Introduction

Wheat, maize and rice occupy the most eminent position among grain crops in terms of production, acreage and source of nutrition, particularly in developing countries. It has been estimated that about 70 % of the land cultivated for food crops is devoted to cereal crops (McDonald and Nicol 2005). By 2030, the world production of cereals is expected to increase to 8 billion tons, whereas world wheat (Triticum aestivum) production is estimated to increase from 584 million tons (1995–1999 average) to 860 million tons (Marathée and Gomez-MacPherson 2001; Hossain and Teixeira Da Silva 2012). However, cereals are exposed to biotic and abiotic stresses. Among the biotic stresses, plant-parasitic nematodes play an important role in decreasing crop yield (Nicol and Rivoal 2008). The cereal cyst nematodes (CCN) are of global importance in cereal production systems. Significant economic losses due to these nematodes have been reported from West Asia, North Africa, Europe, Australia and the United States of America (Rivoal and Cook 1993; Nicol and Rivoal 2008; Sahin et al. 2009). Twelve out of 80 Heterodera species affect roots of cereals and grasses (Subbotin et al. 2010). Of these, H. avenae, H. filipjevi and H. latipons are considered the most economically important, and sometimes coexist (Rivoal and Cook 1993; Abidou et al. 2005a; McDonald and Nicol 2005; Yan and Smiley 2009; Toumi et al. 2012).

The traditional identification of Heterodera species using morphological characteristics is time-consuming and requires specialized skill, especially in the case of mixtures. Because a correct identification of the species is of major importance in nematode control strategies, the development of molecular tools allowing the identification and discrimination between species is warranted (Romero et al. 1996; Rumpenhorst et al. 1996; Rivoal et al. 2003; Subbotin et al. 2003; Waeyenberge et al. 2009). The internal transcribed spacer region (ITS) is possibly the most widely used genetic marker for nematode identification and phylogenetic studies at species level (Vrain et al. 1992; Wendt et al. 1993; Zijlstra et al. 1995). Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the ITS-regions has provided a reliable instrument for a precise identification of cyst nematode species and subspecies (Subbotin et al. 2000; Rivoal et al. 2003; Madani et al. 2004; Abidou et al. 2005b; Smiley et al. 2008; Yan and Smiley 2010). This technique requires a combination of restriction enzymes to distinguish between Heterodera species. It has been reported that 23 restriction enzymes could separate all species belonging to the avenae-group with the exception of H. avenae from H. arenaria (Subbotin et al. 2000). Further, problems with partly digested recognition sites, lack of specificity, and insufficient resolution of small fragments on agarose gels can interfere with a clear interpretation of the results (Waeyenberge et al. 2009). The ITS-region was also used to develop species-specific primers detecting Heterodera species in a simple PCR-reaction, e.g. H. glycines (Subbotin et al. 2001) and H. schachtii (Amiri et al. 2002). However, the analysis of the ITS sequences reveals limited variations for discriminating H. avenae and closely related species (Fu et al. 2011). Moreover, polymorphism between ribosomal DNA (rDNA) repeats can occur within one species, e.g. H. avenae (Bekal et al. 1997; Zhao et al. 2011) and H. filipjevi (Subbotin et al. 2000; Subbotin et al. 2003). This polymorphism makes the design of a species-specific primer based on ITS-sequences very difficult.

The mitochondrial cytochrome oxidase subunit 1 (COI) gene was successfully used to discriminate between many species of free-living marine nematodes (Derycke et al. 2010). Similarly, the gene has been used to generate DNA-barcodes for a variety of biological species, from bacteria to mammals (Hebert et al. 2003). Recently, it was reported that the gene was useful in discriminating between plant-parasitic quarantine nematode species (M. Holterman, personal communication). Against this background, we decided to investigate the COI gene to determine its usefulness for the development of species-specific primers for H. avenae and H. filipjevi.

Materials and methods

Nematodes

Fifty-four populations belonging to 14 Heterodera species and one population of Punctodera punctata were obtained from 18 different countries and 11 hosts (Table 1). Several populations represented species of the avenae-group, viz. H. avenae, H. filipjevi, H. latipons and H. hordecalis. They originated from different hosts and countries, particularly from Syria and Turkey. The suppliers provided full details of the species identity, their host, and origin. We confirmed the species identity of all populations molecularly. For each population, DNA was extracted from five J2 of one cyst, using the method developed by Holterman et al. (2006). The ITS region was amplified by adding 1 μl DNA to the PCR reaction mixture containing 23 μl ddH2O, 25 μl of Dream Taq PCR Master Mix (2×) (Fermentas Life Sciences, Germany), 1 μM of forward primer 5′-CGT AAC AAG GTA GCT GTA G-3′ and 1 μM of the reverse primer 5′-TCC TCC GCT AAA TGA TAT G-3′ (Ferris et al. 1993). The PCR products were purified following the instructions included in the Wizard SV Gel and PCR Clean-Up System kit (Promega Benelux, Leiden, The Netherlands), sequenced in both directions, edited, analysed and compared with sequences in GenBank (http://www.ncbi.nlm.nih.gov/).

Table 1 Cyst nematode species and populations used in this study with their origin, provider and accession number of their ITS and COI sequences in GenBank
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Development of species-specific primer sets

Selection of species-specific primers

Nine Heterodera spp. (H. filipjevi, H. avenae, H. glycines, H. daverti, H. latipons, H. hordecalis, H. schachtii, H. pratensis, H. ciceri) represented by one population each and two populations of both H. avenae and H. filipjevi, along with one population of P. punctata, were selected for designing two species-specific primer sets for detecting H. avenae and H. filipjevi, in separate PCRs. COI was amplified by adding 1 μl DNA of the selected species to the PCR reaction mixture containing 25 μl of Dream Taq PCR Master Mix (2×) (Fermentas Life Sciences, Germany), 1 μM of forward primer JB3F (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′), 1 μM of the reverse primer JB5R (5′-AGC ACC TAA ACT TAA AAC ATA ATG AAA ATG-3′) (Derycke et al. 2005), and ddH2O up to a final volume of 50 μl. The PCR-program settings were as follows: initial denaturation step at 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 41 °C for 30 s and 72 °C for 45 s; and an additional amplification step at 72 °C for 8 min (QBOL protocol 2011; http://www.qbol.wur.nl/UK/). Loading dye (6× Orange DNA, Fermentas) was added to the PCR products. Electrophoresis of PCR products was carried out in a 1.5 % TAE buffered agarose gel (40 min, 100 V), stained in an ethidium bromide bath (1 mg/l) for 30 min and photographed under UV light. Where a positive result was evident, the remainder of the PCR product was purified (Promega Benelux, Leiden, The Netherlands), and sequenced (Macrogen, the Netherlands) in both directions. Finally, the sequences were edited and analysed using software packages Chromas 2.00 (Technelysium, Helensvale, QLD, Australia) and BioEdit 7.0.4.1 (Hall 1999). Software AlleleID 7.73 was used to investigate putative species-specific DNA fragments that could be used as primers for the identification of H. avenae or H. filipjevi. Additionally, an in silico study was done to check the potential of secondary structures, self-primer-dimer and hetero primer-dimer formation within two primers in one set and between two primers from different sets in case of the primers mix (https://www.idtdna.com/analyzer/Applications/OligoAnalyzer/). The sequences of the potential species-specific primers were further screened by searching for their presence in sequences stored in GenBank (BlastN option, http://www.ncbi.nlm.nih.gov/). Finally, one primer set was selected for each species.

Optimization and specificity of species-specific PCRs

A gradient PCR was performed to determine the optimum annealing temperature (Ta) for each primer set. DNA of H. avenae population Fa1 and H. filipjevi population Did15 was used as template DNA. One μl DNA was added to the PCR master mix used for COI gene amplification (see primer selection above). For H. avenae, the designed forward primer (AVEN-COIF) was (5′-GGG TTT TCG GTT ATT TGG-3′), and the designed reverse primer (AVEN-COIR) was (5′-CGC CTA TCT AAA TCT ATA CCA-3′). For H. filipjevi, the designed forward primer (FILI-COIF) (5′-GTA GGA ATA GAT TTA GAT AGT C-3′), and the designed reverse primer (FILI-COIR) (5′-TGA GCA ACA ACA TAA TAA G-3′) were used. The Ta varied between 53 °C and 65 °C. The highest temperature still showing a bright, single and clean PCR-product without secondary products such as primer-dimers for both species-specific PCRs was selected as final Ta.

To check the specificity of both primers sets (AVEN-COI and FILI-COI), separate PCRs with DNA from each Heterodera spp. and P. punctata (Table 1) were carried out using optimized conditions. An additional test was performed to ensure that the primers sets AVEN-COI and FILI-COI were valid for all H. avenae and H. filipjevi populations in our collection, respectively.

Sensitivity test of species-specific PCRs

To check the sensitivity of the primers, DNA was extracted from 100 J2 obtained from one cyst of H. latipons (Fa7A1). The juveniles were placed in a 0.5 ml tube containing 300 μl lysis buffer (Holterman et al. 2006). The tube was incubated as for previous DNA extractions and 1 μl of crude DNA-extract was used for PCR.

The sensitivity test of the AVEN-COI primers set consisted of a species-specific PCR (see above) using 1 μl DNA earlier extracted from 5 J2 of H. avenae (Fa1) (see section Nematodes) mixed with 1 μl DNA extracted from 5 or 100 J2 H. latipons. Similarly, the sensitivity test of the FILI-COI primers set involved a species-specific PCR using 1 μl DNA previously extracted from 5 J2 of H. filipjevi (Fa125) mixed with 1 μl DNA extracted from 5 or 100 J2 H. latipons.

Also, 1 μl of four dilutions (1/5, 1/10, 1/50 and 1/100) of DNA extracted from 5 J2 of both species was used in a species-specific PCR. Equally, 1 μl of two dilutions (1/5 and 1/10) of DNA extracted from 5 J2 of both species was mixed with 1 μl DNA extracted from 100 J2 of H. latipons and used in a species-specific PCR.

Results

Nematode identification

Amplification of the rDNA-ITS region was successful for all samples. Sequencing of both DNA strands of the purified PCR products confirmed the morphological identification of all samples. The sequences were deposited in GenBank; the accession numbers are presented in Table 1.

Species-specific PCRs development

Species-specific primers selection

Amplification of the COI gene was done successfully for all populations, and a single band with fragment size of 470 bp was produced (Fig. 1). No PCR products were obtained in the negative control without nematode DNA template.

Fig. 1
figure 1

Amplification results with the COI gene primers (JB3 & JB5) on a selection of Heterodera spp. and Punctodera punctata. L: 100 bp DNA ladder (Fermentas Life Sciences), 1: Heterodera pratensis (DCP1041A), 2: Punctodera punctata (DCP1041B), 3 & 4: H. filipjevi (Did15 & Fa125), 5: H. glycines (Did38), 6: H. daverti (HD11), 7: H. latipons (Fa7A1), 8 & 9: H. avenae (Fa1 & Fa19), 10: H. ciceri (Fac3), 11: H. hordecalis (TuniB), 12: H. schachtii (HSPol), 13: Negative control

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The in silico investigation revealed no secondary structures and no possible primer dimer formation in both newly designed species-specific primer sets, even in case of mixing different primers. In silico comparative search with the designed primers revealed no match or potential primer binding sites with any of the sequences of Heterodera spp. and Punctodera spp. available in GenBank (http://blast.ncbi.nlm.nih.gov/), and with sequences that we had obtained, with the obvious exception of the H. avenae and H. filipjevi sequences. We therefore, kept the primers sets AVEN-COI and FILI-COI for further experiments. Positions of both species-specific primer sets are shown in the alignment of the sequence of the COI gene of 9 Heterodera spp. and P. punctata (Fig. 2).

Fig. 2
figure 2

Alignment of a selection of our COI-mtDNA sequences (see Table 1 for codes). Position for the species-specific primer set for H. avenae (AVEN-COI) is underlined and bold, and for H. filipjevi (FILI-COI) underlined and italic

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Species-specific PCR(s) optimization and validation

The gradient PCR run with both primers sets and template DNA from H. avenae Fa1 and H. filipjevi Did15, respectively, showed a single and bright band for a range of annealing temperatures between 53 °C and 60 °C (Figures 3 and 4). No primer-dimers were noticed over that range. The highest annealing temperature yielding the brightest specific band without additional band(s) was retained for each species. Because the annealing temperature was optimum at 58 °C for both species, we selected this temperature for further PCRs. The optimised PCR with an initial denaturation step at 95 °C for 5 min, 30 cycles at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s, followed by 72 °C for 8 min, repeatedly resulted in a single band of 109 bp for all H. avenae populations (Fig. 5), and of 245 bp for all H. filipjevi populations (Fig. 6), but did not generate a band for any of the other tested species.

Fig. 3
figure 3

Gradient PCR (temperatures indicated) with the AVEN-COI primers set using Heterodera avenae (Fa1). Temperature range from 53 °C to 65 °C. L: 100 bp DNA ladder (Fermentas Life Sciences)

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Fig. 4
figure 4

Gradient PCR (temperatures indicated) with the FILI-COI primers set using Heterodera filipjevi (Did15). Temperature ranged from 53 °C to 65 °C. L: 100 bp DNA ladder (Fermentas Life Sciences)

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Fig. 5
figure 5

Results of the optimised PCR using the AVEN-COI primers set. L: 100 bp DNA ladder (Fermentas Life Sciences). 1: Heterodera pratensis (DCP1041A), 2: Punctodera punctata (DCP1041B), 3: H. filipjevi (Did23b), 4: H. hordecalis (E69), 5: H. glycines (HGRiggs), 6: H. schachtii (HSPol), 7: H. betae (DCP1248), 8: H. daverti (HD11), 9: H. goettingiana (MP1), 10: H. humuli (MP5), 11: H. ciceri (FaC3), 12: H. trifolii (HT9), 13: H. latipons (Fa3), 14: H. carotae (DCP1734), 15–22: different populations of H. avenae 15: Fa1, 16: Did29, 17: Did33, 18: Did11, 19: Tuni6, 20: Did49, 21: Mus21, 22: Fa19, 23: Negative control

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Fig. 6
figure 6

Results of the optimised PCR using the FILI-COI primers set. L: 100 bp DNA ladder (Fermentas Life Sciences). 1: Heterodera pratensis (DCP1041A), 2: Punctodera punctata (DCP1041B), 3: H. avenae (Fa1), 4: H. hordecalis (E69), 5: H. glycines (HGRiggs), 6: H. schachtii (HSPol), 7: H. betae (DCP1248), 8: H. latipons (Fa3), 9: H. goettingiana (MP1), 10: H. humuli (MP5), 11: H. ciceri (FaC3), 12: H. trifolii (HT9), 13: H. carotae (DCP1734), 14: H. daverti (HD11), 15–22: different populations of H. filipjevi 15: Did15, 16: Did23, 17: E88, 18: Did42b, 19: Did42c, 20: Fa125, 21: Fa126, 22: HFUSA, 23: Negative control

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Sensitivity test of species-specific PCRs

The sensitivity of the PCR assays using the AVEN-COI and FILI-COI primer sets was satisfactory. Both assays were able to detect the DNA extracted from 5 J2 of H. avenae (Fig. 7) or 5 J2 of H. filipjevi (Fig. 8) when mixed with the DNA obtained from 100 J2 of H. latipons. Both assays were also able to detect DNA in four dilutions (1/5, 1/10, 1/50 and 1/100) from DNA originating from 5 J2 of H. avenae or H. filipjevi. Moreover, detection of both species was still possible when 1 μl of the diluted (1/5 and 1/10) target DNA was mixed with 1 μl DNA extracted from 100 J2 of H. latipons.

Fig. 7
figure 7

Result of the sensitivity test using the AVEN-COI primers set in a PCR with 1 μl undiluted or diluted DNA of Heterodera avenae (HA) mixed with 1 μl DNA from H. latipons (HL). 1: negative control, 2–3: 5 J2 of HA, 4–5: 5 J2 of HL, 6–7: 100 J2 of HL, 8–9: 1/5 dilution of 5 J2 of HA, 10–11: 1/10 dilution of 5 J2 of HA, 12–13: 1/50 dilution of 5 J2 of HA, 14–15: 1/100 dilution of 5 J2 of HA, 16–17: 5 J2 of HA and 5 J2 of HL, 18–19: 5 J2 of HA and 100 J2 of HL, 20–21: 1/5 dilution of 5 J2 of HA and 100 J2 of HL, 22–23: 1/10 dilution of 5 J2 of HA and 100 J2 of HL, L: 100 bp DNA ladder (Fermentas Life Sciences)

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Fig. 8
figure 8

Result of the sensitivity test of the FILI-COI primers set in a PCR with 1 μl undiluted or diluted DNA of H. filipjevi (HF) mixed with 1 μl DNA from H. latipons (HL). 1–2: 5 J2 of HF, 3–4: 5 J2 of HL, 5–6: 100 J2 of HL, 7–8: 1/5 dilution of 5 J2 of HF, 9–10: 1/10 dilution of 5 J2 of HF, 11–12: 1/50 dilution of 5 J2 of HF, 13–14: 1/100 dilution of 5 J2 of HF, 15–16: 5 J2 of HF and 5 J2 of HL, 17–18: 5 J2 of HF and 100 J2 of HL, 19–20: 1/5 dilution of 5 J2 of HF and 100 J2 of HL, 21–22: 1/10 dilution of 5 J2 of HF and 100 J2 of HL, 23: negative control, L: 100 bp DNA ladder (Fermentas Life Sciences)

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Discussion

In the last decade, several surveys assessed the distribution of CCN in the main wheat and barley growing areas in Syria (Abidou et al. 2005a) and Turkey (Sahin et al. 2009; Yavuzaslanoglu et al. 2012). The result showed that mixtures of two (H. avenae with H. latipons or H. avenae with H. filipjevi) and sometimes even three species coexist in the same field. Species mixtures increase the difficulties to reach a precise identification. The use of molecular tools having the capacity to accurately identify and separate the species offers an attractive option.

So far, two publications reported the development and use of species-specific primers for the molecular identification of Heterodera spp., i.e. H. glycines (Subbotin et al. 2001) and H. schachtii (Amiri et al. 2002). Amiri et al. (2002) designed a species-specific primer (SHF6) to detect H. schachtii based on polymorphism in using the ITS-rDNA sequences. However, Chemeda et al. (unpublished) found the primer (SHF6) was not able to detect some H. schachtii populations suggesting that this polymorphism is not present in all H. schachtii populations. Heteroplasmy in mtDNA can cause the primer to fail recognising its binding place or to bind a non-specific target. However, during our study we did not encounter any problems as the two primers sets were species specific and were able to detect all representatives of the targeted species originating from different countries and regions. Hence, it can be suggested that the selected COI primer binding places are present in all the H. avenae and H. filipjevi populations.

Designing species-specific primers for H. avenae and H. filipjevi starting from ITS sequences was difficult because of the polymorphism within each species. In addition, the in silico comparative analysis of all our sequences separately with the already available ITS sequences in GenBank (BlastN) showed sometimes high similarity, e.g. 99 % similarity between H. avenae (Fa1) and H. mani (AY148377), H. arenaria (AF274396) and H. australis (AY148395), 97 % between H. filipjevi (Did15) and H. ustinovi (AY148406.1), and 97 % between H. filipjevi (E88) and H. avenae (HM560755.1). Also, the in silico comparison showed comparable similarities in actin gene sequences to occur between Heterodera species; actin gene sequences of H. filipjevi, H. avenae and H. hordecalis are 95 % to 96 % similar. Hence, the actin gene could not be used to design species-specific primers detecting H. avenae or H. filipjevi.

Both newly designed primers were able to detect successfully all H. filipjevi and H. avenae populations that were used in this study. No positive reaction was observed for any of the other cyst nematode species we examined. PCR optimization showed that both primers sets are useful and specific on a range of annealing temperatures (56–60 °C). Because of the very clear and unambiguous specific band obtained at 58 °C, we selected this temperature as annealing temperature. Since this temperature is suitable in PCR reactions with both primers sets, we considered the detection of both species in one run.

The in silico study showed no reason to anticipate that the mixing of the two species-specific primer sets would have the potential for hetero primer-dimer formation when used in the same reaction. However, primer-dimers were observed when the two primers sets were mixed in one PCR using different primers concentrations (data not shown). Because primer-dimers can influence the efficiency and hence also the sensitivity of the PCR, it is not recommended to use both primers sets in a duplex PCR.

However, when used in separate PCRs, both primers sets were able to detected successfully five J2 of H. avenae or H. filipjevi either alone or in a mixture with 100 J2 of H. latipons. The sensitivity is even higher than five J2 since the equivalent of 1/10 of the DNA of five J2 yielded a clear band. From these results it can be concluded that 0.5 J2 of H. avenae or H. filipjevi can be detected among 100 J2 of H. latipons.

According to our result we can conclude that the designed species-specific PCRs are reliable, fast and sensitive identification tools. The method is particularly important for agricultural extension services where the skills to identify the species might be limited.