野生五唇兰根部内生真菌多样性研究

兰科植物根部的内生真菌在兰科植物的整个生活史中起着重要的作用, 为了解不同生境不同类型的兰科植物内生真菌菌群的多样性, 作者于2004年7月至2005年10月, 以海南岛霸王岭自然保护区内的野生五唇兰(Doritis pulcherrima)作为实验材料, 对不同生境、不同形态的五唇兰植株根部的内生真菌群落多样性进行了研究。从附生于石上及生于杂木林或灌丛中、叶背绿色及叶背紫红色的五唇兰植株新鲜营养根段中共分离出83株内生真菌, 鉴定为19个属, 其中包括培养基筛选实验中分离出的30株14属。镰刀菌属(Fusarium)(24.1%)和丝核菌属 (Rhizocto-nia)(14.5%)为优势属。两种叶色的五唇兰内生真菌群落丰富度较为一致; 而两种不同生境中的五唇兰内生真菌群落丰富度则表现出较大的差异: 附生于灌木的五唇兰内生真菌群落Shannon多样性指数远高于附生岩石的。研究结果表明五唇兰内生真菌多样性更多地受生境的影响而不是受植株形态类型的影响。     

四种杓兰的菌根结构及其周年动态

对黄花杓兰、云南杓兰、西藏杓兰和紫点杓兰四种高山杓兰成年植株的根进行了一个生长周期的切片观察。发现四种杓兰的的菌根结构及其在一个生长周期中的动态变化有以下共同特征:在其成年生活期的各个时期,都有真菌入侵形成内生菌根结构,菌丝在其根部皮层细胞内有四种存在状态:(a)咖啡色或黄色、菌丝扭结缠绕而成的大菌丝结;(b)灰褐色或黄色、由片断菌丝组成的大菌丝结;(c)无色、零散的菌丝;(d)橘红色和黄色、菌丝形态模糊的小菌丝结。真菌的入侵-消解在其成年生活期中周而复始地进行,四种状态的菌丝是处于入侵-消解循环不同阶段的菌丝。并由此对高海拔环境下二者的共生关系进行了初步的探讨:真菌侵入杓兰根部以获得自身生存和繁衍所需的营养物质,杓兰利用真菌菌丝消解后的营养物质,进行混合营养。     

实验室条件下五唇兰菌根真菌专一性研究

利用从高原温带兰科植物菌根中获得的22个菌根真菌菌株, 对五唇兰(Doritis pulcherrima)进行了室内种子萌发、原球茎分化和组培苗回接试验, 从交叉回接的角度对附生兰科植物与菌根真菌的生理专一性进行了探讨。经过20周的共生培养, 只有编号为Cf1和Mm1的两个菌株使种子表现出种胚明显膨大的萌发迹象; 9个菌株能够促使原球茎较好地分化发育出根叶; 11个菌株处理苗的平均鲜重增长率高于对照组(156.25%), 其中Mm1的效果达到极显著水平(p = 0.01)。通过根切片显微观察, 在原球茎分化根和回接效果良好的处理苗的根皮层组织发现典型的菌丝团结构, 表明菌根体系已成功建立。温带地生兰菌根真菌对五唇兰种子萌发、原球茎发育和幼苗生长等3个重要生长阶段影响的试验显示, 五唇兰的种子和菌根真菌的共生萌发效果不佳, 而原球茎及幼株更容易与之建立良好的共生关系。同时, 也没有发现同一个真菌菌株能够对五唇兰的种子、原球茎和幼苗均产生促进作用。研究结果表明, 五唇兰的菌根真菌专一性因生理生长阶段的不同而存在差异。     

四川黄龙沟优势兰科植物菌根真菌多样性及其季节变化

在自然条件下,兰科菌根真菌对兰花的种子萌发和植株生长都是必不可少的。为了解高原兰科植物菌根真菌的多样性状况及其季节性变化规律,选取了四川黄龙沟的两种生境中生长的8种优势兰科植物,分别于植株的萌芽期(4月份)、生长期(7月份)和果期(9月份)采集营养根进行菌根真菌的多样性研究。其中,黄花杓兰(Cypripedium flavum)、少花鹤顶兰(Phaiusdelavayi)、二叶匍茎兰(Galearis diantha)和广布小蝶兰(Ponerorchis chusua)分布在开阔生境;筒距兰(Tipularia szechuanica)、小花舌唇兰(Platanthera minutiflora)、珊瑚兰(Corallorhiza trifida)和尖唇鸟巢兰(Neottia acuminate)则分布在密林生境。通过对分离所得的50个菌株进行形态观察和ITS序列测定相结合的鉴定,共获得菌根真菌41种。对担子菌和子囊菌分别进行的系统发育树构建结果显示,子囊菌为优势种类(35种),以柔膜菌目(Helotiales)、炭角菌目(Xylariales)和肉座菌目(Hypocreales)内的种类为主,担子菌则以胶膜菌(Tulasnellaceaesp.)为主。在8种兰科植物中,二叶匐茎兰表现出极高的专一性,其菌根真菌均属于Hypocrea。其余兰科植物的菌根真菌分别属于不同的科,专一性相对较低。物种丰富度和Simpson多样性指数分析结果表明,密林生境的兰科植物的菌根真菌多样性在各生长季节基本高于开阔生境。此外,两种生境的优势兰科植物的菌根真菌物种多样性随生长季节转变所呈现的变化规律是相似的:萌发期和生长期的多样性均较高,峰值出现在生长期,到果期时则大幅下降。这与高原兰科植物的生长特性及营养供求规律基本相符。     

海南霸王岭五唇兰两种生态型生态位特征

东亚特有种五唇兰自然状态下存在叶背红色和绿色两种生态型。对海南岛霸王岭地区的五唇兰群落中草本层生态位特征进行测定和分析,探讨两种生态型五唇兰在群落中的地位和作用以及彼此间的竞争关系。结果表明: 狭穗草、叶背红色型五唇兰和毛俭草在该层占据较大的重要值。叶背红色型五唇兰较叶背绿色型具有更广的生态位宽度; 拟金草和叶背红色型五唇兰以相似的生态位宽度值居该层物种生态位宽度的首位。两种生态型五唇兰均与该层其他大部分物种具有较大生态位重叠值,两者间的生态位重叠亦很大。这说明五唇兰对其生境有很好的适应性,两种生态型间可能存在较大的竞争。     

三种白及属植物不同生长发育时期的菌根显微结构

采用石蜡切片技术对白及Bletilla striata、黄花白及B. ochracea和小白及B. formosana的栽培种在生长期、花期、果期和休眠期的菌根解剖结构特征、菌根真菌入侵方式和菌丝特征等进行观察研究,以进一步了解菌根真菌与白及属植物的共生关系。结果表明,3种白及属植物的菌根真菌均是通过通道细胞侵入根皮层薄壁细胞,侵入后菌丝靠近皮层细胞的细胞核分布,最终在皮层细胞形成菌丝团;真菌侵染率和菌丝形态随着植物生长发育变化而变化,3种白及属植物均表现为花期和生长期的真菌侵染率较高,以丝状菌丝团为主,而果期和休眠期较低,以团块状菌丝团居多;同一时期不同植物类型的菌根特征无显著差异。     

黄花杓兰与菌根真菌共生关系研究

对黄花杓兰(Cypripedlium flavum P.F.Hunt et Summerh.)植株的根进行了一个生长周期的采集和切片观察,发现黄花杓兰具有相应的菌根结构,菌丝的存在状态在各物候期有所不同,可分为5个时期：(1)4月份联通菌丝时期;(2)5月份菌丝团时期Ⅰ;(3)6、7月份无菌丝团时期I;(4)8、9月份菌丝团时期Ⅱ;(5)10月份无菌丝团时期Ⅱ。可以看出,在植株生活转变期,根细胞内出现菌丝团;在植株完成正常的生命过程中,真菌营养是光合营养的补充;真菌菌丝团也作为植物营养的储存器,菌丝团消解释放营养供给植株。在此基础上探讨了菌根真菌与黄花杓兰的共生关系的一个方面：植株通过联通菌丝沟通各个细胞的物质交换,菌根真菌利用植株体内物质完成生命活动,菌丝团消解,使真菌所储藏的营养物转化,为植株所利用。     

不同生境下五唇兰根部可培养内生细菌多样性研究

兰科植物内生细菌与菌根真菌的协作对宿主植物的生长、抗病、抗逆及植物修复环境能力等具有重要意义,揭示其内生细菌多样性及与生境之间的关系有助于阐明兰科植物的适应与进化机制。本研究基于16SrDNA序列分析探讨了不同生境下东南亚特有种五唇兰根部可培养内生细菌多样性及其空间异质性。结果表明:从不同生境下五唇兰根部共分离出内生细菌59株,其中从土生型五唇兰根部分离出内生细菌45株(76.27%),从石生型五唇兰根部分离出内生细菌14株(23.73%);基于内生细菌16SrDNA序列同源性分析及构建的系统发育树显示,五唇兰根部内生细菌分属于7属,即芽孢杆菌属(Bacillus)、伯克氏菌属(Burkholderia)、草酸菌属(Pandoraea)、土壤杆菌属(Agrobacterium)、类芽孢杆菌属(Paenibacillus)、泛菌属(Pantoea)、欧文氏菌属(Erwinia),其中优势属为芽孢杆菌属,次优势属为泛菌属和伯克氏菌属;多样性分析显示,土生型五唇兰根部内生细菌群落的Shannon多样性指数大于石生型五唇兰,不同生境下五唇兰根部内生细菌群落结构差异极显著(P0.01)。土生型五唇兰根部内生细菌群落优势属为芽孢杆菌属和泛菌属,石生型五唇兰根部内生细菌群落优势属为芽孢杆菌属和伯克氏菌属。     

不同外源氮对丛枝菌根真菌Rhizophagus irregularis侵染棉花植株和氮磷转运的影响

探究供应外源氮对接种AM真菌的棉花植株的侵染率和氮磷转运的影响。本文以棉花为研究对象,接种丛枝菌根真菌(Rhizophagus irregularis),向根外菌丝额外供应不同外源氮,测AM真菌的侵染率、棉花植株株高、地上部和地下部鲜重、叶绿素含量、菌根精氨酸含量、地上部的氮磷含量。试验结果显示:不同外源氮条件下,AM真菌对棉花植株的生物量无显著性影响;外源氮的供应均提高了AM真菌的侵染率和棉花植株地上部的氮含量,但硫酸铵和硝酸钾更能促进AM真菌侵染宿主植物,提高宿主植物菌根精氨酸水平和地上部氮含量;除了尿素,其他氮源处理均能明显提高棉花植株地上部磷含量,其中,精氨酸最为显著。说明在AM共生系统中外源氮的供应对宿主植物生长无显著作用,但促进AM真菌侵染宿主植物,并能提高宿主植物氮磷含量。     

宿主植物栽培密度对AM真菌生长发育的影响

温室盆栽条件下宿主植物高粱(SorghumvulgarePers.)的栽培密度对丛枝菌根(Arbuscularmycorrhizae,AM)真菌Glomusmosseae(Nicol.&Gerd.)Gerdemann&Trappe生长发育的影响试验结果表明:60株/盆密度处理的根外菌丝量及孢子数均高于其它处理。在一定栽培密度下(20~60株/盆),植株根系可溶性糖浓度与根外菌丝量呈显著负相关,与菌根侵染率呈显著正相关。植株根中磷浓度与根外菌丝量、根外菌丝量与孢子数均呈显著正相关。植株根中磷浓度与菌根侵染率呈显著负相关。结果说明:适当密植虽对植株生长有一定影响,但却促进了真菌的生长,此时菌根共生体有可能由互惠共生开始向偏利共生或弱寄生转化。密植作为一种调控手段,在菌剂生产中能获得较大数量的侵染根段、菌丝及孢子等繁殖体。     

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.     

Studies of Mycorrhizal Fungi of Chinese Orchids and Their Role in Orchid Conservation in China—A Review

China has over 1,200 species of native orchids in nearly 173 genera. About one fourth of native species are of horticultural merit. Some species are of Chinese medicinal value. In fact, the demand on orchid species with high Chinese medicinal values such as Gastrodia elata, Dendrobium offcinale, along with demands on species of cultural importance, such as those in the genus of Cymbidium, is a major factor causing wild populations to diminish and in some cases, drive wild populations to the brink of extinction. These market demands have also driven studies on the role of mycorrhizal fungi in orchid seed germination, seedling and adult growth, and reproduction. Most of these mycorrhizal studies of Chinese orchids, however, are published in Chinese, some in medical journals, and thus overlooked by the mainstream orchid mycorrhizal publications. Yet some of these studies contained interesting discoveries on the nature of the mycorrhizal relationships between orchids and fungi. We present a review of some of these neglected publications. The most important discovery comes from the mycorrhizal studies on G. elata, in which the researchers concluded that those fungi species required to stimulate seed germination are different from those that facilitate the growth of G. elata beyond seedling stages. In addition, presence of the mycorrhizal fungi associated with vegetative growth of post-seedling G. elata hindered the germination of seeds. These phenomena were unreported prior to these studies. Furthermore, orchid mycorrhizal studies in China differ from the mainstream orchid studies in that many epiphytic species (in the genus of Dendrobium, as medicinal herbs) were investigated as well as terrestrial orchids (mostly in the genus Cymbidium, as traditional horticultural species). The different responses between epiphytic and terrestrial orchid seeds to fungi derived from roots suggest that epiphytic orchids may have a more general mycorrhizal relationship with fungi than do terrestrial orchid species during the seed germination stage. To date, orchid mycorrhizal research in China has had a strongly commercial purpose. We suggest that this continuing research on orchid mycorrhizal relationships are a solid foundation for further research that includes more rare and endangered taxa, and more in-situ studies to assist conservation and restoration of the endangered orchids. Knowledge on the identities and roles of mycorrhizal fungi of orchids holds one of the keys to successful restoration and sustainable use of Chinese orchids.     

Fungal specificity bottlenecks during orchid germination and development

Fungus-subsidized growth through the seedling stage is the most critical feature of the life history for the thousands of mycorrhizal plant species that propagate by means of 'dust seeds.' We investigated the extent of specificity towards fungi shown by orchids in the genera Cephalanthera and Epipactis at three stages of their life cycle: (i) initiation of germination, (ii) during seedling development, and (iii) in the mature photosynthetic plant. It is known that in the mature phase, plants of these genera can be mycorrhizal with a number of fungi that are simultaneously ectomycorrhizal with the roots of neighbouring forest trees. The extent to which earlier developmental stages use the same or a distinctive suite of fungi was unclear. To address this question, a total of 1500 packets containing orchid seeds were buried for up to 3 years in diverse European forest sites which either supported or lacked populations of helleborine orchids. After harvest, the fungi associated with the three developmental stages, and with tree roots, were identified via cultivation-independent molecular methods. While our results show that most fungal symbionts are ectomycorrhizal, differences were observed between orchids in the representation of fungi at the three life stages. In Cephalanthera damasonium and C. longifolia , the fungi detected in seedlings were only a subset of the wider range seen in germinating seeds and mature plants. In Epipactis atrorubens , the fungi detected were similar at all three life stages, but different fungal lineages produced a difference in seedling germination performance. Our results demonstrate that there can be a narrow checkpoint for mycorrhizal range during seedling growth relative to the more promiscuous germination and mature stages of these plants' life cycle.     

Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids

Dust seeds that germinate by obtaining nutrients from symbiotic fungi have evolved independently in orchids and 11 other plant lineages. The fungi involved in this 'mycoheterotrophic' germination have been identified in some orchids and non-photosynthetic Ericaceae, and proved identical to mycorrhizal fungi of adult plants. We investigated a third lineage, the Pyroleae, chlorophyllous Ericaceae species whose partial mycoheterotrophy at adulthood has recently attracted much attention. We observed experimental Pyrola asarifolia germination at four Japanese sites and investigated the germination pattern and symbiotic fungi, which we compared to mycorrhizal fungi of adult plants. Adult P. asarifolia, like other Pyroleae, associated with diverse fungal species that were a subset of those mycorrhizal on surrounding trees. Conversely, seedlings specifically associated with a lineage of Sebacinales clade B (endophytic Basidiomycetes) revealed an intriguing evolutionary convergence with orchids, some of which also germinate with Sebacinales clade B. Congruently, seedlings clustered spatially together, but not with adults. This unexpected transition in specificity and ecology of partners could support the developmental transition from full to partial mycoheterotrophy, but probably challenges survival and distribution during development. We discuss the physiological and ecological traits that predisposed to the repeated recruitment of Sebacinales clade B for dust seed germination.     

Mycorrhizal association and morphology in orchids

We investigated the mycorrhizal associations in 31 adult wild or cultivated green orchids (22 epiphytic, 8 terrestrial, and 1 species with both epiphytic and lithophytic life-forms) from different vegetation types of Western Ghats, southern India. All the orchids examined were mycorrhizal with the extent of colonization varying with species and life-forms. Mycorrhizal association has been reported for the first time in 25 orchids. The entry of mycorrhizal fungi into the roots was mostly through root hairs. In certain epiphytic species, the fungal entry was directly through the epidermis. The fungi formed highly coiled hyphal structures (pelotons) within the root cortex, and their size was related to the cell size. The fungal invasion of the cortical cells was through cell-to-cell penetration. The cortical cells contained intact and lysed pelotons, and their ratio varied with species and life-forms. No significant relationship existed between root hair characteristics and the extent of colonization. Chlamydospores and microsclerotia-like structure were frequently found within the cortical and root hair cells. The liberation of fungal reproductive structures was by spiral dehiscence of the root hairs.     

Untangling above‐ and belowground mycorrhizal fungal networks in tropical orchids

Orchids typically depend on fungi for establishment from seeds, forming mycorrhizal associations with basidiomycete fungal partners in the polyphyletic group rhizoctonia from early stages of germination, sometimes with very high specificity. This has raised important questions about the roles of plant and fungal phylogenetics, and their habitat preferences, in controlling which fungi associate with which plants. In this issue of Molecular Ecology, Martos et al. (2012) report the largest network analysis to date for orchids and their mycorrhizal fungi, sampling a total of over 450 plants from nearly half the 150 tropical orchid species on Reunion Island, encompassing its main terrestrial and epiphytic orchid genera. The authors found a total of 95 operational taxonomic units of mycorrhizal fungi and investigated the architecture and nestedness of their bipartite networks with 73 orchid species. The most striking finding was a major ecological barrier between above‐ and belowground mycorrhizal fungal networks, despite both epiphytic and terrestrial orchids often associating with closely related taxa across all three major lineages of rhizoctonia fungi. The fungal partnerships of the epiphytes and terrestrial species involved a diversity of fungal taxa in a modular network architecture, with only about one in ten mycorrhizal fungi partnering orchids in both groups. In contrast, plant and fungal phylogenetics had weak or no effects on the network. This highlights the power of recently developed ecological network analyses to give new insights into controls on plant–fungal symbioses and raises exciting new hypotheses about the differences in properties and functioning of mycorrhiza in epiphytic and terrestrial orchids.     

Arbuscular mycorrhizal fungal communities change among three stages of primary sand dune succession but do not alter plant growth

Plant interactions with soil biota could have a significant impact on plant successional trajectory by benefiting plants in a particular successional stage over others. The influence of soil mutualists such as mycorrhizal fungi is thought to be an important feedback component, yet they have shown benefits to both early and late successional plants that could either retard or accelerate succession. Here we first determine if arbuscular mycorrhizal (AM) fungi differ among three stages of primary sand dune succession and then if they alter growth of plants from particular successional stages. We isolated AM fungal inoculum from early, intermediate or late stages of a primary dune succession and compared them using cloning and sequencing. We then grew eight plant species that dominate within each of these successional stages with each AM fungal inoculum. We measured fungal growth to assess potential AM functional differences and plant growth to determine if AM fungi positively or negatively affect plants. AM fungi isolated from early succession were more phylogenetically diverse relative to intermediate and late succession while late successional fungi consistently produced more soil hyphae and arbuscules. Despite these differences, inocula from different successional stages had similar effects on the growth of all plant species. Host plant biomass was not affected by mycorrhizal inoculation relative to un‐inoculated controls. Although mycorrhizal communities differ among primary dune successional stages and formed different fungal structures, these differences did not directly affect the growth of plants from different dune successional stages in our experiment and therefore may be less likely to directly contribute to plant succession in sand dunes.     

菌根真菌与兰科植物氮营养关系的研究进展

兰科植物是典型的菌根植物。兰菌根是兰科植物根与真菌形成的菌根共生体。兰菌根真菌的营养来源影响宿主植物的生活方式和营养水平。氮是植物生长的主要限制因子。兰科植物具有富集氮的特征, 其组织和器官的氮含量通常高于同生境中的其他植物。该文综述了兰菌根真菌类别、兰科植物氮营养特征和兰菌根的氮转移机制等的研究进展, 以期为兰科植物资源的保护、再生及可持续利用的相关研究提供参考和借鉴。     

Mycorrhizal acquisition of inorganic phosphorus by the green-leaved terrestrial orchid Goodyera repens

BACKGROUND AND AIMS: Mycorrhizal fungi play a vital role in providing a carbon subsidy to support the germination and establishment of orchids from tiny seeds, but their roles in adult orchids have not been adequately characterized. Recent evidence that carbon is supplied by Goodyera repens to its fungal partner in return for nitrogen has established the mutualistic nature of the symbiosis in this orchid. In this paper the role of the fungus in the capture and transfer of inorganic phosphorus (P) to the orchid is unequivocally demonstrated for the first time. METHODS: Mycorrhiza-mediated uptake of phosphorus in G. repens was investigated using spatially separated, two-dimensional agar-based microcosms. RESULTS: External mycelium growing from this green orchid is shown to be effective in assimilating and transporting the radiotracer (33)P orthophosphate into the plant. After 7 d of exposure, over 10 % of the P supplied was transported over a diffusion barrier by the fungus and to the plants, more than half of this to the shoots. CONCLUSIONS: Goodyera repens can obtain significant amounts of P from its mycorrhizal partner. These results provide further support for the view that mycorrhizal associations in some adult green orchids are mutualistic.