REPRODUCTION AND IDENTIFICATION OF ROOT-KNOT ON PERENNIAL ORNAMENTAL IN FLORIDA

By

ROI LEVIN

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

UNIVERSITY OF FLORIDA

2005

Copyright 2005

by

Roi Levin

ACKNOWLEDGMENTS

I would like to thank my chair, Dr. W. T. Crow, and my committee members, Dr.

J. A. Brito, Dr. R. K. Schoellhorn, and Dr. A. F. Wysocki, for their guidance and support of this work. I am honored to have worked under their supervision and commend them for their efforts and contributions to their respective fields.

I would also like to thank my parents. Through my childhood and adult years, they have continuously encouraged me to pursue my interests and dreams, and, under their guidance, gave me the freedom to steer opportunities, curiosities, and decisions as I saw fit.

Most of all, I would like to thank my fiancée, Melissa A. Weichert. Over the past few years, she has supported, encouraged, and loved me, through good times and bad. I will always remember her dedication, patience, and sacrifice while I was working on this study. I would not be the person I am today without our relationship and love.

iii TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iii

LIST OF TABLES...... vi

LIST OF FIGURES ...... ix

ABSTRACT...... xiii

CHAPTERS

1 INTRODUCTION AND LITERATURE REVIEW ...... 1

Introduction...... 1 Meloidogyne spp...... 2 Relationship with Ornamentals...... 14 Objectives ...... 30

2 REPRODUCTION OF FOUR MELOIDOGYNE SPP. ON SEVERAL OF PERENNIAL ORNAMENTAL PLANTS...... 31

Introduction...... 31 Materials and Methods ...... 33 Results...... 40 Discussion...... 60

3 IDENTIFICATION OF ROOT-KNOT NEMATODES ...... 67

Introduction...... 67 Objectives ...... 69 Materials and Methods ...... 70 Results...... 95 Discussion...... 103

APPENDIX

A PICTURES OF MELOIDOGYNE SPP. ESTERASE AND MALATE DEHYDROGENASE ISOZYME PHENOTYPES UNVEILED THROUGH POLYACRYLAMIDE GEL ELECTROPHORESIS ON PHASTSYSTEM AND MINI-PORTEIN 3 CELL APPARATUSES...... 107

iv B COLLECTIVE RECORD OF THE HOST STATUS OF ORNAMENTAL PLANTS TO , M. JAVANICA, M. ARENARIA, AND M. HAPLA...... 123

REFERENCES ...... 184

BIOGRAPHICAL SKETCH ...... 197

v

LIST OF TABLES

Table page

2-1 Crop and source of liners used for growth room and greenhouse experiments ...... 34

2-2 Crops, experimental sites, liner planting dates, inoculation dates, and study lengths for all crops in the growth room and greenhouse Meloidogyne spp. studies carried out at the University of Florida during 2003 to 2005 ...... 38

2-3 Treatments, root-gall indexes, root weights, number of eggs per , number of eggs per g of roots, and dry shoot weights from the first cv. Evergreen Giant growth room trial ...... 41

2-4 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Liriope muscari cv. Evergreen Giant growth room trial ...... 42

2-5 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Liriope muscari cv. Evergreen Giant greenhouse experiment ...... 44

2-6 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Pittosporum tobira cv. Variegata growth room trial ...... 45

2-7 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Pittosporum tobira cv. Variegata growth room trial ...... 46

2-8 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Pittosporum tobira cv. Variegata greenhouse experiment ...... 47

2-9 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first leucantha growth room trial ...... 49

vi 2-10 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Salvia leucantha growth room trial...... 50

2-11 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the purple-corolla Salvia leucantha greenhouse experiment...... 52

2-12 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the white-corolla Salvia leucantha greenhouse experiment...... 53

2-13 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first cuspidatum growth room trial ...... 55

2-14 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second growth room trial ...... 56

2-15 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g roots, and dry shoot weights from the Odontonema cuspidatum greenhouse experiment...... 57

2-16 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Musa acuminata ssp. zebrina growth room trial...... 58

2-17 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Musa acuminata ssp. zebrina growth room trial...... 59

2-18 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Musa acuminata ssp. zebrina greenhouse experiment...... 61

2-19 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first variegatum cv. Gold Dust trial ...... 62

2-20 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Codiaeum variegatum cv. Gold Dust growth room trial ...... 63

2-21 Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Codiaeum variegatum cv. Gold Dust greenhouse experiment ...... 64

vii 3-1 Known Meloidogyne spp. malate dehydrogenase and esterase relative migrations that were used as references against those that were revealed electrophoretically from females collected from several counties in Florida ...... 91

3-2 Enzyme stain concoctions used in staining malate dehydrogenase and esterase following electrophoresis using the PhastSystem ...... 94

3-3 Enzyme stain concoctions used in staining malate dehydrogenase and esterase following electrophoresis using the Mini-Protean 3 Cell...... 96

3-4 Plant species, family, county, relative migration, isozyme phenotype, number of samples, and Meloidogyne spp. identified from ornamental plants collected in Florida and processed using the PhastSystem and Mini-Protean 3 Cell ...... 97

B-1 Sources and citations of publications referred to in Appendix B...... 124

B-2 Collective record of the host status of ornamental plants to Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla ...... 125

viii

LIST OF FIGURES

Figure page

3-1 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of , as reported by several authors ...... 71

3-2 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne incognita, as reported by several authors ...... 73

3-3 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of , as reported by Esbenshade and Triantaphyllou (1985c)...... 75

3-4 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne arenaria, as reported by several authors ...... 77

3-5 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne hapla...... 79

3-6 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne spp...... 81

3-7 Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of unidentified Meloidogyne spp...... 85

3-8 Malate dehydrogenase (Mdh) and esterase (Est) isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of females extracted from Carya illinoensis from Jefferson County on 31 March 2004 ...... 101

3-9 Malate dehydrogenase (Mdh) and esterase (Est) isozyme phenotype, revealed using the mini-protean 3 cell apparatus, of a Meloidogyne querciana female extracted from Viburnum odoratissimum cv. Awabuki from Hillsborough County on 25 January 2005...... 102

A-1 PhastSystem gels exhibiting smeared malate dehydrogenase isozyme phenotypes whose relative migration could not be accurately measured ...... 107

A-2 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Hibiscus rosa- sinensis cv. Pink Versicolor from Alachua County on 02 April 2003...... 108

ix A-3 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Rosmarinus officinalis from Suwannee County on 30 April 2003 ...... 108

A-4 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Callistemon viminalis from Lee County on 01 May 2003 ...... 109

A-5 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Syagrus romanzoffiana from Lee County on 27 May 2003...... 109

A-6 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Callistemon viminalis from Lee County on 27 May 2003 ...... 110

A-7 Eterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Callistemon viminalis from Lee County on 27 May 2003...... 110

A-8 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira from Alachua County on 18 December 2003 ...... 111

A-9 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira from Alachua County on 18 December 2003...... 111

A-10 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 19 December 2003 ...... 112

A-11 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 19 December 2003 ...... 112

A-12 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Buddleia davidii from Pinellas County on 30 September 2003 ...... 113

A-13 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Buddleia davidii from Pinellas County on 30 September 2003...... 113

A-14 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Brassica rapa cv. Shogoin from Alachua County on 06 January 2004 ...... 114

x A-15 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Brassica rapa cv. Shogoin from Alachua County on 06 January 2004 ...... 114

A-16 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira cv. Variegata from Lake County on 11 February 2004 ...... 115

A-17 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira cv. Variegata from Lake County on 11 February 2004...... 115

A-18 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 22 February 2004 ...... 116

A-19 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 22 February 2004 ...... 116

A-20 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Liriope muscari cv. Evergreen Giant from Hillsborough County on 01 July 2004 ...... 117

A-21 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Liriope muscari cv. Evergreen Giant from Hillsborough County on 01 July 2004...... 117

A-22 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ophiopogon japonicus from Orange County on 16 August 2004 ...... 118

A-23 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ophiopogon japonicus from Orange County on 16 August 2004...... 118

A-24 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Jesticia carnia from Hillsborough County on 20 August 2004 ...... 119

A-25 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Jesticia carnia from Hillsborough County on 20 August 2004...... 119

A-26 Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Solenostemon scutellarioides cv. Elfers from Hillsborough County on 15 September 2004 ...... 120

xi A-27 Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Solenostemon scutellarioides cv. Elfers from Hillsborough County on 15 September 2004 ...... 120

A-28 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Brassica oleracea from Orange County on 11 January 2004 ...... 121

A-29 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Beta vulgaris from Orange County on 11 January 2004 ...... 121

A-30 Malate dehydrogenase and esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Phaseolus vulgaris from Hillsborough County on 25 January 2005...... 122

xii

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

REPRODUCTION AND IDENTIFICATION OF ROOT-KNOT NEMATODES ON PERENNIAL ORNAMENTAL PLANTS IN FLORIDA

By

Roi Levin

May 2005

Chair: W. T. Crow Major Department: Entomology and Nematology

Meloidogyne spp. (root-knot nematodes) are serious pathogens of perennial and woody ornamental plants. Meloidogyne spp. directly limit plant vigor and, by the mechanical action and physiological responses to their feeding, expose their hosts to an array of pathogenic fungi and bacteria. The evaluation of the host status of perennial ornamental plants to root-knot nematodes can identify root-knot resistant plant material, which may be used to replace infected hosts in landscapes. Six perennial ornamental species were evaluated for their host status to M. incognita race 2, M. javanica, M. arenaria race 1, and M. mayaguensis in separate growth room and greenhouse experiments. Data from these experiments indicate that Liriope muscari cv.

Evergreen Giant is a good host to M. incognita race 2, M. javanica, and M. mayaguensis, and a poor host to M. arenaria race 1. In addition, a purple-corolla form of Salvia leucantha and Musa acuminata ssp. zebrina cv. Rowe red are good hosts to the

Meloidogyne spp. evaluated. Pittosporum tobira cv. Variegata, Odontonema cuspidatum,

xiii and Codiaeum variegatum cv. Gold Dust are nonhosts to the Meloidogyne spp. isolates evaluated.

Twenty ornamental plants were identified as hosts to several Meloidogyne spp. via the speciation of root-knot nematode females that were dissected directly from their hosts’ roots. These females were identified primarily by evaluation of esterase (Est) and malate dehydrogenase (Mdh) isozyme phenotypes, unveiled following polyacrylamide gel electrophoresis. Resolved isozyme phenotypes indicate that Rosmarinus officinalis

(rosemary), Syagrus romanzoffiana (queen palm), P. tobira, Brassica rapa (turnip) cv.

Shogoin, Brassica oleracea (kale), Phaseolus vulgaris (bean), L. muscari cv. Evergreen

Giant, and Ophiopogon japonicus (mondo grass) are hosts to M. incognita. Hibiscus rosa-sinensis (hibiscus) cv. Pink Versicolor, B. rapa cv. Shogoin, Ruscus aculeatus

(ruscus), Beta vulgaris (chard), and Viburnum odoratissimum (Viburnum) cv. Awabuki are hosts to M. javanica. Ruscus aculeatus and P. vulgaris are hosts to M. arenaria.

Callistemon viminalis (bottle brush), S. romanzoffiana, and Solenostemon scutellarioides

(coleus) cv. Elfers are hosts to M. mayaguensis. Carya illinoensis (pecan) is a host to M. partityla. In addition, Meloidogyne spp. that could not be identified on the basis of their

Est and Mdh isozyme phenotypes were isolated from the following ornamental plants:

Buddleia davidii (butterfly bush), P. tobira cv. Variegata, L. muscari cv. Evergreen

Giant, and O. japonicus, and Justicia carnea (flamingo plant).

xiv CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW

Introduction

Perennial ornamentals are an important component of the nursery and floriculture industry. According to Hodges and Haydu (2003), the gross wholesale value for U.S.- grown floriculture and nursery crops in 2001 reached $13.3 billion, of which $1.6 billion was produced in Florida alone. Over 2,500 species in roughly 500 genera are included in this category, and are widely distributed in the United States, Canada, and

(LaMondia, 1995, 1997). Widespread dissemination of perennial ornamentals presents an important avenue for distribution of root-knot nematodes (Meloidogyne spp.) and

other plant-pathogenic organisms (LaMondia, 1995). Root-knot nematodes cause

estimated crop losses of 5 to 10% in major crops, and are considered the most widespread

and destructive of all plant-pathogenic nematodes (Haseeb et al., 1984; Stokes, 1977;

Walker et al., 1994). The lack of information regarding perennial ornamental crop losses

due to plant-parasitic nematodes is attributed to the demand for research pertaining to

agronomic crops, the long time period required for crop loss assessment on horticultural

crops, and the vast array and interchangeability of available cultivated plant material

(Walker and Melin, 1998b). Information regarding resistance of ornamental plants to

root-knot nematodes is needed by extension personnel, the landscape industry, plant

producers, and gardeners (Walker et al., 1994; Walker and Melin, 1998a). Furthermore,

knowledge of the susceptibility of cultivated perennials to root-knot may alleviate post-

installation damage associated with these pathogens by the avoidance of highly

1 2 susceptible and increased development and use of resistant cultivars (Giblin-

Davis et al., 1992). Incorporation of resistance into the genomes of perennial cultivars would greatly benefit the industry (Walker and Melin, 1998a), as rotation with resistant species has been shown to successfully control root-knot nematodes in infested nursery fields (LaMondia, 1995). High demand and extensive shipments impact the rate of inspection for root-knot nematodes. Therefore, root-knot nematode management programs that are based on sanitation, resistance, tolerance, rotation, and exclusion, should be applied and developed in nurseries and landscapes to alleviate product losses, and to reduce the spread of the pathogens into uninfested field-grown nurseries and landscapes (Benson and Barker, 1985; LaMondia, 1997; Walker, 1980;

Walker and Melin, 1998b). Care should be taken with the cultivation of tolerant perennials in root-knot nematode infested sites since annuals planted adjacent to such plants may become infected with root-knot nematodes that thrive on tolerant perennials

(McSorley and Dunn, 1990; Rohde, 1972).

Meloidogyne spp.

Historical Background

The first account of root-knot nematodes was by Berkeley, who in 1855 observed galls on roots of greenhouse-grown cucumber plants in England (Hartman and Sasser,

1985). Cornu first coined the name Anguillula marioni Cornu for root-knot nematodes, after observing root galls on Onobrychis sp. (sainfoin). Subsequently, root-knot nematodes were classified in the genera or Anguillula. In 1884, Müller classified root-knot nematodes as H. radicicola and in 1887 the type species Meloidogyne exigua was described by Göldi. In 1932, Goodey reclassified root-knot nematodes as H.

3 marioni (Müller) Goodey. Differences in host responses led Chitwood to redescribe

Meloidogyne exigua Göldi, M. incognita (Kofoid and White, 1919) Chitwood, M. javanica (Treub, 1885) Chitwood, and M. arenaria (Neal, 1889) Chitwood, and to describe M. hapla Chitwood and M. incognita acrita Chitwood, as a new species and variety, respectively (Christie, 1959). In his description of M. hapla, Chitwood cited

Abelia × grandiflora as a host (Bernard and Witte, 1987). Chitwood also gave a general description of the Meloidogyne and differentiated it from the genus Heterodera

(Hirschmann, 1985). Chitwood’s diagnoses of the redescribed and newly described species were based on examinations of morphological features and morphometrics from all life stages of the evaluated species (Eisenback, 1985). These features included the perineal pattern, stylet morphology, and distance from the base of the stylet knobs to the dorsal esophageal gland opening (DEGO) (Christie, 1959). To date, more than 80

Meloidogyne spp. have been described (Randig et al., 2002). The current taxonomic status for the Meloidogyne spp. reviewed in this study is: phylum Nemata, order

Tylenchida, suborder Tylenchina, superfamily , family , subfamily Meloidogyninae, species M. incognita, M. javanica, M. arenaria, and M. mayaguensis Rammah and Hirschmann, 1988 (Andrássy, 1976; Rammah and

Hirschmann, 1988; Thorne, 1961). Meloidogyne incognita, M. javanica, and M. arenaria have been frequently encountered in Florida for many years. Meloidogyne mayaguensis, however, was not detected frequently in Florida until the use of electrophoresis for the identification of root-knot nematodes became commonplace. Meloidogyne mayaguensis was described as a result of the reevaluation of a population that was tentatively identified as M. arenaria at North Carolina State University. Differentiating biological

4 and morphological characters that part this species from the four common Meloidogyne species include a somatic chromosome number of 2n = 44 to 45 and unique enzyme phenotypes revealed through electrophoresis (Rammah and Hirschmann, 1988).

Although the M. mayaguensis type species exhibits a morphologically unique perineal pattern, investigations by Brito et al. (2004) revealed M. mayaguensis isolates having a perineal pattern similar to that of M. incognita in Florida, making this feature unreliable for the identification of M. mayaguensis.

Identification

The accurate identification of root-knot nematodes to species and host races is

essential for their control and is a prerequisite to meaningful research. Many

Meloidogyne species are easily identified based on distinct morphological characters and

restricted host ranges. Several species are difficult to identify due to their similarity to

other species and poor taxonomic descriptions. The four most common root-knot

nematode species, composing 98% of all worldwide populations, are M. incognita, M.

javanica, M. arenaria, and M. hapla (Hussey, 1985a). Other Meloidogyne species, such

as M. mayaguensis, become increasingly important due to their uncommon virulence and

increasing occurrence. Difficulty in identifying root-knot nematodes may result from

morphological variations within and between populations from a same species. Since the

reevaluation of Meloidogyne spp. by Chitwood in 1949, female perineal patterns became

the dominant diagnostic character of the four most common Meloidogyne species. The

perineal pattern presents several benefits that render it a valuable diagnostic tool. Aside

from minor variations, perineal patterns are constant within populations and their source

(females) is abundant in infected host roots. Other diagnostic features used in taxonomic

5 identification include female stylets, male heads and stylets, and second-stage juvenile

(J2) heads and stylets. Morphological features of the perineal patterns of M. incognita,

M. javanica, M. arenaria, and M. mayaguensis are describes as follows:

Meloidogyne incognita. Striae are smooth, wavy, sometimes in a zigzag pattern.

Lateral lines are absent. A squarish, high dorsal arch containing a distinct whorl around the tail terminus is the most conspicuous diagnostic character of this perineal pattern

(Eisenback, 1985).

Meloidogyne javanica. Striae are smooth and somewhat wavy. The dorsal arch is

often low and rounded but may be high and squarish, frequently possessing a whorl in the

tail terminus area. Unique to this species are distinct lateral ridges that run across the

pattern, fading away around the tail terminus (Eisenback, 1985).

Meloidogyne arenaria. Striae are smooth and slightly wavy, often extended laterally, forming wings on one or both lateral sides of the pattern. Distinctive lateral ridges are absent, but are marked by forked, irregular lateral fields. The dorsal arch is low and indented near the lateral fields, forming rounded shoulders (Eisenback, 1985).

Meloidogyne mayaguensis. Striae are fine, continuous, and widely spaced.

Lateral lines are inconspicuous or a single lateral line may be present on one side of the pattern. Dorsal arch is rounded, with a circular, striae-free tail terminus (Rammah and

Hirschmann, 1988). Thirty percent of three isolates from Florida possess perineal patterns that differ from the type species and depict perineal patterns typical of M. incognita (Brito et al., 2004).

Inconsistencies in host-parasite relationships lead to the erection of species- specific races based on their infection of specific crops, namely Gossypium hirsutum

6

(cotton) cv. Deltapine 61, Nicotiana tabacum (tobacco) cv. NC 95, Capsicum frutescens

(pepper) cv. California Wonder, Citrullus vulgaris (watermelon) cv. Charleston Gray,

Arachis hypogaea () cv. Florunner, and Lycopersicon esculentum (tomato) cv.

Rutgers. Based on their susceptibility to the differential hosts, M. incognita and M. arenaria were assigned four and two races, respectively. This host differential system allows for the rotation of the differential hosts as a means of maintaining low nematode populations and thus reduces crop losses. However, the identification of root-knot nematode species solely on the basis of the differential host test is unreliable due to the possibility of mixed populations, and should be used in conjunction with morphological, morphometric, and biochemical evaluations to determine root-knot nematode species

(Hartman and Sasser, 1985).

Problems in the morphological identification of Meloidogyne species, such as intra-species merging of morphological characters, rarely-seen characters, and lack of apparent differences between species, has encouraged much interest in the utilization of biochemical techniques as a complementary, routine method for the identification of root- knot nematodes (Hansen and Buecher, 1970). Enzyme phenotypes, unveiled through staining of polyacrylamide gel slabs following electrophoresis, have become a reliable, less subjective approach to identification of root-knot nematodes (Hussey, 1985a). The first study on root-knot nematode protein profile stability and its utilization in the identification of root-knot nematodes was conducted by Dickson et al. (1970), who established the usefulness of disc-electrophoresis in identification of root-knot nematodes. This early work also verified that protein profiles were stable within nematode species collected from different parts of the world and that infect an array of

7 hosts. Further work by Dickson et al. (1971) examined lactate dehydrogenase (LDH), malate dehydrogenase (MDH), α-glycerophosphate dehydrogenase (GDH), glucose-6-

phosphate dehydrogenase (G-6-PDH), acid phosphatase (AcP), alkaline phosphatase

(AlkP), and esterase (EST) enzyme profiles from Meloidogyne javanica, M. arenaria, and M. hapla females, as well as from three life stages of M. incognita. Dickson et al.

(1971) revealed that enzyme profiles for GDH, MDH, G-6-PDH, and EST differed among the four studied Meloidogyne species. Furthermore, G-6-PDH enzymatic profiles from all evaluated species and GDH enzymatic profiles from M. incognita, M. hapla, and

M. arenaria were monomorphic, while enzymatic profiles from all the remaining species evaluated were polymorphic. Among the enzymes evaluated for the characterization of

Meloidogyne species, MDH and EST enzyme profiles were most variable with respect to electrophoretic mobility and therefore are most useful for differentiation of the four species evaluated by this method. However, MDH enzyme profiles did not differ between M. javanica and M. incognita and EST isoenzymes were detected from M. incognita and M. hapla isolates. Enzymatic profiles from the three life stages of M. incognita resulted in variable band numbers and electrophoretic migration for MDH and

EST. The use of disk-electrophoresis for the assessment of Meloidogyne species protein

phenotypes by Dickson et al. (1971) required the analysis of several specimens of the

same species for the elucidation of a single protein phenotype. This method, therefore,

rendered genetic analysis at the intra- and interspecific levels impossible (Dalmasso and

Berge, 1978). A breakthrough in biochemical speciation of Meloidogyne species was the

use acrylamide gels as thin slabs (0.7-mm-thick) to electrophoretically separate proteins

from individual Meloidogyne females. First used by Dalmasso and Berge (1978), the

8 thin-slab method for polyacrylamide gel electrophoresis (PAGE) has proved to be useful in the identification of root-knot nematodes. This method allowed the speciation of 20 to

25 individual specimens from the same or different populations on single gels.

Analyzing 22,000 specimens of root-knot nematodes using microscale electrophoresis,

Dalmasso and Berge (1978) found that, of the enzymes elucidated, EST were the most useful for the differentiation of the common Meloidogyne species, primarily due to their polymorphic nature (Hussey, 1985a).

Morphological characters, particularly female perineal patterns, are the primary method for routine root-knot nematode identification. However, perineal patterns are variable, and may lead to misidentification of aberrant populations and uncommon species. Conversely, biochemical analyses, particularly esterase phenotypes of young, egg-laying females, in conjunction with morphological and morphometric examinations, allow for precise, accurate diagnoses, thereby alleviating the confusion associated with morphological characters (Hussey, 1985a). In addition to morphological and morphometric analysis and the use of PAGE for the identification of root-knot nematodes, the use of single eggs or J2 for species identification via restriction fragment length polymorphism (RFLP) (Fargette et al, 1996), random amplified polymorphic deoxyribose nucleic acid (DNA) (RAPD) (Blok et al., 1997b), ribosomal DNA amplification (Blok et al., 1997a), and mitochondrial DNA (mtDNA) amplification (Blok et al., 2002), has been reported.

9

Biology and Ecology

Root-knot nematodes occur throughout the world and infect most major plant crops, and are responsible for substantial yield losses and reduced crop quality (Sasser and Carter, 1985). In Florida, root-knot nematodes are the most damaging plant-parasitic nematodes on agricultural crops (McSorley and Dunn, 1989). The life cycle of root-knot nematode is largely indifferent with respect to individual species’ host-parasite relationships and physiological characteristics (Christie, 1959). Root-knot nematode eggs are protected within a gelatinous egg mass produced by the female (Maggenti,

1987). Inside the egg, a first-stage root-knot nematode juvenile (J1) molts once prior to hatching into a J2. While egg hatching is usually spontaneous and does not correlate with plant-root stimuli, root diffusates have been shown to stimulate hatching (Hussey,

1985b). Once hatched, the now J2 move though the soil in search of a suitable feeding site (Christie, 1959). Root penetration by the pathogen involves the mechanical disruption of host tissues. However, cellulose and pectin-dissolving enzymes may also aid in the penetration process (Hussey, 1987). Upon penetration, J2 move within the root in a vertical manner, and often migrate toward and away from the root surface. Although penetration may occur anywhere in the root system, J2 are often observed aggregating and penetrating behind the root cap, near the meristematic zone. Other penetration sites include cracks and lesions of mature roots and areas of secondary root formation (Lewis,

1987). Furthermore, the site of one nematode’s penetration often becomes attractive for other J2, leading to multiple infections in confined areas (Hussey, 1985b). Once established within the plant tissue, root-knot nematodes become sedentary endoparasites, and halt further movement or migration (Christie, 1959). Second-stage juveniles are

10 often observed in cortical tissues about the zone of differentiation, their bodies vertically parallel with the root axis and heads settled in the periphery of the vascular tissue

(Hussey, 1985b). Upon establishment of a suitable feeding site, sexually-undifferentiated

J2 begin to modify the host’s physiology by transforming healthy, undifferentiated cells into specialized feeding sites referred to as giant cells. Modified cells exhibit nuclear, nucleolar, and surface hypertrophy, an increase in cytoplasmic density, organelle hyperplasia, and disappearance of the central vacuoles. While the nematode’s two subventral glands are involved in giant cell initiation, dorsal gland secretions maintain giant cell development (Lewis, 1987). The ability of J2 to invade roots differs with respect to the root-knot nematode species, but symptoms of root-knot nematode penetration are often depicted as root-tip enlargement and root-growth retardation. While gall development is not essential for root-knot nematode survival, the development and maintenance of giant cells is critical for root-knot nematode development. This host- parasite relationship requires the developing root-knot nematode to feed on five to six viable giant cells. The inability to elicit a giant cell response results in nematode death or, if early in the J2 stage, migration out of the root in search of a suitable feeding site

(Hussey, 1985b). Late in the J2 stage, following feeding initiation and giant cell formation, an increase in J2 width is observed, and the genital primordia attain their sexual characteristics prior to the second molt. The third-stage juvenile (J3) of both sexes is depicted by the J2 cuticle surrounding the J3, the loss of the stylet and the median esophageal valve, and the loss of the tail spike, which becomes rounded. The J3 stage passes in a few hours, at which time a third molt gives rise to the fourth-stage juvenile (J4). In this stage, which lasts longer than the third stage, the median esophageal

11 bulb valve is reformed and the excretory pore opens. The female rectal glands, uterus and vagina, and male vas deferens differentiate and enlarge, and the male undergoes metamorphosis, attaining an elongated, cylindrical shape. Following gonad differentiation in the fourth stage, a fourth and final molt reveals the adult nematodes enclosed in the three previous juvenile cuticles. At this stage the stylet reappears in both sexes, the perineal pattern is observed in females, and sperm production is initiated in males prior to the disappearance of the previously-molted cuticles (Triantaphyllou and

Hirschmann, 1960). While male production occurs in most root-knot nematode species,

M. hapla, which reproduces through facultative meiotic parthenogenesis, produce relatively more males than M. incognita, M. javanica, and M. arenaria, which reproduce through obligate mitotic parthenogenesis (Triantaphyllou, 1985). Similarly, M. mayaguensis reproduces through mitotic parthenogenesis (Rammah and Hirschmann,

1988). The pathogen’s parthenogenic reproductive behavior is adaptive to its unstable environmental and physiological conditions, namely the halt in female motility, short generation time, limiting habitat, and male infrequency. Parthenogenesis allows for the establishment of varying phenotypes through polyploidy, thereby increasing adaptation to unfavorable environmental conditions, including, but not limited to, fluctuating thermal gradients, drought, and low oxygen concentrations. Furthermore, such adaptations may allow for rapid generation turnover through rapid maturity of developing juvenile stages

(Maggenti, 1987). Regardless of their reproductive behavior, Meloidogyne spp. produce less eggs and more males in response to increasing populations within a root system

(Lewis, 1987). Upon maturation, females produce a gelatinous matrix through their rectal glands, into which they deposit eggs. The gelatinous matrix provides eggs with

12 physical protection (Maggenti, 1987) and acts as a barrier to temperature fluctuations and water evaporation from eggs (Van Gundy, 1985). Meloidogyne spp. viability and

development is influenced by various environmental and physical stresses, including

temperature, soil texture, moisture, aeration, osmotic potential, and host suitability.

Temperature has the greatest influence on egg development and hatching, growth,

reproduction, and survival. Optimal temperatures for egg development of M. incognita,

M. javanica, and M. arenaria are 10 to 15 °C, and approximately 9 °C for M. hapla.

Conversely, optimal temperatures for growth and development of juvenile and adult

stages of M. incognita, M. javanica, and M. arenaria are 25 to 30 °C. Although

temperature extremes may inhibit reproduction, individual populations within certain

species acclimatize to local temperature regimes. In general, relative cold tolerance is

exhibited by M. hapla, M. incognita, M. arenaria, and M. javanica, in decreasing order,

with M. hapla surviving subzero temperatures and M. javanica not surviving

temperatures below 10 °C. While Meloidogyne eggs do not survive in soils beyond one

year, varying egg mass colors, from white to brown, have been attributed to egg

dormancy and overwintering. Soil texture, moisture, and aeration compose a dynamic,

complex environment, and influence Meloidogyne activity, reproduction, and pathogenicity. Continuously changing, soil texture is a solid-phase component of the soil environment. Vertically and horizontally shifting soil particles are interconnected by the liquid and gas phases of this dynamic system. Optimal temperature regimes and moisture levels of 40 to 60% of field capacity present the most advantageous conditions for nematode activity and metabolism. Moisture levels that are lower or higher than optimal reduce nematode activity due to drying soils and limiting oxygen concentrations,

13 respectively (Van Gundy, 1985). It is widely accepted that Meloidogyne spp. activity is optimal in sandy loam soils and reproduction is greatest in fine sand (Wallace, 1969), primarily due to the low water holding capacity of such soils (Benson and Barker, 1985).

However, Whitehead (1969) reported that eastern African population viability of M. incognita and M. javanica exhibited no correlation with soil texture. In addition,

O’Bannon and Reynolds (1961) reported on heavy M. incognita infestations in coarse- textured soils planted to cotton in Arizona. Root-knot nematode females, examined in roots from different hosts, were variable in size. Highly susceptible plants exhibited large, robust females while less susceptible plants supported smaller females (Pant et al.,

1983). Furthermore, Niblack and Bernard (1985) observed that M. hapla densities were

positively correlated with Cornus florida and Acer rubrum nursery tree age.

Meloidogyne spp. behavior is altered under various soil solution osmotic

potentials. In drying, well-fertilized soil, nematodes are subjected to high osmotic

pressures as the osmotic potential increases. Wallace (1969) observed the highest and

lowest reproduction of M. javanica on Lycopersicon peruvianum (tomato) cv. Tatura

Dwarf when subjected to high and low nutrient levels, respectively. However, M. javanica infectivity decreased with increased electrical conductivity of the soil solution, and J2 migration was observed from high to low salt concentrations (Van Gundy, 1985).

The interacting environmental and biological factors that influence Meloidogyne spp. development, reproduction, and pathogenicity are complex and difficult to evaluate.

Such factors as soil homogeneity, moisture, and temperature, in addition to changing osmotic and water potentials, interact with nematode development, reproduction, and pathogenicity, as well as various metabolic and developmental aspects of the host plant.

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Interactions between nematodes and plants, further complicated by inoculum densities and developmental levels, lead to the inevitable intricacy of this plant-parasite and biological relationship. In evaluating the effect of nematode populations on host plants, factors influencing the experimental environment are minimized to the studied components by controlling variables as temperature, moisture, lighting, host nutrition, initial inoculum density, and initial host development, in hope to reveal a significant relationship between pathogen and host, with minimum interference by unstudied factors

(Van Gudny, 1985).

Relationships with Ornamentals

One of the most serious groups of pathogens to limit agricultural productivity, root-knot nematodes have been reported to infect many plants throughout the world

(Zarina and Abid, 1995), and are considered the most damaging group of plant-parasitic nematodes in Florida (McSorley and Dunn, 1989). The woody ornamental/floriculture industry is one of the U.S.’s fastest growing agricultural segments (Bernard and Witte,

1987). As serious pathogens of numerous woody ornamentals (Barker and Benson,

1977), Meloidogyne spp. have the potential to damage many important nursery crops

(Benson and Barker, 1985) and form disease complexes with certain soil-borne fungal

pathogens, thus increasing their hosts’ susceptibility to such pathogens (Nigh, 1972;

Santamour and Riedel, 1993; Walker and Melin, 1998b). Unlike annual plants, damage

thresholds levels do not apply to perennials, since low Meloidogyne spp. populations

have the potential to increase and cause severe damage over a period of several years

post-planting (LaMondia, 1995).

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In the U.S., root-knot nematodes have been reported to cause damage and reduced yields on ornamental crops in Alabama (Heald, 1967), Arizona (Nigh, 1972; Tarjan,

1952), California (Santo and Lear, 1976; Viglierchio, 1979), Connecticut (LaMondia,

1995, 1996, 1997), Florida (Giblin-Davis et al., 1992; Lehman, 1984a, 1984b; Lehman and Barnard, 1982; McSorley and Dunn, 1989, 1990; McSorley and Marlatt, 1983;

Stokes, 1977, 1982), Georgia (Heald, 1967; Motsinger et al., 1977; Walker et al., 1994;

Walker and Melin, 1998a, 1998b), Hawaii (Sher, 1954), North Carolina (Barker et al.,

1979; Barker and Benson, 1977; Benson and Barker, 1982; Haasis et al., 1961; Rickard and Dupree, 1978, Sasser et al., 1966), Oklahoma (Nemec and Morrison, 1972; Nemec and Struble, 1968), Tennessee (Bernard and Witte, 1987; Bernard et al., 1994; Niblack and Bernard, 1985), Virginia (Eisenback, 1987), and Washington, DC (Santamour, 1992;

Santamour and Riedel, 1993, 1995). Internationally, Meloidogyne spp. have been reported to damage ornamental crops in Australia (Wallace, 1969), Belgium (Coolen and

Hendrickx, 1972; Stoffelen et al., 2000), Egypt (Montasser, 1995), France (De Waele and

Davide, 1998), India (Ahuja and Arora, 1980; Haseeb et al., 1984, 1985; Khanna et al.,

1998; Mishra and Mishra, 1997; Mishra and Misra, 1993; Misra and Mishra, 1997; Misra et al., 2002; Pant et al., 1983; Singh et al., 2000; Singh and Gupta, 1993), (Singh and

Majeed, 1991), Ivory Coast (Adiko, 1988), Korea (Cho et al., 1996), Nigeria (Caveness and Wilson, 1977), Pakistan (Zarina and Abid, 1995), Saudi Arabia (Ibrahim and Al-

Yahya, 2002), Spain (Jaizme-Vega et al., 1997), Tamil Nadu (Rajendran et al., 1975), and Trinidad and Tobago (Bala and Hosein, 1996). Although commonly observed, the potential for damage to many perennial ornamental crops by root-knot nematodes is unrecognized (Bernard and Witte, 1987).

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Meloidogyne spp. have been attributed as causing decline to growth of ornamental plants and thus hinder production and lead to reduced returns (Stokes, 1977).

Meloidogyne incognita has been attributed to such effects on the production of Anthurium andraeanum and other tropical ornamentals (Bala and Hosein, 1996), certain (Montasser, 1995), and several Ilex spp. (Heald, 1967). In addition, production

losses in field-grown Rosa spp. have been attributed to M. hapla (Santo and Lear, 1976).

Furthermore, Meloidogyne spp. have been reported to reduce Dianthus caryophyllus production worldwide by 10 to 20% (Cho et al., 1996) and cause qualitative and quantitative decline in Indian Gladiolus × hortulanus production (Khanna et al., 1998).

Furthermore, in the middle Tennessee nursery-growing region, Meloidogyne hapla

occurs in approximately 25% of nursery blocks (Bernard et al., 1994).

Goff (1936) was one of the first researchers to conduct an extensive survey of the

susceptibility of ornamental plants to Meloidogyne spp. In his survey, Goff noted the

varying degrees of susceptibility among the tested plant species. Root-knot infected

plants often exhibit symptoms that include root galls and root rots, shoot yellowing and

chlorosis, stunted growth, and other symptoms commonly associated with nutritional

deficiencies (Bala and Hosein, 1996; Bird, 1974; Misra et al., 2002, Santo and Lear,

1976; Zarina and Abid, 1995), resulting in general decline (Nigh, 1972), poor yield, and

wilt diseases (Rajendran et al., 1975). Furthermore, photosynthetic rate reduction has

been observed in response to root-knot nematode infections. Often, the ratio of food

resources provided by the host plant and the root-knot nematode density determines the

degree of host response to infection (Bird, 1974). However, Walker and Melin (1998b)

observed greater plant growth in the presence of low plant-parasitic nematode

17 populations than in their absence. Susceptible hosts severely infected with root-knot nematodes often decline as a linear function of time (Barker and Benson, 1977), and such decline is accelerated under unfavorable climatic conditions (Haasis et al., 1961), leading to severe disease symptoms in late summer, fall, and during periods of severe dry periods

(Lehman, 1984a). The degree of root galling is dependant upon the infected plant species and/or and the root-knot nematode species, race, population, or even isolate

(Bird, 1992; LaMondia, 1995; Rohde, 1972). Certain ornamentals infected with root- knot nematodes exhibit unique symptoms. Such plants include Sansevieria cylindrica, which developed leaf discoloration and tip necrosis 4 to 5 months post-infection with M. incognita (Mishra and Mishra, 1997), Philodendron selloum, which exhibits a reduction in leaf size when infected with M. incognita (Mishra and Misra, 1993), and Juniperus horizontalis var. Plumosa and Thuja orientalis cv. Dwarf Greenspike, which exhibit thickened roots and slight galling post-infection with Meloidogyne spp. (Nemec and

Morrison, 1972). Furthermore, Gladiolus × hortulanus plants infected with M. incognita race 2 exhibited leaf drying, reduction in floral stalk height and girth, and reduced number of florets (Khanna et al, 1998). Some plants exhibit minute galls following infection with Meloidogyne spp. In such conditions, root-knot nematode females can be seen protruding from the infected roots. Other plants, such as Rheum spp., Begonia spp., and Thunbergia spp. produce large galls, measuring up to 0.6 m in the latter case (Bird,

1974). In testing the susceptibility of numerous herbaceous perennial ornamentals to M. hapla, however, LaMondia (1995, 1996) did not observe egg mass production in the absence of cellular hypertrophy.

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Mixed populations of Meloidogyne spp. are often observed parasitizing perennial ornamentals. In Pakistan, mixed populations of M. javanica and M. incognita have been reported on Solanum nigrum, Cucumis melo ssp. melo var. flexuosus, and Rosa indica

(Zarina and Abid, 1995). Pant et al. (1983) did not observe infection signs or symptoms

on Matthiola spp., Tagetes spp., Gaillardia spp., Chrysanthemum spp., and Zinnia spp.

after inoculation of these plants with M. incognita. Likewise, no signs or symptoms were

observed in Areca catechu nine months after inoculation with Meloidogyne arenaria race

1, M. incognita races 1 and 3, M. javanica, and M. hapla (McSorley and Dunn, 1989). In testing for pathogenicity of M. incognita on flower bulbs, Montasser (1995) rendered

Amarylis vittata, Clivia miniata, Crinum longifolium, and Narcissus tazetta, which belong to Amaryllidaceae, and Agapanthus umbellatus, Hysacinthus orientalis, Lilium longiflorum, and suaveolens, which belong to , highly resistant. Likewise,

Rajendran et al. (1975) found Barleria prionitis free of M. incognita infections in a pathogenicity experiment. Osborne and Jenkins (1963) observed M. hapla juveniles and light galling in Forsythia intermedia, but indicated that invading juveniles failed to mature. Similar observations were reported by Bernard and Witte (1987) for Ligustrum sinense and Nandina domestica, in which giant cells failed to develop following infection with M. hapla. In addition, N. domestica was a nonhost for M. arenaria (Benson and

Barker, 1982). Several landscape ornamentals were tested for their susceptibility to M. arenaria, M. incognita races 1, 2, and 3, and M. javanica. Of these, Photinia fraseri was tolerant to all the M. incognita isolates as well as to M. javanica. Furthermore, egg mass indices for all evaluated Meloidogyne spp. except for M. javanica were low on Dracaena marginata, while Ficus benjamina was highly susceptible to all Meloidogyne spp. except

19

M. hapla (McSorley and Dunn, 1990). Several herbaceous perennials were evaluated for susceptibility to Meloidogyne spp. by Walker and Melin (1998a). Fragaria × ananassa cv. Pink Panda, Monarda didyma cv. Blue Stockings, Phlox paniculata cv. Eva Cullum,

Franz Shubert, and Oakington Blue, and Polygonum affine cv. Dimity did not support M.

arenaria and M. incognita populations six weeks after inoculation. psilostemon × procurrens cv. Ann Folkard did not support populations of M. arenaria, while G. cinereum cv. Laurence Flatman did not support populations of M. incognita.

Evaluating galling of several herbaceous perennials by Meloidogyne spp., Walker and

Melin (1998a) found no or very few galls on Aethionema cordifolium, Echinacea purpurea, Moranda citriodora, or Patrinia scabiosifolia. Similarly, Santo and Lear

(1976) found Rosa noisettiana cv. Manetti a poor host to M. hapla, while Coolen and

Hendrickx (1972) found R. canina cv. Succes and Heinsohn’s Rekord poor hosts for M.

hapla. In testing the susceptibility of several annual bedding plants to Meloidogyne spp.,

McSorley and Frederick (1994) found that M. incognita race 1 caused no galls on

Ageratum houstonianum cv. Blue Mink, Tagetes patula cv. Dwarf Primrose, Vinca rosea

cv. Little Bright Eye, and Salvia splendens cv. Bonfire, and very light galling on Verbena

× hybrida cv. Florist and Zinnia elegans cv. Scarlet. In the same experiments, Ageratum

houstonianum cv. Blue Mink, Lobularia maritime cv. Rosie O’Day, and T. patula cv.

Dwarf Primrose exhibited no and little infection symptoms post inoculation with M.

javanica and M. arenaria race 1, respectively. Dianthus chinensis cv. Baby Doll Mix

was slightly infected by M. javanica and M. arenaria, while V. rosea cv. Little Bright

Eye and Z. elegans cv. Scarlet were minimally infected by M. javanica and M. arenaria,

respectively. In evaluating carnation cultivars for M. incognita resistance, Fawzy et al.

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(1991) and Cho et al. (1996) reported on the resistance of several cultivars of Dianthus caryophyllus to M. incognita. Bernard and Witte (1987) reported that M. hapla failed to reproduce on several species of Prunus and Rhododendron, although Haasis et al. (1961) reported that Rhododendoron spp. and Camellia spp. supported light Meloidogyne spp. populations in field experiments. Giblin-Davis et al. (1992) evaluated the susceptibility of several Ixora spp. cultivars to M. incognita race 1 and M. javanica. Although all Ixora

spp. were susceptible to the Meloidogyne spp. isolates evaluated, above-ground disease

symptoms were not observed 20 weeks after inoculation. Giblin-Davis et al. (1992)

hypothesized that either the Ixora spp. cultivars tested were tolerant to the inoculated

Meloidogyne spp. isolates, or that 20 weeks was not enough time for above-ground

disease symptoms manifestation. LaMondia (1995, 1996) conducted extensive studies

that evaluated the susceptibility of an array of perennial herbaceous ornamentals to M.

hapla. Approximately 30% of tested perennials were resistant to the isolate evaluated. In

India, Khan and Khan (1989) reported on the resistance of Verbena bipinnatifida to M.

incognita and presented nematicidal properties associated with root exudates from this

plant. Santamour and Riedel (1993) tested 23 landscape trees for root-knot nematode

resistance, of which six were nonhosts to M. arenaria races 1 and 2, M. hapla, M.

incognita, and M. javanica, including Magnolia grandiflora and Gleditsia triacanthos,

which are widely used native American landscape trees. Niblack and Bernard (1985) and

Lehman and Barnard (1982) reported on M. hapla and M. incognita infecting Cornus

florida, and Niblack and Bernard (1985) reported on M. hapla infecting Acer rubrum and

Prunus persica. Several gymnosperms were nonhosts to M. hapla (Bernard and Witte,

1987) and resistant to M. incognita (Nemec and Struble, 1968). Walker and Melin

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(1998b) reported × Cupressocyparis leylandi, Cedrus deodara, and Taxodium distichum to be nonhosts to M. incognita. Bernard and Witte (1987) found no M. hapla reproduction on Acer palmatum and A. saccharum. In Washington, Santamour and

Riedel (1995) reported that Cercis canadensis, C. chinensis, and C. yunnanensis seedlings were severely galled by M. arenaria races 1 and 2, M. incognita, M. javanica, and M. hapla, but only supported populations of M. hapla. Reporting on the susceptibility of Acer spp. to Meloidogyne spp., Santamour (1992) found several Acer spp. resistant or tolerant to M. arenaria races 1 and 2, M. hapla, M. incognita, M. javanica, and M. querciana, and reported a wide range in susceptibility, even among progeny of a single tree.

Several authors documented variable root gall sizes and shapes on different susceptible ornamental crops. Bernard and Witte (1987) documented that M. hapla- induced galls were minute and difficult to detect in Abelia × grandiflora, Photinia × fraseri, Spirae × bumalda cv. Froebelii, and S. × vanhouttei. Galls were intercalary and spindle-shaped on thick, fleshy roots of Hydrangea paniculata and Viburnum carlesii, and spherical and terminal on Cornus florida roots infected with M. hapla. However, C. florida infected with M. incognita exhibited spindle-shaped, intercalary galls.

In testing certain cultivars of Tagetes patula to M. incognita, M. arenaria, and M. hapla, Motsinger et al. (1977) found that only one cultivar, Tangerine, was free of root galls or egg masses. They further suggested that certain T. patula cultivars act as trap crops rather than produce nematicidal agents from their roots. Similarly, pathogenicity studies conducted in India found only T. erecta cv. Crackerjack to be resistant to M. javanica as no galls or penetration were observed on this cultivar following inoculation

22 with the pathogen (Singh and Gupta, 1993). In addition, Eisenback (1987) tested eleven populations of M. hapla of different cytological forms and from different geographic origins for their pathogenicity on T. erecta cv. Carnation. Although most M. hapla populations did not reproduce on this host, several populations varied in their pathogenicity on this host, causing varying degrees of root galling and root proliferation in response to infection.

Perhaps no group of plants has been studied more for their susceptibility to root- knot nematodes as those in the genus Ilex. Susceptibility studies involving Ilex spp. generate conflicting results that may be attributed to nematode variants and differing host susceptibilities. Symptoms often observed in root-knot infected Ilex spp. include poor growth and vigor, foliage yellowing and bronzing, dieback of branches, root-system distortion, and in severe infections, death (Haasis et al., 1961). Furthermore, Ilex spp.

foliage chlorosis is often associated with root galling (Heald, 1967). Meloidogyne

incognita is frequently observed parasitizing Ilex spp. (Bernard et al., 1994). Nemec and

Struble (1968) documented the pathogenicity of M. incognita on I. cornuta cv. Burfordii,

I. crenata cv. Hetz, and I. cassine var. angustifolia. Haasis et al. (1961) found I. crenata to be severely damaged by M. incognita and M. hapla. Similarly, Barker and Benson

(1977) found I. crenata cv. Convexa, Helleri, and Rotundifolia to be susceptible to M. arenaria, and Stokes (1982) documented I. crenata to be frequently infected by M. javanica and M. incognita in commercial nurseries. On the contrary, Barker et al. (1979) found I. cornuta cv. Burfordii and I. vomitoria cv. Nana to be resistant and tolerant to M. arenaria, respectively, and I. cornuta cv. Rotunda a poor host to M. arenaria. McSorley and Dunn (1990) found I. cornuta cv. Burfordii to be a nonhost to M. arenaria, M.

23 incognita races 1 and 3, and M. javanica. Sasser et al. (1966) observed no galling of I. crenata cv. Convexa, Latifolia, and Helleri, or I. cornuta cv. Burfordii following inoculation with M. hapla, but I. crenata cv. Convexa, Latifolia, and Helleri were highly susceptible to M. incognita, M. javanica, and M. arenaria. Heald (1967) indicated that I. crenata cv. Helleri was susceptible to M. incognita, M. javanica, M. arenaria, and M. hapla in greenhouse experiments, in which galled roots were observed and plant weights were reduced in response to root-knot nematode infections. However, in these experiments symptoms of M. hapla were less apparent than those caused by the other root-knot nematode species. Other holly cultivars that did not support M. hapla reproduction include I. × attanuata cv. Foster No. 2, I. crenata cv. Hetzii, and I. cornuta

× aquifolium cv. Nellie R. Stevens (Bernard and Witte, 1987). Accounting for these

susceptibility variations among the various Ilex spp. cultivars, Heald (1967) suggested

that plant selection variations and differing isolates of M. hapla may account for such

inconsistencies. In an attempt to correlate galling inconsistencies with differing

nematode isolates, Bernard et al. (1994) compared galling induced by one isolate of M.

incognita from Tennessee and two isolates of M. hapla from Tennessee and North

Carolina on numerous Ilex spp. cultivars. While all Ilex spp. cultivars supported

reproduction of the M. incognita isolate, the M. hapla isolates differed in their

pathogenicity on the various Ilex spp. cultivars, inducing variable gall numbers and sizes.

This variability was attributed to geographic and host-adaptive variability of the M. hapla

isolates. In examining the effect of root-knot nematode pathogenicity on shoot growth,

Nemec and Struble (1968) found no differences in shoot growth between infected and

non-infected plants after eight weeks in a greenhouse.

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Host plant resistance (HPR) to Meloidogyne spp. may involve several mechanisms, including hypersensitivity reactions, delay in Meloidogyne spp. juvenile maturation, reduction in numbers of giant cells, and reduction in cortical hypertrophy

(Nemec and Morrison, 1972). Botanically, however, HPR also infers to the plant’s ability to withstand, lessen, oppose, or overcome nematode infections, while an immune plant suffers no injury (Rohde, 1972). The increasing availability of plant germplasm and advances in plant science technology may allow for the development of HPR for plant-parasitic nematode control. Such advancements are crucial with the progressing withdrawal of chemical nematicides (Roberts, 1992). An ornamental plant used as a rootstock in a Meloidogyne spp. infested area should exhibit resistance to infection by these pathogens. For example, several Hibiscus rosa-sinensis cultivars with the potential for use as rootstocks based on their reaction to inoculations with M. javanica and M.

incognita have been identified (McSorley and Marlatt, 1983).

Codiaeum variegatum

Codiaeum variegatum (L.) Blume, commonly referred to as , is an

evergreen, glabrous shrub in the family that grows ≥ 2 m tall. Its petioled

leaves are spirally-arranged, alternate, simple and entire or lobed, ovate-lanceolate and

are marked with yellow, white, or red variegation (Bailey, 1958; Gilman, 1999a). While

occur in racemes from leaf axils, they are inconspicuous (Watkins and Sheehan,

1977). Codiaeum variegatum thrives in USDA zones 9b to 11, where large specimens

may be used as foundation plants, hedges, and borders, while smaller specimen may be

used as annual bedding plants. Additionally, it is commonly used as an interior foliage

plant throughout the United States. Codiaeum variegatum is commercially propagated

25 mostly through tip cuttings, and to a lesser extent through air layering and propagation. Potting media used for croton production should be light, slightly acidic, and possess good drainage. In the landscape, crotons grow best in well-draining sandy soils incorporated with organic matter. Croton producers must balance fertilizing and light requirements to produce a saleable plant, as too much fertilizer for the amount of light produces a leggy, weak plant, while too little fertilizer produces a woody, stunted plant. While most croton cultivars require high light, many cultivars that tolerate less light, including C. variegatum cv. Gold Dust, are available, and the market for indoor- thriving cultivars is increasing. Insect pests of crotons include Tetranychus spp. (spider mites), Pseudococcus longispinus (long tailed mealybug), (citrus mealybug), Maconellicoccus hirsutus (pink hibiscus mealybug), Ferrisia virgata (striped mealybug), Polyphagotarsonemus latus (broad mite), and numerous scale species. While stem galls, rots, rusts, scabs, and blights are sometimes encountered on crotons, they are not serious problems (Gilman, 1999a; Henny et al., 1991; Reeves and Bell, 1988; Stamps and Osborne, 2003). Nematode parasites of croton include Paratylenchus spp. (Ibrahim and Al-Yahya, 2002) and spp. (Sher, 1954).

Salvia leucantha

Salvia leucantha Cav. is a 1-m-tall herbaceous perennial shrub, commonly known

as Mexican sage, which belongs to the family Labiatae. This plant possesses cylindrical,

somewhat tapering white branches that are covered with fine wool. Its leaves, silver-gray

colored and arranged in a whorl, possess a short and are 5 to 15 cm long,

lanceolate-linear, crenate, and are pubescent above and tomentose beneath. The plant

exhibits many flowers that occur on 15 to 25-cm-long racemes that appear in the summer.

26

Flowers possess a funnel-shaped, violet to lavender calyx and a white or purple corolla, to 2 cm long (Bailey, 1958; Gilman and Marshall, 1999; Anonymous, 2000). Salvia leucantha thrives in USDA zones 7 to 11 and is used in mass plantings, borders, and edging. In zones 9 to 11, flowers persist longer than in zones 7 and 8. The plant grows well in full sun and prefers well-drained, rich, sandy soils. Mexican sage is commercially propagated through cuttings and divisions (Gilman and Marshall, 1999). Buhrer (1938) reported on Meloidogyne spp. parasitizing S. leucantha. On the contrary, the plant was reported resistant to M. incognita and M. javanica in India (Krishnaprasad, 1979).

Liriope muscari

Liriope muscari Bailey, commonly known as lilyturf, is a grass-like herbaceous perennial in the family Ruscaceae (Gilman, 1999b; Judd, 2003). The plant possesses a short, thick by which it spreads. The stemless, dark green leaves are stiff, long, linear, with an acute tip, and do not exceed the floral scapes, which are 25 to 70 cm high and occur in spring and summer. Scapes possess 10 or more whorls of up to seven flowers, each 0.6 to 0.7 cm across (Bailey, 1958). The plant grows in tufts of various heights, depending on the cultivar. Liriope muscari is often used as a ground cover or for edging, and prefers fertile, well-drained soils. Commercially, this plant is propagated though divisions and seldom through seed (Watkins and Sheehan, 1977). It is also often propagated through tissue culture. Liriope muscari thrives in USDA zones 6 to 10 in shady areas, but tolerates full sun except in the hottest areas of the south, as severe leaf tip burn occurs in such conditions. Various cultivars of lilyturf are available, including variegated foliage (L. muscari cv. Variegata) and white and purple-colored flower varieties. Liriope muscari cv. Evergreen Giant grows to 30-cm-high and spreads slower

27 than other cultivars. Recently, a leaf and crown rot disease, caused by Phytophthora palmivora has taken its toll on L. muscari in landscapes and nurseries, to the extent that

L. muscari cv. Evergreen Giant may lose its popularity in the landscape industry (Leahy and Davison, 1999).

Pittosporum tobira

Pittosporum tobira Ait., also known as Japanese pittosporum, is a 3 to 6-m-high

flowering evergreen shrub to small tree in the family Pittosporaceae. Its whorled, thick,

leathery leaves are 5 to 10 cm long and are glabrous, with revolute margins. Japanese

pittosporum is winter-flowering, possessing 1-cm-long white, fragrant flowers in terminal

umbels. Pittosporum tobira is often grown as a shrub for foundations, hedges, mass

plantings, as a screen, in planter boxes, and may also be trained as a small tree (Bailey,

1958; Gilman, 1999c, 1999d; Stamps, 2002). Furthermore, salt tolerance makes it useful

in seaside plantings, where it is widely used as a hedge and windbreaker (Rinallo and

Bennici, 1989). Since it is damaged by temperatures below -6 °C, P. tobira is restricted

to USDA zones 8 to 10. It prefers fertile, slightly acidic soils, is fairly drought tolerant,

and requires minimal fertilization for optimal growth. Pittosporum tobira establishes

well in partial shade to full-sun conditions (Stamps, 2002). Commercially, this plant is

propagated through semi-hardwood cuttings under mist and seldom by seed (Bailey,

1958; Watkins and Sheehan, 1977). Several cultivars that have either been produced or

assigned to the species include P. tobira cv. Compacta, Variegata, and Wheeler’s Dwarf,

of which P. tobira cv. Variegata possesses thinner leaves than the type species that are

variegated with white, and is heavily cultivated in Florida and California, mainly for

landscape use and for floral designs (Bailey, 1928; Stamps, 1987, 2002). Diseases that

28 often occur on P. tobira include angular leaf spot, caused by Cercospora pittospori,

Rhizoctonia aerial blight, caused by Rhizoctonia ramicola or R. solani, southern blight, caused by Sclerotium rolfsii, root rots, caused by spp. and Rhyzoctonia spp., and

dieback, caused by Agrobacterium spp., Diaporthe spp., Diplodia spp., Nectriella spp.,

homopsis spp., and Sphaeropsis spp. Other, less common diseases include Alternaria leaf

spot, caused by Alternaria tenuissima, mushroom root rot, caused by Armillariella

tabescens, corticium limb blight, caused by Corticium salmonicolor, rough bark disease,

which may be caused by a virus, and several others (Chase and Simone, 2001).

Pittosporum tobira is also a host for the tomato spotted wilt virus (Gera et al., 2000).

This plant often exhibits symptoms of magnesium, iron, manganese, and copper

deficiencies, especially when cultivated in high-pH soils (Dehgan, 1998). Insect pests

often encountered on P. tobira include Aphis gossypii (melon aphid) and Icerya purchasi

(cottony cushion scale) (Hamon and Fasulo, 1998). Buhrer (1938) observed

Meloidogyne spp. infected P. tobira plants, and M. arenaria was detected on a declining

hedge of P. tobira in Florida (Bureau of Nematology, 1989). The ectoparasitic nematode

Belonolaimus longicaudatus has been associated with P. tobira cv. Variegatum (syn. P.

tobira cv. Variegata) decline (Rhoades, 1989).

Odontonema cuspidatum

Odontonema cuspidatum (Nees) Kuntze (syn. O. strictum (Nees) Kuntze), commonly referred to as firespike, is a herbaceous perennial in the family , which is hardy in USDA zones 8B to 11. It is a glabrous, erect shrub, 1 to 2-m-high. that forms clumps that generate from root suckers. It possesses simple, opposite, acuminate, short-petioled leaves that are entire and undulate, 10 to 30 cm long. Its tubular flowers

29 are bright red, pink, white, or lavender, up to 2.5 cm long, and occur in long racemes.

Flowers occur year-round in tropical climates and in the fall in temperate climates. This erect, compact shrub is often used in mass plantings, as a background plant, or a hedge, and often attracts butterflies and hummingbirds. Following the first frost, it dies to the ground, but comes back in the spring. For best flower development and persistence, O. cuspidatum is planted in full sun in fertile, sandy soil. This plant is commercially propagated by cuttings and divisions. Aside from Pseudococcus spp. (mealy bugs), no pests or pathogens have been reported on this plant (Bailey, 1958; Francis, 2004; Gilman and Delvalle, 1999; Watkins and Sheehan, 1977).

Musa acuminata ssp. zebrina

Musa acuminata Colla. ssp. zebrina Van Houtte ex Planch cv. Rowe Red (syn.

Musa sumatrana Baccari cv. Rowe Red) is a cultivated banana in the family Musaceae

that is used as a decorative ornamental. Descriptions surrounding this species have been

erroneous and confused due to the age of specimens at the time of description.

Additionally, the description of wrongly-named specimen, as evident by differing floral

and fruit characters among the described specimen documented, may have contributed to

the taxonomic confusion (Cheesman, 1985). Recent evidence has led to the

reclassification of this plant as M. acuminata ssp. zebrina. The cultivar Rowe Red is not described in the literature, and may have been designated to the species by its cultivators.

Musa acuminata ssp. zebrina is rhizomatous and possesses red-ornamented green pseudostems ≥ 2.5 m high, which are formed via sheathing leaf bases, and continuously sucker from pseudostem bases. Its leaves are spirally arranged, ≤ 1.8 m long and 0.5 m wide, rounded at the base, glaucous, purple beneath, and irregularly purple-patched

30 above, with a 0.3-m-long petiole. The floral spikes droop slightly, and the rachis on which they appear is pubescent. The fruit is inedible, dry, cylindrical and curved, ≤ 7 cm

long and 1.3 cm diam. (Baker, 1893; Cheesman, 1985; Griffiths, 1994; Ricker, 1937).

Musa spp. thrive through USDA zone 9, as long as they are protected in northern regions of zone 9b. Plants in this genus prefer moist, fertile soils and ample fertilization. Musa

acuminata spp. zebrina cv. Rowe Red may be propagated via sucker divisions, but is

produced commercially via tissue culture (Dehgan, 1998). Most Musa spp. are susceptible to Black Sigatoka, caused by Mycosphaerella fijiensis, Yellow Sigatoka, caused by Mycosphaerella musicola, and Panama Disease, caused by Fusarium oxysporum f. sp. cubense. Plant-parasitic nematode pathogens of Musa spp. include M. incognita, M. javanica, M. arenaria, and other Meloidogyne spp., Radopholus simillis,

Pratylenchus goodeyi, P. coffeae, and multicinctus (Adiko, 1988; De

Waele and Davide, 1998; Jaizme-Vega et al., 1997; Stoffelen et al., 2000).

Objectives

The objectives of this research were to:

1. Evaluate the host status of several woody and perennial ornamental plants to

Meloidogyne incognita race 2, M. javanica, M. arenaria race 1, and M.

mayaguensis, in separate experiments.

2. Differentiate malate dehydrogenase and esterase phenotypes of Meloidogyne

spp. that were collected from infected ornamental plants in Florida using

PAGE.

3. Evaluate the usefulness of PAGE in differentiating Meloidogyne spp. for

routine extension diagnostic purposes.

CHAPTER 2

REPRODUCTION OF FOUR MELOIDOGYNE SPP. ON SEVERAL SPECIES OF PERENNIAL ORNAMENTAL PLANTS

Introduction

Root-knot nematodes (Meloidogyne spp.) are the most damaging group of plant- parasitic nematodes to ornamental plants in Florida (McSorley and Dunn, 1989). As serious pathogens of many woody ornamental species, root-knot nematodes limit productivity by damaging numerous nursery crops directly and by forming disease complexes with certain soil-borne fungal pathogens (Barker and Benson, 1977; Benson and Barker, 1985; Nigh, 1972; Santamour and Riedel, 1993; Walker and Melin, 1998b;

Zarina and Abid, 1995). Furthermore, since root-knot nematode populations often thrive and cause damage on perennial hosts for many months and years, damage threshold levels do not apply for such plants (LaMondia, 1995).

Symptoms associated with root-knot nematode infection include root galls and root rots, shoot chlorosis, stunted growth, and other symptoms commonly associated with nutritional deficiencies (Bala and Hosein, 1996; Bird, 1974; Misra et al., 2002; Santo and

Lear, 1976; Zarina and Abid, 1995). Such symptoms are often associated with general decline (Nigh, 1972), poor yield, and wilt diseases (Rajendran et al., 1975).

Published work on the susceptibility of woody ornamentals to Meloidogyne spp. is limited. In Florida, several authors (Giblin-Davis et al., 1992; Lehman, 1984a, 1984b;

Lehman and Barnard, 1982; McSorley and Dunn, 1989, 1990; McSorley and Marlatt,

31 32

1983; Stokes, 1982) reported on the pathogenicity of a number of Meloidogyne spp. to several perennial ornamental plants. Other reports of root-knot infection of ornamentals from Alabama (Heald, 1967), Arizona (Nigh, 1972), California (Santo and Lear, 1976;

Viglierchio, 1979), Connecticut (LaMondia, 1995, 1996, 1997), Georgia (Heald, 1967;

Walker and Melin, 1998a, 1998b), New Jersey (Davis and Jenkins, 1960), North Carolina

(Barker et al., 1979; Barker and Benson, 1977; Benson and Barker, 1982; Haasis et al.,

1961), Oklahoma (Nemec and Morrison, 1972; Nemec and Struble, 1968), Tennessee

(Bernard and Witte, 1987; Bernard et al., 1994; Niblack and Bernard, 1985), and

Washington, DC (Santamour, 1992; Santamour and Riedel, 1993, 1995) have been published. Compared to research on the pathogenicity of Meloidogyne spp. on agronomic crops, and taking into account the vast array of perennial species cultivated, research on the susceptibility of woody and perennial ornamental plants to root-knot nematodes is minimal.

The objectives of these studies were to evaluate the host status of several woody and perennial ornamental plants to Meloidogyne incognita race 2, M. javanica, M. arenaria race 1, and M. mayaguensis, in separate experiments. The experiments were carried out in a controlled environmental chamber (growth room) and a greenhouse at the

University of Florida. The plant species evaluated were Liriope muscari (Lilyturf) cv.

Evergreen Giant, Pittosporum tobira (Pittosporum) cv. Variegata, Odontonema cuspidatum (Firespike), Codiaeum variegatum (Croton) cv. Gold Dust, Musa acuminata ssp. zebrina (ornamental banana) cv. Rowe Red, and Salvia leucantha (Mexican Sage).

While cultivars of Salvia leucantha do not exist, two forms, one with a purple corolla and another with a white corolla, were tested in the greenhouse experiment.

33

Materials and Methods

General Cultivation Practices

Planting. Experimental plants were planted as liners in 15.24-cm plastic (Model

AZE0600, ITML Horticultural Products, Ontario, CA) and clay pots for growth room and greenhouse experiments, respectively, each containing 800 cm3 of planting media. Liners

were obtained from commercial propagators (Table 2-1). Several liners were tested for

the presence of plant-parasitic nematodes by the rapid centrifugal-flotation technique

(Jenkins, 1964) prior to planting.

Planting media. Experiments were identical with respect to the soil mixture used for planting experimental plants and maintaining nematode inocula. The soil mixture was a 2:1 ratio of sand and potting mix (Jungle Growth Professional Growers Mix,

Statham, GA), respectively. The medium was tested for the presence of plant-parasitic nematodes via the rapid centrifugal-flotation method prior to the initiation of each experiment.

Treatments. Five root-knot nematode species were used in the growth room and greenhouse experiments. The nematodes were: (i) M. incognita race 2, (ii) M. javanica,

(iii) M. arenaria race 1, (iv) M. mayaguensis, and (v) non-inoculated control. Exceptions include the 5 June 2003-inoculated Liriope muscari cv. Evergreen Giant and Pittosporum tobira cv. Variegata trials, in which no non-inoculated control treatments were included.

Meloidogyne spp. The Meloidogyne spp. utilized in these experiments were

originally obtained from J. A. Brito, Florida Department of Agriculture and Consumer

Services, Division of Plant Industry, Gainesville, FL. They originated from single egg

34

Table 2-1. Crop and source of liners used for growth room and greenhouse experiments.

Crop Source

Liriope muscari cv. Evergreen Giant Agri-Starts III, Inc., Eustis, FL

Pittosporum tobira cv. Variegata Jon’s Nursery, Inc., Eustis, FL

Liner Source, Inc., Mount Dora, FL

Salvia leucantha – purple corolla Hatchett Creek Farms, LLC, Gainesville, FL

Robrick Nursery, Inc., Hawthorne, FL

Salvia leucantha – white corolla Yoder Brothers, Inc., Lancaster, PA

Odontonema cuspidatum Robrick Nursery, Inc., Hawthorne, FL

Codiaeum variegatum cv. Gold Dust Parrish Nurseries, Inc., Parkland, FL

Musa acuminata ssp. zebrina cv. Rowe Red Agri-Starts I, Inc., Apopka, FL

35 masses, and their species designation was confirmed using perineal patterns and isozyme phenotypes, resolved on polyacrylamide gel slabs following electrophoresis.

Meloidogyne spp. Extraction and Inoculation

Meloidogyne spp. egg and second-stage juvenile (J2) inocula were extracted from

Lycopersicon esculentum Mill. (tomato) cv. Rutgers by the sodium hypochlorite (NaOCl) procedure (Hussey and Barker, 1973), via the shaking of infected roots in 0.53% NaOCl

(Regular Ultra Bleach, Publix Super Markets, Lakeland, FL) solution for 30 seconds, followed by the immediate rinsing of the suspension with 10 liters of water.

Meloidogyne spp. eggs and J2 were inoculated onto test and tomato plants for experimental and inoculum-increase purposes on the same day that the eggs were extracted from infected tomato roots. Meloidogyne spp. inocula were transported to the growth room or greenhouse in sealed 500-ml Erlenmeyer flasks. An aquarium air pump equipped with a 1-ml, 22.8-cm-long, disposable glass Pasteur pipet that was inserted into the Erlenmeyer flask, was used to keep the root-knot nematode inocula thoroughly and evenly suspended. Five thousand eggs and J2 were pipetted into three equidistant holes approximately 3-cm-deep in the pre-moistened soil surrounding the base of each plant.

Test tomato plants were included in every experiment as a control for inoculum viability.

Immediately after inoculation, the holes were covered with the same soil mixture used throughout the experiment. The bench area used for inoculation was thoroughly disinfected with 6.0% regular ultra bleach, and all pipets were replaced between inoculations of the respective Meloidogyne spp. All pots were kept on inverted clay

drainage saucers for the duration of the experiment to avoid Meloidogyne spp.

contamination among pots.

36

Fertilization and watering. In all the experiments, a solution of 0.21 g N, 0.09 g

P, and 0.17 g K (0.21 g 20-20-20 soluble fertilizer, Grace-Sierra Horticultural Products,

Milpitas, CA) in 200 ml water was applied to each pot weekly and plants were watered as needed.

Pesticides. Tetranychus spp. (spider mites) were encountered parasitizing P. tobira in these experiments. Bifenthrin was sprayed at a rate of 0.13 ml a.i./liter when needed. In addition, all tomato plants and all greenhouse-grown crops in these experiments were treated with imidacloprid at a rate of 12.5 mg a.i. per pot, for Bemisia spp. (whitefly).

Photoperiod and temperature. Growth room light was provided by 400-W general lighting metal halide lamps (Osram Sylvania, Danvers, MA), directed toward the experimental plants, which were situated on 1-m-tall benches. Lights were suspended 1 m above the bench tops, and light intensity was recorded at 256.18 ± 52.82 µmol⋅s-1⋅m-2 at a distance of 35 cm above the bench top using a photometer (LI-COR, Model LI-189,

Lincoln, NE). Plants were on a 14-hr light cycle, from 7:00 PM to 9:00 AM. Air and media temperatures in the growth room were maintained using an air conditioner and were measured using a standard thermometer at 26 to 32 °C and 24 to 26 °C, respectively.

Experimental Design

Growth room and greenhouse experiments were arranged in a randomized complete block design, with six and three replications in the growth room and greenhouse, respectively. Each experiment was conducted twice in the growth room and once in the greenhouse. While growth room experiments were harvested approximately

37 two months post-inoculation, greenhouse experiments were conducted over five months or longer, as outlined in Table 2-2. The S. leucantha growth room trials differed with

respect to the plant flower-color forms used. Plants used in the first growth room trial

possessed a mixture of purple- and white-colored corolla S. leucantha plants, while plants

used in the second growth room trial were limited to the purple-corolla form.

Experimental Plants Processing and Meloidogyne spp. Egg Extraction and Counting

All experiments were processed by their respective replications. While entire root systems were processed in the growth room experiments, 10 g of each root system were evaluated for plants in greenhouse experiments due to the extensive root system of these plants. Egg numbers from greenhouse-experimental plants were calculated based on total root weight of respective plants.

Experimental plants were moved, by replication, from the growth room or greenhouse to the nematode assay laboratory at the University of Florida. Each plant was completely processed separately prior to processing of the remaining plants in the respective replication. Each plant was removed from the plastic pot and the soil surrounding its roots was gently shaken into a container. The above-ground portion of each plant was then cut and placed into a pre-weighed 25.4-cm x 33.0-cm manila envelope (Sparco,

Moorestown, NJ), which was placed in a 70 °C oven. Drying times were determined by weighing envelopes every 24 hours until no further weight change was detected. The remaining root system was then immersed in water to remove any adhering soil. The soil-free root system was patted dry and fresh root system weights and gall ratings

(Taylor and Sasser, 1978) were determined and recorded. Eggs were extracted using

0.53% NaOCl (Hussey and Barker, 1973) by a procedure modified by Boneti and Ferraz

Table 2-2. Crops, experimental sites, liner planting dates, inoculation dates, and study lengths for all crops in the growth room and greenhouse Meloidogyne spp. studies carried out at the University of Florida during 2003 to 2005.

Experimental Study length Crop Liner planting date Inoculation date site (days) Liriope muscari cv. Evergreen Giant Ca 22 May 2003 5 June 2003 65

Liriope muscari cv. Evergreen Giant C 26 August 2003 9 September 2003 63

Liriope muscari cv. Evergreen Giant G 19 February 2004 4 March 2004 354

Pittosporum tobira cv. Variegata C 22 May 2003 5 June 2003 70

Pittosporum tobira cv. Variegata C 26 August 2003 9 September 2003 67

Pittosporum tobira cv. Variegata G 19 February 2004 4 March 2004 336

Salvia leucantha – purple and white corolla C 25 October 2003 8 November 2003 68 mix Salvia leucantha – purple corolla C 15 January 2004 29 January 2004 70

Salvia leucantha – purple corolla G 19 February 2004 4 March 2004 154

Salvia leucantha – white corolla G 19 February 2004 4 March 2004 182

Odontonema cuspidatum C 25 October 2003 8 November 2003 81

38

Table 2-2. Continued

Experimental Study length Crop Liner planting date Inoculation date site (days) Odontonema cuspidatum C 15 January 2004 29 January 2004 78

Odontonema cuspidatum G 19 February 2004 4 March 2004 263

Codiaeum variegatum cv. Gold Dust C 22 June 2004 6 July 2004 79

Codiaeum variegatum cv. Gold Dust C 8 September 2004 22 September 2004 96

Codiaeum variegatum cv. Gold Dust G 22 June 2004 6 July 2004 239

Musa acuminata ssp. zebrina cv. Rowe Red C 22 June 2004 6 July 2004 72

Musa acuminata ssp. zebrina cv. Rowe Red C 8 September 2004 22 September 2004 106

Musa acuminata ssp. zebrina cv. Rowe Red G 22 June 2004 6 July 2004 205

aC = Growth-room, G = Greenhouse 39

40

(1981). One ml of solution was drawn out of each treatment and put into a 1-ml, 48- division equine egg parasite counting slide (Advanced Equine Products, Issaquah, WA), which was counted in its entirety. Three 1-ml aliquots were counted per treatment, and the average of the three counts was used for statistical analysis.

Statistical Analysis

Data were subjected to analysis using Analysis of Variance (ANOVA). Mean

comparisons among the treatments were performed with Duncan’s multiple range test

using SAS software (SAS Institute, Cary, NC).

Host Status Classification

The host status of the tested perennials was determined based on the reproduction factor (Rf), which was calculated by dividing the final root-knot nematode density per plant (Pf) by the inoculated root-knot density of 5,000 eggs and J2 (Pi). A Rf ≥ 1.0 was designated as a good host, 1.0 ≥ Rf ≥ 0.1 a poor host, and Rf < 0.1 a nonhost (Sasser et al., 1984).

Results

Liriope muscari cv. Evergreen Giant

Results for the two L. muscari cv. Evergreen Giant growth room trials were

heterogeneous and are therefore presented separately (Tables 2-3 and 2-4). In the first

trial, there were no differences (P ≤ 0.05) among treatments for root-gall index, root

weight, or dry shoot weight. Differences (P ≤ 0.05) were observed among treatments for

number of eggs per plant and number of eggs per g of roots, where fewer eggs were

produced by M. arenaria than by the other three species. In the second trial, there were

no differences (P ≤ 0.05) among treatments for root-gall index, root weight, or shoot dry

Table 2-3. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Liriope muscari cv. Evergreen Giant growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot (g) per plant eggs per g roots Weight (g) Control ------M. incognita race 2 1.17 ± 0.82a 35.95 ± 19.71 31,920.00 ± 44,748.39 ab 848.43 ± 1,047.04 a 7.62 ± 4.26 M. javanica 1.33 ± 1.63 36.33 ± 23.16 19,897.78 ± 36,366.07 a 492.63 ± 608.77 a 8.25 ± 3.17 M. arenaria race 1 0.83 ± 0.82 37.67 ± 20.08 377.78 ± 1,131.08 b 9.11 ± 26.02 b 8.30 ± 3.70 M. mayaguensis 1.00 ± 0.00 37.73 ± 14.28 13,822.22 ± 12,905.42 a 373.04 ± 320.68 a 7.47 ± 4.23 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 41

Table 2-4. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Liriope muscari cv. Evergreen Giant growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs per Mean number of Dry Shoot (0-5) (g) plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 39.18 ± 14.76 0.00 ± 0.00 cb 0.00 ± 0.00 c 12.77 ± 4.22 M. incognita race 2 0.00 ± 0.00 40.00 ± 21.73 231.11 ± 418.14 b 5.36 ± 8.27 b 10.20 ± 3.51 M. javanica 0.33 ± 1.03 37.95 ± 16.83 684.44 ± 829.44 a 20.93 ± 33.18 a 9.73 ± 5.94 M. arenaria race 1 0.00 ± 0.00 38.05 ± 22.35 8.89 ± 27.54 c 0.26 ± 0.81 c 10.63 ± 6.35 M. mayaguensis 0.00 ± 0.00 36.70 ± 24.73 1,000.00 ± 833.61 a 30.51 ± 38.39 a 10.93 ± 8.26 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 42

43 weight. Differences (P ≤ 0.05) were observed among treatments for number of eggs per plant and number of eggs per g of roots, where the number of eggs produced by M.

arenaria was not different (P ≤ 0.05) from the nematode control. Furthermore, M.

javanica and M. mayaguensis produced more eggs than M. incognita, which produced

more eggs than M. arenaria.

In the greenhouse experiment (Table 2-5), differences (P ≤ 0.05) were observed across all

parameters. In this experiment, M. incognita and M. mayaguensis produced more galls

than the other treatments, and the control treatment had a significantly higher root weight

than all other treatments. Meloidogyne incognita, M. javanica, and M. mayaguensis

produced more eggs per plant than M. arenaria and the control treatment, from which no

eggs were recovered. However, M. incognita and M. mayaguensis produced more eggs

per g of roots than M. javanica, which produced more eggs per g of roots than M. arenaria. Finally, the control and M. incognita treatments had higher dry shoot weights than the M. javanica and M. arenaria treatments, and the M. mayaguensis treatment dry shoot weights were not different (P ≤ 0.05) than any of the other treatment. The data suggest that L. muscari cv. Evergreen Giant is a good host to M. incognita race 2 (Rf =

97.1), M. javanica (Rf = 16.6), and M. mayaguensis (Rf = 91.0), and a poor host to M. arenaria race 1 (Rf = 0.12).

Pittosporum tobira cv. Variegata

Results for the two P. tobira cv. Variegata growth room trials were heterogeneous and are therefore presented separately in tables 2-6 and 2-7. The results from the greenhouse experiment are presented in table 2-8. Root galls were not observed on any of the plants in the three studies. No differences (P ≤ 0.05) were observed among treatments for root-

Table 2-5. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Liriope muscari cv. Evergreen Giant greenhouse experiment.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00 bab 141.50 ± 55.65 a 0.00 ± 0.00 b 0.00 ± 0.00 c 30.80 ± 3.83 a M. incognita race 2 5.00 ± 0.00 a 104.33 ± 15.10 b 485,507.38 ± 184,451.74 a 4,646.22 ± 1,555.40 a 29.33 ± 10.05 a M. javanica 0.00 ± 0.00 b 70.93 ± 8.11 b 83,099.47 ± 283,116.92 a 1,100.44 ± 3,742.78 b 21.40 ± 4.40 b M. arenaria race 1 0.00 ± 0.00 b 81.70 ± 34.01 b 597.33 ± 2,069.22 b 6.22 ± 1.55 c 19.70 ± 4.13 b M. mayaguensis 5.00 ± 0.00 a 82.33 ± 37.12 b 454,887.02 ± 412,521.08 a 5,467.56 ± 3,903.75 a 25.20 ± 17.97 ab a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of three replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown.

44

Table 2-6. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Pittosporum tobira cv. Variegata growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot Weight (0-5) (g) per plant eggs per g roots (g) Control ------M. incognita race 2 0.00 ± 0.00a 11.67 ± 6.91 13.33 ± 44.62 abb 1.40 ± 5.07 ab 11.67 ± 2.44 M. javanica 0.00 ± 0.00 12.62 ± 4.63 53.33 ± 168.65 a 4.32 ± 13.10 a 12.67 ± 2.33 M. arenaria race 1 0.00 ± 0.00 12.10 ± 8.07 13.33 ± 44.62 ab 0.79 ± 2.51 ab 12.05 ± 3.63 M. mayaguensis 0.00 ± 0.00 12.23 ± 7.68 0.00 ± 0.00 b 0.00 ± 0.00 b 11.83 ± 3.69 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 45

Table 2-7. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Pittosporum tobira cv. Variegata growth room trial.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 11.57 ± 3.78 0.00 ± 0.00 0.00 ± 0.00 11.42 ± 1.29 M. incognita race 2 0.00 ± 0.00 12.22 ± 4.16 71.11 ± 322.95 5.19 ± 23.57 11.82 ± 1.34 M. javanica 0.00 ± 0.00 11.65 ± 3.05 8.89 ± 43.55 0.67 ± 3.30 11.07 ± 4.05 M. arenaria race 1 0.00 ± 0.00 11.82 ± 1.24 8.89 ± 43.55 0.74 ± 3.63 11.58 ± 1.27 M. mayaguensis 0.00 ± 0.00 10.08 ± 2.99 31.11 ± 78.50 3.56 ± 9.28 10.37 ± 1.14 Data are means of six replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978).

46

Table 2-8. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Pittosporum tobira cv. Variegata greenhouse experiment.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 24.83 ± 14.61 0.00 ± 0.00 0.00 ± 0.00 45.10 ± 19.08 M. incognita race 2 0.00 ± 0.00 23.13 ± 5.32 0.00 ± 0.00 0.00 ± 0.00 49.47 ± 8.32 M. javanica 0.00 ± 0.00 26.87 ± 8.26 0.00 ± 0.00 0.00 ± 0.00 46.37 ± 6.84 M. arenaria race 1 0.00 ± 0.00 25.30 ± 23.30 0.00 ± 0.00 0.00 ± 0.00 43.40 ± 6.54 M. mayaguensis 0.00 ± 0.00 13.80 ± 0.87 0.00 ± 0.00 0.00 ± 0.00 35.03 ± 9.47 Data are means of three replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). 47

48 gall index, root weight, or dry shoot weight in either of the growth room trials or the greenhouse experiment. There were differences (P ≤ 0.05) among treatments for number

of eggs per plant and number of eggs per g of roots in the first growth room trial, where

M. javanica produced more eggs than M. mayaguensis, which produced no eggs.

However, in the second growth room trial and the greenhouse experiment no differences

(P ≤ 0.05) were found. The data suggests that P. tobira cv. Variegata is a nonhost to the

Meloidogyne spp. isolates evaluated (Rf < 0.1).

Salvia leucantha

The results from the first and second purple-corolla S. leucantha growth room

trials are heterogeneous, and are presented in tables 2-9 and 2-10, respectively. Root-

knot nematode galls were not detected on any treatment in the first growth room trial

(Table 2-9). Furthermore, no differences (P ≤ 0.05) were observed among treatments for

root weight or dry shoot weight. Differences (P ≤ 0.05) were observed among treatments

for the number of eggs per plant and the number of eggs per g of roots. The number of

eggs per plant was not different (P ≤ 0.05) between M. javanica and M. mayaguensis.

However, these two species produced more eggs than M.

arenaria and M. incognita. The number of eggs per g of roots produced by M.

javanica was greater than the other species. Results for the second growth room trial

(Table 2-10) indicate differences (P ≤ 0.05) among treatments for root-gall index, number

of eggs per plant, and number of eggs per g of roots. There were no differences (P ≤

0.05) among treatments for root weight and dry shoot weight in this trial. Root galls were

only observed in the M. javanica treatment. The root-gall index for M. javanica was

greater than that for the other species, in which root galls were not detected. For the

Table 2-9. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Salvia leucantha growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs per Mean number of Dry Shoot (0-5) (g) plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 20.15 ± 7.09 0.00 ± 0.00 bb 0.00 ± 0.00 b 14.35 ± 2.59 M. incognita race 2 0.00 ± 0.00 22.42 ± 13.45 0.00 ± 0.00 b 0.00 ± 0.00 b 13.72 ± 2.62 M. javanica 0.00 ± 0.00 20.03 ± 12.97 5,906.67 ± 19,410.10 a 463.25 ± 1,527.17 a 13.35 ± 3.09 M. arenaria race 1 0.00 ± 0.00 21.68 ± 9.22 0.00 ± 0.00 b 0.00 ± 0.00 b 12.50 ± 5.02 M. mayaguensis 0.00 ± 0.00 18.68 ± 9.16 2,053.33 ± 9,207.13 a 94.03 ± 402.60 b 11.75 ± 8.95 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown.

49

Table 2-10. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Salvia leucantha growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00 bab 16.05 ± 4.03 0.00 ± 0.00 d 0.00 ± 0.00 d 9.17 ± 2.57 M. incognita race 2 0.00 ± 0.00 b 17.23 ± 6.35 5,915.56 ± 11,225.64 bc 326.02 ± 568.49 bc 9.52 ± 1.87 M. javanica 1.00 ± 1.26 a 18.42 ± 7.73 113,048.89 ± 164,013.68 a 5,836.49 ± 7,267.12 a 9.10 ± 3.05 M. arenaria race 1 0.00 ± 0.00 b 17.18 ± 8.07 4,537.78 ± 12,798.02 c 239.18 ± 661.38 c 9.35 ± 2.94 M. mayaguensis 0.00 ± 0.00 b 16.57 ± 4.88 36,333.33 ± 126,118.27 b 2,298.54 ± 8,382.65 b 9.20 ± 2.10 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 50

51 number of eggs per plant and the number of eggs per g of roots, the number of eggs produced followed the following order, from high to low: M. javanica, M. mayaguensis, and M. arenaria.

In the purple-corolla S. leucantha greenhouse experiment (Table 2-11), there were

differences (P ≤ 0.05) among treatments for root-gall index, number of eggs per plant,

and number of eggs per g of roots. No differences (P ≤ 0.05) were observed among

treatments for root weight or shoot dry weight. No root-knot nematode galls were

observed on the control and M. incognita treatments, but were observed on the M.

javanica, M. arenaria, and M. mayaguensis treatments. All four root-knot nematodes

reproduced, and the number of eggs per plant and number of eggs per g of roots were not

different (P ≤ 0.05) among species. The data from the two growth room trials and the

purple-corolla S. leucantha greenhouse suggest that this plant is a good host to the four

Meloidogyne spp. evaluated.

In the white-corolla S. leucantha greenhouse experiment (Table 2-12), there were

differences (P ≤ 0.05) among treatments for number of eggs per plant and number of eggs

per g of roots. In both categories, M. incognita produced more eggs than M.

mayaguensis. Egg production was not observed in M. arenaria and M. javanica-

inoculated plants. No differences (P ≤ 0.05) were observed among treatments for the

root-gall index, root weight, or dry shoot weight categories. The data from this

experiment suggests that the white-corolla S. leucantha is a good host to Meloidogyne

incognita and M. mayaguensis.

Table 2-11. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the purple-corolla Salvia leucantha greenhouse experiment.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00 bab 27.43 ± 20.13 0.00 ± 0.00 b 0.00 ± 0.00 b 26.20 ± 2.31 M. incognita race 2 0.00 ± 0.00 b 22.63 ± 7.58 162,494.58 ± 112,613.54 a 7,050.67 ± 2,469.93 a 22.97 ± 13.46 M. javanica 1.83 ± 1.53 a 23.67 ± 11.01 447,239.47 ± 1,014,030.26 a 16,539.56 ± 32,408.80 a 28.17 ± 3.52 M. arenaria race 1 1.33 ± 3.06 a 32.83 ± 20.99 159,567.56 ± 181,554.78 a 4,521.78 ± 3,312.55 a 25.40 ± 10.67 M. mayaguensis 0.67 ± 1.15 ab 27.17 ± 7.62 227,586.04 ± 416,191.95 a 9,284.44 ± 19,449.29 a 25.57 ± 8.31 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of three replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown.

52

Table 2-12. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the white-corolla Salvia leucantha greenhouse experiment.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 39.87 ± 12.23 0.00 ± 0.00 cb 0.00 ± 0.00 c 27.17 ± 5.19 M. incognita race 2 0.00 ± 0.00 39.10 ± 1.78 898,208.53 ± 1,159,608.89 a 22,911.11 ± 29,747.46 a 27.57 ± 6.71 M. javanica 0.00 ± 0.00 36.10 ± 23.95 0.00 ± 0.00 c 0.00 ± 0.00 c 26.33 ± 2.83 M. arenaria race 1 0.00 ± 0.00 37.07 ± 14.53 0.00 ± 0.00 c 0.00 ± 0.00 c 29.67 ± 5.95 M. mayaguensis 0.33 ± 1.15 37.63 ± 19.86 256,939.64 ± 192,705.08 b 7,000.89 ± 2,825.53 b 26.97 ± 4.92 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of three replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 53

54

Odontonema cuspidatum

Results for the two O. cuspidatum growth room trials were heterogeneous and are

therefore presented separately in tables 2-13 and 2-14. The results from the greenhouse

experiment are presented in table 2-15. Root galls were not observed on any of the plants

in the three studies. No differences (P ≤ 0.05) were observed among treatments for root- gall index, root weight, number of eggs per plant, number of eggs per g of roots, or dry shoot weight in either of the growth room trials or the greenhouse experiment. The data suggest that O. cuspidatum is a nonhost to the four Meloidogyne spp. evaluated.

Musa acuminata ssp. zebrina

Results for the two M. acuminata ssp. zebrina growth room trials were heterogeneous and are therefore presented separately in tables 2-16 and 2-17. Root galls and reproduction were observed for all Meloidogyne species in the three experiments.

Differences (P ≤ 0.05) among treatments for root weight or dry shoot weight were not

detected in the first growth room trial (Table 2-16). The root galls and number of eggs

per plant were not different (P ≤ 0.05) among the root-knot nematode species. However,

M. arenaria produced more eggs per g of roots than the other root-knot nematode

species. In the second growth room trial (Table 2-17), M. mayaguensis produced more

galls than M. javanica, which produced more galls than M. arenaria and M. incognita.

Plants inoculated with M. mayaguensis had higher root weights than the other treatments,

and M. arenaria and M. mayaguensis produced more eggs per plant and eggs per g of

roots than M. javanica and M. incognita. The dry shoot weight of the control treatment

was greater than that of the root-knot nematode treatments.

Table 2-13. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Odontonema cuspidatum growth room trial.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 33.85 ± 18.01 0.00 ± 0.00 0.00 ± 0.00 20.07 ± 4.42 M. incognita race 2 0.00 ± 0.00 32.22 ± 9.12 0.00 ± 0.00 0.00 ± 0.00 19.00 ± 1.62 M. javanica 0.00 ± 0.00 29.78 ± 21.93 4.44 ± 21.77 0.44 ± 2.13 18.58 ± 5.79 M. arenaria race 1 0.00 ± 0.00 33.42 ± 22.44 0.00 ± 0.00 0.00 ± 0.00 18.95 ± 5.80 M. mayaguensis 0.00 ± 0.00 26.37 ± 16.32 4.44 ± 21.77 0.22 ± 1.08 15.53 ± 9.36 Data are means of six replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978).

55

Table 2-14. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Odontonema cuspidatum growth room trial.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 11.57 ± 3.78 0.00 ± 0.00 0.00 ± 0.00 16.22 ± 6.90 M. incognita race 2 0.00 ± 0.00 12.22 ± 4.16 31.11 ± 152.41 2.19 ± 10.73 17.85 ± 4.25 M. javanica 0.00 ± 0.00 11.65 ± 3.05 0.00 ± 0.00 0.00 ± 0.00 14.53 ± 15.36 M. arenaria race 1 0.00 ± 0.00 11.82 ± 1.24 0.00 ± 0.00 0.00 ± 0.00 18.17 ± 3.50 M. mayaguensis 0.00 ± 0.00 10.08 ± 2.99 0.00 ± 0.00 0.00 ± 0.00 18.63 ± 2.27 Data are means of six replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978).

56

Table 2-15. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g roots, and dry shoot weights from the Odontonema cuspidatum greenhouse experiment.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 96.40 ± 18.20 0.00 ± 0.00 0.00 ± 0.00 44.03 ± 5.64 M. incognita race 2 0.00 ± 0.00 89.37 ± 6.50 8.89 ± 30.79 0.89 ± 3.08 43.80 ± 12.87 M. javanica 0.00 ± 0.00 89.53 ± 26.26 0.00 ± 0.00 0.00 ± 0.00 44.77 ± 6.63 M. arenaria race 1 0.00 ± 0.00 106.26 ± 1.24 0.00 ± 0.00 0.00 ± 0.00 49.47 ± 16.30 M. mayaguensis 0.00 ± 0.00 87.10 ± 21.38 0.00 ± 0.00 0.00 ± 0.00 42.67 ± 6.47 Data are means of three replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978

57

Table 2-16. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Musa acuminata ssp. zebrina growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of Dry Shoot (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00 bab 23.63 ± 5.44 0.00 ± 0.00 b 0.00 ± 0.00 c 9.52 ± 1.66 M. incognita race 2 4.33 ± 2.07 a 26.55 ± 6.74 189,306.67 ± 114,226.82 a 7,039.58 ± 2,385.42 ab 7.70 ± 6.60 M. javanica 5.00 ± 0.00 a 26.07 ± 5.01 139,053.33 ± 90,863.31 a 5,292.61 ± 3,214.97 b 9.72 ± 1.80 M. arenaria race 1 4.67 ± 1.63 a 24.62 ± 4.47 199,737.78 ± 142,003.68 a 8,119.14 ± 5,689.47 a 9.75 ± 2.50 M. mayaguensis 5.00 ± 0.00 a 26.20 ± 6.42 190,946.67 ± 124,960.19 a 7,477.72 ± 5,774.92 ab 9.32 ± 0.93 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 58

Table 2-17. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Musa acuminata ssp. zebrina growth room trial.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of eggs Dry Shoot (0-5) (g) per plant per g roots Weight (g) Control 0.00 ± 0.00 dab 11.87 ± 3.29 b 0.00 ± 0.00 d 0.00 ± 0.00 c 9.25 ± 1.55 a M. incognita race 2 2.17 ± 1.97 c 13.45 ± 6.83 ab 130,472.22 ± 83,342.51 c 9,684.54 ± 4,138.28 b 7.37 ± 1.53 b M. javanica 3.17 ± 0.82 b 14.47 ± 1.79 ab 170,611.11 ± 92,654.84 bc 11,707.26 ± 5,311.64 ab 7.30 ± 1.91 b M. arenaria race 1 2.00 ± 2.19 c 12.25 ± 4.02 b 180,138.89 ± 95,931.79 ab 15,126.25 ± 9,704.36 a 7.45 ± 1.66 b M. mayaguensis 4.67 ± 1.03 a 15.83 ± 4.34 a 239,500.00 ± 108,061.51 a 15,167.20 ± 6,381.53 a 7.50 ± 1.99 b a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of six replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown. 59

60

In the greenhouse experiment (Table 2-18), no differences (P ≤ 0.05) among treatments were observed for root weight and dry shoot weight, and no differences (P ≤

0.05) among root-knot nematode treatments were observed for number of eggs per plant or number of eggs per g of roots. However, M. mayaguensis-inoculated plants had a higher root-gall index than the other root-knot nematode-infected plants. The data suggest that M. acuminata ssp. zebrina cv. Rowe Red is a good host to the four

Meloidogyne spp. evaluated.

Codieaum variegatum cv. Gold Dust

Results for the two C. variegatum cv. Gold Dust growth room trials were

heterogeneous and are presented in tables 2-19 and 2-20, respectively. The results for the

greenhouse experiment are presented in table 2-21. Root galls were not observed on any

of the plants in the three studies. No differences (P ≤ 0.05) were observed among

treatments for root-gall index, root weight, number of eggs per plant, number of eggs per

g of roots, or dry shoot weight in either of the growth room trials or the greenhouse

experiment. The data suggest that C. variegatum cv. Gold Dust is a nonhost to the

Meloidogyne spp. evaluated.

Discussion

The assignment of host statues to perennial ornamentals is ambiguous. Unlike annual crops, root-knot nematode populations may thrive on perennial hosts for many months and years. Therefore, studies on the host status of root-knot nematodes on perennials that result in the classification an immune or a poor host may be disproved by conducting longer-term studies that allow ample time for pathogen reproduction. The plants evaluated in this study were homogeneous in their response to the four

Table 2-18. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Musa acuminata ssp. zebrina greenhouse experiment.

Treatment Root-gall index Root Weight Mean number of eggs Mean number of eggs Dry Shoot (0-5) (g) per plant per g roots Weight (g) Control 0.00 ± 0.00 cab 26.83 ± 17.92 0.00 ± 0.00 b 0.00 ± 0.00 b 24.00 ± 14.76 M. incognita race 2 2.33 ± 1.15 b 32.30 ± 44.06 277,331.67 ± 389,211.65 a 8,344.33 ± 9,516.67 a 19.77 ± 13.63 M. javanica 4.00 ± 3.64 ab 58.30 ± 27.61 454,100.00 ± 433,463.29 a 8,522.22 ± 10,650.06 a 25.60 ± 8.63 M. arenaria race 1 3.33 ± 2.31 ab 29.10 ± 3.02 318,673.89 ± 470,556.48 a 10,794.44 ± 15,023.29 a 20.23 ± 5.28 M. mayaguensis 4.67 ± 1.15 a 39.50 ± 36.43 236,578.33 ± 515,546.34 a 5,350.00 ± 10,144.02 a 18.67 ± 10.07 a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). bData are means of three replications. Means in columns followed by a common letter are not different (P ≤ 0.05) according to Duncan’s multiple range test. Egg numbers were log-transformed prior to data analysis, but non-transformed numbers are shown.

61

Table 2-19. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the first Codiaeum variegatum cv. Gold Dust trial.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 11.93 ± 3.44 0.00 ± 0.00 0.00 ± 0.00 11.58 ± 3.24 M. incognita race 2 0.20 ± 0.82 13.32 ± 11.05 37.40 ± 97.86 2.78 ± 7.17 11.46 ± 10.18 M. javanica 0.17 ± 0.82 11.32 ± 3.49 8.83 ± 43.55 0.93 ± 4.54 10.78 ± 4.97 M. arenaria race 1 0.17 ± 0.82 14.52 ± 5.59 4.50 ± 21.77 0.41 ± 2.03 13.40 ± 3.68 M. mayaguensis 0.00 ± 0.00 12.10 ± 3.33 26.67 ± 130.64 1.92 ± 9.40 11.33 ± 2.99 Data are means of six replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978).

62

Table 2-20. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the second Codiaeum variegatum cv. Gold Dust growth room trial.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 9.87 ± 2.47 0.00 ± 0.00 0.00 ± 0.00 9.22 ± 2.36 M. incognita race 2 0.17 ± 0.82 10.25 ± 5.80 0.00 ± 0.00 0.00 ± 0.00 8.37 ± 3.42 M. javanica 0.33 ± 1.03 8.05 ± 3.39 0.00 ± 0.00 0.00 ± 0.00 7.53 ± 3.80 M. arenaria race 1 0.33 ± 1.03 8.37 ± 6.41 0.00 ± 0.00 0.00 ± 0.00 7.07 ± 3.52 M. mayaguensis 0.00 ± 0.00 8.82 ± 2.06 0.00 ± 0.00 0.00 ± 0.00 9.05 ± 5.67 Data are means of six replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978). 63

Table 2-21. Treatments, root-gall indexes, root weights, number of eggs per plant, number of eggs per g of roots, and dry shoot weights from the Codiaeum variegatum cv. Gold Dust greenhouse experiment.

Treatment Root-gall Root Weight Mean number of eggs Mean number of Dry Shoot index (0-5) (g) per plant eggs per g roots Weight (g) Control 0.00 ± 0.00a 37.03 ± 8.88 0.00 ± 0.00 0.00 ± 0.00 33.17 ± 10.58 M. incognita race 2 0.00 ± 0.00 35.80 ± 21.20 0.00 ± 0.00 0.00 ± 0.00 36.93 ± 2.90 M. javanica 0.00 ± 0.00 44.00 ± 49.36 0.00 ± 0.00 0.00 ± 0.00 34.27 ± 15.01 M. arenaria race 1 0.00 ± 0.00 39.47 ± 17.76 0.00 ± 0.00 0.00 ± 0.00 35.53 ± 19.60 M. mayaguensis 0.00 ± 0.00 54.80 ± 17.16 0.00 ± 0.00 0.00 ± 0.00 42.33 ± 6.86 Data are means of three replications. No differences (P ≤ 0.05) were observed according to Duncan’s multiple range test. a0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; 5 = more than 100 galls per root system (Taylor and Sasser, 1978).

64

65

Meloidogyne spp. evaluated, with the exception of L. muscari cv. Evergreen Giant, which was a good host to M. incognita race 2, M. javanica, and M. mayaguensis, and a poor host to M. arenaria race 1. Liriope muscari was classified as a nonhost to M. arenaria race 1 in the first and second growth room experiments, yielding Rf values of 0.07 and

0.002, respectively. However, its host status to this pathogen changed to poor (Rf = 0.12) in the greenhouse experiment. It is unclear whether M. arenaria race 1 is a good host under longer term field or greenhouse studies. Pittosporum tobira cv. Variegata was a nonhost to all the Meloidogyne spp. evaluated. Liriope muscari cv. Variegata was previously reported susceptible to M. hapla (LaMondia, 1996). This is the first report of any Meloidogyne spp. on L. muscari cv. Evergreen Giant.

Pittosporum tobira cv. Variegata was a nonhost to the Meloidogyne spp. evaluated in this study. This cultivar was found infected with Meloidogyne spp. in Lake

County, FL (chapter 3). Meloidogyne incognita, M. arenaria, and Meloidogyne sp. were previously reported on P. tobira by Nigh (1972), Bureau of Nematology (1989), and

Goodey et al. (1965), respectively. However, P. tobira cv. Variegata is frequently infected by Meloidogyne spp., as evident by root galls and egg masses that are observed

on infected plant roots (Levin, R., personal observation). It is possible that P. tobira cv.

Variegata is a nonhost to the Meloidogyne spp. isolates evaluated. It is unlikely that the

reproductive period in these studies was limiting, since a small number of eggs (Rf ≤

0.01), which probably remained from the initial inocula, was retrieved from plants in the

two growth room experiments, and no eggs were isolated from plants in the greenhouse

experiment. Differences in host response and results that differ from field observations in

this study may be attributed to the Meloidogyne spp. isolates evaluated. Additional work,

66

involving the isolation, culture, and identification of root-knot nematodes from infected

P. tobira cv. Variegata plants, is required to unveil the host status of this cultivar to

Meloidogyne spp.

The purple-corolla form of S. leucantha and M. sumatrana ssp. zebrina cv. Rowe

Red were good hosts to the Meloidogyne spp. evaluated in these experiments. The white-

corolla form of S. leucantha was a good host to the M. incognita and M. mayaguensis

isolates evaluated, and a nonhost to the M. javanica and M. arenaria isolates evaluated.

Meloidogyne sp. was previously reported on Salvia leucantha (Goodey and Franklin,

1956, Goodey et al., 1965). Although Meloidogyne arenaria and M. incognita have been

reported on numerous Musa acuminata cultivars (Goodey et al., 1965), the host status of

M. acuminata ssp. zebrina cv. Rowe Red to the Meloidogyne spp. evaluated is reported

here for the first time.

Odontonema cuspidatum and Codiaeum variegatum cv. Gold Dust were nonhosts

to the Meloidogyne spp. evaluated in these experiments. Odontonema cuspidatum and C.

variegatum were previously reported as hosts to Radopholus similis and Hoplolaimus sp.,

respectively (Goodey et al., 1965). Since many C. variegatum cultivars are frequently

encountered infected by Meloidogyne spp. (Levin, R., personal observation), the host

status of C. variegatum cv. Gold Dust to the Meloidogyne spp. evaluated and to

additional root-knot nematode species and races needs to be verified and evaluated. In

addition, the usefulness of C. variegatum cv. Gold Dust as a root-knot nematode resistant

rootstock for many Meloidogyne spp.-susceptible C. variegatum cultivars needs to be

investigated.

CHAPTER 3 IDENTIFICATION OF ROOT-KNOT NEMATODES

Introduction

The usefulness of Meloidogyne spp. isozymes, resolved by electrophoresis, as a tool for the identification of root-knot nematode species, has increased dramatically over the last 40 years. Most Meloidogyne spp. show species-specific esterase (Est) phenotypes. Some species, including M. exigua and M. naasi, show nonspecific Est

phenotypes. Therefore, the elucidation of a second enzyme phenotype, malate

dehydrogenase (Mdh), is necessary for differentiation of such species. Dickson et al.

(1971) proved that Est and Mdh phenotypes, resolved following disk electrophoresis,

provide a reliable means for speciating Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla. Disk electrophoresis analysis of Meloidogyne females by Dickson et al.

(1970, 1971) and Hussey et al. (1972) utilized several nematodes of the same species per

isozyme phenotype elucidated, rendering genetic analysis at the intra- and interspecific

levels impossible (Dalmasso and Berge, 1978). The use of polyacrylamide gel

electrophoresis (PAGE) by Dalmasso and Berge (1978) provided the means to unveil

single Meloidogyne female isozyme phenotypes following electrophoresis in a 0.7-mm thick slab gel. Later works (Carneiro et al., 1996, 1998, 2000; Dalmasso and Berge,

1978; Esbenshade and Triantaphyllou, 1985a, 1985b, 1985c, 1987; Fargette, 1987a,

1987b; Fargette and Braaksma, 1990; Pais and Abrantes, 1989; Starr et al, 1996;

Yongfang et al., 1998) utilized innovations of thin-slab gel electrophoresis for the

67 68

identification of single Meloidogyne females. Unfortunately, standard methodology for

such techniques is not universal, and published works utilize various gel-forming

apparatuses and methodologies, an array of enzyme stain concoctions, and varying

electrophoresis run times. In addition, many authors fail to accurately describe their

methods and results in detail.

Esbenshade and Triantaphyllou (1985b) presented numerous Mdh and Est

phenotypes and their associated relative electrophoretic migration (Rm) values (distance

of protein in question relative to the migration distance of the bromophenol-blue dye).

Each band was designated a number, representing its migration relative to other bands,

and the numbers were grouped into categories represented by letters. Combinations of

letters and numbers elucidate particular phenotypic patterns. The letter-number system,

referred to in most works that describe Meloidogyne spp. Mdh and Est phenotypes, along with a M. javanica or M. hapla standard on the same gel, is useful for routine identification of several Meloidogyne species. However, isozyme phenotypes are limited in their usefulness when compared on different gels. Enzyme phenotypes may vary with environmental conditions, nematode life stage, and different populations or isolates

(Caswell-Chen et al., 1993). Furthermore, since variability is inevitable between electrophoretic runs due to human error and unavoidable environmental differences at the time of gel and enzyme stain preparations, slight variations in isozyme phenotypes may lead to improper speciation. Therefore, until procedural materials and methods, evaluation methods, and the accurate reporting of Rm values are standardized, information including gel formulations and sizes, staining methods, electrophoresis run times, and precisely-measured Rm must be included in works that elucidate known or

69 unknown Meloidogyne spp. isozyme phenotypes (Esbenshade and Triantaphyllou, 1985a,

1985b, 1985c; Evans, 1971). Isozyme phenotypes, Rm values, and methodological information have been described accurately (Dalmasso and Berge, 1978; Esbenshade and

Triantaphyllou, 1985a, 1985b, 1985c; Fargette, 1987a, 1987b; Hussey et al., 1972;

Yongfang et al., 1998), and are presented for M. javanica (Figure 3-1), M. incognita

(Figure 3-2), M. arenaria (Figure 3-3 and 3-4), M. hapla (Figure 3-5), various

Meloidogyne spp. (Figure 3-6), and for several unidentified Meloidogyne spp. isolates

(Figure 3-7).

Yongfang et al. (1998) and Cetintas et al. (2003) elucidated Mdh and Est isozyme patterns using a PhastSystem apparatus (Pharmacia Biotech AB, Uppsala, Sweden) and a

Mini Protean 3 Cell apparatus, respectively, for routine identification of root-knot nematodes. Yongfang et al. (1998) determined that the PhastSystem is ideal for the rapid identification of root-knot nematode species. Furthermore, the authors reported that band pattern stability withstands differing root-knot nematode host species, host nutrition, sample origin and cultivation practices, and sample dosage. Although Mdh and Est patterns relative Rm were identical to previously-reported figures (Esbenshade and

Triantaphyllou, 1985a, 1985b, 1985c), Yongfang et al. (1998) reported on a previously unobserved Est phenotype, referred to as J2 (Figure 3-1). A similar phenotype was reported for M. javanica from soybean in Brazil (Castro et al., 2003).

Objectives

The objectives of this study were to

1. Differentiate malate dehydrogenase and esterase phenotypes of Meloidogyne spp. that

were collected from infected ornamental plants in Florida using PAGE.

70

2. Evaluate the usefulness of PAGE in differentiating Meloidogyne spp. for routine

extension diagnostic purposes.

Materials and Methods

Nematode Populations

A total of 20 root samples of ornamental plants were collected from nurseries, botanical gardens, and residential plantings from nine counties in Florida. For root-knot nematode identification, young egg-laying females were dissected from naturally infected roots of each plant.

Isozyme Analysis

Root-knot nematode females, dissected from different galls of the root systems, were used for isozyme analyses following electrophoresis, using either PhastSystem or

Mini-Protean 3 Cell. Meloidogyne spp. were identified by comparing specimen Mdh and

Est phenotypes to those of previously published root-knot nematode species (Dalmasso and Berge, 1978; Esbenshade and Triantaphyllou, 1985c, 1990; Fargette, 1987a, 1987b;

Hussey et al., 1972; Yongfang et al; 1998). The Mdh and Est Rm of known M. incognita,

M. javanica, M. arenaria, M. mayaguensis, and M. partityla isolates are presented in table 3-1 (Dalmasso and Berge, 1978; Esbenshade and Triantaphyllou, 1985c; Fargette,

1987a, 1987b; Hussey et al., 1972; Yongfang et al., 1998). At least 26 females from each root sample were examined for species identification with the Mini-Protean 3 Cell, except for the Carya illinoensis sample, from which eight females were examined.

However, either six or ten females from each root sample were examined for species

identification with the PhastSystem. PhastSystem-run samples were processed on the

Rm (×100) Band Phenotype Designation Key 0 a Enzyme Band Rm (×100) b MDH N1 23.0 20 MDH N3 23.0, 27.0, 30.0

MDH J4 19.0, 24.0, 30.0, 34.0 c EST J3 46.0, 54.5, 58.9 40 EST J2 47.0, 59.0 EST J3b 30.0, 36.0, 38.0

EST P4 61.0, 81.0, 89.0

60

80

100 MDH N1 N3 N1 J4 N/Ad

EST J3 J3 J2 J3b P4e Sourcef 1 1 2 3 4

Systemg 1 1 2 3 4 71

Figure 3-1. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne javanica, as reported by several authors. aRelative electrophoretic migration. bMalate dehaydrogenase. cEsterase. dNo phenotype specified. eFargette (1987b) reported that esterase phenotype P4 is identical to esterase phenotype J3, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Esbenshade and Triantaphyllou, 1985c; 2 = Yongfang et al., 1998; 3 = Fargette, 1987b. g1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = PhastSystem, 10-15% gel; 3 = Pharmacia apparatus, 7% gel; 4 = unspecified.

Rm (×100) Band Phenotype Designation Key

a 0 Enzyme Band Rm (×100)

MDHb N1 23.0 MDH N3b 23.0, 28.3, 35.0 20 ESTc I1 47.0

EST I1a 39.0 EST P1 71.0, 76.0 40 EST S1 43.8

EST P7 65.0, 71.0

60

80

100 d MDH N1 N3b N1 N1 N/A N/A N/A EST I1 I1 S1 S1 P1e P7e I1a f Source 1 1 1 2 3 3 4 Systemg 1 1 1 1 2 2 3

73

Figure 3-2. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne incognita, as reported by several authors. aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. eFargette (1987a, 1987b) reported that esterase phenotypes P1 and P7 are identical to esterase phenotypes I1 and S1, respectively, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Esbenshade and Triantaphyllou, 1985c; 2 = Dalmasso and Berge, 1978; 3 = Fargette, 1987b; 4 = Hussey et al., 1972. g1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = Pharmacia apparatus, 7% gel; 3 = Polyanalyst apparatus, 7% gel.

Rm Band Phenotype Designation Key (×100) 0 Enzyme Band Rma (×100) MDHb N1 23.0

MDH N3 23.0, 27.0, 30.0 20 MDH N3b 23.0, 28.3, 35.0 ESTc A1 53.3 EST A2 53.3, 56.3

EST P3 50.7, 53.3, 56.3 40 EST S1-M1 43.8, 47.0

EST S2-M1 41.3, 43.8, 47.0 EST M3-F1 47.0, 50.0, 53.3, 56.8 60

80

100

MDH N1 N3 N1 N1 N3 N1 N1 N3b N1 EST A2 A2 A3 A1 S1-M1 S2-M1 S1-M1 A2 M3-F1 d Source 1 1 1 1 1 1 1 1 1 Systeme 1 1 1 1 1 1 1 1 1

75

Figure 3-3. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne arenaria, as reported by Esbenshade and Triantaphyllou (1985c). aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. d1 = Esbenshade and Triantaphyllou, 1985c. e1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively.

Rm Band Phenotype Designation Key (×100) Enzyme Band Rma (×100) 0 b MDH N1 23.0 MDH A5 19.0, 24.0, 30.0, 35.0, 40.0 c 20 EST A1 54.0 EST A2a 43.0, 46.0 EST A3a 30.0, 36.0, 38.0 EST P2 65.0, 71.0 40 EST P5 79.0, 85.0

EST P8 59.0, 65.0, 71.0

60

80

100

d MDH N1 A5 N/A N/A N/A N/A EST A1 A3a A2a P5e P2e P8e f Source 1 2 3 4 4 4 Systemg 1 2 3 4 4 4

77

Figure 3-4. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne arenaria, as reported by several authors. aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. eFargette (1987a, 1987b) reported that esterase phenotypes P2, P5, and P8 are identical to esterase phenotypes S1-M1, A2, and S2-M1, respectively, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Yongfang et al., 1998; 2 = Dalmasso and Berge, 1978; 3 = Hussey et al., 1972; 4 = Fargette, 1987b. g1 = PhastSystem; 2 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 3 = Polyanalyst apparatus, 7% gel; 4 = Pharmacia apparatus, 7% gel.

Rm (×100)

0 Band Phenotype Designation Key Enzyme Band Rma (×100)

MDHb H1 37.0 20 MDH H1a 50.0 c EST H1 50.0 EST H1a 33.0 40 EST A1 53.3

60

80

100

MDH H1 H1 H1a N/Ad EST H1 A1 H1a H1

Sourcee 1,2 1 3 1 Systemf 1,2 1 3 1

79

Figure 3-5. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne hapla. aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. e1 = Esbenshade and Triantaphyllou, 1985c; 2 = Yongfang et al., 1998; 3 = Delmasso and Berge, 1978. f1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = PhastSystem; 3 = Pharmacia GE- 2/4 apparatus, separating and stacking gels 7% and 4%, respectively.

Rm Band Phenotype Designation Key (×100) Enzyme Band Rma (×100) 0 b MDH H1 37.0 . carolinensis . chitwoodi . chitwoodi . cruciani . enterolobii . enterolobii . graminicola . hispanica . hispanica

M M M M M M M M M MDH N1 23.0

20 MDH N1a 30.0 c EST VS1 38.0, 43.8 EST VS1-S1a 38.0, 45.0

40 EST S2-M1 41.3, 43.8, 47.0 EST M3a 47.0, 52.5, 55.7

EST S1 43.8

60 EST P6 51.0, 56.0, 65.0, 71.0 EST P7 65.0, 71.0 EST P8 59.0, 65.0, 71.0

80

100 d MDH H1 N1a N/A N1 N1a N/A N1a N1 N/A e EST VS1-S1a S1 P7 M3a VS1-S1 P6e VS1 S2-M1 P8e Sourcef 1 1 2 1 1 2 1 1 2 System g 1 1 2 1 1 2 1 1 2

81

Figure 3-6. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of Meloidogyne spp. aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. eFargette (1987b) reported that esterase phenotypes P6, P7, and P8 are identical to esterase phenotypes VS1-S1, S1, and S2- M1, respectively, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Esbenshade and Triantaphyllou, 1985c; 2 = Fargette, 1987b. g1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = Polyanalyst apparatus, 7% gel.

Band Phenotype Designation Key

Enzyme Band Rma (×100) Rm b (×100) MDH N1 23.0

0 . microcephala . microtyla . nassi . naasi . oryzae . platani . platani . querciana MDH H1 37.0

M M M M M M M M MDH N1a 30.0

MDH N1b 35.0 20 MDH N3a 23.0, 25.5, 28.3 ESTc A1 53.3

EST M1 47.0, 50.0

40 EST VF1 65.8 EST VF1a 46.0 EST VS1 38.0, 43.8

60 EST S1 43.8

EST P7 75.0, 71.0 EST F1 56.8

80

100

MDH N1 H1 N1b N1a N1a N1a N/Ad N3a e EST A1 M1 VF1a VF1 VS1 S1 P7 F1 Sourcef 1 1 2 1 1 1 3 1 g System 1 1 2 1 1 1 3 1

83

Figure 3-6. Continued aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. eFargette (1987b) claims that esterase phenotype P7 is identical to esterase phenotype S1, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Esbenshade and Triantaphyllou, 1985c; 2 = Delmasso and Berge, 1978; 3 = Fargette, 1987b. g1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 3 = Polyanalyst apparatus, 7% gel.

Rm (×100)

0 Band Phenotype Designation Key

Enzyme Band Rma (×100)

MDHb N1 23.0 20 MDH N3 23.0, 27.0, 30.0

MDH N3c 23.0, 30.0, 32.5 ESTc F1 56.8 40 EST P3 86.0 EST VS1 38.0, 43.8

EST VS1-S1 36.0, 43.8 60 EST VS1-M2 40.0, 53.3, 56.3

80

100

MDH N1 N/Ad N1 N3 N3c N3c e EST F1 P3 VS1 VS1 VS1-S1 VS1-M2 Sourcef 1 2 1 1 1 1 g System 1 2 1 1 1 1

85

Figure 3-7. Malate dehydrogenase and esterase relative electrophoretic migrations and enzyme phenotypes of unidentified Meloidogyne spp. aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. eFargette (1987b) claims that esterase phenotype P3 is identical to esterase phenotype F1, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Esbenshade and Triantaphyllou, 1985c; 2 = Fargette, 1987b. g1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = Polyanalyst apparatus, 7% gel.

Rm (×100) 0 Band Phenotype Designation Key

Enzyme Band Rma (×100)

MDHb N1 23.0

20 MDH N3 23.0, 27.0, 30.0

MDH N3c 23.0, 30.0, 32.5 ESTc F1 56.8

40 EST P3 86.0

EST VS1 38.0, 43.8 EST VS1-S1 36.0, 43.8

EST VS1-M2 40.0, 53.3, 56.3 60

80

100

d MDH N1 N/A N1 N3 N3c N3c EST F1 P3e VS1 VS1 VS1-S1 VS1-M2 f Source 1 2 1 1 1 1 Systemg 1 2 1 1 1 1

87

Figure 3-7. Continued aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. dNo phenotypes specified. eFargette (1987b) claims that esterase phenotype P3 is identical to esterase phenotype F1, with Rm differences attributed to electrophoresis apparatus variations (Esbenshade and Triantaphyllou, 1985c). f1 = Esbenshade and Triantaphyllou, 1985c; 2 = Fargette, 1987b. g1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively; 2 = Polyanalyst apparatus, 7% gel.

Rm (×100) 0 Band Phenotype Designation Key Enzyme Band Rma (×100)

b 20 MDH N1 23.0 MDH N1a 30.0 MDH N5 23.0, 25.5, 28.3, 30.0, 32.5 ESTc VS1-M2 40.0, 53.3, 56.3 40 EST S1-M1 43.8, 47.0 EST S2-M1 41.3, 43.8, 47.0 EST M3 47.0, 50.7, 55.7 60 EST A2 53.3, 56.3

80

100 MDH N1 N1a N1a N1 N5

EST VS1-M2 S1-M1 S2-M1 M3 A2 Sourced 1 1 1 1 1 Systeme 1 1 1 1 1 89

Figure 3-7. Continued aRelative electrophoretic migration. bMalate dehydrogenase. cEsterase. d1 = Esbenshade and Triantaphyllou, 1985c. e1 = Pharmacia GE-2/4 apparatus, separating and stacking gels 7% and 4%, respectively.

91

Table 3-1. Known Meloidogyne spp. malate dehydrogenase and esterase relative migrations that were used as references against those that were revealed electrophoretically from females collected from several counties in Florida.

Meloidogyne spp. Relative migration

M. incognita MDHa 21.4 ESTb 42.9 M. javanica MDH 22.7 EST 41.8, 49.0, 54.5 M. arenaria MDH 18.0, 20.0, 22.0 EST 38.0, 40.0 M. mayaguensis MDH 29.0, 32.0, 35.0 EST 31.0, 41.0 M. partityla MDH 31.0 EST 38.0, 49.0, 51.0 aMalate dehydrogenase bEsterase

92 same day of female extraction, while females extracted for processing in the Mini-

Protean 3 Cell apparatus were stored at -4 °C for up to six months without apparent effects on band resolution. One M. javanica female was included in every PhastSystem- run gel while two females were included in every Mini-Protean 3 Cell-run gel as controls.

The PhastSystem apparatus was set according to the PhastSystem user manual

(Pharmacia Biotech AB, 1995) as follows: first step – 400 volt (V) limit, 10.0 current

(mA) limit, 2.5 power (W) limit, bed temperature 15 °C, duration 10.0 volthours (Vh); second step – 400 V limit, 10.0 mA limit, 1.5 W limit, bed temperature 15 °C, duration

2.0 Vh; third step – 400 V limit, 10.0 mA limit, 1.5 W limit, bed temperature 15 °C, duration 100.0 Vh. Meloidogyne females were dissected from infected roots and placed in a BPI dish containing 0.85% sodium chloride (NaCl) at room temperature. Similarly, two M. javanica females were dissected from infected tomato cv. Rutgers roots and placed in 0.85% NaCl in a separate BPI dish. A piece of Parafilm (American National

Can, Chicago, IL) was pressed against the wells of an eight or 12-well sample applicator

(Pharmacia Biotech AB, Uppsala, Sweden) until the well impressions were clearly visible on the Parafilm. Five microliters of extraction buffer (20% w/v sucrose, 2% v/v Triton

X-100, 0.01% w/v bromophenol blue) were pipetted into each well and one Meloidogyne female was removed from the NaCl solution and placed in each well, for a total of 8- or

12-one Meloidogyne spp. samples, including the two M. javanica samples, depending on the sample applicator used (eight or 12 wells). A separate sterile plastic toothpick

(Armonds Manufacturing Company, Bogart, GA) was then used to homogenate each

Meloidogyne female. An 8- or 12-point applicator (Pharmacia Biotech AB, Uppsala,

Sweden), depending on the sample applicator used, was pressed on the wells to upload

93 the samples and immediately placed in the appropriate slot. The system was activated and stopped when the light emitting diode (LED) display read 100 Vh. Gels were removed from the apparatus and immediately washed with dionized water for one minute. Gels were then incubated in the dark in freshly prepared Mdh stain for 15 minutes in a 37 °C Isotemp incubator (Fisher Scientific, Hampton, NH), rinsed with dionized water for one minute, and then placed into freshly prepared Est stain for 30 to

45 minutes in the incubator at 37 °C in the dark (Table 3-2). Gels were rinsed again with dionized water for one minute and then incubated in 50 ml fixative solution (10% v/v glycerol, 10% v/v acetic acid) for at least 24 hrs at room temperature.

Sample preparation and enzyme separation and development using Mini-Protean

3 Cell was carried out as follows: Meloidogyne females were dissected from infected roots and separately placed in 0.6-ml eppendorf tubes (Fisher Scientific, Hampton, NH) containing 10 µl sample buffer (BioRad, Hercules, CA). The tubes, each containing one female, were kept on ice while Meloidogyne females were extracted, and then placed at -

4 °C until electrophoresis was conducted. Females were macerated and 10 µl of the supernatants were loaded into the appropriate wells of the polyacrylamide gel [4% stacking gel (pH 6.8), 8% separating gel (pH 8.8) with Tris-glycine buffer (BioRad,

Hercules, CA)]. Two-ten µl of Meloidogyne javanica homogenates were loaded onto each of two separate wells of the same gel as controls.

Electrophoresis was conducted in a 0.75-mm-thick, 8% running and 4% stacking gel, in a Mini-Protean 3 Cell (BioRad, Hercules, CA). Electrophoresis was performed in

5 °C at 80 V for the first 15 minutes, and then increased to 200 V until the bromophenol blue tracking dye migrated 53 cm from the bottom of the wells, approximately 33

94

Table 3-2. Enzyme stain concoctions used in staining malate dehydrogenase and esterase following electrophoresis using the PhastSystem.

Malate Dehydrogenase Stain Esterase Stain

Stock Solution Stock Solutions

The following were mixed and stored The following were mixed, adjusted to in the dark at room temperature: pH 7.4, and stored in the dark at room temperature: 50.0 ml 1.0 M Tris-HCl, pH 8.0 Sodium Phosphate Buffer 50.0 ml 0.3 M DL-Malic Acid, pH 7.5 9.28 g Na2HPO4, Monobasic, anhydrous 370.0 ml dionized water 3.21 g Na2HPO4, Dibasic, anhydrous Stain Solution 0.3 g EDTA The following were mixed in the dark with a magnetic stirrer for five minutes 400.0 ml dionized water immediately prior to gel staining: Approximately 20.0 ml 4 M NaOH (for 47.0 ml stock solution pH adjustment)

0.007 g β-Nicotinamide Adenine The following were stored at -4 °C: Dinucleotide (β-NAD) 50.0 ml 1 g α-Naphthyl Acetate in 50.0 0.5 ml 0.1 M Nitroblue Tetrazolium ml acetone (NBT) 0.015 g Fast Blue RR Salt 0.5 ml 0.1 M Phenazine Methosulfate (PMS) Stain Solution

The following were mixed in the dark for five minutes immediately prior to gel staining:

25.0 ml Sodium Phosphate Buffer

0.5 ml Naphthyl acetate in acetone

0.015 g Fast Blue RR Salt

95 minutes. Malate dehydrogenase and Est stain concoctions were freshly prepared (Table

3-3). Gels were incubated in the dark in either Mdh stain for approximately 15 minutes or Est stain for approximately 30 minutes or until bands appeared, or stained for both enzymes at the same time intervals, in a 37 °C Isotemp incubator. After incubation, gels were gently washed in deionized water and then placed in 50 ml freshly prepared fixative solution (20% v/v ethanol, 10% v/v glycerol) (BioRad, Hercules, CA) for at least 24 hours. Gels were preserved between two cellophane sheets in 14-cm x 14-cm gel drying frames (Sigma-Aldrich, St. Louise, MO).

Results

A total of 20 root samples were infected with root-knot nematodes. Six species of root-knot nematodes were identified, primarily by isozyme (Est and Mdh) analysis. The major root-knot nematode species identified and their percentage were M. incognita, M.

javanica, M. mayaguensis, M. arenaria, M. partityla, M. querciana, and Meloidogyne

spp. (Table 3-4). Meloidoygne querciana was identified from only one female.

Morphological and morphometric analysis shold be conducted to confirm this species

identification. Mixed populations were found on 40% of the samples. While Rm were

measured for Mini-Protean 3 Cell-run samples, such measurements could not be obtained

from PhastSystem-run samples since the bromophenol blue tracking dye did not persist

on the gels following incubation in the fixative solution. Meloidogyne incognita was

found infecting the following: Rosmarinus officinalis (rosemary) in , Syagrus romanzoffiana (queen palm) in Arecaceae, Pittosporum tobira in Pittosporaceae (Mdh

Rm 0.19, Est Rm 0.38), Brassica rapa (turnip) cv. Shogoin (Mdh Rm 0.21, Est Rm 0.43) and Brassica oleracea (kale) (Mdh Rm 0.21, Est Rm 0.43) in Brassicaceae, Phaseolus

96

Table 3-3. Enzyme stain concoctions used in staining malate dehydrogenase and esterase following electrophoresis using the Mini-Protean 3 Cell.

Malate Dehydrogenase Stain Esterase Stain

Stock Solution Stock Solutions

The following were mixed and stored The following were mixed, adjusted to in the dark at 5 °C: pH 6.0, and stored at room temperature:

1000.0 ml 0.05 M TRIS-HCl, pH 8.6 0.05 M Potassium Phosphate Buffer, pH 6.0

Stain Solution 720.0 ml of 50.0 ml Potassium Phospahte monobasic in 1.0 liter The following were mixed in the dark dionized water, adjusted to pH 6.0 with a magnetic stirrer for five minutes immediately prior to gel staining: 180.0 ml of 25.0 ml Potassium Phosphate dibasic in 500 ml dionized 50.0 ml 0.05 M TRIS-HCl, pH 8.6 water, adjusted to pH 6.0

0.013 g β-Nicotinamide Adenine Stain Solution Dinucleotide (β-NAD) The following were mixed in the dark 0.01 g Nitroblue Tetrazolium (NBT) for five minutes and filtered immediately prior to gel staining: 0.003 g Phenazine Methosulfate (PMS) 50.0 ml 0.05 M Potassium Phosphate buffer, pH 6.0

0.05 g Fast Blue RR Salt

The following were mixed together and added to the stain solution immediately prior to gel staining:

2.5 ml acetone

2.5 ml dionized water

0.05 g α-Naphthyl Acetate

Table 3-4. Plant species, family, county, esterase isozyme phenotype, number of samples, and Meloidogyne spp. identified from ornamental plants collected in Florida and processed using the PhastSystem and Mini-Protean 3 Cell.

Host species Family County Esterase isozyme phenotype Number of samples Meloidogyne spp.

Hibiscus rosa-sinensisa Malvaceae Alachua J3 1 M. javanica

Rosmarinus officinalisa Lamiaceae Suwannee J1 1 M. incognita

Callistemon viminalisa Myrtaceae Lee VS1-S1 1 M. mayaguensis

Syagrus romanzoffianaa Arecaceae Lee VS1-S1 1 M. mayaguensis I1 M. incognita Callistemon viminalisb Myrtaceae Lee VS1-S1 1 M. mayaguensis

Pittosporum tobirab Pittosporaceae Alachua I1 1 M. incognita

Ruscus aculeatusb Ruscaceae Volusia A2 1 M. arenaria

Buddleia davidiib Loganiaceae Pinellas N/A 1 Meloidogyne spp.

97

Table 3-4. Continued

Host species Family County Esterase isozyme Number of samples Meloidogyne spp. phenotype Brassica rapab Brassicaceae Alachua J3 1 M. javanica I1 M. incognita Pittosporum tobirab Pittosporaceae Lake N/A 1 Meloidogyne spp.

Ruscus aculeatusb Ruscaceae Volusia J3 1 M. javanica

Carya illinoesisb Juglandaceae Jefferson Ci3 1 M. partityla

Liriope muscarib Ruscaceae Hillsborough I1 1 M. incognita N/A Meloidogyne spp. Ophiopogon japonicus Ruscaceae Orange I1 1 M. incognita N/A Meloidogyne spp. Justicia carneab Acanthaceae Hillsborough N/A 1 Meloidogyne spp.

Solenostemon Lamiaceae Hillsborough VS1-S1 1 M. mayaguensis scutellarioidesb

98

Table 3-4. Continued

Host species Family County Esterase isozyme Number of samples Meloidogyne spp. phenotype Brassica oleraceab Brassicaceae Orange I1 1 M. incognita

Beta vulgarisb Chenopodiaceae Orange J3 1 M. javanica

Viburnum Caprifoliaceae Hillsborough J3 1 M. javanica b odoratissimum c F1 M. querciana Phaseolus vulgarisb Fabaceae Hillsborough I1 1 M. incognita A2 M. arenaria aProcessed with PhastSystem bProcessed with Mini-Protean 3 Cell cBased on MDH and EST phenotypes from one female 99

100 vulgaris (bean) (Mdh Rm 0.16, 0.20, 0.22, Est Rm 0.38) in Fabaceae, and Liriope muscari (lilyturf) cv. Evergreen Giant (Mdh Rm 0.24, Est Rm 0.27) and Ophiopogon japonicus (mondo grass) (Mdh Rm 0.22, Est Rm 0.42) in Ruscaceae. Meloidogyne javanica was found infecting the following: Hibiscus rosa-sinensis (hibiscus) cv. Pink

Versicolor in Malvaceae, B. rapa cv. Shogoin (Mdh Rm 0.21, Est Rm 0.42, 0.50, 0.54) in

Brassicaceae, Ruscus aculeatus (ruscus) (Mdh Rm 0.20, Est Rm 0.40, 0.48, 0.52) in

Ruscaceae, Beta vulgaris (chard) (Mdh Rm 0.23, Est Rm 0.42, 0.49, 0.55) in

Chenopodiaceae, and Viburnum odoratissimum (Viburnum) cv. Awabuki (Mdh Rm 0.17,

0.20, 0.23, Est Rm 0.37, 0.44, 0.48) in Caprifoliaceae. Meloidogyne arenaria was found infecting R. aculeatus (Mdh Rm 0.18, 0.20, 0.22, Est Rm 0.38, 0.40) and P. vulgaris

(Mdh Rm 0.17, 0.23, 0.27, Est Rm 0.44, 0.46). Meloidogyne mayaguensis was found infecting Callistemon viminalis (bottle brush) (Mdh Rm 0.29, 0.32, 0.35, Est Rm 0.31,

0.41) in Myrtaceae, S. romanzoffiana in Arecaceae, and Solenostemon scutellarioides

(coleus) cv. Elfers (Mdh Rm 0.30, 0.33, 0.37, Est Rm 0.32, 0.39) in Lamiaceae.

Meloidogyne partityla was found infecting Carya illinoensis (pecan) (Mdh Rm 0.31, Est

Rm 0.38, 0.49, 0.51) in Juglandaceae. The phenotype designation Ci3, named after the host’s species and number of bands, was assigned to the Est isozyme phenotype resolved from the Meloidogyne females that were isolated from C. illinoensis (Figure 3-8).

Meloidogyne querciana was found infecting V. odoratissimum cv. Awabuki (Mdh Rm

0.20, 0.24, 0.26, Est Rm 0.47) in Caprifoliaceae (Figure 3-9). Finally, several

Meloidogyne spp. whose Mdh and Est patterns could not be discerned were found infecting Buddleia davidii (butterfly bush) (Mdh Rm 0.19; 0.21; 0.19, 0.23, Est Rm 0.46;

0.43, 0.46; 0.25, 0.43, 0.46; 0.43, 0.46, 0.49; 0.25, 0.43, 0.46, 0.49) in Loganiaceae, P.

101

Rm

Mdh N1 N1a N1a N1a N1a N1

Rm

Est J3 Ci3 Ci3 Ci3 Ci3

Figure 3-8. Malate dehydrogenase (Mdh) and esterase (Est) isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne partityla females extracted from Carya illinoensis from Jefferson County on 31 March 2004.

102

Rm

Mdh N3 N3a Est J3 F1

Figure 3-9. Malate dehydrogenase (Mdh) and esterase (Est) isozyme phenotype, revealed using the mini-protean 3 cell apparatus, of a Meloidogyne querciana female extracted from Viburnum odoratissimum cv. Awabuki from Hillsborough County on 25 January 2005.

103 tobira cv. Variegata (Mdh Rm 0.23, Est Rm 0.47, 0.50; 0.21, 0.28; 0.28, 0.47, 0.50; 0.48,

0.51, 0.54, 0.56; 0.47) in Pittosporaceae, L. muscari cv. Evergreen Giant (Mdh Rm 0.24,

Est Rm 0.27, 0.48) and O. japonicus (Est Rm 0.28, 0.42, 0.45) in Ruscaceae, and Justicia carnea (flamingo plant) (Mdh Rm 0.29, 0.33, 0.37; 0.30; 0.29, 0.33, Est Rm 0.29, 0.37;

0.49, 0.52) in Acanthaceae (Table 3-4).

Discussion

Malate dehydrogenase and Est isozyme phenotypes, unveiled following PAGE, present a method for the speciation of Meloidogyne spp. that is less subjective than perineal patterns and other such morphologically- and morphometrically-based identification techniques. In most cases, Est isozyme phenotypes are species-specific.

Only four Meloidogyne spp. gels that clearly revealed Mdh and Est phenotypes were successfully run using the PhastSystem apparatus. Gels run after 17 May 2003 exhibited smeared Mdh bands whose Rm measurements could not be accurately measured.

Altering many parameters used in gel processing, extraction buffer, and electrophoretic apparatus settings on the PhastSystem and using freshly prepared reagent solutions whose lot numbers were confirmed as useful, many M. javanica females were run after 17 May

2003 in an attempt to resolve the problem surrounding the smeared Mdh bands, but none revealed clear Mdh phenotypes (Figure A-1). In addition, the bromophenol blue migrating line did not persist on PhastSystem-run gels after incubation in the fixative solution, making measurement of Rm impossible. Thus, it was decided to use the Mini-

Protean 3 Cell to carry out PAGE. Based on this work, it is concluded that, if isozymes are resolved well, the PhastSystem apparatus is useful for routine identification of M. incognita, M. javanica, and M. arenaria Mdh and Est isozyme phenotypes, based on their

104 migration in respect to M. javanica Mdh and Est phenotypes only. However, since Rm cannot be measured, identification of uncommon Meloidogyne spp. on the PhastSystem

apparatus is misleading and may result in incorrect speciation.

Mini-Protean 3 Cell-run gels in this work clearly reveal Mdh and Est isozyme

phenotypes after staining for these enzymes. Gels were stained separately for Mdh or

Est, or the enzymes were stained on the same gel. The clearly observed bromophenol

blue dye band and the relatively large gel size (60.5-mm x 91.0-mm) allowed for accurate

Rm measurements. Based on this work, it is concluded that the Mini-Protean 3 Cell

apparatus is useful for the speciation of Meloidogyne spp., based on unknown sample Rm

measurements and isozyme phenotype position, which can be compared with Rm

measurements and isozyme phenotypes of known Meloidogyne spp.

Meloidogyne spp. Mdh and Est isozyme phenotypes presented in this study were

referenced against perineal pattern-confirmed M. incognita, M. javanica, M. arenaria,

and M. mayaguensis, which were run electrophoretically using the same methodology as

the samples in questions (Table 3-1). In addition, cultured M. partityla females, provided

by A. P. Nyczepir, United States Department of Agriculture, ARS, Southeastern Fruit and

Tree Nut Research Laboratory, Byron, GA, on Carya illinoensis roots, were run

electrophoretically using the same methodology as all other samples, and their Mdh and

Est phenotypes and Rm measurements (Table 3-1) were used as a references against

isozyme phenotypes in question. Since human error and environmental conditions vary

slightly in the preparation, staining, and measuring of each gel, Rm values of

Meloidogyne spp. presented in table 3-4 do not consistently conform to those presented in

table 3-1. However, relative distances within isozyme phenotypes and band migrations

105 relative to that of M. javanica controls do conform to the referenced Meloidogyne spp.

presented in table 3-1, or are labeled as unidentified isozyme patterns.

Meloidogyne incognita and Meloidogyne spp. were identified from P. tobira and

P. tobira cv. Variegata, respectively. Meloidogyne incognita, M. arenaria, and

Meloidogyne spp. were previously reported on P. tobira (Bureau of Nematology, 1989;

Goodey and Franklin, 1956; Goodey et al., 1965; Nigh, 1972). In addition, Meloidogyne

spp. were isolated from P. tobira cv. Variegata in this study. However, growth room and

greenhouse studies conducted at the University of Florida (chapter 2) suggest that P.

tobira cv. Variegata is a nonhost to M. incognita race 2, M. javanica, M. arenaria race 1,

and M. mayaguensis. It is possible that P. tobira cv. Variegata is a nonhost to the

Meloidogyne spp. isolates tested in these studies. Further investigation on the

susceptibility of P. tobira cultivars to Meloidogyne spp. is required to alleviate this

conflict. In addition, M. incognita and Meloidogyne spp. were isolated from O.

japonicus. Meloidogyne incognita acrita was previously reported on this plant (Goodey

et al., 1965). Meloidogyne javanica and M. arenaria were isolated from R. aculeatus.

Meloidogyne spp. were also isolated from J. carnea. This is the first report of any

Meloidogyne spp. infecting these plants. Meloidogyne mayaguensis was identified from

C. viminalis and S. romanzoffiana roots. This is the first report of M. mayaguensis

infecting these plants, as well as the first report of M. mayaguensis in Lee and

Hillsborough Counties. Meloidogyne partityla was found infecting C. illinoensis in

Madison County in Florida. This is the first report of this root-knot nematode species in

Florida.

106

Uncommon Meloidogyne spp. may exhibit isozymes that may not be stable between or within populations, or possess phenotypic bands that stain at various intensities depending on the quantity of enzyme present. Therefore, until universal methodologies are accepted for the electrophoretic identification of Meloidogyne spp. via

Mdh and Est phenotypes revealed following electrophoresis, the identification of uncommon Meloidogyne spp. must not involve enzyme phenotype comparisons among works that utilize different methodologies. Isozyme band phenotypes and Rm measurements must only be compared and referenced to M. javanica or M. hapla controls that are run concurrently with the unknown samples, and to isozyme phenotypes revealed through identical methodologies as those used to unveil the unknown samples. All isozyme phenotypes published must include well-described gel formulation and enzyme stain concoction methodologies, accurately-measured gel sizes, and Rm measurements of isozyme phenotypes, revealed on gels processed through identical methodologies, to be compared regardless of gel size.

APPENDIX A PICTURES OF MELOIDOGYNE SPP. ESTERASE AND MALATE DEHYDROGENASE ISOZYME PHENOTYPES UNVEILED THROUGH POLYACRYLAMIDE GEL ELECTROPHORESIS ON PHASTSYSTEM AND MINI- PORTEIN 3 CELL APPARATUSES

Malate

dehydrogenase

Esterase

Figure A-1. PhastSystem gels exhibiting smeared malate dehydrogenase isozyme phenotypes whose relative migration could not be accurately measured.

107 108

PhastSystem-Run Gels

Malate

dehydrogenase

Esterase

Figure A-2. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Hibiscus rosa- sinensis cv. Pink Versicolor from Alachua County on 02 April 2003.

Figure A-3. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Rosmarinus officinalis from Suwannee County on 30 April 2003.

109

Figure A-4. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Callistemon viminalis from Lee County on 01 May 2003.

Figure A-5. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the PhastSystem apparatus, of Meloidogyne females extracted from Syagrus romanzoffiana from Lee County on 27 May 2003.

110

Mini-Protean 3 Cell-Run Gels

Figure A-6. Malate dehydrogenase isozyme phenotypes, revealed using the mini- protean 3 cell apparatus, of Meloidogyne females extracted from Callistemon viminalis from Lee County on 27 May 2003.

Figure A-7. Eterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Callistemon viminalis from Lee County on 27 May 2003.

111

Figure A-8. Malate dehydrogenase isozyme phenotypes, revealed using the mini- protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira from Alachua County on 18 December 2003.

Figure A-9. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira from Alachua County on 18 December 2003.

112

Figure A-10. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 19 December 2003.

Figure A-11. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 19 December 2003.

113

Figure A-12. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Buddleia davidii from Pinellas County on 30 September 2003.

Figure A-13. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Buddleia davidii from Pinellas County on 30 September 2003.

114

Figure A-14. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Brassica rapa cv. Shogoin from Alachua County on 06 January 2004.

Figure A-15. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Brassica rapa cv. Shogoin from Alachua County on 06 January 2004.

115

Figure A-16. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira cv. Variegata from Lake County on 11 February 2004.

Figure A-17. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Pittosporum tobira cv. Variegata from Lake County on 11 February 2004.

116

Figure A-18. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 22 February 2004.

Figure A-19. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ruscus aculeatus from Volusia County on 22 February 2004.

117

Figure A-20. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Liriope muscari cv. Evergreen Giant from Hillsborough County on 01 July 2004.

Figure A-21. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Liriope muscari cv. Evergreen Giant from Hillsborough County on 01 July 2004.

118

Figure A-22. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ophiopogon japonicus from Orange County on 16 August 2004.

Figure A-23. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Ophiopogon japonicus from Orange County on 16 August 2004.

119

Figure A-24. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Jesticia carnea from Hillsborough County on 20 August 2004.

Figure A-25. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Jesticia carnea from Hillsborough County on 20 August 2004.

120

Figure A-26. Malate dehydrogenase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Solenostemon scutellarioides cv. Elfers from Hillsborough County on 15 September 2004.

Figure A-27. Esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Solenostemon scutellarioides cv. Elfers from Hillsborough County on 15 September 2004.

121

Malate dehydrogenase

Esterase

Figure A-28. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Brassica oleracea from Orange County on 11 January 2004.

Figure A-29. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Beta vulgaris from Orange County on 11 January 2004.

122

Figure A-30. Malate dehydrogenase and esterase isozyme phenotypes, revealed using the mini-protean 3 cell apparatus, of Meloidogyne females extracted from Phaseolus vulgaris from Hillsborough County on 25 January 2005.

APPENDIX B COLLECTIVE RECORD OF THE HOST STATUS OF ORNAMENTAL PLANTS TO MELOIDOGYNE INCOGNITA, M. JAVANICA, M. ARENARIA, AND M. HAPLA

The following list (Table B-2) is a collective record of the host status of ornamental plants to Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla, as reported by several authors and gathered from this study. The host statuses of listed plants are reported herein as they are in the original works from which they are reported. Scientific names were verified and misspelled names were corrected as listed by the International

Plant Names Index (IPNI) (www.inpi.org). Referenced publications, referred to as

‘Source’ in table B-2, are cited in table B-1.

123 124

Table B-1. Sources and citations of publications referred to in Appendix B.

Source Citation Source Citation 1 Ahuja and Arora, 1980 29 McSorley and Marlatt, 1983 2 Bala and Hosein, 1996 30 McSorley, 1994 3 Barker and Benson, 1977 31 Mishra and Mishra, 1997 4 Barker et al., 1979 32 Mishra and Misra, 1993 5 Benson and Barker, 1982 33 Misra and Mishra, 1997 6 Bernard and Witte, 1987 34 Misra et al., 2002 7 Bernard et al., 1994 35 Montasser, 1995 8 Bureau of Nematology, 1989 36 Moreno et al., 1992 9 Caveness and Wilson, 1977 37 Motsinger et al., 1977 10 Cho et al., 1996 38 Nemec and Morrison, 1972 11 Coolen and Hendrickx, 1972 39 Nemec and Struble, 1968 12 Davis and Jenkins, 1960 40 Niblack and Bernard, 1985 13 Eisenback, 1987 41 Nigh, 1972 14 Giblin-Davis et al., 1992 42 Pant et al., 1983 15 Haasis et al., 1961 43 Rajendran et al., 1975 16 Haseeb and Pandey, 1987 44 Santamour and Riedel, 1993 17 Heald, 1967 45 Santamour and Riedel, 1995 18 Khan and Khan, 1989 46 Santamour, 1992 19 Khanna et al., 1998 47 Santo and Lear, 1976 20 Kirby, 1978 48 Sasser et al., 1966 21 LaMondia, 1995 49 Singh and Gupta, 1993 22 LaMondia, 1996 50 Singh and Majeed, 1991 23 LaMondia, 1997 51 Tarjan, 1952 24 Lehman, 1984b 52 Viglierchio, 1979 25 McSorley and Dunn, 1989 53 Walker and Melin, 1998b 26 McSorley and Dunn, 1990 54 Walker et al., 1994 27 McSorley and Frederick, 1994 55 Walker, 1980 28 McSorley and Frederick, 2001

Table B-2. Collective record of the host status of ornamental plants to Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla.

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Abelia × grandiflora --- Glossy abelia Sa 6 Acanthus spinosissimus --- Bears breeches S 21

Acer campestre --- Hedge maple S S S S 46

Acer davidii --- Maple S S S S S 46

Acer francheti --- Maple R R S R 46

Acer grandidentatum --- Bigtooth maple S R S R 46

Acer grosseri --- Grosser’s maple S S S S S 46

Acer macrophyllum --- Big leaf maple S S S S S 46

Acer mono --- Painted maple S S S S 46

Acer negundo --- Boxelder S S S S S 46

Acer palmatum --- Japanese maple R 6

Acer palmatum --- Japanese maple S S S S S 46 125

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Acer pentaphyllum --- Maple R R S R 46 Acer platanoides --- Norway maple SS SS R 46

Acer pseudoplatanus --- Sycamore maple RS SS S 46

Acer rubrum --- Red maple S 40

Acer rubrum --- Red maple SR SS R 46

Acer saccharinum --- Silver maple SS SS R 46

Acer saccharum --- Sugar maple R 6

Acer saccharum --- Sugar maple SR SS R 46

Acer tartaricum --- Tartarian maple SS SS S 46

Acer truncatum --- Shantung maple SS SS S 46

Acer velutinum --- Maple SS SS R 46

Achillea × Anthea Yarrow S S 53

Achillea × Coronation Gold Yarrow R 21 126

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Aconitum carmichaelii Arendsii Monkshood S 21 Aconitum lycoctonum --- Monkshood S 23 ssp. ranunculifolium

Acorus calamus --- Calamus S 16

Acroclinium roseum --- Paper flower R 1

Acroclinium roseum --- Paper flower S 42

Adenophora confusa --- Ladybells S 22

Aesculus flava --- Yellow buckeye SS SS 44

Aethionema cordifolium --- Stone cress R R 53

Agapanthus umbellatus --- Agapanthus R 35

Ageratum conyzoides --- Tropical whiteweed S 50

Ageratum houstonianum --- Ageratum S 1

Ageratum houstonianum --- Ageratum S 50 127

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Ageratum houstonianum Blue Blazer Ageratum R 54 Ageratum houstonianum Blue Danube Ageratum R 54

Ageratum houstonianum Blue Mink Ageratum R R R 27

Ageratum houstonianum Hawaii White Ageratum R 54

Ageratum houstonianum Royal Delft Ageratum R 54

Aglaonema commutatum Treubii Philippine evergreen S 33

Ailanthus altissima --- Tree of heaven R R R R R 44

Ajuga reptans --- Common bugle S S S S S 26

Ajuga reptans Bronze Beauty Bugleweed S 23

Ajuga reptans Burgundy Glow Bugleweed S 21

Alcea rosea --- Hollyhock S 50

Alcea rosea Chater's Doubles Hollyhock SL 21

Alchemilla mollis Improved Form Lady's mantle SL 21 128

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Aloe perryi --- Perry's aloe S S 16 Aloe vera --- Aloe vera S S 16

Aloe vera --- Aloe vera S 33

Aloe vera --- Aloe vera S 2

Alpinia galanga --- Greater galangal S 16

Amaryllis vittata --- Amaryllis R 35

Amberboa moschata --- Sweet sultan S 1

Anchusa azurea Dropmore Alkanet S 22

Andrographis paniculata --- Andrographis S 16

Anemone × Queen Charlotte Windflower S 22

Anemone coronaria --- Cut-leaf anemone S 35

Anemone hupehensis var. --- Japanese thimbleweed S 23 japonica

129

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Antirrhinum majus --- Antirrhinum S 1 Antirrhinum majus --- Snapdragon S 42

Antirrhinum majus --- Snapdragon S 50

Antirrhinum majus First Ladies Snapdragon S S S 27

Antirrhinum majus Margaret Snapdragon S S S S 51

Aquilegia caerulea Blue Star Columbine S 21

Arabis caucasica Compinkie Rock cress SL 21

Arctotis stoechadifolia --- African daisy S 1

Arctotis stoechadifolia --- African daisy S 50

Arctotis venusta --- Blue eyed African daisy S 50

Areca catechu --- Betel palm R R R R R 25

Argyreia nervosa --- Elephant creeper S 16

Artemisia schmidtiana Silver Mound Silver mound S 21 130

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Asclepias curassavica --- Blood flower S S 16 Asclepias tuberosa --- Butterfly weed R 22

Asparagus densiflorus --- Sprenger's asparagus fern S 33

Aspidistra elatior --- Cast iron plant S 33

Aster novae-angliae Harrington's Pink New England aster R 21

Aster novae-angliae September Ruby New England aster R 21

Aster novi-belgii Mt. Everest New York aster R 23

Astilbe × arendsii Peach Blossom Feather flower S 21

Astrantia major Rose Symphony Great masterwort S 22

Asystasia gangetica --- Chinese violet S 43

Aucuba japonica Variegata Acuba S 4

Balsamita major --- Costmary R 50

Barleria prionitis --- Porcupine flower R 43 131

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Barringtonia acutangula --- Freshwater mangrove S S 16 Basella alba --- Ceylon spinach S 33

Begonia × Cocktail Gin Begonia S 54

Begonia × Cocktail Vodka Begonia SL 54

Begonia × Encore White Bronze Begonia S 54

Begonia × Party Love Begonia S 54

Begonia × Pizzazz Deep Rose Begonia S 54

Belamcanda chinensis --- Blackberry lily R 21

Bellis perennis --- Lawn daisy S 1

Bellis perennis --- Lawn daisy S 42

Bellis perennis --- Lawn daisy S 50

Betula nigra Heritage River birch S 44

132

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Betula platyphylla var. Japanese white birch S S S S 44 japonica Betula populifolia --- Gray birch SS SS 44

Boltonia asteroides Pink Beauty White doll’s daisy S 22

Borago officinalis --- Common borage S S S S 36

Brassica oleracea var. Kale S 1 acephala Buxus harlandii --- Japanese boxwood SL 6

Buxus microphylla --- Littleleaf boxwood S S 56

Buxus microphylla Japonica Littleleaf boxwood S 5

Buxus sempervirens --- Common boxwood SL 6

Calathea zebrina --- Zebra plant S 33

Calendula officinalis --- Pot marigold S 1

Calendula officinalis --- Pot marigold S 42 133

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Calendula officinalis --- Pot marigold S 50 Calendula persica --- Calendula S 50

Callistemon citrinus --- Crimson bottlebrush S 16

Callistemon lanceolatus --- Crimson bottlebrush S S 16

Callistephus chinensis --- China aster S 1

Callistephus chinensis --- China aster S 50

Camellia japonica --- Common camellia S S 15

Camellia sasanqua --- Sasanqua camellia S S 15

Camellia sasanqua --- Sasanque camellia S 39

Campanula --- Bellflower S 21 poscharskyana Canna indica --- Indian shot R 35

Catharanthus roseus Blush Cooler Madagascar periwinkle S S 28 134

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Catharanthus roseus Carpet Dawn Madagascar periwinkle SL 54 Catharanthus roseus Grape Cooler Madagascar periwinkle SL R 28

Catharanthus roseus Little Blanche Madagascar periwinkle SL 54

Catharanthus roseus Little Bright Eyes Madagascar periwinkle S S 28

Catharanthus roseus Little Delicata Madagascar periwinkle SL 54

Catharanthus roseus Little Mixed Colors Madagascar periwinkle S S 28

Catharanthus roseus Peppermint Cooler Madagascar periwinkle S S 28

Catharanthus roseus Polka Dot Madagascar periwinkle SL 54

Cedrus deodara --- Deodar cedar R 39

Celastrus dependens --- Magzsudhi S 16

Celosia argentea --- Silver cock's comb S 42

Celosia argentea Accession no. 8 Silver cock's comb S S 9

Celosia argentea Century Mix Silver cock's comb S S S 27 135

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Celosia cristata --- Crested cock’s comb S 50 Celosia plumosa Apricot Brandy Celosia S 54

Celosia plumosa Castle Scarlet Celosia S 54

Celosia plumosa Fireglow Celosia S 54

Celosia plumosa Kimona Cream Celosia S 54

Celtis occidentalis --- Common hackberry SS SS R 44

Centaurea cyanus --- Corn flower S 50

Centaurea cyanus --- Cornflower S 1

Centranthus ruber Albus Jupiter’s beard S 22

Cercis Canadensis --- Eastern redbud tree S S S S S 45

Cercis canadensis var. --- Texas redbud SS SS S 45 texensis Cercis chinensis --- Chinese redbud SS SS S 45

136

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Cercis racemosa --- Chain flowered redbud SS SS S 45 Cercis siliquastrum --- Judas tree SS SS S 45

Cercis yunnanensis --- Yunnan redbud SS SS S 45

Chamaecyparis pisifera --- Sawara cypress R 39

Cheiranthus cheiri --- Aegean wallflower S 1

Cheiranthus cheiri --- Aegean wallflower S 50

Chelone obliqua --- Red turtlehead R 21

Chlorophytum comosum --- Spider plant S 33

Chrysanthemum --- Tricolor daisy R 42 carinatum Chrysanthemum --- Tricolor daisy R 50 carinatum Chrysanthemum Giant Hybrids Pyrethum daisy S 21 coccineum

137

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Chrysanthemum --- Crowndaisy R 1 coronarium Cimicifuga acerina --- Fairy candles S 21

Cimicifuga dahurica --- Asian bugbane S 21

Cimicifuga simplex White Pearl Single-stem bugbane S 21

Cissus quadrangularis --- Veld grape S 16

Clarkia unguiculata --- Elegant fairyfan S 1

Clarkia unguiculata --- Elegant fairyfan S 50

Clematis × Hagley Hybrid Clematis S 21

Clivia miniata --- Kaffir lily S 33

Clivia miniata --- Kaffir lily R 35

Codiaeum variegatum Gold Dust Croton R R R Chapter 2 --- Ti plant S 33 138

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Coreopsis stillmanii --- Stillman’s tickseed R 1 Coreopsis tinctoria --- Golden tickseed R 50

Coreopsis verticillata Moonbeam Tickseed S 21

Cornus florida --- Eastern flowering dogwood S 6

Cornus florida --- Eastern flowering dogwood S 40

Cosmos bipinnatus --- Garden cosmos R 1

Cosmos bipinnatus --- Garden cosmos R 50

Cosmos sulphunus --- Sulphur cosmos SL 42

Costus speciosus --- Wild ginger S S 16

Cotoneaster horizontalis --- Rockspray cotoneaster SL 39

Crataeva nurvala --- Three-leaf caper S 16

Crinum longifolium --- Hardy swamplily R 35

Crossandra undulifolia Orange Firecracker flower S 43 139

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Crossandra undulifolia Red Firecracker flower S 43 Croton sparsiflorus --- Croton S 42

Cucumis melo ssp. Flexuosus Snakemelon S S 57 melo Dahlia pinnata --- Pinnate dahlia S 42

Dahlia variabilis --- Dahlia S 1

Dahlia variabilis --- Dahlia R 35

Dahlia variabilis --- Dahlia S 50

Delphinium ajacis --- Larkspur S 1

Delphinium ajacis --- Larkspur S 50

Delphinium Blue Mirror Siberian larkspur S 21 grandiflorum Dendranthema indicum --- Chrysanthemum R 50

140

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Dendranthema --- Florist’s daisy R 1 morifolium Dianella caerulea --- Cerulean flaxlily S 33

Dianthus barbatus --- Sweet william R 1

Dianthus barbatus --- Sweet william S 50

Dianthus barbatus Indian Carpet Sweet william R 21

Dianthus caryophyllus --- Carnation S 1

Dianthus caryophyllus --- Carnation R 50

Dianthus caryophyllus Antalia Carnation SL 10

Dianthus caryophyllus Astra Carnation S 10

Dianthus caryophyllus Beta Carnation S 10

Dianthus caryophyllus Carmit Carnation SL 10

Dianthus caryophyllus Castelaro Carnation SL 10

141

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Dianthus caryophyllus Darling Carnation S 10 Dianthus caryophyllus Desio Carnation SL 10

Dianthus caryophyllus Echo Carnation SL 10

Dianthus caryophyllus Elegance Korea Carnation S 10

Dianthus caryophyllus Espana Carnation SL 10

Dianthus caryophyllus Galil Carnation S 10

Dianthus caryophyllus Imperial White Sim Carnation S 10

Dianthus caryophyllus Izu Pink Carnation SL 10

Dianthus caryophyllus Kappa Carnation R 10

Dianthus caryophyllus Lena Carnation S 10

Dianthus caryophyllus Mars Carnation S 10

Dianthus caryophyllus Mercury Carnation SL 10

Dianthus caryophyllus Rachel Carnation SL 10 142

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Dianthus caryophyllus Rara Carnation SL 10 Dianthus caryophyllus Red Corso Carnation SL 10

Dianthus caryophyllus Red Lena Carnation S 10

Dianthus caryophyllus Roland Carnation S 10

Dianthus caryophyllus Rony Carnation SL 10

Dianthus caryophyllus Sarinah Carnation S 10

Dianthus caryophyllus Saturn Carnation SL 10

Dianthus caryophyllus Saturnus Carnation SL 10

Dianthus caryophyllus Scarlet Elegance Carnation S 10

Dianthus caryophyllus Shinkibo Carnation S 10

Dianthus caryophyllus Target Carnation SL 10

Dianthus caryophyllus Tasman Carnation S 10

Dianthus caryophyllus Virgo Carnation S 10 143

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Dianthus caryophyllus White Royalitee Carnation SL 10 Dianthus caryophyllus Yellow Dusty Carnation S 10

Dianthus chinensis --- Rainbow pink S 42

Dianthus chinensis --- Rainbow pink R 50

Dianthus chinensis Baby Doll Mix Rainbow pink S SL S 30

Dianthus chinensis Princess Scarlet Rainbow pink S 54

Dicentra spectabilis Alba White bleeding heart S 21

Dieffenbachia amoena Tropic Snow Dumb cane S 33

Digitalis ambigua --- Yellow foxglove SL 21

Digitalis lanata --- Grecian foxglove S S 16

Digitalis purpurea --- Common foxglove S 16

Digitalis purpurea Excelsior Common foxglove SL 21

Doronicum orientale Magnificum Oriental false leopardbane S 21 144

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Dracaena marginata --- Dragontree SL SL S SL R 26 Echinacea purpurea --- Purple coneflower R R 53

Echinacea purpurea Leuchtstern Purple coneflower R 22

Echinops bannaticus Taplow Blue Globe thistle SL 22

Elaeagnus pungens --- Elaeagnus R 39

Elytraria acaulis --- Scalystem S 16

Epimedium versicolor Sulphureum Yellow barrenwort R 22

Eriobotrya japonica --- Loquat S S 41

Eschscholtzia californica --- California poppy S 50

Eschscholtzia californica --- Californian poppy S 1

Euonymus alatus Compacta Winged spindletree R 6

Euonymus japonicus --- Japanese spindletree S 41

Euphorbia thymifolia --- Gulf sandmat S 16 145

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Evolulus glomeratus Blue Daze Evolvulus S 8 Fagus grandifolia --- American beech R R S S R 44

Ficus benjamina --- Weeping fig S S S S R 26

Ficus benjamina --- Weeping fig S 33

Ficus elastica Decora Indian rubber tree S 33

Filipendula rubra Venusta Magnifica Queen of the prairie SL 21

Flacourtia indica --- Governor’s plum S 16

Fragaria × ananassa Pink Panda Hybrid strawberry R R 53

Freesia × --- Freesia S 35

Gaillardia × grandiflora Goblin Blanket flower R 21

Gaillardia pulchella Picta Blanket flower R 42

Gardenia jasminoides --- Gardenia S S 12

Gardenia jasminoides --- Gardenia S S S 24 146

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Gardenia jasminoides Radicans Gardenia SL 5 Gentiana sp. Benichidori Gentian S 22

Geranium × Ann Folkard Cranesbill S S 53

Geranium × magnificum --- Cranesbill S 22

Geranium × oxonianum Thurstonianum Cranesbill S 22

Geranium cinereum Laurence Flatman Cranesbill S S 53

Geranium dalmaticum --- Dalmatian cranesbill S 21

Geranium endressii Wargrave Pink Cranesbill S 23

Gerbera jamesonii Nain-Crimson Barberton daisy SL 54

Ginkgo biloba --- Maidenhair tree SS SS S 44

Gladiolus × Oscar Gladiola S 19

Gladiolus × Pink Friendship Gladiola S 19

Gladiolus × Snow Princess Gladiola S 19 147

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Gladiolus × Sylvia Gladiola S 19 Gladiolus × Vinks Glory Gladiola S 19

Gladiolus × White Friendship Gladiola S 19

Gladiolus × gandavensis --- Gladiola S 35

Gladiolus spp. --- Gladiola S 33

Gleditsia triacanthos --- Threespined honeyocust R R R R R 44

Gymnema sylvestre --- Miracle fruit S 16

Gypsophila elegans Covent Garden Showy baby’s breath S 30

Hedera helix --- English ivy SL 39

Hedychium coronarium --- White gingerlily SL 35

Helenium autumnale Brilliant Common sneezeweed R 21

Helianthus annuus --- Common sunflower S 50

Helichrysum bracteatum --- Bracted strawflower S 50 148

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Helictotrichon --- Helictotrichon S 22 sempervirens Heliopsis helianthoides Karat Sunflower heliopsis S 21

Hemerocallis × Bright Banner Daylily SL 22

Hemerocallis aurantiaca --- Hardy daylily SL 35

Hemerocallis fulva Marion Vaughn Common daylily S 33

Heuchera cylindrica Green Ivory Roundleaf alum root S S 53

× Heucherella alba Bridget Bloom Foamy bells S S 53

Hibiscus rosa-sinensis --- Hibiscus S S 41

Hibiscus rosa-sinensis Anderson Crepe Hibiscus SL SL 29

Hibiscus rosa-sinensis Delight Hibiscus SL R 29

Hibiscus rosa-sinensis Fancy Lady Hibiscus S SL 29

Hibiscus rosa-sinensis Florida Sunset Hibiscus SL R 29 149

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Hibiscus rosa-sinensis Go-Go Girls Hibiscus SL SL 29 Hibiscus rosa-sinensis Kona Hibiscus S 29

Hibiscus rosa-sinensis Minerva Hibiscus S SL 29

Hibiscus rosa-sinensis Old Gold Hibiscus SL SL 29

Hibiscus rosa-sinensis Painted Lady Hibiscus S SL 29

Hibiscus rosa-sinensis Painted Lady Hibiscus S S 29

Hibiscus rosa-sinensis Philipino Hibiscus S SL 29

Hibiscus rosa-sinensis President Hibiscus SL SL 29

Hibiscus rosa-sinensis President Hibiscus SL SL 29

Hibiscus rosa-sinensis Pride of Hankins Hibiscus S S 29

Hibiscus rosa-sinensis Rowena Wedding Hibiscus S SL 29

Hibiscus rosa-sinensis Versicolor Pink Hibiscus S Chapter 3

150

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Hyacinthus orientalis --- Garden hyacinth R 35 Hydrangea paniculata Grandiflora Old fashioned snowball S 6

Hygrophila auriculata --- Hygrophila S S 16

Hypericum polyphyllum --- Saint John's wort S 21

Iberis amara --- Iberis S 42

Ilex × Calina Holly S S 7

Ilex × Little Red Holly S S 56

Ilex × attenuata Foster No. 2 Attenuate holly R 6

Ilex × attenuata Foster No. 2 Attenuate holly S S 7

Ilex × meserveae Blue Boy Blue holly S S 7

Ilex × meserveae Blue Girl Blue holly S S 7

Ilex cassine Lowei Dahoon holly S S 7

151

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Ilex cassine var. --- Dahoon holly S 39 angustifolia Ilex cornuta Burfordii Chinese holly R R R R R 26

Ilex cornuta Burfordii Chinese holly S 39

Ilex cornuta Burfordii Chinese holly S S R R 48

Ilex cornuta Burfordii Holly R 4

Ilex cornuta Carissa Chinese holly R SL 56

Ilex cornuta Needlepoint Chinese holly S S 7

Ilex cornuta Rotunda Chinese holly S 4

Ilex cornuta × I. Nellie R. Stevens Holly S S 7 aquifolium Ilex cornuta × I. Nellie R. Stevens Holly R 6 aquifolium Ilex cornuta × I. pernyi Lydia Morris Holly S S 7

152

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Ilex crenata Bennett’s Compacta Japanese holly S S 7 Ilex crenata Compacta Japanese holly S 5

Ilex crenata Compacta Japanese holly S S 56

Ilex crenata Convexa Japanese holly S 3

Ilex crenata Convexa Japanese holly S S 7

Ilex crenata Convexa Japanese holly SS SR 48

Ilex crenata Green Luster Japanese holly S S 56

Ilex crenata Green Lustre Japanese holly S S 7

Ilex crenata Helleri Japanese holly S 3

Ilex crenata Helleri Japanese holly SS SS 17

Ilex crenata Helleri Japanese holly SS SR 48

Ilex crenata Helleri Japanese holly S S 56

Ilex crenata Hetz Japanese holly S 39 153

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Ilex crenata Hetzii Japanese holly R 6 Ilex crenata Latifolia Japanese holly S S S R 48

Ilex crenata Rotundifolia Japanese holly S 3

Ilex crenata Rotundifolia Japanese holly S S 7

Ilex glabra Compacta Inkberry S S 7

Ilex glabra Nordic Inkberry S S 7

Ilex glabra Shamrock Inkberry R R 56

Ilex integra × I. pernyi Elegance Holly S S 7

Ilex opaca Jersey Princess American holly S 7

Ilex vomitoria Nana Yaupon holly S 4

Ilex vomitoria Schelling's Dwarf Yaupon holly R R 56

Ilex vomitoria Stokes Dwarf Yaupon holly S S 7

Impatiens balsamina --- Spotted snapweed S 1 154

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Impatiens balsamina --- Spotted snapweed S 42 Impatiens balsamina --- Spotted snapweed S 50

Impatiens holstii Aztec Impatiens S 55

Impatiens holstii Chickasaw Impatiens S 55

Impatiens holstii Chippewa Impatiens S 55

Impatiens holstii Creek Impatiens S 55

Impatiens holstii Fuchsia Impatiens S 55

Impatiens holstii Futura Red Impatiens S 55

Impatiens holstii Futura White Impatiens S 55

Impatiens holstii Garden Blue Impatiens S 55

Impatiens holstii Hopi Impatiens S 55

Impatiens holstii Maya Impatiens S 55

Impatiens holstii Navajo Impatiens S 55 155

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Impatiens holstii PI 354261 Impatiens S 55 Impatiens holstii PI 354264 Impatiens S 55

Impatiens holstii PI 354265 Impatiens S 55

Impatiens holstii Scarlet Impatiens S 55

Impatiens holstii Scarlet Baby Impatiens S 55

Impatiens holstii Series F1 (Pink) Impatiens S 55

Impatiens holstii Series F1 (Rose) Impatiens S 55

Impatiens holstii Series F1 (Salmon) Impatiens S 55

Impatiens holstii Shawnee Impatiens S 55

Impatiens holstii Twinkles Impatiens S 55

Iris germanica Afternoon Delight German iris S 21

Iris pumila Elfin Queen Dwarf iris SL 21

Iris sibirica Maranantha Siberian iris R 21 156

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Iris tingitana --- Morocco iris S 35 Ixora arborea --- Jungleflame S 16

Ixora casei Super King Ixora R R 14

Ixora coccinea --- Scarlet jungleflame S S 14

Ixora coccinea Maui Scarlet jungleflame S S 14

Ixora coccinea Nora Grant Scarlet jungleflame R R 14

Ixora coccinea Petite Red Scarlet jungleflame S S 14

Ixora coccinea Petite Yellow Scarlet jungleflame SL S 14

Ixora coccinea Singapore Scarlet jungleflame SL S 14

Ixora coccinea × I. Bonnie Lynn Ixora S S 14 Chinensis Jasminum humile --- Italian yellow jasmine S 16

Jasminum nudiflorum --- Winter jasmine S 39

157

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Juglans nigra --- Black walnut R R R R R 44 Juniperus chinensis Hetzii Glauca Blue hetzi juniper R 6

Juniperus conferta Blue Pacific Blue pacific shore juniper R 6

Juniperus excelsa Stricta Greek juniper SL 5

Juniperus horizontalis --- Creeping juniper R 5

Juniperus horizontalis Douglasii Creeping juniper SL 39

Juniperus horizontalis Plumosa Creeping juniper R R 6

Juniperus horizontalis Plumosa Creeping juniper R R 37

Juniperus horizontalis Plumosa Creeping juniper R 39

Justicia betonica --- Squirrel’s tail S 43

Kalanchoe blossfeldiana --- Madgascar widow’s thrill S 33

Kalanchoe fedtschenkoi --- South American air plant S 34

Kochia trichophylla --- Kochia R 50 158

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Koelreuteria paniculata --- Golden rain tree S S S S S 44 Lantana camara Miss Huff South American lantana R R 56

Lantana camara New Gold South American lantana SL R 56

Lathyrus latifolis --- Perennial seet pea S 21

Lathyrus odoratus --- Sweet pea S 1

Lathyrus odoratus --- Sweet pea S 50

Lavandula angustifolia Munstead Dwarf English lavender S 21

Lavendula spica --- Lavender S S S S 36

Leucanthemum × Exhibition Shasta daisy R 21 superbum Leucanthemum × Polaris Shasta daisy S 21 superbum Leucanthemum maximum Alaska Shasta daisy S 30 159

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Liatris scariosa White Spires Devil’s bite R 21 Ligularia dentata Desdemona Strain Leopard plant S 21

Ligustrum sinense Variegatum Chinese privet S 6

Lilium longiflorum --- Easter lily R 35

Linaria bipartita --- Clovenlip toadflax R 1

Linaria bipartita --- Clovenlip toadflax R 50

Linaria cymbalaria --- Toadflax S S 53

Linum grandiflorum --- Flowering flax R 1

Linum grandiflorum Cocineum Flowering flax R 50

Liquidambar styraciflua --- Sweetgum S R R S R 44

Liriope muscari --- Lilyturf S 33

Liriope muscari Evergreen Giant Liliturf S Chapter 3

160

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Liriope muscari Evergreen Giant Liliturf S S SL Chapter 2 Liriope muscari Variegata Lilyturf R 22

Lithospermum diffusum Grace Ward Stoneseed R 22

Lobelia × gerardi Vedrariensis Lobelia R 23

Lobelia cardinalis Complement Scarlet Cardinal flower S 21

Lobelia erinus --- Edging lobelia S 1

Lobelia erinus --- Edging lobelia S 50

Lobularia maritima --- Sweet alyssum S 1

Lobularia maritima --- Sweet alyssum S 50

Lobularia maritima Rosie O'Day Sweet alyssum R R 27

Loropetalum chinense --- Razzle bush S 39

Lupinus hartwegii --- Hartweg’s bluebonnet S 1 161

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Lupinus polyphyllus Russel Hybrids Bigleaf lupin S 21 Lysimachia clethroides --- Gooseneck yellow S 22 loosestrife Lythrum sp. Morden's Pink Loosestrife S 21

Maclura pomifera --- Osageorange RR RR R 44

Magnolia × soulangiana Alexandrina Saucer magnolia R 6

Magnolia grandiflora --- Southern magnolia RR RR R 44

Malva alcea Fastigiata Vervain mallow S 22

Malva moschata Alba Musk mallow SL 21

Marguerita sp. ------S 42

Matthiola incana --- Tensweeks stock R 42

Matthiola incana --- Tenweeks stock S 50

Melia azedarach --- Chinaberrytree S S 41

162

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Melissa officinalis --- Common balm S 16 Mesembranthemum --- Ice plant S 50 tricolor Mesembranthemum --- Ice plant S 1 tricolor Metasequoia --- Dawn redwood R 6 glyptostroboides Miscanthus Silberfeder Silver feather S 22 sinensis Moluccella laevis --- Bells of Ireland R 1

Monarda citriodora --- Lemon bee balm R R 53 Monarda didyma Cambridge Scarlet Scarlet bee balm R 21

Monarda didyma Cambridge Scarlet Scarlet bee balm R R 53

Morus alba --- White mulberry S S 41

Murraya koenigii --- Curry leaf tree S 33 163

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Musa sumatrana ssp. Rowe Red Ornamental banana S S S Chapter zebrina 2 Myosotis alpestris Indigo Blue Forget me not R 22

Nandina domestica --- Heavenly bamboo R 5

Nandina domestica --- Heavenly bamboo S 6

Narcissus tazetta --- Cream narcissus R 35

Nasturtium majus --- Nasturtium S 50

Nepeta nervosa --- Clear blue catmint S S 53

Nicotiana --- Tex-mex tobacco S 16 plumbaginifolia Nierembergia --- Dwarf cupflower S S 53 hippomanica Nigella damascena --- Devil in the bush S 1

Nigella damascena --- Devil in the bush S 50 164

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Ocimum basilicum --- Sweet basil S S S S 36 Ocimum canum --- Hoary basil S S 16

Ocimum gratissimum --- African basil S S 16

Ocimum --- Hoary basil S S 16 kilimandscharicum Odontonema cuspidatum --- Firespike R R R Chapter 2 Operculina turpethum --- St. Thomas lidpot S 16

Ophiopogon japonicus --- Mondo grass S Chapter 3 Origanum majorana --- Sweet marjoram S R R R 36

Origanum onites --- Pot marjoram S S S S 36

Origanum vulgare --- Oregano S R S S 36

Osmanthus × fortunei --- Fortune’s osmanthus S 39

165

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Pachysandra --- Montain spurge R 21 procumbens Pachysandra terminalis --- Japanese spurge S 21

Paederia foetida --- Stinkvine S S 16

Papaver dubium --- Blindeyes S 50

Papaver orientale Carousel Oriental poppy R 21

Papaver orientale Oriental Red Poppy S 30 Perennial Papaver rhoeas --- Corn poppy S 1

Papaver rhoeas --- Corn poppy S 50

Passiflora edulis var. --- Yellow passion fruit R R R 20 flavicarpa Patrinia scabiosaefolia --- Patrinia SL R 53

Penstemon × Purple Passion Talus slope penstemon S S 53

166

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Penstemon × Ruby Talus slope penstemon S S 53 Penstemon × Sour Grapes Talus slope penstemon S S 53

Penstemon digitalis Husker Red Talus slope penstemon S 22

Peperomia --- Peperomia S 33 magnoliaefolia Perovskia atriplicifolia --- Russian sage S 22

Petunia × hybrida --- Garden petunia S 1

Petunia × hybrida --- Garden petunia S 18 Petunia × hybrida --- Garden petunia S 50

Petunia × hybrida Dwarf Bedding Garden petunia S S S 27

Petunia × hybrida Fire Chief Garden petunia S SL S 30

Petunia violacea --- Petunia SL 42

Philodendron laciniatum --- Philodendron S 33

167

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Philodendron selloum --- Philodendron S 32 Philodendron selloum --- Philodendron S 33

Phlox drummondii --- Annual phlox R 1

Phlox drummondii --- Annual phlox R 50

Phlox paniculata Eva Cullum Garden phlox R R 53

Phlox paniculata Fairest One Garden phlox R 21

Phlox paniculata Franz Shubert Garden phlox R R 53

Phlox paniculata Oakington Blue Garden phlox R R 53

Phlox stolonifera Bruce's White Creeping phlox R 21

Photinia × fraseri --- Copper tip photinia S 6

Photinia × fraseri --- Copper tip photinia SL SL S SL 26

Physostegia virginiana Summer Snow Obedient plant SL 22

Pinus ponderosa --- Ponderosa pine R R R 52 168

Table B-1. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Pinus strobus --- Eastern white pine R 6 Pinus virginiana --- Virginia pine R 6

Pisum sativum --- Garden pea S 50

Pittosporum tobira --- Pittosporum S Chapter 3 Pittosporum tobira --- Pittosporum S 8

Pittosporum tobira --- Pittosporum S 41

Pittosporum tobira Variegata Variegated pittosporum R R R Chapter 2 Polemonium reptans Firmament Greek valerian S 21

Polianthes tuberosa --- Tuberose S 35

Polianthes tuberosa Double Tuberose S 33

Polianthes tuberosa Single Tuberose S 33

Polygonum affine Dimity Polygonum R R 53 169

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Poncirus trifoliatus --- Hardy orange S 39 Portulaca grandiflora --- Rose moss R 50

Potentilla nepalensis Miss Wilmott Red potentilla S 21

Primula japonica Red Field Hybrids Primrose R 23

Primula polyantha Crescendo Mix Primrose R 21

Prunus × cistena --- Cherry R 6

Prunus × yedoensis --- Tokyo cherry R 6

Prunus avium --- Sweet cherry S S S S S 44

Prunus cerasifera --- Cherry plum S S S S S 44

Prunus cerasifera Atropurpurea Cherry plum R 6

Prunus glandulosa --- Flowering almond R 6

Prunus laurocerasus var. --- Cherry laurel SL 39 zabeliana

170

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Prunus mahaleb --- Mahaleb cherry S S S S S 44 Prunus serrulata Kwanzan Japanese flowering cherry R 6

Pyrus calleryana --- Callery pear R R R R R 44

Rhododendron × Cannon's Double Azalea R 6

Rhododendron × Girard's Rose Azalea R 6

Rhododendron Boursalt Catawba rosebay R 6 catawbiense Rhoeo discolor --- Boat lily S 33

Robinia pseudoacacia --- Black locust S S S S S 44

Rosa × Dr. Huey Rose S 47

Rosa canina --- Dog rose S 11

Rosa canina Brögs Stachellose Dog rose S 11

Rosa canina Heinsohn’s Rekord Dog rose S 11

171

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Rosa canina Inermis Dog rose S 11 Rosa canina Pfänder Dog rose S 11

Rosa canina Pollmers Dog rose S 11

Rosa canina Schmids Ideal Dog rose S 11

Rosa canina Succes Dog rose S 11

Rosa dumetorum Laxa Corymb rose S 11

Rosa indica --- Cyme rose S S 57

Rosa multiflora --- Japanese rose S 11

Rosa multiflora --- Japanese rose S 47

Rosa noisettiana Manetti Rose SL 47

Rosa odorata --- Tea rose S 47

Rosa rubiginosa --- Sweetbriar rose S 11

Rosa sp. --- Rose S S 41 172

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Rosemarinus officinalis --- Rosemary S S S S 36 Rudbeckia fulgida Goldsturm Orange coneflower R 23

Rudbeckia laciniata Gold Drop Cutleaf coneflower R 21

Ruscus aculeatus --- Butcher’s broom S S Chapter 3 Salvia azurea Grandiflora Azure blue sage S 21

Salvia haematodes --- Sage S 21

Salvia jurisicii --- Sage S 21

Salvia leucantha --- Mexican bush sage S S S Chapter 2 Salvia nemorosa Miss Indigo Sage S S 53

Salvia officinalis --- Common sage S S S S 36

Salvia splendens --- Scarlet sage R 1

Salvia splendens Bonfire Scarlet sage R R SL 27 173

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Salvia splendens Carabiniere Red Scarlet sage SL 54 Salvia splendens Flare Scarlet sage R R 28

Salvia splendens Hotline Red Scarlet sage SL 54

Salvia splendens Hotline White Scarlet sage SL 54

Salvia splendens Lady in Red Scarlet sage R R 28

Salvia splendens Oxford Blue Scarlet sage S S 28

Salvia splendens Rhea Scarlet sage R 54

Salvia splendens Sea Breeze Scarlet sage SL R 28

Salvia splendens Victoria Scarlet sage R SL 28

Salvia splendens Victoria Blue Scarlet sage R 54

Sanguisorba obtusa --- Burnet S 22

Sansevieria cylindrica --- African bowstring hemp S 31

Sansevieria cylindrica --- African bowstring hemp S 33 174

Table B-1. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Sansevieria trifasciata Laurentii Viper’s bowstring hemp S 33 Sassafras albidum --- Sassafras R R R R R 44

Satureja hortensis --- Summer savory S S S S 36

Satureja montana --- Winter savory S S S S 36

Scabiosa caucasica Fama Pincushion flower S 21

Scadoxus multiflorus ssp. --- Blood lily S 33 katherinae Scindapsus aureus --- Centipede tongavine S 33

Scoparia dulcis --- Licorice weed S 16

Sidalcea × Party Girl Checkermallow R 22

Solanum nigrum --- Black nightshade S S 57

Solenostemon Rainbow Coleus S S S 27 scutellarioides Solidago sphacelata Golden Fleece Autumn goldenrod R 22 175

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Sophora japonica --- Japanese pagoda tree S S 44 Spiraea × bumalda Froebelii Spirea S 6

Spiraea × vanhouttei --- Van Houtte’s spirea S 6

Stachys byzantina Lanatna Lamb's ear S 21

Stokesia laevis Blue Danube Stokes’ aster SL 21

Syagrus romanzoffiana --- Queen palm S Chapter 3 Syngonium podophyllum Variegatum American evergreen S 33

Syringa persica --- Persian lilac S 39

Tagetes erecta --- African marigold R 1

Tagetes erecta --- African marigold R 42

Tagetes erecta --- African marigold R 50

Tagetes erecta Carnation African marigold S 13

176

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Tagetes erecta Inca Gold African marigold R 54 Tagetes patula --- French marigold R 1

Tagetes patula --- French marigold R 50

Tagetes patula Dwarf Primose French marigold R R R 27

Tagetes patula Golden Gate French marigold R 54

Tagetes patula Goldie French marigold S S S 37

Tagetes patula Petite Gold French marigold S S S 37

Tagetes patula Petite Harmony French marigold S S S 37

Tagetes patula Tangerine French marigold R R R 37

Tagetes sp. Bupree’s First Whites Marigold S 49

Tagetes sp. Chrysanthemum Marigold S 49 Charm Tagetes sp. Climax Marigold S 49 177

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Tagetes sp. Crackerjack Marigold R 49 Tagetes sp. FM 584 Marigold S 49

Tagetes sp. Giant Double African Marigold S 49 (FM 560) Tagetes sp. Giant Double African Marigold S 49 (FM 561) Tagetes sp. Giant Double African Marigold S 49 (FM 562) Tagetes sp. Golden Age Marigold S 49 (FM 581) Tagetes sp. Local Marigold S 49

Tagetes sp. Marigold Cinnabar Marigold S 49 (FM 608) Tagetes sp. Red Brocade Marigold S 49 (FM 597) Tagetes sp. Spanish Brocade Marigold S 49 (FM 597) 178

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Tagetes sp. Spry Hybrid Marigold S 49 Tagetes sp. Spun Gold Marigold S 49

Tagetes sp. Suttons White Marigold S 49 (FM 370) Tamarix gallica --- French tamarisk S 16

Tanacetum parthenium --- Feverfew S 21

Thalictrum --- Meadow rue S 21 speciosissimum Thuja occidentalis --- Eastern arborvitae SL 39

Thuja occidentalis Globosa Eastern arborvitae R 6

Thuja occidentalis Pyramidalis Nigra Eastern arborvitae R 6

Thuja orientalis Berkmanns Oriental arborvitae SL 39

Thuja orientalis Dwarf Greenspike Oriental arborvitae R 38

Thuja orientalis Dwarf Greenspike Oriental arborvitae S 39 179

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Thymus serpyllum Album Lemon thyme S 22 Tradescantia sp. J. C. Weguelin Spiderwort R 21

Trollius × Lemon Queen Globe flower S 22

Trollius chinensis Golden Queen Globe flower S 23

Tropaeolum majus --- Garden nasturtium S 1

Tsuga canadensis --- Canadian hemlock R 6

Tulipa suaveolens --- Tulip R 35

Ulmus parvifolia --- Chinese elm S S S S R 44

Uraria picta --- Indian gooseberry S S 16

Venidium fastuosum --- Monarch of the veld S 1

Verbascum phoeniceum Benary's Hybrid Purple mullein SL 21

Verbena × hybrida Deep Blue Verbena SL 54

Verbena × hybrida Florist Verbena S S S 30 180

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Verbena × hybrida Showtime Blaze Verbena S 54 Verbena bipinnatifida --- Dakota mock vervain R 18

Verbena hybrida --- Verbena S 1

Verbena hybrida --- Verbena S 50

Verbena officinalis --- Common verbena S 42

Veronica spicata Icicle Spiked speedwell S 21

Viburnum carlesii --- Viburnum S 6

Viburnum odoratissimum Awabuki Sweet arrowwood S Chapter 3 Vinca minor Bowles Variety Common periwinkle R 21

Vinca rosea Little Bright Eye Madagascar periwinkle R SL S 27

Viola cucullata Priceana Marsh blue violet S 21

Viola tricolor --- Johnny jump up S 1

181

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source

Viola tricolor --- Johnny jump up S 50 Viola tricolor Jolly Joker Johnny jump up S 30

Viola wittrockiana Coronation Gold Pansy S 54

Viola wittrockiana Jolly Joker Pansy S 54

Viola wittrockiana Padparadja Pansy S 54

Vitex agnus-castus --- Chaste tree S 39

Washingtonia filifera --- California fan palm S S 41

Woodfordia fruticosa --- Dhaytaki S S 16

Zantedeschia aethiopica --- White calla S 35

Zelkova serrata --- Calla lily S S R S R 44

Zinnia elegans --- Common zinnia S 1

Zinnia elegans --- Common zinnia S 50

Zinnia elegans Scarlet Common zinnia SL S SL 27 182

Table B-2. Continued

M. incognita M. M. M. race javanica arenaria hapla race Plant species Cultivar Common Name 1 2 3 4 ? 1 2 ? Source Zinnia linearis --- Narrow leaved zinnia R 42 aHost Response Key: R = resistant S = susceptible SL = slightly susceptible 183

REFERENCES

Adiko, A. 1988. Plant-parasitic nematodes associated with plantain, Musa paradisiaca (AAB), in the Ivory Coast. Revue de Nématology 11:109-113.

Ahuja, S., and J. S. Arora. 1980. Susceptibility of flowering annuals to root-knot nematode (Meloidogyne incognita). Tropical Pest Management 26:293-295.

Andrássy, I. 1976. Evolution as a basis for the systematization of nematodes. Pitman Publishing, Budapest

Anonymous. 2000. Southern living annuals and perennials. L. T. Chaplin, ed. Oxmoor House, Birmingham, AL.

Bailey, L. H. 1928. The standard encyclopedia of horticulture. vol. III. New York, NY: The Macmillan Company Press.

Bailey, L. H. 1958. Manual of cultivated plants. The Macmillan Company Press, New York

Baker, J. G. 1893. A synopsis of the genera and species of Museae. Annals of Botany 7:189-229.

Bala, G., and F. Hosein. 1996. Plant parasitic nematodes associated with anthuriums and other tropical ornamentals. Nematropica 26:9-14.

Barker, K. R. and D. M. Benson. 1977. Japanese hollies: intolerant hosts of Meloidogyne arenaria in microplots. Journal of Nematology 9:330-334.

Barker, K. R., D. M. Benson, and R. K. Jones. 1979. Interactions of Burfordi, Rotunda, and dwarf yaupon hollies and aucuba with selected plant-parasitic nematodes. Plant Disease Reporter 63:113-116.

Benson, D. M., and K. R. Barker. 1982. Susceptibility of Japanese boxwood, dwarf gardenia, Compacta (Japanese) holly, Spiny Greek and Blue Rug junipers, and nandina to four nematode species. Plant Disease 66:1176-1179.

Benson, D. M., and K. R. Barker. 1985. Nematodes--a threat to ornamental plants in the nursery and landscape. Plant Disease 69:97-100.

Bernard, E. C., and W. T. Witte. 1987. Parasitism of woody ornamentals by Meloidogyne hapla. Annals of Applied Nematology 1:41-45.

185

Bernard, E. C., W. T. Witte, M. M. Dee, and P. L. Jennings. 1994. Parasitism of holly cultivars by three root-knot nematode isolates. Holly Society Journal 12:12-17.

Bird, A. F. 1974. Plant response to root-knot nematode. Annual Review of Phytopathology 12:69-85.

Blok, V. C., M. S. Phillips, and M. Fargette. 1997a. Comparison of sequences from the ribosomal DNA intergenic region of Meloidogyne mayaguensis and other major root-knot nematodes. Journal of Nematology 29:16-22.

Blok, V. C., M. S. Phillips, J. W. McNicol, and M. Fargette. 1997b. Genetic variation in tropical Meloidogyne spp. as shown by RAPD-PCR. Fundamental and Applied Nematology 20:127-133.

Blok, V. C., J. Wishart, M. Fargette, K. Berthier, and M. S. Phillips. 2002. Mitochondrial DNA differences distinguishing Meloidogyne mayaguensis from the major species of tropical root-knot nematodes. Nematology 4:773-781.

Boneti, J. I. S., and S. Ferraz. 1981. Modificação do método de Hussey e Barker para extração de ovos de Meloidogyne exigua de cafeeiro. Fitopatologia Brasileira 6:553.

Brito, J., T. O. Powers, P. G. Mullin, R. N. Inserra, and D. W. Dickson. 2004. Morphological and molecular characterization of Meloidogyne mayaguensis isolates from Florida. Journal of Nematology 36:232-240.

Buhrer, E. M., 1938. Additions to the list of plants attacked by the root-knot nematode (Heterodera marioni). Plant Disease Reporter 22:216-234.

Bureau of Nematology. 1989. Tri-ology Technical Report 28(7):6-7. Bureau of Nematology, Florida Department of Agriculture and Consumer Services, Gainesville, FL, U.S.A.

Carneiro, R. M. D., A. R. A. Almeida, and R. G. Carneiro. 1996. Enzyme phenotypes of Brazilian isolates of Meloidogyne spp. Fundamental and Applied Nematology 19:555-560.

Carneiro, R. M. D. G., M. R. A. Almeida, and P. Quénéhervé. 2000. Enzyme phenotypes of Meloidogyne spp. populations. Nematology 2:645-654.

Carneiro, R. M. D. G., P. Castagnone-Sereno, and D. W. Dickson. 1998. Variability among four populations of Meloidogyne javanica from Brazil. Fundamental and Applied Nematology 21:319-326.

186

Castro, J. M. C., R. D. Lima and R. M. D. G. Carneiro. 2003. Isoenzymatic variability in Brazilian populations of Meloidogyne spp. from soybean. Nematologia Brasileira 27:1-12.

Caswell-Chen, E. P., V. M. Williamson, and B. B. Westerdahl. 1993. Applied biotechnology in nematology. Journal of Nematology 25:719-730.

Caveness, F. E., and G. F. Wilson. 1977. Effect of root-knot nematodes on growth and development of Celosia argentea L. Acta Horticulturae 53:71-73.

Cetintas, R., R. D. Lima, M. L. Mendes, J. A. Brito, and D. W. Dickson. 2003. Meloidogyne javanica on peanut in Florida. Journal of Nematology 35:433-436.

Chase, A. R., and G. W. Simone. 2001. Diseases of pittosporum in Florida. Plant Pathology Fact Sheet PP-29. University of Florida Cooperative Extension Service, Gainesville, FL.

Cheesman, E. E. 1985. Classification of the bananas. III. Critical notes on species. b. Musa acuminata Colla. Kew Bulletin 3:17-28.

Cho, M. R., J. Y. Kim, C. Song, J. Y. Ko, S. Y. Na, and M. S. Yiem. 1996. Screening of carnation cultivars for resistance to Meloidogyne incognita. Journal of Nematology 28:639-642.

Christie, J. R. 1959. Plant nematodes: Their bionomics and control. Agricultural Experiment Station, University of Florida, Gainesville, FL.

Coolen, W. A. and G. J. Hendrickx. 1972. Investigations on the resistance of rose root- stocks to Meloidogyne hapla and penetrans. Nematologica 18: 2, 155-158.

Dalmasso, A., and J. B. Berge. 1978. Molecular polymorphism and phylogenetic relationship in some Meloidogyne spp.: Application to the of Meloidogyne. Journal of Nematology 4:323-332.

Davis, R. A., and W. R. Jenkins. 1960. Histopathology of gardenia (Gardenia jasminoides Veitchi), infected with three species of Meloidogyne. Nematologica 5:228-230.

Dehgan, B. 1998. Landscape plants for subtropical climates. University Press of Florida, Gainesville, FL.

De Waele, D., and R. G. Davide. 1998. The root-knot nematodes of banana. Musa pest factsheet No. 3. International Network for the Improvement of Banana and Plantain, France.

187

Dickson, D. W., D. Huisingh, and J. N. Sasser. 1971. Dehydrogenases, acid and alkaline phosphatases, and esterases for chemotaxonomy of selected Meloidogyne, , Heterodera and Aphelenchus spp. Journal of Nematology 3:1-16.

Dickson, D. W., J. N. Sasser, and D. Huisingh. 1970. Comparative disc-electrophoretic protein analyses of selected Meloidogyne, Ditylenchus, Heterodera, and Aphelenchus spp. Journal of Nematology 2:286-293.

Eisenback, J. D. 1985. Diagnostic characters useful in the identification of the four most common species of root-knot nematodes (Meloidogyne spp.). Pp. 95-112 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne, vol. 1. Biology and control. North Carolina State University Graphics, Raleigh.

Eisenback, J. D. 1987. Reproduction of northern root-knot nematode (Meloidogyne hapla) on marigolds. Plant Disease 71:281.

Esbenshade, P. R., and A. C. Triantaphyllou. 1985a. Electrophoretic methods for the study of root-knot nematode enzymes. Pp. 115-123 in K. R. Barker, C. C. Carter, and J. N. Sasser, eds. An advanced treatise on Meloidogyne, vol. 2. Methodology. North Carolina State University Graphics, Raleigh.

Esbenshade, P. R., and A. C. Triantaphyllou. 1985b. Identification of major Meloidogyne species employing enzyme phenotypes as differentiating characters. Pp. 135-407 in J. N. Sasser and C. C. Carter, eds. An Advanced Treatise on Meloidogyne, vol 1. Biology and control. North Carolina State University Graphics, Raleigh.

Esbenshade, P. R., and A. C. Triantaphyllou. 1985c. Use of enzyme phenotypes for identification of Meloidogyne species (Nematoda: ). Journal of Nematology 17:6-20.

Esbenshade, P. R., and A. C. Triantaphyllou. 1987. Enzymatic relationships and evolution in the genus Meloidogyne (Nematoda: Tylenchida). Journal of Nematology 19:8-18.

Esbenshade, P. R., and A. C. Triantaphyllou. 1990. Isozyme phenotypes for the identification of Meloidogyne species. Journal of Nematology 22:10-15.

Evans, A. A. F. 1971. Taxonomic value of gel electrophoresis of proteins from mycophagous and plant-parasitic nematodes. International Journal of Biochemistry 2:72-79.

Fargette, M. 1987a. Use of esterase phenotypes in the taxonomy of the genus Meloidogyne. 1. Stability of the esterase phenotype. Revue de Nématologie 10:39-43.

188

Fargette, M. 1987b. Use of esterase phenotypes in the taxonomy of the genus Meloidogyne. 2. Esterase phenotypes observed in West African populations and their characterization. Revue de Nématologie 10:45-56.

Fargette, M., and R. Braaksma. 1990. Use of the esterase phenotype in the taxonomy of the genus Meloidoyge. 3. A study of some “B” race lines and their taxonomic position. Revue de Nématology 13:375-386.

Fargette, M., M. S. Phillips, V. C. Blok, R. Waugh, and D. L. Trudgill. 1996. An RFLP study of relationships between species, populations and resistance breaking lines of tropical Meloidogyne spp. Fundamental and Applied Nematology 19:903-912.

Fawzy, R. N., F. W. Riad, and L. R. Rizhallh. 1991. Screening of carnation resistance for root-knot nematode, Meloidogyne incognita. Annals of Agricultural Science 29:797-810.

Francis, J. K. 2004. Odontonema cuspidatum (Nees in DC.) Kuntze. International Institute of Tropical Forestry, United States Department of Agriculture, Forest Service.

Gera, A., A. Kritzman, and J. Cohen. 2000. Pittosporum tobira: a new host for tomato spotted wilt virus. Plant Disease 84:491, 2000.

Giblin-Davis, R. M., A. W. Meerow, and F. G. Bliz. 1992. Host suitability of Ixora spp. for the root-knot nematodes Meloidogyne incognita race 1 and M. javanica. Journal of Nematology 24:722-728.

Gilman, E. F. 1999a. Codiaeum variegatum. University of Florida Fact Sheet FPS-134. University of Florida, Gainesville, FL.

Gilman, E. F. 1999b. Liriope muscari ‘Evergreen Giant’. University of Florida Fact Sheet FPS-348. University of Florida, Gainesville, FL.

Gilman, E. F. 1999c. Pittosporum tobira. University of Florida Fact Sheet FPS-483. University of Florida, Gainesville, FL.

Gilman, E. F. 1999d. Pittosporum tobira ‘Variegata’. University of Florida Fact Sheet FPS-484. University of Florida, Gainesville, FL.

Gilman, E. F., and T. Delvalle. 1999. . University of Florida Fact Sheet FPS-445. University of Florida, Gainesville, FL.

Gilman, E. F. and D. Marshall. 1999. Salvia leucantha. University of Florida Fact Sheet FPS-527. University of Florida, Gainesville, FL.

189

Goff, C. C. 1936. Relative susceptibility of some annual ornamentals to root-knot. Agricultural Experiment Station Bulletin 291, University of Florida, Gainesville, FL.

Goodey, J. B., and M. T. Franklin. 1956. T. Goodey’s the nematode parasites of plants catalogued under their hosts. Commonwealth Agricultural Bureaux, Farnham, UK.

Goodey, J. B., M. T. Franklin, and D. J. Hooper. 1965. The nematode parasites of plants catalogued under their hosts. Third ed. Commonwealth Agricultural Bureaux, Farnham, UK.

Griffiths, M. 1994. The new royal horticultural society dictionary index of garden plants. Macmillan, London.

Haasis, F. A., J. C. Wells, and C. J. Nusbaum. 1961. Plant parasitic nematodes associated with decline of woody ornamentals in North Carolina and their control by soil treatment. Plant Disease Reporter 45:491-496.

Hamon, A. B., and T. R. Fasulo. 1998. Cottony cushion scale, Icerya purchasi Maskell. University of Florida Cooperative Extension Service, Document EENY-034. University of Florida, Gainesville, FL.

Hansen, E. L., and E. J. Buecher. 1970. Symposium: Current approaches to the problems of systematic nematology. Journal of Nematology 2:1-6.

Hartman, K. M. and J. N. Sasser. 1985. Identification of Meloidogye species on the basis of differential host test and perineal-pattern morphology. Pp. 69-76 in K. R. Barker, C. C. Carter, and J. N. Sasser, eds. An advanced treatise on Meloidogyne, vol. 2. Methodology. North Carolina State University Graphics, Raleigh.

Haseeb, A., R. Pandey, and A. Husain. 1985. New host records of the root-knot nematode, Meloidogyne javanica. FAO Plant Protection Bulletin 33:123.

Haseeb, A., R. Pandey, and A. Husain. 1984. Some new host records of the root-knot nematode, Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949. Current Science 53:1151-1152.

Haseeb, A., and R. Pandey. 1987. Incidence of root-knot nematodes in medicinal and aromatic plants-new host records. Nematropica 17:209-212.

Heald, C. M. 1967. Pathogenicity of five root-knot nematode species on Ilex crenata ‘Helleri’. Plant Disease Reporter 51:581-583.

Henny, R. J., L. S. Osborne, and A. R. Chase. 1991. Croton. CFREC-A Foliage Plant Research Note RH-91-12. University of Florida, Gainesville, FL.

190

Hirschmann, H. 1985. The genus Meloidogyne and morphological characters differentiating its species. Pp. 79-92 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne, vol. 1. Biology and control. North Carolina State University Graphics, Raleigh.

Hodges, A. W., and J. J. Haydu. 2003. Commodity outlook 2003: U.S. and Florida ornamental plant markets. Extension Data Information Source FE374. Food and Resource Economics Department, University of Florida, Gainesville, FL.

Hussey, R. S. 1987. Secretions of esophageal glands of Tylenchida nematodes. Pp. 221- 228 in J. A. Veech and D. W. Dickson, eds. Vistas on nematology. Society of Nematologists, Hyattsville, Maryland.

Hussey, R. S. 1985a. Biochemistry as a tool in identification and its probable usefulness in understanding the nature of parasitism. Pp. 127-133 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne, vol. 1. Biology and control. North Carolina State University Graphics, Raleigh.

Hussey, R. S. 1985b. Host-parasite relationships and associated physiological changes. Pp. 143-153 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne, vol. 1. Biology and control. North Carolina State University Graphics, Raleigh.

Hussey, R. S., and K. R. Barker. 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reporter 57:1025- 1028.

Hussey, R. S., J. N. Sasser, and D. Huisingh. 1972. Disk-electrophoretic studies of soluble proteins and enzymes of Meloidogyne incognita and M. arenaria. Journal of Nematology 4:183-189.

Ibrahim, A. A. M., and F. A. Al-Yahya. 2002. Phytoparasitic nematodes associated with ornamental plants in Riyadh region, central Saudi Arabia. Alexandria Journal of Agricultural Research 47:157-167.

Jaizme-Vega, M. C., P. Tenoury, J. Pinochet, and M. Jaumot. 1997. Interactions between the root-knot nematode Meloidogyne incognita and Glomus mosseae in banana. Plant and Soil 196:27-35.

Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692.

Judd, W. S. 2003. The genera of Ruscaceae in the southeastern United States. Harvard Papers in Botany 7:93-149.

191

Khan, M. R., and M. W. Khan. 1989. Verbena bipinnatifida as a biological agent for root-knot nematodes. International Nematology Network Newsletter 6(4):25-26.

Khanna, A. S., S. S. Chandel, and R. Malhotra. 1998. Evaluation of varietal resistance in gladiolus to Meloidogyne incognita (race 2). Pest Management and Economic Zoology 6:27-30.

Kirby, M. F. 1978. Reniform and root knot nematodes on passionfruit in Fiji. Nematropica 8:21-25.

Krishnaprasad, P. R. 1979. Studies on the root-knot nematodes infecting patchouli (Pogostemon cablin Benth). Mysore Journal of Agricultural Sciences 13:367- 368.

LaMondia, J. A. 1995. Response of perennial herbaceous ornamentals to Meloidogyne hapla. Journal of Nematology 27:645-648.

LaMondia, J. A. 1996. Response of additional herbaceous perennial ornamentals to Meloidogyne hapla. Journal of Nematology 28:636-638.

LaMondia, J. A. 1997. Management of Meloidogyne hapla in herbaceous perennial ornamentals by sanitation and resistance. Journal of Nematology 29:717-720.

Leahy, R. M., and D. A. Davison. 1999. Leaf and crown rot of Liriope muscari. Plant Pathology Circular No. 392. Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL.

Lehman, P. S. 1984a. Nematodes causing decline of boxwood. Nematology Circular 108. Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville.

Lehman, P. S. 1984b. Nematodes which cause decline of gardenia. Nematology Circular 111. Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville.

Lehman, P. S. and E. L. Barnard. 1982. A root-knot nematode pathogenic to flowering dogwood Cornus florida. Nematology Circular 84. Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville.

Lewis, S. A. 1987. Nematode-plant compatibility. Pp. 246-252 in J. A. Veech and D. W. Dickson, eds. Vistas on nematology. Society of Nematologists, Hyattsville, Maryland.

Maggenti, A. R. 1987. Adaptive biology of nematode parasites. Pp. 188-195 in J. A. Veech and D. W. Dickson, eds. Vistas on nematology. Society of Nematologists, Hyattsville, Maryland.

192

McSorley R. 1994. Susceptibility of common bedding plants to root-knot nematodes. Proceedings of the Florida State Horticultural Society 107:430-432.

McSorley, R., and R. A. Dunn. 1989. Effects of root-knot nematodes on Areca catechu. Journal of Nematology 21:717-719.

McSorley, R., and R. A. Dunn. 1990. Infection of five species of landscape ornamentals by root-knot nematodes (Meloidogyne spp.). Proceedings of the Soil Science Society of Florida 49:227-230.

McSorley, R., and J. J. Frederick. 1994. Response of some common annual bedding plants to three species of Meloidogyne. Journal of Nematology 26:773-777.

McSorley, R., and J. J. Frederick. 2001. Host suitability of some vinca and salvia cultivars to two isolates of root-knot nematodes. Proceedings of the Florida State Horticultural Society 114:239-241.

McSorley, R., and R. B. Marlatt. 1983. Reaction of Hibiscus rosa-sinensis cultivars to two species of root knot nematodes. HortScience 18:85-86.

Mishra, R. L., and S. D. Mishra. 1997. Sansevieria cylindrica Rojer--a new host of root- knot nematode (Meloidogyne incognita). Progressive Horticulture 29:196.

Mishra, S. D., and R. L. Misra. 1993. New record on the occurrence of root-knot nematode, Meloidogyne incognita on Philodendron selloum C. Koch. Current Nematology 4:245-246.

Misra, R. L., and S. D. Mishra. 1997. Screening of certain ornamentals against root-knot nematode (Meloidogyne incognita). Annals of Agricultural Research 18:234- 236.

Misra, R. L., S. D. Mishra, and S. Misra. 2002. Root-knot nematode infestation in Kalanchoe fedtschenkoi Hamet & Pirr. Journal of Ornamental Horticulture, New Series 5:61.

Montasser, S. A. 1995. Reaction of certain flower bulb plants to root-knot nematode, Meloidogyne incognita. Pakistan Journal of Nematology 13:99-102.

Moreno, J. E., J. R. Rich, E. C. French, G. M. Prine, and R. A. Dunn. 1992. Reactions of selected herbs to three Meloidogyne spp. Nematropica 22:217-225.

Motsinger, R. E., E. H. Moody, and C. M. Gay. 1977. Reaction of certain French marigold (Tagetes patula) cultivars to three Meloidogyne spp. Journal of Nematology 9:278.

193

Nemec, S., and L. S. Morrison. 1972. Histopathology of Thuga orientalis and Juniperus plumose infected with Meloidogyne incognita. Journal of Nematology 4:72-74.

Nemec, S., and B. Struble. 1968. Response of certain woody ornamental plants to Meloidogyne incognita. Phytopathology 58:1700-1703.

Niblack, T. L., and E. C. Bernard. 1985. Plant-parasitic nematode communities in dogwood, maple, and peach nurseries in Tennessee. Journal of Nematology 17:132-139.

Nigh, E. L. Jr. 1972. Susceptibility of Arizona-grown ornamentals to attack by several nematode species. Plant Disease Reporter 56:914-918.

O’Bannon, J. H., and H. W. Reynolds. 1961. Root-knot nematode damage and cotton yields in relation to certain soil properties. Soil Science 92:384-386.

Osborne, W. W., and W. R. Jenkins. 1963. Host-parasite relationships of Meloidogyne hapla, M. incognita acrita, and on Forsythia intermedia. Plant Disease Reporter 47:354-358.

Pais, C. S., and I. M. de Abrantes. 1989. Esterase and malate dehydrogenase phenotypes in Portuguese populations of Meloidoygne species. Journal of Nematology 21:342-346.

Pant, V., S. Hakim, S. P. Singh, and S. K. Saxena. 1983. Susceptibility of different ornamental plants to root knot nematode and morphometrics of females of Meloidogyne incognita. Indian Journal of Nematology 13:217-222.

Rajendran, G., P. Veluswamy, T. S. Muthukrishnan, and R. Sivagami. 1975. Susceptibility of Crossandra and related genera to the root-knot nematode, Meloidogyne incognita (Kofoid, 1889) Chitwood, 1949. South Indian Horticulture 23:147-148.

Rammah, A., and H. Hirschmann. 1988. Meloidogyne mayaguensis n. sp. (Meloidogynidae), a root-knot nematode from Puerto Rico. Journal of Nematology 20:58-69.

Randig, O., M. Bongiovanni, R. M. D. G. Carneiro, and P. Castagnone-Sereno. 2002. Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species. Genome 45:862-870.

Reeves, R. G. and A. Bell. 1988. Codiaeum variegaum pictum--A foliage favorite with new possibilities. Florida Nurseryman 35:69-78.

Rhoades, H. L. 1989. Susceptibility of Pittosporum tobira ‘Variegaum’ to longicaudatus. Nematropica 19:89-92.

194

Rickard, D. A., and A. W. Dupree. 1978. The effectiveness of ten kinds of marigolds and five other treatments for control of four Meloidogyne spp. Journal of Nematology 10:296-297.

Ricker, P. L. 1937. Pp. 2079 in L. H. Bailey. The standard cyclopedia of horticulture. vol 2. The Macmillan Company, New York.

Rinallo, C., and A. Bennici. 1989. Structural changes in Pittosporum tobira Ait. leaves exposed to sea spray. Advances in Horticultural Science 3:63-67.

Roberts, P. A. 1992. Current status of the availability, development, and use of host plant resistance to nematodes. Journal of Nematology 24:213-227.

Rohde, R. A. 1972. Expression of resistance in plants to nematodes. Annual Review of Phytopathology 10:233-252.

Santamour, F. S. Jr. 1992. Susceptibility of maples to root-knot nematodes. Journal of Arboriculture 18:262-265.

Santamour, F. S. Jr., and L. G. H. Riedel. 1993. Susceptibility of various landscape trees to root-knot nematodes. Journal of Arboriculture 19:257-259.

Santamour, F. S. Jr., and L. G. H. Riedel. 1995. Susceptibility of redbuds (Cercis) to root-knot nematodes. Journal of Arboriculture 21:37-40.

Santo, G. S., and B. Lear. 1976. Influence of Pratylenchus vulnus and Meloidogyne hapla on the growth of rootstocks of rose. Journal of Nematology 8:18-23.

Sasser, J. N., and C. C. Carter. 1985. Overview of the international Meloidogyne project 1975-1984. Pp. 18-24 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne, vol. 1. Biology and Control. North Carolina State University Graphics, Raleigh.

Sasser, J. N., C. C. Carter, and K. M. Hartman. 1984. Standardization of host suitability studies and reporting of resistance to root-knot nematodes. NC: North Carolina State University Graphics, Raleigh.

Sasser, J. N., F. A. Haasis, and T. F. Cannon. 1966. Pathogenicity of Meloidogyne species on Ilex. Plant Disease Reporter 50:664-668.

Sher, S. A. 1954. Observations on plant-parasitic nematodes in Hawaii. Plant Disease Reporter 38:687-689.

Singh, D. and D. C. Gupta. 1993. Evaluation of marigold cultivars/hybrids for resistance against Meloidogyne javanica. Haryana Agricultural University Journal of Research 23:156-159.

195

Singh, J. P., and Q. Majeed. 1991. Screening of ornamental annuals to the root-knot nematode, Meloidogyne incognita, in Iraq. Indian Phytopathology 44:179-184.

Singh, Y. P., S. Zafar, S. N. Khan, D. N. Joshi, and Z. Sultan. 2000. Spilanthes acmella and Spilanthes oleracea: new hosts of root-knot nematode. Annals of Forestry 8: 2, 282-284.

Stamps, R. H. 1987. Major Florida cut foliage crops classified according to floral design and landscape usage. Proceedings of the Florida State Horticultural Society 100:179-180.

Stamps, R. H. 2002. Japanese pittosporum/tobira production and use. University of Florida Circular ENH861. University of Florida, Gainesville, FL.

Stamps, R. H., and L. S. Osborne. 2003 Croton production and use. University of Florida Circular ENH878. University of Florida, Gainesville, FL.

Starr, J. L., E. K. Tomaszewski, M. Mundo-Ocampo, and J. G. Baldwin. 1996. Meloidogyne partityla on pecan: isozyme phenotypes and other hosts. Journal of Nematology 28:565-568.

Stoffelen, R., R. Verlinden, J. Pinochet, R. Swenen, and D. De Waele. 2000. Host plant response of Fusarium wilt resistant Musa genotypes to Radopholus similis and . International Journal of Pest Management 46:289-293.

Stokes, D. E. 1977. Effects of root-knot nematodes on ornamental plants. Nematology Circular 24. Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville.

Stokes, D. E. 1982. The effects of root-knot nematodes, Meloidogyne spp. on Japanese holly. Nematology Circular 83. Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville.

Tarjan, A. C. 1952. Comparative studies of some root-knot nematodes infecting the common snapdragon, Antirrhinum majus L. Phytopathology 42:641-644.

Taylor, A. L. and J. N. Sasser. 1978. Biology, identification, and control of root-knot nematodes (Meloidogyne species). North Carolina State University Graphics, Raleigh.

Thorne, G. E. 1961. Principles of nematology. McGraw-Hill Book Co., Inc., New York.

Triantaphyllou, A. C. 1985. Cytogenetics, cytotaxonomy, and phylogeny of root-knot nematodes. Pp. 113-126 in J. N. Sasser and A. C. Carter, eds. An advanced treatise on Meloidogyne. vol 1. Biology and control. orth Carolina State University Graphics, Raleigh.

196

Triantaphyllou, A. C., and H. Hirschmann. 1960. Post-infection development of Meloidogyne incognita Chitwood 1949 (Nematoda: Heteroderidae). Annales de l'Institut Phytopathologique Benaki 3:3-11.

Van Gundy, 1985. Ecology of Meloidogyne spp. – emphasis on environmental factors affecting survival and partitioning. Pp. 177-182 in J. N. Sasser and A. C. Carter, eds. An advanced treatise on Meloidogyne. vol 1. Biology and control. North Carolina State University Graphics, Raleigh.

Viglierchio, D. R. 1979. Response of Pinus ponderosa seedlings to stylet-bearing nematodes. Journal of Nematology 11:377-387.

Walker, J. T. 1980. Susceptibility of Impatiens cultivars to root-knot nematode, Meloidogyne arenaria. Plant Disease 64:184-185.

Walker, J. T., and J. B. Melin. 1998a. Growth of selected woody ornamentals in the field following inoculation with root-knot nematodes. Georgia Agricultural Experiment Stations Research Report No. 651.

Walker, J. T., and J. B. Melin. 1998b. Host status of herbaceous perennials to Meloidogyne incognita and M. arenaria. Journal of Nematology 30:607-610.

Walker, J. T., J. B. Melin, and J. Davis. 1994. Sensitivity of bedding plants to southern root-knot nematode, Meloidogyne incognita race 3. Journal of Nematology 26:778-781.

Wallace, H. R. 1969. The influence of nematode numbers and of soil particle size, nutrients and temperature on the reproduction of Meloidogyne javanica. Nematologica 15:55-64.

Watkins, J. V. and T. J. Sheehan. 1977. Florida landscape plants. University Press of Florida, Gainesville, FL.

Whitehead, A. G. 1969. The distribution of root-knot nematodes (Meloidogyne spp.) in tropical Africa. Nematologica 15:315-333.

Yongfang, C., W. Jianyu, X. Xianqi, and Y. Shengfu. 1998. Using PhastSystem for rapid identification of root-knot nematodes. Acta Phytopathologica Sinica 28:73- 77.

Zarina, B., and M. Abid. 1995. New host records of root-knot nematodes (Meloidogyne species) in Pakistan. Pakistan Journal of Nematology 13:49-50.

BIOGRAPHICAL SKETCH

Roi Levin was born 30 March, 1979, in Tel-Aviv, Israel. At the age of 14, he had moved to Florida, where he graduated from Marjory Stoneman Douglas High School in

Parkland, Florida, in 1997. He began his studies at the University of Florida, Gainesville,

Florida, in 1997, and earned a Bachelor of Science degree in sciences, with an emphasis on animal biology, in 2002. He began studies for his Master of Science degree in entomology and nematology at the Univeristy of Florida, under W. T. Crow, in 2002.

The title of his thesis is “Reproduction and identification of root-knot nematodes on perennial ornamental plants in Florida.”

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

______W. T. Crow, Chair Assistant Professor of Entomology and Nematology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

______J. A. Brito Biological Scientist IV

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

______R. K. Schoellhorn Associate Professor of Horticultural Science

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

______A. F. Wysocki Associate Professor of Food and Resource Economics

This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

May 2005 ______Dean, College of Agricultural and Life Sciences

______Dean, Graduate School