基于气相色谱-质谱法检测禾谷镰孢菌丝胞内外的脱氧雪腐镰刀菌烯醇和15-乙酰基脱氧雪腐镰刀菌烯醇

Detection of Intracellular and Extracellular Deoxynivalenol and 15-Acetyl Deoxynivalenol of Fusarium graminearum by GC-MS

禾谷镰孢(Fusarium graminearum)是引起小麦赤霉病的主要病原真菌,不仅会造成严重的作物减产,还会产生脱氧雪腐镰刀菌烯醇(DON)等真菌毒素污染谷物,威胁人畜健康。为探究禾谷镰孢产毒菌丝胞内外的毒素含量,本文建立了气相色谱-质谱联用法同时检测DON和15-乙酰基脱氧雪腐镰刀菌烯醇(15-ADON)。通过质谱特征扫描,DON和15-ADON均获得了较高离子化效应的离子。在单离子检测(SIM)模式下,利用m/z295、235和193离子定性分析DON,m/z 392、235和193离子定性分析15-ADON。选择其中响应强度最高的m/z 235离子定量分析DON,m/z 193离子定量分析15-ADON。利用该方法检测禾谷镰孢野生型PH-1和DON合成缺陷突变体tri5胞内外的DON和15-ADON含量,PH-1胞内的DON和15-ADON含量分别为(149.13±9.15)μg/g和(1833.31±185.33)μg/g,胞外含量分别为(5910.35±468.23)μg/g和(45222.12±2726.81)μg/g;tri5突变体的胞内外均未检测到DON和15-ADON。该方法可用于菌丝胞内外DON和15-ADON的同时分析。

Fusarium graminearum is the main pathogenic fungus causing wheat head blight.It not only causes severe crop yield reduction,but also produces fungal toxins such as deoxynivalenol(DON)and 15-acetyl deoxynivalenol(15-ADON),which are dangerous food pollutants to people and livestock.For the genetic functional study of Fusarium graminearum,it is often necessary to culture mycelium on LTB medium in the laboratory to investigate its regulatory effect on DON and 15-ADON synthesis.However,the detection of DON and 15-ADON in the mycelium is focused on extracellular,with less focus on the detection of intracellular.Simultaneous detection of intracellular and extracellular DON and 15-ADON can provide more in-depth information of fungal toxin production and secretion functions.In the study,a method based on gas chromatography-mass spectrometry(GC-MS)for simultaneous detection of DON and 15-ADON was developed.The solvents for extracting DON and 15-ADON can be completely separated and the calibration curves of each extract show good linear relationship within a certain range.The mass spectrometric characteristic scan shows high ionization efficiency for both DON and 15-ADON.The retention time of DON and 15-ADON in chromatogram are 6.98 and 7.68 min,respectively.Qualitative analysis of DON was performed using m/z 295,235,and 193 under single ion monitor(SIM)mode,while m/z 392,235,and 193 were used for 15-ADON.The highest intensity ion m/z 235 was selected for the quantification of DON,and m/z 193 for the quantification of 15-ADON.This method was used to detect the intracellular and extracellular levels of DON and 15-ADON in wild-type PH-1 and tri5 mutant with DON biosynthesis deficiency.The intracellular DON and 15-ADON levels in PH-1 are determined to be(149.13±9.15)μg/g and(1833.31±185.33)μg/g,extracellular levels are(5910.35±468.23)μg/g and(45222.12±2726.81)μg/g,respectively.The study identified a mutant strain with the deletion of the first enzyme involved in the synthesis pathway of DON and 15-ADON,both DON and 15-ADON are not detected in the extracellular and intracellular of the tri5 mutant.Additionally,this research confirms that after 7 days of cultivation in LTB medium,the wild-type strain exhibits higher levels of 15-ADON in both intracellular and extracellular compared to DON,with extracellular levels of DON and 15-ADON being higher than intracellular levels.This method can provide a methodological reference for the functional study of toxin synthesis in Fusarium graminearum.