European Journal of Wood and Wood Products

, Volume 77, Issue 2, pp 301–309 | Cite as

Pinewood nematode presence and survival in commercial pallets of different ages

  • P. NavesEmail author
  • M. L. Inácio
  • F. Nóbrega
  • E. Sousa
  • M. Michielsen


The pinewood nematode (PWN) Bursaphelenchus xylophilus is an important forestry quarantine organism in the world and can be disseminated through the international trade of infested wood packaging materials. Although heat treatment (HT) and kiln-drying (KD) are used to disinfest the wood and reduce its moisture, doubt remains if treated wood can be re-infested and support viable populations of the nematode. In this study, the risk associated with disseminating the PWN with kiln-dry pallet timber was evaluated by sampling commercial pallets of different ages and by inoculating B. xylophilus into treated wood to assess the nematode’s survival. A total of 229 timber samples (boards and blocks) were randomly sampled, finding no Bursaphelenchus species. Nematodes of the families Aphelenchidae, Aphelenchoididae, Diplogasteridae, Rhabditidae and Tylenchidae were found in 45% of the samples, being absent from timber with less than 6 months but present with increasing frequency in older pallets. Fungi were encountered frequently, with the genus Trichoderma dominant. In the second trial, artificial inoculations of B. xylophilus were made in pallet timber of different age and assessed after 7, 14, 28, 56 and 84 days. The PWN was recovered in the first sampling, but infestation rates and nematode numbers decreased until disappearing in the subsequent samplings. The results confirm that ISPM 15 treatments effectively sanitize wood, and older, drier wood does not support re-infestation with B. xylophilus, while other saprophitic nematodes and fungi colonise the treated timber. Future research should evaluate the risk associated with KD timber subjected to rewetting and its ability to support viable populations of the PWN.



The authors would like to express gratitude to Mrs. Christine Vael (Brambles/CHEP Europe, UK,) for technical assistance during the experiments, and to INIAV technicians Margarida Fontes, Adérito Bispo, Vitor Gonçalves, Francisco Martins and Marina Soares for valuable collaboration during the experiments. We would also like to acknowledge the contribution of two anonymous reviewers who greatly contributed to improving the paper.


Studies were commissioned to INIAV and financed by Brambles/CHEP Europe, UK, within the protocol of scientific collaboration CHEP-INIAV 2013–2014. Pedro Naves is currently funded by the Fundação para a Ciência e Tecnologia—FCT (contract IF/00471/2013/CP1203/CT0001), Portugal.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Allen EA, Humble LM (2002) Nonindigenous species introductions: a threat to Canada’s forests and forest economy. Can J Plant Pathol 24:103–110CrossRefGoogle Scholar
  2. Anonymous (2009) Decree law no. 1460/2009, 31st December. D.R. no. 252, series I., PortugalGoogle Scholar
  3. Bengtsson J, Logie J (2015) Life cycle assessment of one-way and pooled pallet alternatives. Procedia CIRP 29:414–419CrossRefGoogle Scholar
  4. Benker U (2012) Monochamus alternatus—the Next Alien Causing Trouble. Forstschutz Aktuell 56:34–37Google Scholar
  5. Braasch H, Schönfeld U (2015) Improved morphological key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937. EPPO Bull 45:73–80. CrossRefGoogle Scholar
  6. Braasch H, Tomiczek C, Metge K, Hoyer U, Burgermeister W, Wulfert I, Schonfeld U (2001) Records of Bursaphelenchus spp. (Nematoda: Parasitaphelenchidae) in coniferous timber imported from the Asian part of Russia. For Pathol 31:129–140CrossRefGoogle Scholar
  7. Braasch H, Burgermeister W, Gu J (2009) Revised intra-generic grouping of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae). J Nematode Morphol Syst 12:65–88Google Scholar
  8. Cutler HG, Crumley FG, Cole PD (1986) 6-Pentyl-α-pyrone from Trichoderma harzianum: its plant growth inhibitory and antimicrobial properties. Agric Biol Chem 50:2943–2945Google Scholar
  9. Davies KG, Spiegel Y (2011) Biological control of plant-parasitic nematodes: towards understanding field variation through molecular mechanisms. In: Jones J, Gheysen G, Fenoll C (eds) Genomics and molecular genetics of plant–nematode interactions. Springer, Heidelberg, pp 493–516CrossRefGoogle Scholar
  10. Dennis C, Webster J (1971) The antagonistic properties of the Trichoderma spp.: production of volatile antibiotics. Trans Br Mycol Soc 57:41–48CrossRefGoogle Scholar
  11. Dowding P (1969) The dispersal and survival of spores of fungi causing bluestain in pine. Trans Brit Mycol Soc 52:125–137CrossRefGoogle Scholar
  12. Dwinell LD (1990) Heat-treating and drying southern pine lumber infested with pinewood nematodes. For Prod J 40:53–56Google Scholar
  13. Dwinell LD (1997) The pine wood nematode: regulation and mitigation. Annu Rev Phytopathol 35:153–166CrossRefGoogle Scholar
  14. Dwinell LD (2004) Mitigating the pine wood nematode and its insect vectors in transported coniferous wood. Nematol Monogr Perspect 2:825–835Google Scholar
  15. Evans HF, McNamara DG, Braasch H, Chadoeuf J, Magnusson C (1996) Pest risk analysis (PRA) for the territories of the European union (as PRA area) on Bursaphelenchus xylophilus and its vectors in the genus Monochamus. EPPO Bull 26:199–249CrossRefGoogle Scholar
  16. Eyre D, Macarthur R, Haack RA, Lu Y, Krehan H (2018) Variation in inspection efficacy by member states of wood packaging material entering the European Union. J Econ Entomol 111:707–715. CrossRefGoogle Scholar
  17. Fukushige H (1991) Propagation of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on fungi growing in pine-shoot segments. Appl Entomol Zool 26:371–376CrossRefGoogle Scholar
  18. Futai K (2013) Pine wood nematode, Bursaphelenchus xylophilus. Annu Rev Phytopathol 51:61–83CrossRefGoogle Scholar
  19. Gams W, Bissett J (1998) Morphology and identification of Trichoderma. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium, vol 1. Taylor and Francis, London, pp 3–34Google Scholar
  20. Ghisalberti E, Sivasithamparam K (1991) Antifungal antibiotics produced by Trichoderma spp. Soil Biol Biochem 23:1011–1020CrossRefGoogle Scholar
  21. Gibbs JN (1993) The biology of Ophiostomatoid fungi causing sapstain in trees and freshly-cut logs. Chapter 17. In: Wingfield JM, Seifert KA, Webber JF (eds) Ceratocystis and Ophiostoma. Taxonomy, ecology, and pathogenicity. The American Phytopathological Society, St. Paul, pp 153–160Google Scholar
  22. Glass SV, Zelinka SL (2010) Moisture relations and physical properties of wood, chap 4. In: Centennial (ed) Wood handbook: wood as an engineering material, General technical report FPL GTR-190. Forest Service, Forest Products Laboratory, US Dept of Agriculture, Madison, pp 4.1–4.19Google Scholar
  23. Gu J, Braasch HM, Burgermeister W, Zhang J (2006) Records of Bursaphelenchus spp. intercepted in importing packaging wood at Ningbo, China. For Pathol 36:323–333CrossRefGoogle Scholar
  24. Haack RA (2006) Exotic bark and wood-boring Coleoptera in the United States: recent establishments and interceptions. Can J For Res 36:269–288CrossRefGoogle Scholar
  25. Haack RA, Petrice TR (2009) Bark-and wood-borer colonization of logs and lumber after heat treatment to ISPM 15 specifications: the role of residual bark. J Econ Entomol 102:1075–1084CrossRefGoogle Scholar
  26. Haack RA, Britton KO, Brockerhoff EG, Cavey JF, Garrett LJ, Kimberley M, Lowenstein F, Nuding A, Olson LJ, Turner J, Vasilaky KN (2014) Effectiveness of the international phytosanitary standard ISPM No. 15 on reducing wood borer infestation rates in wood packaging material entering the United States. PLoS One 9:e96611. CrossRefGoogle Scholar
  27. Halik S, Bergdahl DR (1990) Development of Bursaphelenchus xylophilus populations in wood chips with different moisture contents. J Nematol 22:113–118Google Scholar
  28. Harman GE (2000) Myths and dogmas of biocontrol. Changes in perceptions derived from research on Trichoderma harzianum T22. Plant Dis 84:377–393CrossRefGoogle Scholar
  29. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56CrossRefGoogle Scholar
  30. Harrington TC (1981) Cycloheximide sensitivity as a taxonomic character in Ceratocystis. Mycologia 73:1123–1129CrossRefGoogle Scholar
  31. Hisai J, Hirata K, Suzuki K (2006) Investigation of survival time of pine wood nematode (Bursaphelenchus xylophilus) in Wood Chips and Wood Blocks. Res Bull Plant Prot Japan 42:53–55 (in Japanese) Google Scholar
  32. Hopf-Biziks A, Schröder T, Schütz S (2017) Long-term survival and non-vector spread of the pinewood nematode, Bursaphelenchus xylophilus, via wood chips. For Pathol 47:e12340. CrossRefGoogle Scholar
  33. Howell CR (2003) Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis 87:4–10CrossRefGoogle Scholar
  34. Inácio ML, Nóbrega F, Vieira P, Bonifácio L, Naves P, Sousa E, Mota M (2015) First detection of Bursaphelenchus xylophilus associated with Pinus nigra in Portugal and in Europe. For Path 45:235–238CrossRefGoogle Scholar
  35. IPPC—International Plant Protection Convention (2009) International standards for phytosanitary measures: revision of ISPM No. 15, regulation of wood packaging material in international trade. Food and Agriculture Organization of the United Nations, RomeGoogle Scholar
  36. Jaklitsch WM (2009) European species of Hypocrea part I. The green-spored species. Stud Mycol 63:1–91CrossRefGoogle Scholar
  37. Kishi Y (1995) Pine wood nematode and the Japanese pine sawyer. Thomas Comp Limited, TokyoGoogle Scholar
  38. Kobayashi T, Sasaki K, Mamiya Y (1974) Fungi associated with Bursaphelenchus lignicolus, the pine wood nematode (I). J Jpn For Soc 56:136–145 (in Japanese) Google Scholar
  39. Kobayashi T, Sasaki K, Mamiya Y (1975) Fungi associated with Bursaphelenchus lignicolus, the pine wood nematode (II). J Jpn For Soc 57:184–193 (in Japanese) Google Scholar
  40. Kubátová A (2000) Neglected Penicillium spp. associated with declining trees. In: Samson RA, Pitt JI (eds) Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Harwood Academic Publishers, Amsterdam, pp 299–307Google Scholar
  41. Lovett GM, Weiss M, Liebhold AM, Holmes TP, Leung B, Lambert KF, Orwig DA, Campbell FT, Rosenthal J, McCullough DG, Wildova R, Ayres MP, Canham CD, Foster DR, LaDeau SL, Weldy T (2016) Nonnative forest insects and pathogens in the United States: impacts and policy options. Ecol Appl 26:1437–1455CrossRefGoogle Scholar
  42. Maehara N (2008) Reduction of Bursaphelenchus xylophilus (Nematoa: Parasita phelenchidae) population by inoculating Trichoderma spp. into pine wilt-killed trees. Biol Control 44:61–66CrossRefGoogle Scholar
  43. Maehara N, Futai K (1996) Factors affecting both the numbers of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae), carried by the Japanese pine sawyer, Monochamus alternatus (Coleoptera: Cerambycidae), and the nematode`s life history. Appl Entomol Zool 31:443–452CrossRefGoogle Scholar
  44. Maehara N, Futai K (2000) Population changes of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae), on fungi growing in pine-branch segments. Appl Entomol Zool 35:413–417CrossRefGoogle Scholar
  45. Maehara N, Futai K (2008) Microbial control of Bursaphelenchus xylophilus by Fungi. In: Mota MM, Vieira PR (eds) Pine wilt disease: a worldwide threat to forest ecosystems. Springer, Netherlands, pp 359–368CrossRefGoogle Scholar
  46. Maehara N, Hata K, Futai K (2005) Effect of blue-stain fungi on the number of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) carried by Monochamus alternatus (Coleoptera: Cerambycidae). Nematology 7:161–167CrossRefGoogle Scholar
  47. Maehara N, Tsuda K, Yamasaki M, Shirakikawa S, Futai K (2006) Effect of fungus inoculation on the number of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) carried by Monochamus alternatus (Coleoptera: Cerambycidae). Nematology 8:59–67CrossRefGoogle Scholar
  48. Meissner H, DeWald P, Jones E, Lemay A, Millar L, Ye W (2014) Pest survey of softwood boxes, with special emphasis on pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae). EPPO Bull 44:37–43. CrossRefGoogle Scholar
  49. Melencion N, Morrell JJ (2007) Performance of antisapstain compounds on kiln-dried Douglas-fir and hem-fir lumber subjected to rewetting. For Prod J 57:111–113Google Scholar
  50. Molina-Murillo S, Smith T, Reichenbach M, Smith R (2005) Impact of international phytosanitary standards on wood packaging materials end users: pre-implementation assessment. For Prod J 55:24–26Google Scholar
  51. Payne C, Petty JA, Woodward S (1998) Fungal spora of softwood timber drying kilns in Scotland. Mater Org (Berlin) 32:109–125Google Scholar
  52. Penas AC, Dias LS, Mota M (2002) Precision and selection of extraction methods of aphelenchid nematodes from maritime pine wood, Pinus pinaster L. J Nematol 34:62–65Google Scholar
  53. Ramsfield TD, Ball RD, Gardner JF, Dick MA (2010) Temperature and time combinations required to cause mortality of a range of fungi colonizing wood. Can J Plant Pathol 32:368–375CrossRefGoogle Scholar
  54. Rossman AY (1996) Morphological and molecular perspectives on systematics of the Hypocreales. Mycologia 88:1–19CrossRefGoogle Scholar
  55. Ryss AY, Vieira P, Mota MM, Kulinich O (2005) A synopsis of the genus Bursaphelenchus Fuchs, 1937 (Aphelenchida: Parasitaphelenchidae) with keys to species. Nematology 7:393–458CrossRefGoogle Scholar
  56. Schubert M, Fink S, Schwarze F (2008) Evaluation of Trichoderma spp. as a biocontrol agent against wood decay fungi in urban trees. Biol Control 45:111–123CrossRefGoogle Scholar
  57. Seifert KA, Frisvad JC (2000) Penicillium on solid wood products. In: Samson RA, Pitt JI (eds) Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Harwood Academic Publishers, Amsterdam, pp 285–292Google Scholar
  58. Simpson WT (1998) Equilibrium moisture content of wood in outdoor locations in the United States and worldwide. In: Res. Note FPL-RN-268. U.S. Department of Agriculture, Forest Service, Forest Products Laboratory, MadisonGoogle Scholar
  59. Smith RS (1992) Eradication of pinewood nematodes in softwood lumber. Proc Can Wood Preserv Assoc 13:185–206Google Scholar
  60. Sousa E, Naves P, Bonifácio L, Henriques J, Inácio ML, Evans H (2011a) Survival of Bursaphelenchus xylophilus and Monochamus galloprovincialis in pine branches and wood packaging material. EPPO Bull 41:203–207CrossRefGoogle Scholar
  61. Sousa E, Naves P, Bonifácio L, Henriques J, Inácio ML, Evans H (2011b) Assessing risks of pine wood nematode Bursaphelenchus xylophilus transfer between wood packaging simulating assembled pallets in service. EPPO Bull 41:423–431. CrossRefGoogle Scholar
  62. Sriwati R, Takemoto S, Futai K (2007) Cohabitation of the pine wood nematode, Bursaphelenchus xylophilus, and fungal species in pine trees inoculated with B. xylophilus. Nematology 9:77–86CrossRefGoogle Scholar
  63. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  64. TIMCON—Timber Packaging and Pallet Confederation (2017) Pallets and packaging, design and features. Accessed 29 Nov 2017
  65. Tomiczek C, Braasch H, Burgermeister W, Metge K, Hoyer U, Brandstetter M (2003) Identification of Bursaphelenchus spp. isolated from Chinese packaging wood imported to Austria. Nematology 5:573–581CrossRefGoogle Scholar
  66. Tomminen J (1991) Pinewood nematode, Bursaphelenchus xylophilus, found in packing case wood. Silva Fenn 25:109–111. CrossRefGoogle Scholar
  67. Uzunovic A, Khadempour L, Leung K (2008) Heat disinfestation of decay fungi found in post-mountain pine beetle wood. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, Victoria. Mountain Pine Beetle Initiative [From project 8.61]. WP2008-14. Cat. no.: Fo144-7/2010E-PDF. ISBN 978-1-100-14610-2, p 16Google Scholar
  68. Webster J, Weber RWS (2007) Introduction to fungi. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  69. Weindling R (1932) Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22:837–845Google Scholar
  70. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand D, Sninsky JJ, White TJ (eds) PCR Protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322Google Scholar
  71. Wu Y, Trepanowski N, Molongoski J, Reagel P, Lingafelter S, Nadel H, Myers S, Ray A (2017) Identification of wood-boring beetles (Cerambycidae and Buprestidae) intercepted in trade-associated solid wood packaging material using DNA barcoding and morphology. Sci Rep 7:40316CrossRefGoogle Scholar
  72. Yang Z-S, Li G-H, Zhao P-J, Zheng X, Luo S-L, Li L, Niu X-M, Zhang K-Q (2010) Nematicidal activity of Trichoderma spp. and isolation of an active compound. World J Microbiol Biotechnol 26:2297–2302CrossRefGoogle Scholar
  73. Yang Z, Yu Z, Lei L, Xia Z, Shao L, Zhang K, Li G (2012) Nematicidal effect of volatiles produced by Trichoderma sp. J Asia Pac Entomol 15:647–650CrossRefGoogle Scholar
  74. Zahid MI, Grgurinovic CA, Walsh DJ (2008) Quarantine risks associated with solid wood packaging materials receiving ISPM 15 treatments. Aust For 71:287–293CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Unidade Estratégica de Sistemas Agrários e Florestais e Sanidade VegetalInstituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV)OeirasPortugal
  2. 2.Director Government and Regulatory Affairs, EMEA at CHEP (Brambles)SurreyUK

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