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Variability of Meloidogyne exigua on Coffee in the Zona da Mata of
Minas Gerais State, Brazil
1
D. S. Oliveira,
2
R. D. L. Oliveira,
2
L. G. Freitas,
2
and R. V. Silva
2
Abstract: Minas Gerais is the major coffee-producing state of Brazil, with 28% of its production coming from the region of Zona
da Mata. Four major species of root-knot nematode attacking coffee (Meloidogyne incognita, M. paranaensis, M. coffeicola, and M.
exigua) have been reported from Brazil. To determine the variability in Meloidogyne spp. occurring in that region, 57 populations
from 20 localities were evaluated for morphological, enzymatic, and physiological characteristics. According to the perineal pattern,
all the populations were identified as M. exigua; however populations from the municipality of Sa˜o Joa˜o do Manhuac¸u exhibited
patterns very similar to M. arenaria. The identity of all the populations was confirmed by the phenotypes of esterase, malate
dehydrogenase, superoxide dismutase, and glutamate-oxaloacetate transaminase. Thirteen populations (22.8%) showed the typical
one-band (E1) esterase phenotype, whereas the others (77.2%) had a novel two-band phenotype (E2). No intraspecies variability
was found in any population. All populations were able to reproduce on tomato, pepper, beans, cacao, and soybean. Reproduction
was greater on tomato and pepper than on coffee seedlings, the susceptible standard.
Key words: Coffea arabica, isozyme analysis, Meloidogyne exigua, root-knot nematode.
Coffee is a major agricultural commodity in the world
market, and Brazil is the largest producer and exporter.
Minas Gerais State produces almost half of the Brazilian
coffee, with 28% produced in the region of Zona da
Mata (CONAB, 2003). The eco-climatic conditions of
this region favor production of fine and special-grade
coffee with a higher market value. Root-knot nema-
todes (RKN), Meloidogyne spp., are among the most im-
portant pathogens for this crop. Due to the extensive
distribution of RKN in coffee plantations and their high
reproductive capacity, coffee productivity in Brazil has
declined since the 1960s (Campos et al., 1990). In some
cases this has led to abandoning of plantations (Car-
neiro, 1995). At present, the economically viable man-
agement practice is the use of RKN resistance, espe-
cially resistant rootstocks. The use of resistance de-
mands knowledge of the RKN species and, in some
cases, races present in the area. Information on occur-
rence and distribution of the root-knot nematodes in
Minas Gerais dates back to the 1980s and is based
mainly on morphological characters (Campos et al.,
1987; Ferraz, 1980). The objective of this study was to
use precise techniques of characterizing Meloidogyne
spp. to update information on the RKN identity and
distribution from the coffee plantations of the Zona da
Mata region of Minas Gerais.
Materials and Methods
Fifty-seven populations of Meloidogyne spp. were col-
lected from the nematode-infested coffee plantations in
20 municipalities of the Zona da Mata region of Minas
Gerais, Brazil (Table 1). Soil and root samples were
collected from four points, to a depth of 30 cm from
under the canopy of selected trees and pooled. The
composite sample of approximately 500 g soil and
200 g roots was placed in plastic bags, labeled, and
transported to the nematology laboratory. The eggs of
Meloidogyne spp. were extracted from the roots accord-
ing to Boneti and Ferraz (1981) and used to inoculate
coffee seedlings (Coffea arabica L. cv. Catuaı´) for nema-
tode multiplication in greenhouse. The collected
nematode populations were maintained on coffee in
the greenhouse for 2 years.
At least 10 females from each population were pre-
pared for perineal pattern analysis (Taylor and
Netscher, 1974). Isozyme characterizations were con-
ducted for esterase (EST), malate dehydrogenase
(MDH), superoxide dismutase (SOD), and glutamate-
oxaloacetate transaminase (GOT). Milky-white, repro-
ductive females were removed from galls on inoculated
coffee seedlings and transferred to micro-centrifuge
tubes containing 3.5 µL protein extraction buffer (Dal-
masso and Berge´, 1978). Females of known greenhouse
isolate of M. javanica were extracted from tomato roots
(Lycopersicon esculentum cv. Santa Clara) and used as a
reference standard. Electrophoresis was carried out in a
continuous buffer system with 7% acrylamide running
gel. The voltage was maintained at 100 V during the
running period (Carneiro et al., 1996a). After electro-
phoresis, the gels were removed and placed in the ap-
propriate reaction mixture to determine EST, MDH,
SOD, and GOT activity (Alfenas et al., 1991). Enzyme
phenotypes were designated by a letter suggestive of the
species it specified and a numeral indicating the num-
ber of bands (Carneiro et al., 2000).
The set of differential hosts for Meloidogyne host races
(Hartman and Sasser, 1985) plus onion (Allium cepa cv.
Baia Periforme), cacao (Theobroma cacao clone SIC 23),
field beans (Phaseolus vulgaris cv. Carioca), and soybean
(Glycine max cv. FT Cristalina) were used for physiologi-
cal characterization of Meloidogyne spp. populations.
Coffee cv. Catuaı´ was used as the susceptible standard.
The seeds of each species were sown in separate trays
Received for publication 2 June 2004.
1
Supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecno-
lo´ gico (CNPq) and Conso´ rcio Brasileiro de Pesquisa e Desenvolvimento do
Cafe´ . A portion of an M. Sc. dissertation by the first author.
2
Graduate Student, Professors, and Undergraduate Student, respectively,
Departamento de Fitopatologia, Universidade Federal de Vic¸osa, 36571-000
Vic¸osa, MG, Brazil.
E-mail: rdlima@ufv.br
This paper was edited by R. T. Robbins.
Journal of Nematology 37(3):323–327. 2005.
© The Society of Nematologists 2005.
323
containing methyl bromide-treated sand, and the seed-
lings at four-leaf stage were transplanted to 3-liter clay
pots containing methyl bromide-treated substrate (soil:
sand, 2;1, v/v). The seedling were inoculated 2 days
after transplant using 5,000 eggs/seedling of either of
the 10 M. exigua populations (5 E1 and 5 E2 esterase
phenotype) from each municipality. The experiment
was a completely randomized factorial design (11 plant
species × 10 populations) with six replications. The
number of eggs per root system was determined 60 days
after inoculation. The egg number was used to deter-
mine the nematode reproduction factor (Oostenbrink,
1966). The data were transformed to √x, and the means
were compared using Tukey’s test (P= 0.05).
Results
The perineal patterns for 91% of the populations
were typical of M. exigua (Fig. 1A,B), with a slightly
plane low dorsal arc, thick and well-spaced striae, non-
perceptible lateral lines normally, demarked by either
bent or interrupted striae. However, females of the
populations collected from Sa˜o Joa˜o do Manhuac¸u had
perineal patterns similar to those of M. arenaria (Fig.
1C), with a rounded dorsal arc, the lateral lines forming
a shoulder, yet thick and well-spaced striae as in M.
exigua. These configurations were considered as atypi-
cal intra-species variation of M. exigua.
The typical EST phenotype of M. exigua, called E1,
presents relative mobility (Rm) of 1.60. This phenotype
was found in only 22.8% of the populations (Fig. 2A),
whereas the remaining populations (77.2%) showed a
new phenotype (designated E2), which was widely dis-
tributed in the sampled areas (Fig. 3). This phenotype
consists of a weak (Rm 1.60) and a strong band (Rm
1.90) (Fig. 2B). The populations from the municipality
of Sa˜o Joa˜o do Manhuac¸u had the perineal patterns
similar to that of M. arenaria, but with an M. exigua EST
phenotype, e.g., four of the five populations had the E2
phenotype and one population had the E1 phenotype
(Table 1). No polymorphism was observed for MDH,
SOD, and GOT phenotypes (Figs. 4, 5, and 6). Charac-
terization using these enzymes confirmed the diagnosis
of each population as M. exigua. Phenotype N1 (Rm
1.00) for MDH (Fig. 4), E1 (Rm 1.10) for GOT (Fig. 5),
and the phenotype N3 (Rm 1.25; 1.30 and 1.40) for
SOD (Fig. 6) were typically exhibited.
Fig. 1. Perineal patterns in Meloidogyne spp. populations collected
from coffee plantations of Zona da Mata of Minas Gerais, Brazil. A,B)
Typical perineal patterns of M. exigua found in the majority of popu-
lations evaluated. C) The perineal patterns found in some popula-
tions from the municipality of Sa˜o Joa˜o do Manhuac¸u.
Fig. 2. Esterase phenotypes of Meloidogyne exigua populations. A)
A single-band phenotype (E1). B) Double-band phenotype (E2). J3 =
Esterase phenotype of M. javanica used as standard for comparisons.
TABLE 1. Esterase phenotypes of Meloidogyne exigua populations
collected from coffee plantations in municipalities of Zona da Mata of
Minas Gerais. E1 is a single-band phenotype with Rm of 1.60; E2 is a
two-banded phenotype with bands at Rm 1.60 and 1.90.
Municipality
Number of
populations
Number of populations
with phenotype
(E1) (E2)
Alto Jequitiba´312
Araponga 4 2 2
Caiana 2 0 2
Canaa˜404
Carangola 2 1 1
Divino 2 0 2
Espera Feliz 3 1 2
Faria Lemos 2 0 2
Fervedouro 2 0 2
Lajinha 2 0 2
Manhuac¸ u/Realeza 12 3 9
Manhumirim 1 0 1
Miradouro 1 0 1
Miraı´211
Muriae´211
Sa˜o Francisco do Glo´ria 2 1 1
Sa˜o Miguel do Anta 3 1 2
Santana do Manhuac¸u 1 0 1
Sa˜o Joa˜o do Manhuac˜u 5 1 4
Vic¸ osa 2 0 2
Total 57 13 44
324 Journal of Nematology, Volume 37, No. 3, September 2005
No physiological variability on the differential hosts
was detected among the populations (P> 0.05) (Table
2). The plant species ×populations interaction was not
significant for number of eggs produced (P> 0.05).
Onion, watermelon, cotton, tobacco, and peanut were
non-hosts of M. exigua, with reproduction factors of
zero (data not shown).
Discussion
As in many other Meloidogyne species, reproduction in
M. exigua occurs through parthenogenesis, and yet in-
tra-species variability in morphological and physiologi-
cal characters has been reported (Eisenback and Tri-
antaphyllou, 1991; Lopes, 1985; Santos et al., 1992).
The M. exigua populations of Sa˜o Joa˜o do Manhuac¸u
were atypical because their perineal patterns were simi-
lar to that of M. arenaria, which could lead to erroneous
identification if based solely on this characteristic. How-
ever, the EST phenotypes confirmed each population
as M. exigua. The perineal patterns had been the main
taxonomical characteristic to identify species of Meloido-
gyne but now are used primarily to clarify doubts origi-
nating from the isoenzyme phenotypes.
Being subjective, perineal patterns have contributed
to the description of a large number of species in this
genus. A good example of subjectivity and low confi-
dence of this character is the description of M. paranae-
nsis, which was detected in Parana´, Brazil, and is aggres-
sive on coffee. The perineal pattern led to an initial
identification as M. incognita, despite difference in
symptoms and aggressiveness on coffee. Due to the lack
of evaluation of more precise characters, it was desig-
nated as M. incognita biotype IAPAR for 22 years. In
1996 this biotype was re-evaluated and described as a
new species on the basis of morphological and morpho-
metric characteristics, the response of differential hosts,
and, principally, EST phenotype (Carneiro et al.,
1996b).
In the past, new species of Meloidogyne were described
erroneously on the bases of small morphological varia-
tions, including in the perineal pattern. It is now well
established that such intra-species variation is common.
Many erroneously identified species that were later con-
sidered as synonyms of well-characterized species in-
clude M. acrita, M. elegans, and M. inornata (which are
considered as synonyms of M. incognita), M. bauruensis
and M. lordelloi (as synonyms of M. javanica), and M.
thamesi (as a synonym of M. arenaria) (Eisenback and
Triantaphyllou, 1991). Thus, morphological character-
istics should be examined in a large number of speci-
mens to determine the range of variation, which would
minimize errors or doubts in identification of a popu-
lation as a known species or as a new species.
The EST was used to characterize 57 populations be-
cause this enzyme shows the highest degree of polymor-
phism and specificity for the main species of the root-
knot nematode (Carneiro et al., 2000; Esbenshade and
Triantaphyllou, 1985). Of the two EST phenotypes
found among the populations, the phenotype E1 of M.
exigua is reported to be widely distributed in Brazilian
coffee plantations (Naves et al., 2001; Santos and Tri-
antaphyllou, 1992), but in this study it was found only
in 22.8% of the populations collected from Zona da
Mata of Minas Gerais. The new phenotype, E2, despite
showing the same relative mobility values as E1b phe-
notype described by Carneiro et al. (2000), differs in
the intensity of bands, where the band of greater inten-
sity has an Rm of 1.90 (compared to the Rm of 1.60 in
E1 phenotype). With the discovery of a new EST phe-
Fig. 3. Zonal distribution of E1 and E2 esterase phenotypes of
Meloidogyne exigua populations in coffee plantations in 20 municipali-
ties of Zona da Mata, Minas Gerais (MG), Brazil. For the name of the
municipalities see Table 1.
Fig. 4. Malate dehydrogenase (MDH) phenotype of Meloidogyne
exigua populations. A) Populations with E1 esterase phenotype. B)
Populations with E2 esterase phenotype. N1 = M. javanica phenotype
used as standard.
Fig. 5. Glutamate oxaloacetate transminase (GOT) phenotype of
Meloidogyne exigua populations. A) Populations with E1 esterase phe-
notype. B) Populations with E2 esterase phenotype. N1 = M. javanica
phenotype used standard.
Fig. 6. Superoxide dismutase (SOD) phenotype of Meloidogyne
exigua populations. A) Populations with E1 esterase phenotype. B)
Populations with esterase E2 phenotype. N1 = M. javanica phenotype
used as standard.
Variability in Meloidogyne exigua: Oliveira et al. 325
notype it is now possible to detect four phenotypes of
M. exigua: E1 (Rm 1.60), Ela (Rm 1.10 and 1.60), E1b
(Rm 1.60 and 1.90), and E2 (Rm 1.60 and 1.90). The
occurrence of more than one phenotype for the same
enzyme is known for other species of Meloidogyne.
Meloidogyne arenaria, for example, has phenotypes with
one, two, and three bands called A1, A2, and A3, re-
spectively (Esbenshade and Triantaphyllou, 1985). A
two-band EST phenotype (I2), with Rm 1.00 and 1.12,
was reported for M. incognita, which until then was
identified only by the phenotype I1 (Santos and Trian-
taphyllou, 1992; Carneiro et al., 1996a).
Because of the limited use of isozyme electrophore-
sis, there is little information about the isozymatic vari-
ability within the species of Meloidogyne. With wider use
of this technique, the discovery of new EST phenotypes
will allow for more precise and less subjective identifi-
cation of species.
The analysis of other enzymes did not detect variabil-
ity among the populations but were important for con-
firming the diagnoses of the M. exigua, because all
populations examined had isozyme phenotypes typical
of this species. The MDH phenotype N1 (Rm 1.00) was
found in all the populations. Although not a specific
phenotype, this enzyme is important for differentiating
M. exigua from M. naasi with phenotype N1a (Rm 1.40),
which has greater mobility than M. exigua (Esbenshade
and Triantaphyllou, 1985). All populations examined
showed the GOT phenotype E1 (Rm 1.10). In M. exigua
populations of Sa˜o Paulo and Minas Gerais, Brazil, the
SOD phenotype N3 (Rm 1.25, 1.30, and 1.40) was re-
ported by Carneiro et al. (2000). Through perineal pat-
terns or by isozyme analyses, all 57 populations of the
root-knot nematode from the coffee plantations of
Zona da Mata-Minas Gerais were characterized as M.
exigua, despite variability in perineal patterns in popu-
lations from Sa˜o Joa˜o do Manhuac¸u. The EST analysis
also showed occurrence of two phenotypes in the popu-
lations, but without any relationship between the two
types of variability exhibited by M. exigua. There was
also no relationship with the reproduction capacity on
different hosts, because all phenotypes produced a
similar number of eggs/plant. Such lack of relationship
between races and the isoenzymatic phenotypes also
has been reported in M. incognita and other species
with physiological races (Carneiro et al., 2000; Janati et
al., 1982).
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Populations
Host
Tomato Pepper Coffee Field beans Cacao Soybean
1 Eggs
1
238.8a 193.2b 93.3c 57.1d 29.5e 18.8e
RF
1
11.4a 7.6b 1.8c 0.7d 0.2e 0.1e
2 Eggs 276.1a 182.7b 86.8c 44.9d 27.0e 17.7e
RF 15.3a 6.8b 1.6c 0.5d 0.2e 0.1e
3 Eggs 279.7a 202.0b 80.8c 52.9d 31.1e 16.1e
RF 16.6a 8.2b 1.5c 0.6d 0.2e 0.1e
4 Eggs 215.4a 172.1b 88.1c 55.3d 30.8e 15.8e
RF 9.5a 6.0b 1.2c 0.7d 0.2e 0.1e
5 Eggs 218.3a 186.0b 97.0c 48.0d 30.1e 15.4e
RF 9.7a 7.2b 1.9c 0.5d 0.2e 0.1e
6 Eggs 254.9a 176.0b 84.5c 51.8d 31.2e 19.1e
RF 13.1a 6.2b 1.4c 0.5d 0.2e 0.1e
7 Eggs 222.6a 184.7b 79.7c 58.5d 30.1e 15.5e
RF 10.1a 6.9b 1.3c 0.7d 0.2e 0.1e
8 Eggs 230.3a 170.6b 92.4c 64.8d 30.9e 17.5e
RF 10.7a 5.8b 1.7c 0.7d 0.2e 0.1e
9 Eggs 219.8a 205.9b 87.0c 57.1d 30.9e 18.0e
RF 9.8a 8.6b 1.5c 0.7d 0.2e 0.1e
10 Eggs 238.5a 193.3b 98.8c 54.3d 31.1e 18.1e
RF 11.5a 7.5b 1.9c 0.6d 0.2e 0.1e
1
Mean of six replicates. The number of eggs per root system data trans-
formed to √x. Means followed by the same letter within a line are not signifi-
cantly different (Tukey, P> 0.05).
326 Journal of Nematology, Volume 37, No. 3, September 2005
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