Bursaphelenchus xylophilus in Pinus sylvestris—The First Report in Europe

Abstract

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is considered an A2 quarantine organism by the European Plant Protection Organisation. In Europe, this nematode has been reported in Pinus pinaster, P. radiata, and P. nigra. In May 2024, severe wilting symptoms were observed in P. sylvestris trees at Serra da Lousã (Coimbra, the central area of continental Portugal). Wood samples were collected from six wilted trees, and the presence of PWN was investigated. From these, B. xylophilus specimens were detected in five out of the six trees. Species identification was performed based on species-specific morphological diagnostic characters, and this was confirmed by real-time PCR using species-specific primers targeting the B. xylophilus satellite DNA region. This study presents the first detection of B. xylophilus in P. sylvestris in Portugal and in Europe.

1. Introduction

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970, an A2 quarantine organism according to the European Plant Protection Organisation (EPPO) is the causal agent of pine wilt disease (PWD) and is a major pathogen of conifers, which has negative impacts on forest health, natural ecosystem stability and on international wood trade. Species belonging to the genus Pinus are the main PWN hosts, and PWN transmission is carried out by insects, mostly belonging to the genus Monochamus (Coleoptera-Cerambycidae) [1]. Pinus mugo Turra, 1764 (dwarf mountain pine), P. nigra J.F. Arnold, 1785 (black pine), P. pinaster Aiton, 1789 (maritime pine) and P. sylvestris Linnaeus, 1753 (Scots pine) are among the most susceptible European Pinus species [2]. The P. sylvestris species are considered at risk in the northern and central areas of the EPPO region, whereas P. mugo, P. nigra, and P. pinaster are threatened in central and southern areas [1].

Bursaphelenchus xylophilus is a native species from North America, and it spread to Japan in the early XXth century and then into China, Korea, and Taiwan [1]. In Europe, the first detection was in 1999, in Continental Portugal, associated with P. pinaster [3]; in 2008, it was detected for the first time in Spain [4,5]; and in 2009, in Madeira Island in P. pinaster [6].

Following the detection of PWN in Portugal and in Europe, strict measures and control actions were immediately designed and implemented to prevent further PWN dispersal into other European countries. According to national and European Union rules, thousands of Pinus spp. wood samples have been collected and analyzed. Since then, the PWN was also reported in P. radiata D. Don, 1836 in Spain [7] and in P. nigra, in Portugal [8].

The Scots pine (P. sylvestris) is the most widespread species of the Pinus genus in the world and is native to Eurasia. It occupies naturally a territorial range from the Iberian Peninsula in the west to the far east of Russia [9,10]. It grows at a wide range of altitudes, from sea level to high mountains, in different soils, and in areas with different mean annual precipitation levels and extreme temperatures. Portugal represents the outermost western limit of Scots pine’s natural range, and this species appears naturally in forest areas of high-altitude mountains from the north and central regions of Portugal (Marão, Peneda-Gerês, Lousã, Estrela) [11].

In the United States of America (USA), Canada, and Japan, B. xylophilus was already detected in P. sylvestris declining trees [12,13,14,15,16,17]. In Europe, to date, several field surveys have been conducted to determine the incidence and distribution of Bursaphelenchus species in P. sylvestris stands. Although there were no records of PWN in P. sylvestris, several other species, including B. mucronatus Mamiya & Enda, 1979 of the Xylophilus group, have been found associated with trees showing different degrees of decline [17,18,19,20]. This study has a main objective to assess the presence of B. xylophilus in P. sylvestris trees displaying wilting symptoms at Serra da Lousã, Portugal, using species-specific morphological diagnostic characters and real-time PCR.

2. Materials and Methods

2.1. Wood Sampling and Nematode Extraction

Six P. sylvestris trees displaying severe wilting symptoms, without needles, were sampled for the presence of PWN at Serra da Lousã, a mountain located at the south-western extremity of the European Central Cordillera with 15,158 ha and with 1204 m at the highest point [21]. Wood samples (one sample per tree) were collected at different points of the main trunk, at breast height, using a low-speed drill, and kept in plastic bags until nematode extraction. Nematodes were extracted by the tray method [22]. After 48 h, nematodes were collected, handpicked for morphological and molecular studies, and transferred to the fungus Botrytis cinerea Persoon, 1794 grown at 25 °C on malt extract agar medium (BD Difco, Franklin Lakes, NJ, USA) to obtain a nematode isolate [23].

2.2. Morphological Characterization

Females and males extracted from the wood samples were killed by heat in a drop of water on a cavity glass slide, mounted in water, immediately photographed, and morphologically characterized based on the species-specific diagnostic characters [17,24,25]. Photographs were taken with a Leica DM 2500 bright field light microscope using a Leica DFC 450 digital camera (Wetzlar, Germany).

2.3. Real-Time PCR

DNA from five nematodes collected from each wood sample was extracted by a simplified procedure described by [26] and included in EPPO standard diagnostic PM 7/4 (4) Bursaphelenchus xylophilus [25]. Real-time PCR reactions were performed using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in 20 µL reactions containing 5 µL of extracted genomic DNA, 10 µL of 2× NZYSpeedy qPCR Probe Master Mix (NZYtech, Lisbon, Portugal) and 200 nM of each primer and probe. Primers BSatF (5′-TGACGGAGTGAATTGACAAGACA-3′) and BSatRV (5′-AAGCTGAAACTTGCCATGCTAAA-3′) and the fluorogenic TaqMan probe BSatS (5′-ACACCATTCGAAAGCTAATCGCCTGAGA-3′) were designed by [27] for B. xylophilus species-specific detection based on the determined sequence of B. xylophilus satellite DNA and were used in this assay for the specific detection of PWN by real-time PCR, as recommended by EPPO [25]. Thermal cycling conditions consisted of 3 min at 95 °C, followed by 30 cycles of 5 s at 95 °C and 30 s at 60 °C. Data were analyzed using Bio-Rad CFX Manager 3.1 software, according to the manufacturer′s instructions. Ct values were calculated by the software by determining the PCR cycle number at which the reporter fluorescence surpassed the background. The following controls included (i) negative isolation control (NIC) to monitor contaminations during DNA extraction—DNA extracted from five nematodes isolated from wood samples belonging to other families; (ii) positive isolation control (PIC) to ensure that DNA of sufficient quantity and quality is isolated—DNA extracted from five cultured B. xylophilus nematodes (isolate BxPt17AS); (iii) negative amplification control (NAC) to discard false positives due to contamination during reaction mix preparation—with molecular grade water used to prepare reaction mixs; and (iv) positive amplification control (PAC) to monitor the efficiency of the amplification—DNA previously extracted from cultured B. xylophilus (isolate BxPt17AS). Triplicate reactions for each control and sample were performed.

3. Results

Morphologically, the presence of B. xylophilus was detected in five out of the six wood samples collected from P. sylvestris trees. In trees 3 and 6, different developmental stages were detected (adults, propagative, and JIII dispersive juvenile stages) (

Table 1. Number of Bursaphelenchus xylophilus and other nematodes/100 g of wood collected from six Pinus sylvestris trees in Serra da Lousã (Coimbra, Portugal).

P. sylvestris	Wood Sample (g)	B. xylophilus/100 g			Other Nematodes/100 g
		Juveniles	Females	Males
Tree 1	140	3 *	----	----	185
Tree 2	145	----	----	----	131
Tree 3	134	212 **	345	370	376
Tree 4	136	4 *	----	----	52
Tree 5	133	5 *	----	----	79
Tree 6	139	91 **	29	28	286

* JIII dispersive juvenile stages; ** propagative and JIII dispersive juvenile stages.

). The males (Figure 1A–D) and females (Figure 1E–G) exhibited the main characteristics of B. xylophilus [17,24,25]: all specimens displayed a high cephalic region offset by a constriction, with six lips; a stylet with weakly developed basal knobs; a large medium bulb (Figure 1B,C); and an excretory pore either at or behind the median bulb (Figure 1C). Males (Figure 1A) had a strongly curved ventral tail, conoid, with a small terminal bursa, which could be seen in the dorso-ventral position, long spicules, a capitulum flattened with small condylus and a distinct rostrum, with a disc-like projection (cucullus) at the distal extremity (Figure 1D). Females presented a long post-uterine sac and vulva, usually at 70%–80% of the body length; a distinct vulval flap (Figure 1F); and a rounded tail with the absence of a mucro was observed (Figure 1G).

In trees 1, 4, and 5, only JIII dispersive juvenile stages were detected. In all samples, other nematodes (bacterivores and fungivores) belonging to the orders of Rhabditida and Panagrolaimida [28] were detected (Table 1).

Real-time PCR amplifications (Figure 2) confirmed the morphological identification. Genomic DNA from the five nematodes extracted from trees 1, 3, 4, 5, and 6 was specifically amplified as in the positive controls (PIC and PAC), while fluorescence remained below the threshold values for the negative controls (NIC and NAC) (Figure 2).

4. Discussion

This study represents the first report of PWN, an A2 EPPO quarantine organism, in P. sylvestris in Europe’s natural environment. The presence of the PWN in Europe exemplifies the severe environmental and economic impacts that an invasive forest organism can have on pine forests and forest-based industries. The detection of PWN in Portugal has prompted severe control measures on the movement of susceptible wood products and coniferous wood within the EU. To prevent the introduction of PWN and its spread to new areas, Portugal has faced strict restrictions on the movement of plants, woody material, and forest products and implemented control and management strategies in forested areas, including (i) prospection and field surveys; (ii) the eradication of infected trees (iii) the management of tree decline and monitoring of the insect vector and other agents of decline; (iv) insect trap installation; and (v) the treatment and processing of host coniferous products [29]. To date, in Europe (Portugal and Spain), the PWN has been reported in P. pinaster, P. radiata, and P. nigra [3,4,5,6,7,8].

Scots pine is one of the most commercially important species, not only in Central Europe but also across Eurasia. The wood is workable and is one of the strongest softwoods. It is widely used in building and construction, furniture, pulp, and paper. The wood is durable in wet conditions, making it historically valuable for mining props, waterwheels, and piles. Additionally, Scots pine is often employed in land reclamation and for stabilizing loose sand due to its tolerance for poor soils [10,30].

The PWN detection in P. sylvestris within the context of climate change presents an ecological and economic threat to European forests due to the high susceptibility of this pine species to the PWN and due to its great distribution in European territories. Warmer temperatures and altered precipitation create more favorable conditions for PWN dissemination. In Europe, under current climate conditions, only 4.7% of P. sylvestris habitats are predicted to be at risk of being infected by the PWN, but by 2070, this percentage will rise to 61.9% on average, according to a predicted climate change scenario [31].

The high susceptibility of this pine species to the PWN has been confirmed in several research studies under artificial conditions [32,33,34,35,36,37]. Moreover, injured roots and stems of P. sylvestris seedlings could be infected with the PWN through nematode-infected sawdust [38], and PWN can persist for at least six years in 20-year-old inoculated P. sylvestris trees [39]. Concerning the insect vector, in Europe, Monochamus galloprovincialis (Olivier, 1795) is the main vector, and the most important hosts of M. galloprovincialis are pines, mainly P. sylvestris, P. nigra, P. halepensis Mill., 1768, and P. pinaster [1,40]. In Portugal, P. pinaster seems to be the favorite host for M. galloprovincialis; however, studies on feeding, oviposition, and attractive volatiles revealed that P. sylvestris can also be a good host for the insect vector [40,41,42,43].

5. Conclusions

This study presents the first detection of PWN in P. sylvestris in Portugal and in Europe. Species identification was based on species-specific morphological diagnostic characters and on real-time PCR, using species-specific primers targeting the B. xylophilus satellite DNA region. This study also adds valuable information to the current situation of the PWN in the EPPO zone, underlines the need for comprehensive PWN surveys across various pine species in Europe, and highlights the need for the implementation of integrated preventive measures by European countries to prevent the spread of PWN to uninfected areas where P. sylvestris is predominant, especially considering the challenges posed by climate change scenarios.