Analysis of differential metabolites and metabolic pathways of mono- and inter- cropped wheat in response to the infection of Blumeria graminis f.sp. tritici
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摘要: 为了解单作、间作小麦响应白粉病侵染的代谢差异、揭示间作提高小麦抗白粉病生理机制, 本文通过盆栽试验设置 75 mg∙kg−1 (N1)、150 mg∙kg−1 (N2)、225 mg∙kg−1 (N3) 3个施氮水平, 研究接种白粉病病原菌后, 小麦单一种植和小麦蚕豆间作下白粉病的发病情况, 并通过广泛靶向代谢组学分析单作、间作小麦响应白粉病菌侵染的差异。结果表明: 氮水平和氮水平×种植模式显著影响小麦白粉病发病率和病情指数; 在3个氮水平下, 间作降低白粉病发病率25.54%~38.81%、降低病情指数20.11%~21.97%, 其中低氮水平控制效果较好。白粉病菌侵染后, 单作、间作小麦叶片中共检测到822种代谢产物, N1、N2和N3水平下分别发现差异代谢物69种、52种和88种。KEGG代谢通路分析发现单间作小麦差异代谢物主要富集在氨基酸的生物合成、代谢途径和次生代谢物的生物合成。其中N1和N2水平下, 差异代谢物富集在代谢途径, N1和N3水平下差异代谢物富集于氨基酸的生物合成。进一步对上调、下调差异倍数前10的代谢物分析发现, 与单作相比, N1水平间作上调了谷胱甘肽还原型、L-色氨酸、L-天冬酰胺和L-谷氨酰胺, N3水平间作上调了L-天冬酰胺、L-高甲硫氨酸和L-色氨酸。除此之外, 少数生物碱类、酚酸类和有机酸类等代谢物质在氮胁迫下也呈现不同程度的变化。总之, 单作和间作小麦响应白粉病病菌侵染的应答过程受氮水平调控。在白粉病病菌侵染中、间作调控差异代谢物如氨基酸及其衍生物类、生物碱类、酚酸类和有机酸类等在植物体内的变化, 可能是间作提高小麦白粉病抗性的机制之一。其中, 氮胁迫条件下间作调控氨基酸及其衍生物与小麦白粉病抗性提高密切相关。Abstract: Wheat and faba bean cropping could alleviate the occurrence and severity of wheat powdery mildew, however, the physiological mechanism of intercropping improved wheat diseases resitance was still unclear. The purpose of this study was to understand the metabolic difference of mono- and inter- cropped wheat in response to the infection of Blumeria graminis f.sp. tritici and to reveal the physiological mechanism of intercropping forimproving wheat resistance to powdery mildew. In this study, three nitrogen (N) application levels were set: 75 mg·kg−1 (N1), 150 mg·kg−1 (N2), and 225 mg·kg−1 (N3). After inoculated with the B. graminis f.sp. tritici, the occurrence of powdery mildew in mono- and inter- cropped wheat was investigated, and the metabolomic analysis of mono- and inter- cropped wheat in response to the infection of B. graminis f.sp. tritici were analyzed with UPLC-MS/MS by widely targeted metabolomics method. The results showed that N levels and N levels×Planting patterns significantly affected the incidence and disease index of wheat powdery mildew. Under all three N levels, wheat intercropped with faba bean averagely reduced the incidence of wheat powdery mildew by 25.54%−38.81% and decreased the disease index by 18.54%−20.11% relative to mono- cropped wheat (MW), and the intercropping control effect under N1 level was better than N2 and N3 conditions. A total of 822 differential metabolites were detected in the mono- and inter-cropped wheat leaves, of which 69, 52 and 88 differential metabolites were found at N1, N2 and N3 levels, respectively. Wheat and faba bean intercropping regulated flavonoids, alkaloids, amino acids and derivatives, and phenolic acids in wheat leaves as compared to MW. The KEGG pathway enrichment analysis from differential metabolites showed that the differential metabolites were mainly enriched in the biosynthesis of amino acids, metabolic pathways, and the biosynthesis of secondary metabolites. Among them, metabolites with significant differences were enriched in the metabolic pathways at N1 and N2 levels, and the metabolites with significant differences were enriched in the amino acid biosynthesis under N stress conditions (N1 and N3). Further analysis of the metabolites from the top 10 up- and down-regulation found that, intercropping up-regulated glutathione (G-SH), L-tryptophan, L-asparagine, and L-glutamine in wheat leaves at N1 level relative to MW, and up-regulated L-asparagine, L-homomethionine, and L-tryptophan in IW leaves were obsereved at N3 level as compared to MW. In addition, a few other metabolites including alkaloids, phenolic acids, and organic acids in wheat leaves were also regulated by intercropping when compared to MW under N1 and N3 levels. In conclusion, the response of wheat to powdery mildew infection was regulated by N levels. The metabolites involving amino acids and their derivatives, alkaloids, phenolic acids, and organic acid in wheat leaves were regualated by intercropping during the infection of B. graminis f.sp. tritici and induced different physiological reaction, which may be one of the mechanisms of intercropping improved wheat powdery mildew resistance. Particularly, intercropping regulated amino acids and their derivatives under N stress were tightly related to wheat powdery mildew resistance. The present study identified the different responses of mono- and inter- cropped wheat to diseases infection via metabolism analysis, which is helpful to fully understanding crop diversity for the management of pests and diseases.
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图 1 单间作小麦和施氮水平对小麦白粉病发生的影响
MW: 单作小麦; IW: 间作小麦; *表示同一氮水平同一时间不同种植模式下差异显著(P<0.05)。MW: monocropped wheat; IW: intercropped wheat; * indicates that there is significant difference between different planting patterns under the same N level at the same sampling time (P<0.05).
Figure 1. Effects of single room cropping and nitrogen application on powdery mildew occurrence in wheat
图 3 单作、间作小麦叶片代谢物正交偏最小二乘法-判别分析得分图(A、B、C)与置换模型检验图(D、E、F)
R2X、R2Y: 模型对X和Y矩阵的解释率; Q2: 模型的预测能力。R2X, R2Y: interpretation rate of the model for X and Y matrix; Q2: The predictive power of models.
Figure 3. Orthogonal Partial least squares method - discriminant analysis score map (A, B, C) and displacement model test map (D, E, F) of leaf metabolites in mono- and inter-cropped wheat
图 4 单间作小麦叶片差异代谢物Venn图
N1-MW VS N1-IW: N1水平下单作、间作小麦之间差异代谢物的比较; N2-MW VS N2-IW: N2水平下单作、间作小麦之间差异代谢物的比较; N3-MW VS N3-IW: N3水平下单作、间作小麦之间差异代谢物的比较。N1-MW VS N1-IW: comparison of differential metabolites between mono- and inter- cropped wheat under N1 level; N2-MW VS N2-IW: comparison of differential metabolites between mono- and inter- cropped wheat under N2 level; N3-MW VS N3-IW: comparison of differential metabolites between mono- and inter- cropped wheat under N3 level.
Figure 4. Venn diagram of differential metabolites in mono- and inter- cropped wheat leaves
图 5 单间作小麦在不同施氮水平的差异代谢物KEGG分类和富集图
图A、B、C分别表示N1、N2和N3水平下单作、间作小麦之间差异代谢物分类图和富集图。Panel A, B and C in the figure 5, the classification and enrichment of different metabolites between mono- and inter-cropped wheat at N1, N2 and N3 levels, respectively.
Figure 5. KEGG classification and enrichment of different metabolites in mono- and inter- cropped wheat under different N levels
图 6 单间作小麦在不同施氮水平的差异代谢物的层次聚类图
X轴中N1、N2、N3表示3个氮水平; MW1、MW2、MW3表示单作小麦的3个生物学重复; IW1、IW2、IW3表示间作小麦的3个生物学重复; Y轴表示差异代谢物名称。N1, N2, N3 represents three N levels in the X-axis respectively; MW1, MW2, MW3 represents three biological replications of monocropped wheat respectively; IW1, IW2, IW3 represents three biological replications of intercropped wheat respectively. The Y-axis shows the names of differential metabolites.
Figure 6. Hierarchical cluster diagram of different metabolites of mono- and inter- cropped wheat at different N levels
表 1 不同发病时期的氮水平、种植模式和氮水平×种植模式对发病率和病情指数影响
Table 1. Effects of nitrogen level, planting pattern and nitrogen level × planting pattern on incidence and disease index in different onset periods
接种后天数
Days after
inoculation (d)相关项
Related items氮水平
N levels
(N)种植模式
Cropping pattern
(C)N×C 3 DI * ns ns DSI ** ns * 4 DI ns ns ns DSI * ns ns 5 DI * ** ** DSI ns ** *** 6 DI * ns *** DSI * ns ** 7 DI * * *** DSI ** ns * 8 DI * ns ** DSI * ns ** 9 DI ** * *** DSI ** ns ** 10 DI * ns * DSI ns ns ns *: P<0.05; **: P<0.01; ***: P<0.001; ns: 不显著。DI: 发病率; DSI: 病情指数。*: P<0.05; **: P<0.01; ***: P<0.001; ns: not significant. DI: incidence rate; DSI: disease index. 表 2 差异代谢物分类
Table 2. Classification of differential metabolites
分类
ClassN1-MW VS N1-IW N2-MW VS N2-IW N3-MW VS N3-IW 上调
Up下调
Down总体
Total上调
Up下调
Down总体
Total上调
Up下调
Down总体
Total黄酮 Flavonoids 2 1 3 5 0 5 0 9 9 萜类 Terpenoids 0 0 0 1 0 1 1 0 1 有机酸 Organic acids 3 5 8 0 5 5 5 5 10 生物碱 Alkaloids 21 1 22 3 3 6 10 10 20 氨基酸及其衍生物 Amino acids and derivatives 14 1 15 1 5 6 11 0 11 酚酸 Phenolic acids 2 3 5 0 7 7 2 21 23 木脂素和香豆素 Lignans and Coumarins 2 0 2 0 2 2 0 3 3 脂质 Lipids 2 2 4 1 1 2 0 0 0 核苷酸及其衍生物 Nucleotides and derivatives 5 5 10 0 15 15 1 2 3 鞣质 Tannins 0 0 0 0 0 0 0 1 1 其他 Others 0 0 0 1 2 3 0 7 7 合计 Total 51 18 69 12 40 52 30 58 88 N1-MW VS N1-IW: N1水平下单作、间作小麦之间差异物的比较; N2-MW VS N2-IW: N2水平下单作、间作小麦之间差异物的比较; N3-MW VS N3-IW: N3水平下单作、间作小麦之间差异物的比较。N1-MW VS N1-IW: comparison of differential metabolites between mono- and inter- cropped wheat under N1 level; N2-MW VS N2-IW: comparison of differential metabolites between mono- and inter- cropped wheat under N2 level; N3-MW VS N3-IW: comparison of differential metabolites between mono- and inter- cropped wheat under N3 level. -
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