臺灣原生蘭科植物內生真菌之分布與其應用

The Distribution and Application of Root Fungal Endophytes in Taiwan Native Orchids

鄭淑芬(Shu-Fen Cheng)

指導教授 ： 張喜寧

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02246

摘要

為了瞭解臺灣原生蘭科植物根部內生真菌之資源，並開發其有益微生物之共生資源，本研究於臺灣北、中、南三地區，自桃園縣復興鄉東眼山、新竹縣尖石鄉及五峰鄉鳥嘴山、苗栗縣南庄、南投縣仁愛鄉春陽、嘉義縣阿里山、屏東縣大漢山等25處，共採集得到分屬於34屬之69種原生蘭科植物，其中包括根節蘭屬(Calanthe)10種、豆蘭屬(Bulbohyllum)9種，及羊耳蘭屬(Liparis)8種等；此外，並進行根部內生真菌的分離與鑑定，共分離到內生真菌500餘株，經形態及分子鑑定結果顯示，以木黴菌(Trichoderma spp.)、鐮孢菌(Fusarium spp.)及炭角菌(Xylaria spp.)等三屬真菌佔優勢，並且也分離出已有較多研究報導的絲核菌屬(Rhizoctonia spp.)之蘭菌。由此資源調查結果得知，同種類的蘭科植物分布在不同的地域環境，其根內內生真菌種類有所不同；而不同的蘭科植物種類，其根部內生真菌的種類也有所不同。
進一步地，利用本研究及本研究室過去自原生蘭科植物分離之蘭菌，進行藥用與觀賞蘭科植物，包括臺灣金線連、綬草及紅鶴頂蘭等之接種試驗，觀察其對種子發芽、種苗生長與發育之影響，並進行蘭菌與生長環境對臺灣金線連藥效之影響評估。於臺大(海拔10公尺)、新店(海拔500公尺)、新竹縣五峰(海拔1,000公尺)等三個不同海拔地區，以塑膠袋栽培法(plastic bag cultivation method; PBCM)將臺灣金線連B(原生種與紅骨種雜交)及R(紅骨種)等兩種不同品系之盆栽，置於完全封閉的塑膠袋內，接種絲核菌屬蘭菌R02及R04，結果顯示以此法栽培7個月之所有B品系及R品系臺灣金線連組培苗，出瓶後之成活率均在80%以上，且可顯著提高植株產量，又不需施用任何化學農藥。此外，種植於臺大之臺灣金線連B及R兩品系，接種R02蘭菌之植株均有較高的總酚類及保肝活性成分金線連苷(AFEE)之含量。
綬草種子接種發芽實驗結果顯示，絲核菌屬蘭菌普遍具有促進綬草種子發芽之能力，其中以接種生長於燕麥培養基之R02蘭菌，其促進發芽之效果最好，也最穏定。接種R02處理者，播種後15天可發芽，較無菌播種者(播種後75天才發芽)，約可提早2個月發芽。將綬草種子播種於預接種R02 15天之馬鈴薯葡萄糖瓊脂培養基，播種前刮除菌絲，可促進綬草種子發芽，40天後發芽率高達80.2%；未刮除菌絲者發芽率僅有11.2%，而未接種者發芽率為0%。將泥炭土與椰纖2：1體積比例，充分混合做為介質，種植綬草三個月有最高的成活率100%，及較佳的營養生長；利用20/15℃之溫度處理86天，以接種R02者所需到花日數最短；綬草於四種不同溫度處理86天後，移往簡易溫室120天，均以接種R15者，可促進地上部的生長，具較多的葉數；接種R19者，則可促進地下部的生長，具較多的根數及較重的根鮮重；於15/13℃及20/15℃下，分別以接種蘭菌R15及R19者，可以促進綬草的生殖生長，增加其花梗數及花穗長。
紅鶴頂蘭根部接種試驗，接種不同蘭菌R01、R02、R15、R19結果顯示，相較於對照組，接種適當的蘭菌可顯著提高紅鶴頂蘭瓶苗之移植成活率27-31%(R15) 及26-30%(R02)，增加株高6.3 公分(R15)、葉長3.5公分(R15)、花軸長度10.2公分(R19)，及每枝花軸之花朵數3.5朵，並增加其花的產量 (R19)。接種絲核菌R02的植株，其光合作用速率平均3.73 μmole m-2 s-1，顯著高於對照組，以及並可增加葉及假球莖之總碳水化合物含量，分別為24.5 mg·g-1及90.9 mg·g-1。
綜合本研究結果得知，臺灣原生蘭科植物根部具有豐富的內生真菌資源，原生蘭科植物所在的生長環境直接影響內生真菌的多樣性，各地區的環境和氣候為影響蘭科植物內生真菌種類的重要因子，蘭科植物的種類則為次要因子。應用絲核菌屬蘭菌於蘭科植物的接種試驗，臺灣金線連利用塑膠袋栽培法，配合蘭菌的接種，種植於海拔1,000公尺(日夜溫22/18℃)，可以快速大量生產無農藥的金線連，並提升其藥用效能；綬草接種蘭菌，可促進其種子發芽及營養生長，配合低溫春化及高溫處理，能促使花芽分化及花軸抽長之生殖生長；而紅鶴頂蘭接種適當種類的絲核菌屬蘭菌，亦可以增加組織培養小苗移植存活率，並藉由提高光合作用速率，進而促進其生殖生長，增加植株之花梗與花朵數。因此，蘭菌接種技術的應用，具有保護與復育原生蘭科植物的目的，並具有開發為商業化大量生產蘭科植物之潛力。

關鍵字

原生蘭科植物 ； 內生真菌 ； 臺灣金線連 ； 綬草 ； 紅鶴頂蘭

並列摘要

To investigate the diversity of endophytic fungi in Taiwan indigenous orchids and explore the resources of symbiotic microorganisms, wild indigenous orchids were extensively collected from 25 sites in the northern, central and southern parts of Taiwan. A total of 69 orchid species belonging to 34 genera in Orchidaceae, including 10 species of Calanthe, 9 species of Bulbophyllum, and 8 species of Liparis, were collected. More than 500 isolates of endophytic fungi from roots were isolated. Morphological and molecular identification suggested that Trichoderma, Fusarium, and Xylaria spp. were predominant fungal species and Rhizoctonia spp., a group of endophytic fungi in orchids, were also isolated. Our surveys reveal that the same species of orchid plants from different geographic areas exhibit different microbial communities in their roots and the roots of different orchid plant species contains diverse and different endophytic fungi.
To evaluate the effects of orchid mycorrhizal fungi on the germination, seedling growth, and development of Taiwan native ornamental and medicinal orchids (including Anoectochilus formosanus, Spiranthes sinensis, and Phaius tankervilleae) and on the contents of active components in A. formosanus, orchid mycorrhizal fungi isolated from this study and prior studies in our laboratory were selected for inoculation tests. Two micropropagated lines, B and R, of A. formosanus, were separately inoculated with orchid mycorrhizal fungi Rhizoctonia spp., R02 and R04, and grown for seven months by plastic bag cultivation method (PBCM) at three different elevations above sea level, including National Taiwan University (NTU, 10 m elevation), Xindian (500 m elevation) and Wufong (1,000 m elevation). The results showed that the survival rates for ex vitro growth were more than 80% and plant production was also significantly increased without the application of agrichemicals. Both lines of A. formosanus, inoculated with R02 and cultivated at National Taiwan University, contained higher levels of phenolic compounds and hepatoprotective agent AFEE (A. formosanus extraction with ethyl acetate) compared to the non-mycorrhizal control.
Seed inoculation with mycorrhizal fungi revealed that most of Rhizoctonia spp. tested enhanced Spiranthes sinensis seed germination. However, R02 isolate displayed the most effective and stable activity for promoting seed germination. The R02-inoculated seeds grown on oatmeal agar readily germinated 15 days after sowing, which was two months earlier than non-symbiotic treatment (usually germinated after 75 days). Inoculation with R02 also increased the germination rate of S. sinensis ex vitro seeds on potato dextrose agar. Spiranthes sinensis seeds sown on PDA that had been grown with R02 for 15 days showed germination rate up to a 80.2% after 40 days when the hyphae grown on the surface of PDA media were scraped off prior to seeding. Germination rate reduced to 11.2% if the hyphae were not scraped off, or to 0% in the non-mycorrhizal control. When S. sinesis cultivated under 20/15℃ day/night temperature treatment, R02 inoculated samples flowered within the shortest period. When S. sinesis plants were grown under four different temperature conditions for 86 days and then moved to greenhouse for a 120 days, samples inoculated with R15 isolate showed the best shoot and leaf growth. On the other hand, R19 isolate enhanced the root growth of S. sinesis, which more root and higher fresh weight were observed. Under 15/13℃ and 20/15℃ day/night temperature treatments, inoculated with R15 and R19 isolates respectively, promoted S. sinesis reproductive growth and more and longer flower stalks were recorded.
Four orchid mycorrhizal fungi, R01, R02, R15 and R19, were further inoculated onto the roots of Phaius tankervilleae. R15 and R02 increased survival rates of 27-31% and 26-30% respectively; R15 increased plant height 6.3 cm and leaf long 3.5 cm; R19 also increased reproductive growth and flower stalk length 10.2 cm and 3.5 flowers per flower stalk were recorded on ex vitro seedlings. When the plants of P. tankervilleae were inoculated with R02, photosynthetic rate and carbohydrate content significantly increased.
In summary, our studies reveal that the roots of Taiwan indigenous orchids are full of rich and diverse endophytic fungi. The distribution of fungal species is directly influenced by host growth environments. The environmental and climate conditions of specific geographic location are the most important factors, whereas orchidaceae species also influence the presence of endophytic species in their roots. Applications of identified Rhizoctonia spp. in different orchidaceous plants reveal dramatic effects on the vegetative and reproductive growth. R02、R04 inoculated A. formosanus cultivated by plastic bag cultivation method at an altitude of 1,000 m under 22/18℃ day and night temperature can produce lots of agrichemical-free plants. The presence of Rhizoctonia sp. also enhances the medicinal effectiveness of this orchid plant. Inoculation of Rhizoctonia spp. (R02, R15, R19) promotes seed germination and vegetative growth of Spiranthes sinensis. In combination of low temperature vernalization and high temperature treatment, mycorrhizal fungi also enhance reproductive growth such as flower bud differentiation and flower stalks elongation. Inoculation of Rhizoctonia isolates (R02, R15 and R19) also promotes transplant survival rates of P. tankervilleae seedlings and increases the number of flower stalk which resulted from the increased photosynthetic rate. Taken together, exploration and application of orchid mycorrhizal fungi not only can preserve and restore indigenous orchid species, but also can be potentially adopted for commercial mass production of orchid plants.

並列關鍵字

indigenous orchids ； endophytic fungi ； Anoectochilus formosanus ； Spiranthes sinensis ； Phaius tankervilleae

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