<%BANNER%>

Biological Comparison of Four Isolates of Meloidogyne floridensis from Florida

Permanent Link: http://ufdc.ufl.edu/UFE0022801/00001

Material Information

Title: Biological Comparison of Four Isolates of Meloidogyne floridensis from Florida
Physical Description: 1 online resource (66 p.)
Language: english
Creator: Stanley, Jason
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

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

Notes

Abstract: The peach root-knot nematode, Meloidogyne floridensis is a recently described species of root-knot nematode that infects and reproduces on peach rootstock cultivars that are resistant to other species of root-knot nematodes. To date, this nematode species has only been reported in Florida. Other than the population of M. floridensis used for the species description, very little is known about this nematode. This species provides valid concern for Florida growers as more emphasis is being placed on the use of root-knot nematode resistant plant cultivars and crop rotation strategies with the diminishing supply of effective nematicides. The objectives of this study were to investigate four field populations of M. floridensis collected in Alachua, Hendry, and Indian River Counties by: (i) characterizing them using morphometrics, biochemical and differential host studies; (ii) determining host status on root-knot nematode resistant and susceptible cultivars; and (iii) determining pathogenicity and virulence in field studies.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jason Stanley.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Dickson, Donald W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022801:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022801/00001

Material Information

Title: Biological Comparison of Four Isolates of Meloidogyne floridensis from Florida
Physical Description: 1 online resource (66 p.)
Language: english
Creator: Stanley, Jason
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

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

Notes

Abstract: The peach root-knot nematode, Meloidogyne floridensis is a recently described species of root-knot nematode that infects and reproduces on peach rootstock cultivars that are resistant to other species of root-knot nematodes. To date, this nematode species has only been reported in Florida. Other than the population of M. floridensis used for the species description, very little is known about this nematode. This species provides valid concern for Florida growers as more emphasis is being placed on the use of root-knot nematode resistant plant cultivars and crop rotation strategies with the diminishing supply of effective nematicides. The objectives of this study were to investigate four field populations of M. floridensis collected in Alachua, Hendry, and Indian River Counties by: (i) characterizing them using morphometrics, biochemical and differential host studies; (ii) determining host status on root-knot nematode resistant and susceptible cultivars; and (iii) determining pathogenicity and virulence in field studies.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jason Stanley.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Dickson, Donald W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022801:00001


This item has the following downloads:


Full Text

PAGE 1

BIOLOGICAL COMPARISON OF FOUR ISOLATES OF Meloidogyne floridensis FROM FLORIDA By JASON D. STANLEY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

PAGE 2

2008 Jason D. Stanley

PAGE 3

To my wife and daughter, Kiahla and Kelsie, for their unconditional love, generosity, and neverending support.

PAGE 4

ACKNOWLEDGMENTS I thank my committee members beginning with my chair Dr. D. W. Dickson who has shown great patience and friends hip during this venture, Dr. Nancy Burelle and Dr. Howard Frank for their continuous support and guidance; as well as Dr. Janete Brito who is an integral member of the committee and a dear friend and sister to me. I have been very fortunate to be supervised by a group of scien tists of such high esteem in their prospective fields. I would especially like to thank Dr. Renato Inserra who, while not being an official member of my committee, taught me more about the science of nematology than I could ever explain in words. His confidence in me provided the motivation for my scientific growth. I will always regard him as my mentor and friend. I would like to thank my parents (William D. a nd Sheila A. Stanley) who have been the two best friends, parents, and grandparents a son could have throughout both my childhood and adult life. They have always provided guidance, support, and absolute love. Finally, I would like to thank my wife and da ughter, Kiahla and Kelsie. They are, without a doubt, my strength in anything I do, and my two be st friends in the world. I am always inspired by them and love them both with all my heart. This degree belongs to them as much as it belongs to me. I hope more than anything else that th is work inspires Kelsie to go far in life. 4

PAGE 5

TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................... ...............9 CHAPTER 1 INTRODUCTION ................................................................................................................ ..11 Root-Knot Nematodes ( Meloidogyne spp.) ............................................................................11 Historical Background .....................................................................................................11 Development and Life Cycle ...........................................................................................12 Symptoms and Signs .......................................................................................................13 Economic Importance ......................................................................................................14 Identification ................................................................................................................ ....15 Morphological identification ....................................................................................15 Biochemical identification .......................................................................................16 Differential hosts ......................................................................................................17 Molecular identification ...........................................................................................17 Meloidogyne floridensis ..........................................................................................................18 History .............................................................................................................................18 Life Cycle and Mode of Reproduction ............................................................................19 Symptoms and Signs .......................................................................................................20 Host Range ......................................................................................................................20 Distribution and Economic Importance ...........................................................................21 Identification ................................................................................................................ ....21 Morphological and morphometric analysis ..............................................................21 Electrophoretic analysis ...........................................................................................22 Intergenetic spacer rDNA analyses ..........................................................................23 The RAPD-PCR analyses .........................................................................................23 High-fidelity PCR-RFLP analyses ...........................................................................23 Objectives .................................................................................................................... ...........24 2 COMPARISON OF FOUR ISOLATES OF Meloidogyne floridensis USING MORPHOMETRICS, ISOZYMES, AND DIFFERENTIAL HOSTS ..................................29 Introduction .................................................................................................................. ...........29 Materials and Methods ...........................................................................................................30 Nematode Source .............................................................................................................30 Development of Single Egg Mass Isolates ......................................................................31 Comparative Morphology ...............................................................................................31 5

PAGE 6

6 Isozyme Analysis .............................................................................................................3 2 Differential Host Test ......................................................................................................32 Results and Discussion ........................................................................................................ ...33 Comparative Morphometrics ...........................................................................................33 Isozyme Analysis .............................................................................................................3 4 Differential Hosts ............................................................................................................ 34 3 COMPARATIVE HOST STATUS OF SELECT ROOT-KNOT NEMATODE RESISTANT AND SUSCEPTIBLE PLANT CU LTIVARS TO FOUR ISOLATES OF Meloidogyne floridensis ..........................................................................................................46 Introduction .................................................................................................................. ...........46 Materials and Methods ...........................................................................................................46 Nematode Source .............................................................................................................46 Host Status of Select R oot-Knot Nematode Resistant and Susceptible Crops to M floridensis .....................................................................................................................46 Host Status of the Root-K not Nematode Resistant Peach Rootstock Nemaguard and Root-Knot Nematode Susceptible Lovell ...........................................................47 Results and Discussion ........................................................................................................ ...48 Select Horticultural and Agronomic Crops Test .............................................................48 Peach Test ........................................................................................................................49 APPENDIX A REPRODUCTIVE POTENTIAL AND PATHOGENICITY COMPARISON OF FOUR ISOLATES OF Meloidogyne floridensis ................................................................................54 Introduction .................................................................................................................. ...........54 Materials and Methods ...........................................................................................................54 Nematode Source .............................................................................................................54 Microplot Test .................................................................................................................54 Results and Discussion ........................................................................................................ ...56 BIOGRAPHICAL SKETCH .........................................................................................................66

PAGE 7

LIST OF TABLES Table page 1-1. Alphabetical listing of currently recognized Meloidogyne species .......................................25 1-2. Differential hosts used to identify species and races of root-knot nematodes. ......................28 2-1. Isolate numbers assigned to the original Florida Depa rtment of Agriculture log numbers for four isolates of Meloidogyne floridensis used throughout this thesis. ...........36 2-2. Number of Meloidogyne floridensis females collected from four hosts and subjected to polyacrylamide gel electrophoresis to de termine their esterase and malate dehydrogenase isozyme phenotype. ...................................................................................36 2-3. Select morphometrics (mean, standard de viation, and range) of second-stage juveniles of four isolates of Meloidogyne floridensis from Floridaa .................................................37 2-4. Select morphometrics (mean, standard deviation, and range) of males of four isolates of Meloidogyne floridensis from Floridaa ..............................................................................39 2-5. Select morphometrics (mea n, standard deviation, and range) of females of four isolates of Meloidogyne floridensis from Florida a .........................................................................41 2-6. Gall and egg mass rating of four isolates of Meloidogyne floridensis on six different hosts a .................................................................................................................................42 3-1. Plant cultivars used for host status studies of four isolates of Meloidogyne floridensis ......50 3-2. Select agronomic a nd horticultural crops testa: reproduction factor, egg mass and gall indices of four isolates of Meloidogyne floridensis on four root-knot nematode resistant plant cultivars: Crista, Charleston Bell, Mp 710, a nd Forrest and four susceptible cultivars: Talladega, Keystone Resistant Giant, Dixie 18, and S64-J1. ..........51 3-3. Peach testa, reproduction factor, egg mass and ga ll indices of four isolates of Meloidogyne floridensis on the root-knot nematode resistant peach cultivar Nemaguard and the susceptible cultivar Lovell. ................................................................52 7

PAGE 8

LIST OF FIGURES Figure page 2-1. Esterase enzyme phenotype MF3 of 11 single females of Meloidogyne floridensis lanes 1 and 12 single females of M. javanica control. .......................................................44 2-2. Malate dehydrogenase phenotyp e N1 of 11 single females of Meloidogyne floridensis lanes 1 and 14 single females of Meloidogyne javanica control, also designated as N1. ........................................................................................................................... ...........44 2-3. Esterase enzyme phenotype MF3 of 11 single females of Meloidogyne floridensis collected from pepper, lanes 1 and 12 single females of Meloidogyne javanica control. ...................................................................................................................... .........44 2-4. Esterase enzyme phenotype MF3 of 11 single females of Meloidogyne floridensis collected from tobacco. ......................................................................................................4 5 3-1. Host status test of select root-knot ne matode resistant and susceptible plant cultivars conducted in a growth room. .............................................................................................53 3-2. Host status test of the root-knot ne matode susceptible peach cultivar Lovell and resistant cultivar Nemaguard conducted in a greenhouse. .................................................53 4-1. Initial preparation of eight 1.2-m wide 14-m long strips by the application of the herbicide glyphosate in a site at the Universi ty of Florida Plant Science Research and Education Unit, Marion County, FL pl anted with Argentine bahiagrass ( Paspalum notatum ). ............................................................................................................................ 58 4-2. Post-plant application of 56.2 g ammonium nitrate and 28.1 g potassium chloride once per week for 10 weeks through th e drip irrigation system.................................................58 4-3. Microplots containing three tomato cv. Ta lladega plants per microplot randomized into a complete block design with eight replications of six tr eatments consisting of four isolates of Meloidogyne floridensis one isolate of M. incognita race 4, and one noninoculated control. ........................................................................................................59 4-4. Individual microplot consisting of a 47-cm-diam. 50-cm deep plastic pot planted with three tomato cv. Talladega seedlings wate red three times daily via irrigation tubing leading to an emitter. ........................................................................................................ ..59 8

PAGE 9

Abstract of Thesis Presente d to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BIOLOGICAL COMPARISON OF FOUR ISOLATES OF Meloidogyne floridensis FROM FLORIDA By Jason D. Stanley December 2008 Chair: Don W. Dickson Major: Entomology and Nematology Meloidogyne floridensis is a recently described species of root-knot nematode that infects and reproduces on the peach rootstock cultivar s Nemaguard, Nemared, Ok inawa, and Guardian. These rootstocks are resistant to M. incognita and M. javanica A 4-year study was conducted to differentiate the morphology, enzymatic profile, host preference, and pa thogenicity of four isolates of M. floridensis collected from different agricultural regions in Florida. Morphometric variability was observed among the four isolates, wh ich in some cases differed in their means but overlapped in their range values. In total, 1,027 females extracted from roots of peach, pepper, tobacco, and tomato represen ting all four isolates of M floridensis did not differ in their isozyme phenotypes for esterase and malate dehydrogenase and these phenotypes matched those reported in the original descriptio n. All four isolates of M. floridensis exhibited the same reaction as that of M. incognita race 2 in host differential tests with hosts being pepper, tobacco, tomato and watermelon, and nonhosts being cotton and peanut. In comparative host status studies, both rootknot nematode resistant and susceptible peach cu ltivars were good hosts for all four isolates of M. floridensis Both resistant and susceptible cultivars of tomato, pepper, corn, and soybean were evaluated. Results indicate th at the four populations of M. floridensis reproduced poorly but were able to overcome the resistance of the Mi-1 gene in tomato cv. Crista. Of the four isolates two 9

PAGE 10

10 reproduced poorly on the resistan t pepper cv. Charleston Belle and two reproduced well on this cultivar. Both the root-knot nematode resistan t corn cv. Mp 710 and susceptible cv. Dixie 18 were good hosts for all four isolates, whereas both the resistant soybean cv. Forrest and susceptible cv. S64-J1 were nonhos ts for all four isolates of M. floridensis The pathogenicity among the four isolates compared with M. incognita was not determined because of high densities of root-knot nematodes in the nontreated control microplot s. In summary, the ability of M. floridensis to break root-knot nematode resistance ge nes in peach cultivars presents a valid concern among peach growers and nurserymen who provide peach rootstocks as the Florida peach industry develops in future. Also, Florida growers who rely on the use of root-knot nematode resistant crops, such as pepper and to mato, may find this nematode causing substantial losses on such crops.

PAGE 11

CHAPTER 1 INTRODUCTION Root-Knot Nematodes (Meloidogyne spp.) Historical Background Root-knot nematodes (Meloidogyne spp.) are among the worlds most important soilborne plant pathogens. Root-knot or galling of plant roots induced by these nematodes is generally easily identified on most plant hos ts. The first detection of rootknot disease of plants was made by Berkeley in 1855 while studying galls on roots of greenhouse grown cucumber ( Cucumis sativus ) in England. Gldi, 1887 was the first to propose the name Meloidogyne for a root-knot nematode he found infecting coffee in Brazil, which he described later as Meloidogyne exigua In 1949 Chitwood re-erected the genus Meloidogyne (Gldi, 1887) to in clude all root-knot nematode species, redescribed the type species M. exigua (Gldi, 1887) and three other major species of root-knot nematodes including M. arenaria (Neal, 1889), M. incognita (Kofoid and White, 1919), and M. javanica (Treub, 1885) (Chitwood, 1949). This work also led to the description of one new species, M. hapla, and one subspecies, M. incognita var. acrita (Hirschmann, 1985). Chitwood adopted Meloidogyne for the genus name and based his description and new species on morphological comparisons. These comparisons included stylet knob morphology, dorsal esophageal gland opening loca tion, and labial disk comparis ons of males and second-stage juveniles (J2) as well as the female perineal pa ttern. The latter became one of the most widely used and dominant morphological diagnostic charac ters following the rediscription of the genus Meloidogyne Many years later it was concluded that M. incognita var. acrita was impossible to separate from M incognita based on morphology (Triantaphyll ou and Sasser, 1960). Taylor and Sasser (1978) reported M. incognita var. acrita to be a host race of M. incognita and designated 11

PAGE 12

it as M incognita race 3. Also, using host differentials these authors identified three additional races of M. incognita and two races of M. arenaria No host races were designated for M. hapla or M. javanica However, two cytological races of M. hapla have been reported based on chromosome number, race A and B (Triantaphyllou, 1966) and four host races of M. javanica have been reported (Carneiro et. al, 1998, Carn eiro et.al, 2003, Rammah and Hirschmann, 1990). During the interval between Berkeleys init ial discovery of root-knot nematode in 1855 and the re-erection of the genus Meloidogyne by Chitwood in 1949, this group of nematodes underwent numerous name changes. Currently the genus Meloidogyne comprises 94 nominal species (Brito et al., 2008) (Table 1-1). Development and Life Cycle The root-knot nematode life cycle is as follows: there is one egg stage, four juvenile stages (J1 to J4), four molts, and the adult stage. Th e single cell egg begins embryogenesis by dividing and leads to the development of the first-stage juvenile (J1). Th e J1 then molts and becomes a second-stage juvenile (J2) inside the egg. The ve rmiform J2 emerges from the egg and begins an exploratory phase in search of roots to feed upon. The search seem s to be random until the J2 is guided by substances emanating from roots (Taylor and Sasser, 1978). Once a penetration site is selected, the J2 infects the roots of a host and moves mostly betw een root cells until a suitable feeding site is established. Th e infective J2 undergoes further morphological changes that result in the J2 developing inside the root. Its body increases in size becoming sausage shaped. The sausage-shaped J2 feeds and induces the formation of giant cells, which are multinucleated with dense cytoplasm and highly invaginated cell walls. Th ere is repeated mitosis of a single nucleus within the same cell (Huang and Maggenti, 1969; Jones a nd Payne, 1978; Eisenback and Triantaphyllou, 1991). The J2 will molt to become a third-stage juvenile (J3), then molt a third time to become a fourth-stage juvenile (J4), bo th of which do not feed, and a final fourth molt 12

PAGE 13

will produce the adult. The female feeds on the giant cells and becomes sexually mature, whereas the male will undergo a type of metamor phoses during its final molt, resulting in an eelshaped mobile nematode. Males are often found within egg masses, root tissue, or in the plant rhizosphere. The female produces a gelatinous matrix that is released by specialized cells near the anus in which eggs will be laid. The gelatinous matrix will generally be located outside of the root surface but in some circumstances will be found internally. The lengt h of the life cycle in root-knot nematodes is greatly influenced by te mperature. The required time to complete a life cycle is generally 16 to 31 days with an optimum temperature range from 15 to 30 C. There is little activity by Meloidogyne species above about 40 C or below 5 C (Taylor and Sasser, 1978). Complete morphological characteristi cs of all life stages of the genus Meloidogyne are reported by Jepson (1987). Root-knot nematodes reproduce by either amphimixis and (or) parthenogenesis. Amphimixis involves insemination of females by males and is more common in adverse situations involving external stressors to a population. This results in an incr ease in the number of males. While many parthe nogenetic species such as M. arenaria M. incognita and M javanica may reproduce by amphimixis under adverse conditions, some Meloidogyne species such as M. spartinae M. megatyla and M microtyla reproduce only by amphimixis (Karssen and Moens, 2006). Parthenogenesis ca n be either meiotic or mito tic, and involves the production of viable eggs by noninseminated females. Symptoms and Signs Plant symptoms caused by root-knot nematode infection can be both above and below ground. Above ground symptoms are a ma nifestation of the degradation of the roots of the plant. These symptoms would be similar to those cau sed by other soilborne pathogens and external stressors such as extreme temperatures, nutrien t deficiency or lack of water. Above ground 13

PAGE 14

symptoms may include patchy, sparse growth, st unting, chlorosis, incipient wilting, defoliation, and loss of yield. The level of plant response depends on the nematode population density, the Meloidogyne species involved, and the plant cult ivar involved. Extreme adverse growing conditions exacerbates the degree of damage e .g., drought or poor soils (Jepson, 1987). Below ground symptoms include both the characteristic ga lling most usually associated with root-knot nematodes, and rotting of roots caused by secondary pathogens that i nvade the galled tissue seeking a rich nutrient source (Jepson, 1987). Mor phological changes in root s are attributable to the formation of the giant cells where there is an increase in cell si ze (hypertrophy) and cell number (hyperplasia). The formation of giant ce lls may vary depending on the virulence of the nematode species and the reaction of the host. In some cases these nematodes may induce the development of numerous adventiti ous roots or severely limit the growth of secondary roots. One may observe signs of root-knot nematodes on infected plant roots. Once plant roots are galled, a careful examination of the root surface may re veal small egg masses approximately 750 m in diameter. These egg masses will initially be transp arent and then become brownish in color and may have particles of sand ingrained in them. Additional signs of rootknot nematode infection include various developmental stages of the nematode inside the root tissue. Economic Importance Crop losses caused by root-knot nematodes occur throughout the world, with the most obvious damage and losses occurring in warm clim ates with long growing season and warm soil temperatures. Plant-parasitic nematodes are estimat ed to cause annual losses to agriculture on the order of several billion dollars in the United States alone (Sasser a nd Carter, 1985). A large portion of this monetary loss is because of root-knot nematodes. These nematodes have a worldwide distribution and a broad host range. Al most all of the plants that account for the majority of the worlds food supply are suscep tible to infection by root-knot nematodes. 14

PAGE 15

Although average crop yield losses are thought to be about 5%, small farmers in developing nations commonly experience much larger losses (Sasser and Carter, 1985). Identification Identification of Meloidogyne spp. is difficult because many species have morphological characters that are variable and many morphometric measur ements overlap among species. Methodologies used in the identification of Meloidogyne spp. include morphology, chromosome number and mode of reproduction, host differential tests, bioche mical and molecular analyses. Morphological identification Morphological identification of Meloidogyne spp. involves both the use of descriptive morphological characteristics and measurements (morpho metrics) of select characters of specific life stages. There is great variability observed in female perineal patterns and body size, but morphometrics of important morphological featur es such as stylet length in both males and females, and J2 tail length are more stable (Jepson, 1987). While there is some morphological variability within a species, variability among species is gene rally greater, thus one can use morphology as a tool to aid w ith identification. However, Meloidogyne spp. cannot generally be identified by morphological characters of an individual specimen because of the variability within a species. Certainly the larger the numbe r of specimens examined the more accurate the identification is likely to be. It is suggested that never less than 10 specimens be observed for proper identification (Taylor and Sasser, 1978). This rationale applies to males, females, and J2. Initially, root-knot nematode morphology was descri ptive rather than comp arative, with more than 140 characters of the egg, J2, female, and ma le examined (Jepson, 1987). Originally only morphological characters were used to describe new Meloidogyne spp. Work was later done to find characters that could differentiate specie s of root-knot nematodes (Esser, 1976; Eisenback and Hirschmann, 1979, 1981; Eisenback et al., 1980, 1981; Jepson, 1987). Currently characters 15

PAGE 16

such as J2 stylet length, tail le ngth, body length and excretory pore location; male stylet length, spicule length, and gubernaculum length; and female vulval slit length, styl et length, and location of excretory pore are used to aid in the identifica tion of root-knot nematode species (Brito et al., 2004; Handoo et al., 2004). Biochemical identification Polyacrylamide gel electrophoresis was reported as a method for identification of root-knot nematodes in 1971 (Dickson et al., 1971). Of the several isozyme phenotypes explored, esterase and malate dehydrogenase (MDH) proved to be usef ul in species identific ation. In this study the authors used mainly mass homogenates of fema les and J2. It was not until 1978 that a method was reported for resolving proteins from singl e individual life stages (Dalmasso and Berg, 1978). Using this technique, the enzyme phenotypes from 291 isolates of 16 species of root-knot nematodes originating from 65 countries were detected. This technique allowed for the comparison of 20 to 25 individual Meloidogyne females within the same gel. This method proved to be very useful to assess the purity or homogenicity of greenhous e cultures, to separate individual Meloidogyne species from mixed field isolates, to purify isolates by using a single egg mass from an individual female for inoculum, and to ensure proper identification of species, which is critical for plant breeding programs to establish root-knot nemat ode resistant cultivars that may or may not be species specific. Many re searchers have demonstrated the importance of isozyme electrophoresis in expedi ting the identification of root-knot nematode species collected in different parts of the wo rld (Brito et al., 2004, 2008; Carneiro et al., 1996, 1998, 2000; Dalmasso and Berge, 1978; Esbenshade and Tr iantaphyllou, 1985, 1990; Fargette, 1987). While advances in biochemical identification have expedited the process of root-knot nematode identification, the main limitation to this approach is that the isozyme analysis requires that mature females be available for electrophoresis. If there are limited specimens available or the 16

PAGE 17

identification must be made more rapidly such as in the case of regulatory programs, mature females may not be available, and the time re quired to produce progeny may not be acceptable. The use of more recently develope d molecular techniques to aid in the identification of root-knot nematodes could alleviate this problem because th ey allow for the use of a single specimen such as a J2, which are normally more readily availa ble. Neither of these methods, however, provides a means for identifying host specificity or hos t races among the various root-knot nematode species. Differential hosts Differential hosts are used to detect pat hogenic variation within root-knot nematode isolates or species. This method was first reported as a means for separating species (Sasser, 1954). It was later refined and proposed as a method to differentiate and identify four Meloidogyn e species and six host races by subjecting the nematodes to certain plant cultivars with a designation of a plus (+) or minus (-) based on root galling and egg mass production (Table 1-2) (Taylor and Sasser, 1978). Molecular identification Recent achievements in molecular characteri zation of nematodes have advanced our knowledge concerning the phylogenetic relationships between nemat ode taxa and systematics. The use of molecular techniques has become a routine practice in nematode identification (Subbotin and Moens, 2006). The integrati on of multiple methodologies (morphological, biochemical and molecular) for the identifica tion of not only root-knot nematodes, but nematodes in general, is consistently deemed to be the best approach. There are several reasons for a rapid move toward the use of molecular techniques. They o ffer several advantages such as: (i) morphological characters may not always remain constant and may at times be influenced by environmental factors whereas DNA is strictly he ritable and usually stable (Subbotin and Moens, 17

PAGE 18

2006); (ii) the process of morphol ogical identification is generally conducted by scientists with wide experience in nematode morphology, whereas DNA protocols have become standardized and commercial kits are availa ble to non-experts in nematode morphology and anatomy; and (iii) DNA analyses can be accomplished using a single specimen of any nematode life-stage, whereas biochemical analyses require selected nematode life-stages, and morphological analyses require numerous specimens. The following molecular techniques ar e used for the identification of Meloidogyne spp.: (i) Polymerase Chain Reaction-Restriction Frag ment Length Polymorphism (PCR-RFLP) a polymerase chain reaction method that separated M. arenaria M. chitwoodi M. hapla, M. incognita and M. javanica (Powers and Harris, 1993); (ii) (PCR-RFLP ITS region) a polymerase chain reaction method that aided in th e identification of a wi de array of nematodes (Powers et al., 1997); (iii) Ra ndom Amplified Polymorphic DNAPCR Sequence Characterized Amplified Region, (RAPD-PCR, SCAR markers) a RAPD-PCR technique that separated Meloidogyne spp. (Zijlstra et al., 2000; Randig et al., 2002); (iv) Ribosomal DNA-Intergenetic Spacer Region (rDNA-IGS region) aided with distinguishing M. floridensis from M. arenaria M. incognita M. javanica and M mayaguensis (Handoo et al., 2004); (v) (RAPD analyses) Multiplex PCR a technique that detects one species or many species in a sample by using two sets of primers. Species-specific primers for multiplex PCR have been developed for Globodera pallida G rostochiensis Heterodera schactii, H. glycines Ditylenchus dipsaci and species of Meloidogyne and Pratylenchus (Subbotin and Moens, 2006). Meloidogyne floridensis History In 1966, a root-knot nematode population was firs t detected by Professor R. H. Sharpe (University of Florida, Fruit Crops Department ) infecting root-knot nematode resistant peach 18

PAGE 19

rootstock cvs. Nemaguard and Okinawa at the Univer sity of Florida, Gainesville, FL (Sharp et al., 1969). When first reported on resistant peach r ootstock this nematode was considered to be M incognita because of its similar morphology. La ter reports listed the nematode as M incognita race 3, apparently because there was re production on cotton (Sherman and Lyrene, 1983). In 1991 and 1998, this nematode also wa s reported parasitizing root-knot nematode resistant Nemared and Guardian peach rootstoc ks (Sherman et al., 1991; Nyczepir et al., 1998). All four of these rootstocks ar e resistant to the southern ( M. incognita ) and Javanese ( M. javanica ) root-knot nematodes (Nyczepir and Beckman, 2000; Sharp et al., 1969; Sherman et al., 1981; Sherman et al., 1991). Further investigation of this nematode suggested that this was in fact a new root-knot nematode species base d on morphology, host range, biochemical and molecular characterization (Nyczepir et al., 1998). This study culminated in the description of the nematode as M. floridensis (Handoo et al., 2004), the pe ach root-knot nematode. The population of the peach root-knot nemat ode used for the species description was originally collected from the peach rootstock Ne maguard in Gainesville, FL and maintained on tomato ( Solanum esculentum Mill. cv. Rutgers) in a greenhous e at the USDA, ARS Southeastern Fruit and Tree Nut Research Laboratory, Byron, GA. Further studies showed that the nematode isolates from peach failed to reproduce on pepper ( Capsicum annuum ), which is a known host for M incognita (Handoo et al., 2004; Kokalis-Burelle and Nyczepir, 2004). Life Cycle and Mode of Reproduction The life cycle for M. floridensis is similar to that of other sp ecies of root-knot nematodes. The oogenesis process of M. floridensis deviates from the meio tic parthenogenetic forms described by Triantaphyllou (1966) and Van de r Beek et al., (1998). No second maturation division was observed in the M. floridensis gonad (Handoo et al., 2004). Cytologically, bivalent formation during meiosis of M. floridensis provides evidence for a meiotic parthenogenic 19

PAGE 20

pathway (Handoo et al., 2004). The occurrence of meiotic parthenogenesis and suppression of the second maturation division could point toward an intermediate type of parthenogenesis, in between the meiotic form with two maturation divisions and mitotic parthenogenesis (Handoo et al., 2004). It seems reasonable to hypothesize that this species may reproduce by meiotic parthenogenesis and (or) am phimixis (Handoo et al., 2004). Symptoms and Signs Symptoms and signs of M. floridensis infection are the same as other species of root-knot nematodes. These symptoms include galling and ro tting of roots below ground; and loss of vigor, stunted growth, and early defoliation above ground. Signs of M. floridensis will be found exclusively underground and include egg masse s attached to the roots and different developmental stages of the nematode inside root tissue. While both symptoms and signs of M. floridensis infection can be used as diagnostic tools; signs of the nematode are the most reliable since many of the apparent above ground symptoms can be caused by other factors. Host Range In addition to the various peach rootstocks, other hosts reported for M. floridensis were amaranthus ( Amaranthus spinosus ), American pokeweed ( Phytolacca americana ), basil ( Ocimum basilicum ), crimson clover ( Trifolium incarnatum ), cucumber ( Cucumis sativus ), cypress vine ( Ipomoea quamoclit ), dichondra (Dichondra repens ), dill (Anethum graveolens), eggplant ( Solanum melongena), English watercress ( Nasturtium officianale ), impatiens ( Impatiens wallerana), lilac tasselflower ( Emilia sonchifolia ), molinillo ( Leonotis nepetaefolia ), morning glory ( Ipomea violacea, Ipomea triloba ), rape ( Brassica napis ), red root pigweed ( Amaranthus retroflexus ), snap bean ( Phaseolus sp.), snapdragon ( Antirrhinum majus), spurge nettle ( Cnidoscolus stimulosus ), squash ( Cucurbita moschata ), tomato ( Solanum esculentum ), velvet leaf ( Abutilon theophrasti ), verbena ( Verbena hybrida ), watermelon ( Citrullus lanatus), 20

PAGE 21

and wild cucumber ( Cucumis anguira ) (Brito et al., 2004; Handoo et al., 2004; Kaur et al., 2006; Kokalis-Burelle and Nyczepir, 2004; Stanley et al., 2006). Distribution and Econ omic Importance In addition to the Gainesville isolates used for the species description, seven additional isolates of M. floridensis have been identified in six Florida counties including: Alachua, Hendry, Hillsborough, Indian River, Seminole, a nd St. Lucie (Brito et al., 2005, 2008; Church, 2005). These isolates of M. floridensis were discovered during a coope rative survey of root-knot nematodes in the state of Florida conducte d by the Nematology Section of the Florida Department of Agriculture and C onsumer Services, Division of Plan t Industry and the University of Florida Entomology and Nematology Departme nt. The populations were found on snap bean in Alachua County; cucumber, lil ac tassel flower, and tomato in Hendry County; eggplant in Hillsborough County, and tomato in Indian River County (Brito et al., 20 08; Brito et al., 2005). M floridensis also was reported on tomato in Semi nole and St. Lucie Counties by Church (2005). The importance regarding M. floridensis as a pathogen of agricultural crops is because of this nematodes ability to overcome root-kno t nematode resistance genes in some peach rootstocks and other crops. Peaches are comm ercially grown throughout the world. In 2007 more than 1 million tons of peaches were produced in the United States alone with a value of 498 million dollars, with California being responsible fo r more than half the to tal crop (United States Department of Agriculture, National Agri cultural Statistics Service, 2005). Identification Morphological and morphometric analysis In the original description of M. floridensis Handoo et al., (2004) provides a morphological and morphometric diagnosis of M. floridensis as having J2 with a mean body 21

PAGE 22

length of 355 (310-390 m), truncate head withou t annulation, stylet length of 10.1 (10-11 m) with small rounded knobs, lateral field with fo ur incisures, tail 39.4 (35-42.5 m) long, hyaline tail terminus 9.8 (8-12 m); female perineal patte rn with coarse to broke n network-like striae in and above anal area, faint lateral lines interrupti ng transverse striae and smooth wavy lines in the outer field; prevulval re gion, typically without striae; vulva and anus sunken, phasmids large and distinct with conspicuous phasmidal canal. Male s with both short and long forms 1,162 (564 m1.7 mm), stylet length of 20 (17-23 m) with rounded posteri orly sloping knobs, spicules 28 (2335 m) long, and gubernaculum length 7.7 (5-10 m ). As with all root-knot nematodes, M. floridensis is sexually dimorphic with males and the infective stage juvenile being vermiform shaped and mobile, whereas late-stage juveniles destined to be females become pyriform shaped and sedentary. M. floridensis closely resembles M. incognita M. christiei M. graminicola and M. hispanica but with light and scanning electron micr oscopic observations it differs from these species either by the body length, shape of head, ta il and tail terminus of second-stage juveniles; body length and shape of spicules in males, and a distinctive female peri neal pattern (Handoo et al., 2004). Electrophoretic analysis The esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD), and glutamate-oxaloacetate transaminase (GOT) phenotypes for M. floridensis were reported (Carneiro et al., 2000). M. floridensis has a unique EST profile that is species specific and has a high diagnostic value different from all othe r known root-knot nematode species and is designated as MF3 (Rm 38.7, 40.69, 44.18) (Brito et al., 2008). It is charact erized as having the presence of three bands with the central band located at about the same position as the upper band of M. javanica. The EST pattern of the latter is us ed worldwide as a control. The MDH 22

PAGE 23

phenotype has been designated as N1 (E sbenshade and Triantaphyllou, 1985) for M. floridensis and is identical to that of M. arenaria M. incognita and M. javanica Intergenetic spacer rDNA analyses The IGS rDNA sequence of M. floridensis was similar to M. arenaria and M. incognita with both having eight nucleotide changes at five and seven positions representing a 1.1% difference from M. floridensis M. javanica had nine nucleotide cha nges at five positions representing a difference of 1.3% (Handoo et al ., 2004). There was initia l concern that this nematode might be a variant of M. mayaguensis which was detected in the United States for the first time in 2004 (Brito et al., 2004), however, M. mayaguensis had 162 changes at 85 positions, representing a 39% differe nce (Handoo et al., 2004). The RAPD-PCR analyses As part of the speci es description of M. floridensis 11 Meloidogyne isolates representing seven species were separated based on amplifi cation product patterns of primer-DNA templates (Handoo et al., 2004). Five hundred and eleven reproducible fr agments were amplified and scored as RAPD markers. All Meloidogyne spp. could be unambiguously identified and visual analysis indicated that M. floridensis was clearly different from the others. High-fidelity PCR-RFLP analyses High fidelity PCR was used to amplify mito chondrial DNA sequences located between the cytochome oxidase subunit II (COII) and 16S rRNA genes from M. mayaguensis (0.7 kb), M. floridensis (1.1 kb), M arenaria (1.1kb), M. incognita (1.5 kb), and M. javanica (1.6 kb) (Jeyaprakash et al., 2006). This technique differentiated M. floridensis from all of the other species except M. arenaria Further studies of the AT-rich region, or the non-coding region using high fidelity PCR diff erentiated all five Meloidogyne species with the following base pair results: 23

PAGE 24

M mayaguensis (167 bp), M. arenaria (573 bp), M. floridensis (603 bp), M. incognita (963 bp), and M. javanica (1,110 bp). Objectives Our objectives were to investig ate four field populations of M. floridensis by (i) characterizing them using morphometrics, polyacr ylamide gel electrophor esis and differential hosts; (ii) determining their hos t status on root-knot nematode resistant and susceptible plant cultivars; and (iii) determining their pathogenici ty and virulence on the tomato cv. Talladega in microplots. 24

PAGE 25

Table 1-1. Alphabetical listi ng of currently recognized Meloidogyne species No. Species Authority Type species M. exigua Gldi, 1892 1 M. acronea Coetzee, 1956 2 M. actinidiae Li & Yu, 1991 3 M. africana Whitehead, 1960 4 M. aquatillis Esbary & Eveleigh, 1983 5 M. arabicida Lpez & Salazar, 1989 6 M. ardenensis Santos, 1968 7 M. arenaria (Neal, 1889) Chitwood, 1949 8 M. artiellia Franklin, 1961 9 M. baetica Castillo, Vovlas, Subbotin & Troccoli, 2003 10 M. brasiliensis Charachar & Eisenback, 2002 11 M. brevicauda Loos, 1953 12 M. californiensis Abdel-Rahman & Maggenti, 1987 13 M. camelliae Golden, 1979 14 M. caraganae Shagalina, Ivanova & Krall, 1985 15 M. carolinensis Eisenback, 1982 16 M. chitwoodi Golden, OBannon, Santo & Finley, 1980 17 M. christiei Golden & Kaplan, 1986 18 M. cirricauda Zhang, 1991 19 M. citri Zhang, Gao & Weng, 1990 20 M. coffeicola Lordello & Zamith, 1960 21 M. cruciani Garcia-Martinez, Taylor & Smart, 1982 22 M. cynariensis Bihn, 1990 23 M. decalineata Whitehead, 1968 24 M. donghaiensis Zheng, Lin & Zheng, 1990 25 M. dunensis Palomares Rius, Vovlas, Troccoli, Libanas, Landa, & Castillo, 2007 26 M. duytsi Karssen, van Aelst & van der Putten, 1998 27 M. enterolobii Yang & Eisenback, 1983 28 M. ethiopica Whitehead, 1968 29 M. fallax Karssen, 1996 30 M. fanzhiensis Chen, Peng & Zheng, 1990 31 M. floridensis Handoo, Nyczepir, Esmenjaud, van de r Beek, Castagnone-Sereno, Carta, Skantar & Higgins, 2004 32 M. fujianensis Pan, 1985 33 M. graminicola Golden & Birchfield, 1965 34 M. graminis (Sledge & Golden, 1964) Whitehead, 1968 35 M. hainanensis Liao & Feng, 1995 36 M. hapla Chitwood, 1949 25

PAGE 26

Table 1-1. Continued No. Species Authority 37 M. haplanaria Eisenback, Bernard, Starr, Lee & Tomaszewski, 2003 38 M. hispanica Hirschmann, 1986 39 M. ichinohei Araki, 1992 40 M. incognita (Kofoid & White, 1919) Chitwood, 1949 41 M. inornata (Lordello, 1956) 42 M. indica Whitehead, 1968 43 M. izalcoensis Carneiro, Almeida, Gomes & Hernandez, 2005 44 M. javanica (Treub, 1885) Chitwood, 1949 45 M. jianyangensis Yang, Hu, Chen & Zhu, 1990 46 M. jinanensis Zhang & Su, 1986 47 M. kikuyensis de Grisse, 1960 48 M. konaensis Eisenback, Bernard & Schmitt, 1994 49 M. kongi Yang, Wang & Feng, 1988 50 M. kralli Jepson, 1984 51 M. litoralis Elmiligy, 1968 52 M. lini Yang, Hu & Xu, 1988 53 M. lusitanica Abrantes & Santos, 1991 54 M. mali Itoh, Oshima & Ichinohe, 1969 55 M. maritima (Jepson, 1987) Karssen, van Aelst & Cook, 1998 56 M. marylandi Jepson & Golden, 1987 57 M. mayaguensis Rammah & Hirschmann, 1988 58 M. megadora Whitehead, 1968 59 M. megatyla Baldwin & Sasser, 1979 60 M. mersa Siddiqi & Booth, 1992 61 M. microcephala Cliff & Hirschmann, 1984 62 M. microtyla Mulvey, Townshend & Potter, 1975 63 M. mingnanica Zhang, 1993 64 M. minor Karssen, Bolk, van Aelst, van den Beld, Kox, Korthals, Molendijk, Zijlstra, Hoof & Cook, 2004 65 M. morocciensis Rammah & Hirschmann, 1990 66 M. naasi Franklin, 1965 67 M. nataliei Golden, Rose & Bird, 1981 68 M. oryzae Maas, Sanders & Dede, 1978 69 M. oteifae Elmiligy, 1968 70 M. ottersoni (Thorne, 1969) Franklin, 1971 71 M. ovalis Riffle, 1963 72 M. panyuensis Liao et al., 2005 73 M. paranaensis Carneiro, Carneiro, Abrantes, Santos & Almeida, 1996 74 M. partityla Kleynhans, 1986 75 M. petuniae Charachar, Eisenback, & Hirschmann, 1999 26

PAGE 27

Table 1-1. Continued No. Species Authority 76 M. pini Eisenback, Yang & Hartman, 1985 77 M. piperi Sahoo, Ganguly & Eapen, 2000 78 M. plantani Hirschmann, 1982 79 M. propora Spaull, 1977 80 M. querciana Golden, 1979 81 M. salasi Lopez, 1984 82 M. sasseri Handoo, Huettel & Golden, 1993 83 M. sewelli Mulvey & Anderson, 1980 84 M. sinensis Zhang, 1983 85 M. spartinae (Rau & Fassuliotis, 1965) Whitehead, 1968 86 M. subartica Bernard, 1981 87 M. suginamiensis Toida & Yaegashi, 1984 88 M. tadshikistanica Kirjanova & Ivanova, 1965 89 M. thailandica Handoo, Nyczepir, Esmenjaud, van der Beek, Castognone-Sereno, Carta, Skantar & Higgins, 2005 90 M. turkestanica Shagalina, Ivanova & Krall, 1985 91 M. trifoliophila Bernard & Eisenback, 1997 92 M. triticoryzae Gaur, Saha & Kahn, 1993 93 M. ulmi Palmisano & Ambrogioni, 2001 94 M. vandervegtei Kleynhans, 1988 27

PAGE 28

Table 1-2. Differential hosts used to identify species and races of root-knot nematodes. Differential hostsa Meloidogyne species and Tobacco Cotton Pepper Watermelon Peanut Tomato race M. incognita Race 1 + + + Race 2 + + + + Race 3 + + + + Race 4 + + + + + M. arenaria Race 1 + + + + + Race 2 + + + M. javanica Race 1 + + + Race 2b + + + + Race 3b + + + + Race 4c + + + + + M. hapla + + + + aCultivars designated by Taylor and Sasser, 1978 for the differential host test include tobacco ( Nicotiana tabacum cv. NC 95), cotton ( Gossypium hirsutum cv. Deltapine 16), pepper ( Capsicum annuum cv. California Wonder), watermelon ( Citrullus lanatus cv. Charleston Gray), peanut ( Arachis hypogaea cv. Florunner), tomato ( Solanum esculentum cv. Rutgers). b M. javanica race 2 and 3 designated by Rammah and Hirschmann, 1990. c M. javanica race 4 designated by Carneiro et al., 2003. 28

PAGE 29

CHAPTER 2 COMPARISON OF FOUR ISOLATES OF Meloidogyne floridensis USING MORPHOMETRICS, ISOZYMES, AND DIFFERENTIAL HOSTS Introduction Meloidogyne floridensis was identified in Florida as a new species in 2004 (Handoo et al., 2004). Since that time seven populations have been identified in six Flor ida counties on various host crops (Brito et al., 2005, 2008; Church 2005). This nematode is important to agriculture because it breaks root-knot nematode resistance in peach (Sharp et al., 1969; Sherman and Lyrene, 1983; Sherman et al., 1991, Nyczepir et al., 1998; Handoo et al., 2004). Due to the lack of proven available nematicides and the removal of methyl bromide from the world marketplace, much emphasis has been and is currently being placed on alternatives to chemical control of root-knot nematodes, such as the use of root-knot nematode resistant plant cultivars. The ability of M. floridensis to break the resistance of several peach rootstocks, such as cvs. Nemaguard, Nemared, and Okinawa, not only led to the desc ription of the species it also causes valid concerns regarding the ability of this nematode to break root-knot nematode resistance in other plant species. Other than the population used fo r the species description, no information is available about othe r populations of M. floridensis Morphology, morphometrics, polacrylamide gel electrophoresis (PAGE) and host differentials were used to aid with the identification of these new isolates of M. floridensis from Florida. Morphol ogical identification of Meloidogyne spp. involves the use of descriptiv e morphological characteristics and measurements of select characters of specific life stages and has been used historically to differe ntiate species of rootknot nematodes (Esser, 1976; Eisenback a nd Hirschmann, 1979, 1981; Eisenback et al., 1980, 1981; Jepson, 1987). The use of polyacrylamide gel electrophores is (PAGE) to resolve isozyme phenotypes such as esterase and malate dehydrogenase (MDH) has proven to be useful and accurate in the 29

PAGE 30

identification of Meloidogyne spp. (Brito et al., 2004, 2008; Carneiro et al., 1996, 1998, 2000; Dalmasso and Berge, 1978; Esbenshade and Triantaphyllou, 1985, 1990; Fargette, 1987). Differential host tests are currently the only technique available to detect pathogenic variation at both the species and sub-species level by distinguishing host races (Taylor and Sasser, 1978). The objectives of this study we re to biologically compare and classify four isolates of M floridensis by (i) morphological evaluation of select ed characters, (ii) characterization of isozyme phenotypes, and (iii) determination of pathogenic variation among isolates using host differentials. Materials and Methods Nematode Source Three of the four isolates of M. floridensis used for this research we re collected as part of a cooperative root-knot nematode survey conducted throughout the state of Florida by the Florida Department of Agriculture and Consumer Services (FDACS), Divi sion of Plant Industry (DPI), Nematology Section, and the Universi ty of Florida Entomology and Nematology Department (Brito et al., 2008). Th e designation and origin of these four nematode isolates were as follows: isolate 1 (N03-01894) was obtained from the population used for the species description (Handoo et al., 2004), which was origin ally found infecting the peach rootstock cv. Nemaguard in Alachua County. Isolate 2 (N03-0 1582) was collected from tomato in Indian River County; isolate 3 (N04-00503) was collected from infected tomato in Hendry County; and isolate 4 (N04-00627) was collected from cucumber in a separate field in Hendry County. The numbers assigned to the four M. floridensis isolates are FDACS, DPI, Nematology Section log numbers (Table 2-1). 30

PAGE 31

Development of Single Egg Mass Isolates Single egg mass isolates were prepared from each M. floridensis population by collecting egg laying females from the original field popul ations and subjecting at least 26 individual females to polyacrylamide gel electrophoresis (Br ito et al., 2008). Root-knot nematode females were collected from field populations by direct dissection from roots under a microscope. The individual female and associated egg mass were each placed into a separate 0.6-mL microcentrifuge tube with 5-l di H20. Once females were identified as M. floridensis by using polyacrylamide gel electrophoresis the associated egg mass for each female was reared on tomato cv. Rutgers in steam pasteurized soil in a 25-cm diam. clay pot inside a greenhouse. Ten to 13 single egg masses were i noculated onto separate tomato plants. All plants were hand watered daily and fertilized week ly with 20-20-20 NPK fertilizer (Peters Professional, Division of United Industries., St. Louis, MO) and treated with fungicides and inse cticides as needed. A tomato plant inoculated with a single egg mass of M. floridensis generally took up to 6 months before the infection level was high enough to produce inoculum for isolate renewal or experimental use. At this point a single representative plant for each isolate derived from a single egg mass was chosen to represent each isolate ba sed on highest level of infection achieved. Eggs were extracted using the 1% NaOCl method (Hu ssey and Barker, 1973) as modified (Boneti and Feraz, 1981). At each subsequent isolate renewal, new tomato seedlings were inoculated with 5,000 J2 and eggs per pot. This inoculum level wa s used to maintain all isolates throughout the study. Generally this nematode took 2 to 4 months to build up inoculum levels high enough for re-inoculation. Comparative Morphology The morphology of the four isolates of M. floridensis was compared to determine their interand intra-specific variability. Selected me asurements were taken from 20 males, females, 31

PAGE 32

and second-stage juveniles from each isolate (B rito et al., 2004; Handoo et al., 2004). Secondstage juveniles and males were collected from root s placed in a Petri dish with a small amount of water. Males and J2 were relaxed with minima l heat and mounted in water agar (Esser, 1986). Females were dissected directly from infected root systems and cut transversely before being mounted on water agar so that measurements coul d be taken. Specific char acters were measured by an ocular micrometer using a compound micros cope. In total, 1,040 J2 measurements were taken with 13 characters per 20 J2 per each of four isolates; 1,120 measurements were taken for males with 14 characters per 20 males per each of four isolates; and 240 measurements were taken for females with three characters per 20 fe males per each of four isolates. In total, 2,400 individual measurements were made. Isozyme Analysis Fresh egg-laying females were collected from tomato cv. Rutgers and peach cvs. Lovell and Nemaguard from each of the four isolates of M. floridensis and subjected to polyacrylamide gel electrophoresis (PAGE) to characterize and verify the esterase a nd malate dehydrogenase profile of each. Electrophoresis was carried out using a Mini-prot ean III (Bio-Rad) (Brito et al., 2004). The total number of females subjected to PAGE from each isolat e grown on tomato as well as other hosts is re ported in Table 2-2. Differential Host Test Differential host studies were carried out using cotton ( Gossypium hirsutum cv. Deltapine 16), peanut ( Arachis hypogaea cv. Florunner), pepper (Capsicum annuum cv. California Wonder), tobacco ( Nicotiana tabacum cv. NC 95), tomato ( Solanum esculentum cv. Rutgers), and watermelon ( Citrullus lanatus cv. Charleston Gray) (Taylor and Sasser, 1978). This experiment consisted of five re plicates per plant variety per M. floridensis isolate with 30 plants per isolate and 120 plants total. All plants were grown from seed and germinated in vermiculite 32

PAGE 33

in plastic trays. Tobacco seed was planted rough ly 30 days before pepper and the rest 14 days after pepper. This delay allowed fo r variable germination rates of the test plants. Once all test plants reached a height of between 10 and 15-cm, they were transplanted to 25-cm diam. clay pots and allowed to grow for 2 weeks in a gree nhouse. The day before inoculation, eggs were extracted as mentioned above. Eggs were quantified by averaging representative counts of the stock solution in a counting chamber under light microscope observati on. All plants were inoculated with 5,000 J2 and eggs per plant on the same day. Plants were maintained in a growth room for 60 days with a temperature range be tween 17.7 and 30 C. Plants were watered daily and fertilized weekly with a 20-20-20 NPK fertilizer (Peters Professional, Division of United Industries., St. Louis, MO). Insecticides and fungicides were used as needed. After 60 days, plants were removed from pots and root system s were washed so that galling and egg mass numbers could be determined. An index scale of 0-5 was used where 0 = no galls and egg masses; 1 = 1-2 galls and egg masses; 2 = 3-10 galls and egg masses; 3 = 11-30 galls and egg masses; 4 = 31-100 galls and egg masses; and 5 = >100 galls and egg masses per root system (Taylor and Sasser 1978). Data were subjecte d to ANOVA using SAS 9.1 (SAS Institute, Cary, NC), and means were compared based on Duncans multiple-range test at P 0.05. Results and Discussion Comparative Morphometrics The morphometrics for J2, males, and females from all four isolates of M. floridensis are reported in Tables 2-3, 2-4, and 2-5. All four isolates were morphologically similar to each other and compared to the original description of M. floridensis (Handoo et al., 2004). Some differences were observed for certain characte rs among the isolates but the ranges of these characters overlapped, thereby indicating that the differences between mean values can be attributed to body size variability within and among isolates ( P > 0.05). The differences between 33

PAGE 34

mean values of nonallometric characters such as stylet length were < 1 m and difficult to quantify using a light microscope. In comp arison to the original description of M. floridensis the measurements of certain morphologi cal characters of the four isolat es in this study appeared to be greater, but in fact are due to the slight difference between measurem ents taken from freshly killed specimens and those that have been fixe d and mounted in glycerin as was done in the species description of M. floridensis The difference in measurements due to fixation shrinkage in certain Pratylenchus species was estimated to be 5 to 7% (Saha and Kahn, 1989). When taking this into consideration the values of the heat relaxed specimens overlapped the range of those in the original description. As menti oned before, the statistical differe nces in individual characters do not outweigh the fact that the ranges overlap. The morphological comparisons of four isolates of M. floridensis and comparison to the original description of this species do not show evidence of differences among and between isolates. Isozyme Analysis The isozyme phenotype identified from all females from four M. floridensis isolates remained constant regardless of host and matched the designation of MF3 (Rm 38.7, 40.69, 44.18) for M. floridensis for esterase (Brito et al., 2008) and the designa tion of N1 for malate dehydrogenase (Esbenshade and Triantaphyllou, 1985) (Figures 2-1 and 22). These results also indicate that M. floridensis does not demonstrate esterase poly morphism based on select hosts as was reported for M. konaensis (Sipes et al., 2005). Differential Hosts Based on the gall and egg mass ratings tomato, pepper, tobacco, and watermelon are hosts for all four isolates, whereas peanut and cotton are nonhosts (Table 2-6). However, there was low reproduction on cotton (gall index 1.4 to 1.8, egg mass index 1.6 to 1.8). The results obtained in this study do not agree with the original description in that tobacco and pepper were susceptible 34

PAGE 35

35 hosts. The only hosts reported in the original description were tomato and watermelon (Handoo et al., 2004). All four isolates reproduced on pepper and tobacco as well as tomato and watermelon (Table 2-6). To confirm these finding s three additional steps were taken. First, the reproductive factor for pepper and tobacco was determined; second, 52 females were removed from both tobacco and pepper and subjected to P AGE to confirm their identity (Figures 2-3, and 2-4), and third, pepper and tobacco were evaluate d again. In all cases tobacco and pepper were determined to be susceptible to all four M. floridensis isolates (Table 2-7). All four isolates of M. floridensis fit the same differential host status profile as that of M. incognita race 2. Susceptible plants were tobacco, tomato, pepper, and watermelon and those considered as nonhosts were cott on and peanut. These data are s upported by and consistent with the results determined by the reproductive factor and esterase and malate dehydrogenase isozyme phenotypes.

PAGE 36

Table 2-1. Isolate numbers assigned to the orig inal Florida Department of Agriculture log number for four isolates of Meloidogyne floridensis used throughout this thesis research. Log number Isolate number Original host Location N03-01894 1 Peach Alachua County N03-01582 2 Tomato Indian River County N04-00503 3 Tomato Hendry County N04-00627 4 Cucumber Hendry County Table 2-2. Number of females of four isolates of Meloidogyne floridensis collected from four hosts and subjected to polyacrylamide gel electrophoresis to determ ine their esterase and malate dehydrogenase isozyme phenotype. Number of females Isolate Tomato a Pepper b Tobacco b Peach c Total 1 117 52 52 26 247 2 130 52 52 26 260 3 143 52 52 26 273 4 117 52 52 26 247 a Females collected from tomato ( Solanum esculentum ) cv. Rutgers during routine isolate screening. b Females collected from pepper ( Capsicum annuum ) cv. California Wonder and tobacco ( Nicotiana tabacum ) cv. NC 95. cFemales collected from peach ( Prunus persica ) cvs. Nemaguard and Lovell. 36

PAGE 37

Table 2-3. Select morphometrics (mean, st andard deviation, and range) of second-st age juveniles of four isolates of Meloidogyne floridensis from Floridaa. Isolates Character 1 2 3 4 Body length 384.0.9 ab 371.7.8 b 387.0.2 a 370.0 b (348-482) (338-393) (352-417) (335-392) Body width 14.9.4 a 14.7.43 b 14.5.5 b 14.5.4 b (14.0-16.0) (13.7-15.6) (13.7-14.8) (13.2-15.2) Stylet length 10.9.1 b 10.1.4 b 10.2.3 b 10.7.3 a (10.0-10.5) (9.8-11.3) (9.8-10.8) (10.2-11.4) DGO, from stylet base 2.9.0 b 3.2.3 a 3.3.3 a 3.2.4 a (2.5-3.0) (2.9-3.9) (2.9-3.9) (2.6-3.9) Center median bulb to 52.0.1 b 54.5.4 a 55.1.9 a 55.7.2 a anterior end (48.0-55.0) (50.0-59.0) (48.2-61.2) (51.4-59.7) 37Excretory pore to 80.9.8 b 79.5.5 b 83.7.8 a 82.1.4 b anterior end (74.5-86.0) (74.0-85.0) (76.4-89.1) (75.4-92.2) Tail length 41.1.8 a 44.0.3 a 43.3.1 a 43.4.4 a (34.0-45.0) (39.0-48.0) (38.2-48.0) (38.2-48.0) Base of esophageal gland 118.0.8 a 113.0.7 a 116.0.9 a 118.3 a to anterior end (96.0-139.0) (103-129) (102-132) (103-131) Hyaline tail terminus length 10.1.1 ab 8.6.1 c 8.5.4 bc 10.8.9 a (8.5-12.0) (5.9-9.8) (5.8-10.7) (8.8-11.8) a 26.0.2 a 25.0.8 a 26.7.2 a 26.0.2 a (23.0-28.0) (24.0-26.0) (24.5-29.0) (23.0-2 8.0) b 3.8.2 a 4.8.2 ab 4.9.5 ab 4.8.2 b (3.5-4.1) (4.4-5.2) (4.2-5.6) (4.3-5.0) b 3.3.3 ab 3.3.2 ab 3.4.3 a 3.1.2 b (2.7-4.0) (2.8-3.7) (3.0-4.0) (2.6-3.6)

PAGE 38

38Table 2-3. Continued Isolates Character 1 2 3 4 c 9.3.6 a 8.5.4 c 8.9.6 b 8.5.33 c (8.1-11.2) (8.0-9.3) (7.7-10.2) (7.8-9.1) a Measurements ( m) were taken using 20 specimens from each isolate. b Means in the same row followed by the same letter are not significantly different according to Duncans multiple-range test (P 0.05).

PAGE 39

Table 2-4. Select morphometrics (mean, standard devi ation, and range) of males of four isolates of Meloidogyne floridensis from Floridaa. Isolates Character 1 2 3 4 Body length 1,514 ab 1,477.8.6 a 1,547.5 a 1,203.6 b (793-2,038) (993-1,875) (1,072-1,867) (838-1,847) Body width 33.2.9 b 34.2.5 a 35.7.7 a 32.9.9 b (23.5-41.2) (28.4-41.0) (28.4-39.2) (27.4-39.2) Stylet length 21.2.7 b 21.4.7 ab 21.9.7 ab 22.1.2 a (18.0-24.0) (17.6-24.5) (20.6-22.8) (20.6-24.5) Stylet knob width 5.1.4 b 5.8.3 a 5.3.3 b 5.1.5 b (4.4-6.0) (4.9-6.3) (4.9-5.7) (4.4-5.9) Stylet knob height 2.9.3 b 3.1 0.2 a 3.1.2 a 3.1.2 a (2.3-3.4) (2.9-3.4) (2.9-3.4) (2.7-3.4) 39DGO 3.2.5 ab 2.81.3 c 0.36.5 a 3.0.5 bc (2.4-4.4) (2.4-3.4) (2.5-4.4) (2.5-4.4) Excretory pore to 162.8.8 ab 151.2 b 175.6.9 a 155.8 b anterior end (105-209) ( 119-183) (132-212) (122-226) Center median bulb to 90.1.2 a 89.8.3 a 91.2.1 a 91.6.9 a anterior end (73.5111) (68.6-106) (71.3-102) (81.3-112) Tail length 11.4.7 b 13.1.0 a 13.2.3 a 10.1.6 c (8.8-15.0) (9.8-18.6) (10.8-15.6) (7.8-13.7) Spicule length 30.7.6 a 28.4.8 b 30.7.5 a 30.0.9 ab (26.4-34.3) (21.5-34.3) (25.4-35.3) (26.5-33.3) Gubernaculum length 8.6.9 a 7.6.1 b 7.7.0 b 7.8.0 b (6.9-9.8) (5.8-9.3) (5.9-9.8) (5.9-9.8) a 44.7.8 a 43.2.4 a 43.9.8 a 36.5.2 a (26.9-58.7) (30.7-56.3) (34.7-52.4) (27.9-54)

PAGE 40

40Table 2-4. Continued. Isolates Character 1 2 3 4 b 12.8.2 a 12.3.2 a 13.5.1 a 9.07.0 b (8.5-16.3) (8.4-16.4) (9.7-17.9) (6.6-14.1) c 132.4.4 a 114.2.2 b 120.9 ab 118.8.9 ab (72.0-174) (84.1-179) (89.3-153) (75.2-154) a Measurements ( m) were taken using 20 specimens from each isolate. b Means in the same row followed by the same letter are not significantly different according to Duncans multiple-range test (P 0.05).

PAGE 41

Table 2-5. Select morphometrics (mean, standard deviation, and range) of females of four isolates of Meloidogyne floridensis from Florida a. Isolates Character 1 2 3 4 Stylet length 14.1.9 abb 14.3.7 ab 13.8.3 b 14.7.7 a (12.7-16.6) (13.0-15.6) (10.8-15.7) (13.5-16.1) DGO 3.1.4 c 4.6.7 a 3.8.5 b 3.9.16 b (2.5-3.9) (3.9-5.9) (2.9-4.7) (3.5-4.4) Vulval slit length 25.6.1 a 22.8.6 b 22.5.8 b 23.4.3 b (21.6-31.3) (21.0-25.9) (19.6-25.5) (21.5-26.4) a Measurements ( m) were taken using 20 specimens from each isolate. b Means in the same row followed by the same letter are not significantly different according to Duncans multiple-range test ( P 0.05 ). 41

PAGE 42

Table 2-6. Gall and egg mass ra ting of four isolates of Meloidogyne floridensis on six different hosts a. Isolate 1 Isolate 2 Isolate 3 Isolate 4 Host Gallb Egg massb Pf/pic Gall Egg mass Pf/pi Gall Egg mass Pf/pi Gall Egg mass Pf/pi index index index index index index index ind ex Tomato 3.8 4.4 nad 4.2 4.2 na 4.0 4.0 na 4.6 4.6 na Cotton 0 0 na 1.4 1.6 na 1.8 1.8 na 0 0 na Peanut 0 0 na 0 0 na 0 0 na 0 0 na Watermelon 3.8 3.8 na 4.6 4.0 na 4.4 4.0 na 4.0 4.4 na Pepper 2.0 3.2 5.2 0.6 2.6 3.3 1.4 2.4 1.2 1.0 2.6 1.0 Tobacco 3.8 4.0 20 4.0 4.0 20 3.6 3.8 13 2.2 2.2 1.0 a Plant cultivars designated by Taylor and Sasser, 1978 for the differential host test include tobacco ( Nicotiana tabacum cv. NC 95), cotton ( Gossypium hirsutum cv. Deltapine 16), pepper ( Capsicum annuum cv. California Wonder), watermelon ( Citrullus lanatus cv. Charleston Gray), peanut ( Arachis hypogaea cv. Florunner), tomato ( Solanum esculentum cv. Rutgers). b Galling and egg mass index = 0-5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 310, 3 = 11-30, 4 = 31-100, 5 = > 100 (Taylor and Sasser, 1978). 42c Reproduction factor calculated for pepper a nd tobacco only final population (pf)/initial population (pi). Plants with a pf/pi > or = 1 are considered good hosts, <1 and > 0.1 poor hosts, and < 0.1 non hosts (Oostenbrink, 1966; Sasser et al., 1984).d Pf/pi not assessed.

PAGE 43

Table 2-7. Gall and egg mass rati ngs of four isolates of Meloidogyne floridensis on tomato, tobacco, and pepper Isolate 1 Isolate 2 Isolate 3 Isolate 4 Host Galla Egg massa Gall Egg mass Gall Egg mass Gall Egg mass index index index index index index index index Tomato 4.0 4.4 4.5 4.2 4.0 4.0 4.0 4.5 Pepper 2.0 3.2 1.0 2.8 1.4 3.2 1.0 2.7 Tobacco 4.0 4.0 4.5 4.4 3.6 3.8 4.8 4.0 a Galling and egg mass index = 0-5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, 5 = > 100 (T aylor and Sasser, 1978). 43

PAGE 44

Mj Mj Figure 2-1. Esterase enzyme phenot ype MF3 of 11 single females of Meloidogyne floridensis lanes 1 and 12 single females of M. javanica control. Mj Mj Mj Figure 2-2. Malate dehydrogenase phe notype N1 of 11 single females of Meloidogyne floridensis lanes 1 and 14 single females of Meloidogyne javanica control, also designated as N1. Mj Mj Figure 2-3. Esterase enzyme phenot ype MF3 of 11 single females of Meloidogyne floridensis collected from pepper, lanes 1 and 12 single females of Meloidogyne javanica control. 44

PAGE 45

Figure 2-4. Esterase enzyme phenot ype MF3 of 11 single females of Meloidogyne floridensis collected from tobacco. 45

PAGE 46

CHAPTER 3 COMPARATIVE HOST STATUS OF SELECT ROOT-KNOT NEMATODE RESISTANT AND SUSCEPTIBLE PLANT CULTIVARS TO FOUR ISOLATES OF Meloidogyne floridensis Introduction Other than the ability to break the resistance in peach rootstocks, lit tle is known of the ability of M. floridensis to overcome root-knot nematode resistance conferred by other economically important plant cultivars. The objective of this study was to determine if select isolates of M. floridensis would be able to overcome the root-knot nematode resi stance gene(s) in selected plant cultivars. Materials and Methods Nematode Source Nematodes were obtained from same sources described in chapter 2. Host Status of Select Root-Knot Nema tode Resistant and Susceptible Crops to M floridensis The reproductive capability of each of four isolates of M. floridensis was compared on root-knot nematode resistant a nd susceptible cultivars of corn pepper, soybean, and tomato (Table 3-1). This experiment consisted of fi ve replicates of eight treatments for each per M. floridensis isolate in a completely randomized desi gn. Two tests were run concurrently in separate growth rooms. All plan ts were grown from seed sown in vermiculite and germinated in plastic trays. Seedlings were transplanted to 25 -cm diam. clay pots ca. 2 months after seeds were planted. The plants were allowed to grow in a greenhouse for 2 weeks before inoculation to ensure development of a healthy root system. At this time all plants were transferred to two separate growth rooms (Figure 31). Plants were inoculated as described in Chapter 2 and were maintained in the growth rooms for 60 days at temperatures averagi ng between 20.5 C to ensure that high soil temperatures would not have an effect on re sistance (Dropkin, 1969, 46

PAGE 47

Ammati et al., 1986; Thies and Fery, 1998). Plants were watered daily and fertilized weekly with a 20-20-20 NPK fertilizer (Peters Professional, St. Louis, Mo). After 60 days the plants were removed from the pots and their root systems were thoroughly washed. Root galling and egg mass indices were determined on a 0-5 scale (Tay lor and Sasser, 1978). Root systems were then subjected to the NaOCL method for egg extrac tion (Hussey and Barker, 1973) as modified (Boneti and Ferraz, 1981) to determ ine the reproductive factor (Rf = Pf/Pi) in which Pf = total egg recovery per root system (final population) and Pi = init ial inoculum level (initial population) (Oostenbrink, 1966; Sasser et al., 1984). Plants with a Rf 1 were considered good hosts, Rf < 1 and > 0.1 were poor hosts, and a Rf < 0.1 were nonhosts. Data were subjected to ANOVA using SAS 9.1 SAS Institute, Cary NC), a nd means were compared based on Duncans multiple-range test at P 0.05. Host Status of the Root-Knot Nematode Resi stant Peach Rootstock Nemaguard and RootKnot Nematode Susceptible Lovell The reproductive capability of four isolates of M. floridensis was compared on the peach rootstocks Nemaguard and Lovell in a greenhouse. The rootstock Nemaguard is resistant (Table 3-1.) to M. incognita and M javanica (Sharp et al., 1969), whereas L ovell is susceptible (Table 3-1.). All peach seedlings used in this experiment were provided by Dr. Andy Nyczepir, Nematologist, USDA/ARS Southeastern Fruit and Nut Tree Research Laboratory, Byron, GA. The seeds were scarified and soaked in water and refrigerated. After 2 to 4 months the seedlings were large enough for transplanti ng into 25-cm clay pots filled w ith pasteurized soil. After 1 month they were inoculated with 5,000 eggs and J2 per plant. Each rootstock was replicated five times for each of the four M. floridensis isolates. The plants were maintained in a greenhouse for 115 days at temperatures ranging between 22-37 C (Figure 3-2). The first test was conducted in 47

PAGE 48

the spring-summer of 2006 a nd was repeated in the spri ng-summer of 2007. Nematode reproduction and statistical analyses were determined as stated above. Results and Discussion Select Horticultural and Agronomic Crops Test Data from both tests were combined based on homogenicity of varian ce test (Table 3-2). The tomato cv. Talladega was a good host for all four isolates of M. floridensis whereas the resistant tomato cv. Crista was a poor host (T able 3-2). The suscepti ble pepper cv. Keystone Resistant Giant was determined to be a good host for isolates 1, 2, and 3; and a poor host for isolate 4, whereas the resistant pepper cv. Char leston Belle was a good host for isolates 2 and 3 and a poor host for 1 and 4. Both the susceptible corn cv. Dixie 18 and the resistant Mp-710 were determined to be good hosts for all four isolates There was no detectab le reproduction by any of the four M. floridensis isolates on either the susceptible cv. S64-J1 or resistant cv. Forrest soybean. These results indicate that ba sed on egg mass indices and reproduc tive factor (pf/pi) (Table 3-2), all four isolates of M. floridensis reproduced poorly but were able to overcome the resistance of the Mi-1gene in Crista tomato. Isolates 1 and 4 reproduced poorly but were able to overcome the N gene resistance in the Charleston Belle pepper, whereas isolates 2 and 4 reproduced well. The unidentified source of resistance in the co rn cv. Mp-710 was able to be broken by all four isolates of M. floridensis The differences observed among these four isolates of M. floridensis may indicate variability among isolates with rega rd to their ability to overcome resistance, as well as general host preference. This indicates the need for furthe r studies to determine if different biotypes or races exist. 48

PAGE 49

Peach Test In both peach tests, the susceptible cv. L ovell and the resistant cv. Nemaguard were good hosts for all four isolates of M. floridensis (Table 3-3). While galli ng and egg mass indicies and reproductive factors were somewhat higher on L ovell than Nemaguard; those parameters were high enough on Nemaguard to consider this a good host for all four nemat ode isolates. All four M floridensis isolates were able to break the unidentified resistance to M. incognita and M. javanica in Nemaguard as was reported in the original description of M floridensis (Handoo et al., 2004), which demonstrates that M. floridensis has the ability to infect and reproduce on both root-knot nematode resistant and susceptible peach cultivars. 49

PAGE 50

50 Table 3-1. Plant cultivars used for host status studies of four isolates of Meloidogyne floridensis Host Susceptible Re sistant Source of Meloidogyne cultivar cultivar resistance speciesa Tomato Talladega Crista Mi-1 gene Mi, Mj, Ma Pepper Keystone Charleston Belle N gene Mi, Mj, Ma Resistant Giant Corn Dixie 18 Mp 710 unidentified Mi, Mj Soybean S64-J1 Forrest Rmi -1 gene Mi Peach Lovell Nemaguard unidentified Mi, Mj a Meloidogyne species that select cultiva rs are resistant to: Mi = Meloidogyne incognita Mj = Meloidogyne javanica Ma = Meloidogyne arenaria

PAGE 51

Table 3-2. Select agronomic and horticultural crops testa, reproduction factor, egg mass and gall indices of four isolates of Meloidogyne floridensis on four root-knot nematode resi stant plant cultivars: Crista, Charleston Bell, Mp 710, and Forrest and four susceptible cultivars: Talladega, Keystone Resistan t Giant, Dixie 18, and S64-J1. Isolate 1 Isolate 2 Isolate 3 Isolate 4 Host Gallb Egg massb pf/pic Gall Egg mass pf/pi Gall Egg mass pf/pi Gall Egg mass pf/pi index index index index index index index i ndex Tomato Cultivar Talladega 3.7 Adae 3.7 Aa 3.9 Ba 4.3 Aa 3.9 Aa 5.5 Aa 4.0 Aa 3.7 Aa 3.9 Ba 3.6 Aa 3.7 Aa 2.9 Ba Crista 2.0 Cb 2.1 Abc 0.5 Acd 3.1 Ab 2.4 Abc 0.5 Acd 2.6 Bb 2.2 Ab 0.6 Ad 2.2 BCb 2.1 Abc 0.2 Bcd Pepper Cultivar Keystone Resistant Giant 2.0 Ac 2.7 ABb 1.3 Ac 2.2 Ac 2.9 Ab 1.5 Ac 2.0 Abc 2.4 Bb 1.3 Ac 1.0 Bc 1.7 Cb 0.5 Bc Charleston Bell 1.5 ABc 1.9 Ac 0.4 Bcd 1.9 Ac 2.0 Ac 1.1 Acd 1.0 Cd 2.1 Ab 1.2 Ac 1.3 BCc 1.4 Bc 0.3 Bcd Corn Cultivar Dixie 18 0.2 Bc 0.3 Bc 1.8 Bb 2.0 Ac 2.3 Ac 2.8 ABb 1.7 Ac 2.0 Ab 2.3 ABb 1.6 Ac 1.8 Ac 4.6 Ab MP 710 0.0 Bd 0.0 Bd 1.3 Bc 0.8 Ad 1.3 Ad 1.5 Bc 0.1 Be 0.1 Bc 1.2 Bc 0.9 Ad 1.3 Ad 3.1 Ac 51 Soybean Cultivar S64-J1 No reproductionf Forrest No reproduction a Means are an average of combined duplicate test s based on the variance of homogenicity test. b Galling and egg mass index = 0-5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, 5 = > 100 (Taylor and Sasser, 1978). c Reproduction factor final population (p f)/initial population (pi). Plants with a pf/pi > or = 1 ar e considered good hosts, <1 and > 0.1 po or hosts, and < 0.1 non hosts (Oostenbri nk, 1966; Sasser et al., 1984). d Means in the same row followed by th e same upper case letter are not different based on Duncans multiple-range test and are to be co mpared horizontally across isolates within a cultivar and withi n the corresponding index (gall, egg mass, and pf/pi) ( P 0.05). eMeans in the same column followed by the same lower case letter are not different based on Duncans multiple-range test and are to be compared vertically, with in an isolate and within the correspondi ng index (gall, egg mass, and pf/pi) ( P 0.05). f No galling or egg masses detected.

PAGE 52

Table 3-3. Peach testa, reproduction factor, egg mass and gall indices of four isolates of Meloidogyne floridensis on the root-knot nematode resistant peach cultivar Nemagua rd and the susceptible cultivar Lovell. Isolate 1 Isolate 2 Isolate 3 Isolate 4 Host Gallb Egg massb pf/pic Gall Egg mass pf/pi Gall Egg mass pf/pi Gall Egg mass pf/pi index index index index index index index i ndex Lovell 3.9 Adae 3.4 Aa 2.2 Aa 4.0 Aa 3.6 Aa 2.2 Aa 3.4 Aa 3.3 Aa 2.0 Aa 4.0 Aa 3.4 Aa 2.2 Aa Nemaguard 3.2 Bb 3.0 Bb 1.7 Ab 3.9 Aa 3.3 Aa 1.7 Ab 3.2 Ba 3.0 Ba 1.6 Ab 3.7 A Ba 3.0 Bb 1.7 Ab a Means are an average of combined duplicate test s based on the variance of homogenicity test. b Galling and egg mass index = 0-5 scale where 0 = no galls or egg masses, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, 5 = >100 (Taylor and Sasser, 1978). c Reproduction factor final population (p f)/initial population (pi). Plants with a pf/pi > or = 1 ar e considered good hosts, <1 and > 0.1 po or hosts, and < 0.1 nonhosts (Oostenbrink, 1966; Sasser et al., 1984). d Means in the same row followed by the same upper case letter are not different based on Duncans multiple-range test and are to be co mpared horizontally across isolates within a cultivar and withi n the corresponding index (gall, egg mass, and pf/pi) ( P 0.05). e Means in the same column followed by the same lower case letter are not different based on Duncans multiple-range test and are to be compared vertically, with in an isolate and within the correspondi ng index (gall, egg mass, and pf/pi) ( P 0.05). 52

PAGE 53

Figure 3-1. Host status test of select root-knot nematode resistan t and susceptible plant cultivars conducted in a growth room. Figure 3-2. Host status test of the root-knot nematode susceptible peach cultivar Lovell and resistant cultivar Nemaguard conducted in a greenhouse. 53

PAGE 54

APPENDIX A REPRODUCTIVE POTENTIAL AND PATHOGENICITY COMPARISON OF FOUR ISOLATES OF Meloidogyne floridensis Introduction Meloidogyne floridensis has been reported infecting several crops in the peninsular portion of Florida and is likely to be f ound wide spread across this region of the state (Bri to et al., 2005; Church et al., 2005). Although M. floridensis has been found infecting agronomic and horticultural crops such as cucumber, eggplant, tomato, and pepper in Florida agricultural production areas, little is known about the reproductive potentia l and pathogenicity of this nematode. M. floridensis is a pathogen of potential concern because it has shown the ability to break resistance in several rootknot nematode resistant peach root stock cultivars as well as other crops (Chapter 3). It is importa nt that we learn more about this newly described species. In order to obtain more information on th e potential damage caused by this nematode, a study was conducted to compare the reproductive potenti al and pathogenicity of four isolates of M. floridensis and one isolate of M. incognita race 4 on tomato in microplots under field conditions. Materials and Methods Nematode Source Nematodes were obtained from same sources described in chapter 2. Microplot Test The microplot test was carried out at the Univ ersity of Florida Plant Science Research and Education Unit, Marion County, FL. The field chosen to conduct the study had been previously planted with Argentine bahiagrass ( Paspalum notatum ). The soil at the experiment site was classified as Arredondo fine sand (97% sand, 0% silt, 3% clay, and < 1% organic matter; pH 7.8) 54

PAGE 55

(Gee and Bauder, 1986). In September 2006 an ap plication of the herbicide glyphosate was applied to eight 1.2-m wide 14-m long strips and approximately 2 weeks later a second application was applied. Each strip was separa ted by a 1-m wide bahiagrass buffer strip (Figure 4-1). In March 2007, 40 soil cores were taken from each of the eight rows in a zig-zag pattern with a cone-shaped 2.5-cm-diam. 30-cm deep sampling tool. The nematodes were extracted by centrifugal-flotation (Jenkins, 1964) The only plant-parasitic nema todes detected were a small number of ring nematodes ( Mesocriconema spp.) which are commonly found in grassy areas throughout Florida. In late March of 2007, 48 holes were dug to install 47-cm-diam. x 50-cmdeep plastic pots. There were si x pots per each of the eight rows Holes were dug at 50-cm-diam. x 40-cm deep to allow ca. 10 cm of the plastic pot to be above ground level. A 20 cm wide hole was cut out of the bottom of each pot before in stallation to allow for maximum drainage. Upon backfilling the pots all soil was screened to remove any grass root debris. A broadcast application of 13.5 g of a 10-1010 N-P-K with micronutrients wa s applied and mixed into the soil of each microplot to provide 25% of th e total 90.7 kg N and 81.7 kg-K required per season. Additionally 10 post-plant applications of fertilizer were applied once per week through the irrigation system at 56 g ammonium nitrate a nd 28 g potassium chlori de (Figure 4-2). The microplots were randomized into a complete block design with six treatments and eight replications (Figure 4-3). The six treatments consisted of four isolates of M. floridensis, one isolate of M. incognita race 4 and a noninoculated control. Inoculum wa s prepared as described in chapter 2. In preparation for inoculation, 15 uniformly spaced holes with depths of 7, 14, and 21 cm were pressed into the soil of each microplot using a template (Cetintas, et al., 2007). On 11 April 2007 microplots were inoculated with eight eggs and J2 per 100-cm3 of soil. Each microplot had a 55

PAGE 56

volume of 56,304 cm3 of soil with an inoculum level of 4,504 eggs and J2 per microplot. Immediately following inoculation three root-knot nematode susceptible tomato cv. Talladega seedlings were transplanted in a triangular pattern w ith a spacing of 15 cm between each (Figure 4-4). The plots were watered three times daily vi a irrigation tubing leading to emitters placed in each microplot. Insecticides and fungi cides were applied as needed. Tomato fruit were harvested from 9 June to 6 July 2007. The yield for each microplot was recorded at each harvest. On 10 July, tomato plants were removed to determine shoot fresh weight, gall and egg mass indicies, eggs per gram fresh root, and J2 per 100 cm3 of soil. Results and Discussion Upon the removal of the tomato root system s on 10 July, significan t galling was observed in five of the eight noninoculated control plots. Before any additional data could be collected 56 females were removed from each of the noninoculated control plots as we ll as several of the inoculated plots and subjected to polyacrylamide gel electrophoresis as described in chapter 2. The results of the enzymatic analysis indicated that there was signifi cant contamination by Meloidogyne javanica A pure population of this species was found in the control plots, whereas in the inoculated plots M. javanica was associated with M floridensis and M. incognita in variable proportions. It was apparent that the contamination did not come from the inoculation since M. javanica was not used for the experiment and the isolates of M. floridensis and M. incognita were determined to be pure by electrophoresis before inoculation. The two re maining possibilities for the source of the contamination are (i) Meloidogyne javanica was present in the soil and not detected by the initial sampling prior to the inst allation of the microplots or (ii) the nematodes were spread by contaminated machinery. The possibil ity of the latter seems to be the most likely given that there was only bahiagrass in the microplot area and an extensive amount of sampling 56

PAGE 57

was conducted prior to installation of the microplot s. These facts combined with the fact that some of the experiments in close proximity of th is test contained various species of root-knot nematode including M. javanica would make it fairly easy to accep t that the equipment used in these areas such as mowers and large overhead ir rigation could provide a means for the spread of the nematode if not properly sanitized. Unfortunately, because of the contamination of M. javanica it was impossible to draw any conclusions or achieve any va luable results. The variable level of contamination of M. javanica made it impossible to draw any conclusions abou t reproductive potential or pathogenicity of M. floridensis It appears that M javanica was more pathogenic and ha d higher population levels than M. floridensis in the contaminated plots. The undete rmined initial popul ation densities of M. javanica in the plots prevented any reliable evaluation of the pathogenicity and competitiveness of the two nematodes present concomitantly in the microplots. Future studies should be conducted under rigorous phytosanitary conditions in order to determine the damage of M. floridensis to tomato under field conditions. 57

PAGE 58

Figure 4-1. Initial preparation of eight 1.2 m wide 14 m long strips by the application of the herbicide glyphosate in a site at the Universi ty of Florida Plant Science Research and Education Unit, Marion County, Florida planted with Argentine bahiagrass ( Paspalum notatum ). Figure 4-2. Post-plant application of 56.2 g am monium nitrate and 28.1 g potassium chloride once per week for 10 weeks thr ough the drip irrigation system. 58

PAGE 59

Figure 4-3. Microplots containing three tomato cv. Talladega plants per microplot randomized into a complete block design with eight re plications of six treatments consisting of four isolates of Meloidogyne floridensis one isolate of M. incognita race 4 and one noninoculated control. Figure 4-4. An individual micr oplot consisting of a 47-cm-diam. 50-cm deep plastic pot planted with three tomato cv. Talladega seedlings watered three times daily via irrigation tubing leading to an emitter. 59

PAGE 60

LITERATURE CITED Ammati, M., Thomason, I. J., and. McKinney, H. E. 1986. Retention of resistance to Meloidogyne incognita in Lycopersicon genotypes at high soil-temperature. Journal of Nematology 18:491-495. Boneti, J. I. S., and Ferraz, S. 1981. Modificao do mtodo de Hussey & Barker para extrao de ovos de Meloidogyne exigua de raizes de cafeeiro. F itopatologia Brasileira 6:553. Brito, J. A., Kaur, R., Cetintas, R., Stanley, J. D., Mendes, M. L., McAvoy, E. J., Powers, T. O., and Dickson, D. W. 2008. Identificat ion and isozyme characterization of Meloidogyne spp. infecting horticultura l and agronomic crops, and weed plants in Florida. Nematology 10:757-776. Brito, J. A., Powers, T. O., Mullen, P. G., Inserra, R. N., and Dickson, D. W. 2004. Morphological and molecular characterization of Meloidogyne mayaguensi s isolates from Florida. Journal of Nematology 36:232-240. Brito, J. A., Stanley, J. D., Cetintas, R., Ham ill, J., and Dickson, D. W. 2005. A new root-knot nematode infecting vegetables in Florida. Journal of Nematology 37:359 (Abstr.). Brito, J. A., Stanley, J. D., Kaur, R., Cetintas, R., Di Vito, M., Thies, J. A., and Dickson, D. W. 2008. Effects of the Mi-1 N and Tabasco genes on infection and reproduction of Meloidogyne mayaguensis on tomato and pepper genotypes. Journal of Nematology 39:327-332. Carneiro, R. M. D.G., Almeida, M. R. A., and Carneiro, R. G. 1996. Enzyme phenotypes of Brazilian isolates of Meloidogyne spp. Fundamental and Applied Nematology 19:555-560. Carneiro, R. M. D. G., Almeida, M. R. A., and Queneherve, P. 2000. Enzyme phenotypes of Meloidogyne spp. isolates. Nematology 2:645-654. Carneiro, R. M. D. G., Carneiro, R. G., Das Neve s, D. I., and Almeida, M. R. A. 2003. New race of Meloidogyne javanica on Arachis pintoi in the state of Paran. Nematologia Brasileira 27:219-221. Carneiro, R. M. D. G., Castagnone-Sereno, P., and Dickson, D. W. 1998. Variability among four isolates of Meloidogyne javanica from Brazil. Fundamental and Applied Nematology 21:319-326. Cetintas, R., Kaur, R., Brito, J. A., Mendes, M. L., Nyczepir, A. P., and Dickson, D. W. 2007. Pathogenicity and reproductive pote ntial of Meloidogyne mayaguensis and M. floridensis compared with three common Meloidogyne spp. Nematropica 37:21-31. Chitwood, B. G. 1949. Root-knot nematodes. Part 1. A revision of the genus Meloidogyne Goeldi, 1887. Proceedings of the Helmint hological Society of Washington 16:90-104. 60

PAGE 61

Church, G. T. 2005. First report of the root-knot nematode Meloidogyne floridensis on tomato ( Lycopersicon esculentum ) in Florida. Plant Disease 89:527. Christie, J. R. 1959. Plant nematodes their biono mics and control. Gainesville, Florida.: Agricultural Experiment Stati ons, University of Florida. Cook, R., and Star, J. L. 2006. Re sistant cultivars. Pp. 370-391 in R. N. Perry, and M. Moens, eds. Plant Nematology. Oxfordshire, UK: CABI Publishing. Dalmasso, A., and Berg. J. B. 1978. Molecula r polymorphism and phylogenetic relationship in some Meloidogyne spp. Journal of Nematology 10:323-332. Dickson, D. W., Huisingh, D., and Sasser, J. N. 1971. Dehydrogenases, acid and alkaline phosphatases, and esterases fo r chemotaxonomy of selected Meloidogyne Ditylenchus Heterodera and Aphelenchus spp. Journal of Nematology 3:1-16. Di Vito, M., and Saccardo, F. 1979. Resistance of Capsicum to Meloidogyne incognita Pp 455456 in F. Lamberti and C. E. Taylor, eds. Root-Knot Nematodes ( Meloidogyne Species) Systematics, Biology and Control. New York: Academic Press. Di Vito, M., and Saccardo. F. 1982. Resistance of Capsicum to root-knot nematodes ( Meloidogyne spp.). Capsicum Newsletter 1:70-71. Di Vito, M., Saccardo, F., and Zaccheo, G. 1991. Response of lines of Capsicum spp. to Italian isolates of four species of Meloidogyne. Nematologia Mediterranea 19:43-46. Dropkin, V. H. 1969. The necrotic reaction of tomatoes and other hosts resistant to Meloidogyne : Reversal by temperatur e. Phytopathology 59:1632-1637. Eisenback, J. D., and Hirschmann, H. 1979. Morphological comparison of s econd-stage juveniles of several Meloidogyne species (root-knot nematodes) by scanning electron microscopy. Scanning Electron Microscopy 3:223-230. Eisenback, J. D., and Hirschmann, H. 1981. Identification of Meloidogyne species on the basis of head shape and stylet morphology of the male. Journal of Nematology 13:413-521. Eisenback, J. D., and Hirschmann-Triant aphyllou, H. 1991. Root-knot nematodes: Meloidogyne species and races. Pp. 191-274. in W. N. Nickle, ed. Manual of Agricultural Nematology. New York: Marcel Dekker. Eisenback, J. D., Hirschmann, H., and Triant aphyllou A. C., 1980. Morphological comparisons of Meloidogyne female head structures, perineal patterns, and stylets. Journal of Nematology 12:300-313. Eisenback, J. D., Sasser, J. N., and Triantaphyllo u, A. C. 1981. A guide to the four most common species of root-knot nematodes, ( Meloidogyne species) with a pictorial key. A Cooperative Publication, Departments of Plant Pathology and Genetics and United States Agency of International Development, Raleigh. 61

PAGE 62

Esbenshade, P. R., and Triantaphyllou, A. C. 198 5. Use of enzyme phenotype for identification of Meloidogyne species. Journal of Nematology 17:6-20. Esbenshade, P. R., and Triantaphyllou, A. C. 1990. Isozyme phenotypes for identification of Meloidogyne species. Journal of Nematology 22:10-15. Esser, R. P. 1986. A water agar en face technique. Proceedings of the Helminthological Society of Washington 53:254-255. Esser, R. P., Perry, V. G., and Taylor, A. L. 1976. A diagnostic compendium of the genus Meloidogyne (Nematoda: Heteroderidae). Proceedings of the Helminthological Society of Washington 43:138-150. Fargette, M. 1987. Use of the esterase phenotype in the taxonomy of the genus Meloidogyne. 2. Esterase phenotypes observed in West African is olates and their characterization. Revue de Nmatologie 10:45-56. Gee, G. W., and Bauder, J. M. 1986. Particle-size analysis. Pp. 383-411 in A. Klute, ed. Methods of soil analysis, Part 1. Physical and mineralogical methods. Agronomy monograph, No. 9. Madison, WI: American Society of Agronomy. Handoo, Z. A., Nyczepir, A. P., Esmenjaud, D., van der Beek, J. G., Castagnone-Sereno, P., Carta, L. K.,. Skantar, A. M., and Higgins, J. A. 2004. Morphological, molecular, and differential-host ch aracterization of Meloidogyne floridensis n. sp. (Nematoda: Meloidogynidae), a root-knot nematode para sitizing peach in Florida. Journal of Nematology 36:20-35. Hirschmann, H. 1985. The genus Meloidogyne and morphological charac ters differentiating its species. Pp.79-93 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne Vol. 1. Raleigh: North Carolin a State University Graphics. Huang, C. S., and Maggenti, A. R. 1969. Mitotic aberrations and nuclear changes of developing giant cells in Vicia faba caused by root-knot nematode, Meloidogyne javanica Phytopathology 59: 447-455. Jenkins, W. R. 1964. A rapid centrifugal flotati on technique for separating nematodes from soil. Plant Disease Reporter 48:62. Jeyaprakash, A., Tigano, M. S., Brito, J., Carn eiro, R. M. D. G., and Dickson, D. W. 2006. Differentiation of Meloidogyne floridensis from M. arenaria using high-fidelity PCR amplified mitochondrial at-ric h sequences. Nematropica 36:1-12. Jepson, S. B. 1987. Identification of root-knot nematodes ( Meloidogyne species). Wallingford, UK: CABI Publishing. Jones, M. G. K., and Payne, H. L. 1978. Early st ages of nematode-induced giant-cell formation in roots of Impatiens balsamina Journal of Nematology 10:70-81. 62

PAGE 63

Karssen, G., and Moens, M. 2006. Genetic engineering for resistance. Pp. 255-272 in R. N. Perry and M. Moens, eds. Plant Nematology. Oxfordshire, UK: CABI Publishing. Kaur, R., Brito, J. A., and Rich, J. R. 2006. Host suitability of selected weed species to five Meloidogyne species. Nematropica 37:107-120. Kokalis-Burelle, N., and Nyczepir, A. P. 2004. Host range studies for Meloidogyne floridensis. Journal of Nematology 36:328 (Abstr.). Nyczepir, A. P., and Beckman, T. G. 2000. Host status of Guardian peach rootstock to Meloidogyne sp. and M. javanica HortScience 35: 772. Nyczepir, A. P., Esmenjaud, D., and Eisenback, J. D. 1998. Pathogenicity of Meloidogyne sp. (FL-isolate) on Prunus in the southeastern United States and France. Journal of Nematology 30:509 (Abstr.). Oostenbrink, M. 1966. Major characteristics of the relation between nematodes and plants. Meded Landbouwhogesch. Wageningen. 66:1-46. Ott, R. L., and Longnecker, M. T. 2001. An introduc tion to statistical met hods and analysis, Fifth Edition. Duxtbury/Thompson. Powers, T. O., and Harris, T. S. 1993. A polymerase chain reaction method for identification of five major Meloidogyne species. Journal of Nematology 25:1-6. Powers, T. O., Todd, T. C., Burnell, A. M., Murray, P. C. B., Fleming, C. C., Szalanski, A. L., Adams, B. J., and Harris, T. S. 1997. The internal described spacer region as a taxonomic marker for nematodes. Journal of Nematology 29:441-450. Rammah, A., and Hirschmann, H. 1990. Morphological comparis on of three host races of Meloidogyne javanica Journal of Nematology 22:56-68. Randig, O., Bongiovanni, M., Carneiro, R. M. D. G., and Castagnone-Sereno, P. 2002. Genetic diversity of root-knot nema todes from Brazil and the de velopment of SCAR markers specific for the coffee-damaging species. Genome 45:862-870. Saha, M., and Khan, E. 1989. Effect of differe nt fixatives and pro cessing techniques on morphometrics of Pratylenchus zeae Indian Journal of Nematology 19:254-260 Sasser, J. N. 1954. Identification and host-parasite relationships of certain root-knot nematodes ( Meloidogyne spp.). University of Maryland Agricultural Experiment Station Bulletin. A77:1-31. Sasser, J. N., and Carter, C. C. 1985. Overview of the international Meloidogyne project 19751984. Pp. 20-24 in J. N. Sasser and C. C. Carter eds. An Advanced Treatise on Meloidogyne vol. I: Biology and Control. Raleig h: North Carolina State University Graphics. 63

PAGE 64

Sasser. J. N., Carter, C. C., and Hartman, K. M. 1984. Standardization of host suitability studies and reporting of resistance to root-knot ne matodes. North Carolina State University Graphics. Raleigh. Sharp, R. H., Hesse, C. O., Lownsberry, B. A., Perry, V. G., and Hansen, C. J. 1969. Breeding peaches for root-knot nematode resistance. Journal of the American Society for Horticultural Sc ience 94:209-212. Sherman, W. B., and Lyrene, P. M. 1983. Improvem ent of peach rootstock resistant to root-knot nematodes. Proceedings of the Florida State Horticultural Society 96:207-208. Sherman, W. B., Lyrene, P. M., and Hansche, P. E. 1981. Breeding peach rootstocks resistant to root-knot nematode. HortScience 64:523-524. Sherman, W. B., Lyrene, P. M., and Sharpe R. H. 1991. Flordaguard peach rootstock. Horticultural Sc ience 26:427-428. Sipes, B. S., Schmitt, D. P., Xu, K., a nd Serracin, M. 2005. Esterase polymorphism in Meloidogyne konaensis Journal of Nematology 37:438-443. Smith, P. G. 1944. Embryo culture of a tomato species hybrid. Proceeding of the American Horticultural Society. 44:413-416. Stanley, J. D., Kokalis-Burelle, N., and Dickson, D. W. 2006. Host status of Meloidogyne floridensis on selected weeds and cover crops common to Florida. Nematropica 36:148 (Abstr.). Star, J. L., and Roberts, P. A. 2004. Resist ance to plant-parasitic nematodes. Pp. 879-908 in Z. X. Chen, S. Y. Chen, and D. W. Dickson, eds. Nematology Advances and Perspectives, vol. 2. Nematode Management and Utiliz ation. Beijing, CABI Publishing. Subbotin, S. A., and Moens, M. 2006. Molecular taxonomy and phylogeny. Pp. 33-58 in R. N. Perry and M. Moens, eds. Plant Nematology. Wallingford, UK: CABI Publishing. Taylor, A. L., and Sasser, J. N. 1978. Biology, iden tification, and control of root-knot nematodes ( Meloidogyne species). North Carolina State University Graphics, Raleigh. Thies, J. A., and Fery, R. L. 1998. Modified expression of the N gene for the southern root-knot nematode resistance in pepper at high soil temperatures. Journal of the American Society of Horticultural Science 123:1012-1015. Thies, J. A., and Fery, R. L. 2000. Char acterization of resistance conferred by N gene to Meloidogyne arenaria races 1 and 2, M. hapla, and M. javanica in two sets of isogenic lines of Capsicum annuum L. Journal of the American Society of Horticultural Science 125:71-75. Thomas, C., and Cottage, A. 2006. Genetic engineering for resistance. Pp. 255-272 in R. N. Perry and M. Moens, eds. Plant Nematology. Wallingford, UK: CABI Publishing. 64

PAGE 65

65 Triantaphyllou, A. C. 1960. Sex determination in Meloidogyne incognita Chitwood, 1949 and intersexuality in M. javanica (Treub, 1885) Chitwood, 1949. Annales de lInstitut Phytopathologique Benaki 3:12-31. Triantaphyllou, A. C. 1966. Polyploidy and repro ductive patterns in the root-knot nematode, Meloidogyne hapla Journal of Morphology 118:403-414. Triantaphyllou, A. C., and Sasser, J. N. 1960. Variation in perineal patterns and host specificity of Meloidogyne incognita. Phytopathology 50:724-735. United States Department of Agriculture. 2008. National Agricultural Stat istics Service. Crop production 1998-2007 summary. Washington, D C. USDA. ( http://www.nass.usda.gov/Publicat ions/Ag_Statistics/2008/Chap05.pdf. ) Last accessed (August 2008). Van der Beek, J. G., Los, J. A., and Pijnacker L. P. 1998. Cytology of parthenogenesis of five Meloidogyne species. Fundamental and Applied Nematology 21:393-399. Zijlstra, C., Donkers-Venne, D.T.H.M., a nd Fargette, M. 2000. Identification of Meloidogyne incognita M. javanica and M. arenaria using sequence character ized amplified region (SCAR) based PCR assays. Nematology 2:847-853.

PAGE 66

BIOGRAPHICAL SKETCH Jason Stanley was born in 1968, in Schenect ady, New York. In 1986, he graduated from Land O Lakes High School in Land O Lakes Florid a. He began his studies at the University of Florida, Gainesville, Florida, in 1999 and earned a Bachelor of Science degree in entomology and nematology with an emphasis on urban ento mology graduating cum laude in 2003. He began his studies for his Master of Sc ience degree in entomology and ne matology at the University of Florida, under D. W. Dickson in 2004. 66