不同培养基对兰科药用植物手参原球茎共生真菌的分离效果

兰科植物手参Gymnadenia conopsea作为国家二级重点保护野生植物具有重要的药用价值。目前手参还未实现人工栽培,但其种子的真菌共生萌发已获成功。为明确除促萌发真菌外,还有哪些土著真菌参与了手参种子的萌发过程,本研究在自然条件下采用促萌发真菌伴播手参种子,获得了种子萌发形成的原球茎,进而对比了6种常见的培养基PDA (马铃薯葡萄糖琼脂培养基)、MMN (改良Melin-Norkrans培养基)、FIM (真菌分离培养基)、MEA (麦芽浸膏琼脂培养基)、CAM (胡萝卜葡萄糖琼脂培养基)和CMA (玉米粉琼脂培养基)对手参原球茎共生真菌分离效果的影响。共从6种培养基上分离获得了75个菌株,其中MMN、CAM、PDA、FIM、MEA、CMA培养基依次分离得到20株、16株、15株、11株、8株、5株菌。此外,真菌的多样性分析结果表明,MMN培养基的Chao 1、Shannon-Wiener和Simpson多样性指数最高,CAM和PDA培养基次之,CMA培养基最低。综上所述,真菌分离效果最好的是MMN培养基,其次是CAM和PDA培养基,而FIM和MEA培养基对真菌的分离效果影响不大,CMA培养基的分离效果最差。本研究结果可为其他兰科植物原球茎共生真菌的分离提供借鉴,所获得的菌株也有望进一步应用于功能菌剂的研发。

Effects of different media on the isolation of symbiotic fungi from protocorm of medicinal orchid Gymnadenia conopsea

The terrestrial orchid Gymnadenia conopsea with important medicinal value has been listed in the National Key Protected Wild Plant (Category Ⅱ). At present, G. conopsea could not be cultivated artificially, however, seed symbiotic germination has been successfully achieved. In order to determine whether other indigenous fungi were also involved in seed germination process besides the initial germination-promoting fungi, isolation of symbiotic fungi from protocorms formed by germination-promoting fungi under natural conditions were performed on six common media PDA (potato dextrose agar medium), MMN (modified Melin-Norkrans medium), FIM (fungal isolation medium), MEA (malt extract ager medium), CAM (carrot agar medium) and CMA (corn meal agar medium), and the relative abundance were compared. In total, 75 fungal isolates were obtained from the 6 media, and the number of fungal isolates obtained from MMN, CAM, PDA, FIM, MEA and CMA was 20, 16, 15, 11, 8 and 5, respectively. The results of fungal diversity analysis showed that fungal diversity index (Chao 1, Shannon-Wiener, and Simpson diversity index) of the isolates from MMN medium was the highest, and that from CAM and PDA medium was less, while that from CMA medium was the lowest. In short, MMN is the most effective, followed by CAM and PDA, and FIM and MEA media have little effect, while CMA is the worst. Our results provide reference for the isolation of symbiotic fungi from protocorms of other orchids. The fungal strains obtained in this study hopefully could be further used for research purpose and development of functional microbial agents.