分離自綬草的菌根菌

Mycorrhizal fungi isolated from Spiranthes sinensis

藤井健太(Kenta Fujii)

指導教授 ： 羅南徳 ； 山岡裕一

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100700

摘要

綬草(Spiranthes sinensis)是台灣最常見的蘭花物種之一。但是，對其菌根真菌的多樣性(mycorrhizal fungi)知之甚少，而菌根真菌專一性或許造就了其普遍性。於本研究中，台灣本土的綬草菌根真菌被完整地調查以此了解真菌共生物之功能及其多樣性。
成年植物於七個地點中採集，其中台北市有五個，花蓮縣有兩個。此外，種子包被埋在台北市的四個地點，並在四個月後收集以獲取原球莖。菌根真菌以細孔分離法進行分離，並結合形態學和分子系統發育學分析進行鑑定，每種物種的代表性分離物也進行了共生種子發芽試驗。
從39株成年植物中分離出473株菌株，於其中有53.3%屬於Tulasnella (Basidiomycota)，46.7%屬於Rhizoctonia (Basidiomycota)，此外僅有2個分離菌株屬於Ochroconis (Ascomycota)。四個Tulasnella屬的物種被鑑定出來，包括了Tulasnella deliquescens clade A、T. cumulopuntioides、T. tubericola與Tulasnella sp. TTW1，以及七個Rhizoctonia屬的物種，包括了Rhizoctonia sp. AG-A、AG-Fb、AG-G、AG-L、RTW1、RTW2與RTW3。Tulasnella與Rhizoctonia分別於64.1%與59.0%的成熟植物中被分離出來，其中T. deliquescens clade A於所有實驗地點皆為分離頻率最高的物種(於56.4%成熟植物中分離到)，分子演化學分析結果顯示T. deliquescens clade A被分成2個演化支且較為難以分辨其物種。出乎意外地，Rhizoctonia 於所有實驗地點皆有被分離到，且於單一地點，所有的分離菌株皆屬於同一個屬，除了單一採樣地點之外，台北和花蓮的研究點之間未發現菌根真菌的物種組成存在顯著差異。13個原球莖中分離出35個分離株，於其中，88.6%的分離菌株屬於Tulasnella，11.4%屬於Rhizoctonia。這些真菌被鑑定為T. deliquescens clade A、Rhizoctonia sp. AG-G與RTW1。於三個實驗地點T. deliquescens clade A為主要物種。於共生種子發芽試驗中所有分離真菌均被證實為菌根真菌除了Ochroconis spp.以外。此外，T. deliquescens clade A、T. cumulopuntioides、T. tubericola與Rhizoctonia sp. AG-A、AG-Fb、AG-G、AG-L、RTW2能夠協助原球莖發育與葉片出苗。根據本研究成果顯示，T. deliquescens clade A無論是在綬草的根或原球莖且為最優勢之菌根真菌，此結果與日本的S. australis之菌根真菌相似。然而，Rhizoctonia spp.亦為綬草最主要的菌根真菌，超過半數的成熟植物中皆能分離到。因此，經過比較後可之日本的S. australis與台灣的S. sinensis之菌根真菌組成是不同的，需要進一步的研究來闡明影響這些物種組成的生物或非生物因素。另一方面，S. sinensis被認為是真正的菌根真菌廣適者，可以與多種不同的真菌相關，這些真菌在功能上是可以互換的。因此，通過與大量不同的真菌相關聯，S. sinensis可能具有廣泛的分佈和大量的種群。

關鍵字

蘭花菌根真菌 ； 分子系統發育學分析 ； Tulasnella ； Rhizoctonia ； 共生種子發芽試驗

並列摘要

Spiranthes sinensis is one of the most frequently seen orchid species in Taiwan. However, little is known about the diversity of its mycorrhizal fungi and whether mycorrhizal fungal specificity may be involved in its commonness. In this study, the mycorrhizal fungi of S. sinensis in Taiwan were investigated for a better understanding of the diversity and ecological functioning of the fungal symbiont.
Adult plants were collected at seven sites, five in Taipei City and two in Hualien County. In addition, seed packets were buried at four sites in Taipei City and retrieved four months later to get protocorms. Mycorrhizal fungi were isolated using the peloton isolation method, and the isolates were identified by combining morphology and molecular phylogenetic analyses. A symbiotic seed germination test was conducted using representative isolates of each fungal species.
From 39 adult plants, 473 isolates were isolated. Among these isolates, 53.3% belonged to the genus Tulasnella (Basidiomycota), 46.7% to Rhizoctonia (Basidiomycota), and only two isolates were species of Ochroconis (Ascomycota). Four species of Tulasnella were identified as Tulasnella deliquescens clade A, T. cumulopuntioides, “T. tubericola”, Tulasnella sp. TTW1, and seven species of Rhizoctonia were recognized as Rhizoctonia sp. AG-A, AG-Fb, AG-G, AG-L, RTW1, RTW2, and RTW3. Tulasnella spp. and Rhizoctonia spp. were isolated from 64.1 % and 59.0 % of adult plants, respectively. Tulasnella deliquescens clade A was most frequently isolated (from 56.4 % of adult plants) and was present in all study sites except from a single one. Molecular phylogenetic analyses of T. deliquescens clade A revealed that this species was divided into two clades, indicating cryptic species. Unexpectedly, strains of Rhizoctonia were isolated from all study sites, and in a single site., all isolates belonged to this genus. Except for this single collection site, significant differences among the species composition of mycorrhizal fungi between study sites of Taipei and Hualien were not observed. From 13 protocorms, 35 isolates were isolated. Among these isolates, 88.6% belonged to Tulasnella and 11.4% to Rhizoctonia. These fungi were identified as T. deliquescens clade A and Rhizoctonia sp. AG-G and RTW1. At three study sites, T. deliquescens clade A was predominant. In symbiotic seed germination test, all these isolated fungi were proven to be mycorrhizal fungi except for Ochroconis spp. In addition, T. deliquescens clade A, T. cumulopuntioides, “T. tubericola” and Rhizoctonia sp. AG-A, AG-Fb, AG-G, AG-L, RTW2 supported the protocorm development and led to leaf emergence.
In this study, it is revealed that T. deliquescens clade A is predominant as mycorrhizal fungi in the roots and in protocorms of S. sinensis in Taiwan, which is similar to the result of studies on mycorrhizal fungi of S. australis in Japan. However, Rhizoctonia spp. are also main mycorrhizal fungi of S. sinensis, which were isolated from more than half of adult plants. Therefore, compared to the species composition of mycorrhizal fungi of S. australis in Japan, the mycorrhizal fungal species composition of S. sinensis in Taiwan is different. Further studies are necessary to elucidate the biotic or abiotic factors which influence these species compositions. On the other hand, S. sinensis is assumed to be a true mycorrhizal generalist, which can associate with a broad range of different fungi and these fungi are functionally interchangeable. Therefore, S. sinensis may be able to have a wide distribution and large population by associating with a large number of different fungi.

並列關鍵字

Orchid mycorrhizal fungi ； Phylogenetic analysis ； Tulasnella ； Rhizoctonia ； Symbiotic seed germination test

參考文獻

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