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. 16 Acta agriculturae slovenica, 83 - 1, junij 2004 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 ß-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 ?-Naphthyl acetate dissolved in 2 ml 18 Acta agriculturae slovenica, 83 - 1, junij 2004 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. Character M. incognita M. hapla Males N Body length [µm] J2 10 404.5 (378.7-416.8) 13.9 (12.7-15.0) Males Females J2 10 418.0 (389.0-455.3) 2 1562.2 (1520.4-1603.9) 42.3 (37.9-46.7) 21.5 (20.8-22.1) Females 5 - Greatest body diameter [µm] 13.9 (12.8-15.0) 9.5 (9.0-9.9) - Body diam at stylet knobs [µm] 9.7 (9.3-10.1) 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) 2.6 (2.3-2.9) - 16.5 (16.0-16.7) 11.2 (10.2-12.2) 17.2 (16.7-17.6) 4.2 (3.6-4.8) - 14.4 (12.5 - 15.9) 3.7 (2.9 - 5.1) 50.2 (45.6 – 54.6) Dorsal gland opening [µm] Metacorpus length [µm] 3.6 (3.1-4.4) 43.1 (35.6-48) 3.1 (2.2-5.1) - 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] Spicule [µm] 12.5 (10.8-14.9) - - 17.0 (13.6-19.9) - 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 c’ 7.9 (7.2-8.9) 5.0 (4.2-6.8) 7.7 (7.3-8.6) 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 reference (lanes 6,7). as Fig. 2: Meloidigyne incogntia female perineal pattern. 20 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. 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