Application of GC-MS in Botrytis cinerea Metabolome Analysis
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KeyWord:Botrytis cinerea  metabolomics  sample pretreatment  silylation
HU Zhi-hong,CHANG Xu-nian,DAI Tan,WU Jia-chun,LIU Peng-fei 中国农业大学植物保护学院
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      The pretreatment conditions,including extraction solvent,derivatization time and temperature,as well as internal standard were optimized for metabolome analysis of Botrytis cinerea(B-cinerea),an important pathogenic fungus that could induce a severe grey mold on plants.The results showed that,using salicin as the internal standard,a mixture of methanol and water(80∶20) with a dosage of 4.0 mL for 0.1 g of mycelium as extraction reagent,a silylation of N,O-bis(trimethylsilyl)trifluoroacetamide with methoxylamine for 6 h of derivation at 37 ℃ was the optimal for most kinds of metabolites in derivatives yield and stability.In B-cinerea hypha metabolome,210 peaks were detected by GC-MS, and the metabolome was identified as a group of metabolites including mainly amino acids,alcohols,organic acids,sugars and others.Among the metabolites,50 of them were identified by NIST 2008 with a match quality of 80% or more, and part of them by standards.As a result,typical 17 metabolites obtained good linearities in the range of 1.0-100 μg/mL with correlation coefficients more than 0.98.The limits of detection and limits of quantitation were in the range of 0.02-10.0 ng/mL and 0.1-34.0 ng/mL,respectively.Investigation on the reproducibility of the optimal method suggested that the RSD for 89.05% metabolites detected were in the range of 0.01%-16.8%.The good reproducibility implied that the method established was suitable for the metabolome detection of mycelial fungi,and could provide a good reference for microorganism metabolomics research in the field of agricultural and medical science.