铁皮石斛组培苗与菌根真菌共培养过程中的相互作用

doi:10.3773/j.issn.1005-264x.2009.03.002

植物生态学报 ›› 2009, Vol. 33 ›› Issue (3): 433-441.DOI: 10.3773/j.issn.1005-264x.2009.03.002

铁皮石斛组培苗与菌根真菌共培养过程中的相互作用

金辉¹^,²^,⁵(), 许忠祥³, 陈金花¹^,⁴, 韩素芬², 葛颂¹, 罗毅波¹^,^*()

1 中国科学院植物研究所系统与分子进化植物学国家重点实验室,北京 100093
2 南京林业大学森林资源与环境学院,南京 210037
3 南京出入境检验检疫局,南京 210001
4 海南大学园艺园林学院,海南儋州 571737
5 中国科学院研究生院,北京 100049

收稿日期:2008-08-15 接受日期:2009-01-13 出版日期:2009-08-15 发布日期:2009-05-31
通讯作者: 罗毅波
作者简介:*E-mail: luoyb@ibcas.ac.cn
第一联系人：
E-mail: comefine@ibcas.ac.cn
基金资助:
国家科技支撑项目珍稀濒危鸟类和植物繁育技术与示范(2008BAC39B05);国家自然科学基金(30371190)

INTERACTION BETWEEN TISSUE-CULTURED SEEDLINGS OF DENDROBIUM OFFICINALE AND MYCORRHIZAL FUNGUS (EPULORHIZA SP.) DURING SYMBIOTIC CULTURE

JIN Hui¹^,²^,⁵(), XU Zhong-Xiang³, CHEN Jin-Hua¹^,⁴, HAN Su-Fen², GE Song¹, LUO Yi-Bo¹^,^*()

¹State Key Laboratory of Systematic and Evolutionary Botany, Chinese Academy of Sciences, Beijing 100093, China
²College of Forest Resources and Environment, Nanjing Forestry University, Nanjing 210037, China
³Entry-Exit Inspection and Quarantine Bureau, Nanjing, 210001
⁴College of Horticulture and Landscape Architecture, Hainan University, Danzhou, Hainan 571737, China
⁵Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Received:2008-08-15 Accepted:2009-01-13 Online:2009-08-15 Published:2009-05-31
Contact: LUO Yi-Bo

摘要/Abstract

摘要：

由于人为的滥采滥挖和野外生境的退化, 使得铁皮石斛 (Dendrobium officinale) 这种名贵的中药材一直处于极度濒危的状态。为了从菌根真菌的角度给人工保育铁皮石斛提供理论指导, 对铁皮石斛的组织培养苗人工接种‘GDB181’菌株 (Epulorhiza sp.) 。培养60d后, 接菌苗平均鲜重增长率比对照苗高出了84.8%。在营养元素含量方面, 接菌苗的B、Si、Fe、Cu和Mn元素含量的净增率分别为780%、533%、192%、191%和128%, 均在100%以上;其他元素含量也有不同程度的增加 (除Zn外), 结果证明两者有效地建立了共生关系。在显微和超微结构的观察中发现:真菌菌丝随机破坏铁皮石斛的根被入侵到外皮层, 并从外皮层细胞不断扩展延伸到皮层的大型细胞, 最后在大型细胞中被分解消化。在真菌侵染过程中, 被侵染的皮层细胞的细胞壁严重扭曲变形, 菌丝在皮层细胞形成菌丝结, 菌丝结常位于细胞核附近或包围细胞核。在皮层的大型细胞中, 菌丝细胞被植物的溶酶体包围, 部分或全部被消解, 出现脱壁或失去细胞质甚至成为空腔等变化, 最终形成衰败的菌丝残骸, 溶酶体也随之消失。溶酶体分布越多的部位, 菌丝细胞消解变形越严重。含有菌丝残骸的皮层细胞可被新侵染的菌丝重新定殖, 这一菌丝侵染被消化再侵染的过程在铁皮石斛生长发育过程中可不断重复发生。

关键词: 相互作用, 共生, 显微结构, 铁皮石斛, 瘤菌根菌属

Abstract:

Aims Dendrobium officinale is an invaluable Chinese medicinal herb in China that has been threatened by over-collection and habitat deterioration. Understanding the relationship between orchids and mycorrhizae is important for the conservation of orchids. Our aim is to examine the rela-tionship between seedling of D. officinale and mycorrhizae.

Methods The strain of GDB181 was identified by internal transcribed spacer (ITS) sequencing. We inoculated tissue culture seedlings of D. officinale with GDB181 (Epulorhiza sp.) and used ICP-AES to measure the mineral element content of the seedlings. We observed slices of the orchid mycorrhizae under light and electron microscopes.

Important findings Seedlings of D. officinale and the inoculation strain formed symbiosis effectively. The growth of seedlings was promoted by the mycorrhizal fungus. As compared with the control, in-oculated seedlings had 84.8% greater average rate of increase of fresh weight. The mineral contents were elevated except Zn; the contents of B, Si, Fe, Cu and Mn in inoculated seedlings were increased by780%, 533%, 192%, 191% and 128%, respectively. Fungal hyphae entered the exodermis by breaking the velamen randomly and infected other cells continuously by means of penetrating the cell wall. Lastly, the hyphae inhabited the large cells of the cortex and were digested. The cell wall of the cortical cells were distorted and deformed by infection by the hyphae, and many pelotons formed in exodermis and cortex. Pelotons were often found near or encircling the nucleus. The invaded fungal hyphae were surrounded and dissolved by lysosomes partly or completely. With changes of the hyphae from losing cell wall or cytoplasm to remaining cavum, the lysosomes disappeared gradually. More hyphae were dissolved and deformed if there were more lysosomes near them. The cortex cells containing degenerated hyphae were frequently recolonized by hyphae, and the hyphal digestion and the reinfection of cortex cells occurred repeatedly throughout all growing stage of D. officinale.

Key words: interaction, symbiosis, microstructure, Dendrobium officinale, Epulorhiza sp.

金辉, 许忠祥, 陈金花, 韩素芬, 葛颂, 罗毅波. 铁皮石斛组培苗与菌根真菌共培养过程中的相互作用. 植物生态学报, 2009, 33(3): 433-441. DOI: 10.3773/j.issn.1005-264x.2009.03.002

JIN Hui, XU Zhong-Xiang, CHEN Jin-Hua, HAN Su-Fen, GE Song, LUO Yi-Bo. INTERACTION BETWEEN TISSUE-CULTURED SEEDLINGS OF DENDROBIUM OFFICINALE AND MYCORRHIZAL FUNGUS (EPULORHIZA SP.) DURING SYMBIOTIC CULTURE. Chinese Journal of Plant Ecology, 2009, 33(3): 433-441. DOI: 10.3773/j.issn.1005-264x.2009.03.002

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链接本文: https://www.plant-ecology.com/CN/10.3773/j.issn.1005-264x.2009.03.002

https://www.plant-ecology.com/CN/Y2009/V33/I3/433

图/表 4

图1 GDB181菌株的DNA ITS PCR扩增图谱及培养60 d后的接菌苗和对照苗 A:示接种菌株DNA上的ITS区域的大小 The length of ITS reigon of the mycorrhizal fungus M:Marker I:引物ITS1/ITS4扩增的片段Amplified by ITS1/ITS4 II:引物ITS4/ITS5扩增的片段Amplified by ITS4/ITS5 B:示接菌60d后的菌根苗 (黑色箭头示接种菌) Seedlings of Dendrobium officinale cultured symbiotically on DE with mycobionts‘GDB181’after 60 d (black arrow shows the mycorrhizal fugus) C:培养60 d后未接菌的对照苗The control seedlings

Fig.1 The electrophoretic picture of ITS PCR and seedlings of Dendrobium officianle

表1 与菌根真菌共培养60 d后铁皮石斛苗营养元素含量

Table 1 Nutrition elements content of the inoculated seedlings

图2 菌根真菌侵染铁皮石斛的过程 AF:有活力的菌丝Active fungus CW:细胞壁Cell wall P:菌丝结Peloton F:真菌菌丝Fungus N:细胞核Nucleus DF:消解衰败菌丝Degenerate fungus Pl:质体Plastid VE:根被Velamina CO:皮层Cortex VB:髓Vascular Bundle A:根被外的菌丝细胞The fungi lie in the out of velamina, ×20 000 B:菌丝随机破坏根被入侵并在皮层中形成菌丝结 (箭头示破损的根被) Shows the hyphae infected randomly and formed pelotons in the cortex cells (black arrow shows the destructed cell wall), ×200 C:菌丝破坏皮层细胞的细胞壁从一个细胞进入另一个细胞The hyphae infected by breaking the cell wall, ×10 000 D:皮层细胞菌丝结及具菌丝由一个细胞进入另一个细胞 (箭头示菌丝穿壁) The pelotons (black arrows show the penetrating hyphae), ×400 E:染菌细胞的细胞壁严重扭曲变形The deformed and distorted cell wall, ×4 000 F:菌丝细胞在细胞核附近被消解, 细胞核变形膨大The degenerated fungus and deformed nucleus, ×6 000 G:边缘松散并开始消解的菌丝结及部分消解的细胞核The degenerated hyphae and nucleus, ×1 000H:未染菌的皮层细胞, 示正常完整的细胞核及其他细胞器The normal root cell, shows the intact nucleus and other cell organelle, ×3 500 I:未染菌的根The normal root slice, ×100

Fig.2 The course of mycorrhizal fungus infect Dendrobium officinale

图3 菌根真菌的消解过程 FCW:菌丝细胞壁Fungal cell wall G:菌丝细胞的淀粉粒Granulose V:液泡Vacuole DS:桶孔隔膜Dolipore septum M:细胞膜Membrane L:溶酶体Lysosome H:菌丝Hypha A:结构完整, 有活力的菌丝细胞Active fungus, ×25 000 B:逐渐开始消解的菌丝细胞Degenerating hypha cell, ×20 000 C:细胞壁及质膜已消失, 原生质体开始释放的菌丝细胞Degenerated hypha cell, ×35 000 D:有溶酶体积聚的菌丝细胞 (纵切) The hypha cell was encircled with lysosomes, ×6 000 E:菌丝细胞崩解, 溶酶体包围并进入内部空腔中The lysosomes encircled and entered the degenerated fungus, ×10 000 F:新入侵菌丝细胞及开始消解的菌丝细胞Active and degenerate hypha in one cortex cell, ×15 000 G:处于不同消解阶段的菌丝细胞Active and degenerate hypha in one cortex cell, ×15 000 H:上张方框区域的放大Active and degenerate hypha in one cortex cell, ×40 000 I:菌丝细胞消解后留下的残骸The degenerated fungus, ×15 000

Fig.3 The ultrastructure of mycorrhizal fungi in the course of being digestd

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铁皮石斛组培苗与菌根真菌共培养过程中的相互作用

INTERACTION BETWEEN TISSUE-CULTURED SEEDLINGS OF DENDROBIUM OFFICINALE AND MYCORRHIZAL FUNGUS (EPULORHIZA SP.) DURING SYMBIOTIC CULTURE

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