Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species-specific SCAR-PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species-specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root-knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.
Meloidogyne ethiopica is a root-knot nematode (RKN) found throughout the tropics (Hunt & Handoo, 2009). Recently, it has been detected in Europe (Širca et al., 2004; Conceição et al., 2012) and has been placed in the 2011 EPPO alert list. Additional information was recently added to the taxonomic status of this nematode based on its detection in Brazil and Chile, parasitizing kiwifruit (Actinidia deliciosa) and grapevine crops (Vitis vinifera), respectively (Carneiro et al., 2003, 2004, 2007). The characteristics of those isolates were compared with the type isolate of M. ethiopica and an isolate originating from Kenya: all isolates showed the characteristics typical for the species (Carneiro et al., 2004).
The identification of M. ethiopica requires a detailed study of a suite of distinctive morphological features of the female, male and second-stage juveniles (J2). However, the perineal patterns of the female are highly variable and are similar to M. arenaria or to M. incognita. Isozyme phenotyping is currently the most useful character for routine differentiation of M. ethiopica from other RKN species as the esterase E3 is species-specific (Carneiro et al., 2004). However, the technique requires fully developed female nematodes in good condition, which are sometimes difficult to obtain from field samples.
In Brazil, M. ethiopica has been reported causing severe infection of root systems of kiwifruit plants, which show defective growth, are distorted by small and large multiple galls, and are devoid of fine roots (Carneiro et al., 2003). It is hypothesized that this nematode was introduced into Brazil in 1989 on kiwifruit seedlings imported from Chile (Carneiro et al., 2003). Furthermore, given the large number of host species, M. ethiopica is considered a difficult nematode to eradicate (Carneiro et al., 2003, 2004). Therefore, it is important to protect uninfected sites by planting certified nematode-free stocks and avoiding other pathways by which this RKN species might be introduced.
The wide distribution of M. ethiopica throughout the various Chilean regions is probably because of the use of contaminated grapevine seedlings. In Chile, the prohibition of producing grapevine seedlings in areas with RKN and on the movement of infested seedlings into new areas seems to be ineffective, as all nurseries surveyed were severely infested with M. ethiopica and commercialization of uncertified plant materials is allowed (Carneiro et al., 2007).
PCR-based analysis of neutral molecular markers, e.g. amplified fragment length polymorphisms (AFLP) and random amplified polymorphic DNA (RAPD), has been very useful to detect genetic variability within RKN species and such methods are sensitive, rapid and relatively simple, showing several loci scattered throughout the genome without requiring prior knowledge of the genetic information of the target species (Welsh & McClelland, 1990; Williams et al., 1990; Cenis, 1993; Castagnone-Sereno et al., 1994; Vos et al., 1995; Carneiro et al., 1998, 2008a; Randig et al., 2002; Fargette et al., 2005; Santos et al., 2012). In addition, differential DNA fragments resulting from these markers have been used to design longer, species-specific PCR primers. Such PCR-based detection tools used for RKN species are attractive because they are simple, fast and can be used routinely on a large number of samples, particularly for quarantine purposes, without requiring prior multiplication of the target nematode or living J2 or females (Zijlstra, 2000; Zijlstra et al., 2000; Randig et al., 2002; Powers, 2004; Blok, 2005; Blok & Powers, 2009; Tigano et al., 2010).
In this study phylogenetic analyses based on AFLP and RAPD markers were used to assess the genetic diversity within M. ethiopica isolates obtained from different hosts and regions of Brazil, Chile and Kenya. In addition, a sequence characterized amplified region (SCAR) marker was developed that can be used individually or in multiplex PCR reactions containing mixtures of species for fast and routine diagnosis of this important nematode species from field samples.
Materials and methods
Fourteen populations of M. ethiopica originating from Brazil and Chile, one M. ethiopica population from Kenya and three other Meloidogyne isolates found on kiwifruit plants (Table 1) were identified and purified using isozyme analyses (esterase and malate dehydrogenase) according to Carneiro et al. (1996) and Carneiro & Almeida (2001). Ten other RKN species, each represented by one single isolate, were also included in the analysis (Table 1). Purified nematode isolates were multiplied on tomato plants (Solanum lycopersicum cv. Santa Clara) under greenhouse conditions.
Table 1. Meloidogyne spp. isolates used in this study
Brazilian states: DF, Distrito Federal; RS, Rio Grande do Sul; SP, São Paulo; PR, Paraná; PE, Pernambuco.
Esterase and malate dehydrogenase phenotyping.
Brasília, DF, Brazil
Farroupilha, RS, Brazil
Itapetininga, SP, Brazil
Braganey, PR, Brazil
Requínoa, Cachapoal, Chile
Casablanca, Santa Rita, Chile
Codigua, Melipilla, Chile
Rengo, Cachapoal, Chile
Fundo El Quadro, Casablanca, Chile
San Clemente, Talca, Chile
Los Robles, Santa Emiliana, Chile
Sagrada Família, Monte Frutal, Chile
Casa Blanca, La Rotunda, Chile
Caxias do Sul, RS, Brazil
Brasília, DF, Brazil
Londrina, PR, Brazil
Londrina, PR, Brazil
Farroupilha, RS, Brazil
Londrina, PR, Brazil
Petrolina, PE, Brazil
Londrina, PR, Brazil
São Paulo, SP, Brazil
Nematode eggs were extracted from infected roots of tomato plants according to McClure et al. (1973) and stored at −80°C. For each nematode isolate, total genomic DNA was extracted and purified from aliquots of 200–300 μL of eggs according to a protocol described by Randig et al. (2002). Genomic DNA was also extracted from a single J2 nematode and/or female following a method described by Castagnone-Sereno et al. (1995).
For each isolate, c. 1 μg of total genomic DNA was digested overnight at 37°C with EcoRI (15 U μL−1; Invitrogen) and ligated to the specific adapters following the method of Suazo & Hall (1999). A total of 30 random 22-mer primers (Integrated DNA Technologies) were used, consisting of the EcoRI adapter core sequences 5′-GACTGCGTACCAATTCAGT-3′ plus the 3′ selective nucleotides (AGT, ACT, AGC, ATT, GGC, CAG, TGG, CCT, ACC, TCG, ATA, AGG, AAT, AAC, GCC, CGA, GGG, CTC, CAT, TTA, TTG, TAC, GAC, GTG, CCG, TCT, GAG, TGC, CGT and CAC). PCR reactions were performed in a 25 μL final volume containing 1 μL (50 ng μL−1) digested DNA, 2·5 μL 10× PCR buffer without magnesium chloride (Invitrogen), 1 μL 50 mm MgCl2, 0·5 μL 10 mm dNTPs, 1 μL 10 μm primer and 0·3 μL Taq DNA polymerase (5 U μL−1; Invitrogen). DNA was amplified using the PTC-100 thermocycler (MJ Research) under the following cycling parameters: 1 min at 95°C; 37 cycles of 1 min at 94°C, 1 min at 56°C, 2·5 min at 72°C; and a final extension of 10 min at 72°C (Suazo & Hall, 1999). PCR products were separated by electrophoresis in a 1·5% (w/v) agarose–synergel (0·7% agarose, 0·4% synergel; Diversified Biotech), stained with ethidium bromide (0·3 μg mL−1) and photographed under UV light. The analysis was repeated three times.
PCR reactions were performed in a 13 μL final volume containing 1·3 μL 10× PCR reaction buffer (Phoneutria Biotecnologia & Serviços), 0·4 μL 10 μm primer (Operon Technologies), 2 μL 1·25 mm dNTPs (Invitrogen), 0·2 μL 5 U μL−1Taq DNA polymerase (Phoneutria Biotecnologia & Serviços) and 3 μL total genomic DNA (3 ng μL−1) of the M. ethiopica isolates, Meloidogyne sp. and M. enterolobii, a species distantly related to M. ethiopica (Table 1). The following 33 random 10-mer oligonucleotide primers (Operon Technologies) were used for assessing the genetic diversity of M. ethiopica: OPA-02, OPA-03, OPA-04, OPA-05, OPA-07, OPA-13, OPA-15, OPA-18, OPB-13, OPC-09, OPC-11, OPC-16, OPC-18, OPD-08, OPD-18, OPD-20, OPE-02, OPE-12, OPE-16, OPE-18, OPG-02, OPG-03, OPG-06, OPJ-10, OPJ-19, OPJ-20, OPK-01, OPK-05, OPN-08, OPP-01, OPP-02, OPR-07 and OPR-08. Another set of 57 RAPD primers was used for screening the M. ethiopica genome to find DNA markers present only in this species as compared to the other RKN species listed in Table 1. These primers comprised: OPA-01, OPA-04, OPA-07, OPA-12, OPA-13, OPA-14, OPA-18, OPAB-02, OPAB-03, OPAB-04, OPAB-06, OPAS-08, OPB-04, OPB-05, OPB-06, OPB-07, OPB-09, OPB-11, OPB-12, OPC-02, OPC-07, OPC-09, OPC-20, OPD-05, OPD-16, OPE-05, OPE-07, OPE-08, OPF-06, OPG-02, OPG-03, OPG-05, OPG-06, OPG-13, OPH-01, OPJ-10, OPJ-20, OPK-01, OPK-07, OPK-09, OPK14, OPK-16, OPK-19, OPK-20, OPL-08, OPL-19, OPM-10, OPM-20, OPN-10, OPO-01, OPO-02, OPO-05, OPQ-12, OPR-03, OPR-04, OPR-07 and OPR-08. The amplification was performed on a PTC-100 thermocycler, using the following settings: 5 min at 94°C; 40 cycles of 30 s at 94°C, 45 s at 36°C, 2 min at 70°C; and a final extension of 10 min at 70°C (Randig et al., 2002). PCR products were separated by electrophoresis in a 1·5% (w/v) agarose gel, stained with ethidium bromide (0·3 μg mL−1) and visualized under UV light. All RAPD analyses were repeated three times.
AFLP and RAPD markers were used to determine the intraspecific genetic diversity among 15 isolates of M. ethiopica and three isolates of Meloidogyne sp., using M. enterolobii as an out-group (Table 1). The experiments were run three times and only DNA fragments consistently present between repeats were recorded as present or absent directly from the gels. DNA fingerprints were converted into a 0–1 binary matrix and phylogenetic reconstruction was performed using the neighbour-joining (NJ) algorithm (Saitou & Nei, 1987) in paup* v. 4b10 (Swofford, 2002), considering the data as unordered with no weighting.
To test the node support of the generated trees, the analysis was performed on 1000 bootstrap replicates and only values above 50% were considered. As the AFLP and RAPD markers could be considered to be independent characters, the two data sets were combined into a global NJ analysis, using the total evidence approach proposed by Huelsenbeck et al. (1996). The same settings used for the individual analysis were used for this combined data set.
Development of species-specific SCAR-PCR
A DNA fragment present only in the M. ethiopica genome was extracted from the RAPD gel using the Wizard SV Gel/PCR Clean Up system (Promega) and cloned into the pGEM-T Easy vector (Promega), following the manufacturer's instructions. Sequencing of the insert was carried out on two independent clones by Macrogen. A pair of species-specific primers, with longer sequences, higher annealing temperature (Tm) and high G+C content compared to the RAPD primer, was designed based on the consensus sequences using primer3 v. 4.0 (Rozen & Skaletsky, 2000) and synthesized by Invitrogen. Specific SCAR primers were designed spanning the RAPD primer sequences. Further analysis using a primer pair designed from OPE-07, which differentially amplified a DNA fragment in M. ethiopica, was performed to test its specificity to this species. The sequences of this fragment were blast searched against the NCBI databases (http://blast.ncbi.nlm.nih.gov). SCAR-PCR was performed in a 25 μL final volume, containing 1× reaction buffer (Phoneutria Biotecnologia & Serviços), 200 μm of each dNTP (dATP, dTTP, dGTP and dCTP; Invitrogen), 10 μm each primer (meth-F 5′-ATGCAGCCGCAGGGAA-CGTAGTTG-3′ and meth-R 5′-TGTTGTTTCATGTGCTTCG-GCATC-3′), 1 U Taq DNA polymerase (Phoneutria Biotecnologia & Serviços) and 6 ng total genomic DNA from M. ethiopica and other RKN species tested (Table 1). Amplifications were performed using a PTC-100 thermocycler with the following cycling conditions: 5 min at 94°C; followed by 33 cycles of 30 s at 94°C, 45 s at 66°C, 1 min at 70°C; and a final extension of 8 min at 70°C. Amplification conditions of SCAR-PCR using a single J2 or female were the same as described above, except that after the denaturation step a total of 39 amplification cycles were used. PCR products were resolved in a 1·2% agarose gel, stained with ethidium bromide (0·3 μg mL−1) and photographed under UV light. PCR products were also resequenced to check whether the same original sequences were obtained.
Because some of the RKN species listed in Table 1 are found together in the field, combinations of multiplex PCR were carried out. Multiplex PCR reactions of M. ethiopica + M. incognita or M. ethiopica + M. arenaria were performed with 5 μm of each primer pair: (i) methF/R (M. ethiopica) plus inc14-F 5′-GGGATGTGTAAATGCTCCTG-3′ and inc14-R 5′-CCCGC-TACACCCTCAACTTC-3′ (M. incognita) (Randig et al., 2002); and (ii) methF/R plus ar-F 5′-TCGGCGATAGAGGTAAATGA-C-3′ and ar-R 5′- TCGGCGATAGACACTACAACT-3′ (M. arenaria) (Zijlstra et al., 2000). Similarly, multiplex PCR reactions of M. ethiopica + M. hapla or M. ethiopica + M. javanica were performed with: (iii) 10 μm methF/R plus 5 μm h-F 5′-TGACGGCGGTGAGTGCGA-3′ and h-R 5′-TGACGGCGGTA-CCTCATAG-3′ (M. hapla) (Zijlstra et al., 2000); and (iv) methF/R plus 5 μm jav-F 5′-GGTGCGCGATTGAACTGAGC-3′ and jav-R 5′-CAGGCCCTTCAGTGGAACTATAC-3′ (M. javanica) (Zijlstra et al., 2000). All multiplex PCR reactions were performed using 6 ng pooled nematode DNA from eggs or DNA extract from a single individual. PCR conditions were the same as described previously for individual SCAR analysis. All experiments were run three times.
Results for the isozyme analysis are shown in Table 1. All isolates of M. ethiopica showed the E3 esterase phenotype (EST) typical for this species (Carneiro et al., 2003, 2004). For the three undescribed Meloidogyne isolates an L3 EST phenotype was obtained. Additional information, such as phylogenetic analysis and morphology, indicated that these latter isolates very probably represent an undescribed species of RKN (authors' unpublished results). All other species showed typical EST phenotypes as previously described: M. arenaria (A2), M. morocciensis (A3), M. javanica (J3), M. hispanica (H3), M. hapla (H1), M. incognita (I2), M. enterolobii (M2), M. exigua (E1), M. paranaensis (P1) and M. inornata (I3). All nematode species listed in Table 1 showed the N1 malate dehydrogenase phenotype, except for M. hapla which showed the H1 phenotype.
Genetic diversity within M. ethiopica isolates
The genetic variability within 15 M. ethiopica isolates was analysed using a total of 30 AFLP and 33 RAPD random primers (Fig. 1). The sizes of amplified fragments ranged from 150 to 2500 base pairs (Fig. 1) and the number of reproducible amplified fragments observed with each marker ranged from 207 to 384 (Table 2). Overall, there was c. 22·5% polymorphism among M. ethiopica isolates and c. 28% polymorphism between M. ethiopica and Meloidogyne sp. (Table 2). Reproducible amplified fragments resulting from these markers were recorded to build a 0–1 matrix from which phylogenetic analysis was performed. The NJ trees obtained for each of the two types of markers were similar. After combining data together into one single analysis, the same tree topology was observed, with all isolates of M. ethiopica belong to a single cluster (82% bootstrap value) and without any supported subspecific clustering of the isolates (Fig. 2). The similar topologies indicate that the tree provides an accurate estimate of the relationships of the isolates studied. The three isolates of Meloidogyne sp., which showed the L3 esterase profile, clustered together.
Table 2. Polymorphisms observed within Meloidogyne ethiopica isolates and related species
Development of a PCR-based detection tool for M. ethiopica
To look for a species-specific DNA marker for M. ethiopica that can be used as a PCR diagnostic tool, M. ethiopica and the other RKN species were screened using a total of 57 10-mer RAPD primers. Amplification with primer OPE-07 resulted in a fragment of 350 bp that was present only in M. ethiopica isolates (Fig. 3). This DNA fragment was cloned, sequenced and converted into a SCAR marker. A blast search against the NCBI databases using this sequence resulted in no hits to any sequences deposited in the database (results not shown).
SCAR-PCR reactions with primer pair methF/R produced a single amplicon of 350 bp for M. ethiopica, in comparison to no amplicons produced from other Meloidogyne spp. tested (Fig. 4). DNA sequencing of this PCR product resulted in the same original sequence as the cloned fragment. The specificity and reliability of this primer pair was also tested against 14 isolates of M. ethiopica, and results revealed amplification of a DNA product of the expected size (Fig. 5). This primer pair was also successful in amplifying DNA from single J2 and individual females from M. ethiopica isolates (Fig. 6) or when used in multiplex PCR reactions containing mixtures of other major RKN species (Fig. 7).
This study demonstrated that M. ethiopica isolates from Brazil and Chile had an overall low genetic diversity of c. 22·5%. In addition, a primer pair that can be used for specific diagnosis of M. ethiopica was developed. The specificity of the primer pair was validated by analysing other RKN species and 14 isolates of M. ethiopica. The sensitivity of the reaction allowed the identification of single individual nematodes of the target species when used in mixtures with other major RKN nematode species associated with grapevine and kiwifruit plants. Thus, these species-specific PCR primers can be used in the diagnosis of this important plant-parasitic nematode.
Meloidogyne ethiopica is a damaging and extremely polyphagous species, having the potential to spread globally and to cause a significant impact on important crops (Carneiro et al., 2003, 2007; Lima et al., 2009; Strajnar et al., 2011; Conceição et al., 2012). Although this nematode is not yet considered to be a quarantine pest in Brazil, it has been placed on the 2011 European alert list as a potential treat to agriculture, following its recent detection on tomato plants in Slovenia (Širca et al., 2004) and on kiwifruit and maize (Zea mays) in Greece (Conceição et al., 2012). Furthermore, the finding that M. ethiopica is able to survive at subzero temperatures in open fields (Strajnar et al., 2011) makes establishment of this pathogen a possibility and hence it could pose a significant threat not only to tropical and subtropical regions but also to places such as Europe. To the authors' knowledge, esterase phenotyping was the only routine reliable method to identify this nematode when the present study was initiated. Unfortunately, few laboratories are able to use this technique. Hence, the PCR-based detection tool described here will contribute to faster and simpler detection of this important nematode and help prevent its further spread worldwide.
The low genetic variability observed among M. ethiopica isolates might be related to the mode of reproduction of M. ethiopica via mitotic parthenogenesis, as this results in clonal progenies (Triantaphyllou, 1985). This finding is similar to other studies, e.g. Randig et al. (2002) reported a diversity of 19·5% and 30·1% for M. javanica and M. incognita, respectively, two species with the same mode of reproduction as M. ethiopica. Conversely, the low genetic diversity observed within M. ethiopica isolates might be because the isolates were mainly from Brazil and Chile, with M. ethiopica probably having been introduced into Brazil from Chile with imported grapevine and kiwifruit seedlings (Carneiro et al., 2003). In this case, it would be necessary to increase the number of isolates from other countries in order to verify the data on the genetic variability of M. ethiopica. Nonetheless, the discovery of this low genetic diversity observed among M. ethiopica isolates could be important for future breeding programmes that ultimately lead to improved control management practices against this pathogen in Brazil and Chile.
The phylogenetic analysis indicated that there was no specific clustering related to original host plant or geographic origin of the isolates tested. Correlations between the geographic location for each isolate of M. ethiopica and the genetic distance between them were inconsistent. This result indicates a nonsignificant geographic–genetic correlation among this group of accessions.
Phylogenetic analysis further supported the grouping of the three isolates that had the same, L3, esterase phenotype. This cluster was successfully separated on the basis of AFLP and RAPD data, suggesting that these three isolates may belong to the same, as yet undescribed, species. The same unidentified RKN have been compared to several of the most common species of Meloidogyne in a phylogenetic study based on mitochondrial DNA sequences, and were shown to be closely related (c. 85% bootstrap value) to isolates that have since been confirmed as M. inornata (Carneiro et al., 2008b) and M. ethiopica (Tigano et al., 2005). However, the SCAR marker developed here for M. ethiopica did not result in any PCR amplification for this Meloidogyne sp. In addition, esterase profiling and morphology (authors' unpublished data) give further support to the classification of isolates from this cluster as belonging to an undescribed RKN species. Altogether, the results presented here suggest the need for further studies using a more comprehensive set of individuals of M. ethiopica to validate the evaluation of the intraspecific variability of this species.
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and EMBRAPA—Recursos Genéticos e Biotecnologia for their financial support. VRC thanks CAPES for his scholarship.