initial population densities and effects of...

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 THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

To everyone who had faith in me

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ACKNOWLEDGMENTS

I give thanks to my mom Rocio, my dad Rodrigo, and my grandparents Rosa

Emilia and Adalberto for supporting me all the time, and inspiring me to go further; to my

brothers Fernando, Diego, and my sisters Mirian, and Katty, for being my emotional

support; to Daniel, for being my friend and partner from the first day of this journey. To

Dr. Mengistu for his guidance, support and patience through all this process, and for

being a good advisor; to all my committee members for enlightening me and made me a

better professional. To my lab mates Gideon, Anil, Alexandros, and Ruhiyyih and lab

staff, Alex, 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 my former advisor, for her

advising, and for teaching me to give my best in everything I do.

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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 Control .................................................................................................... 19 Plant Resistance ..................................................................................................... 20 Resistance Management and Mi-Gene................................................................... 21

2 DAMAGE POTENTIAL OF M. HAPLANARIA ON THE MI-GENE CONTAINING RESISTANT TOMATO CULTIVAR “SANIBEL” ...................................................... 25

Introduction ............................................................................................................. 25 Materials and Methods............................................................................................ 27

Plant Material ................................................................................................... 27 Inoculum Preparation ....................................................................................... 27 Pot Preparation and Inoculation of M. haplanaria ............................................. 28

Data Collection ................................................................................................. 28 Results .................................................................................................................... 29 Discussion .............................................................................................................. 30

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. ....................................... 36


Inoculum Preparation ....................................................................................... 38

Plant Material ................................................................................................... 38 Inoculum Preparation ....................................................................................... 39

Data collection .................................................................................................. 39

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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


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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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 inoculation 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 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. ............. 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. Plants 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) 24ᵒC, B) 28ᵒC and C) 32ᵒC. 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 24ᵒ, 28ᵒ. .. 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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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 24ᵒ, 28ᵒ and 32ᵒC. ........................ 47

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 inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC.. ........................................................................................................... 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 24ᵒ, 28ᵒ and 32ᵒC. .......................................... 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 24ᵒ, 28ᵒ and 32ᵒC. ........................ 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 24ᵒ, 28ᵒ and 32ᵒC. .......................................................... 49

3-9 Linear regression analysis of temperature on egg masses on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. .......................................... 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 in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. .............................................. 50



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 after inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. .......................................... 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 24ᵒ, 28ᵒ and 32ᵒC.. ......................................... 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 growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC.. ......................................... 53

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3-16 Effect of temperature 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 24ᵒ, 28ᵒ and 32ᵒC. 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




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

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 tools 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. The Mi-gene is widely used in different tomato cultivars to provide resistance

to a 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 such as high; temperatures, initial

population density and gene dosage can interfere with the expression of this gene and

limit its use in Florida.

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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 on tomato.

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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 US., fresh 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 producer of tomato in the US, contributing approximately 36% of the total

production of fresh market tomatoes in the US. (USDA-NASS, 2016). The majority of

the Florida 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 of 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 sucking

their juices and weakening the plants. In addition, some insect pests serve as vectors

for several viruses and pathogens. Other insects like caterpillars can cause severe

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defoliation of the plants, this compromises the photosynthetic capacity of the plant,

reducing total yield (Webb et al., 2013).

The environment has a strong influence on the severity and incidence of pests

and diseases. Temperature, light and humidity are related to the development of

diseases by influencing spore germination, infection and propagation of pathogens

(Huber and Gillespie, 1992). For 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 spp.,

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 than 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 males maintain

their vermiform form though all their life cycle. Mature females lay eggs into gelatinous

masses composed of a glycoprotein matrix produced from rectal glands; this matrix

keeps the eggs together and protected from extreme environmental conditions and

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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 necessary (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 site serves as a nutrient sink for

the developing J2. Nematode growth and reproduction entirely depend on the

development of giant cells. Under favorable 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; adult females continue to

feed and enlarge to become round to pear-shaped.

Root-knot nematodes exhibit variations in reproductive 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 the 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 generations in one cropping season. Many Meloidogyne

species have a broad host range. The overall host range of Meloidogyne species

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encompasses from 3,000 to 5,500 plant species (Abad et al., 2003; Mitkowski and

Abawi, 2003).

Symptoms are expressions of root dysfunction, the above ground symptoms of

root knot nematodes are characterized as a patchy distribution of chlorotic, stunted,

necrotic, and wilted plants. The belowground symptoms typically include root galling,

however other nematode species such as Nacobbus spp. or the false root-knot

nematode are able to cause extensive root galling. Crop yield reduction is commonly

associated with high initial population densities of nematodes in the soil, and of the loss

of root function resulting from population increase and reinfection. Population growth is

favored by favorable environmental conditions that promote the early appearance of

symptoms and increase the damage severity (Noling, 1999; Ploeg, 2002).

Economic Importance of Root-Knot Nematodes

Root-knot nematodes are economically important pests on a wide range of

vegetables throughout the world (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 economic 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 ability 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 nematodes (Manzanilla-

Lopez and Starr, 2009).

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Meloidogyne incognita, M. javanica, M. arenaria and M. hapla are considered the

most important root-knot nematode species worldwide, given their wide host range and

their global distribution (Moens et al., 2009). Some of these species are especially

important to tomato production. Yield reductions in tomato from root-knot nematodes

have been reported globally 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 reduced 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

resistance 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 considered 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 requirement to maintain nematode

population densities under the economic threshold and to minimize the potential

damage on the crop (Barker and Olthof, 1976). Cropping history and information about

common nematode species in the field and previous crops planted is the first thing to do

before establishing a crop in order to determine the best management practices that fit

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in the agricultural system, followed by a continuous monitoring of the soil and roots 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, host 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 crop, nematode populations usually are maintained below

damaging levels.

Other techniques such as flooding and solarization 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

investment before successful implemented within the field.

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Chemical Control

The management of plant-parasitic nematodes in the soil by chemical means is

dependent upon bringing the nematicide into contact with the nematode in

concentrations high enough to affect them. The use of nematicides is generally

recommended in situations such as; high initial population density, 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 increasing concerns about adverse effects and

environmental impacts, many have been removed from the market. For example,

methyl bromide was the predominant product for the control of nematodes and many

other soil-borne pests and pathogens in high value horticultural crops. In 2001,

production of methyl bromide was halted due to its adverse effects to the environment

(Ristaino and Thomas, 1996).

Chemical control of nematodes 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 vaporizes 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 nematodes, whereas other compounds primarily target

other organism such as soil-borne pathogens and weed seeds (Noling, 1999). Non-

fumigants can have contact or systemic activity, and are applied as liquid or granular

formulations and are typically incorporated in the soil or applied through chemigation.

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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-dichloropropene, metam potassium, dimethyl disulfide, and chloropicrin, but

none are as effective as methyl bromide (Zasada et al., 2010). Most studies conducted

for evaluating non-fumigant nematicides have shown that they are less consistent 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 in maintaining another organism density at a lower

average than would occur in their absence”. There are several organisms such as

viruses, fungi, predatory nematodes, and mites that attack nematodes in the soil.

Mechanisms of biocontrol of nematodes include 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 characteristics that an

organism has, the better candidate it is as a biological control.

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Certain bacteria 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 bacteria 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 of plant-parasitic nematodes that attack many

agricultural crops. These gram-positive, mycelial, endospore-forming bacteria are

mainly obligate parasites of nematodes in that they cannot survive without their host.

Pasteuria species are ubiquitous in most environments and are found in different parts

of the world. Pasteuria penetrans is 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 becoming 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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economical method for managing nematodes in both high- and low-cash value cropping

systems.

Several resistant cultivars are commercially available for management of root-

knot nematodes in tomato. Resistant 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 to 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 has increased during recent years. This

technique allows growers to use nematode resistant rootstocks and introduce scions

with desirable fruit characters. Some of these rootstocks carry the Mi-gene in their

genome providing resistance to certain species 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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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 shown 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 tomato 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 28oC (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 25oC and completely lose their resistance at 32oC.

New thermostable genes are under study and evaluation 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. With

the continuous spread and establishment of nematode species into new geographical

areas, other root-knot nematode species that affect tomato and are not affected by the

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Mi-gene are becoming increasingly important (Liu and Williamson, 2006; Kiewnick et al.,

2009).

In August 2015, the UF/IFAS Nematode 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 techniques 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 could

have on Florida’s agriculture, including potential impacts to a variety of fruits, vegetables

and agronomic crops.

Information on crop-nematode relationships is vital for growers to decide on

economically viable management 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 initial population densities and temperatures on the

response of different Mi-gene resistant tomato cultivars towards M. haplanaria will be

studies. We will establish a baseline threshold to be used in further research of

management practices to reduce damage associated with this nematode. The

objectives of this study are:

1. To determine the damage thresholds of M. haplanaria on the Mi-gene containing resistant tomato cultivar “Sanibel”.

2. To determine the impact of temperature on stability of the Mi-gene and compare the developmental rates of M. haplanaria, M. incognita and M. enterolobii.

24

3. To compare the response of Mi-gene resistant cultivars and rootstocks to infection by M. haplanaria, M. incognita, and M. enterolobii.

25

CHAPTER 2 DAMAGE POTENTIAL OF M. HAPLANARIA ON THE MI-GENE CONTAINING

RESISTANT TOMATO CULTIVAR “SANIBEL”

Introduction

Root-knot nematodes (Meloidogyne spp.) are 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 known 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 shown to infect M.

arenaria-susceptible cultivars of peanut, garden pea, and radish (Bendezu et al., 2004).

The degree of damage caused by a particular 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). Damage threshold is the population density in which measurable plant damage

or yield loss occurs. These thresholds depend on nematode species, crop, soil type and

environmental conditions (Ferris, 1978; Trudgill and Phillips, 1997). For example, the

Nematode Assay Lab at the University of Florida considers the damage threshold for

root-knot nematodes on tomato under field conditions at 1/100 cm3 of soil, whereas for

corn it is 80/100 cm3 (Unpublished data). This variability on the damage threshold is

applicable for each plant-parasitic nematode attacking any particular crop.

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 is important to understand how initial population densities can affect the normal

course 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 can be confused with nutrient deficiencies, and which ultimately,

compromise tomato yield. These symptoms are the physiological response of the plant

to the limited uptake of nutrients and water caused by the presence of nematodes inside

the root system. 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

conditions 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

Maleita et al. (2012) suggest that the Mi-gene only provides partial resistance; in the

scenario of high population densities the plant and root growth is reduced and

reproductive parameters for the nematodes increase.

In order to develop and predict potential damage from new species of nematodes

it is important to consider the relation between initial population densities and plant

growth and yield. Meloidogyne haplanaria is a recently discovered root-knot nematode

species and so far, the impact of M. haplanaria initial population density on yield

reduction of tomato is unknown. The objective of this research was to identify an initial

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 Seeds; Saint Leonard, Maryland), and Rutgers

(Burpee & Co, Warminster, Pennsylvania) were used in this study. Seedlings were

grown on Miracle-Gro® Potting mix (The Scotts Company, Marysville, Ohio) and were

maintained inside a growth chamber at 26ᵒC 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-infested tomato roots were used to extract

eggs using the NaOCl method described by Hussey and Barker (1973). Clean 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 thoroughly for 2

minutes. The suspension was poured onto nested 75 µm and 25 µm 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 small 25 µm mesh, rinsed with tap and collected in a clean tube for counting.

28

Pot Preparation and Inoculation of M. haplanaria

Four-week old tomato seedlings were transplanted into 15.24 cm-diameter pots

filled with 1000 cm3 autoclaved 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/ cm3 of soil) onto

resistant (Sanibel) and a susceptible (Rutgers) tomato by pipetting inoculum 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 temperature 28ºC ± 2.

Pots were arranged on greenhouse tables using a randomized block design with

eight replicates per treatment. Plants were watered daily and fertilized 20 days after

transplanting with 3 g 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 were harvested, and root gall index was assessed using the

rating scale from 0 to 10 described by Zeck (1971). Total numbers of eggs 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.

Collected data was analyzed according to the general lineal model and if

required, mean separation test was made using Tukey’s test at P ≤ 0.05 using SAS

9.1.3 software (SAS Institute Inc.; Cary, North Carolina) and R studio (RStudio, Inc.;

Boston, Massachusetts) . Regression analysis was performed on reproductive

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 Pi ≤ T), m is the minimum relative yield

(the value of y at very large Pi), P (= Pi) is the initial nematode population 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 an effect (P ≤ 0.05) on

reproductive parameters. Significant differences across the Pi’s 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 figures 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/ cm3 of soil.

The regression analysis on reproductive factor displayed on figure 2-5 showed that

Rutgers was significantly high compared to Sanibel at the lowest initial population

density (0.25 eggs J2/ cm3) (P < 0.0001). The regression analysis presented a negative

slope for Rutgers and Sanibel (-0.5246; -0.2129). The Seinhorst’s model fitted for shoot

height (Figure 2-6) and root length (Figure 2-8) for both Rutgers and Sanibel, but shoot

30

weight fitted only for Rutgers (Figure 2-7). The tolerance limit (T) was determine at 1

and 3 eggs J2/ cm3 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 cultivars

Rutgers and Sanibel, indicating that both tomato cultivars are hosts for 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 increase with the Pi inoculum level of M. haplanaria, indicating

their virulence on the tested tomato cultivars. This is in agreement with several other

studies (Eisenback 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 and 3 nematodes/ cm3 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 consistent 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

was also observed that the carrying capacity for the nematode infection was around 32

31

eggs J2/ cm3 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/ cm3 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).

Therefore, the data collected on this study provided a preliminary tolerance value to be

used for future micro plots or field experiments. Additionally, the reproductive factor (Rf)

for both cultivars showed a negative slope across the different population densities,

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 plot conditions

than in the hail-net-cage experiment.

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 R² = 0.311 P < 0.0001

y = 47.204ln(x) + 117.27 R² = 0.3473 P < 0.0001

0

100

200

300

400

500

600

700

0 10 20 30 40 50 60

Eg

g m

asses

Initial population densities

Rutgers

Sanibel

y = 82077ln(x) + 61580 R² = 0.5028 P < 0.0001

y = 66529ln(x) + 29975 R² = 0.4811 P < 0.0001

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

0 10 20 30 40 50 60

Eg

gs


Rutgers

Sanibel

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 R² = 0.8346 P < 0.0001

y = 1.6654ln(x) + 2.748 R² = 0.8173 P < 0.0001

0

2

4

6

8

10

0 10 20 30 40 50 60

RG

I


Rutgers

Sanibel

y = 4119.3ln(x) + 1813.4 R² = 0.4268 P < 0.0001

y = 2628.4ln(x) + 1309.9 R² = 0.5164 P < 0.0001

0

10000

20000

30000

40000

50000

60000

0 10 20 30 40 50 60

Eg

gs

pe

r g

ram

of

roo

t


Rutgers

Sanibel

34

Figure 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 greenhouse conditions.

Figure 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 of 8 replications and the line is the predicted function obtained when the data was fitted to the Seinhorst model. The parameters obtained were for Rutgers: 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 R² = 0.2479 P < 0.0001

y = -0.2129x + 17.755 R² = 0.1615 P < 0.0001

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

Re

pro

du

cti

ve

fa

cto

r (P

f/P

i)


Rutgers

Sanibel

35

Figure 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 mean of 8 replications and the line is the predicted function obtained when the data were fitted to the Seinhorst model. The parameters obtained were for Rutgers: Y= 64.65; m= 0.15; T= 7.9; and for Sanibel Y= 66.27; m= 0.83; T= 0.64.

Figure 2-8. Relationship between initial population density (Pi) 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 model. 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.

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 (López-Pérez 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 affected 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 (López-Pérez et al.,

2006).

Temperature has a direct effect on plant growth, root-knot nematode life cycle,

and the interaction between 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 15.4ᵒC to 35ᵒC (Dropkin, 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 tomato plants.

Several experiments demonstrated that exposure to long periods of high temperatures

37

above 28ᵒC 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 creates

the ideal conditions for nematodes to attack the plant and increases 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 temperatures can cause

an irreversible loss of Mi-gene resistance at high soil temperatures (>28ᵒC). Efficacy of

the Mi-gene can be hamper by mutation(s) on it or a gene required in the Mi-mediated

resistance 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 ᵒC 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 31ᵒC; maintain partial resistance at 34ᵒC, and completely losses the

resistance at 37ᵒC (Dropkin et al., 1969; Abdul-Baki et al., 1996; Verdejo-Lucas et al.,

2013; Marques de Carvalho et al., 2015).

The objectives of this study were to understand the interaction between the

resistant cultivar Sanibel bearing the Mi-gene and the susceptible Rutgers with M.

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 newly

discovered root-knot nematode species to temperature extremes and compare damage

to other Meloidogyne species endemic to Florida.



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-infested tomato roots were used to extract

eggs using the NaOCl method described by Hussey and Barker (1973). Clean 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 thoroughly for 2

minutes. The suspension was poured onto nested 75 µm and 25µm 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 small 25 µm mesh, rinsed with tap water and collected in a clean tube for counting.

Plant Material

Tomato cultivars Sanibel (Reimer Seeds; Saint Leonard, 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 26ᵒC.

39


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). Clean 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 thoroughly for 2 minutes. The suspension was poured onto nested

75 µm and 25µm 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 small 25 µm mesh, rinsed with tap water and collected in a

clean tube for counting. Four-week old tomato seedlings were then transplanted into a

3.1-cm-diam. and 21.6-cm-deep plastic cones filled with 120 cm3 of autoclaved sandy

loamy soil. After 48 hours, each cone was inoculated with 360 eggs and J2, and the

cones were randomly distributed on racks. Cones with the treatments were placed into

separate temperature controlled growth chambers at 24ᵒC, 28ᵒC, and 32ᵒC, and

maintained at 60% relative humidity and 14L: 10D photoperiod. Plants were watered

daily and fertilized 20 days post inoculation 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; data 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 Bybd et al. (1983) in order

40

to enumerate egg masses and observe the developmental stages of root knot-

nematodes inside the roots. 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 differences in developmental stages described by Moens et al.

(2009). This experiment was repeated one time.

Collected data were analyzed using SAS 9.1.3 software (SAS Institute Inc.; Cary,

North Carolina,). Nematode development and infectivity assessment data were

analyzed after counts were log (x + 1) transformed for analysis to fulfil the criteria for

normality. Mean separation among treatments was done using Tukey’s test (P ≤ 0.05).

Regression analysis was also performed to determine the response of the different

nematode species at the increase of temperature.

Results


parameters, so the data from the repetitions was combined for analysis. Visual

observations of both cultivars Rutgers and Sanibel maintained at 24 and 28ᵒC showed

healthy growth (Figure 3-1 A and B), whereas plants that were kept at 32ᵒC expressed

symptoms of heat stress such as stunting, wilting, necrosis, and reduced leaf area

(Figure 3-1 C). The effects of temperature on total number of eggs, total egg masses,

root gall index and eggs / g of root for both tomato cultivars and the three root-knot

nematode species (M. incognita, M, enterolobii and M. haplanaria) are presented in

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 the tomato cultivars

Rutgers and Sanibel and exposed at temperatures of 24, 28 and 32ºC. These results

41

present significant differences within each group, and M. haplanaria exhibit the highest

results across the different treatments in both cultivars, except in Rutgers at 24ºC where

M. enterolobii presented the highest number of egg masses.

Egg production is observed on figure 3-3. It is noticeable that M. enterolobii has a

larger production of eggs on Sanibel at 24ºC, Rutgers at 28ºC, and both cultivars at

32ºC. Figure 3-4 presents the results for RGI. From here it is possible to notice a

variation on this parameter for each nematode species. At 24ºC, M. incognita and M.

enterolobii reported the largest galling in both tomato cultivars, whereas M. haplanaria

in Sanibel had the largest galling at 24 ºC. At 28ºC it was observed a similar pattern on

Rutgers, but in Sanibel M. haplanaria and M. enterolobii had similar galling. Meanwhile,

at 32ºC M. enterolobii 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 24ºC, whereas M. enterolobii and M. haplanaria

on Sanibel had a better reproduction at 28ºC. Meloidogyne enterolobii was significantly

higher than M. haplanaria and M. incognita in Rutgers, but for Sanibel, M. haplanaria

had the largest result. At 32ºC, M. enterolobii presented higher results on Rutgers and

Sanibel.

Figure 3-6 presents the results of the linear regression analysis on egg masses

for M. enterolobii, and shows that the slope for the regression 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 eggs/ g of root in which Rutgers had a larger slope than

42

Sanibel (120.74; 27.29), and RGI where Sanibel showed a larger slope in comparison to

Rutgers (0.270; 0.083) (Figure 3-8).

A different response was observed on the slopes of the egg masses (Figure 3-9)

where the linear regression resulted with a negative slope on the cultivar Rutgers (-

12.969), whereas the slope for 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 negative slope was

observed for cultivar Rutgers (-55.104; -29.487), while the cultivar Sanibel resulted in

positive 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) (Figure 3-12).

Reproductive parameters of M. haplanaria such as eggs, 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 J2s/ g of root of

nematode species M. enterolobii, M, haplanaria and M. incognita. Results show that the

total number of J2s was significantly higher at 32ᵒC 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 32ᵒC.

43

The total number J3s and J4s / g of root were not significant for any of the treatments

and cultivars (data not shown).

Discussion

Rutgers and Sanibel were both negatively impacted by constant temperatures of

32ᵒC. In addition, temperature had a profound effect on nematode development and

reproduction, although differences among nematode species and tomato cultivars were

reported. Meloidogyne enterolobii, is known for naturally infest resistant cultivars of

tomato and is considered highly virulent (Kiewnick et al., 2009), it was able to reproduce

both Rutgers (susceptible) and root-knot nematode resistant Sanibel. However, 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 28ᵒC and 30ᵒC. The same effect, but at a reduced level, was observed

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.

Information 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 28ᵒC. This study reports similar results at a

44

constant temperature of 24ᵒC for Rutgers, but Sanibel presented a slightly lower value

at the same temperature but the root gall index increased with temperature.

Observations on roots and counting of different nematode developmental stages

at temperatures of 24, 28 and 32ᵒC, 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 32ᵒC, 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 32ᵒC 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 32.2ᵒC mature females could be observed 14 days

after inoculation. Therefore, 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 infection 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 was slower than M. enterolobii and M. haplanaria. In this study the presence

of J3s and J4s were not observed with M. incognita at 32ᵒC in Rutgers and Sanibel.

45

In conclusion, M. haplanaria was 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 32ᵒC; this is in comparison to the virulent control M.

enterolobii and the widely distributed M. incognita.

46

Figure 3-1. Impact of temperature on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, M. haplanaria, and M. incognita at A) 24ᵒC, B) 28ᵒC and C) 32ᵒC. Photographs by Lisbeth Espinoza.

Figure 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 24ᵒ, 28ᵒ and 32ᵒC. 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

24ᵒC 28ᵒC 32ᵒC

Eg

g m

asses (

log

x +

1)

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 inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. 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 inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. Letters represent 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




To

tal eg

gs (

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




RG

I

48

Figure 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 inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. Letters represent significant differences at 5% within the cultivar group.

Figure 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 24ᵒ, 28ᵒ and 32ᵒC.

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




Eg

gs p

er

gra

m o

f ro

ot

(l

og

(X

+1)

y = 237.86x - 4855.1 R² = 0.27681 P = 0.0009

y = 21.427x - 204.15 R² = 0.1852 P = 0.008

0

1000

2000

3000

4000

5000

6000

7000

8000

22 24 26 28 30 32 34

Eg

gs

Temperatures ºC

Rutgers

Sanibel

49

Figure 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 24ᵒ, 28ᵒ and 32ᵒC.

Figure 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 24ᵒ, 28ᵒ and 32ᵒC.

y = 120.74x - 2469.4 R² = 0.3278 P = 0.0002

y = 27.298x - 501.02 R² = 0.2535 P = 0.001

0

500

1000

1500

2000

2500

3000

22 24 26 28 30 32 34

Eg

gs p

er

g o

f ro

ot

Temperatures ᵒC

Rutgers

Sanibel

50

Figure 3-9. Linear regression analysis of temperature on egg masses on tomato cultivars Rutgers and Sanibel inoculated with M. enterolobii, 40 days after inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC.

Figure 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 in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC.

y = -12.969x + 440.07 R² = 0.7469 P < 0.0001

y = 3.3958x - 56.111 R² = 0.4235 P < 0.0001

0

20

40

60

80

100

120

140

160

180

200

22 24 26 28 30 32 34

Eg

g m

asses

Temperature ºC

Rutgers

Sanibel

y = -55.104x + 2047.6 R² = 0.3615 P = 0.0001

y = 9.5417x - 178.97 R² = 0.8147 P < 0.0001

0

200

400

600

800

1000

1200

22 24 26 28 30 32 34

Eg

gs

Temperature ºC

Rutgers

Sanibel

51



y = -29.487x + 1165.2 R² = 0.208 P = 0.005

y = 10.058x - 227.25 R² = 0.6493 P < 0.0001

0

100

200

300

400

500

600

700

800

22 24 26 28 30 32 34

Eg

gs p

er

g o

f ro

ot

Temperatures ᵒC

Rutgers

Sanibel

y = -0.1771x + 8.3194 R² = 0.593 P < 0.001

y = -0.0521x + 2.6528 R² = 0.1847 P = 0.008

0

1

2

3

4

5

6

22 24 26 28 30 32 34

RG

I

Temperatures ᵒC

Rutgers

Sanibel

52

Figure 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 after inoculation in growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC.

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 24ᵒ, 28ᵒ and 32ᵒC. Letters represent significant differences at 5% within the cultivar group.

y = 0.0938x + 0.8472 R² = 0.1251 P = 0.034

y = 0.2594x - 3.8495 R² = 0.69778 P < 0.0001

0

1

2

3

4

5

6

22 24 26 28 30 32 34

RG

I

Temperatures ᵒC

Rutgers

Sanibel

C

B

A

C

B

A

0

5

10

15

20

25

30

35

40

24ᵒC 28ᵒC 32ᵒC 24ᵒC 28ᵒC 32ᵒC

Rutgers Sanibel

J2s

/ g

of

roo

t

53

Figure 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 growth chamber maintained at 24ᵒ, 28ᵒ and 32ᵒC. Letters represent significant differences at 5% within the cultivar group.

Figure 3-16. Effect of temperature 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 24ᵒ, 28ᵒ and 32ᵒC. Letters represent 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

roo

t

C

B

A

B A

B 0

20

40

60

80

100

120

140

160

180


Rutgers Sanibel

J2s

/ g

of

roo

t

54

CHAPTER 4 THE RESPONSE OF MI-GENE RESISTANT TOMATO CULTIVARS AND

ROOTSTOCKS TO ROOT INFECTION BY MELOIDOGYNE HAPLANARIA, MELOIDOGYNE INCOGNITA, AND MELOIDOGYNE ENTEROLOBII.

Introduction

Resistance to nematodes can result from the fusion of different genes and traits

within a large genetic diversity among wildtype species. However, resistance to root-

knot nematodes in tomato is conferred by a single dominant gene known as the Mi-

gene located 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 the

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 30ᵒC have been identified. However,

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) some 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

root-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

55

nematode populations that are able to overcome the resistance and 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 production.

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 pathogens and nematodes. Studies have been

conducted to 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

rootstocks materials presented a reduced nematode galling, whereas others had

demonstrated little effect, but final yields were not compromised (López-Pérez 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


Plant Material

Resistant tomato cultivars Sanibel (Reimer seeds Saint Leonard, Maryland), and

Amelia (Harris Seeds; Rochester, New York); the resistant rootstocks, Estamino, and

Maxifort (MiMi) (Johnny’s seeds; Winslow, Main); were compared with the susceptible

Rutgers (mimi) (Burpee & Co, Warminster, Pennsylvania) and Monica (mimi) (Johnny’s

seeds; Winslow, Main). Seedlings were propagated in Miracle Gro® Potting mix (The

Scotts Company, Marysville, Ohio) and were maintained inside a temperature controlled

growth chamber at 26ᵒC and watered daily.

56


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 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 thoroughly for 2 minutes. The suspension was

poured onto nested 75 µm and 25µm 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 small 25 µm 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 into 15.24-cm-diameter pots filled with 1.000 cm3 autoclaved sandy loam

soil. Pots were inoculated with 3 eggs and J2s/ cm3 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 28ºC ±

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 were harvested, and root gall index was assessed using the

rating scale from 0 to 10 described by Zeck (1971). (1971). Total numbers of eggs

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 fulfill the criteria for normality. Mean separation among treatments was done using

Tukey’s test (P < 0.05).

Results


parameters, so the data from the repetitions was combined for analysis. The root-knot

nematode species M. enterolobii, M. haplanaria, and M. incognita were able to

reproduced on 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 plant

parameter such as shoot and root length and 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. enterolobii (P < 0.0001), M. haplanaria (P < 0.0001), and M.

58

incognita (P < 0.0001). Among the cultivars inoculated with M. enterolobii, Sanibel

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); Tomato cultivars Sanibel (1.56 ± 0.13) and Amelia (1.60 ±

0.08) showed the lowest number of egg masses compared with the control (Figure 4-1).

Total number of eggs differed among 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, presented 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).

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. Cultivars 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), whereas the lower results were observed on Sanibel (2.14 ± 0.10).

Inoculations 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 production of eggs (Figure 4-4).

Discussion

In this study, the host suitability of commercially available tomato cultivars was

tested for their reaction to infection by M. enterolobii, M. haplanaria, and M. incognita.

Our results 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 severe damage reflected on the shoot,

in all the treatments some plants presented symptoms of wilting, yellowing, necrosis,

even some of them were death.

Results showed that all the cultivars tested were highly susceptible to M.

enterolobii. This affirmation can be noticed particularly on the parameters RGI and total

eggs. 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 results showed that both were suitable for high

root-knot nematode reproduction.

60

Maxifort is a cross from Solanum lycopersicum x S. habrochaites known to be a

homozygous resistant cultivar (MiMi), but our experiment showed 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 can interfere

on the normal development 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 than 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 trait 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 gene dosage on nematode infection.

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 differences 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 inoculation 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

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

M.enterolobii M.haplanaria M. incognita

To

tal eg

g m

asses

(lo

g (

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

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el


To

tal eg

gs

(

log

(x+

1))

62



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

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el


RG

I

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

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el

Am

elia

Esta

min

o

Ma

xifort

Mo

nic

a

Ru

tgers

Sanib

el


Eg

gs /

g o

f ro

ot

(l

og

x +

1)

63

CHAPTER 5 CONCLUSIONS

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 hatching 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 reproduce 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 focus 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 species.

64

LIST OF REFERENCES

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Abad, P., Gouzy, J., Aury, J. M., Castagnone-Sereno, P., Danchin, E. G. J., Deleury, E., Perfus-Barbeoch, L., Wincker, P. 2008. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology 26:909–915.

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BIOGRAPHICAL SKETCH

Lisbeth Espinoza-Lozano is originally from Ecuador, where she obtained her

undergrad degree in agriculture. After graduation, she became part of the Biotechnology

Research Center of Ecuador (CIBE) developing research work on plant pathology and

extension. Currently, Lisbeth is dually enrolled in the Doctor of Plant Medicine, and the

master's in entomology and nematology under the supervision of Dr. Tesfa Mengistu.

Lisbeth's has served as teaching assistant for the nematode diagnostics class, and also

has actively participated in outreach, and extension event performed by the nematode

assay lab. Lisbeth’s research has focused on understanding biological aspects of new

emergent specie of root-knot nematode in Florida.

initial population densities and effects of...

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