白木香内生真菌的分离鉴定及诱导结香作用研究

Isolation and Identification of Endophytic Fungi from Aquilaria sinensis and Agarwood Induction

目的:分离、纯化经通体结香技术诱导的白木香木质部真菌,筛选能够高效促进白木香产生优质沉香的菌株。方法:采用组织块分离法分离、纯化菌株,通过形态学特征结合内转录间隔区(ITS)序列分析鉴定真菌;将真菌菌液接种到白木香树4个月后进行功能验证;依据《中华人民共和国药典》(以下简称《中国药典》) 2020年版沉香项下薄层色谱、浸出物含量测定、沉香四醇含量测定及特征图谱方法分析菌株诱导效果。结果:从白木香木质部中分离出48株真菌,优势菌株为镰刀菌;经刺盘孢菌属(Colletotrichum sp.)、黑曲霉Aspergillus niger、渐进绿木霉Trichoderma paraviridescens诱导的沉香薄层色谱斑点与对照药材一致;经镰刀菌属(Fusarium sp.)、黑曲霉、渐进绿木霉诱导的沉香浸出物质量分数均大于10%,与对照组差异有统计学意义(P<0.05);经可可毛二孢菌Lasiodiplodia theobromae、黑曲霉、渐进绿木霉等6株菌诱导的沉香特征图谱与对照药材一致,沉香四醇质量分数均大于0.1%,与对照组差异有统计学意义(P<0.05)。结论:从白木香木质部分离出的内生真菌可诱导沉香形成,其中黑曲霉和渐进绿木霉能够较快速诱导白木香结香,结香所得的树脂品质符合《中国药典》2020年版标准。

Objective:To isolate and purify the xylem fungi from Aquilaria sinensis induced by whole-tree agarwood-inducing technique(Agar-WIT),and screen the specific strains that effectively promoted the production of high-quality agarwood.Methods:The strains were isolated and purified by tissue separation method,and the fungi were identified by morphological characteristics combined with internal transcribed spacer(ITS)sequencing.Fungi solution was inoculated into A.sinensis for 4 months for functional verification.According to the agarwood standards in the Pharmacopoeia of the People's Republic of China(hereinafter referred to as the Chinese Pharmacopoeia,2020 edition),the effect of strain-induced agarwood was analyzed by thin layer chromatography,extract determination,agarotetrol determination and characteristic chromatogram.Results:A total of 48 strains of fungi were isolated from the xylem of A.sinensis,and the dominant strain was Fusarium sp.The thin-layer chromatogram of the agarwood induced by Colletotrichum sp.,A.niger and T.paraviridescens were consistent with those of the reference medicinal material.The extract content of agarwood induced by Fusarium sp.,A.niger and T.paraviridescens were more than 10%,and the difference was statistically significant when compared with that in the control group(P<0.05).The characteristic chromatograms of agarwood induced by 6 strains such as L.laosensis,A.niger and T.paraviridescens were in accordance with those of the reference medicinal materials,and the content of agarotetrol was more than 0.1%,higher than that in the control group(P<0.05).Conclusion:Endophytic fungi isolated from the xylem of A.sinensis could induce the formation of agarwood,among which A.niger and T.paraviridescens induced the formation in a more quickly way.The quality of resin obtained by agarwood formation conformed to the standards in the Chinese Pharmacopoeia(2020 edition).