摘要
兜兰属(Paphiopedilum)植物,又称为“拖鞋兰”,在园艺界负有盛名。兜兰作为兰科植物中最诱人的花卉之一,在世界各国拥有为数众多的爱好者。目前,因为自然生境的破坏和人为过度采集,野生兜兰种群受到巨大威胁。所有的野生兜兰植物都被列为《野生动植物濒危物种国际贸易公约》(CITES)附录1中的濒危物种。兰花是真菌异养的植物。在自然条件下,只有合适的共生真菌侵染种子,为其提供碳素和无机营养,粉尘状的兰花种子才能成功萌发。而且,兰花幼苗的发育,成年兰花的生长和繁殖都需要菌根真菌的共生。根据生境、分布、生态型和分类地位的不同,本研究选取了5种野生兜兰植物作为研究对象,包括紫纹兜兰(P. purpuratum)、长瓣兜兰(P. dianthum)、带叶兜兰(P. hirsutissimum)、硬叶兜兰(P. micranthum)和麻栗坡兜兰(P. malipoense)。通过野外采样,利用光学显微镜和扫描电镜观察5种野生成年兜兰植物菌根的形体与结构,改进了兜兰植物菌根真菌的分离方法,并对5种兜兰植物菌根真菌进行分离和鉴定,分析了5种兜兰植物菌根真菌的多样性。通过原地萌发和室内共生萌发试验,探讨了兜兰分布与菌根真菌的关系,并筛选出可用于兜兰植物保育的有效共生真菌。主要结果如下：
1.通过野外原地观察,发现紫纹兜兰、硬叶兜兰和麻栗坡兜兰3种地生型兜兰的根部浅生于腐殖质层,半附生型的带叶兜兰和石上附生型的长瓣兜兰的根部生于岩石表面积土中,5种兜兰的根部外形都明显区别于真正的地生兰和石上附生兰。通过光学显微镜和扫描电镜观察,5种兜兰均具有典型的单子叶植物根的基本结构,3种地生型兜兰的皮层数目多于2种石上附生型兜兰,维管束数目则少于石上附生型兜兰；长瓣兜兰的外皮层具有通道细胞,而其它4种兜兰的外皮层没有通道细胞。5种野生兜兰植物根部皮层细胞中都分布着其菌根真菌形成的“菌丝团”结构,菌丝团在皮层细胞中呈现不同的形态,相邻皮层细胞中的菌丝团通过联通菌丝相连,当菌丝团消解时,皮层细胞内出现大量淀粉粒。
2.改进了菌丝团分离法及分离培养基,具体分为以下步骤：(1)POA分离培养基的制备；(2)根段的处理和筛选；(3)菌丝团悬浮液的制备；(4)共生真菌的分离和纯化。选择质量和长度相同,并有菌根真菌侵染的野生带叶兜兰根段,与其它3种分离方法和2种分离培养基进行了分离比较试验,使用本研究建立的菌丝团分离法和培养基能获得菌株数较多的类丝核菌(Rhizoctonia-like fungi)纯培养。
3.利用菌丝团分离法,从5种野生成年兜兰植物根部分离得到16株类丝核菌(Rhizoctonia-like fungi),ITS序列的系统发育分析显示,它们都属于胶膜菌属Tulasnella (瘤菌根菌属Epulorhiza的有性态),分属于8个类群(Type);每一种兜兰都能两种类群或两种类群以上的真菌共生。花岗岩地区的紫纹兜兰的菌根真菌属于Type3,4,8,与石灰岩地区的兜兰的共生真菌是不同的类群；石灰岩地区的带叶兜兰、长瓣兜兰、麻栗坡兜兰都能与Type2勺真菌共生,同时又能与Type1或Type7的真菌共生；而同样生长于石灰岩地区的硬叶兜兰却有自己独特的菌根真菌Type5,6。
4.野外原地萌发试验结果显示,兜兰种子在野外萌发困难,萌发率极低,在广东野外有紫纹兜兰分布的区域,紫纹兜兰种子原地共生萌发12个月后,其萌发率约为1.06×10-4。在广西雅长自然保护区带叶兜兰分布的区域,带叶兜兰种子的萌发率约为3.8×10-5。在深圳的野生兰科植物迁地保护园和广西雅长自然保护区无兰区种子没有一粒萌发,这表明有成年兰科植物分布的地区,其生长环境更利于兰花种子萌发。室内共生萌发试验结果显示,分布范围广,种群数量较大的带叶兜兰种子对共生真菌的选择范围广,不仅能与宿主兰花的菌根真菌共生,还能能与来自其它兜兰的成年植株根部菌根真菌共生萌发,这表明带叶兜兰在种子萌发成苗阶段对共生真菌的兼容性较强,这可能是其分布较广的原因之一。
5.通过室内共生萌发试验,多株菌根真菌对带叶兜兰种子萌发有促进作用,最高萌发率为21.0%,其中一株共生真菌己获得专利保护。分别以燕麦培养基和原生境腐殖质作为共生培养基质,首次利用共生萌发技术获得可用于移栽的带叶兜兰共生苗。
The genus Paphiopedilum is very well known as lady's slipper orchid in horticultural science. Paphiopedilum orchids, as one of the most attractive flowers in the Orchidaceae, have numerous fans in the world. At present, wild populations of Paphiopedilum orchids are under great threat as a result of habitat destruction and over-collection for their beautiful and unique flowers. All species of this genus have been classified as endangered species in Appendix I of the CITES. Orchids are myco-heterotrophic plants. In nature, dust-seeds of orchid can germinate only when they are infected with appropriate symbiotic fungi which supply carbon and inorganic nutrients. Furthermore, orchid seedling development, adult growth and reproduction also need mycorrhizal fungi. Based on the different habitat, distribution, ecotype and classification status, P. purpuratum, P. dianthum, P. hirsutissimum, P. micranthum and P. malipoense were selected as samples. This study was tried to investigate the mycorrhizal structure, the mycorrhizal fungi diversity and the relationship between orchid distribution and symbiotic fungi. Meanwhile this study was also tried to establish more effective isolation method and symbiotic cultivation technology. The main results were as follows:
1. The roots of P. purpuratum, P. micranthum and P. malipoense grew in the litter on the face of soil. The root external morphology of these three species was significantly different from the real terrestrial orchid. The roots of semi-epiphytic P. hirsutissimum and epiphytic P. dianthum grew in the litter on the surface of rocks and also different from the real epiphytic orchid. The basic structure of monocotyledonous plant roots was observed in these five species by optical microscopy and scanning electron microscopy. The exodermis structure, the number of cortical and vascular bundles of P. purpuratum, P. micranthum and P. malipoense was different from two lithophytic species. The exodermis structure of semi-epiphytic P. hirsutissimum was similar to three terrestrial species. The cortical and vascular bundles of this species, however, were similar to epiphytic P. dianthum. There were many pelotons that presented different forms in the cortical cells of these five species. The pelotons in the adjacent cortical cells linked through communication hyphae. A large number of starch grains were observed in cortical cells when the pelotons were digested.
2. The technique of pelotons isolation from orchid mycorrhiza was improved. The four steps were as:(1) preparation of isolating medium disks,(2) treatment and selection of roots,(3) preparation of the suspension of pelotons,(4) isolation and purification of mycorrhizal fungi. The other three kinds of separation method and two kinds of the separation medium were compared in this study. More Rhizoctonia-like fungi were obtained using our technique of pelotons isolation and separation medium.
3. Sixteen strains of Rhizoctonia-like fungi were isolated from the root pelotons of these five species and identified by morphological and molecular characteristics. All these strains belonged to Epulorhiza (anamorph of Tulasnella). Based on the phylogenetic analysis of the ITS-5.8S rDNA, sixteen isolates of Epulorhiza were separated into eight types. The fungi isolated from the roots of P. hirsutissimum and P. malipoense belonged to Type1and Type2. The fungi isolated from the roots of P. dianthum belonged to Type2and Type7. The strains associated with P. purpuratum were divided into Type3, Type4and Type8. The fungi isolated from the roots of P. micranthum belonged to Type5and Type6. These results showed the species in common habitats, like P. hirsutissimum, P. dianthum and P. malipoense, shared the same fungal taxa and P. purpuratum from distinctive habitat was associated with special fungal species of Epulorhiza. The molecular phylogenetic analyses of Cypripedioideae orchids and their mycorrhizal fungi indicated that specific relationship between them was complex. Cypripedium and Paphiopedilum species had its own unique mycorrhizal fungi. They also shared mycorrhizal fungi in the same clade.
4. In situ and in vitro symbiotic seed germination of P. purpuratum, P. dianthum, P. hirsutissimum and P. malipoense were carried out. The results showed that seed germination of Paphiopedilum were difficult in nature. The germination rate of P. purpuratum in its habitat was1.06×10-4after buried12months. The germination rate of P. hirsutissimum in its habitat was3.8×10-5. The seeds placed in the site which grown Paphiopedilum adult plants were easier to germinate. The widespread P. hirsutissimum was generalist in its association with fungal symbionts compared to other three restricted species in vitro symbiotic seed germination test. This may be one of the reasons for its wide distribution.
5. Several strains can promote seed germination of P. hirsutissimum in vitro symbiotic seed germination test, one of which has patent protection. It is the first report of obtaining Paphiopedilum symbiotic seedlings which can be used for transplanting by two kind of symbiotic seed germination techniques.
引文
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