|其他摘要||The genera Paphiopedilum and Cypripedium both belong to the same subfamily Cypripedioideae of the family Orchidaceae. Although they are closely related to each other, there are great differences in ecological distributions, growth habitats and life forms between the two genera. The former is terrestrial or epiphytic, distributed in the tropics or sub-tropics, and usually evergreen, whereas the latter is terrestrial, deciduous, and distributed across most of temperate Northern Hemisphere, and has an “adult dormancy” period every year. The asymbiotic seed germination is much easier in Paphiopedilum than in Cypripedium, as is the case with seedlings plantation and the survival of adult plants transplanted from the wild. It is well recognized that mycorrhiza plays a vital role in the orchid life, and the development of mycorrhizal relationship is also a crucial event in the evolution of the Orchidaceae. In this thesis, studies on mycorrhizal structure and mycorrhizal fungi of three Paphiopedilum species and three Cypripedium species, seedling mycorrhization of P. armeniacum in semi-natural conditions, and some “opportunistic fungi” in the roots of two Paphiopedilum species were carried out. The main results are summarized as follows:
1. The root cross-sections of P. micranthum, P. armeniacum, P. dianthum, C. flavum, C. guttatum and C. tibeticum were observed, and mycorrhizae were found in adult plants of all six orchid species. The epidermis of Paphiopedilum root zones was a multiseriate velamen filled with many hyphae while roots of Cypripedium had a simple epidermis of living cells. In the roots of Paphiopedilum, mycorrhizal infection was confined to a proportion of cortex cells, while Cypripedium exhibited a pattern of dense infection. Lots of starch granules were found in the cortex of Cypripedium but seldom in Paphiopedilum. The velamen cells benefit Paphiopedilum plants by holding water from their habitats where soil water supplies sometimes changed dramatically. The density of mycorrihzal infection might indicate that deciduous Cypripedium plants need more mycotrophy than evergreen Paphiopedilum species, and starch granules in the cortex helped Cypripedium over the winter.
2. Mycorrhizal fungi of six orchids were studied by the method of isolation of mycobionts combined with molecular sequencing (ITS and mtLSU) of fungi directly from the mycorrhizae. 175 ITS and 80 mtLSU fungal sequences from over 200 DNA samples of orchid roots were obtained. It was found that most of them (160 ITS sequences and 76 mtLSU sequences) belonged to the family Tulasnellaceae, while only a few were other fungi (“opportunistic fungi”), such as Fusarium solani, Cladosporium lignicola, Penicillium sp., etc. The phylogenetic analysis revealed for the first time that Paphiopedilum mycorrhizal fungi were members of the fungal family Tulasnellaceae, just the same as those in Cypripedium, having a strong specificity. The phylogenetic tree constructed from ITS sequences of mycorrhizal fungi from Cypripedium species and Paphiopedilum species could be divided into 11 clades, and each clade might represent one or two or even three related fungal species. Among all the mycorrhizal fungal clades, every orchid studied was associated with more than two fungal clades. The ability to be associated with several fungi improves the orchid adaptation to the environment variations.
3. Based on the phylogenetic tree constructed from ITS sequences, it was found for the first time that each orchid had one or two main fungal clades among several fungal clades associated, which we called “dominant mycorrhizal fungi”. Different species of Paphiopedilum or Cypripedium having similar habitats might have some identical mycorrhizal fungi, yet the dominant mycorrhizal fungi for each orchid species were not the same. Which fungi could be the dominant ones are determined by the plant, yet influenced with the habitats.
4. The seeds of P. armeniacum could germinate freely and develop into seedlings in the absence of mycorrhizal fungi when sufficient nutrients provided. In semi-natural conditions, P. armeniacum seedlings developed from asymbiotic seed germination could be successfully transplanted to an orchid nursery in a greenhouse. For a certain period of time after transplantation, new roots were found to be mycorrhized while the old ones not. We traced the process of P. armeniacum seedling mycorrhization and identified mycorrhizal fungi to be its specificity fungi (such as Clade 8 and Clade 6) by molecular method for the first time.
It was newly found that the dominant mycorrhizal fungi of P. micranthum switched from fungal Clade 11 to Clade 8 (Tulasnella calospora) when transplanted from the wild to the orchid greenhouse in Kunming or Xingyi. T. calospora is the mycorrhizal fungus of Paphiopedilum and many other known orchids, but not Cypripedium. The reason why adult Cypripedium plants grew poorly when transplanted might be due to the shortage of their specificity mycorrhizal fungi in the new environments or due to the new environments incompatible with their fungal partners.
The dominant mycorrhizal fungi switch in P. micanthum when transplanted and successful mycorrhization of P. armeniacum seedlings in semi-natural conditions may be responsible for the easiness of Paphiopedilum plantations as compared with Cypripedium, and also throw some light on the axenic seedlings mycorrhization and conservation of the family.
5. Apart from Tulasnellaceae, there existed some “opportunistic fungi” in the roots of Cypripedium and Paphiopedilum. The relationships between orchids and them are still largely unknown. By co-cultivations of four “opportunistic fungi” with axenic P. micranthum seedlings, and seven with P. armeniacum seedlings, it was found that some of the fungi caused death of the seedlings while others not. Two harmless opportunistic fungi, M2 and M7, identified as Chaetomium sp. and Umbelopsis sp. respectively by ITS sequences, were selected to perform a further study. Data showed that both fungi stimulated the seedling growth. It was found that M2 could supply seedlings with some nitrogen nutrients. However, the reason for M7 remains unclear. The fungi living in the orchid are very diverse and abundant, and their relationships with their host plants are much more complicated. To understand their mutual actions between orchid and fungi, the opportunistic fungi should not be neglected.|