Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes

Authors

  • S. Kumari,

    1. Crop Research Institute, Division of Plant Health, Drnovská 507, Ruzyně, 16106 Prague 6, Czech Republic
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  • S. A. Subbotin

    Corresponding author
    1. Plant Pest Diagnostics Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA 95832-1448, USA
    2. Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Leninskii Prospect 33, Moscow 117071, Russia
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E-mail: subbotin@ucr.edu

Abstract

Plant parasitic nematodes of the family Trichodoridae cause substantial yield losses in many agricultural crops. Rapid and accurate identification of trichodorids to the species level is critical for selection of appropriate measures for control. This study analysed 99 sequences of the D2–D3 expansion segments of the 28S rRNA gene and 131 sequences of the 18S rRNA gene from the stubby nematodes belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. Species delimiting was based on the integration of morphological identification, which is not provided in the present article, and molecular-based phylogenetic inference and sequence analysis. Twenty-two valid species and several species complexes were identified among nematodes included in the analysis. PCR-RFLPs of the partial 18S rDNA and the D2–D3 expansion segments of the 28S rDNA were tested and proposed for identification of these nematodes. Gel PCR-RFLP profiles and tables with restriction fragment lengths for several diagnostic enzymes are provided for identification. Some problems of taxonomy and phylogeny of nematodes of the family Trichodoridae are also discussed.

Introduction

Nematodes of the family Trichodoridae are widely distributed in Europe and North America and are also reported from other parts of the world. These nematodes can cause substantial crop losses by acting as plant pathogens and as vectors for plant viruses. Because trichodorid nematode feeding can cause stunting of the roots, they are referred to as stubby root nematodes. One hundred and two species belonging to five genera, Allotrichodorus, Monotrichodorus, Nanidorus, Paratrichodorus and Trichodorus are currently recognized in the Trichodoridae family. Several stubby nematode species are known to vector tobraviruses, which cause economically important diseases in several crops (Decraemer, 1995; Decraemer & Robbins, 2007). Viruses belonging to the genus Tobravirus include Tobacco rattle virus, Pea early-browning virus and Pepper ringspot virus. There is a highly specific relationship between virus and nematode vector, so that particular virus isolates are transmitted only by certain nematode species.

Rapid and accurate identification of trichodorids to the species level is the first critical step for selection of appropriate measures for control of these nematodes. Traditional identification of trichodorids is based on analysis of morphological and morphometrical characters, which often have high intraspecific variability, are complex, difficult and time-consuming. Recently, DNA-based approaches have been successfully adapted for the molecular diagnostics of trichodorids. Blaxter et al. (1998), Boutsika et al. (2004b), Riga et al. (2007), Van Megen et al. (2009) and Duarte et al. (2010) have published sequences of 18S rRNA and ITS rRNA genes for several agriculturally important species of trichodorids. Using sequence data, Boutsika et al. (2004a) and Riga et al. (2007) developed a PCR with specific primers for diagnostics of P. allius, P. macrostylus, P. pachydermus, P. teres, T. primitivus and T. similis. Holeva et al. (2006) designed a real-time PCR assay for detection and quantification of P. pachydermus and T. similis in field samples. Recently, Duarte et al. (2011) developed a PCR-RFLP assay based on the 18S rRNA gene for rapid identification of 12 trichodorid species belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. DNA techniques have been successfully applied to diagnostics of several stubby nematode species. However, many species remain uncharacterized at the molecular level.

The main objectives of this study were to: (i) verify species identification of trichodorid nematodes collected in the Czech Republic, USA and India by using phylogenetic analysis of rRNA gene sequences; (ii) develop a PCR-RFLP assay using D2–D3 expansion fragments of the 28S rRNA gene for diagnostics of stubby nematodes; and (iii) test the PCR-RFLP assay developed by Duarte et al. (2011) with a wider range of trichodorid samples.

Materials and methods

Nematode populations

Nematode populations used in this study were obtained from soil samples collected from the Czech Republic, India and USA (California) (Table 1). The nematodes from the USA were extracted from samples using a centrifugal flotation technique (Coolen, 1979; Hooper, 1986a) and the nematodes from the Czech Republic and India were extracted by a sieving and decanting method (Brown & Boag, 1988). Specimens were killed by gentle heat, fixed in 4% formalin or triethanolamine–formalin (TAF, 2% triethanolamine, 7% formaldehyde solution, 91% water) and mounted in anhydrous glycerin for examination (Hooper, 1986b). Morphological identification of specimens was done using keys provided by Decraemer (1995) and Decraemer & Baujard (1998), with corresponding species descriptions. Some of the species from the Czech Republic used here were described morphologically by Kumari (2010) and Kumari & Decraemer (2011). In this study, the species were defined and delimited based on an integrated approach that considered morphological evaluation, molecular based phylogenetic inference (tree based methods) and sequence analysis (genetic distance methods) (Sites & Marshall, 2004).

Table 1.   Species and populations of trichodorids and outgroup taxon sequenced in the present study
Identification based on morphology and rRNA sequencesLocalityHost plantCollection coderRNA genesaSource of materials
18SITS1ITS2D2–D3 of 28S
  1. a–, not sequenced; NA not acquired.

  2. bAfter Kumari et al. (2007).

  3. cAfter Kumari & Decraemer (2009).

Nanidorus minor Lodi, San Joaquin County, CA, USAAlfalfaCD465 JN123364 JN123380 JN123393, JN123394Subbotin S.A.
N. minor Kerman, Fresno County, CA, USACottonCD474 JN123365 JN123381 JN123395, JN123396Subbotin S.A.
N. renifer Goulds, FL, USABromeliadCD541 JN123366 JN123397, JN123398Subbotin S.A.
Paratrichodorus pachydermus‘B’Dobříš, Czech RepublicBeechXP1 JN123399 Kumari S.
P. pachydermus‘B’Jeseň, Czech RepublicMaplePP1 JN123367 JN123376 JN123382 JN123400 Kumari S.
P. pachydermus‘B’Lhenice, Czech RepublicSweet cherryXP2 JN123401 Kumari S.
P. pachydermus‘B’Maroltov, Czech RepublicMapleXP3 JN123402 Kumari S.
P. pachydermus‘B’Obory, Czech RepublicOakXP4 JN123403 Kumari S.
P. pachydermus‘B’Rájov, Czech RepublicUnkownXP5 JN123404 Kumari S.
P. porosus‘C’Incline, Mariposa County, CA, USATreesCD606 JN123368 JN123383 JN123405, JN123406Subbotin S.A.
Trichodorus pakistanensis‘A’Rajot, HP, IndiaSacred figTINP JN123369 NA JN123384 JN123407 Kumari S.
T. primitivus Ratiškovice, Czech RepublicPeachTP2 JN123370 NA JN123385 JN123408 Kumari S.
T. similis Červeny vrch, Czech RepublicOakTS3 JN123371 JN123377 JN123386 DQ832183 b Kumari S.
T. sparsus‘A’Borek, Czech RepublicGrassXT6 JN123409 Kumari S.
T. sparsus‘A’Brodce, Czech RepublicBeechXT7 JN123410 Kumari S.
T. sparsus‘A’Hlínoviště, Czech RepublicBeechXT8 JN123411 Kumari S.
T. sparsus‘A’Chloumek, Czech RepublicOakXT9 JN123412 Kumari S.
T. sparsus‘A’Janov, Czech RepublicBeechXT10 JN123413 Kumari S.
T. sparsus‘A’Jetřichovice, Czech RepublicGrassXT11 JN123414 Kumari S.
T. sparsus‘A’Kámen, Czech RepublicBeechXT12 JN123415 Kumari S.
T. sparsus‘A’Kozí Hory, Czech RepublicBeechXT13 JN123416 Kumari S.
T. sparsus‘A’Lysá nad Labem, Czech RepublicUnkownXT14 JN123417 Kumari S.
T. sparsus‘A’Mcely, Czech RepublicUnkownXT15 JN123418 Kumari S.
T. sparsus‘A’Mezní Louka, Czech RepublicBeechXT16 JN123419 Kumari S.
T. sparsus‘A’Obrubce, Czech RepublicPineTS4 JN123372 JN123378 JN123387 JN123420 Kumari S.
T. sparsus‘A’Ohaveč, Czech RepublicOakXT17 JN123421 Kumari S.
T. sparsus‘A’Staré splavy, Czech RepublicBirchXT18 JN123422 Kumari S.
T. sparsus‘A’Vojtěchov, Czech RepublicBeechXT19 JN123423 Kumari S.
T. sparsus‘B’Borek, Czech RepublicGrassTS5 JN123373 JN123379 JN123388 JN123424 Kumari S.
T. sparsus‘B’Přelovice, Czech RepublicStrawberryXT20 JN123425 Kumari S.
T. variopapillatus Křivoklát, Czech RepublicElmTV6 GQ148719 b NA JN123389 GQ148718 c Kumari S.
T. viruliferus Bílé podolí, Czech RepublicAppleXT21 JN123426 Kumari S.
T. viruliferus Ratiškovice, Czech RepublicPeachTV7NA JN123390 JN123427 Kumari S.
T. viruliferus Výsoká, Czech RepublicMapleTV8 JN123374 NA JN123391 JN123428 Kumari S.
Trichodorus sp. CCA, USAUnknownCA103 JN123375 JN123392 JN123429 Subbotin S.A.
Trichodorus sp. DKingsburg, Kings County, CA, USAPeachCD488 JN123430, JN123431Subbotin S.A.
Alaimus sp.Bílé Podolí, Czech RepublicAppleFLN JN123432 Kumari S.

DNA isolation, amplification, cloning and sequencing

Molecular studies of trichodorid samples from the Czech Republic, India and California, USA were conducted using slightly different protocols at the Crop Research Institute (Czech Republic) and at the California Department of Food and Agriculture (USA). Trichodorid nematodes collected from the Czech Republic and India were stored in 1 m NaCl before analysis. Total genomic DNA from individual nematodes was extracted according to the rapid method of Stanton et al. (1998). Four regions, 18S, ITS1, ITS2 and partial 28S of rRNA genes, were amplified using nematode universal primers (Table 2). The 18S rRNA gene was amplified into three fragments. Primer combinations were as follows: first fragment SSU_F_04 + SSU_R_09, second fragment SSU_F_22 + SSU_R_13, and third fragment SSU_F_23 + SSU_R_81. PCR was performed in a 25 μL total volume containing one PCR bead (GE Healthcare), 20·5 μL double distilled sterile water and 2·0 μL each primer (10 pmol μL−1) (synthesized by Generi Biotech). To this, 0·5 μL of DNA was added as a template for PCR. A negative control (sterilized water) was included in all PCR experiments. All PCR reactions were performed on a DNA Engine PTC–1148 thermal cycler (Bio-Rad). The cycling profile for all four markers was as follows: initial denaturation for 3 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, with a final extension at 72°C for 10 min. Amplicons were analysed by electrophoresis and the remaining products were purified using the High Pure Product Purification kit (Roche Diagnostics GmbH) and sequenced in both directions using each primer pair (Macrogen). sequencher 4.8 (Gene codes Corp.) was used to assemble and view each sequence and check for base-calling errors.

Table 2.   Primer combinations used in the present study
GenePrimer codeDirectionPrimer sequence 5′–3′Reference
18S rRNASSU_F_04ForwardGCT TGT CTC AAA GAT TAA GCC Blaxter et al. (1998)
18S rRNASSU_R_09ReverseAGC TGG AAT TAC CGC GGC TG Blaxter et al. (1998)
18S rRNASSU_F_22ForwardTCC AAG GAA GGC AGC AGG C Blaxter et al. (1998)
18S rRNASSU_R_13ReverseGGG CAT CAC AGA CCT GTT A Blaxter et al. (1998)
18S rRNASSU_F_23ForwardATT CCG ATA ACG AGC GAG A Blaxter et al. (1998)
18S rRNASSU_R_81ReverseTGA TCC WKC YGC AGG TTC AC Blaxter et al. (1998)
18S rRNA1091-FForwardAGG AAT TGA CGG AAG GGC AC Duarte et al. (2010)
18S rRNA1671-RReverseTCC TCT AAG TAA ATC CCA TTG G Duarte et al. (2010)
ITS1 rRNABL18ForwardCCC GTC GMT ACT ACC GAT T Boutsika et al. (2004a)
ITS1 rRNA5818ReverseACG ARC CGA GTG ATC CAC Boutsika et al. (2004a)
ITS2 rRNAITSAForwardATC GAT GAA GAA CGC AGC Boutsika et al. (2004a)
ITS2 rRNAPXb481ReverseTTT CAC TCG CCG TTA CTA AGG Vrain et al. (1992)
28S rRNAD2AForwardACA AGT ACC GTG AGG GAA AGT TG Nunn (1992)
28S rRNAD3BReverseTCG GAA GGA ACC AGC TAC TA Nunn (1992)

For stubby nematode species collected in the USA, DNA was extracted using the proteinase K protocol. Several specimens from each sample were put into a drop of water on a glass slide and cut under a binocular microscope. Each nematode specimen was transferred to an Eppendorf tube containing 25 μL double distilled water, 2 μL 10 × PCR buffer and 3 μL proteinase K (600 μg mL−1) (Promega). Tubes were incubated at 65°C for 1 h and then at 95°C for 15 min. Detailed protocols for PCR, cloning and sequencing were as described by Tanha Maafi et al. (2003). The PCR product was purified using the QIAquick Gel Extraction Kit (QIAGEN). The primer sets used for amplification and sequencing of ribosomal RNA gene fragments are given in Table 2. PCR products were purified and run on a DNA multicapillary sequencer at the University of California, Riverside.

Sequences were submitted to GenBank under accession numbers as indicated in Table 1.

PCR-RFLPs

The PCR product of the 18S rRNA gene was digested with TaqI, SatI, BseNI or TscAI. The PCR product of D2–D3 expansion fragments of the 28S rRNA gene was digested with BseNI, PstI, PvuII or RsaI. Three to five microlitres of purified PCR products were digested with each of the restriction enzymes. RFLPs were separated by electrophoresis using TAE-buffered gels, stained with ethidium bromide, visualized using a UV transilluminator and photographed. The length of each restriction fragment was obtained by virtual digestion of each sequence using WebCutter 2.0 (http://www.firstmarket.com/cutter/cut2.html).

Sequence and phylogenetic analysis

The new sequences of the 18S rRNA gene and D2–D3 expansion fragments of 28S rRNA gene were aligned using clustalX 1.83 (Thompson et al., 1997) using default parameters of corresponding gene sequences (Blaxter et al., 1998; Boutsika et al., 2004a; Van Megen et al., 2009; Duarte et al., 2010; RC Holeva, MS Phillips, FG Wright, DJ Brown and VC Blok, the James Hutton Institute, Dundee, UK, unpublished data, XQ Li and JW Zheng, Institute of Biotechnology, Zhejiang University, China, unpublished data). Outgroup representatives of the genera Tripyla and Alaimus for the D2–D3 and Tobrilus, Prismatolaimus, Tripyla, Tylolaimophorus and Diphtherophora for 18S data sets were chosen using previous published data (Van Megen et al., 2009). Sequence data sets were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). BI analysis under the GTR + I + G model for each gene was initiated with a random starting tree and was run with four chains for 1 × 106 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The log-likelihood values of the sample points stabilized after approximately 1000 generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given for appropriate clades. Sequence differences between samples were calculated with paup* 4b10 (Swofford, 2003) as an absolute distance matrix and the percentage was adjusted for missing data.

Results

Species identification and delimiting

Ninety-nine and 131 sequences from trichodorids were included in the analyses of the D2–D3 of 28S and 18S rRNA genes, respectively. Forty sequences of the 28S and 12 sequences of the 18S rRNA gene were obtained in the present study. Using traditional morphological characters and molecular criteria (apomorphies and DNA distances), the following 10 species were distinguished within the samples: Nanidorus minor, N. renifer, Paratrichodorus pachydermus, P. porosus, Trichodorus pakistanensis, T. primitivus, T. similis, T. sparsus, T. variopapillatus and T. viruliferus. Two representatives of Trichodorus (Trichodorus sp. C and Trichodorus sp. D) from California were not identified to a species level. Several samples identified as representative of the same morpho-species showed differences in molecular characteristics, and were thus classified here as different species types: Trichodorus sparsus type ‘A’, ‘B’, ‘C’, ‘D’; T. pakistanensis‘A’, ‘B’; P. porosus‘A’, ‘B’, ‘C’; P. pachydermus‘A’, ‘B’; P. teres‘A’, ‘B’, ‘C’; P. hispanus‘A’, ‘B’. The analysis of the D2–D3 of the 28S rDNA sequence data set revealed four unidentified species of Trichodorus, and the partial 18S rDNA sequence data set distinguished four unidentified species of Trichodorus and two unidentified species of Paratrichodorus. Morphological descriptions and identifications of these nematodes are not available. More detailed morphological and molecular analysis is required to further evaluate and identify these samples. A total of 22 valid known species (Trichodorus– 11 species; Nanidorus– three species; Paratrichodorus– eight species) were identified and included in the analyses.

Sequence and phylogenetic analysis

18S rDNA

The alignment for the partial 18S rDNA included 136 sequences and was 1137 bases long. Fourteen Trichodorus, three Nanidorus and 10 Paratrichodorus nominal and putative species were included in the analysis.

Intraspecific sequence variations for some species were: P. porosus‘C’, 0–0·5% (0–6 nt); N. minor, 0–0·6% (0–7 nt); N. renifer, 0–0·4% (0–4 nt); T. primitivus, 0–0·3% (0–3 nt); Tpakistanensis, 0–0·7% (0–8 nt); T. sparsus‘A’, 0·2 (2 nt); T. sparsus‘B’, 0–0·3% (0–4 nt); T. similis, 0–0·5% (0–5 nt); and T. nanjingensis, 0–0·9% (0–11 nt). The 18S BI tree included a major weakly supported clade with all Trichodorus species, one highly supported clade with most Paratrichodorus samples, a weakly supported clade with P. porosus‘A’ and ‘B’, and two distinct clades with Nanidorus renifer and N. nanus, respectively, and a group of N. minor sequences (Fig. 1). The genus Trichodorus was monophyletic, whereas Paratrichodorus and Nanidorus were shown to be paraphyletic. Relationships between the major clades were not well resolved. Trichodorus sparsus‘A’, ‘B’, ‘C’, ‘D’, P. teres‘A’, ‘B’, ‘C’ and P. hispanus‘A’, ‘B’ formed corresponding groups of related clades, whereas there were no sister relationships for T. pakistanensis‘A’ with ‘B’ and P. porosus‘C’ with ‘A’+‘B’, respectively.

Figure 1.

 Phylogenetic relationships within trichodorid nematodes: Bayesian 50% majority rule consensus tree from two runs as inferred from partial 18S rRNA gene sequence alignments under the GTR + I + G model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are indicated by bold letters. *Originally identified as ‘uncultured nematode’; **Originally identified as Trichodorus variopapillatus.

D2–D3 of 28S rDNA

The alignment for the D2–D3 of the 28S rDNA included 102 sequences and was 819 nucleotides in length. Thirteen Trichodorus, two Nanidorus and three Paratrichodorus nominal and putative species were included in this analysis.

Intraspecific sequence variations for some species were: P. porosus‘C’, 0–0·6% (0–5 nt); P. pachydermus‘B’, 0% (0 nt); T. sparsus‘A’, 0% (0 nt); T. pakistanensis‘A’, 0–0·6% (0–5 nt); T. primitivus, 0·3% (2 nt); N. renifer, 0–1·6% (0–10 nt); N. minor, 0–1·6% (0–11 nt). In the D2–D3 BI tree Trichodorus samples were distributed among six moderate or highly supported major clades, and Nanidorus and Paratrichodorus represented two and three clades, respectively (Fig. 2). The genus Trichodorus was paraphyletic, whereas Paratrichodorus and Nanidorus were monophyletic.

Figure 2.

 Phylogenetic relationships within trichodorid nematodes: Bayesian 50% majority rule consensus tree from two runs as inferred from D2 to D3 of 28S rRNA gene sequence alignments under the GTR + I + G model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are indicated by bold letters. *Only D3 of 28S rRNA gene sequence used.

ITS1 of rDNA

The ITS1 sequences were obtained from four species. Comparison of the sequences revealed the following differences: for P. pachydermus [Czech Republic and UK (AJ439513)], 16% (76 nt); for T. similis [Czech Republic and UK (AJ439523)], 1% (13 nt); and for T. sparsus (‘A’ and ‘B’), 20% (220 nt).

ITS2 of rDNA

The ITS2 sequences were obtained from 13 samples. Sequences of Nanidorus minor, P. porosus‘C’ from California, and T. pakistanensis‘A’ from India showed a high level of similarity (>99%) with sequences of corresponding species from China. Sequences of T. similis from the Czech Republic and UK were also similar. Surprisingly, sequences of T. primitivus from the Czech Republic and UK differed by 37%, whereas in the D2–D3 sequences these samples differed only by 0·3%.

PCR-RFLP study

The results of RFLPs of the partial 18S rDNA for Nanidorus, Paratrichodorus and Trichodorus using four restriction enzymes are given in Figure 3. Lengths of restriction fragments after digestion of PCR products with six enzymes are presented in Table 3. Restriction of PCR products by BseNI clearly distinguished Nanidorus, Paratrichodorus (except for P. porosus‘C’) and Trichodorus from each other, in numbers and lengths of fragments (Table 3). Restriction of P. porosus‘C’ by this enzyme resulted in two fragments of similar lengths for some Trichodorus species. However, AvaI clearly differentiated this species from all Trichodorus. Three species of Nanidorus are distinguished by SatI, although the differences between N. minor and N. renifer are based on fragments of less than 60 bp, which may not be clearly visible on agarose gels. PCR-RFLP and virtual digestion of 18S rDNA sequences revealed that six enzymes differentiated P. anemones, P. hispanicus‘B’, P. porosus‘C’, P. teres‘A’ and Paratrichodorus sp. ‘C’ from each other and other Paratrichodorus species. Paratrichodorus hispanus‘A’, P. macrostylus and P. pachydermus‘B’ were indistinguishable from each other by any of the enzymes, as well as Paratrichodorus allius from P. teres‘C’. The six enzymes differentiated most Trichodorus species, except for T. beirensis, T. sparsus‘A’ and ‘B’, T. viruliferus and Trichodorus sp. ‘A’, which generated similar RFLP profiles.

Figure 3.

 PCR-RFLP of the partial 18S rRNA gene for trichodorid nematodes. (a), TaqI; (b), SatI; (c), BseNI; (d), TscAI. Lanes: M, 100 bp DNA marker (Promega); 1, Paratrichodorus pachydermus‘B’; 2, Trichodorus pakistanensis‘A’; 3, T. similis; 4, T. sparsus‘A’; 5, T. variopapillatus; 6, T. viruliferus; 7, T. primitivus; 8, T. sparsus‘B’; 9, Nanidorus renifer; 10, Trichodorus sp. C; 11, N. minor; 12, P. porosus‘C’.

Table 3.   Length (bp) of restriction fragments after digestion of PCR products obtained from the partial 18S rRNA gene for Nanidorus, Paratrichodorus and Trichodorus
SpeciesUnrestricted PCRaRestriction enzyme
AatII AvaI BseNI (BsrI) SatI (Fnu4HI) TaqI TscAI (TspRI)
  1. aBold numbers – fragment verified by PCR-RFLP in Duarte et al. (2010) and/or this study.

Nanidorus minor 610 610 610 546, 64 275, 240, 39, 36, 20 610 278, 217, 115
Nanidorus renifer 610 610610 546, 64 275, 240, 56, 39 610 278, 217, 115
Nanidorus nanus 610610610546, 64240, 214, 61, 56, 39610278, 217, 115
Paratrichodorus allius 612 354, 258 463, 149 612 298, 275, 39 612 280, 246, 86
Paratrichodorus anemones 611 353, 258 611 611 297, 275, 39 526, 85 332, 279
Paratrichodorus divergens 612 354, 258 612 612 298, 275, 39 361, 251 246, 220, 86, 60
Paratrichodorus hispanus‘A’612612612612298, 275, 39612280, 246, 86
Paratrichodorus hispanus‘B’ 612 354, 258 612 612 298, 275, 39 612 280, 246, 86
Paratrichodorus macrostylus 612612612612298, 275, 39612280, 246, 86
Paratrichodorus pachydermus‘B’ 612 612 612 612 298, 275, 39 612 280, 246, 86
Paratrichodorus porosus‘C’ 612 612 463, 149 446, 166 298, 275, 39 612 280, 246, 86
Paratrichodorus teres‘A’612612612612298, 275, 39558, 54280, 246, 86
Paratrichodorus teres‘C’612354, 258463, 149612298, 275, 39612280, 246, 86
Paratrichodorus sp. B612354, 258612612298, 275, 39361, 251246, 220, 86, 60
Paratrichodorus sp. C 612 354, 258 612 612 275, 241, 46, 39, 11 251, 246, 115 280, 246, 86
Trichodorus beirensis 612 354, 258 612 446, 100, 66 298, 275, 39 612 332, 280
Trichodorus cylindricus‘A’612354, 258612446, 100, 66275, 275, 39, 23612280, 246, 86
Trichodorus cylindricus‘B’612354, 258612446, 166298, 192, 83, 39612280, 207, 86, 39
Trichodorus lusitanicus 612 612 612 446, 100, 66 298, 275, 39 612 332, 280
Trichodorus nanjingensis 612354, 258612219, 215, 100, 66, 12,298, 275, 39612332, 280
Trichodorus pakistanensis‘A’ 612 354, 258612 446, 100, 66 298, 275, 39 388, 224 280, 246, 86
Trichodorus pakistanensis‘B’612354, 258612215, 219, 100, 66, 12298, 192, 83, 39612280, 246, 86
Trichodorus primitivus 612 354, 258 612 446, 100, 66 298, 192, 83, 39 612 280, 246, 86
Trichodorus similis 612 354, 258612 446, 166 298, 192, 83, 39 612 332, 280
Trichodorus sparsus‘A’ 612 354, 258612 446, 100, 66 298, 275, 39 612 332, 280
Trichodorus sparsus‘B’ 612 354, 258612 446, 100, 66 298, 275, 39 612 332, 280
Trichodorus sparsus‘C’612354, 258612446, 100, 66275, 241, 57, 39612332, 280
Trichodorus sparsus‘D’612612612446, 166298, 275, 39612332, 280
Trichodorus variopapillatus 612 354, 258612 263, 183, 100, 66 298, 275, 39 612 280, 246, 86
Trichodorus viruliferus 612 354, 258612 446, 100, 66 298, 275, 39 612 332, 280
Trichodorus sp. A 612 354, 258 612 446, 100, 66 298, 275, 39 612 332, 280
Trichodorus sp. B 612 354, 258 612 446, 100, 66 298, 192, 83, 39 612 332, 280
Trichodorus sp. C 611 354, 257611 445, 116 298, 274, 39 611 280, 245, 86

The D2–D3 PCR-RFLP profiles generated by four enzymes for 12 species of trichodorids are given in Figure 4. Lengths of restriction fragments from RFLP for the D2–D3 fragment of the 28S rDNA for Nanidorus, Paratrichodorus and Trichodorus are presented in Table 4. The four restriction enzymes BseNI, PstI, PvuII and RsaI separated all valid and putative species. The results of PCR-RFLP analysis based on all enzymes studied were identical to those expected from in silico analysis.

Figure 4.

 PCR-RFLP of the D2–D3 of 28S rRNA gene for trichodorid nematodes. (a), Nanidorus minor; (b), N. renifer; (c), Paratrichodorus pachydermus‘B’; (d), P. porosus‘C’; (e), Trichodorus pakistanensis‘A’; (f), T. primitivus; (g), T. sparsus‘A’; (h), T. sparsus‘B’; I, T. variopapillatus; (j), T. viruliferus; (k), Trichodorus sp. C; (l), Trichodorus sp. D. Lanes: M, 100 bp DNA marker (Promega); U, unrestricted PCR product; 1, BseNI; 2, PstI; 3, PvuII; 4, RsaI.

Table 4.   Length (bp) of restriction fragments after digestion of PCR products obtained from the D2–D3 of the 28S rRNA gene for Nanidorus, Paratrichodorus and Trichodorus
SpeciesUnrestricted PCRaRestriction enzymes
BseNI PstI PvuII RsaI
  1. aBold number – fragment verified by PCR-RFLP in this study; italics number – addition fragments.

  2. bPresence of several additional bands on a gel not considered after virtual restriction.

Nanidorus minor 800 405, 214, 181 800 691, 109 444, 189, 161, 6
Nanidorus renifer 797 797 797 688, 109 630, 161, 6
Paratrichodorus anemones 794680, 114794794627, 161, 6
Paratrichodorus pachydermus‘A’796796796734, 62501, 161, 128, 6
Paratrichodorus pachydermus‘B’ 796 348, 234, 214 796 583, 110, 103 629, 161, 6
Paratrichodorus porosus‘C’ 795 441, 182, 172 529, 266 581, 110, 104 246, 223, 161, 128, 21, 6
Trichodorus cedarus 797797797400, 397630, 161, 6
Trichodorus cylindricus 797483, 314547, 250797472, 161, 158, 6
Trichodorus nanjingensis 796 796, 516, 280796686, 110354, 161, 275, 6
Trichodorus pakistanensis‘A’ 797 797 547, 250 797 472, 161, 158, 6
Trichodorus primitivus 796 455, 181, 121, 12 796 796 286, 227, 161, 116, 6
Trichodorus similis 796 482, 314 796 796 402, 227, 161, 6
Trichodorus sparsus‘A’ 796 350, 232, 214 796 796 471, 161, 158, 6
Trichodorus sparsus‘B’ 796 446, 350 530, 266 686, 110 471, 161, 158, 6
Trichodorus variopapillatus 796 268, 214, 133, 115, 66 546, 250 734, 62 419, 210, 161, 6
Trichodorus viruliferus 794 480, 181, 133 794 794 243, 226, 161, 158, 6
Trichodorus sp. C 797 455, 161, 115, 66 797, 431, 366 797 630, 161, 6 b
Trichodorus sp. D 796 616, 115, 65 796 796 629, 161, 6 b
Trichodorus sp. E798315, 233, 214, 36548, 250798260, 222, 161, 149, 6

Discussion

Diagnostics of trichodorid nematodes is often difficult because of high intra- and interspecific variability of many morphological and morphometrical characters. Results of phylogenetic and sequence analyses may provide additional criteria to help identify and delimit species. In this study, Bayesian inference was used for phylogenetical reconstruction and species delimiting. Although the results show agreement between molecular and morphological identification for many trichodorid species, identification of some samples remains uncertain because of the presence of different sequences under the same specific name in GenBank. In this study a letter code was assigned for samples clustered separately in phylogenetic trees and morphologically identified as representatives of a single trichodorid species.

Phylogenetic and sequence analysis of the partial 18S and 28S rRNA gene sequences revealed a group of related sequences morphologically identified as representatives of the species T. sparsus (types ‘A’, ‘B’, ‘C’, ‘D’). A high level of variation of morphological and morphometric characters has been reported between populations of T. sparsus from different countries (Loof, 1973; Peneva, 1988; Decraemer, 1995). Recently, Decraemer et al. (2008) distinguished three morphotypes of T. sparsus from Serbia based upon a combination of morphological characters and morphometrics, and noticed that separation between different morphotypes was not straightforward. Probably, the T. sparsus group consists of several sibling (or cryptic) species or subspecies. Final identification of T. sparsus isolates and other samples marked in this study as T. pakistanensis‘A’, ‘B’, P. porosus‘A’, ‘B’, ‘C’ and P. pachydermus‘A’, ‘B’ will be possible after a more thorough morphological characterization of populations. Collection and molecular characterization of nematode materials from the type localities may be critical to resolving these identification problems. Species delimitation is controversial and should rely on the consensus of several data sets and criteria. Additional morphological, molecular and biogeographical data should be used to confirm the delimitation of species made in this and other studies.

PCR-RFLP analysis of ribosomal RNA gene sequences is one of the most effective methods of identification of different nematode groups comprising many species. The PCR-RFLP technique is a simple, rapid and cost-effective technique in comparison with other techniques. Because it is assumed that the sequence of the rRNA gene is conserved within a species, but diverse between species, selection of an appropriate gene marker for identification is crucial in developing a diagnostic. Duarte et al. (2011) designed a PCR-RFLP assay for identification of 12 trichodorids using a fragment of the 18S rRNA gene. The present analysis showed that not all species can be identified using this region. The D2–D3 of the 28S rRNA gene has higher interspecific variation and evolves more rapidly than the 18S rRNA gene, and therefore appears to be a more appropriate marker for identification of multiple species when compared with the 18S rRNA gene. Restriction of D2–D3 amplicons by four enzymes produces species-specific restriction patterns for all species analysed. Even though this study demonstrates advantages of PCR-RFLP analysis for the identification of stubby nematodes, ribosomal RNA gene markers may exhibit intraspecific polymorphism, which will cause variation in restriction maps in different geographically distinct populations. Thus, it will be necessary to investigate the variation of sequences from different geographically separated populations for some species before application of this technique.

Phylogenetic relationships within trichodorids were recently studied by Van Megen et al. (2009) and Duarte et al. (2010) using full length 18S rRNA sequences, with only 17 and 15 stubby nematode species included in these analyses, respectively. The present study, which included more species and compared a smaller fragment of the 18S rRNA gene, showed similar relationships between taxa as previous studies. However, this analysis generated low supports for relationships between major clades and also revealed artifactual paraphylies for Paratrichodorus and Nanidorus. Major differences in topologies between trees were observed mainly in positions of poorly supported clades. Observed paraphyly for Trichodorus in D2–D3 of the 28S rRNA gene tree here and the 18S rRNA gene tree by Van Megen et al. (2009), and for Paratrichodorus and Nanidorus in the 18S rRNA gene tree here, might be explained by increasing for absolute total number of species on the branches and nodes with more descendent species, which lead to an unbalanced tree. The increase in imbalance is consistent with a cumulative effect of differences in diversification rates between branches (Holman, 2005). Validity of the genus Nanidorus has been the subject of some debate (Siddiqi, 1980, 2002) and only recently Duarte et al. (2010) proposed accepting Nanidorus as a valid genus based on morphological features and the results of molecular analysis of the 18S rRNA gene. Although the 18S rRNA gene analysis in the present study does not generate enough resolution to understand relationships between Nanidorus, the D2–D3 of the 28S rRNA gene, having a higher phylogenetic signal, gives clear evidence for its monophyly and also confirms closer relationships of this genus with the genus Trichodorus rather than with Paratrichodorus.

Observed clustering of trichodorid species in the tree was generally in agreement with reported phenotypic similarity of male characters (Decraemer, 1995; Decraemer & Baujard, 1998). Sorting of three prime characters (number of ventromedian precloacal supplements, number of ventromedian cervical pappillae and habitus), Decraemer & Baujard (1998) distinguished 13 male groups within Trichodoridae. In the trees here, T. lusitanicus, T. viruliferus, T. beirensis, T. similis, T. primitivus and T. variopapillatus, all belonging to the male Group 12, and T. cylindricus from Group 11, clustered together. Trichodorus sparsus (Group 10) was in a separate clade on D2–D3 of the 28S rRNA and 18S RNA gene trees. Two major subclades of Paratrichodorus on the 18S rRNA gene tree also corresponded to the male groupings: (i) P. allius (Group 2), P. porosus (Group 3), P. teres (Group 4); and (ii) P. anemones, P. pachydermus, P. hispanus, P. macrostylus (Group 6).

Thus, PCR-RFLP and sequencing of ribosomal RNA markers appear to be a useful and appropriate method for characterization and accurate identification of stubby nematodes. However, larger numbers of species and populations from diverse origins and other alternative gene markers should be included and analysed in future studies to confirm the findings made in this work.

Acknowledgements

The authors acknowledge Professor F. Decraemer for help with identification of some trichodorid samples and Dr C. Blomquist for critical reading of the manuscript draft. The work was partly supported by the Ministry of Agriculture of the Czech Republic, project number MZe – 0002700604, etapa 09 and the Russian Foundation for Basic Research, project number 11-04-01514.

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