions. Experiments performed by Fourie et al (2010) evaluated the host suitability of a the susceptible and resistant cultivars Prima2000 and LS5995 in hail net cage and micro plot conditions, and the Rf they reported also presented a reduction of this factor as the initial populations increased; they also reported higher results on micro plo t conditions than in the hail net cage experiment.

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32 Figure 2 1 Effect of initial population densities on total egg masses of M. haplanaria on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16 32 and 64 eggs and J2/ g of soil in greenhouse conditions. Figure 2 2 Effect of initial population densities on total production of M. haplanaria eggs on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16, 32 and 64 eggs and J2 / g of soil in greenhouse conditions y = 52.921ln(x) + 161.32 R = 0.311 P < 0.0001 y = 47.204ln(x) + 117.27 R = 0.3473 P < 0.0001 0 100 200 300 400 500 600 700 0 10 20 30 40 50 60 Egg masses Initial population densities Rutgers Sanibel y = 82077ln(x) + 61580 R = 0.5028 P < 0.0001 y = 66529ln(x) + 29975 R = 0.4811 P < 0.0001 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1000000 0 10 20 30 40 50 60 Eggs Initial population densities Rutgers Sanibel

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33 Figure 2 3 Effect of initial population densities on root gall index of M. haplanaria on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16 32 and 64 eggs and J2/ g of soil in greenhouse conditions. Figure 2 4 Effect of initial population densities on the production of M. haplanaria eggs / g of root on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16 32 and 64 eggs and J2/ g of soil in greenhouse conditions. y = 1.3758ln(x) + 4.4226 R = 0.8346 P < 0.0001 y = 1.6654ln(x) + 2.748 R = 0.8173 P < 0.0001 0 2 4 6 8 10 0 10 20 30 40 50 60 RGI Initial population densities Rutgers Sanibel y = 4119.3ln(x) + 1813.4 R = 0.4268 P < 0.0001 y = 2628.4ln(x) + 1309.9 R = 0.5164 P < 0.0001 0 10000 20000 30000 40000 50000 60000 0 10 20 30 40 50 60 Eggs per gram of root Initial population densities Rutgers Sanibel

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34 Figure 2 5 Effect of initial popula tion densities on the reproductive factor of M. haplanaria / g of root on tomato cultivars Rutgers and Sanibel, 60 days after inoculation of 0, 0.25, 1, 2, 4, 8, 16 32 and 64 eggs and J2/ g of soil in greenhouse conditions. Figure 2 6 R elationship betwe en initial population density (P i) of M. haplanaria and relative shoot height (cm) on tomato cultivars Rutgers and Sanibel. Plants were harvested after 60 days and each point in the graph represents a mean of 8 replications and the line is the predicted function obtained when the data was fitted to the Seinhor st model The parameters obtained were for Rutge rs: Y = 64.64; m = 0.14 ; T = 3.25; and for Sanibel Y = 66.27; m = 0.83; T = 3.14. y = 0.5246x + 34.881 R = 0.2479 P < 0.0001 y = 0.2129x + 17.755 R = 0.1615 P < 0.0001 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 Reproductive factor (Pf/Pi) Initial population densities Rutgers Sanibel

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35 Figure 2 7 R elationship betwe en initial population density (P i) of M. haplanaria and relative shoot fresh weight (gm) on tomato cultivars Rutgers and Sanibel. Plants were harvested after 60 days and each point in the graph represents a mean of 8 replications and the line is the predicted function obtained when the data were fitted to the Seinhor st model. The parameters obtained were for Rutg ers: Y = 64.65; m = 0.15; T = 7.9; and for Sanibel Y = 66.27; m = 0.83; T = 0.64 Figure 2 8 R elationship between initia l popul ation density (P i) of M. haplanaria and relative root length (cm) on tomato cultivars Rutgers and Sanibel. Plants were harvested after 60 days and each point in the graph represents a mean of 8 replications and the line is the predicted function obtained when the data were fitted to the Seinhorst mode l. The parameters obtained were for Rutgers: Y = 24.02 ; m = 14.55 ; T = 0 .9; and for Sanibel Y = 24.86 ; m = 13.74 ; T = 1.2

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36 CHAPTER 3 IMPACT OF TEMPERATURE ON THE STABILITY OF THE MI GENE, AND COMPARISON OF THE DIFFERENT DEVELOPMENTAL PROCESSES OF M. HAPLANARIA M. INCOGNITA AND M. ENTEROLOBII Introduction Resistance against Meloidogyne species has been reported in many agricultural crops. Tomato is one of the few crops in which Meloidogyne resistance has been widely used, and commercial resistance cultivars and rootstocks are available for tomato production (Lpez Prez et al., 2006; Danso et al., 2011; Cortada et al. 2012) Resistance against M. incognita M. javanica and M. arenaria has been developed in the widely used tomato cultivars bearing the Mi gene (Ornat et al., 2001) However, expression of resistance is affecte d by different factors such as soil temperature, species and populations of Meloidogyne gene dosage, and tomato genetic background (Ornat and Sorribas, 2008) Thus, tomato cultivars should be carefully chosen, particularly when they are followed by a nematode susceptible crop (Lpez Prez et al., 2006) Temperature has a direct effect on plant growth, root knot nematode life cycle, and the interaction bet ween resistant plants and nematode populations. The time to complete the life cycle of M. incognita varies from 17 to 57 days depending temperature ( Dropkin, 1963) and it has been observed that M. incognita survival and reproduction occurs within the temperature range of 15.4 C to 35 C (Dropki n, 1963) However, these values can vary from Meloidogyne species to species and from host to host (Wong and Mai, 1973) High temperatures and heat stress can physiologically affect t omato plants. Several experiments demonstrated that exposure to long periods of high temperatures

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37 above 28 C can affect the normal development of the plant causing heat stress and interfering with the normal physiological processes of the plant such as photosynthesis, assimilates partitioning and fruit setting (Camejo et al., 2005) This heat stress creat es the ideal conditions for nematodes to attack the plant and increase s the severity of the damage by compromising plant development (Haroon et al., 1993; Ornat and Sorribas, 2008) In addition to affecting vital processes of the plant, high temperature and heat stress can also interfere with the stability of the Mi gene. High temp eratures can cause an irreversi ble loss of Mi gene resi s tance at high soil temp e ratures (>28 C ). Efficacy of the Mi gene can be hamper by m utation(s) on it or a gene required in the Mi mediated resistan ce pathway and a failure on transcription due to DNA methylation (Dropkin et al., 1969) Nevertheless, this relationship between temperature and breaking of resistance has shown some inconsistency on the results Some studies have suggested that exposure to short periods of temperatures above 30 C can partially reduce Mi gene expression and resistance can be recovered However, if exposure is greater than 4 days, there will be a complete breakdown of the resistance is complete and irreversible (Marques de Carvalho, et al., 2015) Results from other in vitro studies conducted on tomato root explants suggest that the Mi gene can persist after exposures to temperatures up to 3 1 C; maintain partial resistance at 34 C and com pletely loss es the resi stance at 37 C (Dropkin et al., 1969; Abdul Baki et al., 199 6; Verdejo Lucas et al., 2013; Marques de Carvalho et al., 2015) The objectives of this study were to understand the interaction betwee n the resistant cultivar Sanibel bearing the Mi gene and the susceptible Rutgers with M.

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38 haplanaria at three different constant temperatures and to determine how these temperatures affect their life cycle compared to other common species of Meloidogyne This information is relevant to understand the damage response from this new ly discovered root knot n ematode species to temperature extremes and compare damage to other Meloidogyne species endemic to Florida Materials and Methods Inoculum Preparation Meloidogyne haplanaria inoculum was obtained from a pure population multiplied on susceptible Rutgers tom ato This population was identified using the protocol described by Joseph, et al. ( 2016 ). Heavily infested tomato roots were used to extr act eggs using the NaOCl method described by Husse y and Barker ( 1973) C lean roots were chopped and blended with tap water for one minute. The egg suspension was then transferred into a glass bottle with 0.5% NaOCl, and mixed thorou ghly for 2 minutes. The suspension was poured onto nested 75 m and 25m sieves respectively and rinsed with tap water to remove all the residu al NaOCl. The suspension was centrifuged at 3500 rpm for 3 minutes, and the supernatant was discharged and refi lled with sucrose solution (454 g/ L) The new sucrose solution was centrifuged at 3500 rpm for 3 minutes and the supernatant with the eggs was poured on a small 25 m mesh, rinsed with tap water and collected in a clean tube for counting. Plant Material Tomato cultivars Sanibel ( Reimer Seeds; Saint L eonard, Maryland), and Rutgers (Burpee & Co, Warminster, Pennsylvania) were used in this study. Seedlings were grown into Miracle Gro Potting Mix (The Scotts Company, Marysville, Ohio) and maintained for four weeks inside growth chamber at 26 C

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39 Inoculum Preparation Nematode populations of M. incognita M. enterolobii and M. haplanaria were extracted from a pure population maintained on susceptible Rutgers tomato Heavily infested tomato roots were used to extract eggs using the NaOCl method described by Hussey and Barker (1973) C lean roots were chopped and blended with tap water for one minu te. The egg suspension was then transferred into a glass bottle with 0.5% NaOCl, and mixed thoroughly for 2 minutes. The suspension was poured onto nested 75 m and 25m sieves, respectively, and rinsed with tap water to remove all the residual NaOCl. The suspension was centrifuged at 3500 rpm for 3 minutes, and the supernatant was discharged and refilled with sucrose solution (454 g/L) The new sucrose solution was centrifuged at 3500 rpm for 3 minutes and the supernatant with the eggs was poured on a smal l 25 m mesh, rinsed with tap water and collect ed in a clean tube for counting Four week old tomato seed lings were then transplanted in to a 3.1 cm dia m and 21.6 cm deep plastic cones filled with 120 cm 3 of autoclaved sandy loamy soil. After 48 hours, eac h cone was inoculated with 360 eggs and J2, and the cones were randomly di stributed on racks. Cones with the t reatments were placed i n to separate temperatu re controlled growth chambers at 24 C, 28 C, and 32 C, and maintained at 60% relative humidity and 14 L: 10D photoperiod. Plants were watered daily and fertilized 20 days post inoc ulation with 20 ml of a Miracle Gro All Purpose Plant Food (24 8 16; N P K) (The Scotts Company, Marysville, Ohio) Data collection Forty days after inoculation plants were harvested ; d ata on root gall index (RGI) was assessed using the rating scale described by Zeck ( 1971 ) Plant roots were cleared and stained using the acid fuchsine method as described by By b d et al. ( 1983) in order

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40 t o enumerate egg masses and observe the developmental stage s o f root knot nematodes inside the roo ts Eggs were extracted using the NaOCl method by Hussey and Barker ( 1973) The number of eggs, J2, J3, and J4 produced / g of root were counted based on the diff erences in developmental stages described by Moens et al. ( 2009) This ex periment was repeated one time. Collected data were analyzed using SAS 9.1 .3 software (SAS Institute Inc. ; Cary, North Carolina ). Nema tode development and infectivity assessment data were analyzed after counts were l og (x + 1) transformed for analysis to fulfil the criteria for normality. Mean separation among treat ments was do test ( P 0.05 ) Regression analysis was also performed to determine the response of the different nematode species at the increase of temp erature. Results The data sets from the repetitions were consistent on all the evaluated parameters, so the data from the repeti tions was combined for analysis Visual observations of both cultivars Rutgers and Sanibel maintained at 24 and 28 C showed healthy growth (Figure 3 1 A and B), whereas plants that were kept at 32 C expressed symptoms of heat stress such as stunting, wilting, necrosis, a nd reduced leaf area (Figure 3 1 C). The effects of temperature on total numbe r of eggs, total egg masses, root gall index and eggs / g of root for both tomato cultivars and the three root knot nematode spec ies ( M. incognita, M, enterolobii and M. haplanaria ) are presented i n figures 3 2, 3 3 3 4 and 3 5 Figure 3 2 shows the transformed data for egg masses of the nematode species M. enterolobii M. haplanaria and M. incognita inoculated on th e tomato cultivars Rutgers and Sanibel and exposed at temperatures of 24, 28 and 32C. These results

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41 present significant differences within each group, and M. haplanaria exhibit the highest re sults across the different treat ments in both cultivars, except in Rutgers at 24C where M. enterolobii presented the highest number of egg masses. E gg prod uction is observed on figure 3 3 I t is noticeable that M. enterolobii has a larger production of eggs on Sanibel at 24 C Rutgers at 28 C and both cultivars at 32 C F igu re 3 4 presents the results for RGI. F rom here it is possible to notice a variation on this parameter for each nematode species. A t 24 C M. incognita and M. enterolobii reported the largest galling in both tomato cultivars whereas M. haplan aria in Sanibel had the largest galling at 24 C At 28 C it was observed a similar pattern on Rutgers, but in Sanibel M. haplanaria and M. enterolobii had similar galling. Meanwhile, at 32 C M. entero lobii and M. haplanaria exhibit the largest results in Rutgers; M. haplanaria produced the largest galls on Sanibel. The results of eggs / g of root presented on figure 3 5 showed that M. incognita had a better reproduction on Rutgers at 24 C whereas M. enterolobii and M. haplanaria on Sanibel had a better reproduction at 28 C Meloidogyne enterolobii was significantly higher than M. haplanaria and M. incognit a in Rutgers, but for Sanibel, M. haplanaria had the largest result. At 32 C M. enterolobii present ed higher results on Rutgers and Sani bel. Figure 3 6 presents t he results of the linear regr ession analysis on egg masses for M. enterolobii and shows that the slope for the regressi on line was higher on Rutgers (237.86) than in Sanibel (21.42). The same tendency was observed in Rutgers ( figure 3 7 ) for the parameter egg s / g of root in which Rutgers had a larger slope than

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42 Sanibel (120.74; 27.29) and RGI where Sanibel showed a larger slope in compar ison to Rutgers (0.270; 0.083) (Figure 3 8). A different res ponse was observed on the slop es of the egg masses (F igure 3 9 ) where the linear regression resulted with a negative slope on the cultivar Rutgers ( 12.969), whereas the slope fo r Sanibel was positive (3.395). Linear regression analysis of M. incognita reproductive parameters eggs and eggs/ g of root at different temperatures on the cultivars Rutgers and Sanibel are presented in figures 3 10 and 3 11 For both parameters, a neg ative slope was observed for cultivar Rutgers ( 55.104; 29.487), while the cultivar Sanibel resulted in posit ive slopes of 9.541 (eggs) and 10.058 (eggs/ g of root) Conversely, RGI presented negative slopes for Rutgers and Sanibel ( 0.177; 0.0521) (Fi gure 3 12 ) Reproductive parameters of M. haplanaria such as egg s, egg masses and eggs/ g of roots were fitted on a polynomial regression analysis; however not significant differences were observed on this model (data not shown) RGI was the only parameter that fitted on a linear regression and presented significant differences for Rutgers and Sanibel; the slopes for these lines were 0.00093 for Rutgers and 0.259 for Sanibel (Figure 3 13 ) Figures 3 14, 3 15 and 3 16 present the average of the J2 s/ g of root of nematode species M. enterolobii M, haplanaria and M. incognita Results show that the total number of J2 s was significantly higher at 32 C on tomato cultivars Rutgers and Sanibel when inoculated with M. enterolobii (Figure 3 14), and M. haplanaria (Figure 3 15) whereas M. incognita (Figure 3 16) successfully produced J2s on Rutgers at 32 C

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43 The total number J3s and J4s / g of root were not significant for any of the treatments and cultivars (d ata not shown) Discussion Rutgers and Sanibel were b oth negatively impacted by con stant temperatures of 32 C In addition, temperature had a profound effect on nematode development and r eproduction, although differences among nematode species and tomato cultivars were reported Meloidogyne enterolobii is known for naturally infest resistant cultivars of tomato a nd is considered highly virulent (Kiewnick et al., 2009) it was able to reproduce both Rutg ers (susceptible) and root knot nematode resistant Sanibel H owever in our experiment total eggs RGI, and eggs/ g of root of M. enterolobii had a better reproduction on Rutgers than in the resistant Sanibel. The increment of this parameters as the temperatures increased, confirms the responses observed by Juanhua et al. ( 2013) who reported a sustained increase in reproduction of M. enterolobii at temperatures of 28 C and 30 C The same effect but at a reduced level was observe d on the resistant cultivar Sanibel. It was noticed that the total egg masses on Rutgers presented an opposite effect; a reduction on the egg masses as the temperature increased, this result could be the response of the nematode to the high temperature and the plant stress. I nformation regarding varietal, physiological and temperature mediated are unclear response to the effect of M. haplanaria is lacking. Bendezu et al. (2004) reported that M. haplanaria was able to successfully reproduce on Rutgers and Motelle a tomato cultivar that carries the Mi gene and was observed to be resistant to M. arenaria with Motelle a RGI of 3.2 for Rutgers and 3.5 was observed with M. haplanaria with air temperatures lower than 28 C This study reports similar results at a

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44 constant temperature of 24 C for Rutgers, but Sanibel presented a slightly lower value at the same temperature b ut the root gall index increased with temperature. Observations on roots and counting o f different nematode developmental stages at temperatures of 24, 28 and 32 C at 40 days after inoculation, have shown significant differences on the response of cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita At 32 C Rutgers and Sanibel had a production of 32.2 and 22.9 J2s / g of root respectively when they were inoculated with M. enterolobii while with M. haplanaria generated 234.46 J2s / g of root on Rutgers, and 203.23 J2s / g of root on Sanibel. In addition, at 32 C M. incognita produced 145.28 J2s / g of root on the susceptible cultivar Rutgers and 0.68 J2s / g of root on the resistant cultivar Sanibel Similar observations were made by Wong and Mai ( 1973) when they evaluated the life cycle of M. hapla on lettuce at different temperatures; they found that at temperature regimes of 26 .0 to 32.2 C mature females could be observed 14 days after inoculation T he refore the results from this study suggest that M. haplanaria can complete their life cycle in a shorter period of time than M. enterolobii indicating that this species could be even more virulent than M. enterolobii on resistant tomato cultivars. The response of Rutgers and Sanibel to in fection by M. incognita was in agreement with previous findings where higher reproduction was observed on Rutgers and lower reproduction on Sanibel. Additionally, we found that the development of M. incognita wa s slower th an M. enterolobii and M. haplanaria In this study the presence of J3s and J4s were not observed with M. incognita at 32 C in Rutgers and Sanibel

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45 In conclusion, M. haplanaria wa s able to successfully complete its life cycle in a shorter period of time and generate more new progeny on both a susceptible and a resistant cultivar at temperatures of 32 C ; this is in comparison to the virulent control M. enterolobii and the widely distributed M. incognit a.

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46 Figure 3 1 Impact of temperature on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita at A) 24 C, B) 28 C and C) 32 C Photographs by Lisbeth Espinoza Figure 3 2 Effect of temperature s on total egg production (log x + 1 ) on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C Letters represent significant differences at 5% within the cultivar group. A C B B A C B A B B A C B A B B A C 0 0.5 1 1.5 2 2.5 Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Rutgers Sanibel Rutgers Sanibel Rutgers Sanibel Egg masses (log x + 1)

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47 Figure 3 3 Effect of temperature on the total egg mass production (log x + 1 ) on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C Letters represent significant differences at 5% within the cultivar group. Figure 3 4 Effect of temperature on RGI on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita 40 days after inoculat ion in gro wth chamber maintained at 24 28 and 32 C Letters r epresent significant differences at 5% within the cultivar group. B C A A A B A B C B A C A B C A B C 0 0.5 1 1.5 2 2.5 3 3.5 4 Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Rutgers Sanibel Rutgers Sanibel Rutgers Sanibel Total eggs (log x + 1) A B A B A B A B A A A B A A B B A C 0 1 2 3 4 5 6 Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Rutgers Sanibel Rutgers Sanibel Rutgers Sanibel RGI

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48 F igure 3 5 Effect of temperature on eggs / g of root (log x + 1 ) on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii M. haplanaria and M. incognita 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C Letters r epresent significant differences at 5% within the cultivar group. Figure 3 6 Line a r regression analysis of temperature on eggs on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C B B A A A B A B C B A C A B B A B C 0 0.5 1 1.5 2 2.5 3 3.5 4 Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Me Mh Mi Rutgers Sanibel Rutgers Sanibel Rutgers Sanibel Eggs per gram of root (log (X+1) y = 237.86x 4855.1 R = 0.27681 P = 0.0009 y = 21.427x 204.15 R = 0.1852 P = 0.008 0 1000 2000 3000 4000 5000 6000 7000 8000 22 24 26 28 30 32 34 Eggs Temperatures C Rutgers Sanibel

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49 Figure 3 7 Line a r regression analysis of temperature on eggs/ g of root on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C Figure 3 8 Line a r regression analysis of temperature on RGI on tomato cultivars Rutgers and Sanibel inoculated with M. ent erolobii 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C y = 120.74x 2469.4 R = 0.3278 P = 0.0002 y = 27.298x 501.02 R = 0.2535 P = 0.001 0 500 1000 1500 2000 2500 3000 22 24 26 28 30 32 34 Eggs per g of root Rutgers Sanibel

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50 Figure 3 9 Line a r regression analysis of temperature on egg masses on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C Figure 3 10 Effect of temperature on eggs / g of root (log x + 1 ) on tomato cultivars Rutg ers and Sanibel inoculated with M. incognita 40 days after inoculation in grow th chamber maintained at 24 28 and 32 C y = 12.969x + 440.07 R = 0.7469 P < 0.0001 y = 3.3958x 56.111 R = 0.4235 P < 0.0001 0 20 40 60 80 100 120 140 160 180 200 22 24 26 28 30 32 34 Egg masses Temperature C Rutgers Sanibel y = 55.104x + 2047.6 R = 0.3615 P = 0.0001 y = 9.5417x 178.97 R = 0.8147 P < 0.0001 0 200 400 600 800 1000 1200 22 24 26 28 30 32 34 Eggs Temperature C Rutgers Sanibel

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51 Figure 3 11. Effect of temperature on eggs / g of root (log x + 1 ) on tomato cultivars Rutg ers and Sanibel inoculated with M. incognita 40 days after inoculation in growth chamber maintained at 24 28 and 32 C Figure 3 12 Effect of temperature on eggs / g of root (log x + 1 ) on tomato cultivars Rutg ers and Sanibel inoculated with M. incognita 40 days after inoculation in growth chamber maintained at 24 28 and 32 C y = 29.487x + 1165.2 R = 0.208 P = 0.005 y = 10.058x 227.25 R = 0.6493 P < 0.0001 0 100 200 300 400 500 600 700 800 22 24 26 28 30 32 34 Eggs per g of root Rutgers Sanibel y = 0.1771x + 8.3194 R = 0.593 P < 0.001 y = 0.0521x + 2.6528 R = 0.1847 P = 0.008 0 1 2 3 4 5 6 22 24 26 28 30 32 34 RGI Rutgers Sanibel

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52 Figure 3 13 Effect of temperature on eggs / g of root (log x + 1 ) on tomato cultivars Rutg ers and Sanibel inoculated with M. haplanaria 40 days after inoculation in growth chamber maintained at 24 28 and 32 C Figure 3 14 Effect of temperature on the total number of J2s / g of root on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii 40 days after inoculation in growth chamber maintained at 24 28 and 32 C Letters r epresent significant differences at 5% within the cultivar group. y = 0.0938x + 0.8472 R = 0.1251 P = 0.034 y = 0.2594x 3.8495 R = 0.69778 P < 0.0001 0 1 2 3 4 5 6 22 24 26 28 30 32 34 RGI Rutgers Sanibel C B A C B A 0 5 10 15 20 25 30 35 40 Rutgers Sanibel J2s/ g of root

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53 Figure 3 15 Effect of tempe rature on the number of total J2s / g of root on tomato cultivars Rutgers and Sanibel inoculated with M. haplanaria 40 days after inoculat ion in growth chamber maintained at 24 28 and 32 C Letters r epresent significant differences at 5% within the culti var group. Figure 3 16 Effect of tempe rature on the number of total J2s / g of root on tomato cultivars Rutgers and Sanibel inoculated with M. incognita 40 days after inoculation in growth chamber maintained at 24 28 and 32 C Letters r epresent significant differences at 5% within the cultivar group. B C A B B A 0 50 100 150 200 250 Rutgers Sanibel J2s/ g of root C B A B A B 0 20 40 60 80 100 120 140 160 180 Rutgers Sanibel J2s/ g of root

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54 CHAPTER 4 THE RESPONSE OF MI GENE RESISTANT TOMATO CULTIVARS AND ROOTSTOCKS TO ROOT INFECTION BY M ELOIDOGYNE HAPLANARIA M ELOIDOGYNE INCOGNITA AND M ELOIDOGYNE ENTEROLOBII Introduction Resistance to nematodes can result from the fusion of different genes and traits wit hin a large genetic diversity among wildtype species However, r esistance to root knot nematodes in tomato is conferred by a single dominant gene known as the Mi gene locat ed in the short arm of chromosome 6 of the wildtype Solanum peruvianum (Roberts et al., 1990) The Mi gene is a constitutive gene that produces a hypersensitive reaction in the nematode invaded area blocking or slowing down their development inside the roots (Milligan et al., 1998) The Mi gene is a member of t he nucleotide binding, leucine rich repeated family; this protein family is known for conferring resistance to root knot nematodes, potato aphids, and plant viruses (Rossi et al., 1998; Brommonschenkel et al., 2000) Other sources of resistance that have proved to be heat stable at temperatures above 30 C have been i dentified H owever commercial plant material with this trait not yet available in the market (Marques de Carvalho et al., 2015) Several nematode resistant tomato cultivars are commercially available and these cultivars carry the resistant Mi gene in homozygous ( MiMi) or heterozygous (Mimi) s ome researchers suggested that the genetic background on tomato and pepper have an influence in the expression of the resistance and in the reproductive ability of roo t knot nematodes (Tzortzakakis et al., 1998; Jacquet et al., 2005; Barbary et al., 2014) In contrast, this affirmation is threatened by the emergence of virulent root knot

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55 nematode populations that are able to overcome the resistance an d successfully complete their life cycle generating a profuse infective progeny (Cortada et al., 2009) To enhance the protection provided by resistant cultivars, grafted plants have also been successfully tested and are commercially available for field p roduction Grafting is an agricultural technic in which a scion with desirable fruit characteristics is inserted onto a resistant rootstock (King et al., 2008) The rootstocks used for grafting are mainly resistant to soil borne patho gens and nematodes. Studies have been conducted t o test the efficacy of different rootstocks grafted with different scions or self grafted under root knot nematodes infestations ; and it was determined that some of the rootsto cks materials presented a reduced nematode galling, whereas others had demonstra ted little effect, but final yields were not compromised (Lpez Prez et al., 2006; Kunwar et al., 2015; Owusu et al., 2016) The objective of this research was to study the development and reproduction of M. haplanaria on different tomato cultivars when a resistant rootstock was used for nematode management Materials a nd Methods Plant Material Resistant tomato cultivars Sanibel (Reimer seeds Saint Leonard, Maryland ), and Amelia (Harris Seeds ; Rochester, New York ); the resistant rootstocks, Estamino, and ; Winslow, Main ); were compared with the susceptible Rutgers (mimi) (Burpee & Co, Warmins ter, Pennsylvania) and Monica (mimi) ( seeds ; Winslow Main ). Seedlings were propagated i n Miracle G ro P otting mix (The Scotts Company, Marysville, Ohio) and were maintained inside a temperature controlled growth ch amber at 26 C and watered daily.

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56 Inoculum Preparation The nematode species M. incognita M. enterolobii and M. haplanaria were used for this study. Heavily infested tomato roots were used to extract eggs using the NaOCl method described by Hussey and Barker (1973). Clean roots were chop ped and blended with tap water for one minute. The egg suspension was then transferred into a glass bottle with 0.5% NaOCl, and mixed thoroughly for 2 minutes. The suspension was poured onto nested 75 m and 25m sieves, respectively, and rinsed with tap w ater to remove all the residual NaOCl. The suspension was centrifuged at 3500 rpm for 3 minutes, and the supernatant was discharged and refilled with sucrose solution (454 g/ L The new sucrose solution was centrifuged at 3500 rpm for 3 minutes and the sup ernatant with the eggs was poured on a small 25 m mesh, rinsed with tap water and collected in a clean tube for counting. Pot Preparation and Inoculation Four weeks old tomato seedlings of the different tomato cultivars were transplanted i nto 15.24 cm dia meter pots filled with 1 .000 cm 3 autoc laved sandy loam soil Pots were inoculated with 3 eggs and J2s / cm 3 soil by pipetting nematode solution into four 3 cm deep holes in the soil of each pot. After 48 hours, the seedlings were transplanted into the pots and maintained in a greenhouse with temperatures of 28C 2. Plants were fertilized 20 days after transplant with 3 g of Osmocote Plus Smart Release Plant Food (15 9 12, N P K) (The Scotts Company, Marysville, Ohio) per pot. Data Collection After 60 days, plants w ere harvested, and r oot gall index was assessed using the rating scale from 0 to 10 described by Zeck ( 1971 ) (1971). Total numbers of eggs

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57 masses were ascertained after staining the whole root with 0.0015% phloxine B for 20 min at room temperature. After recording total egg mass count, eggs were extracted using the NaOCl method as described above. Eggs were counted from 1 ml aliquot of egg suspension under an inverted microscope (Olympus CK30; Center Valley, Pennsylvania). The reproductive factor was obtained from the division of the final population by the initial population. This experiment was repeated one time. All count data was analyzed using the statistical software SAS 9.1.3 software (SAS Institute Inc. ; Cary, North Carolina ) A general lineal model (Proc GLM) was used to determine difference within the treatments. Nematode reproductive parameters and root infection count were analyzed using a logarithmic transformation (log x + 1) of the counting data to ful f ill the criteria for normality. Mean separation among treatments was done u sing Tukey test ( P < 0.05 ) Results The data sets from the repetitions were consistent on all the evaluated parameters, so the data from th e repetitions was combined for analysis. The root knot nematode species M. enterolobii M. haplanaria and M. incognita were able to reproduced o n all Mi gene bearing resistant and susceptible cultivars ; Amelia, Estamino, Maxifort, Monica, Rutgers and Sanibel. The cultivars Monica and Amelia were particularly susceptible to infection by all nematode species, showing symptoms of yellowing, wilting, even death by the end of the experiment. As results some p lant parameter such as shoot and root length an d weight were excluded from analysis. No symptoms or plant mortality was observed in the nematode free controls Significant differences were observed in the number of egg masses within the nematode species M. e nterolobii ( P < 0.0001 ) M haplanaria ( P < 0.0001 ) and M.

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58 incognita ( P < 0.0001 ) Among the cultivars inoculated with M. enterolobii Sanib el presented the largest number of egg masses (2 .22 0.03) On the other hand, inoculations with M. haplanaria resulted with the largest production of egg masses on Rutgers (2.35 0.05) and the smallest on Maxifort (1.28 0.10 ) Rutgers infected with M. incognita were used as a susceptible control and they showed the highest number of egg masses (2.26 0.07) ; T omato cultivars Sanibel (1.56 0.13) and Amelia (1.60 0.08) showed the lowest number of egg masses comp ared with the control (Figure 4 1). Total number of eggs diffe red a mong the cultivars when inoculated with M. enterolobii ( P = 0.0009), M. haplanaria ( P < 0 .0001), and M. incognita ( P < 0 .0001). Tomato cultivars inoculated with M. enterolobii p resented differences on the total number of eggs with values between 4.94 0.22 for Estamino and 4.64 0.16 for the cultivar Maxifort. Conversely, in cultivars infected with M. haplanaria the largest number of eggs was observed in Monica (5.19 0.09) whereas the lowest was reported on Sanibel (4.17 0 .06) As response of inoculations with M. incognita Maxifort (4.55 0.09) and Sanibel (4.06 0 .10) had the highest and the lowest number of eggs, respectively (Figure 4 2) The RGI parameter was highly affected by nematode species M. enterolobii ( P < 0 .0001), M. haplanaria ( P < 0 .0001), and M. incognita ( P < 0 .0001). All the cultivars inoculated with M. enterolobii presented a RGI between 8 and 9. Inoculations of the cultivars with M. haplanaria produced a RGI of 7.5 on Amelia and 3 on Sanibel The cultivar Rutgers inoculated with M. incognita showed the largest RGI of 7.3 whereas the lower was reported on Amelia (Figure 4 3).

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59 The number of eggs/ g of root were different among the cultivars Plants infected with M. enterolobii had variation in the number of eggs / g root. C ultivar s Amelia (3.42 0.33) and Rutgers (3.36 0.19) presented the largest number of eggs/ g of root, whereas Maxifort had the lowest number (2.96 0.37) When cultivars were inoculated with M. haplanaria a larger infestation was reported on Amelia (3.78 0.28) or Monica (3.75 0.33) where as the lower results were observed on Sanibel (2.14 0.10) Inoculation s with M. incognita resulted in a low production of eggs/ g of root on cultivar Sanibel (2.02 0.13), meanwhile Maxifort (3.10 0.29) and Amelia (3.10 0.14) had a high prod uction of eggs (Figure 4 4). Discussion In this study, the host suitability of commercially availabl e tomato cultivars w as tested for their reaction to infection by M. enterolobii M. haplanaria and M. incognita Our result s showed that Amelia (root knot nematode resistant) and Monica (root knot nematode susceptible) were the most susceptible to the tested nematode species compared with the other cultivars because of the sev ere damage reflected on the shoot in all the treatments some pl ants presented symptoms of wilting, yellowing, necrosis, even some of them were death R esu l t s showed that all the cultivars tested were highly susceptible to M. enterolobii This affirmation can be noticed particularly on the parameters RGI and total egg s. Sanibel is a root knot nematode resistant and heat tolerant cultivar and presented lower results on the parameters total eggs, RGI and eggs/ g of root when was inoculated with M. haplanaria and M. incognita Estamino and Maxifort are used as nematode resistant rootstock but our result s showed that both were suitable for high roo t knot nematode reproduction.

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60 Maxifort is a cross from Solanum lycopersicum x S. habrochaites known to be a homozygous resistant cultivar (MiMi), but our experiment sho wed infection by M. incognita M. enterolobii and M. haplanaria Cortada et al (2009 ) evaluated the susceptibility of this rootstock against several root knot nematode species such as M. javanica M. arenaria and M. incognita and reported that other homozygous crosses (MiMi) were evaluated on the same experiment and presented a reduced infection of the root knot nematodes. Other authors suggest that the homozygosis and heterozygosis of a cultivar has a direct effect on the gene expression and c an interfere on the normal develop ment of the nematode (Trudgill and Phillips, 1997; Tzortzakakis et al., 1998; Jacquet et al., 2005) From our study the data was not conclusive to determine the resistance or tolerance of any of the evaluated cultivars to M. haplanaria It is recognized that M. enterolobii is a naturally virulent root knot species, and can overcome to the Mi resistance; from the previous experiments we hypothesize that M. haplanaria is also highly virulent, and could possibly be more virulent tha n M. enterolobii We found that none of the evaluated cultivars had resistance against any of the nematode populations tested. We hypothesize that the heat tolerance trai t present on Sanibel may have a partial effect on the resistance of this cultivar to root knot nematodes. Further studies on known tomato resistant crosses should be developed therefore it will be clearer the effect of the gen e dosage on nematode infection.

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61 Figure 4 1. Effect of M. enterolobii M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions. Letters represent significant d ifferences at P < 0.05 within nematode species group Figure 4 2 Effect of M. enterolobii M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after i noculation in greenhouse conditions. Letters represent significant differences at P < 0.05 within nematode species group B B B B B A C D E B A B D B C B A B 0 0.5 1 1.5 2 2.5 3 Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel M.enterolobii M.haplanaria M. incognita Total egg masses (log (x+1)) A A B AB A AB A A B AB A AB B C C A A D 0 1 2 3 4 5 6 Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel M.enterolobii M.haplanaria M. incognita Total eggs (log (x+1))

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62 Figure 4 3 Effect of M. enterolobii M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions. Letters represent significant differences at P < 0.05 within nematode species group Figure 4 4 Effect of M. enterolobii M. haplanaria and M. incognita on the total egg mass production of the tomato cultivars: Amelia, Estamino, Maxifort, Monica, Rutgers, and Sanibel 60 days after inoculation in greenhouse conditions. Letters represent s ignificant differences at P < 0.05 within nematode species group B A AB B B A B A AB B B A A A A B B D 0 1 2 3 4 5 6 7 8 9 10 Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel M.enterolobii M.haplanaria M. incognita RGI A AB B AB A AB A B AB AB A AB A B B A AB C 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel Amelia Estamino Maxifort Monica Rutgers Sanibel M.enterolobii M.haplanaria M. incognita Eggs / g of root (log x + 1)

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63 CHAPTER 5 C ONCLUSIONS In this study, the damage potential of Meloidogyne haplanaria and the effects of temperature and tomato cultivars on reproduction were studied. The result of this study led to the following conclusions: 1. The preliminary damage threshold for M. haplanaria under greenhouse and in vitro conditions was 1 egg and J2/g of soil. 2. Higher temperatures can reduce the life cycle of M. haplanaria and stimulates an early egg hatc hing compared to another virulent species of root knot nematodes such as M. enterolobii 3. The high virulence of M. haplanaria can have an impact on integrated management decisions. 4. M. haplanaria can be highly virulent and it was able to successfully repr oduce on the Mi gene bearing cultivars such as Amelia Sanibel, Maxifort and Estamino regardless their genetic background or their gene dosage. This study demonstrated the basic biology and damage threshold functions M. haplanaria Future research should f ocus damage threshold studies under microplot and field conditions. It is also important to understand the distribution of this species in Florida and design effective management strategies to reduce the negative impact of this new virulent nematode specie s.

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64 LIST OF REFERENCE S Abad, P., Favery, B., Rosso, M. N., and Castagnone Sereno, P. 2003. Root knot nematode parasitism and host response: molecular basis of a sophisticated interaction. Molecular Plant Pathology 4:217 224. Abad, P., Gouzy, J., Aury, J. M., Castagnone Sereno, P., Danchin, E. G. J., Del eury, E., Perfus Barbeoch, L., Wincker, P. 2008. Genome sequence of the metazoan plant parasitic nematode Meloidogyne incognita Nature Biotechnology 26:909 915. Abdul Baki, A. A., Haroon, S. A., and Chitwood, D. J. 1996. Temperature effects on resistance to Meloidogyne spp in excised tomato roots. HortScience 31:147 149. Araujo, M. T., Dickson, D. W., Augustine, J. J., and Bassett, M. J. 1982. Optimum initial ino culum levels for evaluation of resistance in tomato to Meloidogyne spp at two different soil temperatures. Journal of N ematology 14:536 539. Barbary, A., Palloix, A., Fazari, A., Marteu, N., Castagnone sereno, P., and Djian caporalino, C. 2014. The plant genetic background affects the efficiency of the pepper m ajor nematode resistance genes M e 1 and M e 3. Theory of Applied Genetics 127:499 507. Barker, K. R., and Olthof, T. H. A. 1976. Relationships between nematode population densities and crop responses. A nnual Review of Phytopathology 14:327 353. Bendezu, I. F., Morgan, E., and Starr, J. L. 2004. Host of Meloidogyne haplanaria Nematropica 34:205 209. Brommonschenkel, S. H., Frary, A., Frary, A., and Tanksley, S. D. 2000. The broad spectrum T ospovirus resi stance gene sw 5 of tomato is a homolog of the root knot nematode resistance gene M i Molecular Plant Microbe Interactions 13:1130 1138. Bybd, D. W., Kirkpatrick, T., Barker, K. R., and Barker, K. R. 1983. An improved technique for clearing and staining pl ant tissues for dete ction of nematodes. Journal of N ematology 15:142 143. Alarcn, J. J. 2005. High temperature effects on photosynthetic activity of two tomato cultivars wi th different heat susceptibility. Journal of Plant Physiology 162:281 289. Castagnone Sereno, P., Danchin, E. G., Perfus Barbeoch, L., and Abad, P. 2013. Diversity and evolution of root knot nematodes, genus Meloidogyne : new insights from the genomic era. Annual Review of Phytopathology 51:203 220.

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70 Verdejo Lucas, S., Blanco, M., Cortada, L., and Sorribas, F. J. 2013. Resi stance of tomato rootstocks to Meloidogyne arenaria and Meloidogyne javanica under intermittent elevated soil temperatures above 28 C. Crop Protection 46:57 62. Verdejo Lucas, S., C ortada, L., Sorribas, F. J., and Ornat, C. 2009. Select ion of virulent populations of Meloidogyne javanica by repeated cultivation of mi resistance gene tomato rootstocks under field conditions. Plant Pathology 58:990 998. Webb, S. E., Stansly, P. a, Schuster, D. J., Funderburk, J. E., Smith, H., and Whitefly, S. 2013. Insect management for tomatoes peppers, and eggpla nt. University of F lorida. 1 40 pp. Widmer, T. L., Mitkowski, N. a, and Abawi, G. S. 2002. Soil organic matter and management of plant p arasitic nematodes. Journal of N ematology 34:289 295. Wong, T. K., and Mai, W. F. 1973. Effect of temperature on growth, development and reproduction of Meloidogyne hapla in lettuce. Journal of N ematology 5:139 142. Zasada, I. A Halbrendt, J. M., Kokalis Burelle, N., LaMondia, J., McKenry, M. V, and Noling, J. W. 2010. Managing nematodes without methyl bromide. Annual R evi ew of P hytopathology 48:311 328. Zeck, W. M. 1971. A rating scale for field evaluation of root knot infestations. Pflanzenschutz Nachri chten Bayer AG 24:141 144.

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71 BIOGRAPHICAL SKETCH Lisbeth Espinoza Lozano is originally from Ecuador, where she ob tained her undergrad degree in a griculture. After graduation, she became part of the Biotechnology Research Center o f Ecuador (CIBE) developing research work on plant pathology and e xtension. Currently, Lisbeth is dually enrolled in the Doctor of Plant Medicine, and the master's i n entomology and nematology under the supervision of Dr. Tesfa Meng istu. Lisbeth's has serve d as teaching assistant for the n ematode diagnostics class and also has actively participated in outreach and extension event performed by the n ematode ed on understanding biological aspects of new emergent specie of root knot nematode in Florida