Identification and intraspecific variability of Steinernema feltiae (Filipjev, 1934) isolates from different localities in Poland

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Summary

Presented study is part of a project aimed at identifying entomopathogenic nematode (EPN) species and analysing their distribution in various habitats of Poland. Here, an attempt was undertaken to determine intraspecific variability of nematodes of the species Steinernema feltiae isolated from seven different localities in central and southern Poland. Molecular characteristic and phylogenetic analysis was performed based on nucleotide sequences in the ITS region.

Research on the occurrence of EPNs in Poland have been conducted since the 1990s but there is still no data verified genetically, as well as data on the intraspecific variability of isolates Steinernema feltiae. This paper reports initial results of intraspecific variability Steinernema feltiae in Poland.

Introduction

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae are worldwide widespread soil organisms. EPNs are obligatory lethal parasites of many insect species associated with soils. Therefore, they are commonly used in the biological plant protection (some species are produces on industrial scale). According to Nguyen and Hunt (2007) there are 60 species of the genus Steinernema but only 16 of the genus Heterorhabditis. One of the most common species in the world is S. feltiae (Filipjev, 1934), which prefers climate of temperate zones. S. feltiae can be found in various habitats: in deciduous forests, meadows, orchards, gardens and croplands (Hominick, 1996; Mráček et al., 2005; Adams et al,. 2006). In Polish studies S. feltiae is reported as most often isolated species (Bednarek, 1990; Dzięgielewska, 2012).

Identification of entomopathogenic nematode species is traditionally based on morphological features but nowadays molecular methods using genetic markers are a more useful tool. In particular, identification of closely related nematode species with traditional method remains difficult and in many instances only the analysis of DNA sequences from species in question can provide an accurate identification (Liu & Berry, 1995). Internal transcribed spacer regions of the ribosomal DNA (rDNA) tandem repeat unit (ITS1 – 5.8S –ITS2) are required to species identification and in phylogenetic studies (Nguyen et al., 2001; Stock et al., 2001; Hominick, 2002; Spiridonow et al., 2004). The ITS region gene sequences has also been used to study among- and intra-population variability of Steinernema feltiae and other Steinernematidis (Nguyen et al., 2001; Yoshida, 2003; Kuwata et al., 2006; Desta et al., 2011).

Research on the occurrence of EPNs in Poland have been conducted since the 1990s but there is still lack of genetically verified data from Poland. To our knowledge this is a first study of intraspecific variability based on ITS rDNA region of Steinernema feltiae in Poland. Research aimed at determine the intraspecific variability of strains Steinernema feltiae will continue and in the future, combined with their virulence.

In this study we amplified and sequenced the ITS region to confirm determination of taxonomic status of seven Steinernematid isolates from different locations in Poland.

The aim of study was to identify intraspecific variability of these isolates and phylogenetic relationships among some Steinernema species living in Europe.

Material and Methods

Soil samples for the study on the occurrence of EPNs in Poland were collected in the years 2010 – 2011. In total, 167 soil samples were taken from 111 localities in Poland along north-south transect.

In the laboratory, EPNs were isolated with the trap method using live bait (larvae of Galleria mellonella L. Lepidoptera: Pyralidae) (Bedding & Akhurst, 1975). Each soil sample was mixed and divided among 5 containers of a volume of 250 cm3. Five larvae of G. mellonella were placed in each container which was then placed in an incubator at 20 °C. The first control was performed after 5 days by replacing dead insects by alive ones. This procedure was repeated every two days until the twentieth day of experiment. White’s traps (White, 1927) were used to collect invasive larvae (IJs) of nematodes. Traps were placed in an incubator at 25 °C. After two weeks, invasive larvae were obtained from dead insects, pipetted to bottles for tissue culture and placed in a water. Bottles were preserved in a refrigerator at 4 °C. IJs larvae obtained from White’s traps were then used to infect the next larvae of G. mellonella to maintain the culture and to identify species and strains. Nematodes were identified using morphological criteria (Poinar, 1990; Adams & Nguyen, 2002). Isolated nematodes classified as S. feltiae (Table 1) were analysed by molecular methods to confirm species affiliation and their intraspecific variability.

Table 1

Sites of S. feltiae isola

IsolateSite characteristic
S feltiae KAT 13field (Miscantus giganteus crop) (Silesia Region)
S. feltiae ZAG 4deciduous forest, the Zagożdżonka River valley (Kozienicka Forest)
S. feltiae ZAG 11deciduous forest, the Zagożdżonka River valley (Kozienicka Forest)
S. feltiae ZWO 4meadow, the Zwoleńka River valley (Kozienicka Forest)
S. feltiae ZWO 21meadow, the Zwoleńka River valley (Kozienicka Forest)
S. feltiae ZWO 23meadow, the Zwoleńka River valley (Kozienicka Forest)
S. feltiae SIEW 1field (wheat crop) (Silesia)

DNA of EPNs isolate was extracted from 100 – 1000 individuals of the invasive juveniles (IJS) by phenol-chloroform method and precipitated with ethanol as described by Tumialis et al. (2014).The ITS 1-5.8S – ITS 2 region of rDNA was amplified with PCR method using 18s and 26s primers as described by Vrain et al. (1992). All PCR reaction consisted of an initial denaturation step of 3 min. at 95 °C, followed by 35 cycles at 95 °C for 30 s, at 50 °C for 30s and at 72 °C for 60s with final extension step of 5 min at 72 °C. Amplification products were purified by ethanol precipitation and directly sequenced with the BidDye Terminator Cycle sequencing Ready Reaction Kit v 3.1 (Life Technologies).

Results and Discussion

Entomopathogenic nematodes were isolated from 53 out of 167 analysed soil samples. The presence of S. feltiae was recorded in 11 samples.

DNA was isolated from 7 out of 11 samples. The ITS region of the rDNA was successfully amplified by PCR reaction and sequenced. To indicate the taxonomic position of the nematode, the ITS sequences of studied isolates were compared with the existing data available in GenBank (www.ncbi.nlm.nih.gov). The BLASTN search in GenBank revealed that Polish isolates had a high similarity (99 – 100 %) with those sequences for Steinernema feltiae. Sequences of other Steinernema species exhibited a lower degree of similarity with the isolates obtained during this study. The ITS sequences of seven studied isolates were aligned using GenDoc with ITS sequences of different strains of Steinernema feltiae and six species of Steinernema occurring in Europe, which have been deposited in GenBank. The aligned sequence data were analyzed by the Neighbour-Joining method using MEGA (Tamura et al. 2011). Phylogenetic analyses of ITS rDNA sequences placed isolates from Poland (ZWO23, ZWO21, ZAG11, ZAG4, KAT13, SIEW1) in a clade with other isolates/strains of Steinernema feltiae (Fig 1).

Fig. 1
Fig. 1

Phylogenetic relationship among Steinernematid species based on the sequences of the ITS region determined by the NJ method. Numbers on branches more than 50% indicated the percentage of 10000 bootstrap replicates. Sequences obtained in this study are marked with black triangle

Citation: Helminthologia 53, 3; 10.1515/helmin-2016-0030

Five Polish isolates (ZWO23, ZWO21, ZAG11, ZAG4) from Mazovian Region (Kozienicka Forest) and one isolate (KAT13) from Silesia Region clustered together with S. feltiae from Czech Republic (Rudolfov), Indonesia and other locations and showed sequence identities between 99 % and 100 %. Isolate SIEW1 from Silesia Region was closer to the isolate from Czech Republic (Rudolfov) (99 % identities) than any other from Poland. The analyzed sequences derived from different locations (Poland, Europe and other places in the world) did not show any relationship with regard to their geographical relationship. Similarity of studied isolates is shown in Table 2.

Table 2

Pairwise similarity of studied isolates (samples)

IsolateIdentities
ZAG4ZAG11ZWO4ZWO21ZWO23KAT13SIEW1
ZAG4-980/980

(100 %)
980/980

(100 %)
980/980

(100 %)
980/980

(100 %)
980/980

(100 %)
904/919

(98 %)
ZAG110/980

(0 %)
-980/980

(100 %)
980/980

(100 %)
980/980

(100 %)
980/980

(100 %)
904/919

(98 %)
ZWO40/980

(0 %)
0/980

(0 %)
-980/980

(100 %)
980/980

(100 %)
980/980

(100 %)
904/919

(98 %)
ZWO210/980

(0 %)
0/980

(0 %)
0/980

(0 %)
-980/980

(100 %)
980/980

(100 %)
904/919

(98 %)
ZWO230/980

(0 %)
0/980

(0 %)
0/980

(0 %)
0/980

(0 %)
-980/980

(100 %)
904/919

(98 %)
KAT130/980

(0 %)
0/980

(0 %)
0/980

(0 %)
0/980

(0 %)
0/980

(0 %)
-904/919

(98 %)
SIEW112/919

(1.3 %)
12/919

(1.3 %)
12/919

(1.3 %)
12/919

(1.3 %)
12/919

(1.3 %)
12/919

(1.3 %)
-
Gaps

The intraspecific variability of ITS rDNA region in S. feltiae (11 isolates) reported by Spiridonov et al. (2004) was in the range of 0 – 2.4 %. The intraspecific variability of ITS sequences for European isolates obtained from Izhersk (Russia), United Kingdom, Merelbeke (Belgium), Czech Republic and San Bernardino (Switzerland) ranged from 0 – 1.6 % and reaching up to 2.4 % between the British (A2) and the Armenian isolates. Desta et al. (2011) studying the ITS rDNA region in four isolates (SCM, SNGD, SNC, Ssp60) of S. feltiae found lower intraspecific variability (0.2 – 0.9 %). In this study intraspecific variability of examined ITS region varied between 0 and 2 %, which was at similar level when compared with European isolates reportered by Spiridonov et al. (2004).

Obtained results confirm species affiliation of studied nematodes and show high similarity among the isolates.

Acknowledgments

The study was carried out in the framework of the Project N N309 4228838 financed by the Ministry of Science and Higher Education, Poland.

References

  • Adams B.J. Fodora A. Koppenhöfer H.S. Stackebrandt E. Stock S.P. Klein M.G. (2006): Biodiversity and systematics of nematode-bacterium entomopathogens. Biol. Control. 37: 32 – 49. DOI:

    • Crossref
    • Export Citation
  • Adams B.J. Nguyen K.B. (2002): Taxonomy and systematics. In: Gaugler R. (Ed) Entomopathogenic Nematology Wallingford UK CABI Publishing pp. 1 – 33

    • Crossref
    • Export Citation
  • Beddinng R.A. Akhurst R.J. (1975): A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21: 109 – 110. DOI:

    • Crossref
    • Export Citation
  • Bednarek A. (1990): Ecological conditions of biological activity of entomophilic nematodes in the environment of agrocenosis soil. Scientific Hearings and Monographs. Warsaw University of Life Sciences-SGGW Press 108 pp. (In Polish)

  • Desta T.A. Mulawarman M. Wayenberge L. Moens M. Viaene N. Ehlers R.U. (2011): Identification and intraspecific variability of Steinernema feltiae strains from Cemoro Lawang village in Eastern Java Indonesia. Russ. J. Nematol. 19: 21 – 29

  • Dzięgielewska M. (2012): Occurrence of entomopathogenic nematodes from family Steinernematidae and Heterorhabditidae in orchards chemically protected and unprotected. Prog. Plant Prot. 52: 415 – 420 (In Polish)

  • Hominick W.M. (2002): Biogeography. In: Gaugler R. (Ed) Entomopatogenic Nematology CABI Publishing Wallingford pp. 115 – 143

  • Hominick W.M. Reid A.P. Bohan D.A. Briscoe B.R. (1996): Entomopathogenic nematodes – biodiversity geographical distribution and the convention on biological diversity. Biocontrol Sci. Techn. 6: 317 – 331. DOI:

    • Crossref
    • Export Citation
  • Kuwata R. Shigematsu M. Yoshiga T. Yoshida M. Kondo E. (2006): Intraspecific variation and phylogenetic relationships of steinernematids isolated from Japan based on the sequences of the ITS region of the nuclear rRNA gene and the partial mitochondrial COI gene. Jpn. J. Nematol. 36: 11 – 21. DOI:

    • Crossref
    • Export Citation
  • Liu J. Berry R.E. (1995): Determination of PCR Conditions for RAPD Analysis in Entomopathogenic Nematodes (Rhabditida: Heterorhabditidae and Steinernematidae). J. Interverterbr. Pathol. 65: 79 – 81. DOI:

    • Crossref
    • Export Citation
  • Mráček Z. Bečvář S. Kindlmann P. Jersáková J. (2005): Habitat preference for entomopathogenic nematodes their insect hosts and new faunistic records for the Czech Republic. Biol. Control 34: 27 – 37. DOI:

    • Crossref
    • Export Citation
  • Nguyen K.B. Hunt D.J. (2007): Heterorhabditidae: species descriptions. In: Nguyen K.B. Hunt D.J. (Eds) Entomopathogenic Nematodes: Systematics Phylogeny and Bacterial Symbionts (Nematology Monographs and Perspectives). Brill Leiden The Netherlands pp. 611 – 692

  • Nguyen K.B. Maruniak J. Adams B.J. (2001): Diagnostic and phylogenetic utility of the rDNA internal transcribed spacer sequences of Steinernema. J. Nematol. 33: 73 – 82

  • Poinar G.O. (1990): Biology and taxonomy of Steinernematidae and Heterorhabditidae. In: Gaugler R. Kaya H. K. (Eds) Entomopathogenic Nematodes in Biological Control CRC Press Boca Raton FL pp. 23 – 58

  • Spiridonow S.E. Reid A.P. Prodrucka K. Subbotin S.A. Moens M. (2004): Phylogenetic relationship within the genus Steinernema (Nematoda: Rhabditida) as inferred from analyses of the ITS1-5.8S-ITS2 region of rDNA and morphological features. Nematology 6: 547 – 566. DOI:

    • Crossref
    • Export Citation
  • Stock S.P. Campbell J.F. Nadler S.A. (2001): Phylogeny of Steinernema Travassos 1927 (Cephalobina: Steinernematide) inferred from ribosomal DNA sequences and morphological characters. J. Parasitol. 87: 877 – 889. DOI: http://dx.doi.org/10.1645/0022-3395(2001)087[0877:POSTCS]2.0.CO;2

    • Crossref
    • Export Citation
  • Tamura K. Peterson D. Peterson N. Stecher G. Nei M. Kumar S. (2011): MEGA5 : Molecular Evolutionary Genetics Analysis using Maximum Likelihood Evolutionary Distance and Maximum Parsimony Methods. Mol. Biol. Evol. 28: 2731 – 2739. DOI:

    • Crossref
    • Export Citation
  • Tumialis D. Gromadka R. Skrzecz I. Pezowicz E. Mazurkiewicz A. Popowska-Nowak E. (2014): Steinernema kraussei (Steiner 1923) (Rhabditida: Steinernematidae) – the first record from Poland. Helminthologia 52: 162 – 166. DOI:

    • Crossref
    • Export Citation
  • White G.F. (1927): A method for obtaining infective nematode larvae from cultures. Science 66: 302 – 303. DOI:

    • Crossref
    • Export Citation
  • Vrain T.C. Wakarchuk D.A. Levesque C.A. Hamilton R.I. (1992): Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fund. Appl. Nematol. 15: 563 – 573

  • Yoshida M. (2003): Intraspecific variation in RFLP patterns and morphological studies of Steinernema feltiae and S.kraussei (Rhabditida: Steinernematidae) from Hokkaido Japan. Nematology 5: 735 – 746. DOI:

    • Crossref
    • Export Citation

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  • Adams B.J. Fodora A. Koppenhöfer H.S. Stackebrandt E. Stock S.P. Klein M.G. (2006): Biodiversity and systematics of nematode-bacterium entomopathogens. Biol. Control. 37: 32 – 49. DOI:

    • Crossref
    • Export Citation
  • Adams B.J. Nguyen K.B. (2002): Taxonomy and systematics. In: Gaugler R. (Ed) Entomopathogenic Nematology Wallingford UK CABI Publishing pp. 1 – 33

    • Crossref
    • Export Citation
  • Beddinng R.A. Akhurst R.J. (1975): A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21: 109 – 110. DOI:

    • Crossref
    • Export Citation
  • Bednarek A. (1990): Ecological conditions of biological activity of entomophilic nematodes in the environment of agrocenosis soil. Scientific Hearings and Monographs. Warsaw University of Life Sciences-SGGW Press 108 pp. (In Polish)

  • Desta T.A. Mulawarman M. Wayenberge L. Moens M. Viaene N. Ehlers R.U. (2011): Identification and intraspecific variability of Steinernema feltiae strains from Cemoro Lawang village in Eastern Java Indonesia. Russ. J. Nematol. 19: 21 – 29

  • Dzięgielewska M. (2012): Occurrence of entomopathogenic nematodes from family Steinernematidae and Heterorhabditidae in orchards chemically protected and unprotected. Prog. Plant Prot. 52: 415 – 420 (In Polish)

  • Hominick W.M. (2002): Biogeography. In: Gaugler R. (Ed) Entomopatogenic Nematology CABI Publishing Wallingford pp. 115 – 143

  • Hominick W.M. Reid A.P. Bohan D.A. Briscoe B.R. (1996): Entomopathogenic nematodes – biodiversity geographical distribution and the convention on biological diversity. Biocontrol Sci. Techn. 6: 317 – 331. DOI:

    • Crossref
    • Export Citation
  • Kuwata R. Shigematsu M. Yoshiga T. Yoshida M. Kondo E. (2006): Intraspecific variation and phylogenetic relationships of steinernematids isolated from Japan based on the sequences of the ITS region of the nuclear rRNA gene and the partial mitochondrial COI gene. Jpn. J. Nematol. 36: 11 – 21. DOI:

    • Crossref
    • Export Citation
  • Liu J. Berry R.E. (1995): Determination of PCR Conditions for RAPD Analysis in Entomopathogenic Nematodes (Rhabditida: Heterorhabditidae and Steinernematidae). J. Interverterbr. Pathol. 65: 79 – 81. DOI:

    • Crossref
    • Export Citation
  • Mráček Z. Bečvář S. Kindlmann P. Jersáková J. (2005): Habitat preference for entomopathogenic nematodes their insect hosts and new faunistic records for the Czech Republic. Biol. Control 34: 27 – 37. DOI:

    • Crossref
    • Export Citation
  • Nguyen K.B. Hunt D.J. (2007): Heterorhabditidae: species descriptions. In: Nguyen K.B. Hunt D.J. (Eds) Entomopathogenic Nematodes: Systematics Phylogeny and Bacterial Symbionts (Nematology Monographs and Perspectives). Brill Leiden The Netherlands pp. 611 – 692

  • Nguyen K.B. Maruniak J. Adams B.J. (2001): Diagnostic and phylogenetic utility of the rDNA internal transcribed spacer sequences of Steinernema. J. Nematol. 33: 73 – 82

  • Poinar G.O. (1990): Biology and taxonomy of Steinernematidae and Heterorhabditidae. In: Gaugler R. Kaya H. K. (Eds) Entomopathogenic Nematodes in Biological Control CRC Press Boca Raton FL pp. 23 – 58

  • Spiridonow S.E. Reid A.P. Prodrucka K. Subbotin S.A. Moens M. (2004): Phylogenetic relationship within the genus Steinernema (Nematoda: Rhabditida) as inferred from analyses of the ITS1-5.8S-ITS2 region of rDNA and morphological features. Nematology 6: 547 – 566. DOI:

    • Crossref
    • Export Citation
  • Stock S.P. Campbell J.F. Nadler S.A. (2001): Phylogeny of Steinernema Travassos 1927 (Cephalobina: Steinernematide) inferred from ribosomal DNA sequences and morphological characters. J. Parasitol. 87: 877 – 889. DOI: http://dx.doi.org/10.1645/0022-3395(2001)087[0877:POSTCS]2.0.CO;2

    • Crossref
    • Export Citation
  • Tamura K. Peterson D. Peterson N. Stecher G. Nei M. Kumar S. (2011): MEGA5 : Molecular Evolutionary Genetics Analysis using Maximum Likelihood Evolutionary Distance and Maximum Parsimony Methods. Mol. Biol. Evol. 28: 2731 – 2739. DOI:

    • Crossref
    • Export Citation
  • Tumialis D. Gromadka R. Skrzecz I. Pezowicz E. Mazurkiewicz A. Popowska-Nowak E. (2014): Steinernema kraussei (Steiner 1923) (Rhabditida: Steinernematidae) – the first record from Poland. Helminthologia 52: 162 – 166. DOI:

    • Crossref
    • Export Citation
  • White G.F. (1927): A method for obtaining infective nematode larvae from cultures. Science 66: 302 – 303. DOI:

    • Crossref
    • Export Citation
  • Vrain T.C. Wakarchuk D.A. Levesque C.A. Hamilton R.I. (1992): Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fund. Appl. Nematol. 15: 563 – 573

  • Yoshida M. (2003): Intraspecific variation in RFLP patterns and morphological studies of Steinernema feltiae and S.kraussei (Rhabditida: Steinernematidae) from Hokkaido Japan. Nematology 5: 735 – 746. DOI:

    • Crossref
    • Export Citation
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    Phylogenetic relationship among Steinernematid species based on the sequences of the ITS region determined by the NJ method. Numbers on branches more than 50% indicated the percentage of 10000 bootstrap replicates. Sequences obtained in this study are marked with black triangle

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