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2010 MELOIDOGYNE SPP. INFECTING ORNAMENTAL IN FLORIDA J. A. Brito Division of Plant Industry, [email protected]

R. Kaur University of Florida

R. Cetintas Kahramanmaras Sutcu Imam University

J. D. Stanley Division of Plant Industry

M. L. Mendes University of Florida

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Brito, J. A.; Kaur, R.; Cetintas, R.; Stanley, J. D.; Mendes, M. L.; Powers, Thomas O.; and Dickson, D. W., "MELOIDOGYNE SPP. INFECTING ORNAMENTAL PLANTS IN FLORIDA" (2010). Papers in Plant Pathology. 441. http://digitalcommons.unl.edu/plantpathpapers/441

This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors J. A. Brito, R. Kaur, R. Cetintas, J. D. Stanley, M. L. Mendes, Thomas O. Powers, and D. W. Dickson

This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/plantpathpapers/441

MELOIDOGYNE SPP. INFECTING ORNAMENTAL PLANTS IN FLORIDA

J. A. Brito1*, R. Kaur2, R. Cetintas3, J. D. Stanley1, M. L. Mendes2, T. O. Powers4, and D. W. Dickson2

1Division of Plant Industry, Gainesville, FL 32614, USA; 2Entomology and Nematology Department, University of Florida, Gainesville, FL 32611, USA; 3Department of Plant Protection, Kahramanmaras Sutcu Imam University, Kahramanmaras, 46060, Turkey; 4Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583, USA. *Corresponding author: [email protected]

ABSTRACT Brito, J. A., R. Kaur, R. Cetintas, J. D. Stanley, M. L. Mendes, T. O. Powers, and D. W. Dickson. 2010. Meloidogyne spp. infecting ornamental plants in Florida. Nematropica 40:87-103. A total of 206 root samples were collected from ornamental plants growing in ornamental nurseries and various landscapes in Florida. Isozyme phenotypes, especially esterase (EST) and malate dehydro- genase (MDH) were the main methods used to identify the root-knot species. When needed, the morphology of female perineal patterns, morphometric characters and mitochondrial DNA were used to aid in the identification. Six Meloidogyne spp., M. arenaria, M. floridensis, M. graminis, M. incognita, M. javanica and M. mayaguensis were found infecting ornamental plants in Florida. As previously report- ed EST activity was of highest diagnostic value to identify Meloidogyne spp. found in this study; however, MDH was helpful to distinguish M. mayaguensis and M. graminis from the other root-knot nematode spe- cies identified. Five new EST phenotypes were detected associated with 17 unidentified root-knot nem- atode populations. To our knowledge, this is the first report of ornamental plants in the genera Dracena and , and Ligustrum and Washingtonia being host of M. floridensis and M. mayaguensis, respective- ly. New plant species host records for M. mayaguensis were Ajuga reptans, Amaranthus tricolor, Buddleja da- vidii, Caryopteris × clandonensis, Clerodendrum × ugandense, Hibiscus grandiflorus, Lagerstroemia indica, Penta lanceolata, Plectranthus scutellarioides, and Solandra maxima. Key words: Esterase isozyme phenotyping, Florida, malate dehydrogenase isozyme phenotyping, Me- loidogyne species, ornamental plants, root-knot nematode.

RESUMEN Brito, J. A., R. Kaur, R. Cetintas, J. D. Stanley, M. L. Mendes, T. O. Powers, and D. W. Dickson. 2010. Meloidogyne spp. en plantas ornamentales en Florida. Nematropica 40:87-103. Se colectaron 206 muestras de raíces de plantas ornamentales cultivadas en viveros y en distintos paisajes en Florida. El principal método para identificar las especies fue el de fenotipo isoenzimático, especialmente de esterasas (EST) y malato deshidrogenasas (MDH). En algunos casos, se comple- mentó la identificación con morfología del patrón perineal de hembras, caracteres morfométricos y ADN mitocondrial. Se encontraron seis especies: M. arenaria, M. floridensis, M. graminis, M. incognita, M. javanica y M. mayaguensis en las plantas ornamentales observadas. La actividad de esterasa fue la de más alta utilidad en el diagnóstico de especies de Meloidogyne en este estudio, pero la actividad de malato deshidrogenasa fue útil para distinguir a M. mayaguensis y M. graminis de otras especies de ne- matodo agallador. Se detectaron cinco nuevos fenotipos de esterasa asociados con 17 poblaciones de nematodo agallador no identificadas. Hasta donde sabemos, este es el primer registro de M. floridensis en plantas de los géneros Dracena e Hibiscus, y de M. mayaguensis en Ligustrum y Washingtonia. Nuevos registros de especies vegetales para M. mayaguensis incluyen Ajuga reptans, Amaranthus tricolor, Buddleja davidii, Caryopteris × clandonensis, Clerodendrum × ugandense, Hibiscus grandiflorus, Lagerstroemia indica, Penta lanceolata, Plectranthus scutellarioides y Solandra maxima. Palabras clave: especies de Meloidogyne, fenotipo isoenzimático de esterasa, fenotipo isoenzimático de malato deshidrogenasa, Florida, nematodo agallador, plantas ornamentales.

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INTRODUCTION 1987a; De Waele and Elsen, 2007;) has made them a useful tool for nematode In 2007, Florida ranked third among identification. Among the isozyme systems, states in the USA in the production and esterase (EST) has the highest diagnostic gross sale of nursery plants and ranked first value because the majority of the pheno- in production of ornamental grasses, types described are nematode-species spe- woody ornamental plants, preparative cific; however, the use of more than one nursery materials and palm trees (Anony- isozyme may be needed as the result of mous, 2007). Florida led the nation in sales intraspecific variability and differences in of potted foliage for indoor use and hang- migrations obtained from different electro- ing baskets and also was the nation’s leader phoresis apparatus and laboratories. in sales of cut cultivated greens in 2005 Isozyme analysis, specifically EST in combi- (Anonymous, 2008). Many of the orna- nation with malate dehydrogenase (MDH) mental plants currently used for landscap- (Dickson et al., 1970; Esbenshade and Tri- ing are susceptible to several pathogens, antaphyllou, 1985), resolved using poly- including root-knot (Meloidog- acrylamide gel electrophoresis (PAGE) has yne spp.) (Barker and Benson, 1977; Ben- proven to be a valuable, fast and reliable son and Barker, 1985; Sinclair et al., 1987; method to identify the most common root- Martinez et al., 2003). knot nematode species collected from dif- Up to March 2010, 97 nominal species of ferent parts of the world (Dickson et al., Meloidogyne have been described. The proper 1970, 1971; Esbenshade and Triantaphyl- identification of Meloidogyne spp. is very lou, 1985; Fargette, 1987a; 1987b; Pais and important for implementation of plant Abrantes, 1989; Carneiro et al., 1996; 2000; breeding, nematode management, and par- Karssen, 2002, Castro et al., 2003; Cofcewicz ticularly for certification and quarantine in et al., 2004, 2005; Molinari et al., 2005); regulatory programs. Species identification is however, novel esterase phenotypes have primarily based on morphological and mor- been discovered in root-knot nematode phometrics characters of the males, females surveys (Esbenshade and Triantaphyllou, and second stage juveniles (Jepson, 1987). 1985; Hernandez et al., 2004; Adam et al., Accurate and reliable identification using 2005; Molinari et al., 2005). To determine morphology and morphometrics is a difficult whether theses novel phenotypes represent and time consuming task that requires well a new root-knot nematode species, a nema- trained personnel. Morphological charac- tode species already described but with an ters, especially female perineal patterns, are unknown EST/MDH phenotype or an one of the major characters used to aid in the aberrant pattern; a combination of mor- identification of root-knot nematodes in rou- phological, morphometric, host range, bio- tine analysis; however, perineal patterns are chemical and molecular studies are variable, and may lead to misidentification of needed. Currently, several DNA-based aberrant populations and new species. Con- methods such as restriction fragment versely, biochemical markers such as isozyme length polymorphisms (RFLP), random phenotypes used in conjunction with mor- amplified polymorphic DNA (RAPD), phological and morphometric, allow a more amplification of the ribosomal DNA in the precise and accurate identification of Meloid- intergenic spacer region (IGS) between ogyne spp. the 18S and 5S gene, amplification of the The relative stability of isozymes pheno- mitochondrial DNA (mtDNA) region types within Meloidogyne spp. (Fargette, between the COII and lRNA genes and the

Ornamental plants as hosts of Meloidogyne species: Brito et al.89

63 bp repeat region have shown to be use- ing the year of collection and the serial ful to aid in the identification of Meloidog- number to maintain sample identity. Root yne spp. (Powers et al., 1986; 1993; 2005; samples with limited infection were cut Blok et al., 1997a; 1997b; 2002; Zijlstra et al., into ca. 2-cm pieces, mixed with pasteur- 2000; Randig et al., 2002; Handoo et al., ized soil and placed into a clay pot in which 2004); however, these methods are still a tomato seedling (Solanum lycopersicum expensive to use in large surveys (Powers et ‘Rutgers’) was transplanted. Plants were al., 2005) as well as in routine analyses in maintained in a greenhouse at 26 ± 1.8°C many nematode diagnostic laboratories until used for nematode identification. due to the costs with equipment, reagents The procedure for isozyme extraction and DNA sequencing. from each nematode female was the same The objectives of the current study were as that reported by Brito et al., 2008. At to i) identify the root-knot nematode spe- least 26 egg-laying females were dissected cies found infecting ornamental plant spe- directly from each root system and isozyme cies in Florida, ii) evaluate the usefulness of profiles determined using polyacrylamide EST and MDH phenotypes in differentiat- gel electrophoresis (PAGE) with two gels ing Meloidogyne spp. for routine diagnostic run at the same time (Brito et al., 2008). purposes, and iii) determine the plant host One gel was stained for both MDH and of each of the Meloidogyne sp. identified. EST activity (Esbenshade and Triantaphyl- lou, 1985), whereas the second one, was MATERIALS AND METHODS stained only for EST. Extracts from single M. javanica females were added separately A total of 206 root samples were col- to individual wells on each gel as standards. lected from ornamental plants in 26 coun- The nematodes species with new or ties in Florida for isolation and unknown EST phenotypes were also identification of root-knot nematode spe- stained for two different isozymes; superox- cies in this study. Species identification was ide dismutase (SOD) and glutamic-oxalo- performed using primarily esterase (EST) acetic transaminase (GOT) activities. phenotypes in combination with malate Relative mobility of isozymes was calculated dehydrogenase (MDH) (Esbenshade and and phenotype designations were assigned Triantaphyllou, 1985); when needed, the according to Esbenshade and Triantaphyl- morphology of females perineal patterns lou (1985) and Fargette (1987a). There (Hartman and Sasser, 1985; Rammah and was no EST phenotype described for M. Hirschman, 1988), morphometric charac- graminis. Therefore, the phenotype Mg1 ters (Jepson, 1987; Rammah and Hir- was designated for this nematode species, schman, 1988) and mitochondrial DNA which represent the first two letters of the (Powers and Harris, 1993) were used to aid Meloidogyne sp. followed by the number of in the identification. major bands of EST activity, as proposed Samples used for this study mainly con- previously (Brito et al., 2008). It is worth sisted of roots collected individually from mentioning that according to Esbenshade ornamental plants growing in several nurs- and Triantaphyllou (1985), the phenotype eries, from various landscapes as well as G1 should be assigned to this species; how- samples submitted to the Nematology Lab- ever, that phenotype had already been oratory, Division of Plant Industry, Gaines- assigned to M. graminicola (Carneiro et al., ville, Florida for certification. Each sample 2000). Preliminary results of this study was given an accession number, represent- have been reported (Brito et al., 2004b). 90 NEMATROPICA Vol. 40, No. 1, 2010

RESULTS AND DISCUSSION The EST phenotype Mf3 (Rm: 38.37, 40.69, 44.18) (Brito et al., 2008), which is A total of six root-knot nematode spe- species- specific for M. floridensis, was iso- cies and six unidentified populations of lated from three populations found repro- Meloidogyne spp. were found infecting 75 ducing in Dracena sp. and Hibiscus sp. ornamental plant species belonging to 36 (Table 1; Fig. 1). This is the first report of botanical families in this study (Tables 1 ornamental plants being host for M. and 2). Only the major bands of EST and floridensis. All populations of this nematode MDH activities were used for phenotype species reported in this study were designation and species identification as detected singly and not as mixed popula- described in previous studies (Dickson et tions with other Meloidogyne spp. Nonethe- al., 1970, 1971; Esbenshade and Trianta- less, M. floridensis has been reported in phyllou, 1985; Fargette, 1987a; Pais and mixed populations with M. incognita and Abrantes, 1989; Carneiro et al., 2000; Kars- M. javanica infecting Phaseolus spp. (Brito et sen, 2002, Castro et al., 2003; Cofcewicz et al., 2008). It is worth mentioning that M. al., 2005; Molinari et al., 2005). A schematic floridensis is known to occur only in Florida representation of EST and MDH pheno- (Handoo et al., 2004). types containing relative migration (Rm) Meloidogyne graminis was the only spe- values and band numbers detected for cies infecting nine root samples of Steno- each Meloidogyne sp. identified in each sam- taphrum secundatum var. Amerishade and S. ple is presented in Figs. 1 and 2. secundatum and exhibited a single EST band with a very slow migration (Rm: 19.2) (Fig. 1). Perineal patterns of females and Esterase phenotypes (EST) morphometrics of J2 were also used to aid Fourteen distinct EST phenotypes and the identification of this nematode species 26 major bands of EST activities were iden- (data not shown). Results were similar to tified among the populations of Meloidog- those reported previously (Jepson, 1987). yne spp. examined (Tables 1 and 2; Fig. 1). The phenotype I1 (Rm: 39.5), which A total of six root-knot nematode species has been consistently identified from M. plus six unidentified populations were incognita collected in several regions found in this study. around the world (Esbenshade and Trian- The phenotype A2 (Rm: 45.35, 48.83) taphyllou, 1985; Pais and Abrantes, 1989; (Tables 1 and 2; Fig. 1) was detected in 44 Carneiro et al., 2004; Brito et al., 2008) was populations of M. arenaria found infecting observed in 59 populations of M. incognita several ornamental plants species either infecting several ornamental plants (Tables alone or in mixed populations with M. incog- 1 and 2; Fig. 1). Nevertheless, four addi- nita, M. javanica or M. mayaguensis (Tables 1 tional populations identified as M. incog- and 2). The A2 phenotype is species-specific nita exhibited the phenotype I2 (Rm: 39.5, for M. arenaria, and has been observed in sev- 41.0) (Table 2; Fig. 1). This phenotype eral populations of this nematode in Brazil shares a common band with phenotype I1 (Carneiro et al., 2000; Castro et al., 2003; at Rm 39.5 (Fig. 1). Similarly, both pheno- Cofcewicz et al., 2004), Guadalupe, French types were detected among populations of Guiana and Martinique (Cofcewicz et al., M. incognita from Brazil (Carneiro et al., 2005), Portugal (Pais and Abrantes, 1989), 1996, 2000, 2004; Castro et al., 2003; Bar- West Africa (Fargette et al., 1987b), and the bosa et al., 2004; Cofcewicz et al., 2004), United States (Brito et al., 2008). Guadalupe, French Guiana and Martin- Ornamental plants as hosts of Meloidogyne species: Brito et al.91 ., 2008. et al Hillsborough and Levy Martin, Orange, Palm Beach, Santa Rosa and Volusia RuscaceaeMalvaceae Dade and Lake Poaceae Rosaceae Araliaceae Araliaceae Arecaceae Zingiberaceae LiliaceaeBalsaminaceae Orange Buddlejaceae Verbenaceae Cyperaceae Euphorbiaceae Rubiaceae Agavaceae Araceae Alachua, Dade, Hillsborough, Botanical families County of origin var. var. Poaceae Alachua, Brevard, var. sp. secundatum × clandonensis Plant hosts sp. × weyeriana Impatiens sp. sp. sp. sp. sp. Hibiscus Stenotaphrum Amerishade Schefflera actinophylla S. arboricola Syagrus romanzoffiana Zingiber officinale Allium schoenoprasum Sibiricum Dracena Caryopteris papyrus Cypress Euphorbia tirucalli Gardenia Hosta Hibiscus rosa-sinensis Rosa Alocasia Buddleia v M. javanica. MDH N1a N3 N1 N1 ., 2008). M. mayaguensis. et al species infecting ornamental plants in Florida. Enzyme phenotypes EST Mg1 A2 Mf3 A2 Meloidogyne spp. sp. 1 found in mixed population with sp. 3 and 4 found in mixed population with MDH phenotype not resolved. Meloidogyne EST = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985) Brito nematode was isolated, fol- New EST phenotypes: designations were assigned using first two letters of the plant host species name from which root-knot Meloidogyne M. graminis M. arenaria M. floridensis M. arenaria lowed by number of major isozyme bands (Brito Table 1. Isozyme phenotypes of Table v w x y z Meloidogyne 92 NEMATROPICA Vol. 40, No. 1, 2010 ., 2008. et al Hardee, Orange, Palm Beach and Volusia Hillsborough and Palm Beach Solanaceae Alachua, Dade, Flagler, Buxaceae Marantaceae Verbenaceae Araceae Moraceae Rubiaceae Balsaminaceae Rubiaceae Liliaceae Arecaceae AraceaeSolanaceae Verbenaceae Alachua, Dade, Verbenaceae Solanaceae Botanical families County of origin sp.

sp. sp. sp. sp. sp. Buddlejaceae Plant hosts sp. Buxaceae spp. sp. S. secundatum Brugmansia Buddleja Buxus Buxus microphylla Calathea rufibarba Duranta erecta, aureum Epipremnum Ficus Gardenia Impatiens Ixora Ophiopogon Syagrus romanzoffiana Amorphophallus Brugmansia suaveolens Clerodendrum ugandense Lantana montevidensis Solanum rantonnetii v M. javanica. species infecting ornamental plants in Florida. MDH N1 ., 2008). M. mayaguensis. et al Meloidogyne Enzyme phenotypes EST I1 N1 J3 spp. sp. 1 found in mixed population with sp. 3 and 4 found in mixed population with MDH phenotype not resolved. Meloidogyne EST = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985) Brito nematode was isolated, fol- New EST phenotypes: designations were assigned using first two letters of the plant host species name from which root-knot Meloidogyne lowed by number of major isozyme bands (Brito Table 1. (Continued)Isozyme phenotypes of Table M. incognita M. javanica v w x y z Meloidogyne Ornamental plants as hosts of Meloidogyne species: Brito et al.93 ., 2008. et al Dade, Duval, Flagler, Gilchrist, Dade, Duval, Flagler, Hardee, Hillsborough Lake, Nassau, Orange, Palm Beach, Pasco and Putnam Alliaceae BuddlejaceaeLythraceae Alachua and Seminole Liliaceae LamiaceaeSolanaceae Collier, Alachua, Citrus, Clay, Solanaceae Buddlejaceae Myrtaceae Myrtaceae Myrtaceae Verbenaceae Rubiaceae Malvaceae Lythraceae Verbenaceae Oleaceae Myricaceae Botanical families County of origin × sunray sp. spp. sp. sp. Plant hosts Tulbaghia violacea Tulbaghia Buddleja davidii indica Lagerstroemia Ophiopogan japonicus Ajuga reptan, Brugmansia Brugmansia Buddleja davidii Callistemon Callistemon citrinus C. viminalis Clerodendrum ugandense Gardenia Hibiscus grandiflorus indica Lagerstroemia Lantana montevidensis Ligustrum Myrica cerifera v M. javanica. species infecting ornamental plants in Florida. MDH ., 2008). M. mayaguensis. et al Meloidogyne Enzyme phenotypes EST J2VS1-S1 N1 N1a sp. 1 found in mixed population with spp. sp. 3 and 4 found in mixed population with MDH phenotype not resolved. Meloidogyne EST = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985) Brito nematode was isolated, fol- New EST phenotypes: designations were assigned using first two letters of the plant host species name from which root-knot Meloidogyne lowed by number of major isozyme bands (Brito M. javanica M. mayaguensis v w x y z Table 1. (Continued)Isozyme phenotypes of Table Meloidogyne 94 NEMATROPICA Vol. 40, No. 1, 2010 ., 2008. et al and Volusia and Volusia Rubiaceae Lamiaceae Salicaceae Solanaceae Bignoniaceae Melastomaceae Melastomaceae Aquifoliaceae Myricaceae Arecaceae Beach Amaranthaceae Palm Arecaceae Botanical families County of origin sp. Arecaceae Pasco × compacta × elegans sp. Araceae Highlands and Palm Beach sp. Balsaminaceae sp. Rubiaceae sp. Begoniaceae Alachua, Marion, Orange, Plant hosts sp. Asclepiadaceae × sepulcralis Penta lanceolata Plectranthus scutellarioides Salix Solandra maxima capensis, Tecomaria Tibouchina Tibouchina Begonia Gardenia Hoya Ilex crenata Impatiens Myrica cerifera Syagrus romanzoffiana Amaranthus tricolor Caladium Phoenix dactylifera Washingtonia v M. javanica. species infecting ornamental plants in Florida. MDH ., 2008). M. mayaguensis. et al Meloidogyne Enzyme phenotypes EST A2;I1 N1;N1 A2; I1; VS1-S1 N1; N1 N1a A2;J3A2;J3 VS1-S1 N1;N1 N1;N1 N1a spp. sp. 1 found in mixed population with sp. 3 and 4 found in mixed population with MDH phenotype not resolved. Meloidogyne EST = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985) Brito nematode was isolated, fol- New EST phenotypes: designations were assigned using first two letters of the plant host species name from which root-knot Meloidogyne lowed by number of major isozyme bands (Brito Table 1. (Continued)Isozyme phenotypes of Table M. arenaria and M. incognita M. arenaria M. incognita, javanica and M. mayaguensis and M. javanica M. arenaria M. javanica M.arenaria, and M. mayaguensis v w x y z Meloidogyne Ornamental plants as hosts of Meloidogyne species: Brito et al.95 ., 2008. et al Madison and Sarasota Myricaceae Nassau VerbenaceaeMyricaceae Dade and Flagler Acanthaceae VerbenaceaeMyricaceae Alachua Rubiacea Flagler and Myricaceae Malvaceae Verbenaceae Verbenaceae Arecaceae Botanical families County of origin sp. Acanthaceae sp. Solanaceae Alachua and Dade sp. Solanaceae Alachua, Hardee, Marion × clandonensis sp. Solanaceae Plant hosts sp. Caprifoliaceae Alachua and Orange Clerodendrum ugandense Myrica cerifera Myrica cerifera Abelia Justicia brandegeeana Petunia Pseuderanthemum Caryopteris Myrica cerifera jasminoides Gardenia Brugmansia Brugamansia Myrica cerifera Hibiscus grandiflorus Lantana camara L. montevidensis Washingtonia v M. javanica. species infecting ornamental plants in Florida. — MDH ., 2008). N1 M. mayaguensis. et al Meloidogyne Enzyme phenotypes EST y y A2; VS1-S1I1;J3 N1; N1a N1;N1 I1;J3; VS1-S1 N1;N1 N1a I1; VS1-S1J3; VS1-S1 N1; N1a N1; N1a Ep2 x sp. 2 Vo1 spp. sp. 1 found in mixed population with sp. 3 and 4 found in mixed population with sp. 1 MDH phenotype not resolved. Meloidogyne EST = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985) Brito nematode was isolated, fol- New EST phenotypes: designations were assigned using first two letters of the plant host species name from which root-knot Meloidogyne M. arenaria and M. mayaguensis M. arenaria Meloidogyne M. incognita and javanica M. incognita, javanica and M. mayaguensis M.incognita and M. mayaguensis M. javanica and mayaguensis lowed by number of major isozyme bands (Brito Table 1. (Continued)Isozyme phenotypes of Table v w x y z Meloidogyne Meloidogyne 96 NEMATROPICA Vol. 40, No. 1, 2010 ., 2008. et al PodocarpaceaeCaprifoliaceae Brevard and Hillsborough Caprifoliaceae CaprifoliaceaeCaprifoliaceae Brevard and Hillsborough RubiaceaeBuxaceae Hardee and Highlands Myrtaceae Rubiaceae Alachua Lamiaceae Liliaceae Botanical families County of origin and . sp. Plant hosts sp Podocarpus Vibernun odoratissimum suspensum V. Vibernun odoratissimum Vibernun suspensum jasminoides Gardenia unidentified ornamental plant Buxus microphylla Callistemon viminalis Gardenia Plectranthus scutellariodes Liriope muscari v M. javanica. species infecting ornamental plants in Florida. MDH N1 N1a N1 ., 2008). M. mayaguensis. et al Meloidogyne y y y Enzyme phenotypes EST Cv2 Vo1 z sp. 3 sp.4sp. 5 Vo2 Gj2 N1 spp. sp. 1 found in mixed population with sp. 3 and 4 found in mixed population with sp. 6 MDH phenotype not resolved. Meloidogyne EST = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985) Brito nematode was isolated, fol- New EST phenotypes: designations were assigned using first two letters of the plant host species name from which root-knot Meloidogyne lowed by number of major isozyme bands (Brito Table 1. (Continued)Isozyme phenotypes of Table Meloidogyne Meloidogyne Meloidogyne Meloidogyne v w x y z Meloidogyne Ornamental plants as hosts of Meloidogyne species: Brito et al.97 County of origin Dade and Palm Beach Calhoun, Dade, Hillsborough, Jackson, Manatee and Palm Beach Palm Beach Dade and Hendry Gadsden, Hillsborough, Lake, Lee and Orange Palm Beach and St. Johns Dade, Hardee, Hendry, Palm Beach Broward Volusia Alachua and Orange Collier Lake . 2 7 1 2 6 5 1 1 1 2 1 1 = M. mayaguensis , Mm Number of Samples = M. javanica y MDH , Mj N1 N1 N2a N1 N1 N1a N1; N1 N1; N1 N1; N1a = M. incognita Enzyme phenotypes , Mi EST A2 I1 I1 I2 J3 VS1-S1 A2; I1 A2; VS1-S1 N1; N1a A2; I1; VS1-S1A2; I1; J3; VS1-S1I2; J3 N1; N1a N3; N1; N1 N1a J3; VS1-S1 = M. arenaria species found infecting unidentified ornamental plants in Florida along with their isozyme phenotypes and county of origin. Ma x Mm spp. spp.: and Mm Meloidogyne and Mi Mm Mj Mm and and and and Meloidogyne Est = Esterase, MDH = Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou (1985). Est = Esterase, MDH Malate dehydrogenase. Phenotype designation according to Esbenshade and Triantaphyllou Ma Mi Mi Mi Mj Mm Ma Ma Ma, Mi Ma, Mi, Mj Mi Mj Table 2. Table x y Meloidogyne 98 NEMATROPICA Vol. 40, No. 1, 2010

Fig. 1. Schematic representation of esterase phenotypes of Meloidogyne spp. infecting ornamentals in Florida. A2 = M. arenaria, Mf3 = M. floridensis, Mg1 = M. graminis, I1 and I2 = M. incognita, J3 and J2 = M. javanica, VS1-S1 = M. mayaguensis, Ep2 = M. sp.1, Mc1 = M. sp.2, Vo1 = M. sp.3, Vo2 = M. sp.4, Gj2 = M. sp.5 and Cv2 = M. sp.6. zRm = Relative mobility ique (Cofcewicz et al., 2005), Portugal (Pais Fig. 1). Likewise, this phenotype was also and Abrantes, 1989) and United States reported from populations of M. javanica (Brito et al., 2008). Phenotypes I1 and I2 infecting Abelmoschus esculentus (Oliveira et were never found together in the same al., 2007) and Musa sp. (Cofcewicz et al., sample in this study; however the detection 2004) in Brazil, and also Musa sp. from of both phenotypes among populations of Guadalupe, French Guiana and Martin- M. incognita could be associated with some ique (Cofcewicz et al., 2005). intraspecific variability. All 72 nematode populations identified The species-specific phenotype J3 as M. mayaguensis exhibited two major bands appeared in 36 populations identified as of EST activity (Rm 29.06; 38.37), consistent M. javanica (Tables 1 and 2; Fig. 1). These with the VS1-S1 phenotype (Fig. 1). At populations occurred singly or mixed with times, each of these bands resolved into two M. arenaria, M. incognita and M. mayaguen- minor bands. Morphology of perineal pat- sis (Tables 1 and 2). These results were con- terns and morphometrics of selected char- sistent with those in previous studies acters were similar to those from the species (Esbenshade and Triantaphyllou, 1985; description (Rammanh and Hisrchmann, Pais and Abrantes, 1989; Tomaszewski et al., 1988) and other isolates of M. mayaguensis 1994; Carneiro et al., 1996, 2000, 2004; Cas- found in Florida (Brito et al., 2004a). Fur- tro et al., 2003; Cofcewicz et al., 2004, 2005; thermore, analysis of the mtDNA region Molinari et al., 2005; Brito et al., 2008). between COII and lrRNA genes was also Three populations of M. javanica infecting used to compare with the EST phenotype. Buddleja davidii and Ophiopogon japonicus Results were in agreement with those exhibited the J2 phenotype (Table 1; reported previously (Brito et al., 2004a). Ornamental plants as hosts of Meloidogyne species: Brito et al.99

have identical sequences of the mtDNA region between COII and lrRNA genes (Xu et al., 2004). Furthermore, sequence data obtained from studies based on COI, ITS, and IGS showed that the Swiss M. enter- olobii populations, and two isolates of M. mayaguensis, each from Brazil and the USA Fig. 2. Schematic representation of malate dehydroge- nase phenotypes of Meloidogyne spp. populations showed 100% similarity (Kiewnick et al., found infecting ornamental plants in Florida. N1 = M. 2007; 2008). Currently, M. mayguensis is arenaria, M. floridensis, M. incognita, M. javanica, M. being synonymized with M. enterolobii (Ger- sp.3, M. sp.4, and M. sp.6; N2a = M. incognita; N3 = M. arenaria, N1a = M. graminis, M. mayaguensis, M. sp.2 rit Karssen, personal communication). and M. sp.5. Phenotypes designation according to Es- Six unique EST profiles were detected benshade and Triantaphyllou (1985). zRm= Relative from 19 unidentified populations of mobility. Meloidogyne spp. infecting different orna- mental plants in this study. These popula- tions were named Meloidogyne sp. 1 to 6. The EST phenotype, VS1-S1 proved to Two populations of Meloidogyne sp. 1 exhib- be of high diagnostic value to distinguish ited two EST major bands Rm: 41.0, 43.70) M. mayaguensis from all other root-knot (Table 1; Fig. 1) similar to a phenotype nematode species identified in this study, (Ep2) already described for a root-knot particularly M. incognita. It is most likely nematode infecting originally Eclipta pros- that M. mayaguensis has been erroneously trata in Florida (Brito et al., 2008), whereas identified as M. incognita in the past in Flor- five new EST phenotypes (Mc1, Cv2, Gj2, ida due to some similarity of the perineal Vo1, and Vo2) were described from the patterns of these two species (Brito et al., remaining 17 unidentified populations 2004a, 2008). In North America, this nem- (Table 1). Phenotype designations used to atode is known to occur only in Florida assign these new phenotypes were the same (Brito et al., 2004a, b). Meloidogyne may- as previously reported (Esbenshade and aguensis was identified from root samples Triantaphyllou, 1985; Brito et al., 2008). A of several ornamental plant species belong- phenotype designated as Mc1 with one ing to 16 botanical families (Table 1). To major band of activity at Rm: 32.0 was our knowledge Ajuga reptans, Amaranthus detected in three populations of Meloidog- tricolor, Buddleja davidii, Caryopteris × clan- yne sp. 2 infecting initially Myrica cerifera donensis, Clerodendrum ugandense, Hibiscus (Table 1; Fig. 1). In a differential host test grandiflorus, Lagerstroemia indica, Penta lan- (Hartman and Sasser, 1985) single egg ceolata, Plectranthus scutellarioides, and Solan- mass isolates obtained from Meloidogyne sp. dra maxima are new host records for M. 2 reproduced on tobacco ‘NC95’, cotton mayaguensis. ‘DPL 16’, watermelon ‘Charleston Grey’, It is worth mentioning that the pheno- pepper ‘California Wonder’ and tomato type VS1-S1 also has been reported for ‘Rutgers’, but not on peanut ‘Florunner’. another root-knot nematode species, M. Furthermore, the isolates also reproduced enterolobii from China (Yang and Eisenback, well on potato but did not reproduce on 1983; Esbenshade and Triantaphyllou, corn or wheat (data not shown). 1985). These two root-knot nematode spe- The phenotype Vo1, with one major cies share not only biochemical and mor- band of activity (Rm 51.16) (Table 1; Fig. 1) phological characteristics, but they also and Vo2, with two major bands (26.0; 51.16) 100 NEMATROPICA Vol. 40, No. 1, 2010

(Table 1; Fig. 1) were observed in mixture the populations of Meloidogyne spp. in this population with Meloidogyne sp. 3 and Meloi- study (Tables 1 and 2; Fig. 2). The pheno- dogyne 4 infecting Vibernum odoratissimum. type N1 (Rm: 20.2) was detected in all pop- These two phenotypes share a major band ulations of M. arenaria, M. floridensis, M. at Rm 51.16 and remained stable when incognita, M. javanica, Meloidogyne sp. 3, nematode isolates were inoculated and Meloidogyne sp. 4 and Meloidogyne sp. 6 reared on tomato ‘Rutgers’. Furthermore, (Tables 1 and 2; Fig. 2), except one popula- nematode isolates with phenotypes Vo1 or tion of M incognita, which exhibited a Vo2 showed the same host reactions when unique phenotype (N2a) with two major inoculated on differential host plant culti- bands of activity at Rm: 20.2: 23.1 (Table 2; vars (Hartman and Sasser, 1985); both iso- Fig. 2) and two populations of M. arenaria, lates reproduced on all plant cultivars which showed the N3 phenotype (Rm: except peanut ‘Florunner’ and pepper ‘Cal- 20.0:23.1:26.0) with three bands of MDH ifornia Wonder’. These nematode isolates activity (Tables 1; Fig. 2). The N1 pheno- showed the same MDH (NI) (Fig. 2), type has been commonly associated with glutamic-oxaloacetic transaminase (NIa) the three major species of Meloidogyne col- and superoxide dismutase (N2b) pheno- lected in other regions of the world types (Esbenshade and Triantaphyllou, (Esbenshade and Triantaphyllou, 1985; 1985) regardless of the EST phenotype. Pais and Abrantes, 1989; Carneiro et al., Another phenotype, Gj2 with two major 2004, Brito et al., 2008). Nonetheless, the bands (Rm 50.6; 52.2) (Table 1; Fig. 1) was phenotype N3 has been detected in some detected in two nematode populations des- populations of M. arenaria in the United ignated as Meloidogyne sp. 5, which were States (Brito et al., 2008) and also from found infecting Gardenia jasminoides and an other geographical regions (Esbenshade unidentified ornamental plant in Hardee and Triantaphyllou, 1985; Pais and and Highlands Counties, respectively. Iso- Abrantes, 1989; Cofcewicz et al., 2005). To lates of these nematodes had the same host our knowledge, this is the first report of the reactions as those of M. javanica race 1 and phenotype N2a identified from M. incog- M. arenaria race 2 (Hartman and Sasser, nita, which could be associated with some 1985). Meloidogyne sp. 6, initially found variability among populations of this nema- reproducing on Callistemon viminalis in Ala- tode species. This phenotype remained sta- chua County, exhibited the phenotype Cv2 ble when a nematode isolate was and had three bands (Rm 28; 40.2) (Table inoculated and reared on tomato ‘Rut- 1; Fig. 1). Isolates obtained from the field gers’. population showed the same differential The phenotype N1a, which showed one host reaction as that of M. incognita race 2. very strong band (Rm: 28.1) of MDH activ- Currently, single egg masses obtained from ity was detected in all the populations of M. all unidentified root-nematode species are graminis, M. mayaguensis, Meloidogyne sp. 2 being reared on tomato ‘Rutgers’ and will and Meloidogyne sp. 5 (Tables 1 and 2; Fig. be used for further investigation as an 2). An identical phenotype has been attempt to identify each nematode species. reported from populations of M. chitwoodi, M. enterolobii, M. naasi, M. oryzae, M. plan- Malate dehydrogenase phenotypes tani (Esbenshade and Triantaphyllou, 1985), and M. partityla and M. graminicola Five bands of MDH activity and four (Brito et al., 2006). Therefore it is of MDH phenotypes were observed among restricted diagnostic value to differentiate Ornamental plants as hosts of Meloidogyne species: Brito et al. 101

these Meloidogyne spp.; however, the N1a usda/current/NursProd/NursProd-09-26- phenotype could aid in the discrimination 2007.pdf. Accessed April 2010. of these root-knot nematode species from Anonymous. 2008. Florida Department of Agriculture and Consumer Services. Overview of Florida Ag- M. arenaria, M. floridensis, M. incognita and riculture. http://www.florida-agriculture.com/ M. javanica. agfacts.htm. Accessed April 2010. The results obtained in this study, in Barbosa, D. H. S. G., H. D. Vieira, R. M. Souza, and C. combination with those already reported P. Silva. 2004. Survey of root-knot nematode (Me- in other studies, clearly show the usefulness loidogyne spp.) in coffee plantations in state of Rio de Janeiro, Brazil. Nematologia Brasileira of isozymes as a valuable tool for identifica- 28:43-47. tion of Meloidogyne spp. in a large number Barker, K. R., and D. M. Benson. 1977. Japanese hol- of samples. The EST and MDH phenotypes lies: intolerant hosts of Meloidogyne arenaria in mi- were useful to detect mixed populations of croplots. Journal of Nematology 9:330-334. Meloidogyne spp. as well as to determine new Benson, D. M., and K. R. Barker. 1985. Nematodes - a threat to ornamental plants in the nursery and host records for root-knot species; however, landscape. Plant Disease 69:97-100. EST had a higher diagnostic value than Brito, J. A., R. Kaur, R. Cetintas, J. D. Stanley, M. L. MDH in the discrimination of the Mendes, E. J. McAvoy, T. O Powers, and D. W. Meloidogyne spp. found infecting ornamen- Dickson. 2008. Identification and isozyme char- tal plants in Florida. acterization of Meloidogyne spp. infecting horti- cultural and agronomic crops, and weed plants in Florida. Nematology 10:757-766. ACKNOWLEDGEMENTS Brito, J. A., R. Kaur, D. W. Dickson, J. R. Rich, and L. A. Halsey. 2006. The pecan root-knot nematode, Meloidogyne partityla Kleynhans, 1986. Nematolo- This research was supported by the gy Circular No. 222, Florida Department of Agri- Tropical and Subtropical Agriculture culture and Consumer Services, Division of Plant Research grant (T-STAR) #2005-34135- Industry, Gainesville, Florida. 15895, Cooperative State Research, Educa- Brito, J. A., T. O. Powers, P. G., Mullin, R. N., Inserra, tion and Extension Services (CSREES), and D. W. Dickson. 2004a. Morphological and molecular characterization of Meloidogyne may- United States Department of Agriculture, aguensis isolates from Florida. Journal of Nema- USA. The authors thank Dr. Richard E. tology 36:232-240. Weaver, Jr., Botany Section, Division of Brito, J. A., J. D. Stanley, R. Cetintas, T. O. Powers, R. Plant Industry, Gainesville, Florida, for N. Inserra, E. J. McAvoy, M. L. Mendes, W. T. identification of weed species and also Ana Crow, and D. W. Dickson. 2004b. Identification and host preference of Meloidogyne mayaguensis, L. Ochoa, Christine A. Zamora, Charles L. and other root-knot nematodes from Florida, Spriggs, Ping Qiao, Matthew W. Brodie and and their susceptibility to Pasteuria penetrans. Sol F. Locker for their support during the Journal of Nematology 36:308-309. field work. Block, V. C., M. S. Phillips, J. W. McNicol, and M. Far- gette. 1997a. Genetic variation in tropical Meloid- ogyne spp. as shown by RAPD-PCR. Fundamental LITERATURE CITED and Applied Nematology 20:127-133. Block, V. C., M. S. Phillips, and M. Fargette. 1997b. Adam, M. A. M., M. S. Phillps, and V. C. Block. 2005. Comparison of sequences from the ribosomal Identification Meloidogyne spp. from northeast DNA intergenic region of Meloidoygne mayaguen- Libya and comparison of their inter- and intro- sis and other major root-knot nematodes. Jour- specific genetic variation using RAPDs. Nematol- nal of Nematology 29:16-22. ogy 7:599-609. Blok, V. C., J. Wishart, M. Fargette, K. Berthier, and M. Anonymous. 2007. The National Agricultural Statis- S. Phillips. 2002. Mitochondrial DNA differences tics Service. Released 9 September 2007, Agricul- distinguishing Meloidogyne mayaguensis from the tural Statistics Board, U.S. Department of Agri- major species of tropical root-knot nematodes. culture. http://usda.mannlib.cornell.edu/ Nematology 4:773-781. 102 NEMATROPICA Vol. 40, No. 1, 2010

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Received: Accepted for publication: 1/IV/2010 13/V/2010 Recibido: Aceptado para publicación