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

Autor: Shu-Fen Cheng, 鄭淑芬
Rok vydání: 2012
Druh dokumentu: 學位論文 ; thesis
Popis: 101
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.
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