草酸青霉C11对地黄生长的影响及其代谢产物分析

doi:10.17521/cjpe.2022.0439

摘要/Abstract

摘要：

分离对地黄(Rehmannia glutinosa)生长有害的菌株并解析其代谢产物对地黄生长的影响, 可为破解地黄连作障碍问题提供理论支持。从地黄重茬土壤中分离霉菌并研究其发酵液对地黄生长的影响, 然后通过不同极性梯度萃取、硅胶柱层析方法分离对地黄危害最严重的代谢产物, 并通过高效液相色谱和液相色谱-质谱联用技术鉴定分离出的物质。结果：筛选出1株草酸青霉(Penicillium oxalicum)菌株C11, 分离鉴定到其有害化感物质是环(精-亮)二肽(分子量为269.347, 分子式为: C₁₂H₂₃N₅O₂)。结果表明草酸青霉C11抑制了地黄产量41.5%, 其有毒代谢产物环(精-亮)二肽可能是造成地黄连作障碍的原因。

关键词: 地黄, 连作障碍, 霉菌, 草酸青霉

Abstract:

Aims The aim of this study was to isolate the harmful strain and analyze the effects of its metabolites on Rehmannia glutinosa, which provided theoretical support for solving R. glutinosa continuous cropping obstacles.

Methods The fungi were isolated from continuous cropping soil. The fungal fermentation liquid metabolites were separated using different polarity gradient extraction and silica gel column chromatography. Metabolites were determined by high performance liquid chromatography and liquid chromatography-mass spectrometry.

Important findings A strain of Penicillium oxalicum C11 was isolated from continuous cropping soil. The toxic metabolite of P. oxalicum C11 was identified as cyclo (L-Arg-L-Leu) dipeptide (molecular weight: 269.347, molecular formula: C₁₂H₂₃N₅O₂). Results indicated P. oxalicum C11 inhibited R. glutinosa yield by 41.5%, suggesting that the toxic metabolite L-Arg-L-Leu could be vital for R. glutinosacontinuous cropping obstacles.

Key words: Rehmannia glutinosa, continuous cropping obstacles, mold, Penicillium oxalicum

郝尚华, 罗梦香, 曹宏丽, 张森, 王明道. 草酸青霉C11对地黄生长的影响及其代谢产物分析. 植物生态学报, 2024, 48(6): 809-816. DOI: 10.17521/cjpe.2022.0439

HAO Shang-Hua, LUO Meng-Xiang, CAO Hong-Li, ZHANG Sen, WANG Ming-Dao. Effects of Penicillium oxalicum C11 on Rehmannia glutinosa growth and its metabolites analysis. Chinese Journal of Plant Ecology, 2024, 48(6): 809-816. DOI: 10.17521/cjpe.2022.0439

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链接本文: https://www.plant-ecology.com/CN/10.17521/cjpe.2022.0439

https://www.plant-ecology.com/CN/Y2024/V48/I6/809

图/表 4

表1 高效液相色谱(HPLC)及电喷雾液相色谱-质谱联用(LC-MS)相关参数和设置

Table 1 Parameters and settings of high-performance liquid chromatography (HPLC) and electrospray liquid chromatography-mass spectrometry (LC-MS)

HPLC	LC-MS
色谱柱 Chromatographic column: ACQUITY UPLC HSS T3	色谱柱 Chromatographic column: ACQUITY UPLC HSS T3
孔径 Aperture: 5 μm	进样量 Sample quantity: 2 μL
进样量 Sample quantity: 15 μL	流速 Velocity: 0.5 mL·min^-1
流动相: A, 0.1%甲酸乙腈; B, 纯水	柱温 Column temperature: 30 ℃
Mobile phase: A, 0.1% formic acid acetonitrile; B, pure water	流通池路径长度 Flow pool path length: 10.000 mm
流速 Velocity: 0.5 mL·min^-1	流通池体积 Flow cell volume: 0.500 μL
柱温 Column temperature: 40 ℃	扫描范围 Scan range: 50-1500 m/z
检测波长 Detection wavelength: 500 nm	低压极限 Low pressure limit: 0 pounds per square inch
进样时间 Sample time: 16.5 min	高压极限 High pressure limit: 15 000 pounds per square inch

图1 有害菌筛选和草酸青霉菌株C11对地黄生长的影响(平均值±标准差)。 A, 地黄苗形态。B, 地黄根质量。C, 地黄根长。D, C11菌株发酵液对地黄愈伤组织的影响。E, 盆栽地黄质量。F, 不同处理的地黄形态。从左至右分别为第一茬地黄、菌株C11处理地黄、连作地黄。G, 田间地黄质量。CK, 对照; C7、C11、F2、F9、F11, 霉菌菌株。不同小写字母表示差异显著(p < 0.05)。**, p < 0.01; ***, p < 0.001。

Fig. 1 Screening of harmful strains and effects of Penicillium oxalate C11 strain on the growth of Rehmannia glutinosa (mean ± SD). A, Seedling morphology of R. glutinosa. B, Mass of R. glutinosa root. C, Length of R. glutinosa root. D, Effect of fermentation broth of C11 strain on callus of R. glutinosa. E, Quality of potted R. glutinosa. F, R. glutinosa morphology of different treatments. They are first crop R. glutinosa, C11 strain treated R. glutinosa, and continuous crop R. glutinosa, respectively, from left to right. G, Mass of field R. glutinosa. CK, control; C7, C11, F2, F9, F11, strains. Different lowercase letters indicate significant differences (p < 0.05). ** p < 0.01; ***, p < 0.001.

图2 草酸青霉菌株C11形态和系统发育树。 A, C11菌落正面。B, C11菌落背面。C, C11菌丝及孢子形态。D, 系统发育树。

Fig. 2 Penicillium oxalicum C11 strain morphology and phylogenetic tree. A, Positive side of C11 colony. B, Reverse side of C11 colony. C, C11 mycelium and spore morphology. D, Phylogenetic tree.

图3 草酸青霉菌株C11有害代谢产物的分离及鉴定。 A, 不同萃取相生物检测结果(平均值±标准误)。B, 萃取水相51-59收集管生物检测结果(平均值±标准误)。C, 地黄苗生物检测结果统计(平均值±标准误)。D, 物质峰的一级质谱图。E, 物质峰的二级质谱图。F, 纯物质与54样品管液相图。CK, 对照。不同小写字母表示差异显著(p < 0.05)。

Fig. 3 Biomeasurement results of different polar extraction phases and silica gel column chromatography of Penicillium oxalicum fermentation broth. A, Bioassay results of different phases (mean ± SE). B, Bioassay results of collected pipes with water phases 51-59 (mean ± SE). C, Statistics of biological test results of R. glutinosa seedlings (mean ± SE). D, First-order mass spectrogram of the material peak. E, Secondary mass spectrogram of the material peak. F, Liquid phase diagram of pure substance and 54 sample tube. CK, control. Different lowercase letters indicate significant differences (p < 0.05).

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