2 enzimas meloidogyne ornamentales

87

MELOIDOGYNE

SPP. INFECTING ORNAMENTAL PLANTS IN FLORIDA

J. A. Brito

1*

, R. Kaur

2

, R. Cetintas

3

, J. D. Stanley

1

, M. L. Mendes

2

, T. O. Powers

4

, and D. W. Dickson

2

1

Division of Plant Industry, Gainesville, FL 32614, USA;

2

Entomology and Nematology Department,University of Florida, Gainesville, FL 32611, USA;

3

Department of Plant Protection, KahramanmarasSutcu Imam University, Kahramanmaras, 46060, Turkey;

4

Department 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 nematode species. When needed,the morphology of female perineal patterns, morphometric characters and mitochondrial DNA wereused 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

Hibiscus

, 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

,

Pentalanceolata

,

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 yADN 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 demás alta utilidad en el diagnóstico de especies de

Meloidogyne

en este estudio, pero la actividad demalato 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 denematodo 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

. Nuevosregistros de especies vegetales para

M. mayaguensis

incluyen

Ajuga reptans

,

Amaranthus tricolor

,

Buddlejadavidii

,

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.

88 NEMATROPICA Vol. 40, No. 1, 2010

INTRODUCTION

In 2007, Florida ranked third amongstates in the USA in the production andgross sale of nursery plants and ranked firstin production of ornamental grasses,woody ornamental plants, preparativenursery materials and palm trees (Anony-mous, 2007). Florida led the nation in salesof potted foliage for indoor use and hang-ing baskets and also was the nation’s leaderin sales of cut cultivated greens in 2005(Anonymous, 2008). Many of the orna-mental plants currently used for landscap-ing are susceptible to several pathogens,including root-knot nematodes (

Meloidog-yne

spp.) (Barker and Benson, 1977; Ben-son and Barker, 1985; Sinclair

et al

., 1987;Martinez

et al

., 2003

).

Up to March 2010, 97 nominal species of

Meloidogyne

have been described. The properidentification of

Meloidogyne

spp. is veryimportant for implementation of plantbreeding, nematode management, and par-ticularly for certification and quarantine inregulatory programs. Species identification isprimarily based on morphological and mor-phometrics characters of the males, femalesand second stage juveniles (Jepson, 1987).Accurate and reliable identification usingmorphology and morphometrics is a difficultand time consuming task that requires welltrained personnel. Morphological charac-ters, especially female perineal patterns, areone of the major characters used to aid in theidentification of root-knot nematodes in rou-tine analysis; however, perineal patterns arevariable, and may lead to misidentification ofaberrant populations and new species. Con-versely, biochemical markers such as isozymephenotypes used in conjunction with mor-phological and morphometric, allow a moreprecise and accurate identification of

Meloid-ogyne

spp.The relative stability of isozymes pheno-

types within

Meloidogyne

spp. (Fargette,

1987a; De Waele and Elsen, 2007;) hasmade them a useful tool for nematodeidentification. Among the isozyme systems,esterase (EST) has the highest diagnosticvalue because the majority of the pheno-types described are nematode-species spe-cific; however, the use of more than oneisozyme may be needed as the result ofintraspecific variability and differences inmigrations obtained from different electro-phoresis apparatus and laboratories.Isozyme analysis, specifically EST in combi-nation with malate dehydrogenase (MDH)(Dickson

et al.

, 1970; Esbenshade and Tri-antaphyllou, 1985), resolved using poly-acrylamide gel electrophoresis (PAGE) hasproven to be a valuable, fast and reliablemethod to identify the most common root-knot nematode species collected from dif-ferent parts of the world (Dickson

et al

.,1970, 1971; Esbenshade and Triantaphyl-lou, 1985; Fargette, 1987a; 1987b; Pais andAbrantes, 1989; Carneiro

et al.

, 1996; 2000;Karssen, 2002, Castro

et al.

, 2003; Cofcewicz

et al.

, 2004, 2005; Molinari

et al.

, 2005);however, novel esterase phenotypes havebeen discovered in root-knot nematodesurveys (Esbenshade and Triantaphyllou,1985; Hernandez

et al.

, 2004; Adam

et al.

,2005; Molinari

et al.

, 2005). To determinewhether theses novel phenotypes representa new root-knot nematode species, a nema-tode species already described but with anunknown EST/MDH phenotype or anaberrant pattern; a combination of mor-phological, morphometric, host range, bio-chemical and molecular studies areneeded. Currently, several DNA-basedmethods such as restriction fragmentlength polymorphisms (RFLP), randomamplified polymorphic DNA (RAPD),amplification of the ribosomal DNA in theintergenic spacer region (IGS) betweenthe 18S and 5S gene, amplification of themitochondrial DNA (mtDNA) regionbetween 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-ful to aid in the identification of

Meloidog-yne

spp. (Powers

et al.

, 1986; 1993; 2005;Blok

et al.

, 1997a; 1997b; 2002; Zijlstra

et al.

,2000; Randig

et al.

, 2002; Handoo

et al.

,2004); however, these methods are stillexpensive to use in large surveys (Powers

etal.

, 2005) as well as in routine analyses inmany nematode diagnostic laboratoriesdue to the costs with equipment, reagentsand DNA sequencing.

The objectives of the current study wereto i) identify the root-knot nematode spe-cies found infecting ornamental plant spe-cies in Florida, ii) evaluate the usefulness ofEST and MDH phenotypes in differentiat-ing

Meloidogyne spp. for routine diagnosticpurposes, and iii) determine the plant hostof each of the Meloidogyne sp. identified.

MATERIALS AND METHODS

A total of 206 root samples were col-lected from ornamental plants in 26 coun-ties in Florida for isolation andidentification of root-knot nematode spe-cies in this study. Species identification wasperformed using primarily esterase (EST)phenotypes in combination with malatedehydrogenase (MDH) (Esbenshade andTriantaphyllou, 1985); when needed, themorphology of females perineal patterns(Hartman and Sasser, 1985; Rammah andHirschman, 1988), morphometric charac-ters (Jepson, 1987; Rammah and Hir-schman, 1988) and mitochondrial DNA(Powers and Harris, 1993) were used to aidin the identification.

Samples used for this study mainly con-sisted of roots collected individually fromornamental plants growing in several nurs-eries, from various landscapes as well assamples submitted to the Nematology Lab-oratory, Division of Plant Industry, Gaines-ville, Florida for certification. Each samplewas given an accession number, represent-

ing the year of collection and the serialnumber to maintain sample identity. Rootsamples with limited infection were cutinto ca. 2-cm pieces, mixed with pasteur-ized soil and placed into a clay pot in whicha tomato seedling (Solanum lycopersicum‘Rutgers’) was transplanted. Plants weremaintained in a greenhouse at 26 ± 1.8°Cuntil used for nematode identification.The procedure for isozyme extractionfrom each nematode female was the sameas that reported by Brito et al., 2008. Atleast 26 egg-laying females were dissecteddirectly from each root system and isozymeprofiles determined using polyacrylamidegel electrophoresis (PAGE) with two gelsrun at the same time (Brito et al., 2008).One gel was stained for both MDH andEST activity (Esbenshade and Triantaphyl-lou, 1985), whereas the second one, wasstained only for EST. Extracts from singleM. javanica females were added separatelyto individual wells on each gel as standards.The nematodes species with new orunknown EST phenotypes were alsostained for two different isozymes; superox-ide dismutase (SOD) and glutamic-oxalo-acetic transaminase (GOT) activities.Relative mobility of isozymes was calculatedand phenotype designations were assignedaccording to Esbenshade and Triantaphyl-lou (1985) and Fargette (1987a). Therewas no EST phenotype described for M.graminis. Therefore, the phenotype Mg1was designated for this nematode species,which represent the first two letters of theMeloidogyne sp. followed by the number ofmajor bands of EST activity, as proposedpreviously (Brito et al., 2008). It is worthmentioning that according to Esbenshadeand Triantaphyllou (1985), the phenotypeG1 should be assigned to this species; how-ever, that phenotype had already beenassigned to M. graminicola (Carneiro et al.,2000). Preliminary results of this studyhave been reported (Brito et al., 2004b).


RESULTS AND DISCUSSION

A total of six root-knot nematode spe-cies and six unidentified populations ofMeloidogyne spp. were found infecting 75ornamental plant species belonging to 36botanical families in this study (Tables 1and 2). Only the major bands of EST andMDH activities were used for phenotypedesignation and species identification asdescribed in previous studies (Dickson etal., 1970, 1971; Esbenshade and Trianta-phyllou, 1985; Fargette, 1987a; Pais andAbrantes, 1989; Carneiro et al., 2000; Kars-sen, 2002, Castro et al., 2003; Cofcewicz etal., 2005; Molinari et al., 2005). A schematicrepresentation of EST and MDH pheno-types containing relative migration (Rm)values and band numbers detected foreach Meloidogyne sp. identified in each sam-ple is presented in Figs. 1 and 2.

Esterase phenotypes (EST)

Fourteen distinct EST phenotypes and26 major bands of EST activities were iden-tified among the populations of Meloidog-yne spp. examined (Tables 1 and 2; Fig. 1).A total of six root-knot nematode speciesplus six unidentified populations werefound in this study.

The phenotype A2 (Rm: 45.35, 48.83)(Tables 1 and 2; Fig. 1) was detected in 44populations of M. arenaria found infectingseveral ornamental plants species eitheralone or in mixed populations with M. incog-nita, M. javanica or M. mayaguensis (Tables 1and 2). The A2 phenotype is species-specificfor M. arenaria, and has been observed in sev-eral populations of this nematode in Brazil(Carneiro et al., 2000; Castro et al., 2003;Cofcewicz et al., 2004), Guadalupe, FrenchGuiana and Martinique (Cofcewicz et al.,2005), Portugal (Pais and Abrantes, 1989),West Africa (Fargette et al., 1987b), and theUnited States (Brito et al., 2008).

The EST phenotype Mf3 (Rm: 38.37,40.69, 44.18) (Brito et al., 2008), which isspecies- specific for M. floridensis, was iso-lated from three populations found repro-ducing in Dracena sp. and Hibiscus sp.(Table 1; Fig. 1). This is the first report ofornamental plants being host for M.floridensis. All populations of this nematodespecies reported in this study weredetected singly and not as mixed popula-tions with other Meloidogyne spp. Nonethe-less, M. floridensis has been reported inmixed populations with M. incognita andM. javanica infecting Phaseolus spp. (Brito etal., 2008). It is worth mentioning that M.floridensis is known to occur only in Florida(Handoo et al., 2004).

Meloidogyne graminis was the only spe-cies infecting nine root samples of Steno-taphrum secundatum var. Amerishade and S.secundatum and exhibited a single ESTband with a very slow migration (Rm: 19.2)(Fig. 1). Perineal patterns of females andmorphometrics of J2 were also used to aidthe identification of this nematode species(data not shown). Results were similar tothose reported previously (Jepson, 1987).

The phenotype I1 (Rm: 39.5), whichhas been consistently identified from M.incognita collected in several regionsaround the world (Esbenshade and Trian-taphyllou, 1985; Pais and Abrantes, 1989;Carneiro et al., 2004; Brito et al., 2008) wasobserved in 59 populations of M. incognitainfecting several ornamental plants (Tables1 and 2; Fig. 1). Nevertheless, four addi-tional populations identified as M. incog-nita exhibited the phenotype I2 (Rm: 39.5,41.0) (Table 2; Fig. 1). This phenotypeshares a common band with phenotype I1at Rm 39.5 (Fig. 1). Similarly, both pheno-types were detected among populations ofM. incognita from Brazil (Carneiro et al.,1996, 2000, 2004; Castro et al., 2003; Bar-bosa et al., 2004; Cofcewicz et al., 2004),Guadalupe, French Guiana and Martin-

Ornamental plants as hosts of Meloidogyne species: Brito et al. 91Ta

ble

1. I

sozy

me

phen

otyp

es o

f Mel

oido

gyne

spe

cies

infe

ctin

g or

nam

enta

l pla

nts

in F

lori

da.

Mel

oido

gyne

spp

.

En

zym

e ph

enot

ypes

v

Plan

t hos

tsB

otan

ical

fam

ilies

Cou

nty

of o

rigi

nE

STM

DH

M. a

ren

aria

A2

N1

Alo

casi

a sp

.A

race

aeA

lach

ua, D

ade,

H

illsb

orou

gh,

Mar

tin

, Ora

nge

, Pal

m B

each

, Sa

nta

Ros

a an

d Vo

lusi

a B

uddl

eia

× w

eyer

iana

Bud

dlej

acea

e

Car

yopt

eris

× c

land

onen

sis

Verb

enac

eae

Cyp

ress

pap

yrus

Cyp

erac

eae

Euph

orbi

a tir

ucal

liE

uph

orbi

acea

e

Gar

deni

a sp

.R

ubia

ceae

Hos

ta s

p.A

gava

ceae

Hib

iscu

s ro

sa-si

nens

isM

alva

ceae

Ros

a sp

.R

osac

eae

Sche

ffler

a ac

tinop

hylla

Ara

liace

ae

S. a

rbor

icol

aA

ralia

ceae

Syag

rus

rom

anzo

ffian

aA

reca

ceae

Zing

iber

offi

cina

leZ

ingi

bera

ceae

M. a

rena

ria

A2

N3

Alli

um s

choe

nopr

asum

var

.L

iliac

eae

Ora

nge

Sibi

ricu

m I

mpa

tiens

sp.

Bal

sam

inac

eae

M. fl

orid

ensi

sM

f3N

1D

race

na s

p.R

usca

ceae

Dad

e an

d L

ake

Hib

iscu

s sp

.M

alva

ceae

M. g

ram

inis

Mg1

N1a

Sten

otap

hrum

sec

unda

tum

var

. Po

acea

eA

lach

ua, B

reva

rd,

Hill

sbor

ough

an

d L

evy

Am

eris

had

ePo

acea

e

v EST

= E

ster

ase,

MD

H =

Mal

ate

deh

ydro

gen

ase.

Ph

enot

ype

desi

gnat

ion

acc

ordi

ng

to E

sben

shad

e an

d T

rian

taph

yllo

u (1

985)

an

d B

rito

et a

l., 2

008.

wM

DH

ph

enot

ype

not

res

olve

d.x M

eloi

dogy

ne s

p. 1

foun

d in

mix

ed p

opul

atio

n w

ith

M. m

ayag

uens

is.

y New

EST

ph

enot

ypes

: des

ign

atio

ns

wer

e as

sign

ed u

sin

g fi

rst t

wo

lett

ers

of th

e pl

ant h

ost s

peci

es n

ame

from

wh

ich

the

root

-kn

ot n

emat

ode

was

isol

ated

, fol

-lo

wed

by

num

ber

of m

ajor

isoz

yme

ban

ds (

Bri

to e

t al.,

200

8).

z Mel

oido

gyne

sp.

3 a

nd

4 fo

und

in m

ixed

pop

ulat

ion

wit

h M

. jav

anic

a.


S. s

ecun

datu

m

M. i

ncog

nita

I1N

1B

rugm

ansi

a sp

. So

lan

acea

eA

lach

ua, D

ade,

Fla

gler

, H

arde

e, O

ran

ge, P

alm

Bea

ch

and

Volu

sia

Bud

dlej

a sp

.B

uddl

ejac

eae

Bux

us s

p.B

uxac

eae

Bux

us m

icro

phyl

laB

uxac

eae

Cal

athe

a ru

fibar

baM

aran

tace

ae

Dur

anta

ere

cta,

Verb

enac

eae

Epip

rem

num

aur

eum

A

race

ae

Ficu

s sp

.M

orac

eae

Gar

deni

a sp

.R

ubia

ceae

Impa

tiens

sp.

Bal

sam

inac

eae

Ixor

a sp

p.R

ubia

ceae

Oph

iopo

gon

sp.

Lili

acea

e

Syag

rus

rom

anzo

ffian

aA

reca

ceae

M. j

avan

ica

J3N

1A

mor

phop

hallu

s sp

.A

race

aeA

lach

ua, D

ade,

H

illsb

orou

gh a

nd

Palm

Bea

chB

rugm

ansi

a su

aveo

lens

Sola

nac

eae

Cle

rode

ndru

m u

gand

ense

Verb

enac

eae

Lan

tana

mon

tevi

dens

isVe

rben

acea

e

Sola

num

ran

tonn

etii

Sola

nac

eae

Tabl

e 1.

(C

onti

nue

d)Is

ozym

e ph

enot

ypes

of M

eloi

dogy

ne s

peci

es in

fect

ing

orn

amen

tal p

lan

ts in

Flo

rida

.

Mel

oido

gyne

spp

.

En

zym

e ph

enot

ypes

v

Plan

t hos

tsB

otan

ical

fam

ilies

Cou

nty

of o

rigi

nE

STM

DH

v EST

= E

ster

ase,

MD

H =

Mal

ate

deh

ydro

gen

ase.

Ph

enot

ype

desi

gnat

ion

acc

ordi

ng

to E

sben

shad

e an

d T

rian

taph

yllo

u (1

985)

an

d B

rito

et a

l., 2

008.

wM

DH

ph

enot

ype

not

res

olve

d.x M

eloi

dogy

ne s

p. 1

foun

d in

mix

ed p

opul

atio

n w

ith

M. m

ayag

uens

is.

y New

EST

ph

enot

ypes

: des

ign

atio

ns

wer

e as

sign

ed u

sin

g fi

rst t

wo

lett

ers

of th

e pl

ant h

ost s

peci

es n

ame

from

wh

ich

the

root

-kn

ot n

emat

ode

was

isol

ated

, fol

-lo

wed

by

num

ber

of m

ajor

isoz

yme

ban

ds (

Bri

to e

t al.,

200

8).

z Mel

oido

gyne

sp.

3 a

nd

4 fo

und

in m

ixed

pop

ulat

ion

wit

h M

. jav

anic

a.

Ornamental plants as hosts of Meloidogyne species: Brito et al. 93

Tulb

aghi

a vi

olac

eaA

lliac

eae

M. j

avan

ica

J2N

1B

uddl

eja

davi

dii

Bud

dlej

acea

eA

lach

ua a

nd

Sem

inol

e

Lag

erst

roem

ia in

dica

Lyt

hra

ceae

Oph

iopo

gan

japo

nicu

sL

iliac

eae

M. m

ayag

uens

isV

S1-S

1N

1aA

juga

rept

an,

Lam

iace

aeA

lach

ua, C

itru

s, C

lay,

Col

lier,

Dad

e, D

uval

, Fla

gler

, Gilc

hri

st,

Har

dee,

Hill

sbor

ough

Lak

e, N

assa

u, O

ran

ge, P

alm

B

each

, Pas

co a

nd

Putn

am

Bru

gman

sia

× su

nray

Sola

nac

eae

Bru

gman

sia

sp.

Sola

nac

eae

Bud

dlej

a da

vidi

iB

uddl

ejac

eae

Cal

liste

mon

spp

.M

yrta

ceae

Cal

liste

mon

citr

inus

Myr

tace

ae

C. v

imin

alis

Myr

tace

ae

Cle

rode

ndru

m u

gand

ense

Verb

enac

eae

Gar

deni

a sp

.R

ubia

ceae

Hib

iscu

s gr

andi

floru

sM

alva

ceae

Lag

erst

roem

ia in

dica

Lyt

hra

ceae

Lan

tana

mon

tevi

dens

isVe

rben

acea

e

Lig

ustr

um s

p.O

leac

eae

Myr

ica

ceri

fera

Myr

icac

eae

Tabl

e 1.

(C

onti

nue

d)Is

ozym

e ph

enot

ypes

of M

eloi

dogy

ne s

peci

es in

fect

ing

orn

amen

tal p

lan

ts in

Flo

rida

.

Mel

oido

gyne

spp

.

En

zym

e ph

enot

ypes

v

Plan

t hos

tsB

otan

ical

fam

ilies

Cou

nty

of o

rigi

nE

STM

DH

v EST

= E

ster

ase,

MD

H =

Mal

ate

deh

ydro

gen

ase.

Ph

enot

ype

desi

gnat

ion

acc

ordi

ng

to E

sben

shad

e an

d T

rian

taph

yllo

u (1

985)

an

d B

rito

et a

l., 2

008.

wM

DH

ph

enot

ype

not

res

olve

d.x M

eloi

dogy

ne s

p. 1

foun

d in

mix

ed p

opul

atio

n w

ith

M. m

ayag

uens

is.

y New

EST

ph

enot

ypes

: des

ign

atio

ns

wer

e as

sign

ed u

sin

g fi

rst t

wo

lett

ers

of th

e pl

ant h

ost s

peci

es n

ame

from

wh

ich

the

root

-kn

ot n

emat

ode

was

isol

ated

, fol

-lo

wed

by

num

ber

of m

ajor

isoz

yme

ban

ds (

Bri

to e

t al.,

200

8).

z Mel

oido

gyne

sp.

3 a

nd

4 fo

und

in m

ixed

pop

ulat

ion

wit

h M

. jav

anic

a.


Pent

a la

nceo

lata

Rub

iace

ae

Plec

tran

thus

scu

tella

rioi

des

Lam

iace

ae

Salix

× s

epul

cral

isSa

licac

eae

Sola

ndra

max

ima

Sola

nac

eae

Teco

mar

ia c

apen

sis,

Big

non

iace

ae

Tib

ouch

ina

× co

mpa

cta

Mel

asto

mac

eae

Tib

ouch

ina

× el

egan

sM

elas

tom

acea

e

M. a

rena

ria

and

M. i

ncog

nita

A2;

I1N

1;N

1B

egon

ia s

p.B

egon

iace

aeA

lach

ua, M

ario

n, O

ran

ge,

and

Volu

sia

Gar

deni

a sp

.R

ubia

ceae

Hoy

a sp

.A

scle

piad

acea

e

Ilex

cre

nata

Aqu

ifol

iace

ae

Impa

tiens

sp.

B

alsa

min

acea

e

Myr

ica

ceri

fera

Myr

icac

eae

Syag

rus

rom

anzo

ffian

aA

reca

ceae

M. i

ncog

nita

, M

. jav

anic

aan

d M

. may

ague

nsis

A2;

I1;

VS1

-S1

N1;

N1

N1a

A

mar

anth

us tr

icol

or

Am

aran

thac

eae

Palm

Bea

ch

M. a

rena

ria

and

M. j

avan

ica

A2;

J3N

1;N

1C

alad

ium

sp.

Ara

ceae

Hig

hla

nds

an

d P

alm

Bea

ch

Phoe

nix

dact

ylife

raA

reca

ceae

M.a

rena

ria,

M. j

avan

ica

and

M. m

ayag

uens

isA

2;J3

VS1

-S1

N1;

N1

N1a

Was

hing

toni

a sp

.A

reca

ceae

Pasc

o

Tabl

e 1.

(C

onti

nue

d)Is

ozym

e ph

enot

ypes

of M

eloi

dogy

ne s

peci

es in

fect

ing

orn

amen

tal p

lan

ts in

Flo

rida

.

Mel

oido

gyne

spp

.

En

zym

e ph

enot

ypes

v

Plan

t hos

tsB

otan

ical

fam

ilies

Cou

nty

of o

rigi

nE

STM

DH

v EST

= E

ster

ase,

MD

H =

Mal

ate

deh

ydro

gen

ase.

Ph

enot

ype

desi

gnat

ion

acc

ordi

ng

to E

sben

shad

e an

d T

rian

taph

yllo

u (1

985)

an

d B

rito

et a

l., 2

008.

wM

DH

ph

enot

ype

not

res

olve

d.x M

eloi

dogy

ne s

p. 1

foun

d in

mix

ed p

opul

atio

n w

ith

M. m

ayag

uens

is.

y New

EST

ph

enot

ypes

: des

ign

atio

ns

wer

e as

sign

ed u

sin

g fi

rst t

wo

lett

ers

of th

e pl

ant h

ost s

peci

es n

ame

from

wh

ich

the

root

-kn

ot n

emat

ode

was

isol

ated

, fol

-lo

wed

by

num

ber

of m

ajor

isoz

yme

ban

ds (

Bri

to e

t al.,

200

8).

z Mel

oido

gyne

sp.

3 a

nd

4 fo

und

in m

ixed

pop

ulat

ion

wit

h M

. jav

anic

a.


M. a

rena

ria

and

M. m

ayag

uens

isA

2; V

S1-S

1N

1; N

1aC

lero

dend

rum

uga

nden

seVe

rben

acea

eD

ade

and

Flag

ler

Myr

ica

ceri

fera

Myr

icac

eae

M. i

ncog

nita

and

M. j

avan

ica

I1;J

3N

1;N

1A

belia

sp.

Cap

rifo

liace

aeA

lach

ua a

nd

Ora

nge

Just

icia

bra

ndeg

eean

aA

can

thac

eae

Petu

nia

sp.

Sola

nac

eae

Pseu

dera

nthe

mum

sp.

Aca

nth

acea

e

M. i

ncog

nita

, M

. jav

anic

a an

d M

. may

ague

nsis

I1;J

3; V

S1-S

1N

1;N

1 N

1aC

aryo

pter

is ×

cla

ndon

ensi

sVe

rben

acea

eA

lach

ua

M.in

cogn

ita a

nd M

. may

ague

nsis

I1; V

S1-S

1N

1; N

1aM

yric

a ce

rife

raM

yric

acea

eFl

agle

r an

d M

adis

onG

arde

nia

jasm

inoi

des

Rub

iace

a

M. j

avan

ica

and

M. m

ayag

uens

isJ3

; VS1

-S1

N1;

N1a

Bru

gman

sia

sp.

Sola

nac

eae

Ala

chua

, Har

dee,

Mar

ion

an

d Sa

raso

taH

ibis

cus

gran

diflo

rus

Mal

vace

ae

Lan

tana

cam

ara

Verb

enac

eae

L. m

onte

vide

nsis

Ve

rben

acea

e

Was

hing

toni

aA

reca

ceae

Mel

oido

gyne

sp.

1x

Ep2

y—

Myr

ica

ceri

fera

Myr

icac

eae

Nas

sau

Mel

oido

gyne

sp.

2Vo

1yN

1B

ruga

man

sia

sp.

Sola

nac

eae

Ala

chua

an

d D

ade

Myr

ica

ceri

fera

Myr

icac

eae

Tabl

e 1.

(C

onti

nue

d)Is

ozym

e ph

enot

ypes

of M

eloi

dogy

ne s

peci

es in

fect

ing

orn

amen

tal p

lan

ts in

Flo

rida

.

Mel

oido

gyne

spp

.

En

zym

e ph

enot

ypes

v

Plan

t hos

tsB

otan

ical

fam

ilies

Cou

nty

of o

rigi

nE

STM

DH

v EST

= E

ster

ase,

MD

H =

Mal

ate

deh

ydro

gen

ase.

Ph

enot

ype

desi

gnat

ion

acc

ordi

ng

to E

sben

shad

e an

d T

rian

taph

yllo

u (1

985)

an

d B

rito

et a

l., 2

008.

wM

DH

ph

enot

ype

not

res

olve

d.x M

eloi

dogy

ne s

p. 1

foun

d in

mix

ed p

opul

atio

n w

ith

M. m

ayag

uens

is.

y New

EST

ph

enot

ypes

: des

ign

atio

ns

wer

e as

sign

ed u

sin

g fi

rst t

wo

lett

ers

of th

e pl

ant h

ost s

peci

es n

ame

from

wh

ich

the

root

-kn

ot n

emat

ode

was

isol

ated

, fol

-lo

wed

by

num

ber

of m

ajor

isoz

yme

ban

ds (

Bri

to e

t al.,

200

8).

z Mel

oido

gyne

sp.

3 a

nd

4 fo

und

in m

ixed

pop

ulat

ion

wit

h M

. jav

anic

a.


Podo

carp

us s

p.

Podo

carp

acea

eB

reva

rd a

nd

Hill

sbor

ough

Mel

oido

gyne

sp.

3z

Vo1y

N1

Vibe

rnun

odo

ratis

sim

um

Cap

rifo

liace

ae

V. s

uspe

nsum

Cap

rifo

liace

ae

Mel

oido

gyne

sp.

4Vo

2N

1Vi

bern

un o

dora

tissi

mum

C

apri

folia

ceae

Bre

vard

an

d H

illsb

orou

gh

Vibe

rnun

sus

pens

umC

apri

folia

ceae

Mel

oido

gyne

sp.

5G

j2y

N1a

Gar

deni

a ja

smin

oide

s an

dun

iden

tifi

ed o

rnam

enta

l pla

nt

Rub

iace

aeH

arde

e an

d H

igh

lan

ds

Mel

oido

gyne

sp.

6C

v2y

N1

Bux

us m

icro

phyl

laB

uxac

eae

Ala

chua

Cal

liste

mon

vim

inal

isM

yrta

ceae

Gar

deni

a sp

.R

ubia

ceae

Plec

tran

thus

scu

tella

riod

esL

amia

ceae

Lir

iope

mus

cari

Lili

acea

e

Tabl

e 1.

(C

onti

nue

d)Is

ozym

e ph

enot

ypes

of M

eloi

dogy

ne s

peci

es in

fect

ing

orn

amen

tal p

lan

ts in

Flo

rida

.

Mel

oido

gyne

spp

.

En

zym

e ph

enot

ypes

v

Plan

t hos

tsB

otan

ical

fam

ilies

Cou

nty

of o

rigi

nE

STM

DH

v EST

= E

ster

ase,

MD

H =

Mal

ate

deh

ydro

gen

ase.

Ph

enot

ype

desi

gnat

ion

acc

ordi

ng

to E

sben

shad

e an

d T

rian

taph

yllo

u (1

985)

an

d B

rito

et a

l., 2

008.

wM

DH

ph

enot

ype

not

res

olve

d.x M

eloi

dogy

ne s

p. 1

foun

d in

mix

ed p

opul

atio

n w

ith

M. m

ayag

uens

is.

y New

EST

ph

enot

ypes

: des

ign

atio

ns

wer

e as

sign

ed u

sin

g fi

rst t

wo

lett

ers

of th

e pl

ant h

ost s

peci

es n

ame

from

wh

ich

the

root

-kn

ot n

emat

ode

was

isol

ated

, fol

-lo

wed

by

num

ber

of m

ajor

isoz

yme

ban

ds (

Bri

to e

t al.,

200

8).

z Mel

oido

gyne

sp.

3 a

nd

4 fo

und

in m

ixed

pop

ulat

ion

wit

h M

. jav

anic

a.


Tabl

e 2.

Mel

oido

gyne

spe

cies

foun

d in

fect

ing

unid

enti

fied

orn

amen

tal p

lan

ts in

Flo

rida

alo

ng

wit

h th

eir

isoz

yme

phen

otyp

es a

nd

coun

ty o

f ori

gin

.

Mel

oido

gyne

spp

.x

En

zym

e ph

enot

ypes

y

Num

ber

of S

ampl

esC

oun

ty o

f ori

gin

EST

MD

H

Ma

A2

N1

2D

ade

and

Palm

Bea

ch

Mi

I1N

17

Cal

hou

n, D

ade,

Hill

sbor

ough

, Jac

kson

, Man

atee

an

d Pa

lm B

each

Mi

I1N

2a1

Palm

Bea

ch

Mi

I2N

12

Dad

e an

d H

endr

y

Mj

J3N

16

Gad

sden

, Hill

sbor

ough

, Lak

e, L

ee a

nd

Ora

nge

Mm

VS1

-S1

N1a

5D

ade,

Har

dee,

Hen

dry,

Pal

m B

each

an

d St

. Joh

ns

Ma

and

Mi

A2;

I1

N1;

N1

1Pa

lm B

each

Ma

and

Mm

A

2; V

S1-S

1N

1; N

1a1

Bro

war

d

Ma,

Mi a

nd

Mm

A

2; I

1; V

S1-S

1N

1; N

1; N

1a1

Volu

sia

Ma,

Mi,

Mj a

nd

Mm

A2;

I1;

J3;

VS1

-S1

N3;

N1;

N1

N1a

1L

ake

Mi a

nd

Mj

I2; J

3N

1; N

12

Ala

chua

an

d O

ran

ge

Mj a

nd

Mm

J3; V

S1-S

1N

1; N

1a1

Col

lier

x Mel

oido

gyne

spp

.: M

a =

M. a

rena

ria,

Mi =

M. i

ncog

nita

, Mj =

M. j

avan

ica,

Mm

= M

. may

ague

nsis

.y E

st =

Est

eras

e, M

DH

= M

alat

e de

hyd

roge

nas

e. P

hen

otyp

e de

sign

atio

n a

ccor

din

g to

Esb

ensh

ade

and

Tri

anta

phyl

lou

(198

5).


ique (Cofcewicz et al., 2005), Portugal (Paisand Abrantes, 1989) and United States(Brito et al., 2008). Phenotypes I1 and I2were never found together in the samesample in this study; however the detectionof both phenotypes among populations ofM. incognita could be associated with someintraspecific variability.

The species-specific phenotype J3appeared in 36 populations identified asM. javanica (Tables 1 and 2; Fig. 1). Thesepopulations occurred singly or mixed withM. arenaria, M. incognita and M. mayaguen-sis (Tables 1 and 2). These results were con-sistent with those in previous studies(Esbenshade and Triantaphyllou, 1985;Pais and Abrantes, 1989; Tomaszewski et al.,1994; Carneiro et al., 1996, 2000, 2004; Cas-tro et al., 2003; Cofcewicz et al., 2004, 2005;Molinari et al., 2005; Brito et al., 2008).Three populations of M. javanica infectingBuddleja davidii and Ophiopogon japonicusexhibited the J2 phenotype (Table 1;

Fig. 1). Likewise, this phenotype was alsoreported from populations of M. javanicainfecting Abelmoschus esculentus (Oliveira etal., 2007) and Musa sp. (Cofcewicz et al.,2004) in Brazil, and also Musa sp. fromGuadalupe, French Guiana and Martin-ique (Cofcewicz et al., 2005).

All 72 nematode populations identifiedas M. mayaguensis exhibited two major bandsof EST activity (Rm 29.06; 38.37), consistentwith the VS1-S1 phenotype (Fig. 1). Attimes, each of these bands resolved into twominor bands. Morphology of perineal pat-terns and morphometrics of selected char-acters were similar to those from the speciesdescription (Rammanh and Hisrchmann,1988) and other isolates of M. mayaguensisfound in Florida (Brito et al., 2004a). Fur-thermore, analysis of the mtDNA regionbetween COII and lrRNA genes was alsoused to compare with the EST phenotype.Results were in agreement with thosereported previously (Brito et al., 2004a).

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


The EST phenotype, VS1-S1 proved tobe of high diagnostic value to distinguishM. mayaguensis from all other root-knotnematode species identified in this study,particularly M. incognita. It is most likelythat M. mayaguensis has been erroneouslyidentified as M. incognita in the past in Flor-ida due to some similarity of the perinealpatterns of these two species (Brito et al.,2004a, 2008). In North America, this nem-atode is known to occur only in Florida(Brito et al., 2004a, b). Meloidogyne may-aguensis was identified from root samplesof several ornamental plant species belong-ing to 16 botanical families (Table 1). Toour knowledge Ajuga reptans, Amaranthustricolor, Buddleja davidii, Caryopteris × clan-donensis, Clerodendrum ugandense, Hibiscusgrandiflorus, Lagerstroemia indica, Penta lan-ceolata, Plectranthus scutellarioides, and Solan-dra maxima are new host records for M.mayaguensis.

It is worth mentioning that the pheno-type VS1-S1 also has been reported foranother root-knot nematode species, M.enterolobii from China (Yang and Eisenback,1983; Esbenshade and Triantaphyllou,1985). These two root-knot nematode spe-cies share not only biochemical and mor-phological characteristics, but they also

have identical sequences of the mtDNAregion between COII and lrRNA genes(Xu et al., 2004). Furthermore, sequencedata 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 USAshowed 100% similarity (Kiewnick et al.,2007; 2008). Currently, M. mayguensis isbeing synonymized with M. enterolobii (Ger-rit Karssen, personal communication).

Six unique EST profiles were detectedfrom 19 unidentified populations ofMeloidogyne spp. infecting different orna-mental plants in this study. These popula-tions were named Meloidogyne sp. 1 to 6.Two populations of Meloidogyne sp. 1 exhib-ited two EST major bands Rm: 41.0, 43.70)(Table 1; Fig. 1) similar to a phenotype(Ep2) already described for a root-knotnematode infecting originally Eclipta pros-trata in Florida (Brito et al., 2008), whereasfive new EST phenotypes (Mc1, Cv2, Gj2,Vo1, and Vo2) were described from theremaining 17 unidentified populations(Table 1). Phenotype designations used toassign these new phenotypes were the sameas previously reported (Esbenshade andTriantaphyllou, 1985; Brito et al., 2008). Aphenotype designated as Mc1 with onemajor band of activity at Rm: 32.0 wasdetected in three populations of Meloidog-yne sp. 2 infecting initially Myrica cerifera(Table 1; Fig. 1). In a differential host test(Hartman and Sasser, 1985) single eggmass isolates obtained from Meloidogyne sp.2 reproduced on tobacco ‘NC95’, cotton‘DPL 16’, watermelon ‘Charleston Grey’,pepper ‘California Wonder’ and tomato‘Rutgers’, but not on peanut ‘Florunner’.Furthermore, the isolates also reproducedwell on potato but did not reproduce oncorn or wheat (data not shown).

The phenotype Vo1, with one majorband of activity (Rm 51.16) (Table 1; Fig. 1)and Vo2, with two major bands (26.0; 51.16)

Fig. 2. Schematic representation of malate dehydroge-nase phenotypes of Meloidogyne spp. populationsfound infecting ornamental plants in Florida. N1 = M.arenaria, M. floridensis, M. incognita, M. javanica, M.sp.3, M. sp.4, and M. sp.6; N2a = M. incognita; N3 = M.arenaria, N1a = M. graminis, M. mayaguensis, M. sp.2and M. sp.5. Phenotypes designation according to Es-benshade and Triantaphyllou (1985). zRm= Relativemobility.


(Table 1; Fig. 1) were observed in mixturepopulation with Meloidogyne sp. 3 and Meloi-dogyne 4 infecting Vibernum odoratissimum.These two phenotypes share a major bandat Rm 51.16 and remained stable whennematode isolates were inoculated andreared on tomato ‘Rutgers’. Furthermore,nematode isolates with phenotypes Vo1 orVo2 showed the same host reactions wheninoculated on differential host plant culti-vars (Hartman and Sasser, 1985); both iso-lates reproduced on all plant cultivarsexcept peanut ‘Florunner’ and pepper ‘Cal-ifornia Wonder’. These nematode isolatesshowed the same MDH (NI) (Fig. 2),glutamic-oxaloacetic transaminase (NIa)and superoxide dismutase (N2b) pheno-types (Esbenshade and Triantaphyllou,1985) regardless of the EST phenotype.

Another phenotype, Gj2 with two majorbands (Rm 50.6; 52.2) (Table 1; Fig. 1) wasdetected in two nematode populations des-ignated as Meloidogyne sp. 5, which werefound infecting Gardenia jasminoides and anunidentified ornamental plant in Hardeeand Highlands Counties, respectively. Iso-lates of these nematodes had the same hostreactions as those of M. javanica race 1 andM. arenaria race 2 (Hartman and Sasser,1985). Meloidogyne sp. 6, initially foundreproducing on Callistemon viminalis in Ala-chua County, exhibited the phenotype Cv2and had three bands (Rm 28; 40.2) (Table1; Fig. 1). Isolates obtained from the fieldpopulation showed the same differentialhost reaction as that of M. incognita race 2.Currently, single egg masses obtained fromall unidentified root-nematode species arebeing reared on tomato ‘Rutgers’ and willbe used for further investigation as anattempt to identify each nematode species.

Malate dehydrogenase phenotypes

Five bands of MDH activity and fourMDH phenotypes were observed among

the populations of Meloidogyne spp. in thisstudy (Tables 1 and 2; Fig. 2). The pheno-type N1 (Rm: 20.2) was detected in all pop-ulations of M. arenaria, M. floridensis, M.incognita, M. javanica, Meloidogyne sp. 3,Meloidogyne sp. 4 and Meloidogyne sp. 6(Tables 1 and 2; Fig. 2), except one popula-tion of M incognita, which exhibited aunique phenotype (N2a) with two majorbands of activity at Rm: 20.2: 23.1 (Table 2;Fig. 2) and two populations of M. arenaria,which showed the N3 phenotype (Rm:20.0:23.1:26.0) with three bands of MDHactivity (Tables 1; Fig. 2). The N1 pheno-type has been commonly associated withthe three major species of Meloidogyne col-lected in other regions of the world(Esbenshade and Triantaphyllou, 1985;Pais and Abrantes, 1989; Carneiro et al.,2004, Brito et al., 2008). Nonetheless, thephenotype N3 has been detected in somepopulations of M. arenaria in the UnitedStates (Brito et al., 2008) and also fromother geographical regions (Esbenshadeand Triantaphyllou, 1985; Pais andAbrantes, 1989; Cofcewicz et al., 2005). Toour knowledge, this is the first report of thephenotype N2a identified from M. incog-nita, which could be associated with somevariability among populations of this nema-tode species. This phenotype remained sta-ble when a nematode isolate wasinoculated and reared on tomato ‘Rut-gers’.

The phenotype N1a, which showed onevery strong band (Rm: 28.1) of MDH activ-ity was detected in all the populations of M.graminis, M. mayaguensis, Meloidogyne sp. 2and Meloidogyne sp. 5 (Tables 1 and 2; Fig.2). An identical phenotype has beenreported from populations of M. chitwoodi,M. enterolobii, M. naasi, M. oryzae, M. plan-tani (Esbenshade and Triantaphyllou,1985), and M. partityla and M. graminicola(Brito et al., 2006). Therefore it is ofrestricted diagnostic value to differentiate


these Meloidogyne spp.; however, the N1aphenotype could aid in the discriminationof these root-knot nematode species fromM. arenaria, M. floridensis, M. incognita andM. javanica.

The results obtained in this study, incombination with those already reportedin other studies, clearly show the usefulnessof isozymes as a valuable tool for identifica-tion of Meloidogyne spp. in a large numberof samples. The EST and MDH phenotypeswere useful to detect mixed populations ofMeloidogyne spp. as well as to determine newhost records for root-knot species; however,EST had a higher diagnostic value thanMDH in the discrimination of theMeloidogyne spp. found infecting ornamen-tal plants in Florida.

ACKNOWLEDGEMENTS

This research was supported by theTropical and Subtropical AgricultureResearch grant (T-STAR) #2005-34135-15895, Cooperative State Research, Educa-tion and Extension Services (CSREES),United States Department of Agriculture,USA. The authors thank Dr. Richard E.Weaver, Jr., Botany Section, Division ofPlant Industry, Gainesville, Florida, foridentification of weed species and also AnaL. Ochoa, Christine A. Zamora, Charles L.Spriggs, Ping Qiao, Matthew W. Brodie andSol F. Locker for their support during thefield work.

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Received: Accepted for publication:1/IV/2010 13/V/2010

Recibido: Aceptado para publicación

2 enzimas meloidogyne ornamentales

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