外生菌根菌侵染红皮云杉微观机制的研究
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摘要
运用林木菌根学、菌物学、分子生物学及石蜡制片技术等试验方法，对2株红皮云杉高效外生菌根菌的培养特性、生理学习性、分类地位的分子生物学确认以及侵染红皮云杉微观机制进行了系统研究，研究结果表明：
菌株025菌丝生长最适温度为25℃，最适碳源为葡萄糖，最适pH值为6.5～7.5；菌株035菌丝生长最适温度为20℃，最适碳源为蔗糖，最适pH值为7.0；2菌株均在无氮下生长最佳，供试氮源对2菌株的菌丝生长均有一定的抑制作用。菌株025生长较菌株035快。
首次利用通用引物ITS1与ITS4从高效菌株025和035中扩增出rDNA ITS序列，2个菌株ITS序列长分别为663bp和662bp，GenBank登陆号分别为EF531717和EF531716。通过比对，初步确定菌株025为Cortinarius sp．，与粘柄丝膜菌[Ccollinitus(AY083181)]相似性为95％；菌株035为Suillus sp．，与点柄乳牛肝菌[S．granulatus(AJ272409)]相似性为94％。
不同外生菌根菌对云杉苗木根部侵染的部位、侵染过程不同，云杉苗木根部皮层细胞的反应亦不同。菌株035首先是菌套内部菌丝作用于根尖组织表皮，使其逐渐消失；通过已消失的表皮孔隙进入到皮层组织内部，与皮层组织细胞作用，依附，蔓延，形成哈蒂氏网，逐渐形成菌根，根尖组织细胞颜色变深，细胞壁增厚。菌株025则是菌套内部菌丝依附在表皮上，菌丝接触皮孔和表皮缝隙，由皮孔和表皮缝隙侵入皮层细胞，在侵入的同时，皮孔和表皮缝隙变形逐渐消失，在菌丝的作用下，表皮才逐渐消失，菌丝进入皮层细胞后，蔓延方式与菌株035相似。供试外生菌根菌株在植物根部形成菌丝套，菌丝体深入红皮云杉根部皮层细胞间形成哈蒂氏网，但不进入到根部内皮层组织细胞中。
不同外生菌根菌侵染苗木后，形成菌根的时间长短不同。人工培养条件下生长快的菌株，形成菌根的时间短。菌株025侵染红皮云杉营养根形成菌根速度远远大于菌株035，苗木接种菌株025后，约21d即可形成菌根；而菌株035侵染速度较慢，接种一个多月后才与红皮云杉根系形成菌根。不同外生菌根菌在红皮云杉根尖形成的菌套厚度与苗木生长有一定的正相关性。
The studies on culture and physiological characteristics, the molecular biological classification and infection mechanism of two ectomycorrhizal fungi of Picea koraiensis were done by the methods of mycorrhizicology, mycology, molecular biology and technique of paraffin section. The results show as follow:
To strain 025 growth, the optimum temperature is 25℃, optimum carbon source is glucose, optimum pH is 6.5～7.5; To strain 035 growth, the optimum temperature is 20℃, optimum carbon source is sucrose, optimum pH is 7.0. Both two strains grow best without nitrogen source, the nitrogen sources have certain inhibitory effects to the growth of 2 strains. The growth of strain 025 is faster than that of strain 035.
The rDNA ITS sequence was amplified from strain 025 and 035 with the universal primers ITS 1 and ITS4 for the first time. Their ITS sequence length are 663bp and 662bp, the GenBank accession number is EF531717 and EF531716 respectively. By comparing with strains in GenBank, it is confirm that strain 025 is Cortinarius sp., the comparability between strain 025 and Cortinarius collinitus(AY083181) is 95%; strain 035 is Suillus sp., the comparability between strain 035 and Suillus granulatus (AJ272409) is 94%.
The infection position and course of the different ectomycorrhizal fungi of Picea koraiensis are different, and the response of root cortex cells of Picea koraiensis seedlings is also different. The mycelium inner mantle of strain 035 firstly effect on the epidermis of root tip, and make them disappeared gradually; then enter into the cortex tissue through the gaps of disappeared cortex tissue, and forming Hartig net and then forming ectomycorrhiza gradually. The colour of root tip cells turn deeper, and the cellwall became thicker. The mycelium inner mantle of strain 025 adhere on the cortex of root, then enter into cortex cells through cortical pore and cortex gap, at same time the cortical pore and cortex gap disappear gradually, and then cortex disappear gradually. 2 ectomycorrhizal fungi can form mantle and Hartig net, but the mycelium can not enter into cells ofendoderm.
The period of forming ectomycorrhiza is different for different ectomycorrhizal fungi. The faster the fungi grow, the shorter the ectomycorrhiza form. To the speed for ectomycorrhiza forming, strain 025 is far greater than strain 035. The thickness of mantle round the root rip has certain relation to seedling growth.
菌株025菌丝生长最适温度为25℃，最适碳源为葡萄糖，最适pH值为6.5～7.5；菌株035菌丝生长最适温度为20℃，最适碳源为蔗糖，最适pH值为7.0；2菌株均在无氮下生长最佳，供试氮源对2菌株的菌丝生长均有一定的抑制作用。菌株025生长较菌株035快。
首次利用通用引物ITS1与ITS4从高效菌株025和035中扩增出rDNA ITS序列，2个菌株ITS序列长分别为663bp和662bp，GenBank登陆号分别为EF531717和EF531716。通过比对，初步确定菌株025为Cortinarius sp．，与粘柄丝膜菌[Ccollinitus(AY083181)]相似性为95％；菌株035为Suillus sp．，与点柄乳牛肝菌[S．granulatus(AJ272409)]相似性为94％。
不同外生菌根菌对云杉苗木根部侵染的部位、侵染过程不同，云杉苗木根部皮层细胞的反应亦不同。菌株035首先是菌套内部菌丝作用于根尖组织表皮，使其逐渐消失；通过已消失的表皮孔隙进入到皮层组织内部，与皮层组织细胞作用，依附，蔓延，形成哈蒂氏网，逐渐形成菌根，根尖组织细胞颜色变深，细胞壁增厚。菌株025则是菌套内部菌丝依附在表皮上，菌丝接触皮孔和表皮缝隙，由皮孔和表皮缝隙侵入皮层细胞，在侵入的同时，皮孔和表皮缝隙变形逐渐消失，在菌丝的作用下，表皮才逐渐消失，菌丝进入皮层细胞后，蔓延方式与菌株035相似。供试外生菌根菌株在植物根部形成菌丝套，菌丝体深入红皮云杉根部皮层细胞间形成哈蒂氏网，但不进入到根部内皮层组织细胞中。
不同外生菌根菌侵染苗木后，形成菌根的时间长短不同。人工培养条件下生长快的菌株，形成菌根的时间短。菌株025侵染红皮云杉营养根形成菌根速度远远大于菌株035，苗木接种菌株025后，约21d即可形成菌根；而菌株035侵染速度较慢，接种一个多月后才与红皮云杉根系形成菌根。不同外生菌根菌在红皮云杉根尖形成的菌套厚度与苗木生长有一定的正相关性。
The studies on culture and physiological characteristics, the molecular biological classification and infection mechanism of two ectomycorrhizal fungi of Picea koraiensis were done by the methods of mycorrhizicology, mycology, molecular biology and technique of paraffin section. The results show as follow:
To strain 025 growth, the optimum temperature is 25℃, optimum carbon source is glucose, optimum pH is 6.5～7.5; To strain 035 growth, the optimum temperature is 20℃, optimum carbon source is sucrose, optimum pH is 7.0. Both two strains grow best without nitrogen source, the nitrogen sources have certain inhibitory effects to the growth of 2 strains. The growth of strain 025 is faster than that of strain 035.
The rDNA ITS sequence was amplified from strain 025 and 035 with the universal primers ITS 1 and ITS4 for the first time. Their ITS sequence length are 663bp and 662bp, the GenBank accession number is EF531717 and EF531716 respectively. By comparing with strains in GenBank, it is confirm that strain 025 is Cortinarius sp., the comparability between strain 025 and Cortinarius collinitus(AY083181) is 95%; strain 035 is Suillus sp., the comparability between strain 035 and Suillus granulatus (AJ272409) is 94%.
The infection position and course of the different ectomycorrhizal fungi of Picea koraiensis are different, and the response of root cortex cells of Picea koraiensis seedlings is also different. The mycelium inner mantle of strain 035 firstly effect on the epidermis of root tip, and make them disappeared gradually; then enter into the cortex tissue through the gaps of disappeared cortex tissue, and forming Hartig net and then forming ectomycorrhiza gradually. The colour of root tip cells turn deeper, and the cellwall became thicker. The mycelium inner mantle of strain 025 adhere on the cortex of root, then enter into cortex cells through cortical pore and cortex gap, at same time the cortical pore and cortex gap disappear gradually, and then cortex disappear gradually. 2 ectomycorrhizal fungi can form mantle and Hartig net, but the mycelium can not enter into cells ofendoderm.
The period of forming ectomycorrhiza is different for different ectomycorrhizal fungi. The faster the fungi grow, the shorter the ectomycorrhiza form. To the speed for ectomycorrhiza forming, strain 025 is far greater than strain 035. The thickness of mantle round the root rip has certain relation to seedling growth.
引文
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[2] 李纯丽，王玲．红皮云杉的采种、催芽及育苗技术[J]．特种经济动植物，2005，(8)：37
[3] 楚孔利，郑秀云．对红皮云杉造林和透光抚育技术的研究[J]．林业勘查设计，2002，(2)：35
[4] Unger, F.. Beitrage Zur Kenntniss der parasitischen pflanzen[J]. Anatom ischphyciologische rheli. (Contribution to knowledge of parasitic plants, Anatomical phyciological part. )Ann. Wien. Mus. Nalurgeschichte, 1840, (2):13～60
[5] Lees, E.. On the parasitic growth of Monotropa hypopitys[J]. The Phytologist, 1841, (1):97～101
[6] Ryland, T. G.. On the nature of the byssoid substance found invensting the roots of Monotropa hypopitys[J].The Phytologist, 1842, (1):341～348
[7] Kamienski, F.M.. Die vegetationsorganen der Monotpopa hypopitys L [J]. Bot. Zei., 1881, (39) :458～461
[8] Frank, A. B.. Ueber die auk Wurzelsymbiose beruhende Ernahrunggewisser Baume dutch Unterirdische pilze[J]. Ber. Deut. 8ot. Ges., 1885, (3): 128～145
[9] 黄亦存，沈崇尧，裘维蕃．外生菌根的形态学、解剖学及分类学研究进展[J]．真菌学报，1992，11(3)：169～181
[10] 吴铁航．VA菌根真菌(球囊霉目)分类研究[J]．土壤学报，1994，(3l增刊)：26～37
[11] Melin, E. Studier over barrtradsplantans utveckling ⅰ rahumusm, Ⅱ Mycorrhizas utbildning hos tallplantan ⅰ olika rahunusformer[J].Medd Skogst Anst. Stockholm, 1927, (23):433～494
[12] Maeda, M.. The meaning of mycorrhiza in regard to systematic botany[J]. Kumamoto J. Sci. B., 1954, (3):57～84
[13] Trappe, J. M.. The fungus associates of ectotrophicmycorrhizae[J].Bot. Rev., 1962, (28):538～606
[14] 孟繁荣等编著．林木菌根学[M]．哈尔滨：东北林业大学出版社，1996
[15] 弓明钦，陈应龙，仲崇禄．菌根研究及应用[M]．第一版．北京：中国林业出版社，1997
[16] 郭秀珍，毕国昌．林木菌根及应用技术[J]．北京：中国林业出版社，1989：1～305
[17] 栾庆书，李立，李希桥．中国外生菌根研究的20年成就[J]．辽宁林业科技，2000，(6)：36～38
[18] Mark, D.H&Beattie, D.J.. Mycorrhizae-promising aid to timber grower[J]. For. Farmer, 1977, (36):6～9
[19] Miller, O.K. Jr. Taxonomy of ecto-and Eeoterdomycorrhizal fungi [J]. In "Methods & Principles of Mycorrhizal Research" (Ed. Schenck)P, 1982, 91～102
[20] 吴人坚，谭惠瓷．23种外生菌根真菌的生态分布[J]．林业科学，1983，19(3)：327～331
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