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马尾松感染松材线虫后的早期诊断指标筛选
郑哲, 李越, 陈凤毛, 李敏, 王梦瑶, 王立超
南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (6) : 81-88.
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马尾松感染松材线虫后的早期诊断指标筛选
Early diagnostic indicator screening after the infection of Pinus massoniana by Bursaphelenchus xylophilus
【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.
松材线虫病 / 松材线虫 / 拟松材线虫 / 马尾松 / 代谢组学 / 7-脱氢胆固醇 / 液相色谱法 / 病害检测
pine wilt disease / Bursaphelenchus xylophilus / B. mucronatus / Pinus massoniana / metabonomics / 7-dehydrocholesterol / liquid chromatography / disease detection
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20(S)-Hydroxyvitamin D3 (20(OH)D3) is an endogenous metabolite produced by the action of CYP11A1 on the side chain of vitamin D3 (D3). 20(OH)D3 can be further hydroxylated by CYP11A1, CYP27A1, CYP24A1 and/or CYP27B1 to several hydroxyderivatives. CYP11A1 also hydroxylates D3 to 22-monohydroxyvitamin D3 (22(OH)D3), which is detectable in the epidermis. 20-Hydroxy-7-dehydrocholesterol (20(OH)-7DHC) has been detected in the human epidermis and can be phototransformed into 20(OH)D3 following the absorption of ultraviolet B (UVB) energy by the B-ring. 20(OH)D3 and its hydroxyderivatives have anti-inflammatory, pro-differentiation and anti-proliferative effects, comparable to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Since cytochromes P450 with 20- or 25-hydroxylase activity are found in insects participating in ecdysone synthesis from 7-dehydrocholesterol (7DHC), we tested whether D3-hydroxyderivatives are present in honey, implying their production in bees. Honey was collected during summer in the Birmingham area of Alabama or purchased commercially and extracted and analyzed using LC-MS. We detected a clear peak of m/z = 423.324 [M + Na]+ for 20(OH)D3 corresponding to a concentration in honey of 256 ng/g. We also detected peaks of m/z = 383.331 [M + H − H2O]+ for 20(OH)-7DHC and 25(OH)D3 with retention times corresponding to the standards. We further detected species with m/z = 407.329 [M + Na]+ corresponding to the RT of 7DHC, D3 and lumisterol3 (L3). Similarly, peaks with m/z = 399.326 [M + H − H2O]+ were detected at the RT of 1,25(OH)2D3 and 1,20-dihydroxyvitamin D3 (1,20(OH)2D3). Species corresponding to 20-monohydroxylumisterol3 (20(OH)L3), 22-monohydroxyvitamin D3 (22(OH)D3), 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), 20,24/25/26-dihydroxyvitamin D3 (20,24/25/26(OH)2D3) and 1,20,23/24/25/26-trihydroxyvitamin D3 (1,20,23/24/25/26(OH)3D3) were not detectable above the background. In conclusion, the presence of 7DHC and D3 and of species corresponding to 20(OH)-7DHC, 20(OH)D3, 1,20(OH)2D3, 25(OH)D3 and 1,25(OH)2D3 in honey implies their production in bees, although the precise biochemistry and photochemistry of these processes remain to be defined.
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As the most severe forestry quarantine disease in several countries, pine wilt disease (PWD) causes substantial economic losses and poses a significant threat to the forest ecosystem. It is necessary to find a rapid and sensitive method for the early diagnosis of the disease to control the development of the disease effectively. This paper investigated the effect of Bursaphelenchus xylophilus (the pinewood nematode; PWN) on the chlorophyll fluorescence kinetic curve (OJIP curve) and the parameters of needles using four-year-old Pinus thunbergii as experimental materials and chlorophyll fluorescence analysis as a technical tool. It was shown by the results in the OJIP curve that the fluorescence intensity of the inoculated plants was significantly increased at points O and I. Additionally, the relative variable fluorescence intensity at points K and J was comparable to that of the control plants. Several chlorophyll fluorescence parameters of the treatment significantly increased or decreased with disease progression. At the same time, the control group had no significant changes in each parameter. Therefore, chlorophyll fluorescence parameters can be used as indicators for the early diagnosis of PWD, among which the DIo/RC parameter was the best. In summary, PWN invasion will produce fluorescence on the PSII of P. thunbergii, and its chlorophyll fluorescence parameters are expected to achieve early PWD diagnosis.
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