Introduction

In 1999, Portugal became the first European country affected by pine wilt disease (PWD). Nine years later, its entire continental territory was declared a demarcated area (Mota et al., 2009Mota MM, Futai K, Vieira P, 2009. Pine wilt disease and the pinewood nematode, Bursaphelenchus xylophilus. In: Integrated Management of Fruit Crops Nematodes; Ciancio A, Mukerji, K, (eds). pp: 253-274. Springer, Dordrecht. 10.1007/978-1-4020-9858-1_11). The same year (2008), the pathogen causing this disease, the pinewood nematode (PWN) Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, was first detected in Spain (Robertson et al., 2011Robertson L, Cobacho Arcos S, Escuer M, et al., 2011. Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner & Buhrer, 1934 (Nickle, 1970) in Spain. Nematology 13: 755-757. 10.1163/138855411X578888). Currently, the disease in this country is limited to three regions bordering Portugal: Salamanca (Castile and Leon), Cáceres (Estremadura) and Pontevedra (Galicia). European Union legislation, specifically Decision 2012/535/EU on emergency measures to prevent the spread of B. xylophilus, established demarcated areas in these territories, including an infested zone (where the presence of the nematode has been confirmed) and a buffer zone (20 km radius around the infested zone). Within these areas, a plan is in place and measures are being implemented to eradicate the nematode and monitor the affected forest to detect possible new outbreaks.

Early detection of the PWN, followed by the cutting and removal of infected trees are essential for preventing its spread to new areas. All EU Member States conduct annual surveys of susceptible plants in non-affected areas to detect possible entries of this pathogen. In demarcated areas, surveys should be performed more frequently and at different sampling intensities, depending on the distance to a positive tree, using a grid-based approach. In the intensive survey zone of the demarcated area in southern Pontevedra (Galicia), sampling is carried out in a 1x1 km grid: once a month (100 – 500 m radius around the infected point) or every two months (500 m – 3 km radius around the infected point) during the flight season of Monochamus galloprovincialis (Olivier 1795) (the only confirmed B. xylophilus vector in the EU to date), and once during winter. In the rest of the demarcated area, sensitive plants are sampled and tested for PWN presence in a 2x2 km grid, once during the flight season and once during winter. This generates a large number of samples, because 46% of the demarcated area is covered by Pinus conifers. Processing and analysing the samples also require technical expertise, so a simple, rapid, preferably on-site detection method is urgently needed to control PWN spread, as an alternative to traditional methods.

Conventionally, wood samples are incubated at 25°C for at least 14 days before nematodes can be detected and identified. The nematodes are then extracted from plant tissues using the Baermann funnel technique and identified by their morphological and morphometric features under a stereomicroscope (EPPO, 2023EPPO, 2023. PM 7/4(4) Bursaphelenchus xylophilus. EPPO Bull 53: 156-183.). Adult female and male B. xylophilus specimens are thus essential for unequivocal identification. The male has a spicule of characteristic shape and size, while the female has a rounded tail and a vulval flap (Ryss et al., 2005Ryss A, Vieira P, Mota M, Kulinich O, 2005. A synopsis of the genus Bursaphelenchus Fuchs, 1937 (Aphelenchida: Parasitaphelenchidae) with keys to species. Nematology 7: 393-458. 10.1163/156854105774355581; Braasch and Schönfeld, 2015Braasch 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. 10.1111/epp.12174). Even so, molecular-based detection techniques may be required to confirm the morphological identification. Many molecular tests are accepted in the European and Mediterranean Plant Protection Organization (EPPO) protocol for nematode identification (EPPO, 2023EPPO, 2023. PM 7/4(4) Bursaphelenchus xylophilus. EPPO Bull 53: 156-183.), including a modification of the Loop-mediated isothermal amplification (LAMP) test developed by Kikuchi et al. (2009Kikuchi T, Aikawa T, Oeda Y, et al., 2009. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology 99: 1365-1369. 10.1094/PHYTO-99-12-1365). This quick, very sensitive method does not require specialized techniques, equipment, or personnel. Commercial LAMP kits show potential for use as field tools in highly infected areas or even as an initial control method in areas under surveillance. The sensitivity of the test could eliminate the recommended 14-day incubation period to maximize the likelihood of detection in wood samples.

The main aim of this study was to evaluate the effectiveness of a commercial kit based on LAMP methodology for the detection of B. xylophilus in Pinus radiata D. Don and Pinus pinaster Aiton. These are the most affected species in Spain and the most important conifers in terms of annual cut volume in Galicia (3.3 Mm³ in 2022; Xunta de Galicia, 2023Xunta de Galicia, 2023. Sistema de indicadores da administración dixital. Producción forestalhttps://indicadores-forestal.xunta.gal/portal-bi-internet/dashboard/Dashboard.action), one of the affected autonomous communities. The specific aims of this study were: (i) to test the effectiveness of the LAMP kit for detecting seven B. xylophilus isolates from different origins in artificially inoculated P. pinaster wood chips; (ii) to test the effectiveness of the LAMP kit for detecting B. xylophilus in samples collected from artificially inoculated dead P. pinaster and P. radiata seedlings; (iii) to determine the test’s ability to detect the nematode at any disease stage or post-inoculation period.

Materials and methods

Three different experiments were performed using two methods for B. xylophilus detection: (i) extraction by the Baermann funnel technique and subsequent morphological identification under stereomicroscope, and (ii) a commercial B. xylophilus kit (Nippon Gene Co. Ltd., Tokyo, Japan) based on the LAMP technique.

In Experiment 1, P. pinaster wood chips were artificially inoculated with seven B. xylophilus isolates from different geographic origins (Japan, United States, Portugal, and Spain) (Menéndez‐Gutiérrez et al., 2021Menéndez‐Gutiérrez M, Villar L, Díaz R, 2021. Virulence of seven pathogenic Bursaphelenchus xylophilus isolates in Pinus pinaster and Pinus radiata seedlings and its relation with multiplication. For Pathol 51: e12677. 10.1111/efp.12677). Wood chips were sampled to detect B. xylophilus at 1 (four samples per isolate), 30 (one sample per isolate), and 90 (four samples per isolate) days after inoculation (DAI) using the LAMP kit. The Baermann funnel technique was performed 30 DAI for nematode detection. The inoculum dose was 10,000 nematodes per ml, and wood chip humidity before inoculation was above 80%. In Experiment 2, inoculations were carried out with the Japanese isolate Ka4 (Filipiak, 2015Filipiak A, 2015. Pathogenicity of selected isolates of the quarantine pinewood nematode Bursaphelenchus xylophilus to Scots pine (Pinus sylvestris L.). J Plant Prot Res 55: 378-382. 10.1111/epp.12915; Hirao et al., 2019Hirao T, Matsunaga K, Hirakawa H, et al., 2019. Construction of genetic linkage map and identification of a novel major locus for resistance to pine wood nematode in Japanese black pine (Pinus thunbergii). BMC Plant Biol 19: 1-13. 10.1186/s12870-019-2045-y) and the Spanish isolate SpPO1 (Menéndez‐Gutiérrez et al., 2021Menéndez‐Gutiérrez M, Villar L, Díaz R, 2021. Virulence of seven pathogenic Bursaphelenchus xylophilus isolates in Pinus pinaster and Pinus radiata seedlings and its relation with multiplication. For Pathol 51: e12677. 10.1111/efp.12677) at a dose of 4,000 nematodes per ml. A total of six two-year-old seedlings, two P. pinaster seedlings and one P. radiata seedling per isolate, were inoculated and kept under controlled conditions. Three months after inoculation, all seedlings were dead and two 1.5-cm segments per seedling, taken from 5 and 20 cm below the inoculation point, were used for detection via LAMP. The remainder of each seedling was processed for extraction using the Baermann funnel technique. Experiment 3 was also performed under greenhouse conditions in three blocks, with two treatments involving eight inoculated and three control P. pinaster seedlings per block. The seedling disease stage was assessed weekly according to the percentage of wilted needles, on a scale of 1 (no external symptoms) to 7 (all needles brown and wilted) (Menéndez-Gutiérrez et al., 2018Menéndez-Gutiérrez M, Alonso M, Jiménez E, et al., 2018. Interspecific variation of constitutive chemical compounds in Pinus spp. xylem and susceptibility to pinewood nematode (Bursaphelenchus xylophilus). Eur J Plant Pathol 150: 939-953. 10.1007/s10658-017-1334-2). Seedlings in each block were processed at 15, 45, and 70 DAI. Two samples were taken from each seedling, one above and one below the inoculation point, and incubated in a culture chamber at 25°C for 14 days. Then two wood chips were taken from each sample to determine the presence of B. xylophilus using the LAMP kit, and the remainder of each seedling was used for nematode extraction using the Baermann funnel method. DNA extracted from the seedling chips on the last sampling date (70 DAI) was quantified by fluorometry using Qubit^TM 4 (Invitrogen, USA).

The commercial LAMP kit was used as described in Kikuchi et al. (2009Kikuchi T, Aikawa T, Oeda Y, et al., 2009. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology 99: 1365-1369. 10.1094/PHYTO-99-12-1365). For DNA extraction, two 1.5 mm wood chips were added to 800 µl of extraction solution and kept at 55°C for 20 minutes, then at 100°C for 10 minutes. After this, the test solution was prepared using a mixture of reagents from the kit. Samples for DNA amplification were prepared by adding 2 µl of the extracted DNA, 18 µl of the reagent mixture and 20 µl of mineral oil. The reaction mixture was incubated at 63°C for 90 minutes for DNA amplification, then for 2 minutes at 80°C for enzyme inactivation. Finally, the presence or absence of PWN in the sample was visually determined using UV light.

Results and discussion

Effective management and prevention of the spread of PWD involve exhaustive surveillance for early detection and rapid implementation of control measures to prevent further spreading of the disease. The LAMP technique provides fast results for B. xylophilus detection in wood chips without the need for advanced skills and sophisticated equipment. Nonetheless, our results showed a decline in PWN detection rates using a commercial LAMP kit from 70 DAI on, and a higher detection level with the Baermann funnel technique. Through this extraction method, we observed a decrease in the number of nematodes from 15 DAI onward; along with a higher number of nematodes in the segment above the inoculation point in the first stages of the disease.

In Experiment 1, the detection rate using the LAMP kit on wood chips artificially inoculated with B. xylophilus was 100 % at 1 (twenty-eight samples analysed) and 30 DAI (seven samples analysed) but decreased to 18% (five positive samples out of twenty-eight samples) at 90 DAI. This contradicts a previous study indicating that the LAMP kit could detect PWNs in pine wood for at least 6 years after tree death, though detection rates varied from 0% to 63% depending on the sampling point (Kanetani et al., 2011Kanetani S, Kikuchi T, Akiba M, et al., 2011. Detection of Bursaphelenchus xylophilus from old discs of dead Pinus armandii var. amamiana trees using a new detection kit. For Pathol 41: 387-391. 10.1111/j.1439-0329.2010.00695.x).

Significantly, our findings in wood chips confirmed for the first time that the LAMP kit was able to detect PWNs from the various origins studied (Table 1). Until now, the commercial LAMP kit had only been tested on Asian isolates (Kikuchi et al., 2009Kikuchi T, Aikawa T, Oeda Y, et al., 2009. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology 99: 1365-1369. 10.1094/PHYTO-99-12-1365; Kanetani et al., 2011Kanetani S, Kikuchi T, Akiba M, et al., 2011. Detection of Bursaphelenchus xylophilus from old discs of dead Pinus armandii var. amamiana trees using a new detection kit. For Pathol 41: 387-391. 10.1111/j.1439-0329.2010.00695.x; Nakabayashi et al., 2018Nakabayashi Y, Aikawa T, Matsushita M, Hoshizaki K, 2018. Sampling design for efficient detection of pine wood nematode, Bursaphelenchus xylophilus, in diseased trees using a DNA detection kit: Variation across branch, trunk and tree. Nematology 20: 641-652. 10.1163/15685411-00003166). Sample humidity at 30 DAI ranged from 33% to 59 % and the number of PWNs per gram of dry wood was quite large in all samples, ranging from 221 to 2,718 nematodes.

In Experiment 2 involving samples collected from dead P. pinaster and P. radiata seedlings, the nematode density obtained from the Baermann funnel method was high in both pine species and for both B. xylophilus isolates, ranging from 636 to 3,311 nematodes per gram of dry wood. The kit detected PWNs in all samples, collected at 5 and 20 cm below the inoculation point in the two pine species. (Table 2).

In Experiment 3, none of the seedlings showed any disease symptoms at 15 DAI, when the first samples were taken (the first symptoms did not occur until 24 DAI). However, in the first sampling, PWNs were recovered from all samples and both sampled segments (Table 3; A, above; B, below the inoculation point) using the Baermann funnel method, with counts ranging from 126 to 7,589 nematodes per gram of dry wood. The number of nematodes was significantly higher in the segment above the inoculation point (χ²=5.83 p=0.0157). The LAMP kit detected B. xylophilus in only nine of the sixteen analysed samples.

On the second sampling date (45 DAI), all seedlings showed disease symptoms ranging from stage 2 to 6. The mean number of nematodes in the two segments had decreased compared to the first date and was slightly higher in the segment below the inoculation point (χ²=0.94 p=0.3329). This time, the LAMP kit detected B. xylophilus in all samples.

By the final sampling date (70 DAI), all seedlings had reached disease stage 7 and were dead (Table 3). In three of the sixteen samples, no PWNs were extracted via the Baermann funnel technique. The number of PWNs extracted was significantly lower than on the other sampling dates (15 vs 70 DAI, χ²=15.97 p<0.0001; 45 vs 70 DAI, χ²=8.87 p=0.0029). However, the LAMP kit did not detect PWNs in any of these samples (Table 3).

Contrary to the findings of other authors (Kanetani et al., 2011Kanetani S, Kikuchi T, Akiba M, et al., 2011. Detection of Bursaphelenchus xylophilus from old discs of dead Pinus armandii var. amamiana trees using a new detection kit. For Pathol 41: 387-391. 10.1111/j.1439-0329.2010.00695.x; Nakabayashi et al., 2018Nakabayashi Y, Aikawa T, Matsushita M, Hoshizaki K, 2018. Sampling design for efficient detection of pine wood nematode, Bursaphelenchus xylophilus, in diseased trees using a DNA detection kit: Variation across branch, trunk and tree. Nematology 20: 641-652. 10.1163/15685411-00003166), in our study, detection levels with the Baermann funnel technique were higher than with the LAMP detection kit. We also found higher detection levels from disease stages 2 to 6, whereas Nakabayashi et al. (2018Nakabayashi Y, Aikawa T, Matsushita M, Hoshizaki K, 2018. Sampling design for efficient detection of pine wood nematode, Bursaphelenchus xylophilus, in diseased trees using a DNA detection kit: Variation across branch, trunk and tree. Nematology 20: 641-652. 10.1163/15685411-00003166) found no effect of disease stage on detection level.

The lower detection levels observed using the LAMP kit with artificially inoculated wood chips and seedlings in our study could be attributed to differences in DNA extraction methods. When we quantified DNA on the last seedling sampling date, all but three samples (two of which had large numbers of other nematode species) were below the LAMP minimum DNA threshold of 51.4 pg/μl, as indicated by Meng et al. (2018Meng F, Wang X, Wang L, et al., 2018. A loop-mediated isothermal amplification-based method for detecting Bursaphelenchus xylophilus from Monochamus alternatus. For Pathol 48: 1-7. 10.1111/efp.12404). We hypothesize two main causes: either the buffer extraction was not maintained at -20ºC during shipping from Japan, resulting in deterioration, or that the method is unsuitable for species with high resin content, such as P. pinaster.

Several studies have highlighted the importance of appropriate sampling design to maximize detection (Kanetani et al., 2011Kanetani S, Kikuchi T, Akiba M, et al., 2011. Detection of Bursaphelenchus xylophilus from old discs of dead Pinus armandii var. amamiana trees using a new detection kit. For Pathol 41: 387-391. 10.1111/j.1439-0329.2010.00695.x; Nakabayashi et al., 2018Nakabayashi Y, Aikawa T, Matsushita M, Hoshizaki K, 2018. Sampling design for efficient detection of pine wood nematode, Bursaphelenchus xylophilus, in diseased trees using a DNA detection kit: Variation across branch, trunk and tree. Nematology 20: 641-652. 10.1163/15685411-00003166). However, we found differences between the two sampling points only on the first sampling date, although other authors have observed within-tree differences for PWN distribution in seedlings (Son et al., 2014Son J, Hogetsu, T, and Moon Y, 2014. The process of epithelial cell death in Pinus thunbergii caused by the pine wood nematode, Bursaphelenchus xylophilus. Nematology 16: 663-668. 10.1163/15685411-00002795). Our results might reflect the higher probability of collecting PWN-infected wood chips from a small seedling than from an adult tree, as the sample collected for the LAMP kit is approximately 0.12 g (Kikuchi et al., 2009Kikuchi T, Aikawa T, Oeda Y, et al., 2009. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology 99: 1365-1369. 10.1094/PHYTO-99-12-1365).

In summary, the LAMP kit successfully detected isolates of different origins in different pine species. However, despite its advantages as a simple method that does not require specialized knowledge or sophisticated equipment, we found it challenging to use in the field because the method requires hygiene and temperature conditions that are very difficult to attain in a natural forest environment. It should be noted that this method was performed with artificially inoculated Pinus seedlings and PWN-infected seedlings are not commonly found in nature. Additionally, when the number of nematodes extracted by Baermann was low in our study, the LAMP kit failed to detect them. This was likely due to a problem with DNA extraction.

Although LAMP technology continues to improve in terms of efficiency and interpretation of reactions (Meng et al., 2022Meng F, Liu Z, Li Y, Zhang X, 2022. Genes Encoding Potential Molecular Mimicry Proteins as the Specific Targets for Detecting Bursaphelenchus xylophilus in PCR and Loop-Mediated Isothermal Amplification Assays. Front Plant Sci 13: 890949. 10.3389/fpls.2022.890949; Wang et al., 2022Wang X, Wang LF, Cao YF, et al., 2022. Bursaphelenchus xylophilus detection and analysis system based on CRISPR - Cas12. Front Plant Sci 13: 1-11. 10.3389/fpls.2022.1075838), Nippon Gene is currently the only company that commercializes LAMP kits for detecting B. xylophilus. Further research is required to determine the causes of the DNA extraction issues and enhance stability regarding temperature conditions. Finally, and crucially, new surveillance techniques for managing the spread of the disease must also be developed and implemented.