Karssen, G.

Sirca, S.

Urek, G.

Name not available

The incidence of the root-knot nematode Meloidogyne incognita and Meloidogyne hapla in Slovenia

Wageningen Yield 

Acta agriculturae slovenica, 83 - 1, junij 2004 str. 15 - 22
Agrovoc descriptors: Meloidogyne incognita, Meloidogyne hapla, plant nematodes, surveys,
identification, electrophoresis,  enzymatic analysis, morphology, phenotypes, Slovenia
Agris category codes: H10
COBISS code 1.01
The incidence of the root-knot nematode Meloidogyne
incognita and Meloidogyne hapla in Slovenia
Saša ŠIRCA1, Gregor UREK2  and Gerrit KARSSEN3
Received: October 24, 2003; accepted: March 2, 2004.
Delo je prispelo 24. oktobra 2003; sprejeto 2. marca 2004.
ABSTRACT
The root knot nematode Meloidogyne incognita (Kofoid and White) Chitwood, 1949 is one of
the most harmful and most ubiquitous species in the genus Meloidogyne. It was detected very
often in the greenhouses of northern Europe, but in the open field it is restricted to the
southern parts of Europe.  While this species has been recorded in almost all parts of the
world and also in some parts of the former Yugoslavia, there is no data about its presence in
Slovenia. M. incognita is able to parasitize about 700 plant hosts and varieties including the
majority of economically important crops. In the late summer of 2002, nematodes of M.
incognita were isolated from the roots of hot pepper plants, Capsicum annum L., from the
greenhouse situated in Portorož at the Adriatic Coast, Slovenia. The nematode was
morphologically identified as M. incognita and confirmed by isozyme gel electrophoresis
(PhastSystem, Pharmacia). To our knowledge, this is the first report of M. incognita in
Slovenia. Beside this finding, M. hapla was also found for the first time in the open field in
Slovenia; until now it has been detected on different host plants grown only in greenhouses.
In October 2002, M. hapla was isolated from galls of the sweet pepper grown in the open field
in Ljubljana situated in the central part of Slovenia.
Key words: identification, isozyme gel electrophoresis, Meloidogyne incognita, M. hapla,
morphology, nematodes
IZVLEČEK
PRVO POROČILO O NAJDBI OGORČICE KORENINSKIH ŠIŠK MELOIDOGYNE
INCOGNITA V SLOVENIJI
Vrsta Meloidogyne incognita (Kofoid and White) Chitwood, 1949, je ena najbolj škodljivih in
najbolj razširjenih ogorčic koreninskih šišk, Meloidogyne spp. V območjih severne Evrope je
bila večkrat ugotovljena v rastlinjakih, na prostem pa se v Evropi pojavlja predvsem v
južnejših, toplejših predelih. Medtem ko so o omenjeni vrsti poročali že iz skoraj vseh območij
sveta, tudi iz nekaterih predelov bivše Jugoslavije, pa o njeni navzočnosti v Sloveniji ni
nobenih podatkov. Ogorčica M. incognita zajeda okoli 700 različnih rastlinskih vrst, vključno z
večino gospodarsko pomembnih rastlin.  Vrsto M. incognita smo pri nas prvič izolirali pozno
                                                
1 B. Sc. Agr., Agricultural Institute of Slovenia, Hacquetova 17, 1001 Ljubjana, Slovenia
2 Ph. D., Agricultural Institute of Slovenia, Hacquetova 17, 1001 Ljubjana, Slovenia
3 Prof., Ph.D., Plant Protection Service, Nematology Section, P.O. Box 9102, 6700 HC
Wageningen, The Netherlands.
Acta agriculturae slovenica, 83 - 1, junij 200416
poleti leta 2002 iz korenin paprike iz rastlinjaka v Portorožu. Ogorčico smo morfološko
določili, identifikacijo pa potrdili z izoencimsko gelsko elektroforezo (PhastSystem,
Pharmacia). Poleg že omenjene vrste smo v preteklem letu iz korenin feferonov v okolici
Ljubljane prvič na prostem pri nas izolirali tudi vrsto M. hapla, o kateri navzočnosti v
rastlinjakih se je v Sloveniji v preteklosti že poročalo.
Ključne besede: identifikacija, izoencimska gelska elektroforeza, Meloidogyne incognita, M.
hapla, morfologija, ogorčice
1 INTRODUCTION
Root-knot nematodes, the genus Meloidogyne, represent a relatively small but
economically important group of obligate plant parasites. They are distributed world
wide and parasitize thousands of higher plant species including monocotyledons,
dicotyledons, and herbaceous and woody plants (Eisenback and Hirschmann, 1991).
They reproduce and feed within the roots and usually cause the formation of knots
and galls on roots of susceptible host plants. The physiology of infected plants is
disordered, crop yield is reduced and the quality of the plant products is affected.
More than eighty nominal species have been described so far, about ten species are
agricultural pests, four are major pests and distributed world wide in agricultural areas
(Karrsen, 2002).
Twenty root-knot nematode species have been detected in Europe so far, thirteen of
them having been described from an European type locality (Karssen et al., 1998).
Most species were described from agricultural areas, except for M. ardenensis, M.
deconincki, M. litoralis, M. kralli, M. maritima and M. duytsi (Karrsen, 2002). From
the five root-knot nematodes detected in former Yugoslavia only one species, M.
hapla, was found in Slovenia until 2002.   
Among all described root-knot nematodes, two species: M. chitwoodi and M. fallax
were recently added to the European list of quarantine organisms, to prevent further
distribution within Europe. They parasitize on monocotyledons and dicotyledons,
including several crop plants as potatoes, carrots and tomatoes (Santo et al., 1980;
O’Bannon et al., 1982; Brinkman et al., 1996; Karssen, 2002).
Knowledge about the dissemination of Meloidogyne  spp. in Slovenia is rather poor.
Infrequently was reported about the presence of root-knot nematodes in Slovenia and
only M. hapla was detected in some glasshouses in the regions of Čatež and
Ljubljana. This was the reason for starting more intensive survey on Meloidoyne
species in Slovenia. So far, two different species of the genus Meloidogyne were
detected. The nematodes were identified with morphological and biochemical
methods as M. incognita and M. hapla. Nematodes of M. incognita were isolated from
the roots of paprika plants, Capsicum annum L., from the greenhouse situated in
Portorož at the Adriatic Coast, Slovenia. M. hapla was found for the first time in the
open field in Slovenia. It was isolated from galls of the sweet pepper grown in the
open field in Ljubljana.
Many enzymatic studies have demonstrated that species within Meloidogyne genus
can be differentiated using polyacriamide-gel electrophoresis (PAGE). PhastSistem
ŠIRCA, S., UREK, G., KARSSEN, G.: The incidence of the root-knot nematode ... 17
(Amersham Biosciences) enables biochemical approach for isozyme phenotyping
using very thin (0.4 mm) polyacrylamide slab gel electrophoresis where relatively
small amounts of enzyme (single female content) can be analysed. Combining malate
dehydrogenase (MDH) and esterase (EST) phenotypes analyses is possible to
distinguish between M. incognita and M. hapla (Dalmasso and Berge, 1987;
Esbenshade and Triantaphyllou, 1985; Karssen et al., 1995).
2 MATERIAL AND METHODS
2.1 Nematodes
Females, males and second stage juveniles of M. incognita and M. hapla were isolated from
the infested Capsicum roots samples taken in greenhouse near Portorož and in open field in
Ljubljana, respectively. Roots were placed in a Petry dish and poured over with 0.9% NaCl to
prevent female bursting. Under dissecting microscope nematodes were isolated using scalpel
and nematological needle.
2.2 Method 1: morphometrical identification
Isolated males and second juvenile stages were heat killed and fixed in trietanolamin-formalin
(TAF) solution while analyzes of female parameters and perineal patterns were made on
fresh isolated females. Nematodes were analyzed under microscope connected to personal
computer (LUCIA image analyser system). From different life stages different morphological
parameters were measured and compared with species characteristic parameters (Karssen,
2002). Identification of the species was made by combining morphometrics and isozyme
phenotypes patterns.
2.3 Method 2: native PAGE and isozyme staining
2.3.1 Preparation of the samples
Six young females of each species were isolated under dissecting microscope. After isolation
females were rinsed with reagent-grade water and transferred to 12 sample-well stamp
placed on ice bath. Each female was placed in sample well containing 0.5µl of extraction
buffer (20% sucrose, 2% Triton X-100, 0.01% Bromphenol Blue) (Esbenshade and
Triantaphyllou, 1985) and squashed with needle to release body content. Samples were
loaded on two 12/0.3 sample applicator which were placed into applicator arms of the
PhastSystem device.  For reference species we used Meloidogyne javanica (lines 6 and 7).
2.3.2 Electrophoresis and enzyme staining
Electrophoresis took place onto PhastGel gradient (8 - 25) gel with buffer system according to
manufacture instructions. The following adapted program was used (Karssen et al., 1995):
Sample appl. down at 3.2 Vh
Sample appl. up at 3.3 Vh
Sep 3.1400 V 10 mA 2.5 W 10ºC 10 Vh
Sep 3.1400 V   1 mA 2.5 W 10ºC   2 Vh
Sep 3.1400 V 10 mA 2.5 W 10ºC      125 Vh
After electrophoresis gel was stained for enzymatic activity in a Petry dish at 37ºC with
different staining solutions. MDH staining solution contained 0.05 g β-NAD, 0.03 g Nitro Blue
Tetrazolium, 0.02 g Phenazine Methosulfate,  5.0 ml 0.5 M Tris pH 7.1, and 7.5 ml stock (10.6
g Na2CO3 + 1.34 g L-malic acid in 100 ml water) dissolved in 70 ml of reagent-grade water.
For EST activity we used staining solution contained 100 ml 0.1 M Phosphate buffer pH 7.3,
0.06 g Fast Blue RR salt, 0.03 g EDTA and 0.04 g α-Naphthyl acetate dissolved in 2 ml
Acta agriculturae slovenica, 83 - 1, junij 200418
acetone (Karssen et al., 1995). Incubation for MDH lasted 5 minutes, after that gel was twice
washed with distilled water and further stained for EST activity for 30 minutes. After isozyme
phenotypes patterns were clearly visible the enzymatic reaction was stopped by rinsing gels
with distilled water and fixed for 5 minutes in a solution of 10% acetic acid, 10% glycerol and
80% distilled water.
3 RESULTS
Isozyme phenotype patterns (see Figure 1) were analyzed by calculating relative
migration rates (Rm). The Rm’s for MDH were 0.32 and 0.47 for M. incognita and M.
hapla., respectively and 3.30 for M. javanica as reference. EST phenotype Rm was
0.56 for M. incognita and 0.57 for M. hapla. M. javanica gave tree banded EST
pattern with Rm’s 0.55, 0.62 and 0.64. Species specific isozyme pattern supported
morphological identification of species done on females perineal patterns (Figure 2
and 3) and measurements of nematode different life stages (see Table 1).
Table 1: Morphometrics (average, minimum and maximum) of M. incognita and M.
hapla isolated from the roots of hot and sweet pepper in Slovenia.
M. incognita M. haplaCharacter
J2 Males Females J2 Males Females
N 10 - 5 10 2 5
Body length [µm] 404.5
(378.7-
416.8)
- 418.0
(389.0-
455.3)
1562.2
(1520.4-
1603.9)
-
Greatest body
diameter [µm]
13.9 (12.7-
15.0)
- 13.9 (12.8-
15.0)
42.3 (37.9-
46.7)
-
Body diam. at
stylet knobs [µm]
9.7 (9.3-
10.1)
- 9.5 (9.0-
9.9)
21.5 (20.8-
22.1)
-
Body diam. at anus
[µm]
10.5 (8.4-
12.0)
- - 10.3 (9.5-
11.2)
- -
Stylet length [µm] 11.3 (10.3-
11.9)
16.5 (16.0-
16.7)
11.2 (10.2-
12.2)
17.2 (16.7-
17.6)
14.4 (12.5 -
15.9)
Dorsal gland
opening [µm]
2.6 (2.3-
2.9)
3.6 (3.1-
4.4)
3.1 (2.2-
5.1)
4.2 (3.6-
4.8)
3.7 (2.9 -
5.1)
Metacorpus length
[µm]
- - 43.1 (35.6-
48)
- - 50.2 (45.6 –
54.6)
Metacorpus
diameter [µm]
- - 39.3 (32-
51)
- - 50.6 (46.0 –
54.2)
Tail length [µm] 51.5 (45.3-
56.8)
- - 54.2 (49.2-
59.6)
- -
Tail terminus
length [µm]
12.5 (10.8-
14.9)
- - 17.0 (13.6-
19.9)
- -
Spicule [µm] - - - 39.2 (35.2-
43.1)
-
a 29.0 (26.7-
32.1)
- 30.1 (28.2-
32.0)
37.2 (34.1-
40.1)
-
c 7.9 (7.2-
8.9)
- - 7.7 (7.3-
8.6)
- -
c’ 5.0 (4.2-
6.8)
- - 5.3 (4.8-
5.6)
- -
ŠIRCA, S., UREK, G., KARSSEN, G.: The incidence of the root-knot nematode ... 19
Fig. 1: Isozyme (EST and MDH) phenotype patterns of individual female of M. hapla
(lanes 1,2,3,4,5), M. incognita (lanes 8,9,10,11,12) and M. javanica as
reference (lanes 6,7).
Fig. 2: Meloidigyne incogntia female perineal pattern.
Acta agriculturae slovenica, 83 - 1, junij 200420
Fig. 3: Meloidigyne hapla female perineal pattern.
4 DISCUSSION
M. J. Berkely (1855) was the first who correlated galls on glasshouse cucumber roots
with nematodes. The first root-knot nematode was described in Europe as Anguillula
marioni on Onobrychis sativa Lam. by Cornu in 1879 which was in 1884
synonymized with Heterodera radicicola by Müller (cited in Karrsen, 2002). At the
end of the 19th century, root-knot nematodes were known from all parts of the world
and their polyphagous behaviour were recognized.
Almost one century after the first report of root-knot nematodes, they were still
marked as a single polyphagous species within the genus Heterodera, despite the
clearly documented differences between root-knot and cyst nematodes by
Marcinowski (1909) and Nagakura (1930) (cited in Klindić, 1968). In 1949 Chitwood
separated the root-knot nematodes from Heterodera, re-erected the genus
Meloidogyne, and described a generic diagnosis for the genus. This was the turning
point in root-knot nematode taxonomy and the end of the period of confusion in
taxonomical studies of this group of nematodes (cited in Klindić, 1968). Between
1949 and 1998 more than eighty nominal Meloidogyne species have been described,
especially from North and South America, Africa, China and Europe (Karrsen, 2002).
M. artiellia was the first root-knot nematode described in Europe. Twenty root-knot
nematode species have been detected in Europe so far, thirteen of them having been
described from an European type locality (Karrsen et al., 1998).
Root-knot nematodes were also studied in several regions of former Yugoslavia.
Protič mentioned the presence of the root-knot nematodes on tomato and egg plant
roots in Hercegovina already in 1926 while Martinović in 1947 established the root-
ŠIRCA, S., UREK, G., KARSSEN, G.: The incidence of the root-knot nematode ... 21
knot nematodes on cucumber plants grown in one of glasshouses near Belgrade in
Serbia (cited in Grujčić, 1971). Klindić (1955) reported about damages on red pepper
caused by root-knot nematodes in Hercegovina. Her investigations were focused
mainly in study of efficacy of DD nematicides. Grujičić (1959) reported about the
presence of the Meloidogyne spp. on tomato (Solanum lycopercisum L.), red pepper
(Capsicum annuum L.), cucumber (Cucumis sativus L.), lettuce (Lactuca sativa L.),
celery (Apium graveolens L.) and the weed species Solanum nigrum L. on the
territory of Serbia where he examined the influence of temperature, moisture and soil
on nematode population dynamics. In 1967 Grujičić established M. naasi on sugar
beet, mangold, wheat and barley in Serbia. With all these crops the nematode
appeared sporadically. Some reports about damages caused by root-knot nematodes,
especially from the regions of Istria and Dalmatia, derived from Croatia; no species
were identified (Maceljski, 1967). In 1969 Klindić isolated M. incognita, M. incognita
var. incognita, M. arenaria, M. hapla and M. javanica from different locations in
Hercegovina. In 1970 Grujičić and Paunović for the first time detected M. hapla in
the open field in Serbia. Grujičič also reported about the presence of M. incognita, M.
arenaria, M. acrita, M. hapla and M. javanica on various vegetable plants,
particularly in glasshouses in Serbia. Krnjaič (1977) reported about the spreading of
M. incognita and M. arenaria in glasshouses in Macedonia.
At Agricultural Institute of Slovenia we have recently started more intensive survey
on presence of Meloidogyne species in Slovenia. So far, we have found new locations
of M. hapla and for the first time M. incognita in Slovenia (Širca et al, 2003). Due to
variety of soil and climate present in Slovenia we expect to find more Meloidogine
species in the future.
5  REFERENCES
Brinkman, H., Goossens, J.M., Van Riel, H.R. Comparative host suitability of selected crop
plants to Meloidogyne chitwoodi Golden et al. 1980 and M. fallax Karssen et al.
1996. Anzeiger für Schädlingskunde Pflanzenschutz Umweltschutz, 96 (1996), 127
– 129.
Dalmasso, A., Berge, J.B. Molecular polymorphism and philogenetic relationship in some
Meloidogyne spp.: Application to the taxonomy of Meloidogyne. Journal of
Nematology,10 (1987), 323 – 332.
Eisenback, J. D., Hirschmann, H. T. Root Knot Nematodes: Meloidogyne Species and Races.-
V: Nickle, W. R. Manual of Agricultural Nematology.- Marcel Dekker, Inc. New
york, Basel, Hong Kong, (1991),  191 – 274.
Esbenshade, P.R., Triantaphyllou, A.C. Use of Enzyme Phenotypes for Identification of
Meloidogyne Species. Journal of Nematology,17 (1) (1985), 6 – 20.
Grujičić, G. Prilog proučavanju ekologije Meloidogyne sp. Zaštita bilja, 54 (1959), 69 – 75.
Grujičić, G. Korenova nematoda (Meloidogyne naasi Franklin) u Srbiji. Zaštita bilja, 93-95
(1967), 193 – 197.
Grujičić, G., Paunović, M. Prilog proučavanju korenove nematode (Meloidogyne hapla
Chitwood), Zaštita bilja, 112 – 113 (1971a), 147 – 151.
Grujičić, G. Korenove nematode (Meloidogyne spp.) na povrtarskim biljkama i mogučnosti
suzbijanja preparatima koji niso fitotoksični, Zaštita bilja, 112 – 113 (1971b), 23 – 34.
Acta agriculturae slovenica, 83 - 1, junij 200422
Karssen, G., Van Hoenselaar, T., Verkerk-Bakker, B., Janssen, R. Species identification of
cyst and root-knot nematodes from potato by electrophoresis of individual females.
Electrophoresis, 16 (1995), 105-109.
Karssen, G., Van Hoenselaar, T. Revision of the genus Meloidogyne Göldi, 1892 (Nematoda:
Heteroderidae) in Europe. Nematologica, 44 (1998), 713-788.
Karssen, G. The plant-parasitic nematode genus Meloidogyne Göldi, 1892 (Tylenchida) in
Europe.- Koninklijke Brill NV, Leiden, The Netherlands, (2002),157 p.
Klindić, O. Korenova nematoda (Heterodera marioni Cornu) i propadanje paprika na
području Trebizata. Zaštita bilja, 32 (1955), 31 – 44.
Klindić, O. Vrste nematoda prouzrokovača korenovih guka - Meloidogyne spp. na području
Južne Hercegovine. Zaštita bilja, 102 (1968),  411 – 430.
Krnjaić, D. Fitopatogene nematode u staklarama SR Makedonije. Zaštita bilja, 142 (1977),
429 – 433.
Maceljski, M. Problem korijenovih nematoda u Hrvatskoj. Biljna zaštita 12 (1967), 285 –
288.
O’Bannon, J.H., Santo, G.S., Nyczepir, A.P. Host range of the Columbia root-knot nematode.
Plant Disease, 66 (1982), 1045 – 1048.
Santo, G.S., O’Bannon, J.H., Finley, A.M., Golden, A.M. Occurrence and host range of a new
root-knot nematode (Meloidogyne chitwoodi) in the Pacific Northwest. Plant
Disease, 64 (1980), 951-952.
Širca, S., Urek, G., Karssen, G. Occurrence of the Root-Knot Nematodes Meloidogyne
incognita and M. hapla in Slovenia. Plant Disease, 87 (9) (2003), 1150.


The incidence of the root-knot nematode Meloidogyne incognita and Meloidogyne hapla in Slovenia

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