蘭科臺灣白及菌根內與根域土真菌多樣性與功能探討

臺灣白及(Bletilla formosana)為蘭科(Orchidaceae)白及屬(Bletilla)，其為臺灣原生的多年生草本蘭花。在自然環境下，蘭花種子萌芽與後續的植株發育必須仰賴適合的菌根菌提供其生長所需的碳源。過去研究認為蘭花菌根菌具有較高的專一性，蘭花菌根菌的共生成為蘭花分布的限制因子。本研究選擇能廣泛分布在不同海拔與生育環境的臺灣白及作為探討葉綠素蘭花的分布與菌根內真菌群落組成關係之研究對象。材料採集臺灣北(新北市坪林區，BPRF、BPS)、中(苗栗縣卓蘭鎮，BZRF、BZS)、南(嘉義縣阿里山鄉，BARF、BAS)與東(花蓮縣豐濱鄉，BSRF、BSS)四個不同自然生育環境的臺灣白及透過ITS1F、ITS4引子對擴增內轉錄間隔區(internal transcribed spacer，ITS)並以PacBio第三代長片段定序技術進行菌根內真菌多樣性分析。本研究共獲得180,676條高品質的真菌ITS全長序列，可歸屬為2門17綱41目72科112屬209種。長片段技術可提高種層級的解析度，本研究結果顯示可得65%完整學名的菌種。真菌群落多樣性指數方面，以北部樣區Shannon值2.2顯著高於其它三個樣區。整體而言，子囊菌門、赤殼菌科為四個臺灣白及樣區共同優勢菌群。在屬的層級，四個樣區的優勢菌群組成有顯著差異(ANOSIM, P=0.002)，以此推測臺灣白及與真菌的專一性較低。這解釋臺灣白及之能廣泛分布於不同生育環境的可能原因之一。本研究同時也將菌根中的真菌分離純化培養並進行分子鑑定，目前共分離純化43株分離株，已完成18株分離株之鑑定，其來自10屬11種真菌。接續將43株分離株與臺灣白及進行共生發芽來初步評估其功能性。其中分離株BZF-9為尖孢鐮刀菌，其能使臺灣白及種子順利發芽與發育成原球莖。Fusarium oxysporum BZF-9共生培養試驗結果表示，接種F. oxysporum BZF-9不會影響臺灣白及小苗的存活率，每盆接種0.334 g的F. oxysporum BZF-9的實驗組栽培3個月後會讓臺灣白及有33.3%的小苗有黃化的外表型性狀，每盆接種0.167 g的F. oxysporum BZF-9共生栽培3個月後，進行徒手切片鏡檢可以觀察到臺灣白及於接種F. oxysporum BZF-9內具有菌絲圈。根域土中鐮刀菌屬(Fusarium)也是優勢菌群，臺灣白及菌根中的鐮刀菌屬可能來自土壤。根據文獻搜尋，本研究為第一篇成功應用第三代長片段定序技術完成野生地生性綠色蘭花菌根內真菌群落結構之分析。長片段定序可讓我們更清楚了解菌根內真菌的多樣性與其之間交互關係，有助於分離純化有益共生菌根菌，其可應用於未來產業共生培養繁殖生產臺灣白及種苗用途與協助完整的野生蘭花復育計畫執行。

關鍵字

共生培養；長片段技術；地生性蘭花；部分真菌異營性蘭花；蘭花菌根菌

並列摘要

Bletilla formosana, a species of Bletilla in Orchidaceae is a protist herbaceous perennial orchid in Taiwan. In the natural environment, the germination and development of orchid seeds must rely on suitable mycorrhizal fungi which can provide the carbon source for their growth. In the previous studies, it was suggested that orchid mycorrhizal fungi had high specificity, and the distribution of orchid mycorrhizal fungi became the limiting factor of orchid distribution. In this study, Bletilla formosana, which is widely distributed at different altitudes and fertility environments, was selected as the research object to explore the relationship between the distribution of chlorophyll orchids and the composition of fungal communities in the mycorrhiza. The Bletilla formosana were collected from four different natural environments including the northern, central, southern, and eastern parts of Taiwan. Fungal internal transcribed spacer (ITS) was amplified by ITS1F and ITS4 primer pair, and sequenced by PacBiolong-read sequencing technology. A total of 180,676 high-quality fungal ITS full-length sequences were obtained, which can be attributed to 2 phyla, 17 orders, 41 orders, 72 families, 112 genera, and 209 species in this study. The long-read technique improves the resolution at the species level, and the results show nearly 65% of ASV with complete scientific names at species level. In terms of fungal community diversity index, the Shannon value of 2.2 in the northern sample area was significantly higher than that in the other three sample areas. Overall, Ascomycota and Nectriaceae are the dominant plyla across the four Bletilla formosana mycorrhiza. At the genus level, the fungal community composition of the four sites was significantly different (ANOSIM, P=0.002), It is speculated that Bletilla formosana has a low specificity with fungi.This may explain one of the possible reasons why Bletilla formosana is widely distributed in different reproductive environments. In addition, Fusarium is a dominant fungal community in four tran sects, Potential mycorrhizal fungi were isolated and identified, a total of 43 isolates have been isolated and purified, and the identification of 18 isolates has been completed, which are from 10 genus and 11 species fungi. 43 isolated fungal strains were germinated symbiotically with Bletilla formosana seeds to initially assess their functionality. Among them, the isolated strain BZF-9 F. oxysporum acusia can successfully germinate and develop into protobulbs from Bletilla formosana seeds. The results of F. oxysporum BZF-9 symbiotic culture test showed that the symbiosis of Bletilla formosana seedlings and BZF-9 did not affect the mortality rate of seedlings, but the experimental group with high concentration (0.334 g/pot) would give Bletilla formosana seedlings 33.3% yellowing after 3 months of cultivation, and the low concentration group (0.167 g/pot) had a symbiotic cultivation for 3 months. Observations can be made to observe the presence of pelotons inside the roots of Bletilla formosana symbiotic culture seedlings. Fusarium in root soil is also the dominant flora. The Fusarium in the Bletilla formosana mycorrhizae probably came from soil. According to literature search, this study is the first to successfully apply Third Generation Sequencing to complete the analysis of fungal community structure in the mycorrhizae of wild Terrestrial with chlorophyll orchids. Third Generation Sequencing (PacBio) allows researchers to better understand the diversity of fungi within the mycorrhizae and their potential interaction and helps to isolate and purify beneficial commensal mycorrhizal fungi, and can be applied on industrial symbiotic culture propagation to produce Bletilla formosana seedlings and assist in the implementation of complete wild orchid rehabilitation programs.