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

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 –80^oC until needed. Each extract was prepared by incubating the tubes at 60^oC for 60 min, followed by 95^oC 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 95^oC for 3 min; 36 cycles of 95^oC for 30 sec, 50^oC for 40 sec, and 72^oC for 70 sec; and final extension at 72^oC for 5 min, for the 28S was 95^oC for 3 min; 36 cycles of 95^oC for 30 sec, 58^oC for 45 sec, and 72^oC for 70 sec; and final extension at 72^oC for 5 min, for the ITS was 95^oC for 3 min; 36 cycles of 95^oC for 30 sec, 55^oC for 60 sec, and 72^oC for 105 sec; and final extension at 72^oC 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 FlashGel^TM 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.

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).

Figure 1

Figure 2

Figure 3

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.