【目的】利用受不同线虫侵染的马尾松(Pinus massoniana)茎干部位代谢组,筛选出适合用于松材线虫病(pine wilt disease,PWD)早期诊断的代谢物指标,以有效提高对松材线虫病的防控。【方法】选择3种具有不同致病力的线虫:强毒力松材线虫(Bursaphelenchus xylophilus)虫株FCBX,强毒力拟松材线虫(Bursaphelenchus mucronatus)虫株BM7,无毒力拟松材线虫(Bursaphelenchus mucronatus)虫株FCBM,均于南京林业大学森林病理实验室分离和培养。将其接种至生长状态相似的4年生马尾松,以验证其对马尾松的毒力,空白对照(CK)接种无菌水。利用超高效液相色谱和串联质谱(ultra-high-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)测定接种3 d后马尾松茎干部位的代谢组分,利用主成分分析对代谢组进行质控,通过代谢物表达量差异P值和投影重要性(variable importance projection, VIP)值相结合的方法筛选出在松材线虫接种后特异性表达的代谢产物,并利用高效液相色谱仪(ultra-performance liquid chromatography, UPLC)对筛选出的特异性代谢物进行靶向检测和验证。【结果】马尾松茎干部位检测代谢产物的结果可靠,3种线虫对马尾松的毒力有明显差异。 7-脱氢胆固醇(7-dehydrocholesterol)是在接种不同线虫的马尾松代谢比较组中共同的代谢物,利用高效液相色谱法测定其在马尾松接种不同线虫3、7、21 d后的含量,该物质只在接种强毒力松材线虫株FCBX处理组中可以检测到,含量(质量分数)分别为4.09、66.77、28.8 μg/g,在无菌水对照处理组(CK)以及接种强毒力和无毒力两种拟松材线虫株BM7、FCBM处理组中无法检测到7-脱氢胆固醇的存在。【结论】在马尾松代谢组中筛选出的7-脱氢胆固醇,是马尾松被松材线虫早期侵染后的一种特异性代谢物,该特异性可以将松材线虫侵染与其他不同种线虫侵染的马尾松区分开,因此检测7-脱氢胆固醇含量可以用于松材线虫病的早期诊断。

【Objective】This study aims to identify suitable metabolites for the early diagnosis of pine wilt disease (PWD). To achieve this, the metabolome of stem segments from Pinus massoniana infected by different nematodes was analyzed, with the goal of improving the prevention and control of PWD.【Method】With four-year-old P. massoniana trees with similar growth characteristics were inoculated with three different nematode species, each with varying pathogenicity. These included the highly virulent Bursaphelenchus xylophilus (nematode strain FCBX), the highly virulent B. mucronatus (nematode strain BM7), and the non-virulent B. mucronatus (nematode strain FCBM). These nematodes were isolated and cultured in the Forest Pathology Laboratory of Nanjing Forestry University. Inoculation of P. massoniana trees with these nematodes were conducted under controlled conditions to assess their virulence. A sterile water control group (CK) was also included. Three days after injection, the metabolome of stem segments from P. massoniana was analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Principal component analysis (PCA) was used to ensure quality control of the metabolomic data. Metabolites with specific expression following B. xylophilus inoculation were identified through a combination of P-values and variable importance in projection (VIP) scores. Targeted detection and validation of these metabolites were conducted using ultra-performance liquid chromatography (UPLC).【Result】The three nematode species exhibited significant differences in virulence toward P. massoniana. Metabolomic analysis of stem segments revealed reliable results. One key finding was that 7-dehydrocholesterol was a common differential metabolite across the comparative metabolomes of P. massoniana inoculated with different nematodes. Levels of this compound were measured using UPLC on the 3rd, 7th, and 21st days after inoculation. It was detectable only in the group inoculated with the highly virulent B. xylophilus (nematode strain FCBX), with concentrations of 4.09, 66.77, and 28.8 μg/g, respectively. 7-dehydrocholesterol was undetectable in the sterile water control group (CK), as well as in the groups treated with either highly virulent B. mucronatus (nematode strain BM7) or non-virulent B. mucronatus (nematode strain FCBM).【Conclusion】7-dehydrocholesterol, identified through the metabolomic profiling of P. massoniana, was found to be a specific metabolite following early infection by B. xylophilus. This metabolite's specificity allows it to distinguish B. xylophilus infection from infections caused by other nematodes. Therefore, the detection of 7-dehydrocholesterol levels could serve as an effective tool for the early diagnosis of pine wilt disease.