Distribution and Identification of Root-knot Nematodes from Turkey

Abstract

Root knot nematodes are causing serious losses in protected cultivation fields in the West Mediterranean region of Turkey. Correct and confident identification of the plant parasitic nematodes is important for vegetable growing and breeding. Therefore, ninety-five populations of plant parasitic nematodes were collected from regional greenhouses. Previously described species-specific primers were used to identify Meloidogyne populations. The present study indicated that SEC-1F/SEC-1R and INCK14F-INCK14R primers for identifying of M. incognita, Fjav/Rjav and DJF/DJR primers for M. javanica and Far/Rar for M. arenaria primers can be effective tools to identify the Turkish root-knot nematode species. Dissemination ratios of the population were 64.2%, 28.4% and 7.3% for Meloidogyne incognita, M. javanica and M. arenaria, respectively. The results showed that M. incognita was the prominent root-knot nematode species in the West Mediterranean coastal areas of Turkey.

Turkey is amongst the leading vegetable producers in the world. Total vegetable production was nearly 22 million metric tones produced from 815.000 ha (Anonymous, 2007). Vegetables are produced both in open fields and protected fields in Turkey. Turkey is geographically divided into seven regions including the Mediterranean that is further divided into two regions which includes the West Mediterranean and East Mediterranean regions. The West Mediterranean region is the centre of vegetable production due to its climatic conditions.

The root-knot nematode group is one of the most important pathogen affecting crops in protected cultivation in the Mediterranean coastal areas of Turkey (Elekçioğlu and Uygun 1994; Elekçioğlu et al., 1994; Devran et al., 2008). Over the past decade, a number of studies have been conducted on root-knot nematodes in Turkey (Elekçioğlu and Uygun, 1994; Elekçioğlu et al., 1994; Mennan and Ecevit, 1996; Söğüt and Elekçioğlu, 2000; Devran et al., 2002; Devran et al., 2008). However, a comprehensive work, so far, has not been done on the distribution and diagnosis of the nematode in the West Mediterranean region of Turkey.

Correct identification of plant parasitic nematodes is of importance in terms of vegetable growing and breeding. Root-knot nematodes have been identified based on morphological characters (Eisenback and Triantaphyllou, 1991), host-plant response (Hartman and Sasser, 1985), isozyme analyses (Esbenshade and Triantaphyllou, 1990) and molecular techniques (Powers and Harris, 1993; Powers et al., 1997; Ziljstra et al., 1995; Zijlstra et al, 2000; Adam et al., 2007). However, identification by morphological characters and host plant response is time consuming and needs extensive labor. Isozymes analysis is an effective method which can be carried out on females of Meloidogyne spp. (Esbenshade and Triantaphyllou, 1990). However, application of this method only on female individuals is a limited factor, is difficult, and can be affected by environmental factors. However, molecular techniques based on DNA can be used in every stage of the nematode's life cycle, and they are rapid, and reliable.

The objectives of this study were to identify root-knot nematodes collected from different locations of the West Mediterranean coastal areas of Turkey using molecular methods, and to select effective primers from previous studies for Meloidogyne species identification in this region. Furthermore, regional distribution of the nematode was determined.

Materials and Methods

Root-knot nematode cultures: Ninety-five populations of Meloidogyne spp. from the Mediterranean costal areas of Turkey were collected from the roots of infested cultivated host plants (Table 1, Figure 1). Egg masses were picked up from roots using a small needle. Each root-knot nematode isolate was established as single mass line in monoxenic pure cultures. Root-knot nematodes were reared on fresh market tomato plants (Tueza F1, Multi Tarım, Turkey). Tomato seeds were germinated in steam-sterilized sandy soil in seed tray, and 2-wk-old seedlings were transplanted singly into 250 ml plastic pots. Plants were inoculated at the fourth true leaf stage with single nematode egg mass.

Table 1.

Root-knot nematodes populations used in this study

Fig. 1.

The West Mediterranean Region map. 1: Tomato, 2: Pepper, 3: Cucumber, 4: Eggplant and 5: Bean.

DNA extraction: DNA was extracted from nematode egg masses, juveniles and females according to DNAeasy Tissue and Blood Kit (Qiagen, Hilden, Germany) following manufacturer' instructions.

Species-Specific PCR: The primers used for DNA analysis of the root-knot nematode species are listed in Table 2. All PCR amplification was performed in a total volume of 25 μl containing 10XPCR Buffer, 0.2 mM dNTP, 0,4 μM of each primer, 2 mM MgCI₂, 20 ng of template DNA and 1 Unit Taq DNA Polymerase (Vivantis). The PCR amplification condition of primers are described in Table 3. PCR was carried out using a thermal cycler DNA Engine PTC-200 Peltier Thermal Cycler (Bio-Rad, Hercules, CA). PCR products were separated by electrophoresis in Tris-EDTA (TAE) buffer with 2 % agarose gel stained with ethidium bromide (0.5 μg/ml) at 100 V for 2.5 h and then visualized under UV light.

Table 2.

Primers used for molecular identification of root-knot nematode

Table 3.

PCR amplification conditions of primers used for molecular identification of root-knot nematode

Results

Molecular Characterization: The primer pairs used for diagnosis of Meloidogyne incognita, M. javanica and M. arenaria were screened for species specificity. The optimized primers resulted in consistent amplifications by DNA obtained from egg masses, juveniles and females. In this study, gene specific and SCAR primers were used for identification of root-knot nematodes. Tesarova et al. (2003) reported that Sec primer was developed from SEC protein gene sequences belonging to M. incognita. C2F3 and 1108 primers were developed from mitochondrial gene sequence (Powers and Harris, 1993). Other primers were species-specific SCAR developed from RAPD markers (Zijlstra et al., 2000; Dong et al., 2001; Randig et al., 2002; Meng et al., 2004).

Although some primer sets resulted in positive amplifications, there were also some primers giving no product, therefore these primer sets were not used for root-knot nematode identification.

Meloidogyne incognita was identified using five different primer sets (Table 2). PCR with the M. incognita species-specific SEC-1F/SEC-1R, inc-K14-F/ inc-K14-R and MIF/MIR produced approximately 500 bp, 400 bp and 1000 bp DNA fragment for all M. incognita populations, respectively (Figs. 2-4). However, Finc/Rinc and DIF/DIR primer sets did not display amplification. Whilst M. javanica was identified by Fjav-Rjav and DJF/DJR primer sets produced 670 bp and 1650 bp fragment from all populations, respectively (Fig. 5A, B), there were no amplification products with C2F3-1108 primers. The Far/Rar primer set produced a 420 bp fragment, which was characteristic in all populations of M. arenaria. However, DAF/DAR and C2F3/1108 primers did not give amplification products in M. arenaria populations (Fig. 6).

Fig. 2.

Amplification products with the M. incognita species-specific SEC-1F/SEC-1R primers. M: 100 bp DNA Ladder (Vivantis), Samples (G2-O5), C: Positive control, H: water

Fig. 4.

Amplification products with the M. incognita species-specific MIF-MIR primers. M: 100 bp DNA Ladder (Vivantis), Samples (G2-O5), C: Positive control, H: water

Fig. 5A.

PCR products with the M. javanica species-specific DJF-DJR primers. M: 100 bp DNA Ladder (Vivantis), Samples (G1-O3), H: water

Fig. 5B. PCR products with the M. javanica species-specific Fjav-Rjav primers M: 100 bp DNA Ladder (Vivantis), Samples (G1-O3), H: water

Fig. 6.

Amplification products with the M. arenaria species-specific Far-Rar primers. M: 100 bp DNA Ladder (Vivantis), Samples (M9-O7), C: Positive control, H: water

Fig. 3.

Amplification products with the M. incognita species-specific inc-K14-F/ inc-K14-R primers. M: 100 bp DNA Ladder (Vivantis), Samples (G2-O5), C: Positive control, H: water

Distribution of the pathogen: Meloidogyne incognita, M. javanica and M. arenaria ratios were 64.2%, 28.4% and 7.3% of the total populations, respectively. Meloidogyne incognita was determined as the most common root-knot nematode species in the Mediterranean coastal areas of Turkey. Meloidogyne incognita was also detected in all districts in which tomato, pepper, cucumber and eggplant cultivars are grown. Meloidogyne javanica was detected in every district except for Serik. Meloidogyne javanica was not detected within samples collected from pepper growing areas. Meloidogyne arenaria was especially found in Ortaca where the tomato is cultivated.

Discussion

Root-knot nematode species are morphologically very similar to each other and identification to the species level is difficult. Moreover, more than one root knot nematode species are sometimes found together in the same plant root. Therefore, fast and accurate identification of root-knot nematodes is needed for management and breeding. Root-knot nematode species, M. incognita, M. javanica and M. arenaria, which cause serious yield losses in protected vegetable areas in Mediterranean coastal areas of Turkey, were identified using species-specific primers in this study. Primers optimized successfully resulted in amplification of DNA obtained from all the nematode sources including egg masses, juveniles and females.

Different primer sets were used in our studies to identify of root-knot nematodes. SEC-1F/SEC-1R, inc-K14-F/inc-K14-R, MIF/MIR for M. incognita, Fjav/Rjav, DJF/DJR and Far/Rar primers for M. arenaria gave successfully PCR products. Our results showed agreement with earlier studies (Zijlstra et al., 2000; Dong et al., 2001; Teserova et al., 2003; Randig et al., 2002; Meng et al., 2004; Adam et al, 2005; Tzortzakakis et al., 2005; Adam et al., 2007). Meloidogyne incognita species-specific MIF/MIR primer effectively gave the expected DNA fragment in all M. incognita populations. Moreover, this primer successfully gave PCR products in some M. javanica populations (A1, A4, A5, AKS2, AKS7). This primer was reported as a specific primer for M. incognita (Meng et al., 2004, Adam et al., 2007). This study showed that this primer was not specific for identification of M. incognita which might be due to differences in the binding sites of the primers.

Although the primers Finc/Rinc (Zijlstra et al., 2000, Adam et al, 2005), DIF/DIR (Dong et al., 2001), C2F3/1108 (Powers and Harris, 1993, Powers et al., 2005) and DAF/DAR (Dong et al., 2001) were previously tested and reported as effective primers, no amplification product was obtained by these primers in our study. Amplification could have failed due to changes in the priming sites and PCR conditions. Obtaining inconsistent product by Finc/Rinc primers has previously been reported (Adam et al., 2007) and our findings are in agreement with Adam et al. (2007).

In previous research, the authors reported that M. incognita was a common species in vegetables growing areas of Turkey (Elekçioğlu and Uygun, 1994; Elekçioğlu et al., 1994; Söğüt and Elekçioğlu, 2000, Mennan and Ecevit, 1996). Söğüt and Elekçioğlu, (2000) informed that M. areneria was rarely detected in protected cultivars. Meloidogyne javanica was also not founf in pepper cultivations of the East Mediterranean region (Söğüt and Elekçioğlu, 2000). Our study showed accordance with these previous findings.

In conclusion, our study reported on the optimization of PCR for routine, rapid and accurate identification of Turkish root-knot nematode species.