Pine is among the most popular timber species worldwide. Its ecological, economic, and social benefits are internationally recognized. Additionally, pine, which is an integral component of many products influencing human lifestyles, is a major forestation species in China (Jones et al., 2008; Soliman et al., 2012). However, it is susceptible to pine wilt disease (PWD) caused by the pine wood nematode Bursaphelenchus xylophilus,(Nickle et al., 1981), which poses a major threat to pine forests (Mamiya and Enda, 1972; Mota et al., 1999; Futai, 2013).

As a global forest quarantine disease, PWD can adversely affect forest ecologies and prevent sustainable timber production in the Americas, Europe, and Asia. Bursaphelenchus xylophilus is native to North America, but it was introduced to Japan in 1905, China in 1982, South Korea in 1988, Portugal in 1999, Spain in 2008, and the Madeira islands in 2010. After it was introduced to Europe, it spread to the Mediterranean coast (Futai, 2013). Since the first outbreak of PWD in China in 1982 (Cheng et al., 1983), the disease has been detected in 726 county-level administrative regions in 19 provinces. In the winter of 2017, PWD was detected in Fushun, Liaoning province, where the annual average temperature is 8.5°C referring to China Meteorological Administration. Almost 100,000 hectares were affected by PWD, resulting in the deaths of more than 50 million pine trees and economic losses exceeding 25 billion RMB. Hence, strengthening the quarantine and preventing the spread of PWD are the primary objectives of PWD disease management program.

The beetle species Monochamus alternatus and M. saltuarius are the principal vectors of B. xylophilus in Asia. These insects carry the dispersal fourth-stage juveniles of B. xylophilus and other nematodes (Ryss and Subbotin, 2017; Kanzaki and Giblin-Davis, 2018). Therefore, reliably discriminating B. xylophilus from other nematodes according to their morphological characteristics is a difficult task. Examining M. alternatus and M. saltuarius for the presence of B. xylophilus juveniles is an important part of an effective disease control strategy. More specifically, accurately identifying the juvenile stages is crucial for halting the spread of PWD.

Nematode species are routinely identified on the basis of their morphological characteristics. More specifically, B. xylophilus is typically identified according to the morphological characteristics of the adult-stage nematode; however, this requires a certain level of expertise and experience. Moreover, it is impossible to accurately identify the larvae. The limitations to morphology-based methods for identifying B. xylophilus have necessitated the development of methods relying on immunological, physiological, biochemical, and genetic characteristics. The current accepted methods for detecting and identifying B. xylophilus primarily involve molecular biology techniques. For example, the PCR(Wang et al., 2009) and loop-mediated isothermal amplification (LAMP) are commonly used detection techniques.

Regarding PCR, which enables the rapid amplification of DNA, two primers specific for the target DNA must be designed. The target sequence is then amplified by DNA polymerase (e.g., Taq DNA polymerase) in three steps (denaturation, renaturation, and extension). The amplified sequence serves as a template for the next cycle. Because each cycle takes 2–4 min to complete, the target sequence can be amplified several million times in 2–3 h. Because of it, the PCR technique is mature for clinical diagnoses and quarantine. The LAMP technique, which can be used to rapidly amplify DNA, requires four primers specific for six regions of the target DNA. The target sequence is then amplified by Bst DNA polymerase at a constant temperature between 60 and 65°C. As a rapid, simple, specific, sensitive, and low-cost technique, LAMP has been exploited for research regarding disease detection and gene chip development (Tomita et al., 2008).

In this study, we developed PCR- and LAMP-based methods for the direct detection of B. xylophilus. The proposed PCR and LAMP techniques use the species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins, including thaumatin-like protein-1 (Bx-tlp-1; accession number KM063438.1), thaumatin-like protein-2 (Bx-tlp-2; accession number MK000287), and a cysteine proteinase inhibitor (Bx-cpi; accession number MK000288). Shinya et al. (2013) performed a proteomic analysis and identified two putative B. xylophilus thaumatin-like proteins (TLPs) and one cysteine proteinase inhibitor with sequences that were highly similar to those of plant proteins. These proteins were subsequently determined to induce cell death in Nicotiana benthamiana (Kirino et al., 2020). Moreover, the TLP sequences in B. xylophilus and Pinus massoniana are reportedly similar (Wang et al., 2014). Additionally, Meng et al. (2017) cloned the gene encoding a P. massoniana TLP (Pm-tlp) and revealed that its expression is associated with the expression of the B. xylophilus Bx-tlp-1. The relatively high sequence similarity between potential molecular mimicry proteins and plant proteins suggests that they may have similar functions. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees indicated that the temporal changes to the α-pinene content in the trees are consistent with the expression levels of the genes encoding a TLP (CPI) in B. xylophilus and P. massoniana. Thus, these genes are likely important for B. xylophilus infections of pine species (Meng et al., 2017, 2020).

Because the potential molecular mimicry proteins are associated with B. xylophilus pathogenicity, we designed primers specific for the B. xylophilus genome, which may be useful for developing new disease prevention and control measures.

The causative agent of PWD, B. xylophilus, is a prevalent organism carried by beetles (i.e., M. alternatus or M. saltuarius). Therefore, the numbers of beetles (M. alternatus or M. saltuarius) in a pine forest is a crucial indicator of the possibility of a PWD outbreak. Unless B. xylophilus from M. alternatus or M. saltuarius is specifically treated in the primary phase, the resulting PWD will often lead to tree death. The efficient and rapid detection of B. xylophilus from M. alternatus or M. saltuarius is necessary for preventing outbreaks via the implementation of specific treatments (Wang et al., 2009; Cardoso et al., 2012). Because death by PWD is very rapid, a novel method for detecting PWD must produce results quickly.

Proteins secreted by pathogens that are structurally or functionally similar to host defense-related proteins are called molecular mimicry proteins. Researchers identified substances released by pathogens that mimic plant defense-related compounds and disrupt physiologically important plant signaling pathways (Winn et al., 2021). Previous studies revealed the relatively high similarity between the Bx-TLP (Bx-CPI) and Pm-TLP (Pm-CPI) sequences, indicative of a similarity in their functions (Wang et al., 2014; Meng et al., 2017, 2020, 2022). Moreover, the expression of B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi) is responsive to α-pinene, which may affect terpene metabolism in pine trees and influence the pathogenicity of B. xylophilus (Meng et al., 2019, 2020, 2022).

In the present study, we designed specific primer sets targeting Bx-tlp-1, Bx-tlp-2, and Bx-cpi for the rapid detection of B. xylophilus and M. alternatus or M. saltuarius carrying B. xylophilus. The PCR and LAMP assays specifically detected all B. xylophilus isolates, with no amplification for all other examined species. The high specificity of the LAMP assay was conferred by the six primers targeting six regions of the Bx-tlp-1 and Bx-cpi sequences, which were specific to B. xylophilus. The specificity of the LAMP primers used in this work was significantly better than that reported by Kikuchi et al. (2009; Supplementary Table S2), who detected B. mucronatus and B. fraudulentus in 90 min. On the other hand, there were no amplified products in other Bursaphelenchus group species, such as B. firmae, B. luxuriosae, and B. sexdentati. Meanwhile, the average concentration of genomic DNA from a single B. xylophilus was about 32 ng/μl (the volume was 10 μl), and the lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively. Moreover, LAMP primers targeting Bx-tlp-1 or Bx-cpi were 5-fold more sensitive than the LAMP primers targeting syg-2 (51.4 pg; Meng et al., 2018) and were able to rapidly detect B. xylophilus from M. alternatus or M. saltuarius. Thus, the key genes associated with B. xylophilus pathogenicity may be excellent targets for the rapid detection of the nematode because they may also be related to the fitness of the hosts, insects, and environment.

Considering the high sensitivity of the LAMP assay, the post-amplification procedures should be performed in a separate room (i.e., away from the PCR and LAMP reagents) to minimize the possibility of contamination (Kikuchi et al., 2009; Zhu et al., 2009; Meng et al., 2018). The LAMP assay requires only a regular water bath or a heat block that maintains the temperature at 63°C. Therefore, it is more cost-effective than a conventional PCR assay. Moreover, the two additional loop primers increase the speed and specificity of the amplification, resulting in faster reactions (relative to a conventional PCR). Additionally, the key genes associated with pathogenicity (e.g., those encoding potential molecular mimicry proteins) can be used as the specific targets for detection, which may be significant for preventing or controlling disease outbreaks. Our LAMP-based method detected B. xylophilus Bx-tlp-1 within 20 min and B. xylophilus from M. alternatus or M. saltuarius within 30 min. These results are better than the LAMP assay results obtained by Meng et al. (2018) who detected the B. xylophilus target gene within 25 min and B. xylophilus from M. alternatus within 50 min. Furthermore, in contrast to a conventional PCR assay, the LAMP assay enables the detection of a positive amplification by the naked eye. The reactions and results can be interpreted simply by observing the color change in the reaction mixture. In addition to eliminating the need for the time-consuming electrophoretic analysis, LAMP assays can be performed without the sophisticated equipment needed for PCR (Kang et al., 2009; Huang et al., 2010; Hu et al., 2011; Kanetani et al., 2011; Meng et al., 2018).

In conclusion, M. alternatus is the main insect vector for B. xylophilus. The number of nematodes in M. alternatus is an important factor related to the distribution of B. xylophilus. Therefore, the fast and efficient detection of M. alternatus carrying B. xylophilus is necessary for the monitoring and early detection of PWD (Kikuchi et al., 2009; Hu et al., 2011; Cardoso et al., 2012; Meng et al., 2018). In this study, we designed PCR and LAMP primers specific for the B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi). The specific amplified fragments for Bx-tlp-1, Bx-tlp-2, and Bx-cpi were 755, 241, and 202 bp, respectively. However, no specific amplification products were detected for the other analyzed nematodes (e.g., B. mucronatus). These findings indicate that Bx-tlp-1, Bx-tlp-2, and Bx-cpi can be used to specifically detect B. xylophilus as well as M. alternatus or M. saltuarius carrying B. xylophilus. Furthermore, designing primers specific for B. xylophilus following an in-depth analysis of the functions of key pathogenicity genes may have important implications for future attempts at developing reliable methods for the early diagnosis and prevention of PWD.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

FM, YL, and XZ: experimental design. FM and ZL: experimental implementation. ZL: material contribution. FM: data analysis and manuscript writing. All authors contributed to the article and approved the submitted version.

The present research was funded by the Fundamental Research Funds for the Central Universities (2021ZY03) and China Postdoctoral Science Foundation (2020M680410).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.