土壤丛枝菌根真菌准离体培养体系的建立

Establishment of an in vitro quasi-asymbiotic culture system for indigenous arbuscular mycorrhizal fungi from soil

摘要:

目的 丛枝菌根(arbuscular mycorrhizal, AM)真菌是植物根围微生物群的关键组成部分，能与72%的陆生植物形成共生体。然而，AM真菌属于植物专性共生土壤真菌，难以进行富集分离和人工纯培养。本研究旨在建立一种基于添加根系分泌物的非植物共生培养体系，以解决AM真菌难以离体培养的问题。方法 利用 “多层三明治” 夹膜培养体系，对土壤AM真菌孢子进行非植物共生的准离体培养，并采用分子系统学方法对培养的AM真菌进行鉴定。结果 利用 “多层三明治” 夹膜培养体系对土壤AM真菌进行非植物共生的准离体培养，发现紫云英的根系分泌物能有效促进AM真菌的菌丝生长。与培养30 d和45 d相比，在培养60 d时产生大量的次生孢子，数量为(951±45)个。进一步的孢子回接试验表明，该培养产生的次生孢子能侵染紫云英幼苗根系。使用分子鉴定法鉴定出适用于 “多层三明治” 夹膜培养体系的2种AM真菌，分别为摩西斗管囊霉(Funneliformis mosseae)和隐类球囊霉(Paraglomus occultum)。使用模拟紫云英根系分泌物成分配制的营养液对AM真菌孢子进行 “多层三明治” 夹膜离体培养，结果表明添加根系分泌物显著促进AM真菌菌丝生长。结论 在植物培护的 “多层三明治” 夹膜培养体系中，紫云英根系分泌物能持续诱导AM真菌在非共生条件下产生菌丝体和具有侵染宿主植物能力的次生孢子。本研究为进一步解决AM真菌离体培养和分离鉴定等问题提供了新方法。

关键词:丛枝菌根真菌;“多层三明治” 培养体系;准离体培养;分离鉴定

Abstract:

Objective Arbuscular mycorrhizal (AM) fungi are crucial components of the plant rhizosphere microbiota, capable of forming symbiotic relationships with 72% of terrestrial plants. However, AM fungi are plant-specific symbiotic fungi in soil, and they are difficult to be enriched for isolation and achieve artificial pure culture. This study aimed to develop a non-plant symbiotic culture system based on the addition of root exudates to solve the problem of difficult in vitro culture of AM fungi.Methods The “multi-layer sandwich” culture system was used for the in vitro quasi-asymbiotic culture of AM fungal spores from soil. Molecular systematics methods were employed to identify the cultured AM fungi.Results A “multi-layer sandwich” culture system was used for the in vitro quasi-asymbiotic culture of AM fungi from soil. It was found that the root exudates of Astragalus sinicus effectively promoted the hyphal growth of AM fungi. A large number (951±45) of secondary spores were produced after 60 days of culture, exceeding those after 30 days and 45 days of culture. Further spore inoculation tests indicated that the secondary spores produced from this culture colonized the roots of A. sinicus seedlings. Two AM fungal species, Funneliformis mosseae and Paraglomus occultum, were identified by molecular characterization as suitable for the “multi-layer sandwich” culture system. Finally, a nutrient solution composed of simulated root exudate components from A. sinicus was used for the “multi-layer sandwich” culture of AM fungal spores. The results showed that the addition of root exudates significantly promoted the hyphal growth of AM fungi.Conclusion In the plant-assisted “multi-layer sandwich” culture system, the root exudates of A. sinicus can continually induce AM fungi to produce hyphae and secondary spores capable of colonizing host plants under non-symbiotic conditions. This study provides a new method for solving the problem related to the in vitro culture, isolation, and identification of AM fungi.

Key words:arbuscular mycorrhizal fungi;“multi-layer sandwich” culture system;quasi-asymbiotic culture;isolation and identification