不同培养基对兰科药用植物手参原球茎共生真菌的分离效果

Effects of different media on the isolation of symbiotic fungi from protocorm of medicinal orchid Gymnadenia conopsea

JIANG Xiling, DING Wanlong, XING Xiaoke , ,*

Institute of Medicinal Plant Development, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100193, China

摘要

兰科植物手参Gymnadenia conopsea作为国家二级重点保护野生植物具有重要的药用价值。目前手参还未实现人工栽培,但其种子的真菌共生萌发已获成功。为明确除促萌发真菌外,还有哪些土著真菌参与了手参种子的萌发过程,本研究在自然条件下采用促萌发真菌伴播手参种子,获得了种子萌发形成的原球茎,进而对比了6种常见的培养基PDA (马铃薯葡萄糖琼脂培养基)、MMN (改良Melin-Norkrans培养基)、FIM (真菌分离培养基)、MEA (麦芽浸膏琼脂培养基)、CAM (胡萝卜葡萄糖琼脂培养基)和CMA (玉米粉琼脂培养基)对手参原球茎共生真菌分离效果的影响。共从6种培养基上分离获得了75个菌株,其中MMN、CAM、PDA、FIM、MEA、CMA培养基依次分离得到20株、16株、15株、11株、8株、5株菌。此外,真菌的多样性分析结果表明,MMN培养基的Chao 1、Shannon-Wiener和Simpson多样性指数最高,CAM和PDA培养基次之,CMA培养基最低。综上所述,真菌分离效果最好的是MMN培养基,其次是CAM和PDA培养基,而FIM和MEA培养基对真菌的分离效果影响不大,CMA培养基的分离效果最差。本研究结果可为其他兰科植物原球茎共生真菌的分离提供借鉴,所获得的菌株也有望进一步应用于功能菌剂的研发。

关键词：兰科种子;共生萌发;共生真菌分离;真菌多样性

Abstract

The terrestrial orchid Gymnadenia conopsea with important medicinal value has been listed in the National Key Protected Wild Plant (Category Ⅱ). At present, G. conopsea could not be cultivated artificially, however, seed symbiotic germination has been successfully achieved. In order to determine whether other indigenous fungi were also involved in seed germination process besides the initial germination-promoting fungi, isolation of symbiotic fungi from protocorms formed by germination-promoting fungi under natural conditions were performed on six common media PDA (potato dextrose agar medium), MMN (modified Melin-Norkrans medium), FIM (fungal isolation medium), MEA (malt extract ager medium), CAM (carrot agar medium) and CMA (corn meal agar medium), and the relative abundance were compared. In total, 75 fungal isolates were obtained from the 6 media, and the number of fungal isolates obtained from MMN, CAM, PDA, FIM, MEA and CMA was 20, 16, 15, 11, 8 and 5, respectively. The results of fungal diversity analysis showed that fungal diversity index (Chao 1, Shannon-Wiener, and Simpson diversity index) of the isolates from MMN medium was the highest, and that from CAM and PDA medium was less, while that from CMA medium was the lowest. In short, MMN is the most effective, followed by CAM and PDA, and FIM and MEA media have little effect, while CMA is the worst. Our results provide reference for the isolation of symbiotic fungi from protocorms of other orchids. The fungal strains obtained in this study hopefully could be further used for research purpose and development of functional microbial agents.

Keywords：orchid seed;symbiotic germination;isolation of symbiotic fungi;fungal diversity

兰科是世界上有花植物中最大的科之一,据估计已超过26 000种(Joppa et al. 2011),可与多种真菌共生,然而兰科植物由于种子微小,无胚乳,在自然条件下萌发困难,因此非常依赖真菌为种子萌发提供必要的营养(Smith & Read 2008;Rafter et al. 2016),从而促进种子萌发和植物的生长发育(Rasmussen & Rasmussen 2009),因而兰科植物与真菌有着天然的共生关系。菌根真菌已被认为是影响兰科植物分布的重要因素之一(McCormick et al. 2018),并在缓解植物生物和非生物胁迫如干旱、盐胁迫、极端温度、抗病等方面发挥着重要作用(McCormick & Jacquemyn 2014;Diagne et al. 2020)。目前认为兰科菌根真菌类群主要包括角担菌科Ceratobasidiaceae、胶膜菌科Tulasnellaceae和Serendipitaceae等(Dearnaley et al. 2012;Jacquemyn et al. 2017)。

为了实现兰科种子的萌发,通常采用的方法是从植物的根部或原球茎中分离共生真菌,这些分离得到的菌株在促进兰科植物种子萌发(Gao et al. 2020)、幼苗生长(Porras-Alfaro & Bayman 2007)以及对兰科植物的种群恢复方面具有重要作用(Li et al. 2021;Zhao et al. 2021),尤其是从原球茎中分离的共生真菌已被证明对种子萌发具有较好的促进效果(Selosse et al. 2017;Meng et al. 2019)。因此,分离和鉴定兰科植物原球茎共生真菌,对于揭示兰科植物共生真菌的多样性,筛选促进种子萌发的菌株,以及进一步研究共生真菌的生态功能等方面均具有重要意义(Yang et al. 2020;Zhao et al. 2021)。然而,原球茎共生真菌的成功分离受到多种因素的影响,如分离部位(Shao et al. 2020)、分离方法(Zhu et al. 2008)和培养基种类(黄芳和张春英 2013)等。其中培养基种类对于兰科植物共生真菌分离效果的影响较大,例如Shimura & Koda (2005)利用5种培养基AWA (酸性水琼脂培养基)、CWA (氯霉素水琼脂培养基)、PSA (马铃薯蔗糖琼脂培养基)、OMA1 (燕麦琼脂培养基)和GPA (绿豌豆琼脂培养基)对杓兰Cypripedium calceolus共生真菌进行分离,仅在CWA和OMA1培养基分离到3种类似丝核菌Rhizoctonia的真菌。由于不同真菌通常具有多样化和特定的营养需求,在缺乏特定营养物质的情况下难以生长,因此不同培养基对真菌分离的效果也通常存在显著差异(Gao & Liu 2010;Qi et al. 2012)。

手参Gymnadenia conopsea (L.) R. Br.是一种广域分布的地生型兰科植物,零星分布于亚洲的温带、亚热带以及欧洲北部地区,在我国主要分布于东北、华北、西北及四川、云南、西藏等地。手参为我国传统中药,也是藏药和蒙药的常用药,具有补肾益精、理气止痛等作用(Shang et al. 2017)。由于手参还未实现人工栽培,其开发和应用完全依赖野生资源,市场需求的不断攀升加之较低的自然繁殖率,其野生种群数量急剧下降,现已被我国《重点保护野生植物名录》列为二级保护植物(国家林草局和农业农村部,2021年9月7日, http://www.gov.cn/zhengce/zhengceku/2021-09/09/content_5636409.htm)。为实现手参资源的可持续利用,在前期研究中我们从手参成年植株根部分离获得了一个菌株Ceratobasidium GS2,并在实验室条件下首次成功实现了手参种子真菌共生萌发(高越等 2019;Gao et al. 2020),进而在自然条件下与该菌株伴播同样实现了手参种子的萌发(结果另文发表)。由于手参自然繁殖率较低,有关手参的有性繁殖还未见相关报道,另外,在野生条件下的土壤中找到其细微如尘的种子自然萌发所形成的原球茎十分困难,而前期在自然条件下手参种子的成功萌发则使我们获得手参的原球茎成为可能。因此,本研究拟明确在自然条件下,除了所使用的GS2菌株外,在种子萌发突破种皮后形成原球茎的过程中,有哪些土著真菌参与其中,因而采用不同的培养基对原球茎中的共生真菌进行了分离,并对比不同培养基对原球茎共生真菌的分离效果,以期为今后开展其他兰科植物原球茎共生真菌的分离提供参考。

1 材料与方法

1.1 手参原球茎来源1.1.1 菌株来源

GS2 (GenBank登录号OK655 751)是从手参根部分离出的一株促萌发真菌,其被证明可以在实验室条件下促进手参种子萌发和早期幼苗发育(高越等 2019;Gao et al. 2020)。

1.1.2 真菌栽培种制备

按照麦麸:树叶:锯末=2:1:1的比例制作固体基质,保持含水量在60%左右,然后将基质装入至500 mL培养瓶的2/3处,并在121 ℃下灭菌3 h。待基质冷却后,在超净工作台中向每个瓶子内接种5个1 cm×1 cm的GS2菌株接种块,用封口膜密封后置于25 ℃的暗培养室中培养1个月左右,待菌丝长满整个培养瓶后即可用作原位种子萌发实验的栽培种。

1.1.3 实验设计

实验用种子袋为8 cm×7 cm的可降解玉米纤维袋,共设计3组实验,每组100个种子袋,每袋装入约150粒种子。A组：接入2 g GS2栽培种;B组：接入2 g无菌基质;C组：仅含种子。

1.1.4 手参种子野外萌发实验

2019年4月在西藏林芝比日神山海拔3 700 m的林缘草地,将所有手参种子袋随机埋在深度为10-13 cm土壤中,并附上塑料标签以便回收。于2020年9月回收种子袋,发现A组中的手参种子已有原球茎形成(图1),而B组和C组中的手参种子均无萌发迹象。将收集到的含有原球茎的种子袋存于冰盒中带回实验室进行共生真菌的分离工作。

图1

图1  自然条件下GS2伴播手参种子的萌发

A：种子突破表皮;B：形成的原球茎. 标尺=500 μm

Fig. 1  Germinating seeds of Gymnadenia conopsea mixed with GS2 inoculum under natural condition.

A: Seeds break though testa; B: Protocorm formation. Bars=500 μm.

1.2 培养基类型

PDA培养基：马铃薯200 g,葡萄糖20 g,琼脂12 g,去离子水1 000 mL,马铃薯切块后加水煮沸20 min,过滤后取滤液定容至1 000 mL,121 ℃灭菌25 min。

MMN培养基：CaCl2 0.05 g,MgSO4 0.15 g,NaCl 0.025 g,FeCl3 (1%) 1.2 mL,KH2PO4 0.5 g,Vitamin B1 100 μg,(NH4)2HPO4 0.25 g,麦芽粉6 g, 葡萄糖10 g,柠檬酸0.2 g,琼脂12 g,去离子水1 000 mL,121 ℃灭菌25 min。

MEA培养基：麦芽粉30 g,大豆蛋白胨3 g,琼脂15 g,去离子水1 000 mL,121 ℃灭菌25 min。

FIM培养基：Ca(NO3)2·4H2O 0.5 g,KH2PO4 0.2 g,KCl 0.1 g,MgSO4·7H2O 0.1 g,酵母膏0.1 g,蔗糖5.0 g,琼脂12 g,去离子水1 000 mL,121 ℃灭菌25 min。

CMA培养基：玉米粉40 g,琼脂12 g,去离子水1 000 mL,121 ℃灭菌25 min。

CAM培养基：胡萝卜200 g,葡萄糖20 g,琼脂12 g,去离子水1 000 mL,胡萝卜切块后加水煮沸20 min,过滤后取滤液定容至1 000 mL,121 ℃灭菌25 min。

1.3 共生真菌的分离和纯化

选取已形成原球茎的5个种子袋,每个种子袋随机取10个原球茎,分离时采用上述6种培养基。制备好的培养基以1 cm直径的打孔器打孔,将培养基片转至新培养皿中,每皿间隔放入7片,每种培养基各制备6皿。将原球茎置于流水下冲洗干净,依次浸没在75%乙醇溶液中1 min,1.5% NaClO溶液1 min,75%乙醇溶液30 s,无菌水清洗6次,重复此操作。将已表面消毒的原球茎置于无菌的培养皿中,倒入少量无菌水,用回形针轻刮原球茎皮层,释放菌丝团,用封口膜封口后置于25 ℃黑暗条件下培养24 h (Zhu et al. 2008)。

真菌的分离采用单菌丝团分离培养法,具体操作如下：在体视显微镜下用移液枪从每份培养液中吸取单个菌丝团分别将其转接到直径1 cm圆形培养基(PDA、MMN、MEA、FIM、CMA和CAM培养基)上,封口膜封口后置于25 ℃黑暗条件下培养3-5 d。

在无菌条件下,用接种针挑取菌丝尖端0.5 cm左右的菌丝,转接至相应的培养基上,25 ℃黑暗条件下培养,反复纯化3次。将纯化后的菌株转接到试管中,4 ℃保存。

1.4 菌株鉴定1.4.1 形态学鉴定

根据《真菌鉴定手册》对从不同培养基分离的共生真菌进行形态学的初步鉴定(魏景超 1979)。

1.4.2 分子生物学鉴定

参照CTAB植物基因组DNA快速提取试剂盒(北京艾德莱生物科技有限公司)使用说明提取菌株的DNA。采用通用引物ITS1-OF/ITS4-OF (Waud et al. 2014)和ITS1/ITS4 (White et al. 1990)扩增ITS序列。25 μL的PCR扩增反应体系包括：12.5 μL PCR Master Mix,引物(10 μmol/L)各1 μL,DNA模板2 mL,用ddH2O补足至25 μL。PCR扩增程序：95 ℃预变性10 min;95 ℃变性15 s,58 ℃退火45 s,72 ℃延伸50 s,共35个循环;72 ℃再延伸7 min,4 ℃保存。取3 μL PCR产物用1%琼脂糖凝胶电泳检测,PCR产物由中美泰和(北京)生物科技有限公司进行双向测序。获得的序列在GenBank中进行BLAT比对,查找相似性最高的序列信息,并结合菌株形态学特征,确定真菌种类。

1.5 数据分析

为研究不同培养基上手参原球茎共生真菌类群的丰富度和多样性,采用相对多度(relative abundance)评估分离的真菌物种对群落总多度贡献的大小,以物种个体数量作为多度的测量指标(马克平 1994);采用Chao丰富度指数(Chao1 richness index)作为度量物种丰富度的指标(Chao 1984),辛普森多样性指数(Simpson diversity index) (Simpson 1949)和香农-威纳多样性指数(Shannon-Wiener diversity index) (Spellerberg & Fedor 2003)分析从不同培养基分离的共生真菌多样性。利用R软件包计算各参数(R Core Team 2016)。采用SPSS 26.0 (IBM SPSS Statistics,USA)进行统计学分析,统计检验为Kruskal-Wallis检验,以α=0.05作为检验水准。

2 结果与分析

2.1 手参原球茎共生真菌的群落组成

利用单菌丝团分离培养法从6种培养基共获得75株真菌,结合形态学和rDNA-ITS序列最终鉴定为30种,隶属12个科,相对多度最高的3个科分别是裂褶菌科Schizophyllaceae (42.86%)、耙齿菌科Irpicaceae (22.86%)和角担菌科Ceratobasidiaceae (11.43%) (图2A),每个菌株在不同培养基上的分离率见表1。

图2

图2  手参原球茎共生真菌科水平的相对多度

A：所有分离到的共生真菌的相对多度;B：每种培养基上分离到的共生真菌的相对多度

Fig. 2  Relative abundance of symbiotic fungi isolated from protocorm of Gymnadenia conopsea at family level in total (A) and each medium (B).

表1  真菌的分子鉴定及不同培养基上的相对多度

Table 1  Molecular identification and relative abundance of fungi on different media

样本
SampleGenBank登录号
GenBank accession No.鉴定结果
Proposed identification相对多度
Relative abundance (%)PDAMMNMEAFIMCMACAMZ-E-4A-1OL471317Basidiomycota sp.12.5Z-M-5A-1OL471298Ceratobasidium sp.1012.5B-F-5B-1OL471297Ceratobasidiaceae sp.13.318.2B-H-5A-1OL693805Chaetomium sp.6.3B-E-5C-1OL471318Coprinopsis sp.6.7B-F-5A-1OL471319Cryptomarasmius sp.9.1Z-H-4A-1OL693804Diatrype sp.6.3Z-E-5A-1OL693807Didymellaceae sp.6.7Z-C-5C-1OL693808Eutypella sp.20B-M-2A-1OL471289Funalia sp.5Z-M-4B-2OL471306Irpex sp. 16.759.16.3Z-M-5D-1OL471304Irpex sp. 26.759.118.8Z-E-4A-2OL471305Irpex sp. 312.5Z-F-4A-3OL471303Irpex sp. 46.71012.59.1Z-M-4C-1OL693802Lemonniera sp.5Z-E-4D-1OL693806Peroneutypa sp.6.752520Z-P-4D-1OL471316Polyporales sp.6.720Z-P-5A-1OL471295Schizophyllum sp. 16.7Z-C-5A-2OL471294Schizophyllum sp. 220Z-C-5B-1OL471292Schizophyllum sp. 320Z-E-4C-1OL471293Schizophyllum sp. 425Z-F-4A-2OL471291Schizophyllum sp. 5203012.527.318.8Z-H-4B-2OL471290Schizophyllum sp. 6159.112.5Z-M-2A-1OL471311Schizophyllum sp. 75Z-H-3A-2OL471296Schizophyllum sp. 89.16.3Z-M-5E-1OL693803Sordariomycetes sp.5Z-H-3C-1OL471314Typhula sp.6.3Z-H-5C-2OL471307Trametes sp. 16.3B-E-5A-1OL471308Trametes sp. 26.7B-E-5B-1OL471309Trametes sp. 36.7

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2.2 不同培养基对手参原球茎共生真菌的分离效果的影响

在从5组手参原球茎分离的75株共生真菌中,其中从MMN培养基上分离得到的真菌菌株数量最多(20株),略高于CAM培养基(16株)和PDA (15株)上分离得到的菌株数量,分离菌株数量最少的是CMA (5株)。对比担子菌与子囊菌的分离效果,共分离得到11株子囊菌,64株担子菌,仅FIM培养基上未分离到子囊菌,其余5种培养基中均分离到子囊菌;从6种培养基中都分离到了担子菌,其中在MMN和CAM培养基中均分离到了原始菌株GS2,在PDA和FIM培养基上分离到了角担菌科真菌B-F-5B-1。此外,在6种培养基中均分离到了裂褶菌科真菌,说明裂褶菌科真菌对培养基的选择性较低,其中MMN培养基对裂褶菌科的分离效果最好,其相对多度最高为30%;FIM对角担菌科真菌的分离效果最好,其相对多度最高为18.2%,CMA培养基对耙齿菌的分离效果较好,其相对丰度最高为18.8% (表1)。

6种培养基真菌的分离结果见表1,从PDA培养基上分离得到的真菌物种数均值最高,为2.40±0.80,其次是MMN (2.20±0.75)和CAM培养基(2.00±1.41),但不具有统计意义上的显著差异(P≥0.05);从CMA培养基中分离得到的真菌物种的均值最低,仅有1.00±0.63。MMN、CAM和PDA培养基上分离得到的真菌菌株平均值分别为4.00±1.27、3.20±1.47和3.00±1.90,FIM、MEA、CMA则较少,分别为2.20±0.98、1.60±1.02、1.00±0.63,6种培养基的分离得到的真菌菌株平均值存在显著差异(P<0.05)。综合真菌分离的物种和菌株数量,MMN培养基在对手参原球茎共生真菌显示了较为充分的分离效果。

2.3 不同培养基分离共生真菌的多样性

从6种培养基分离共生真菌的多样性具有显著性差异(Simpson指数,P<0.05;Shannon- Wiener指数,P<0.05),其中MMN培养基的Shannon-Wiener和Simpson指数最高,表明其具有较好的均衡性和多样性,其次是CAM和PDA培养基(表2)。CMA培养基的Shannon-Wiener和Simpson指数最低,这可能是CMA培养基对真菌的选择性较高,说明CMA培养基适用于分离和培养特定种属的真菌,例如,仅在CMA培养基上分离到了Diaporthaceae真菌。此外,不同培养基的真菌丰富度具有显著性差异(P<0.05) (表2),MMN培养基的Chao 1指数仍高于其他培养基。此外,6种培养基分离的真菌物种组成和相对多度略有差异,其中MMN和PDA的相对多度最高,而MEA和CMA培养基的相对多度最低(图2B),这可能是由于真菌对营养的要求不同所导致的对培养基的偏好性选择。因此,就真菌的物种丰富度和物种多样性而言,对真菌分离效果最好的培养基为MMN培养基,其次是CAM和PDA培养基。

表2  不同培养基对手参原球茎共生真菌的多样性影响

Table 2  Effects of different media on diversity of symbiotic fungi isolated from germinating seeds of Gymnadenia conopsea

培养基
Media物种数均值
Observed species菌株个体数均值
Number of isolatesChao 1丰富度指数
Chao1 richness index香农-维纳指数
Shannon-Wiener index辛普森指数
Simpson’s indexPDA2.40±0.80a3.00±1.90b3.80±2.41b0.79±0.41b0.62±0.11aMMN2.20±0.75a4.00±1.27a4.94±1.43a1.10±0.34a0.66±0.10aCAM2.00±1.41a3.20±1.47b4.20±1.08a0.98±0.26ab0.63±0.10aFIM1.60±1.02b2.20±0.98c3.00±1.76c0.70±0.35b0.48±0.26bMEA1.20±0.75b1.60±1.36c2.38±1.39c0.54±0.30c0.39±0.25cCMA1.00±0.63c1.00±0.63d1.88±1.24c0.42±0.07d0.33±0.24d

注：同一列标有不同小写字母的表明组间差异显著(P<0.05),相同小写字母的表明组间差异不显著(P≥0.05)

Note: Different letters in the same column indicate significantly difference (P<0.05), same letters in the same column indicate no significant difference (P≥0.05).

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3 讨论

本研究通过对比不同培养基对分离共生真菌多样性的影响,发现手参原球茎中共生真菌分离效果与所使用的培养基存在一定的关联性,另外还发现手参原球茎中的共生真菌具有较高的多样性。除了本研究中常见的6种培养基外,SDA (沙氏葡萄糖琼脂培养基)、AWA、CWA、SAM (蔗糖琼脂培养基)、OMA和GPA也可作为分离兰科共生真菌的基础培养基(Qayyum et al. 2016;Zettler & Corey 2018)。由于不同真菌对营养的多样化需求,不同培养基成分对真菌的分离效果不同,如MMN和PDA对成年兰科共生真菌有着非常好的分离效果(Stewart et al. 2003;Adeoyo et al. 2019),而CWA和OMA培养基对杓兰中的共生真菌表现出较好的分离效果(Shimura & Koda 2005;Shimura et al. 2009)。本研究发现MMN、CAM及PDA培养基对手参原球茎共生真菌的分离效果较好,这可能由于这些培养基中的高碳水化合物含量(例如PDA=20 g葡萄糖/L)更受对营养需求高、生长速度较快的原球茎共生真菌(例如裂褶菌属Schizophyllum sp.和角担菌属Ceratobasidium sp.真菌)等的青睐。Yokoya et al. (2015)利用FIM成功地从马达加斯加的附生、陆生和石生兰科植物中分离出了一些广谱的内生真菌,尽管FIM培养的碳水化合物含量较低(FIM=5 g蔗糖/L),但在分离其他兰科植物(例如鬼兰Dendrophylax lindenii和大花万代兰Vanda coerulea)的共生真菌方面也有着不错的效果(Aggarwal et al. 2012;Hoang et al. 2017)。此外,含有丰富碳水化合物的CMA培养基和MMN培养基在分离兰科共生真菌方面也具有良好的分离效果(Marx 1969;Currah et al. 1997;Pereira et al. 2005;Sousa et al. 2019;Xi et al. 2020)。然而在本研究中FIM、MEA和CMA培养基可能由于其碳水化合物含量较少(例如FIM=5 g蔗糖/L)或培养基中缺少某些原球茎共生真菌喜好的营养物质导致其分离效果不佳,这表明成年兰科共生真菌类群和原球茎共生真菌类群对营养物质的需求存在差异性。

本研究综合分离的真菌物种数、菌株数量、真菌相对多度和多样性指数对6种常见培养基对手参原球茎共生真菌分离效果进行评估,结果表明6种培养基对手参原球茎共生真菌的分离效果存在显著性差异,其中MMN的分离效果最好,CAM和PDA次之,FIM和MEA培养基对真菌的分离效果影响不大,CMA培养基的分离效果最差。今后在其他兰科植物原球茎共生真菌的分离中可借鉴参考。另外,从手参原球茎中分离到的其他共生真菌,其在种子萌发过程中所起的作用及可能的生态功能还需在今后的研究中逐渐阐明。

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