First report of Bursaphelenchus antoniae from Pinus strobus in the U.S.

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VOLUME 50 , ISSUE 4 (December 2018) > List of articles

First report of Bursaphelenchus antoniae from Pinus strobus in the U.S.

Lynn K. Carta * / R. L. Wick

Keywords : DNA extraction, Nematode taxonomy, Molecular identification

Citation Information : Journal of Nematology. Volume 50, Issue 4, Pages 473-478, DOI: https://doi.org/10.21307/jofnem-2018-052

License : (PUBLISHER)

Published Online: 03-December-2018

ARTICLE

ABSTRACT

Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. The specimens were identified as Bursaphelenchus antoniae Penas et al., 2006 based on 18S rDNA molecular sequence vs only 95% similarity with PWN B. xylophilus. Compared to the previously described Portuguese population of B. antoniae, the sequences generated for the MA population were 98.3% similar in the ITS1, 2 rDNA and 99.9% similar for 28S rDNA. There was 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae. This population has morphology consistent with that of Penas et al., 2006; however, the female tail on this MA pine population is mucronate and more attenuated than in B. antoniae from Portuguese P. pinaster found in association with Hylobius sp. Ecological associations of both populations of B. antoniae are discussed.

Graphical ABSTRACT

Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. The white pine specimen was submitted to the University of Massachusetts Nematology Lab to examine for the pine wood nematode (PWN), Bursaphelenchus xylophilus, as required for shipment of pine logs to an Asian trading partner. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. Female nematodes having a vulval flap but an acute tail did not agree with PWN B. xylophilus that has a rounded tail. Specimens were characterized microscopically and with four molecular markers to identify this population.

Materials and methods

Individual specimens from white pine trees in Massachusetts, and specimens of B. antoniae from Portugal were mechanically disrupted in 20 µl of extraction buffer (Thomas 2011) then stored in PCR tube at –80oC until needed. Each extract was prepared by incubating the tubes at 60oC for 60 min, followed by 95oC for 15 min to deactivate proteinase K.

PCR amplification: Each 25 µl PCR reaction was prepared with 2 µl of the extract and 23 µl of the PCR master mix containing 0.625U TaKaRa EX Taq (Takara Bio USA, Inc., Mountain View, CA) according to the manufacturer’s protocol. The ribosomal 18S SSU DNA, ribosomal 28S LSU DNA, internal transcribe spacer (ITS) and cytochrome c oxidase I (COI) were amplified by PCR with the primer sets described in Table 1. The PCR condition for the 18S was 95oC for 3 min; 36 cycles of 95oC for 30 sec, 50oC for 40 sec, and 72oC for 70 sec; and final extension at 72oC for 5 min, for the 28S was 95oC for 3 min; 36 cycles of 95oC for 30 sec, 58oC for 45 sec, and 72oC for 70 sec; and final extension at 72oC for 5 min, for the ITS was 95oC for 3 min; 36 cycles of 95oC for 30 sec, 55oC for 60 sec, and 72oC for 105 sec; and final extension at 72oC for 5 min, and for the COI was 1X (94°C for 1 min), 5 X (94°C for 40 sec, 45°C 45 sec, 72°C 1 min), 35 X (94°C for 40 sec, 51°C 45 sec, 72°C 1 min), and final extension 72°C for 5 min. PCR products were visualized with the Lonza FlashGelTM DNA system (VWR International, Radnor, PA) and then treated with ExoSAP-IT reagent (Affymetrix, Inc, Santa Clara, CA) according to the manufacturer’s protocol. Direct DNA sequencing was performed bidirectionally with the primers (Table 1) and an ABI BigDye Terminator v3.1 kit and in an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) owned by the USDA Systematic Entomology Lab, Beltsville, MD.

Table 1

Primers used for PCR and sequencing.

10.21307_jofnem-2018-052-t001.jpg

Phylogenetic analysis was performed with Geneious ver. 7.1.7 (Biomatters, Auckland, NZ), using Clustal W alignment (Thompson et al., 1994) with default parameters and Bayesian likelihood tree constructed with the MRBAYES plugin (Huelsenbeck and Ronquist, 2001). Sequences from GenBank used in phylogenetic trees for 18S rDNA and 28S rDNA are given in Tables 2,3. Sequences generated were submitted to GenBank under accession numbers (18S: MK160127, MK160128, 28S: MK160125, MK160126, ITS: MK160122, COI MA: MK160123, MK160124, COI Portugal: MK174262, MK174263).

Table 2

Summary of 18S rDNA sequences in Figure 2 tree.

10.21307_jofnem-2018-052-t002.jpg
Table 3

Summary of 28S rDNA sequences in Figure 3 tree

10.21307_jofnem-2018-052-t003.jpg
Figure 1

A. Female body, B. Male body, C. Female Tail, D. Male Tail.

10.21307_jofnem-2018-052-f001.jpg
Figure 2

18S, MrBayes tree with posterior probabilities on branches of Bursaphelenchus antoniae and close relatives within the ‘B. hylobianum species group’ (in Clade I of Kanzaki et al., 2015) based on a Clustal W alignment implemented in Geneious ver. 7.1.7 (Biomatters, Auckland, NZ) using the MRBAYES plugin with Chain Length 1,100,000, Burnin 110,000, mean -LnL - 7438.56.

10.21307_jofnem-2018-052-f002.jpg
Figure 3

28S MrBayes tree with posterior probabilities on branches of B. antoniae based on a Clustal W alignment implemented in Geneious ver. 7.1.7 (Biomatters, Auckland, NZ) with Chain Length 1,100,000, Burnin 110,000, mean -LnL 3407.0.

10.21307_jofnem-2018-052-f003.jpg

Results and discussion

Bursaphelenchus antoniae females (Fig. 1A) and males (Fig. 1B) were found for the first and only time in North America since its species description from Portugal (Penas et al., 2006a). All standard morphometric measurements were within the bounds of the original population from Europe.

Female n = 5: L = 597.5 ± 44.6 (527.5–650.5) µm, body width = 21.6 ± 1.3 (19.8–23.1) µm, pharynx length = 67.8 ± 3.7 (63.2–73.2) µm, tail length = 44.2 ± 2.1 (41.3–46.8) µm, ABD = 11.0 ± 1.6 (8.6–12.3) µm, stylet length= 14.3 ± 0.6 (13.3–14.8) µm, a = 27.7 ± 1.9 (26.4–30.7), b = 8.8 ± 0.6 (7.9–9.6), c = 13.5 ± 0.8 (12.3–14.4), c’ = 4.1 ± 0.5 (3.7–5.0), V = 71 ± 1.1 (69–72)%.

Male n = 5: L = 568 ± 71 463–654) µm, body width = 20.3 0.4 (20.1–20.6) µm, e = 71.4 ± 1.8 (70.2–72.7) µm, tail length = 30.0 ± 3.5 (28.3–36.6) µm, ABD = 17.0 ± 0.0 (17.0–17.0) µm, stylet length = 17 ± 1 (13–15) µm, spicule length = 15± 1.0 (41–21) µm, a = 30.2 ± 2.22 (28.7–31.8), b = 8.6 ± 1.00 (76.5–9.3), c = 16.9 ± 1.75 (15.7–18.2), c’ = 2.3 ± 0.2 (2.11–2.6).

This population is part of a species complex within a clade of other weevil-vectored Bursaphelenchus (Penas et al., 2006a, 2006b, 2007) within the Hylobius species group of Bursaphelenchus species associated with weevil vectors. This group is phylogenetically distinct from the Xylophilus group (Kanzaki et al., 2015).

The female tail tip in B. antoniae was clearly pointed (Penas et al., 2006a) while in this US population the tail tip was mucronate (Fig. 1C) and not acute. The closely related species B. parantoniae (Munawar et al., 2015) had a bluntly rounded tail tip. These female tail tip shapes may represent genetic, epigenetic or environmental polyphenisms (Duncan et al., 2014; Susoy et al., 2015). These possibilities would be clarified if cultures of both populations could be crossed to assess the stability of these phenotypes. In North America, the pathogenic form of Bursaphelenchus xylophilus “r” has a round tail and usually occurs in pine species (Bolla et al., 1986). The generally non-pathogenic form “m” (or mucro) has a pointed tail. However, since this form can be environmentally induced (Tsai et al., 2016), and mucronate, pathogenic populations exist (Gu et al., 2011), tail form is not a very reliable indicator of potential pathogenicity of an isolate. Therefore the stability of these tail variations is important to understand in greater detail.

The 18S sequence was 99.9% similar to the Portuguese population of B. antoniae and 99.7% similar to and B. parantoniae (Fig. 2). The 28S sequence showed 97.8% similarity to B. parantoniae (Fig. 3). The ITS rDNA was 98.3% similar to B. antoniae Portugal. There were 7/834 bp differences and 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae. The COI sequence was only 88% similar to B. mucronatus. simply because there are very few COI sequence accessions for Bursaphelenchus species in GenBank.

Determining whether a given species is native or introduced is an important question when dealing with an apparently known species occurring on a new continent. Bursaphelenchus luxuriosae described in Japan was identified in Portugal. This was the third member of the xylophilus group in Portugal “It is difficult to ascertain whether B. luxuriosae was introduced, together with its insect vector, or already occurred as a native species (Inácio et al., 2017).” There may be an endemic association of US B. antoniae with another Hylobius in the USA, (Salom, 1997) such as the relatively common pales weevil, H. pales, in eastern North America (www.na.fs.fed.us/spfo/pubs/fidls/pales/fidl-pales.htmPales weevil). Alternatively, the nematode may have been introduced with the regulated ecological counterpart H. abietis, commonly found in Europe (Leather et al., 1999). Many Hylobius spp. have been intercepted at US borders over recent years (USDA-APHIS, AQAS database), and others may have managed to get through yet remain undetected. Beetle-targeted surveys in MA/CT are needed to determine whether the pales weevil actually carries B. antoniae in the USA. If B. antoniae was an introduced species it might conceivably be pathogenic to some US pines.

Acknowledgements

The authors thank Dr Manuel Mota, Universidade de Évora, Évora, Portugal for specimens of B. antoniae to generate COI sequence. Thanks also to Shiguang Li, USDA-ARS MNGDBL, for excellent technical help. Mention of a trade name or commercial product in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

References


  1. Bolla, R. I., Winter, R. E. K., Fitzsimmons, K., and Linit, M. J.. 1986. Pathotypes of the pinewood nematode Bursaphelenchus xylophilus. Journal of Nematology 18: 230–238.
  2. Carta, L. K., and Li, S.. 2019. Improved 18S small subunit rDNA primers for problematic nematode amplification. Journal of Nematology. 50 4: 533–542.
    [CROSSREF]
  3. Cherry, T., Szalanski, A. L., Todd, T. C., and Powers., T. O.. 1997. The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology 29: 23–29.
    [PUBMED]
  4. Duncan, E. J., Gluckman, P. D., and Dearden, P. K.. 2014. Epigenetics, plasticity and evolution: how do we link epigenetic change to phenotype?. Journal of Experimental Zoology (Mol. Dev. Evol.) 322B: 208–220.
    [CROSSREF]
  5. Gu, J., Wang, J., Braasch, H., Burgermeister, W., and Schroder, T.. 2011. Morphological and molecular characterisation of mucronate isolates (M form) of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae). Russian Journal of Nematology 19: 103–120.
  6. Holterman, M., Wurff, A. V. R., Elsen, S. V. D., Megen, H. V., Bongers, T., Holovachov, O., Bakker, J., and Helder, J.. 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution 23: 1792–1800.
    [CROSSREF]
  7. Huelsenbeck, J. P., and Ronquist, F.. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 17: 754–755.
    [CROSSREF]
  8. Inácio, M. L., Nóbrega, F., Mota, M., and Vieira, P.. 2017. First detection of Bursaphelenchus luxuriosae associated with Pinus pinaster in Portugal and in Europe. Forest Pathology 47: e12296.
    [CROSSREF]
  9. Kanzaki, N., and Futai, K.. 2002. A PCR primer set for determination of phylogenetic relationships of Bursaphelenchus species within the xylophilus group. Nematology 4: 35–41.
    [CROSSREF]
  10. Kanzaki, N., Okabe, K., and Kobori, Y.. 2015. Bursaphelenchus sakishimanus n. sp. (Tylenchomorpha: Aphelenchoididae) isolated from a stag beetle, Dorcus titanus sakishimanus Nomura (Coleoptera: Lucanidae), on Ishigaki Island, Japan. Nematology 17: 531–542.
    [CROSSREF]
  11. Leather, S. R., Day, K. R., and Salisbury, A. N.. 1999. The biology and ecology of the large pine weevil, Hylobius abietis (Coleoptera: Curculionidae): a problem of dispersal?. Bulletin of Entomological Research 89: 3–16.
    [CROSSREF]
  12. Munawar, M., Fang, Y., He, J., Gu, J.-F., and Li, H.-M.. 2015. Bursaphelenchus parantoniae n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from Belgium. Nematology 17: 1141–1152.
    [CROSSREF]
  13. Nunn, G. B., Theisen, B. F., Christensen, P., and Arctander, P.. 1996. Simplicity correlated size growth of the nuclear 28S ribosomal D3 expansion segment in the crustacean order Isopoda. Journal of Molecular Evolution 42: 211–223.
    [CROSSREF]
  14. Penas, A. C., Metge, K., Mota, M., and Valadas, V.. 2006a. Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchinae) associated with Hylobius sp. from Pinus pinaster in Portugal. Nematology 8: 659–669.
    [CROSSREF]
  15. Penas, A.C., Bravo, M. A., Naves, P., Bonifacio, L., Sousa, E., and Mota, M.. 2006b. Species of Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) and other nematode genera associated with insects from Pinus pinaster in Portugal. Annals of Applied Biology 148: 121–131.
    [CROSSREF]
  16. Penas, A. C., Bravo, M. A., Valadas, V., and Mota, M.. 2007. Detailed morphobiometric studies of Bursaphelenchus xylophilus and characterisation of other Bursaphelenchus species (Nematoda: Parasitaphelenchidae) associated with Pinus pinaster in Portugal. Journal of Nematode Morphology and Systematics 10: 137–163.
  17. Salom, S. M.. 1997. Status and management of pales weevil in the eastern United States. Tree Planters’ Notes 48: 4–11.
  18. Susoy, V., Ragsdale, E. J., Kanzaki, N., and Sommer, R. J.. 2015. Rapid diversification associated with a macroevolutionary pulse of developmental plasticity. eLife 2015 4: 1–39.
  19. Thomas, W. K.. 2011. Molecular techniques. in International Seabed Authority (Eds), , Marine benthic nematode molecular protocol handbook (Nematode Barcoding), Technical Study No. 7, ISA Technical study series, pp. 22–37.
  20. Thompson, J. D., Higgins, D. G., and Gibson, T. J.. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673–4680.
    [CROSSREF]
  21. Tsai, I. J., Tanaka, R., Kanzaki, N., Akiba, M., Yokoi, T., Espada, M., Jones, J. T., and Kikuchi, T.. 2016. Transcriptional and morphological changes in the transition from mycetophagous to phytophagous phase in the plant-parasitic nematode Bursaphelenchus xylophilus. Molecular Plant Pathology 17: 77–83.
    [CROSSREF]
  22. Vrain, T. C., Wakarchuk, D. A., Lévesque, A. C., and Hamilton, R. I.. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15: 563–573.
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FIGURES & TABLES

Figure 1

A. Female body, B. Male body, C. Female Tail, D. Male Tail.

Full Size   |   Slide (.pptx)

Figure 2

18S, MrBayes tree with posterior probabilities on branches of Bursaphelenchus antoniae and close relatives within the ‘B. hylobianum species group’ (in Clade I of Kanzaki et al., 2015) based on a Clustal W alignment implemented in Geneious ver. 7.1.7 (Biomatters, Auckland, NZ) using the MRBAYES plugin with Chain Length 1,100,000, Burnin 110,000, mean -LnL - 7438.56.

Full Size   |   Slide (.pptx)

Figure 3

28S MrBayes tree with posterior probabilities on branches of B. antoniae based on a Clustal W alignment implemented in Geneious ver. 7.1.7 (Biomatters, Auckland, NZ) with Chain Length 1,100,000, Burnin 110,000, mean -LnL 3407.0.

Full Size   |   Slide (.pptx)

REFERENCES

  1. Bolla, R. I., Winter, R. E. K., Fitzsimmons, K., and Linit, M. J.. 1986. Pathotypes of the pinewood nematode Bursaphelenchus xylophilus. Journal of Nematology 18: 230–238.
  2. Carta, L. K., and Li, S.. 2019. Improved 18S small subunit rDNA primers for problematic nematode amplification. Journal of Nematology. 50 4: 533–542.
    [CROSSREF]
  3. Cherry, T., Szalanski, A. L., Todd, T. C., and Powers., T. O.. 1997. The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology 29: 23–29.
    [PUBMED]
  4. Duncan, E. J., Gluckman, P. D., and Dearden, P. K.. 2014. Epigenetics, plasticity and evolution: how do we link epigenetic change to phenotype?. Journal of Experimental Zoology (Mol. Dev. Evol.) 322B: 208–220.
    [CROSSREF]
  5. Gu, J., Wang, J., Braasch, H., Burgermeister, W., and Schroder, T.. 2011. Morphological and molecular characterisation of mucronate isolates (M form) of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae). Russian Journal of Nematology 19: 103–120.
  6. Holterman, M., Wurff, A. V. R., Elsen, S. V. D., Megen, H. V., Bongers, T., Holovachov, O., Bakker, J., and Helder, J.. 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution 23: 1792–1800.
    [CROSSREF]
  7. Huelsenbeck, J. P., and Ronquist, F.. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 17: 754–755.
    [CROSSREF]
  8. Inácio, M. L., Nóbrega, F., Mota, M., and Vieira, P.. 2017. First detection of Bursaphelenchus luxuriosae associated with Pinus pinaster in Portugal and in Europe. Forest Pathology 47: e12296.
    [CROSSREF]
  9. Kanzaki, N., and Futai, K.. 2002. A PCR primer set for determination of phylogenetic relationships of Bursaphelenchus species within the xylophilus group. Nematology 4: 35–41.
    [CROSSREF]
  10. Kanzaki, N., Okabe, K., and Kobori, Y.. 2015. Bursaphelenchus sakishimanus n. sp. (Tylenchomorpha: Aphelenchoididae) isolated from a stag beetle, Dorcus titanus sakishimanus Nomura (Coleoptera: Lucanidae), on Ishigaki Island, Japan. Nematology 17: 531–542.
    [CROSSREF]
  11. Leather, S. R., Day, K. R., and Salisbury, A. N.. 1999. The biology and ecology of the large pine weevil, Hylobius abietis (Coleoptera: Curculionidae): a problem of dispersal?. Bulletin of Entomological Research 89: 3–16.
    [CROSSREF]
  12. Munawar, M., Fang, Y., He, J., Gu, J.-F., and Li, H.-M.. 2015. Bursaphelenchus parantoniae n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from Belgium. Nematology 17: 1141–1152.
    [CROSSREF]
  13. Nunn, G. B., Theisen, B. F., Christensen, P., and Arctander, P.. 1996. Simplicity correlated size growth of the nuclear 28S ribosomal D3 expansion segment in the crustacean order Isopoda. Journal of Molecular Evolution 42: 211–223.
    [CROSSREF]
  14. Penas, A. C., Metge, K., Mota, M., and Valadas, V.. 2006a. Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchinae) associated with Hylobius sp. from Pinus pinaster in Portugal. Nematology 8: 659–669.
    [CROSSREF]
  15. Penas, A.C., Bravo, M. A., Naves, P., Bonifacio, L., Sousa, E., and Mota, M.. 2006b. Species of Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) and other nematode genera associated with insects from Pinus pinaster in Portugal. Annals of Applied Biology 148: 121–131.
    [CROSSREF]
  16. Penas, A. C., Bravo, M. A., Valadas, V., and Mota, M.. 2007. Detailed morphobiometric studies of Bursaphelenchus xylophilus and characterisation of other Bursaphelenchus species (Nematoda: Parasitaphelenchidae) associated with Pinus pinaster in Portugal. Journal of Nematode Morphology and Systematics 10: 137–163.
  17. Salom, S. M.. 1997. Status and management of pales weevil in the eastern United States. Tree Planters’ Notes 48: 4–11.
  18. Susoy, V., Ragsdale, E. J., Kanzaki, N., and Sommer, R. J.. 2015. Rapid diversification associated with a macroevolutionary pulse of developmental plasticity. eLife 2015 4: 1–39.
  19. Thomas, W. K.. 2011. Molecular techniques. in International Seabed Authority (Eds), , Marine benthic nematode molecular protocol handbook (Nematode Barcoding), Technical Study No. 7, ISA Technical study series, pp. 22–37.
  20. Thompson, J. D., Higgins, D. G., and Gibson, T. J.. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673–4680.
    [CROSSREF]
  21. Tsai, I. J., Tanaka, R., Kanzaki, N., Akiba, M., Yokoi, T., Espada, M., Jones, J. T., and Kikuchi, T.. 2016. Transcriptional and morphological changes in the transition from mycetophagous to phytophagous phase in the plant-parasitic nematode Bursaphelenchus xylophilus. Molecular Plant Pathology 17: 77–83.
    [CROSSREF]
  22. Vrain, T. C., Wakarchuk, D. A., Lévesque, A. C., and Hamilton, R. I.. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15: 563–573.

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