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。     

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。     

独蒜兰种子共生萌发研究

采用从独蒜兰分离的菌根真菌与其种子在燕麦培养基上共生萌发。结果表明GN21、GN23和GN24的作用效果较好, 其中GN21萌发率84.6%, 高于对照7个百分点, 达到显著性。同时也表明, 从同一兰科植物分离到的属于同一属的不同菌根真菌种, 有的能明显促进种子萌发, 有的却不能促进萌发, 甚至产生病斑, 引起原球茎死亡。     

菌根真菌对白及种子萌发和幼苗生根的作用

自然条件下,兰科植物需要依赖菌根真菌获得营养才能萌发。本研究对白及根和原球茎中分离的4株菌根真菌（WQ17-33、WQ17-43、JST-3和SL15-7）进行分子鉴定,并评价光照和黑暗条件下不同菌株促白及种子萌发和幼苗生根的效果。结果表明,4个菌株分别隶属于鬼伞属Coprinus、胶膜菌属Tulasnella、腊壳菌属Sebacina和Serendipita。在种子萌发前期（未形成叶子）进行暗培养较光照对菌株JST-13和SL15-7处理组原球茎阶段萌发具有显著的促进作用。不同菌株共生萌发效果不同,菌株SL15-7较其他处理原球茎和幼苗发育阶段的萌发率高。菌株JST-13和SL15-7处理组形成的幼苗较其他处理组强壮,定殖的菌丝团也较多,其幼苗生根效果也较对照组好。该研究表明白及可与多种不同类群的菌根真菌菌株形成共生关系,这些真菌在促进白及种子萌发和生根能力方面存在差异。     

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。     

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。     

独蒜兰种子共生萌发研究

采用从独蒜兰分离的菌根真菌与其种子在燕麦培养基上共生萌发.结果表明GN21、GN23和GN24的作用效果较好,其中GN21萌发率84.6%,高于对照7个百分点,达到显著性.同时也表明,从同一兰科植物分离到的属于同一属的不同菌根真菌种,有的能明显促进种子萌发,有的却不能促进萌发,甚至产生病斑,引起原球茎死亡.     

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。     

不同消毒和培养基条件对兰花菌根真菌分离的影响

研究了不同的消毒时间和培养基类型对兰花菌根真菌分离效果的影响,以期获得兰花菌根真菌的最佳分离方法.采用了次氯酸钠和酒精作为消毒试剂,发现依次浸没于75 %酒精60 s、2 %次氯酸钠60 和75 %酒精30 s的组合消毒条件下分离效果最佳;选择了3种培养基（PDA、CZA和MEA）,结果表明MEA培养基的分离效果最为稳定.通过组织分离法分离兰花菌根真菌,筛选得到最佳的分离培养条件,旨在为兰花菌根真菌的多样性以及共生效应研究提供研究基础.     

胶膜菌属真菌S7(Tulasnella sp.)及提取物对铁皮石斛种子萌发的影响

兰科植物种子细小,无胚乳,自然条件下依赖共生的真菌为其提供营养实现种子萌发.前期研究表明,在燕麦琼脂(oatmeal agar, OMA)培养基上与兰科铁皮石斛种子共生培养时,胶膜菌属真菌S7(Tulasnella sp.)能促进种子萌发.本文用S7培养物在6种基质上进行播种实验,考察S7的田间使用效果;将S7提取物分别加入OMA和MS培养基中,考察S7提取物对铁皮石斛种子萌发的影响.研究发现:(ⅰ)不使用S7,种子在3种基质上能够出芽,最高出芽率为1.96%±1.26%,在1种基质上能形成幼苗,最高成苗率为0.96%±1.07%.使用S7,种子在6种基质上都能够出芽并形成幼苗;种子在泥炭土:锯末(1:1)基质上的萌发效果最好,出芽率和成苗率分别达到26.68%±6.26%和20.97%±6.93%.这些结果验证了S7具有促进铁皮石斛种子萌发作用,可应用于田间播种,同时说明播种基质能够影响S7的作用.(ⅱ) OMA培养基中添加高浓度S7菌丝体提取物后,培养50天形成原球茎,比对照组提前了20天; 60天时形成叶片,对照组原球茎未见继续发育;培养80天种子萌发率为54.90%±2.46%,与对照组相比提高了57.6%(P0.05),出芽率为9.11%±4.17%,对照组仅为0.41%±0.38%.这些结果提示, S7菌丝体提取物中含有可以促进种子萌发与原球茎发育的物质.本研究为进一步探索该菌的实际应用价值,并探索共生真菌中促进兰科种子萌发的活性代谢产物奠定了基础,为兰科种子共生萌发的机制研究开拓了新的思路.     

Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae)—preliminary evidence for the symbiotic culture advantage

Eulophia alta (Linnaeus) Fawcett & Rendle seeds collected from the Florida Panther National Wildlife Refuge (Collier County, FL; FPNWR) were used in a screen of five asymbiotic orchid seed germination media to determine their effectiveness in promoting germination and protocorm development. In addition, 10 fungal isolates collected from the roots of E. alta at sites in the FPNWR, Highlands County (FL), and Goethe State Forest (Levy County, FL; GSF), and a fungal isolate from the roots of Spiranthes brevilabris collected from GSF were screened for their effectiveness at promoting in vitro symbiotic germination of E. alta seeds. After 18 weeks asymbiotic culture, seeds sown on PhytoTechnology Orchid Seed Sowing Medium germinated to a higher percentage (87.9%) and had a higher percentage of protocorms with developing protomeristems (32.7%) than seeds cultured on Knudson C, Malmgren Modified Terrestrial Orchid Medium, ?-strength Murashige & Skoog, or Vacin & Went Modified Orchid Medium. Significantly more leaf-bearing protocorms were observed on PhytoTechnology Orchid Seed Sowing Medium (0.8%) and Vacin & Went Modified Orchid Medium (1.3%) than other media tested. Of the fungi tested, one fungal isolate (Ealt-396) promoted germination to 69.0%, two isolates promoted germination to less than 0.75% and did not support further protocorm development, and eight isolates did not support germination. Seeds co-cultured in darkness with Ealt-396 grew more rapidly than asymbiotic seedlings following germination. In addition, co-cultured (=symbiotic) seedlings continued to develop more rapidly than asymbiotic seedlings upon transfer to 16/8 h light/dark photoperiod. Symbiotic seed culture of E. alta may be a more desirable method of propagation since protocorms develop more rapidly than seeds sown on asymbiotic media. Symbiotic seedlings may be more appropriate for reintroduction to natural areas than asymbiotic seedlings since symbiotic seedlings could serve to inoculate soils with a germination promoting mycobiont.     

In vitro propagation and mass scale multiplication of a critically endangered epiphytic orchid, Gastrochilus calceolaris (Buch.-Ham ex J.E.Sm.) D.Don. using immature seeds

In vitro asymbiotic seed germination potential of its immature seeds (36 weeks after pollination) of G. calceolaris was successfully tested on three different agar gelled nutrient media i.e. Murashige and Skoog (MS), Mitra et al. (M) and potato dextrose agar (PDA). Seeds germinated within 15.75+/-0.75 to 35.75+/-0.75 days in the three different media. The protocorms developed therefrom subsequently differentiated into first leaf and root primordia, and complete seedlings were obtained within 111.25+/-1.25 to 141.25+/-1.25 days on MS and M media. The protocorms, though failed to differentiate further on basal PDA medium, despite repeated subculturings, incorporation of peptone (P; 1 gl(-1)), yeast extract (YE; 2 gl(-1)) and coconut water (CW; 20%) in the medium proved beneficial in inducing differentiation, in these germinating entities. Additional use of growth additives (P/YE/CW), in general, favoured better germination, protocorm formation and seedling development. The optimal nutritional combination during seed germination, protocorm growth and multiplication and seedling development was found to be CW (10%) enriched MS medium.     

兰科植物种子共生萌发真菌多样性及共生萌发机制研究进展

自然环境下,兰科植物种子细小无胚乳,需要和适宜的真菌共生才能萌发,因而与真菌有天然的共生关系。自身繁殖率低加之近年来栖息地环境破坏导致兰科植物资源更加濒危,而通过筛选适合的真菌进行种子的共生萌发可以有效地实现兰科植物的种质保育及濒危种类野生居群的生态恢复。本文对地生型、附生型以及腐生型等兰科植物已发现的萌发真菌的多样性进行了系统地梳理,发现担子菌门的胶膜菌科、角担菌科以及蜡壳耳目真菌为已报道共生萌发真菌的主要类群;同时对兰科植物种子的共生萌发机制,包括形态学机制、营养机制和分子机制等方面的相关研究进行了归纳论述,但是当前关于兰科植物和真菌互作机制方面的研究还相对较少,许多问题需要进一步明确。本文对共生萌发真菌在兰科植物保育和繁育中的应用以及共生萌发机制的研究等方面具有一定的参考价值。     

In-vitro predatory activity of nematophagous fungi from Costa Rica with potential use for controlling sheep and goat parasitic nematodes

In tropical and subtropical regions of the world, parasitic diseases are a main cause of losses in livestock productivity. The increased acquired resistence to anthelmintics by gastrointestinal nematodes, requires biological control be considered as a potential feasible and effective alternative. The most effective natural soil enemies of nematodes are nematophagous fungi. In order to collect and identify predator nematophagous fungi (PNF), samples were obtained from 51 farms distributed throughout the seven provinces of Costa Rica. The origin samples included: soil from different crops (potatoes, tomatoes, bananas, ornamental plants, squash and coffee); animal feces (cattle, sheep, goat and horse); soil and fallen leaves from forest; and plants with signs of nematode infection. Each sample was processed using three techniques for the extraction of fungi from soil: sprinkling technique, soil dilution and humidity chamber. Twenty four strains of nematophagous fungi were found in 19 farms; 83.3% of the fungi were isolated by sprinkling technique. The following fungi were identified: Arthrobotrys oligospora (n = 13); Candelabrella musiformis (n = 9); and for the first time there was isolation of A. conoides (n = 1) and A. dactyloides (n = 1) in the country. Moreover, 16 strains from Trichoderma (n=13), Beauveria (n = 1), Clonostachys (n = 1) and Lecanicillium (n = 1) were obtained. In addition, pH of each possible fungal isolation source was measured, and it varied from 5.2 to 9.9, however PNF isolates fell within the range of 5.6 to 7.5. The PNF strains were cultivated in four different media for the production of chhlamydospores: potato dextrose agar (PDA); corn meal agar (CMA); malt extract agar (MEA) and potato carrot agar (PCA). Out of these cultures, 95.8% of the strains formed chlamydospores primarily in the PCA. Of these strains, the profilic spore producers were subjected to ruminant artificial gastrointestinal conditions. A total of 14 fungi were tested, out of which 42.9% survived the digestive analysis. Neither A. conoides nor A. dactyloides were viable following the in vitro gastrointestinal test. The PNF isolated in this study demonstrated an action against ovine and caprine gastrointestinal nematodes and are candidates for use in biological control of these organisms. Among these microorganisms, Candelabrella musiformis appears to be the most promising fungi for use as a biological control agent in Costa Rica.     

构树可培养内生真菌的多样性初探

构树Broussonetia papyrifera为桑科重要的资源植物。为调查逆境构树内生真菌的多样性,我们对铅锌矿区构树根、茎、叶和果实各部位进行内生真菌分离鉴定,从中分离得到187株内生真菌,根据形态学与分子生物学方法鉴定为20属,其中链格孢属Alternaria（50.27%）、镰孢属Fusarium（11.76%）为优势属。不同培养基的内生真菌分离率结果表明,马铃薯葡萄糖琼脂培养基（PDA）分离出的菌株数最多,占总数的73.96%;构树不同部位的菌株分离数目显示,叶中分离出的菌株较多,占38.02%。在属的分类水平上,根、茎、叶、果实不同部位的真菌数量也存在明显差异。Shannon-Wiener多样性指数和Simpson多样性指数分析结果提示不同样地的构树内生真菌的多样性也存在差异。Chao1指数和ACE指数也提示构树内生真菌丰富度与样地和组织有关。     

Enhanced symbiotic seed germination of Cypripedium macranthos var. rebunense following inoculation after cold treatment

Cypripedium macranthos var. rebunense is a well-known wild orchid in Japan, and is considered to be a symbol for rare plant conservation. A fungus isolated from roots of C. macranthos var. rebunense induced symbiotic germination of the species in vitro. Cold treatment of the seeds at 4°C prior to fungal inoculation was required for the symbiotic germination. Changing the timing of inoculation of the fungus to the seeds greatly improved germination frequency. Maximum germination was attained after seeds were inoculated just after the cold treatment for 12 weeks, and approximately 20% of the seeds developed into protocorms more than 1 mm long. These results suggest that fungal inoculation takes place at the beginning of spring in nature, and the tough impervious seed coat may preserve the seed from the infection during autumn and winter seasons. The lengthy culture period of more than 16 weeks at 20°C on the same medium with the fungus caused gradual browning and rot of the protocorms. By elimination of the fungus with a fungicide and by transfer to a nutrient rich medium, approximately 20% of the protocorms developed into healthy plantlets. The methods obtained here appear to be applicable to symbiotic germination of many other threatened Cypripedium spp.