First Report of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on Monochamus galloprovincialis (Coleoptera: Cerambycidae) in Spain

Abstract

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, was found to be the causal agent of pine wilt disease in Pinus in Japan in 1971 (Mamiya 1987). In Europe, the PWN was first detected in Portugal in 1999 (Mota et al. 1999) and then in Spain in 2008 (Robertson et al. 2011). In 2001, pine sawyer beetle (Monochamus galloprovincialis Olivier) was associated with the PWN in Portugal (Sousa et al. 2001). In As Neves (Galicia, northwest Spain), the PWN was detected in 2010 (Abelleira et al. 2011), resulting in the establishment of a demarcated zone (DZ) 20 km wide in all directions. By 2018, the PWN was detected from four more municipalities (Salvaterra de Mino, Ponteareas, Tui, and Porrino), resulting in further expansion of the size of the DZ. In an attempt to detect M. galloprovincialis as a possible vector of the PWN in the DZ, surveys were conducted during spring and summer of 2018. Fifty multiple-funnel traps baited with aggregation pheromone were hung on a 2-m-high iron structure at the edge of adult pine stands to determine the distribution of M. galloprovincialis and maximize their capture. The traps were checked weekly from May to October, and the insects inside the traps were collected and visually identified to the species level. A total of 265 M. galloprovincialis were captured from the traps. Each insect was placed in an individual Petri dish containing sterile distilled water, cut into small pieces, and kept at room temperature (18 to 22 C°) for 24 h to extract dauer juveniles of the nematode. The dauer juveniles obtained from the insect were identified morphologically and further substantiated by DNA-based methods. For morphological study, 100 dauer juveniles were mounted on temporary slides in Amman’s lactophenol and observed under a light microscope (Eclipse 80i, Nikon Instruments, Tokyo, Japan) at different magnifications (200× to 1,000×). The dauer juveniles had a dome-shaped head, weak stylet, poorly defined median bulb, and an elongated conoid tail, with the mucronate terminus similar to that described by Penas et al. (2008). The population density of the dauer juveniles ranged from 1 to 33,000 individuals per plate. The total percentage of M. galloprovincialis carrying dauer juveniles of the PWN was 28%. To study morphological characteristics of the adults, dauer juveniles were reared on Botrytis cinerea Pers. in potato dextrose agar plates for 15 days at 25°C. All morphological characters of adults (n = 50) corresponded with B. xylophilus (Penas et al. 2008). The DNA was extracted from dauer juveniles and adults using a QIAamp DNA Micro Kit (Qiagen, Germany). A region of ribosomal DNA (rDNA), containing the internal transcribed spacer regions ITS1 and ITS2 were amplified using the method described by Burgermeister et al. (2009). Half of the PCR-amplified product was used for sequencing in our laboratory, and the other half was subjected to restriction enzyme digestion using AluI, HaeIII, HinfI, MspI, and RsaI. The restriction patterns corresponded to those described for B. xylophilus by Burgermeister et al. (2009). The nucleotide sequences generated in this study were compared with nonredundant sequences in the GenBank using the MegaBLAST algorithm in the BLASTn program and had 99 to 100% homology with B. xylophilus (accession nos. JQ288086 and KF025322). Two representative sequences were submitted to GenBank (accession nos. MK789297 and MK789298). This is the first report of the presence of dauer juveniles of PWN on M. galloprovincialis in Spain. Our study further substantiates the importance of M. galloprovincialis as a vector of B. xylophilus in Europe.