硬叶兰种子的迁地共生萌发及有效共生真菌的分离和鉴定

兰科植物的种子原地和迁地共生萌发技术是近年发展起来的开展兰科植物种子和共生真菌研究的有效方法。该研究对兰属(Cymbidium)附生植物硬叶兰(C. mannii)开展了种子的迁地共生萌发研究, 试图获得其种子萌发的有效真菌。利用硬叶兰成年植株根部周围的树皮、苔藓、枯枝落叶、腐殖质等作为培养基质, 进行种子的共生培养。在培养133天后, 成功地获得了处于不同阶段的已萌发种子、原球茎和幼苗, 并从原球茎中分离得到一种瘤菌根菌属(Epulorhiza)真菌。用所分离到的FCb4菌株和一种从兜唇石斛(Dendrobium aphyllum)分离到的胶膜菌属(Tulasnella) FDaI7菌株和硬叶兰种子在燕麦琼脂培养基上进行共生萌发, 设置不接菌作为对照处理, 以检验FCb4菌株对硬叶兰种子萌发的有效性。经过58天的培养, 不接菌的对照处理中种子没有萌发, 接种FCb4和FDaI7菌株的处理都有很高的种子萌发率, 两种接菌处理在不同光照条件下的种子萌发率均无显著性差异。但暗培养条件下, 种子萌发形成原球茎后, 表现出生长停滞的趋势, 仅有很少的原球茎继续生长达到幼苗阶段, 说明原球茎发育后期与幼苗发育阶段需要光照。在光照条件下, 接种FCb4菌株处理中达到幼苗阶段种子的比例为(25.67 ± 9.27)%, 显著高于接种FDaI7菌株处理的(3.04 ± 2.27)% (W = 56, p = 0.026, Mann-Whitney U-test), 表明此研究中分离到的瘤菌根菌属真菌能有效地促使硬叶兰种子萌发并生长发育到幼苗阶段。     

共生真菌对兰科植物种间杂交后代种子萌发的效应

石斛属(Dendrobium)植物在种子共生萌发过程中与真菌有着较为专一的共生关系, 为探讨这种共生关系在种间杂交后代上的进化和适应, 深入理解兰科植物和真菌共生关系的形成机制, 该研究利用能有效促进铁皮石斛(Dendrobium officinale)和齿瓣石斛(D. devonianum)种子萌发形成幼苗, 并具有较强专一性的胶膜菌属(Tulasnella)真菌SSCDO-5和瘤菌根菌属(Epulorhiza)真菌FDd1, 开展真菌对铁皮石斛和D. tortile种间杂交种子萌发效应的研究。结果表明, 在真菌与种子共生培养68天时, SSCDO-5菌株和FDd1菌株都能有效地促进杂交种子形成原球茎和幼苗, 两个接菌处理之间无显著差异, 来源于铁皮石斛的SSCDO-5菌株不但没有表现出优势, 反而在杂交石斛幼苗形成率上低于来源于齿瓣石斛的FDd1菌株(SSCDO-5: (22.13 ± 6.62)%; FDd1: (29.53 ± 5.51)%); SSCDO-5菌株和铁皮石斛在幼苗形成和发育阶段的共生专一性并没有在杂交后代上得到遗传或表现, 或者说是杂交打破了这种专一性的共生关系, 使得杂交后代能够和不同的真菌建立新的共生关系。该结果不支持关于共生真菌专一性是石斛属植物杂交后代形成的重要限制因素的假设, 推测石斛属植物在幼苗分化和发育阶段与真菌这种专一性的共生关系是在适应特定生态环境的过程中形成和建立的。     

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。     

共生真菌对兰科植物种间杂交后代种子萌发的效应

石斛属(Dendrobium)植物在种子共生萌发过程中与真菌有着较为专一的共生关系,为探讨这种共生关系在种间杂交后代上的进化和适应,深入理解兰科植物和真菌共生关系的形成机制,该研究利用能有效促进铁皮石斛(Dendrobium officinale)和齿瓣石斛(D.devonianum)种子萌发形成幼苗,并具有较强专一性的胶膜菌属(Tulasnella)真菌SSCDO-5和瘤菌根菌属(Epulorhiza)真菌FDd1,开展真菌对铁皮石斛和D. tortile种间杂交种子萌发效应的研究。结果表明,在真菌与种子共生培养68天时,SSCDO-5菌株和FDd1菌株都能有效地促进杂交种子形成原球茎和幼苗,两个接菌处理之间无显著差异,来源于铁皮石斛的SSCDO-5菌株不但没有表现出优势,反而在杂交石斛幼苗形成率上低于来源于齿瓣石斛的FDd1菌株(SSCDO-5:(22.13±6.62)%; FDd1:(29.53±5.51)%); SSCDO-5菌株和铁皮石斛在幼苗形成和发育阶段的共生专一性并没有在杂交后代上得到遗传或表现,或者说是杂交打破了这种专一性的共生关系,使得杂交后代能够和不同的真菌建立新的共生关系。该结果不支持关于共生真菌专一性是石斛属植物杂交后代形成的重要限制因素的假设,推测石斛属植物在幼苗分化和发育阶段与真菌这种专一性的共生关系是在适应特定生态环境的过程中形成和建立的。     

带叶兜兰种子原地共生萌发及有效菌根真菌的分离与鉴定

为获得带叶兜兰(Paphiopedilum hirsutissimum)种子萌发的共生真菌,采用原地共生萌发技术获得了2株自然萌发的小幼苗,并分离和筛选出了有效的种子萌发共生菌——瘤菌根菌(Epulorhiza sp.)。为验证分离菌株对带叶兜兰种子萌发的有效性,将Phs34号菌株与带叶兜兰种子在灭菌后的原生境基质上进行室内共生萌发试验,结果表明,经过6周的培养,对照组没有观察到种子的萌发;接菌的种子胚明显膨大,突破种皮,形成原球茎,平均萌发率为(58.35±3.41)%。这表明分离得到的瘤菌根菌能促进带叶兜兰的种子萌发。     

云南杓兰菌根真菌组成及共生关系研究

杓兰属(Cypripedium)植物因具有较高的观赏和药用价值而长期被过度采集,已成为濒危植物。利用菌根技术进行杓兰属植物的保护和人工栽培,需要获得其可培养的菌根真菌。该研究采用分离培养法和共生回接方法,研究了云南杓兰菌根真菌菌群组成及其共生关系。结果表明:(1)从10株云南杓兰300块毛根组织中分离获得126株内生真菌,归属为3个菌属,分别是胶膜菌属(Tulasnella)73株、伏革菌属(Corticium)36株、角担菌属(Ceratobasidium)17株。其中,胶膜菌属为优势菌群,占总菌株数量的57.94%。(2)6株供试菌株中,4株菌株可显著缩短种子的萌发过程,6株菌株对幼苗的生长有显著的促进作用。(3)从中筛选获得一株CY-18高效促生真菌,对云南杓兰种子共生萌发和幼苗共生生长有极显著的促进作用。该研究结果为更好地利用菌根技术进行杓兰属植物资源的保护与可持续利用奠定了基础。     

兰科药用植物手参种子的真菌共生萌发

手参Gymnadenia conopsea是一种地生型兰科植物,是我国的传统中药,同时也是蒙药、藏药常用药,在西藏地区亦作食用。现代药理学研究表明手参具有非常好的药理活性,然而当前手参还不能进行人工栽培,市场需求完全依赖野生资源,因此资源短缺仍然是制约该种药用植物进一步开发利用的瓶颈。兰科植物种子萌发及早期的幼苗生长均需真菌参与,基于此,本文从手参根系中进行了真菌分离,并获得一株菌株,分子鉴定为角担菌属Ceratobasidium GS2。将GS2菌株与手参种子进行共培养,可明显促进手参种子的原球茎的形成并最终分化成幼苗。手参种子共生萌发的成功对于实现手参的种质保育、人工栽培和野生居群的生态恢复均具有重要的意义。     

铁皮石斛种子的室内共生萌发

由于人为的采挖和原生境的破坏,使得铁皮石斛野生资源已濒临灭绝。因此,保护铁皮石斛野生资源及其生境,加快其野生资源的繁殖显得非常重要。以铁皮石斛种子(TTC染色显示种子生活力为77.65%)为材料,与分离自2种野生兰科植物根部的4株共生真菌 (C20来自铁皮石斛,L12,L24b 和 L28来自美花石斛)在燕麦培养基上进行共生萌发。经过18周的共生培养,4株真菌均不同程度地促进了铁皮石斛种子的萌发,其中菌株L24b (Epulorhiza)和L28 (Epulorhiza)显著提高了种子的萌发率,分别比对照高出26.51%和12.20%,但未形成幼苗,只是处于原生分生组织阶段(阶段3);菌株C20(Epulorhiza)和L12 (Alternaria) 虽没有显著提高种子的萌发率,但对原球茎的发育和幼苗的生长有明显的促进作用;而对照的种子仍然处于膨大转绿期,即萌发阶段(阶段2)。同时发现,TTC染色显示的铁皮石斛种子生活力要高于种子共生萌发的萌发率(除了菌株L24b)。研究结果表明:种子生活力染色检测的活力值只代表种子所具有的潜在的萌发能力,而不能代表实际的萌发率。在异地条件下,铁皮石斛与共生真菌间没有严格的专一性,可以与瘤菌根菌属、链格孢属真菌形成共生关系。菌株C20和L12能促进萌发后的种子进一步分化成幼苗。这两个菌株为铁皮石斛的人工优质栽培和野外种群的建立提供了可能。     

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。     

东北红松纯林菌根外生菌根真菌群落与环境因子的相关性

外生菌根是木本植物根系与真菌形成的共生结构,外生菌根真菌在红松等外生菌根树种的定植与森林生态系统的保持方面起到至关重要的作用。明确菌根系统内外生菌根真菌群落组成是揭示菌根共生机制的前提条件。本研究利用Illumina Hiseq测序平台对生长季内红松纯林内根围土壤及菌根样品ITS2区进行高通量测序,分析其外生菌根真菌群落结构随季节的变化规律,同时通过统计学的方法分析了红松根系微生态中外生菌根真菌群落结构组成变化与其他生物因素、非生物因素的相关性。结果如下：（1）从6月份到10月份,5个月的菌根样品测序共得到741个真菌OTUs,利用FUNGuild数据库分析,其中85个OTUs归类为外生菌根真菌,优势属（相对丰度>5）为蜡壳菌属Sebacina、乳牛肝菌属Suillus、Meliniomyces、红菇属Russula、棉革菌属Tomentella、须腹菌属Rhizopogon和缘腺革菌属Amphinema。6月份菌根中外生菌根真菌的多样性最大,显著高于其他月份。（2）红松林外生菌根真菌群落组成受到土壤pH、有效磷含量、有效钾含量和土壤有效氮含量的影响,它们与外生菌根真菌优势属相对丰度呈现正相关或负相关。（3）根围土壤内真菌是影响红松根系外生菌根真菌相对丰度的另一重要因素,其中,包括普可尼亚属Pochonia、产丝齿菌属Hyphodontia、镰刀菌属Fusarium、Collembolispora、枝穗霉属Clonostachys、Apodus、鹅膏属Amanita在内的土壤真菌与根内外生菌根真菌的相对丰度呈线性关系。同时,超过85%的根内外生菌根真菌与同一取样地的土壤共有,可以认为侵染和扩散是红松根内外生菌根真菌群落形成的主要方式,同时兼有植物根系的选择,因为根内并不包括所有土壤中存在的外生菌根真菌,其机制需要进一步人工模拟试验验证。     

Addition of fungal inoculum increases germination of orchid seeds in restored grasslands

Grasslands restored on arable land often retain high residual nutrients, modified soil biota, and lower plant species diversity. Establishment of rare plant species with complex multitrophic interactions, typical of undisturbed nutrient-poor environments, may be hindered by the absence of interacting organisms. We hypothesised that the addition of a mycorrhizal symbiont improves the seed germination of orchids that crucially depend on fungi. We focused on grasslands restored on arable land 1–15 years ago featuring residual mineral nutrients and low organic matter contents compared to semi-natural grasslands and on four orchid species differing in the level of mycorrhizal specificity: high – Anacamptis pyramidalis and Orchis mascula – and low – Platanthera bifolia and Gymnadenia conopsea. Five fungal isolates obtained from non-green underground mycorrhizal orchid seedlings (protocorms) or adults' roots were tested for orchid-fungus compatibility under conditions in vitro. Orchid seeds inserted in retrievable seed packets were subsequently co-introduced with selected fungal isolates grown either on agar or sterilized hay into the soil of nine restored grasslands and incubated for twelve months. The identity of mycorrhizal fungi in retrieved protocorms was verified by molecular methods. The isolates that supported protocorm establishment in vitro enabled also protocorm formation in situ, but success rates differed among orchid species. While mycorrhizal specialists produced most protocorms after inoculation, the mycorrhizal generalists took advantage of naturally occurring fungi and produced some protocorms both in inoculated and uninoculated treatments. We showed that the addition of mycorrhizal fungi enhanced protocorm formation regardless of the modified soil environment, especially in mycorrhizal specialist orchids. This method may help to restore populations of native orchid species in their former distribution ranges, including farming-altered habitats.     

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid,Dendrobium aphyllum (Orchidaceae)

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P?Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P?P?Trichoderma suppressed seed germination by 26.4 % (P?Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P?Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.     

In vitro symbiotic seed germination of South Indian endemic orchid Coelogyne nervosa

Study on the dependence of orchids on fungi for seed germination and seedling development provides a mean for understanding the role of fungi in the orchid development process. The epiphytic orchid Coelogyne nervosa endemic to south India is exploited in an unsustainable manner for its therapeutic value. So a protocol for symbiotic seed germination was established for C. nervosa. We isolated a fungus by plating mycorrhizal root discs of the terrestrial orchid Eulophia epidendreae and identified it as Epulorhiza sp., by sequencing the internal transcribed spacer (ITS) regions of the ribosomal RNA gene. Germination of C. nervosa seeds was higher when inoculated with Epulorhiza sp. Uninoculated seeds of C. nervosa ceased to develop soon after the initiation of germination, and the embryo failed to rupture the seed testa. The isolated fungal hyphae entered the germinating seeds either through the pores in-between the integuments, or through the rhizoids. After the fungal establishment (peloton formation) in embryonic cells, the embryo transformed into a protocorm and after 45 days, 66% of the germinated seeds were transformed into protocorms. Nevertheless, promeristem formation occurred only after fungal association. Sixty-three percent of the protocorms developed their first leaf by 90 days and 62% of these produced a second leaf by 120 days after fungal inoculation. All the seedlings in green leaf stage produced roots and contained fungal pelotons. Our results suggest that the Epulorhiza sp. could be successfully used in the in vitro production of C. nervosa for their reintroduction into its natural environment.     

同色兜兰的非共生萌发与快速繁殖研究

以授粉后不同发育时期的同色兜兰种子为材料,观察其形态特征和萌发过程,并探讨建立同色兜兰高效快繁体系的最佳条件。结果表明,种子发育中后期即授粉后210~240d为较适宜的采收期,授粉后210d的种子萌发率最高(达77.79%);1/4 MS和1/2MS为同色兜兰适宜的基本培养基,添加100mL/L椰乳或1g/L蛋白胨对种子萌发及原球茎生长和分化有明显的促进作用;添加1g/L活性炭对原球茎褐化有一定的抑制作用,但添加剂量不宜过大;添加香蕉汁和苹果汁对同色兜兰种子萌发和原球茎生长分化有抑制作用;暗处理对同色兜兰种子萌发无影响;分化后的原球茎在壮苗和生根培养基上培养120d即可得到4~5片叶、高3~5cm的同色兜兰健壮试管苗。     

Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand

Symbiotic seed germination of Pecteilis susannae (L.) Rafin was investigated using 11 fungal isolates recovered from roots of four Thai terrestrial orchids (P. susannae, Eulophia spectabilis, Paphiopedilum bellatulum and Spathoglottis affinis). Seed germination and protocorm development were evaluated up to 133 days after sowing. Protocorm development was most advanced, up to stage 5 (elongation of the first leaf), when seeds were cultured with 4 Epulorhiza isolates obtained from roots of P. susannae (CMU-Aug 028, 4.3%, CMU-Aug 007, 4.2%, and CMU-Aug 013, 2.2%) and E. spectabilis (CMU-STE 014, 3.9%). Moreover, stage 4 protocorm development (emergence of the first leaf) occurred with fungal isolates CMU-STE 011, 5.7%, (Epulorhiza sp.) and CMU-AU 212, 4.3%, (Tulasnella sp.) obtained from roots of E. spectabilis and S. affinis respectively. When seed was incubated without fungi (control), development was limited to stage 3 of protocorm development (appearance of promeristem). This is the first report of protocorm stage 5 development in P. susannae using compatible fungal symbionts. Optimization of seed germination and seedling fitness will assist the conservation and propagation of this orchid species and other terrestrial orchids in Thailand.     

Endophytic fungi from Pecteilis susannae (L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand

Eight endophytic fungi were isolated from roots of the threatened terrestrial orchid, Pecteilis susannae (L.) Rafin. Phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that seven isolates belonged to the genus Epulorhiza and one to Fusarium. All fungal isolates were cultured with orchid seeds collected from three field sites near Doi Suthep-Pui National Park, Chiang Mai, Thailand. Seed germination and protocorm development were evaluated up to 70 days after sowing. Percent symbiotic seed germination was highest (86.2%) when seeds were cultured with Epulorhiza (CMU-Aug 013). The protocorm development was the most advanced up to stage 2, continued embryo enlargement, or rupture of the testa, and the highest percentage was 17.8% when seeds were cultured with Epulorhiza (CMU-Aug 007). Without fungi, seed germination and protocorm development were 62.1% and 11.1%, respectively. The dependency of P. susannae on fungal symbionts for early seedling development is yet to be determined. Optimizing seed germination and seedling fitness will assist the conservation of this threatened orchid in Thailand.     

Variation in mycorrhizal performance in the epiphytic orchid <Emphasis Type="Italic">Tolumnia variegata in vitro</Emphasis>: the potential for natural selection

Symbiotic seed germination is a critical stage in orchid life histories. Natural selection may act to favor plants that efficiently use mycorrhizal fungi. However, the necessary conditions for natural selection – variation, heritability, and differences in fitness – have not been demonstrated for either orchid or fungus. With the epiphytic orchid Tolumnia variegata as a model system, we ask the following questions: (1) Do seeds from different individuals in a population differ in germination and seedling development in the presence of the same fungi? (2) Do different mycorrhizal fungi (Ceratobasidium spp.) differ in ability to stimulate seed germination and growth in T. variegata? And (3) are the Ceratobasidium isolates that best induce seed germination and seedling development more closely related to each other than to isolates that are less effective? We performed symbiotic seed germination experiments in vitro. The experiments were done using mycorrhizal fungi isolated from T. variegata; relationships among the fungi were inferred from nuclear ribosomal ITS sequences. We found significant variation for both symbiotic germination and seedling growth among biparental seed crops obtained from a population of T. variegata plants. Differences among Ceratobasidium fungi in seed germination were significant. The fungi that induced highest seed germination and seedling development belonged to two of four clades of Ceratobasidium. The two experiments show that there is potential for natural selection to act on orchid–fungus relationships. Given that orchids vary in performance, and that mycorrhizal fungi are not geographically distributed homogeneously, mycorrhizae may affect population size, distribution and evolution of orchids.