Nematol. medit. (2001), 29: 91-97

Agricultural Research Centre, Department of Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke,

MELOIDOGYNE CHITWOODI AND M. FALLAX IN BELGIUM

by L. WAEYENBERGE 1 and M. MOENS

Summary. During a Meloidogyne survey in Belgium a total of 2877 soil and root samples were taken. Root­ knot were found in 9.2% of the samples. The species were identified according to morphological, morphometrical, biochemical, and molecular characters. Ten percent of the positive samples contained M. cbit­ woodi and/or M.fallax. Among the enzymes used, only Tru 91 clearly separated M. cbitwoodi, M.fallax and M. bapla from each other. RAPD was used to examine the molecular diversity between the populations. The tree constructed on the basis of the RAPD patterns grouped the populations according to the species. It revealed considerable intraspecific variation supporting the idea that M. cbitwoodi and M. fallax have been present in Belgium for a long time.

Root-knot nematodes are widely distributed In the present paper the distribution and the and cause substantial reduction of crop yield molecular diversity of both M. chitwoodi and M. and quality. In 1974 a root-knot species was fallax populations in Belgium are reported. found on tubers in North-America and grouped in the Meloidogyne hapla complex. Later, the population was described Material and methods as a new species, M. chitwoodi (Golden et aI., 1980). Since its description, M. chitwoodi has Soil and root samples, in total 2877, were been reported in several European countries: collected throughout Belgium during 1996-1997. the (Brinkman and Van Riel, 1990), Potato and vegetable producing areas were Germany (Heinicke, 1993; Mliller et aI., 1996) sampled more intensively and the number of and France (Marzin, pers. com.). In 1992 it was samples was increased in areas where M. chit­ detected for the first time in Belgium (M. woodi had previously been detected. Soil sam­ Moens, unpublished). ples were put in pots and planted with tomato During the period 1992-1995 a root-knot ne­ (cvs Marmande or Moneymaker). Two months matode population closely related to M. chit­ later, roots were washed and examined for the woodi was detected in the Netherlands. Be­ presence of root-knots. cause of differences in morphology and To increase the possibility for detecting isozyme patterns, this population was later de­ mixed populations 30 young females were col­ scribed as M. fallax (Karssen, 1996). lected from each population and kept for bio-

1 Corresponding author: [email protected].

- 91- chemical identification. Corresponding egg microlitre DNA extract was used for the PCR. masses were used to build up monospecific The PCR mixture contained 3 pI lOX PCR populations. This was done on potato tubers (c, buffer, 4 mM MgCb, 250 11M of each dNTP, 0.2 Bintje) in closed plastic containers filled with pM of a ten-mer primer of random sequence sterilised sand (method originally developed by (OPA2, OPA3, OPA4, OPA7, OPA9, OPAI0, J. J. Van De Haar, The Netherlands). OPAll, OPAI2, OPA13, OPE9, OPEI9, OPK2, }1;1. chitwoodi Golden, 0' Bannon, Santo et OPMI2, Operon Technologies, Alameda, USA), Finley and M. fal/ax Karssen, were morphologi­ 2 units Taq DNA-polymerase (Qiagen, Crawley, cally and morphometrically identified according West Sussex, UK) and double distilled water up to Jepson (1987) and Karssen (1999). The tech­ to a total volume of 30 pI. The PCR programme nique described by Karssen et ai. (1995) was was carried out in a DNA thermal cycler (PTC used for biochemical characterisation. 200, Biozym, Landgraaf, The Netherlands) with DNA was extracted from five juveniles of the an initial denaturation at 93°C for 5 min, fol­ same egg mass according to Joyce et at. (1994) lowed by 45 cycles of 93 °C for 30 s, 36°C for and used in PCR. Two 21 nucleotide sequences 1 min, and 72 °C for 1 min and a final exten­ complementary to conserved sequences in the sion at 72°C for 8 min. Ten microlitres of PCR 18S and 26S rONA genes of Xipbinerna mixture were used for electrophoresis in a 2% bricoiensis (Vrain et at., 1992) served as primers. agarose gel containing ethidium bromide. Fol­ Upon completion, 5 ml of the PCR mixture was lowing electrophoresis, DNA fragments were vi­ used for electrophoresis in a 1% agarose gel sualised under UV light and photographed. The containing ethidium bromide. Following elec­ patterns were recorded manually as a binary trophoresis, DNA fragments were visualised un­ matrix. The RAP Distance Programs, version 1 der Lv light and photographed. The concentra­ (Armstrong J., Gibbs A., Peakall Rod and tion of the DNA bands was estimated with a Weiller G. of the Australian .\rational University, DNA mass ladder on the gels. Equal concentra­ Canberra, Australia) was used for the analysis of tions of each PCR product were digested with the' RAPD fragments. Genetic distances among different restriction enzymes (AluL Bsh1236I, the 22 populations were calculated by means of BsiZI, CfoI, HinfI, lipaII, MvaI and Tru9l) in the pairwise comparison using the Dice coefficient. appropriate buffer according to manufacturer's Cluster analysis was performed using TREE­ instructions. RFLP patterns were visualised un­ VIEW (Page, 1996). der UV light after electrophoresis on a 2% agarose gel with ethidium bromide and pho­ tographed. To check repeatedly the identity and Results and discussion purity of the monospecific cultures, PCR with species-specific primers for M. chitwoodi and M. Identification of root-knot nematodes using fallax (Petersen et ai., 1997) were used. morphological and morphometrical characteris­ To study the molecular diversity of M. cbit­ tics requires skill, is time-consuming and often woodi and M. fallax, total DNA was extracted inconclusive. Biochemical techniques using es­ from egg masses with the Puregene DNA isola­ terase and malate dehydrogenase patterns have tion Kit ( Tissue Protocol, Biozym, Land­ proven to be suitable for the characterisation of graaf, The Netherlands). In addition to the col­ A1eloidogyne species (Esbenshade and Trianta­ lected Belgian populations, DNA was also ex­ phyllou, 1985; Pais and Abrantes, 1989; Esben­ tracted fromiVf. chitwoodi race 1, 2 and 3 shade and Triantaphyllou, 1990; Ibrahim and (USA), M. chitwoodi (Rips, The Netherlands) Perry, 1993). Although Karssen et al. (1995) and iVI. fallax (Baexem, The Netherlands). One adapted this technique to an automated elec-

- 92- trophoretic apparatus it still has its limits as it requires young females. The technique was adopted because it allowed the use of the cor­ responding egg masses of identified females to initiate pure cultures. Molecular tecniques are potentially more powerful for nematode diagnosis. Comparitive analysis of coding and non-coding regions of ri­ bosomal DNA (rDNA) became a popular tool for species and subspecies identification of plant­ parasitic nematodes from many genera (Vrain et al., 1992; Wendt et al., 1993; Zijlstra et aI., 1995; Subbotin et al., 2000; Waeyenberge et al., 2000). Studies on the genus Meloidogyne by Zijlstra et al. (1995) and Schmitz et at. (1998) revealed that only one (RsaI) out of 33 restriction en­ Fig. 2 - Locations of M. chitwoodi ( . ) and M. fallax (T) zymes differentiated M. chitwoodi from M . fal­ populations detected in Belgium. lax. However, the RsaI pattern of M. fallax was not clearly distinguishable from that of M. hap­ Root-knot nematodes were present in 9.2% lao Tru9I undoubtedly separated the three of the samples and M. hapla was the predomi­ species (Fig. 1). PCR with the species specific nant species. M. chitwoodi and/ or M. fallax primers designed by Petersen et al. (1997) re­ were found in 10% of the positive samples. In peatedly confirmed the purity of the cultures certain areas M. chitwoodi and M. fallax were and proved that these primers are valid for the widespread and caused more problems than M. identification of Belgian populations of M. chit­ hapla. M. chitwoodi and M. fallax were detect­ woodi and M. fallax. ed mainly in areas of intensive vegetable grow­ ing for the canning industlY in the provinces of and (Fig. 2, Table I and II). In these parts of Belgium, non-infected fields are becoming rare. M. chitwoodi was also found in the heart of a potato producing area of the a province of West-Flanders. The nematodes were extracted from sandy to b sandy-loam soils which were slightly acidic. Both species were isolated from different hosts (Table I and II). A note in the FAO Plant Protection Bul­ c letin (Anonymus, 1991) suggests that M. chitwoo­ di was already present in The Netherlands in 1930. The nematode would have been able to remain undetected because of its resemblance to M. hapla. The same situation might also have oc­ *HCCCC F C C F F * 1 12 11 10 9 9 1 2 1 2 curred in Belgium as M. chitwoodi juveniles were isolated from oak forest soil (code C1). Fig. 1 - Tru9I RFLP pattern of Meloidogyne species - see Table III for codes; ": lOObp DNA-ladder (a = l.5Kb, b = For all populations listed in Table III, repro­ 500bp, c = lOObp). ducible RAPD patterns were obtained with all

- 93- TABLE I Origin qlMeloidogyne chitwoodi populations detected in Belgium.

Province Location Soil Host Antwerp Sandy 5.92 maize Sandy 5.83 black salsify Poederlee Sandy 5.02 fallow Postel Sandy 5.55 black salsify Postel (*) Sandy 6.21 black salsify Postel Sandy 5.72 black salsify Postel Sandy 5.83 black salsify Postel Sandy 5.56 black salsify Postel Sandy 5.80 bean Postel (*) Sandy 5.78 Postel Sandy 6.82 fallow Sandy-loam 5.49 bean Limburg Hasselt Sandy 6.81 sugar beet Houthalen Sandy 5.92 oak forest Sandy 6.43 maize West-Flanders Waregem Sandy-loam 6.04 fallow

* Population mixed with

TABLE II - Origin ofMeloidogyne fallax populations detected in Belgium.

Province Location Soil Host Antwerp Sandy sugarbeet Kasterlee Sandy 6.59 maize Postel (*) Sandy 5.78 carrot Postel Sandy 5.39 grassland Postel Sandy 5.67 grassland Postel Sandy 5.30 carrot Postel Sandy 6.21 black salsify Limburg Hasselt Sandy 6.81 sugarbeet Lommel Sandy 5.79 grassland Stokkem Sandy-loam 6.82 maize

(*) mixed with M. chitwoodi. of the primers tested. The RAPD patterns re­ fallax (Fig. Sub-clusters are not related to vealed considerable intraspecific variation any geographic origin. Studies by Schmitz et at. among the populations (Fig. 3). The tree is (1998) grouped the three American M. chitwoo­ composed of three main branches grouping the di populations in one cluster. In our results AI. populations of M. hapla, M. chitwoodi or M. chitwoodi races 1 and 2 are separated from TABLE III - List ofMeloiclogyne populations 'used for RAPD.

Species Origin Code M. hapZa Belgium, Lommel HI M. chitwoodi Belgium, Houthalen CI M. chitwoodi Belgium, Postel C2 M. chitwoodi Belgium, Waregem C3 l\II. chitwoodi Belgium, Hasselt C4 l\II. chitwoodi Belgium, Putte C5 M. chitwoodi Belgium, Kasterlee C6 M. chitwoodi Belgium, Lommel C7 l\II. chitwoodi Belgium, Balen C8 M. chitwoodi The Netherlands, Rips C9 l\II. chitwoodi race 1 USA, Oregon CIO l\II. chitwoodi race 2 USA, Oregon Cll l\II. chitwoodi race 3 USA, California CI2 l\II.jallax Belgium, Hasselt FI M.jallax Belgium, Stokkem F2 l\II.jallax Belgium, Postel F3 M.jallax Belgium, Postel F4 l\II.jallax Belgium, Postel F5 l\II.jallax Belgium, Lommel F6 l\II.jallax Belgium, Arendonk F7 l\II.jallax Belgium, Kasterlee F8 l\II.jallax The Netherlands, Baexem F9

• F F F F He e e e e • F F F H C e e e e c e • F FFHccece 1 2 3 4 1 1 2 5 8 9 3 4 5 1 1 2 3 6 7 8 12 6 7 8 1 4 7 8 9 10

Fig. 3 - RAPD patterns for Meloidogyne species. (A) OPA2, (B) OPA3, (C) OPA4; see Table III for codes; .: lKb DNA-ladder (a = lOKb, b = 3Kb, c = 1Kb).

- 95- ,--______H1 Acknowledgements. This research was

,.------C12 nanced by "Begrotingsfonds voor de productie

...-----C8 en bescherming van planten en plantaardige producten", "Ministerie van Middenstand en .------C2 Landbouw - Bestuur voor Onderzoek en On­ ...------C1 twikkeling" and "N. V. La Corbeille". We are grateful to Drs H. Mojrahedi, G. S. Santo and G.

C4 Karssen for providing Meloidogyne populations from the USA and the Netherlands.

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Accepted for publication on 12 February 2001.

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