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1 微生物学通报 Microbiology China JAN 20, 2011, 38(1): by Institute of Microbiology, CAS 研究报告春兰菌根真菌 CL-3 菌株的鉴定及共生体系的构建 * 陈敏庞基良杨洁琼周月兰 ( ) 摘要 : 采用分离自野生春兰 (Cymbidium goeringii) 根部的真菌 CL-3 菌株, 进行了春兰内生菌根真菌的人工接种再分离及其共生培养研究通过对 CL-3 菌株形态学观察和 ITS 序列同源性分析, 发现该菌株 ITS 序列与 Acremonium strictum 的亲缘关系最为接近, 序列同源性为 100% 用 CL-3 菌株接种春兰组培苗, 接种后 2 个月可从组培苗中再分离获得该菌株, 且 CL-3 菌株处理苗的鲜重增长率达 80.5%, 经方差分析, 与对照相比有显著差异通过石蜡切片和染色, 在已接种的组培苗的根部组织中可观察到 CL-3 菌株存在, 表明 CL-3 菌株能与组培幼苗成功建立共生培养体系关键词 : 春兰, 菌根真菌, ITS 序列, 共生培养 A mycorrhizal fungi CL-3 from Cymbidium goeringii: characterization and establishment of symbiotic associations CHEN Min * PANG Ji-Liang YANG Jie-Qiong ZHOU Yue-Lan (College of Life and Environmental Science, Hangzhou Normal University, Hangzhou, Zhejiang , China) Abstract: The fungal strain CL-3 was isolated from the root of wild Cymbidium goeringii with using conventional isolation method. The inoculum of strain CL-3 was used to study its interaction with its host plant. According to the morphological features examination and rdna ITS sequence analysis, the closest relative of the strain was Acremonium strictum, with the similarity of 100%. After two months of symbioses culture, the fresh biomass of C. goeringii inoculated with CL-3 increased by 80.5%, and the endophytic CL-3 was re-isolated from the seedlings. The statistics showed that the difference between inoculation group and control group was significant. The presence of the fungus in the tissues was studied with paraffin-cut section. The results showed that the fungus was observed in inner tissues of roots, indicating that the symbiosis relationship between Cymbidium goeringii and fungi could be established. 基金项目 : (No. Z ) * 通讯作者 :Tel: ; : mchen63@163.com 收稿日期 : ; 接受日期 :

2 30 微生物学通报 2011, Vol.38, No.1 Keywords: Cymbidium goeringii, Mycorrhizal fungi, ITS sequence, Symbiosis culture (Cymbidium georingii) (Cymbidium),,,,, [1],,,, [2],,, ( ),,, [3],,,, [4], CLB111 CLB113 MLX102, 70.6% 70.2% 68.6% [5],,, Richardson [6] EDS (Energy-dispersive spectrometry) :, Mg 2+ Ca 2+ K + CL-3, ITS, (Acremonium strictum), CL-3, CL-3 1 材料与方法 1.1 实验材料 : CL-3, (Cymbidium goeringii), : 1.2 培养基 PDA : 200 g 1 cm 3, 30 min, 20 g, 20 g, 1 L,,, Pa 30 min, ph (1 L): 10 g, 20 g, 5 g, IBA 1.0 g, NAA 0.5 g, ml, ml, ml, 1 mol/l KNO 3 15 ml, 1 mol/l NH 4 NO 3 8 ml, 1 mol/l CaCl ml, 1 mol/l KH 2 PO ml, 1 mol/l MgSO ml, ph 5.8, Pa 20 min - I (1 L): 5 g, 10 g, 5 g, ml, ml, ml, 1 mol/l KNO 3 15 ml, 1 mol/l NH 4 NO 3 8 ml, 1 mol/l CaCl ml, 1 mol/l KH 2 PO ml, 1 mol/l MgSO ml, ph, Pa 20 min - II (1 L): 10 g, - III (1 L): 15 g, 1.3 试剂 Taq PCR 1.4 形态学观察及鉴定 [7], 1.5 PCR 扩增和 DNA 测序菌种在 PDA 培养基上 25 C 培养 3 5 d, 取新

3 : CL-3 31 [8] 鲜培养物, 参照龙良鲲等的方法提取总 DNA ITS (ITS1: 5 -TCCGTAG GTGAACCTGCGG-3, ITS4: 5 -TCCTCCGCTTATT GATATGC-3 ) PCR (50 µl): 5 μl 10 Taq buffer, 4 µl dntps (2.5 mmol/l), 0.5 µl Taq DNA (5 U/µL), 5 µl MgCl 2 (25 mmol/l), 0.5 µl (10 µmol/l), 0.5 µl (10 µmol/l), 0.5 µl, 50 µl PCR : 94 C 5 min; 94 C 30 s, 54 C 45 s, 72 C 60 s, 30 ; 72 C 10 min PCR 5 µl, 1.0%, UVI 1.6 GenBank 比对 GenBank (BLAST), (ATCC) (DSM) 1.7 兰花组培苗接种培养和生物量的测定,,,, 3, 1,, CL-3 PDA ( 0.5 cm),, 1, ( ), 5 25 C, Lux, h 3,,,,, (%), ( ) 100/ [9] 1.8 菌根真菌的重分离 2 mm, PDA, 25 C 5 7 d,,,,, 1.9 菌根石蜡切片的制作 4 cm,, 5 mm, FAA,,,, 8 µm,,, [10] 2 结果 2.1 CL-3 菌株的形态鉴定 CL-3 PDA 25 C 7 d, 4.5 cm,,,,,,,,, ( 1), [7], (Acremomium) 图 1 CL-3 菌株在 PDA 培养基的菌落形态 (A) 及菌体特征 (B) Fig. 1 Colony (A) and conidiophores (B) of CL-3 strain on PDA medium 2.2 CL-3 菌株的分子鉴定 CL-3 DNA ITS, GenBank ( AY083232) BLAST,, Acremonium strictum 100%, GenBank 6

4 32 微生物学通报 2011, Vol.38, No.1 ITS, Treedrawing ( 2), CL-3 Acremonium strictum,, CL-3 Acremonium strictum 2.3 CL-3 菌株和兰花组培苗共培养体系 CL-3 3 -, CL-3 1, 71% 100%,,,, 2, % 80.5% 44.7%,, (P<0.05), - Ⅱ (P<0.01), ( 1) ITS ( ),, CL-3 100%, CL-3 ( ), CL-3, CL 处理苗菌根结构显微观察 CL-3 ( 3A),,,, ;, ( 3B), [11] 3 讨论 3.1 兰科菌根真菌的分离与鉴定,,,,, [12] 19 Fig. 2 图 2 基于 rdna ITS 序列的系统发育树 Phylogenetic tree of Acremonium spp. based on the rdna ITS sequence Strain Table 1 表 1 CL-3 菌株和兰花组培苗在共生培养基上的共培养结果 The co-culture result of seedlings and fungus on symbiotic culture medium Survival rate (%) Increment of fresh biomass (%) Re-isolated strains Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ CL * 80.5** 44.7* CK Note: * P<0.05; ** P<0.01.

5 : CL-3 33 图 3 组培苗菌根的显微结构 Fig. 3 Microstructure of the mycorrhizal root of tissue-cultured seedling : A: ; B:. VE: ; CO: ; P: ; N:. Note: A: Cortex with pelotons; B: Higher magnification of the cortical cells infected by fungi. VE: Velamen; CO: Cortex; P: Peloton; N: Nucleus. 26,,,, Ceratorhiza Epulorhiza Moniliopsis [13], 41,, [14], (Pleospora), [15], 5,, [16] CL-3, (Acremomium) (Acremonium strictum),, 3.2 兰科植物与菌根真菌的共生关系 Cameron [17], N P, 14 C,, 14 C,,,,,, [10] % 67.3% 62.4%, CL-3, 2,,, 80.5%, 24.2%,,,,,,, (Rhizoctonia. solani) (Rhizoctonia goodyeraerepentis),, 3

6 34 微生物学通报 2011, Vol.38, No.1 MS, 3,,,,,, [18],, CL-3,, CL-3 - II,,,, 80.5%, 参考文献 [1]. [M]. 2. :, 1993: [2],. [M]. 1. :, 2004: 433. [3] Lee SS, Park SS, Kim TJ, et al. Effect of orchid habitat soil on growth of tissue cultured Cymbidium konran and C. goeringii and root infection of orchid mycorrhizal fungus[j]. Journal of the Korean Society for Hoticultural Science, 1997, 38(2): [4],,,. [J]., 2007, 27(3): [5],,. [J]., 2005, 25(2): [6] Richardson KA, Peterson RL, Currah RS. Seed reserves and early symbiotic protocorm development of Plantanthera hyperborean (Orchidaceae)[J]. Canadian Journal of Botany, 2003, 70: [7]. [M]. :, 1979: 567. [8],,,. AM DNA PCR-DGGE [J]., 2005, 24(4): [9],,,. [J]., 1996, 15(4): [10],,,. [J]., 2002, 21(3): [11] Andersen TF. A comparative taxonomic study of Rhizoctonia sensulato employing morphological, ultrastructural and molecular methods[j]. Mycological Research, 1996, 100(9): [12],,. [J]., 1998, 19(4): [13],,,. [J]., 2010, 30(13): [14],,,. [J]., 2010, 23(3): [15],,,. [J]., 2008, 30(3): [16],,,. [J]., 2008, 36(17): , [17] Cameron DD, Leake JR, Read DJ. Mutualistic mycorrhiza in orchids: evidence from plant-fungus carbon and nitrogen transfers in the green-leaved terrestrial orchid Goodyera repens[j]. New Phytologist, 2006, 171(2): [18] Dijk E, Eck ND. Effects of mycorrhizal fungi on in vitro nitrogen response of some Dutch indigenous orchid species[j]. Canadian Journal of Botany, 1995, 73:

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