Bursaphelenchus mucronatus (Nematoda: Parasitaphelenchidae) associated with Monochamus galloprovincialis from Bosnia and Herzegovina and Georgia

Pheromone-baited black cross traps (Ecconex/Galloprotect Pack) were established as a part of the workshops focused on general surveys for forest quarantine species, including the pine wood nematode and its insect vectors. Two traps were hanged in the crowns of most abundant pine trees (approximately 20 m high), one in 2017 in the area of Banja Luka (BiH) on Pinus nigra, second in 2018 in Mamkoda park (Georgia) on P. sylvestris. Traps were checked for insects weekly (from June to September). Insects caught in the trap containers were transferred into plastic bottles, labelled, and transported to the laboratory. Although also other insects were caught in the traps, this study focused only on the identification and screening of known insect vectors of the pine wood nematode (i.e., Monochamus spp.). All collected vectors were identified morphologically to the species level before nematode analysis.

Nematodes were extracted by dissection and maceration of insects in sterile water followed by three hours incubation at room temperature. The presence or absence of nematodes was checked under the stereomicroscope (IPPC, 2016). The extracted nematode “dauer larvae” were fixed in 4 % formalin and transferred to pure glycerin according to De Grisse (1969) or prepared for molecular analysis. Where a large number of nematodes was available, a subset of living nematodes was transferred onto the plates containing the sporulating form of the fungus Monilinia sp., instead of Botryotinia fuckeliana (anamorph: Botrytis cinerea) in accordance with IPPC (2016), and cultured. Three weeks later, adults were fixed and transferred into glycerin as mentioned above.

Morphological and morphometric analyses were based on the main diagnostic features for the genus Bursaphelenchus as described by Ryss et al. (2005) and Braasch et al. (2009). Specimens mounted in permanent slides were deposited in the nematological collection of the Division of Plant Pest Diagnostics in Olomouc, Czech Republic.

For molecular identification, DNA from single nematode specimens (“dauer larvae”) was extracted and processed as follows: nematodes were homogenised in 50 μL of nematode lysis buffer (10mM Tris-HCl, pH 8.8; 1mM EDTA; 1 % Triton X-100 (v/v); 100 μg ml⁻¹ proteinase K) in a 1.5 mL Eppendorf tube using a micropestle. Samples were incubated at 55 °C for 1 h and subsequently at 95 °C for 10 min. The resulting DNA extracts were used as a template for each PCR reaction. The identification of individual nematodes was performed using two different loci, i.e., the ITS1-5.8S-ITS2 region of ribosomal DNA and the D2–D3 region of the 28S rDNA. The ITS1-5.8S-ITS2 region was amplified using the forward primer 5′-CGTAACAAGGTAGCTGTAG-3′ (Ferris et al., 1993) and the reverse primer 5′-TTTCACTCGCCGTTACTAAGG-3′ (26S, Vrain, 1993). For amplification of the D2–D3 region the forward primer D2A 5′-ACAAGTACCGTGAGGGAAAGTTG-3′ and the reverse primer D3B 5′-TCGGAAGGAACCAGCTACTA-3′ were used (De Ley et al., 1999).

The ITS1-5.8S-ITS2 PCR reaction was performed in a total volume of 50 μl containing 1x PCR buffer (75mM Tris-HCl pH 9.0; 50mM KCl; 20mM (NH₄)₂SO₄, 4mM MgCl₂, 400μM dNTPs, 600nM primers, 2 U of DNA polymerase (Biotools, Madrid, Spain) and 10 μl of undiluted template DNA, in a GenePro thermal cycler (Bioer Technology Co., LTD) using an initial denaturation step for 5 min at 94 °C, followed by 40 reaction cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 48 °C and extension for 2 min at 72 °C, with a final 10min extension at 72 °C. Six μl of the PCR product were digested for at least 2 hours at 37 °C, using 1x restriction buffer and 5 U of enzyme (RsaI, HaeIII, MspI, HinfI and AluI) in the total reaction volume of 10 μl. Restriction products were run on the Midori Green Advance (Nippon Genetics) stained 3 % TBE-buffered agarose gel (65 V, 60 min).

Amplification of the D2–D3 region was performed in a total volume of 25 μl using 1x PCRBIO Taq Mix (PCR Biosystems, London, UK), 400nM primers and 2 μl of undiluted template DNA, in a GenePro thermal cycler (Bioer Technology Co., LTD) using an initial denaturation step for 1 min at 95 °C, followed by 5 reaction cycles of denaturation for 15 s at 95 °C, annealing for 15 s at 45 °C and extension for 30 s at 72 °C and 35 cycles of 15 s at 95 °C, 15 s at 54 °C and 30 s at 72 °C.

After ExoSAP purification, PCR products of both regions were submitted for direct Sanger sequencing to the Centre of Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany (Olomouc, Czech Republic) using the corresponding forward and reverse primers. Resulting sequence data were analysed using Geneious Bioinformatics software platform (Biomatters). The online bioinformatic tool RestrictionMapper (www.restrictionmapper.org) was used for in-silico calculations of ITS-restriction fragment sizes and virtual RFLP pattern, using five above mentioned restriction enzymes, which are known to generate species-specific ITS-RFLP profiles for the genus Bursaphelenchus (Burgermeister et al., 2009).

The D2–D3 sequences obtained from nematodes collected in BiH and Georgia, as well as 10 sequences downloaded from the GenBank database (https://www.ncbi.nlm.nih.gov/), were used to reconstruct the phylogenetic relationships with other Bursaphelenchus species. Aphelenchoides besseyi (AY508109.1) was used as outgroup taxa. Phylogenetic analysis was conducted in MEGA X (Kumar et al., 2018). DNA sequences were aligned using ClustalW with default options (Thompson et al., 1994). The final alignment was visually checked. The optimal nucleotide substitution model for the Maximum Likelihood (ML) method was chosen according the Akaike Information Criterion (AIC). The evolutionary history was inferred by using the ML method based on the General Time Reversible (GTR) model with a discrete Gamma distribution of evolutionary rate differences among sites (five categories, + G) and invariant sites (+ I) (Nei & Kumar, 2000). The bootstrap consensus tree was inferred from 100 replicates (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50 % bootstrap replicates were collapsed.

As mentioned above, the main goal of our study was to detect potential insect vectors of pine wood nematode in BiH and Georgia, and therefore our analyses focused primarilly on the genus Monochamus. Forty specimens of Monochamus galloprovincialis were captured and processed in BiH. In Georgia, a total of 82 individuals of M. galloprovincialis were captured, of which 32 were analyzed. The processed M. galloprovincialis specimens collected in BiH and Georgia contained only “dauer larvae” of nematodes settled in tracheae of the sawyer beetles. Morphometric analysis of dauer larvae from both countries is summarized in Table 1, while morphological diagnostic characters are shown in Figure 1. Morphological and morphometric analysis of adult stages of Georgian population recovered from the fungal cultures confirmed the species identification of these nematodes as B. mucronatus (Fig. 1 and Table 1).

Fig. 1.

For both BiH and Georgian populations found during the survey, a molecular characterization was performed using the ITS1-5.8S-ITS2 and D2–D3 region of 28S of the ribosomal DNA generated from single dauer larvae specimens.

The amplification and sequencing of ITS1-5.8S-ITS2 region yielded an 884 bp sequence (corresponding to the entire PCR product sequence after primer trimming) for both BiH and Georgian populations (OK500292 and OK500293, respectively) sharing 99.7 % identity to each other: BiH sequence included one ambiguous base (A/G=R) at position 161 and G at 642 (which prevented amplicon cutting with RsaI restriction enzyme at this position) while Georgian sequence had an A in position 642 (cutting motif for RsaI) and R at position 805. Both R bases come from an A+R double-peak presence in all raw data reads. From BiH population two specimens were analysed separately and sequenced twice in both directions resulting in 7 out of 8 raw data reads useful for consensus sequence generation. In all 7 of them a clear double-peak was observed. From Georgian population a single specimen was sequenced in both directions resulting in a double-peak presence in both raw data. Both BiH and Georgian sequences show high similarity (99 % for BiH due to R base at 161 position and 100 % for Georgian whose R is located out of section aligned with sequences deposited at NCBI (sequences AM396572.1 and MK584707.1, see Fig. 2).

Fig. 2.

The ITS-RFLP pattern was calculated virtually for the ITS sequences generated for both populations collected in BiH and Georgia. The virtual restriction profile generated for four enzymes were similar for both populations (Table 2) and showed the same reference ITS-RFLP pattern established for the B. mucronatus European type by Burgermeister et al. (2009). The only difference found between both populations was in the pattern generated by the RsaI restriction enzyme (Table 2). The restriction profile of RsaI of the population from Georgia corresponded to the profile of East-Asian type of B. mucronatus and differed from the reference profile for the European type of B. mucronatus (Burgermeister et al., 2009).

In case of D2–D3 region amplification and sequencing resulted in a sequence of 743 bp (corresponding to the entire PCR product sequence after primer trimming) for the population collected in BiH (OK523378) and partial 720 bp for the population collected in Georgia (OK523379). Both sequences shared 100 % identity to each other and also to the population of B. mucronatus kolymensis (MK584707) from Romania (Calin et al., 2020) and other sequence of B. mucronatus (AM396572.1) from Germany (data not shown). All four sequences clustered together in phylogenetic analysis (Fig. 2).