植物菌根块菌分子鉴定及菌丝培养基优化的研究

Molecular identification and optimization of hyphae culture medium in plant mycorrhizal tubers

关键词:  块菌属, ITS, 菌丝, 固体培养基, 优化

Key words:  Tuber, ITS, hyphae, solid medium, optimization

摘要:

块菌属(Tuber)是由真菌感染其适宜植物根系形成,部分块菌如法国黑孢块菌(T. melanosporum)和中国印度块菌(T. indicum)等为珍稀药食两用菌,具有很高的经济价值。对块菌的过度挖掘造成野生块菌资源匮乏,块菌人工栽培技术则是实现块菌产业可持续性发展的重要基础。目前国内外人工栽培主要采用块菌子实体匀浆制成的子囊孢子悬浮液,与人工种植苗木根系共生。利用块菌菌丝替代子实体孢子悬浮液进行接种,具有减少对块菌子实体的依赖性和生产成本、保护林下块菌野生资源、促进块菌人工繁育进程的积极意义,但需要成熟高效的菌丝培养技术。该研究从云南、陕西等地林下采取块菌样品,在ITS分子鉴定并纯化得到纯种菌丝的基础上,以菌丝直径为指标,经单因素实验和4因素3水平正交实验L₉(3⁴),筛选得出最适宜的菌丝生长培养基。结果表明:(1)形态和ITS分子鉴定得到的13份块菌样品分属印度块菌(T. indicum)和假凹陷块菌(T. pseudoexcavatum)两种。(2)纯化的印度块菌菌丝培养基的最佳碳源为马铃薯浸出液,最佳氮源为酵母膏,VB1对印度块菌菌丝生长并无显著性促进作用。(3)最佳的碳源、氮源、无机盐组合为马铃薯浸出液150 g·L^-1、酵母膏3 g·L^-1、MgSO₄ 2 g·L^-1、KH₂PO₄ 2 g·L^-1。25 ℃,自然pH下培养8 d菌丝直径可达49.44 mm。

Abstract:

Tuber is a type of fungus underground, formed by infecting suitable plant roots. And some species such as T. melanosporum and T. indicum are rare and valuable for both medicine and food, and have high economic values. The over excavation of Tuber has caused a shortage of wild Tuber resources, and the artificial cultivation technology of Tuber is an important foundation for the sustainable development of the Tuber industry. At present, the ascosporic suspension made from the homogenate of the fruiting body, is mainly used in the artificial cultivation, which is symbiotic with the roots of the planting seedlings. It is valuable for reducing the dependence on the fruiting body of Tuber and the production cost, protecting the wild resources of Tuber under the forest and promoting the process of artificial propagation of Tuber, to use the hyphae of Tuber instead of the spore suspension for inoculation, but it needs mature and efficient technology for hyphae culture. Tuber samples in this study were taken from the forests of Yunnan, Shaanxi and other places. Then, ITS sequence analysis was carried out to identify species and purify hyphaes. The most suitable hyphae growth medium was selected by the L₉(3⁴)orthogonal test(four factors with three levels), using the hyphae diameter as an index. The results were as follows:(1)The 13 Tuber samples identified by morphological and ITS molecular identification belonged to T. indicum and T. pseudoexcavatum.(2)The optimum carbon source for T. indicum hyphae was potato extract, and the optimum nitrogen source was yeast extract. However, VB1 had no significant promotion effect on the hyphae growth of T. indicum.(3)The optimal combination of carbon, nitrogen and inorganic salts was potato extract 150 g·L^-1, yeast extract 3 g·L^-1, MgSO₄ 2 g·L^-1, and KH₂PO₄ 2 g·L^-1. The diameter of hyphae can reach 49.44 mm, which is cultured at 25 ℃ and natural pH for 8 d.