生态学报  2014, Vol. 34 Issue (24): 7300-7310

文章信息

吕立新, 王宏伟, 梁雪飞, 郝思静, 杜威, 朱虹, 戴传超
LÜ Lixin, WANG Hongwei, LIANG Xuefei, HAO Sijing, DU Wei, ZHU Hong, DAI Chuanchao
不同化学型和季节变化对茅苍术内生真菌群落多样性的影响
Effects of different chemotypes and seasonal dynamic variation on the species diversity of endophytic fungal communities harbored in Atractylodes lancea
生态学报, 2014, 34(24): 7300-7310
Acta Ecologica Sinica, 2014, 34(24): 7300-7310
http://dx.doi.org/10.5846/stxb201303200468

文章历史

收稿日期:2013-03-20
网络出版日期:2014-03-19
不同化学型和季节变化对茅苍术内生真菌群落多样性的影响
吕立新, 王宏伟, 梁雪飞, 郝思静, 杜威, 朱虹, 戴传超     
南京师范大学生命科学学院, 江苏省微生物资源产业化工程技术研究中心, 江苏省微生物与功能基因组学重点实验室, 南京 210023
摘要:对茅山型茅苍术(MA)和湖北型茅苍术(HA)的内生真菌多样性进行调查,分析二者之间优势菌群和特异菌群的差异.将两种化学型的茅苍术移栽至同一环境中,分别于春季、夏季、秋季3个季节采集叶片进行内生真菌的分离、鉴定和多样性分析.调查一共得到530株内生真菌,总分离率为58.89%,通过形态学特征和ITS序列鉴定归结为20个属.MA内生真菌分离率要高于HA,二者的真菌多样性构成有一定差异性.MA和HA内生真菌的优势菌群呈基本重合现象,主要集中于交链孢属、球座菌属、刺盘孢属等几个属;而二者之间的特异菌群显示出差异性,MA的特异菌群为Leptospora属、支顶孢属、壳针孢属等,HA特异菌群为白僵菌属、轮枝孢属、毛壳菌属等.随着季节的变化,内生真菌群落显示出一定的演替规律.夏季内生真菌的多样性高于春季和秋季.
关键词茅苍术    内生真菌多样性    特异菌群    化学型    季节    
Effects of different chemotypes and seasonal dynamic variation on the species diversity of endophytic fungal communities harbored in Atractylodes lancea
LÜ Lixin, WANG Hongwei, LIANG Xuefei, HAO Sijing, DU Wei, ZHU Hong, DAI Chuanchao     
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
Abstract:Atractylodes lancea (Thunb.) DC is a Chinese medicinal herb. The Maoshan mountain area is famous for producing the authentic medicinal herb of A. lancea. Volatile oils from A. lancea show antimicrobial activity and include the characteristic atractylone, SymbolbA@ -eudesmol, hinesol, and atractylodin. Because the active compositions are different, A. lancea samples from different places are divided into the Maoshan chemotype and the Hubei chemotype.To study the formation of the authentic medicinal herb, the endophytic fungal diversity of the two chemotypes of A. lancea was analyzed. Maoshan chemotype A. lancea (MA) and Hubei chemotype A. lancea (HA) were transplanted from each habitat and cultivated in the same environmental conditions. The leaves were selected, and endophytic fungi were isolated and identified during spring, summer, and autumn in 2011.The results indicated that plentiful fungal diversity was present in A. lancea. A total of 530 endophytic fungi were isolated from the leaves of A. lancea, with an isolation rate of 58.89%. All of the isolates were identified based on their morphological characteristics and molecular phylogeny; the isolates belonged to 20 genera. The species in Alternaria, Colletotrichum, and Guignardia were the predominant groups of fungi, and their relative isolation frequencies were 36.04%, 19.43%, and 9.62%, respectively, which were much greater than the isolation frequencies of other genera.The isolation rate (IR) of endophytic fungi from MA was 70.22%, and that from HA with 47.53%. The diversity index, which was determined by the Shannon-Wiener index (H'), revealed that the diversity of endophytic fungi in MA (H' = 2.16) is somehow higher than that in HA (H'= 1.83). Sorenson's similarity index between MA and HA was 0.67, which indicated that the community composition of endophytic fungi from the two types of A. lancea was different to some extent. The dominant genera of endophytic fungi in the two chemotypes of A. lancea showed basic similarities; these genera included Alternaria, Guignardia, and Colletotrichum. However, some genera were chemotype-specific. The MA-specific genera included Leptospora, Acremonium, and Septoria; the HA-specific genera included Beauveria, Verticillium, and Chaetomium.The diversity of endophytic fungi among different seasons presented certain successive patterns.The IR in summer was the highest (IR = 71.33%) and was followed by autumn (IR = 63.00%) and spring (IR = 42.33%). This suggested that the isolation of endophytic fungi increased from spring to summer and then declined slightly in autumn. The Shannon-Wiener index (H') indicated that greater endophytic fungal diversity was observed during summer (H' = 2.20) than spring (H' = 1.64) and autumn (H' = 1.88). With respect to seasonal factors, Sorenson's similarity index between spring and summer was 0.71, and that between summer and autumn was 0.76, while that between spring and autumn was 0.64. This indicated that there were some differences in the community composition of endophytic fungi among different seasons, but the differences were not significant. The dominant genera of endophytic fungi of A. lancea isolated in different seasons included Alternaria, Guignardia, and Colletotrichum; all of these genera were observed during every season, showing a wide seasonal distribution.The results of this study offer information about the differences in endophytic fungal diversity of two chemotypes of A. lancea under the same environmental conditions; these results also reveal seasonal variation in the endophytic fungi of A. lancea. Differences in the community composition of endophytic fungi between two chemotypes of A. lancea may have an important effect on the genuineness of the medicinal herb. These results are important for understanding the formation of the authentic medicinal herb of A. lancea.
Key words: Atractylodes lancea (Thunb.) DC.    endophytic fungal diversity    specific endophytic fungi    chemotypes    season    

植物内生菌是一类在其全部或部分生活周期内,定殖在植物组织内部并且不引起明显病害的微生物[1]。植物与内生菌各自的获益依赖于二者共同作用,在互利共生的关系中,植物体为内生菌提供生长必需的能量和营养,而内生菌又可通过自身的代谢产物或借助于信号传导作用对植物体产生影响[2]。许多植物内生菌具有重要的生物学和生态学功能,如可以促进植物生长[3, 4],增强抗病能力[5, 6, 7],提高植物对温度、干旱和盐碱的抵抗力[8, 9, 10],并能够影响药用植物活性次级代谢产物的积累[11, 12, 13, 14]

茅苍术(Atractylodes lancea (Thunb.) DC.)为菊科苍术属多年生草本植物,是重要的传统中药材。茅苍术在我国分布广泛,江苏茅山是苍术药材的道地产区。郭兰萍等[15]在前人研究的基础上,提出将茅苍术按照有效成分含量的差异分为两种化学型:茅山苍术型(MA)和湖北苍术型(HA)。MA以茅山茅苍术为代表,主要产地是江苏、山东、河南北部等地区,以江苏茅山为道地产区;HA以湖北茅苍术为代表,主要位于湖北、陕西、安徽、河南南部等地,药用价值要低于前者。目前针对影响茅苍术道地性形成的因素已经有大量研究[16, 17, 18, 19]

本研究分别在春,夏,秋3个季节从两种不同化学型的茅苍术中分离内生真菌,分析化学型的不同以及季节的变化对茅苍术的优势菌群和特异菌群的影响,以期全面阐明茅苍术内生真菌的多样性,为后续研究内生真菌的菌群结构对茅苍术药用活性成分的影响提供重要依据;同时分离出并收集大量内生真菌资源,为进一步筛选新型化合物奠定基础。

1 材料和方法

在本研究的实验体系中,将来自不同地域的两种化学型的茅苍术(MA和HA)移栽到相同的环境中进行培育,排除了其原产地不同气候、土壤和光照等环境因素对植物生长造成的不同影响,同时也避免了茅苍术因外界环境的差异而感染不同内生菌群的可能。在此条件下,确定内生真菌菌群的不同源于不同化学型茅苍术体内共生的真菌,如果存在外界内生真菌感染的情况,也可归结于宿主本身基因型对内生真菌的选择作用。

1.1 植物材料的采集、鉴定

实验在江苏省南京市南京师范大学植物园开展,试验地位于南京市栖霞区,属亚热带季风气候,年平均温度15.4 °C,年平均降水量1106 mm,海拔40 m。选用产自江苏茅山的茅苍术为MA型的代表,产自陕西地区的茅苍术为HA型的代表,分别于2011年3月中旬将两种化学型茅苍术的多年生块根移栽至南京师范大学植物园内,每种化学型栽种两个小区。小区为5 m×5 m样方,相隔4 m距离,保持排水和土壤相互隔离。

于2011年春季(5月)、夏季(8月)和秋季(10月)选择健康的茅苍术,采集无病斑和虫洞的叶片,每种化学型各采集150片完整叶片,取样后立即放入保鲜袋中密封,并贴上标签,运回实验室4 ℃保藏,并在24 h内处理完毕。

1.2 实验方法 1.2.1 内生真菌的分离及表面消毒效果分析

参照Guo等[20]和Kusari等[21]的方法对植物组织进行表面消毒。将新鲜叶片用表面活性剂浸泡5 min,自来水冲洗10 min,除去表面附着物。在超净台内将植物材料浸入75%的酒精消毒;1 min后取出,放入0.1%的升汞中消毒7—8 min;无菌水冲洗3次,每次涮洗2 min左右。将叶片剪成0.5 cm×0.5 cm大小,每一组处理随机选取150个组织块,置于加有硫酸链霉素(50 mg/L)的PDA平板(90 mm)上,封口后27 ℃恒温培养14 d。每隔12 h观察,待截断处菌丝长出,切取菌丝转入新的PDA平板上继续培养。纯化2—3次后得到单一菌种。

通过3种表面消毒处理方法后,观测结果如下:

(1)漂洗液检验法验证

将最后一次漂洗消毒材料的漂洗液吸取0.2—0.3 mL 涂布于PDA固体平板上进行培养。如果周围无任何菌长出,证明得到的真菌来源于植物组织内部。

(2)组织印迹法的检验

用灭过菌的镊子夹住已切好的组织块,使其表面与固体平板接触3—5 min后取出,置于相同条件下培养3—7 d后,若未发现平皿中有菌落出现,证明该样品表面灭菌彻底。

1.2.2 内生真菌的形态学鉴定及保藏

观察平板上菌落特征,对产孢的菌种根据产孢结构和孢子形态等归结为不同的形态型,参照《真菌鉴定手册》对分离得到的茅苍术内生菌进行形态学鉴定。纯化已鉴定菌种后,接入PDA斜面,4 ℃冰箱中保藏。菌种保藏于南京师范大学生命科学学院微生物菌种标本保藏室。

1.2.3 内生真菌形态型菌株基因组的提取

本研究采用CTAB法提取菌株的基因组DNA,挑取分离纯化后菌株的少许菌丝体转接到新的PDA培养基上进行培养,培养温度为25 ℃,根据所需菌株菌丝生物量多少决定培养时间。

提取过程如下:用无菌手术刀刮下无培养基的真菌培养物约100 mg于无菌研钵中,倒入液氮,用研钵将菌丝充分研磨,以无菌药勺将研磨出的菌丝粉末转入1.5 mL的离心管;在离心管中加入1 mL预热的65 ℃的CTAB抽提缓冲液(1×,DNA抽提),100 μL 10% SDS;65 ℃温浴30—60 min,不时震荡;10000 r/min离心5 min;分3层,从下往上依次为沉淀,中清,浑浊,取中层;加等体积氯仿异戊醇(24:1)混匀10 min;10000 r/min 10 min,取上清;用氯仿异戊醇(24:1)抽提离心,反复多次,直到液面清晰;上清液加入等体积异丙醇-20 ℃沉淀30—60 min;12000 r/min 10 min预冷100%乙醇洗一次;室温下蒸发溶于20 μL TE中,琼脂糖电泳检测,4 ℃或-20 ℃冰箱保存备用。

1.2.4 内生真菌形态型菌株的ITS的PCR扩增、检测和测序

在每个形态型中随机取样测定菌株ITS序列。采用通用引物ITSl/ITS4扩增真菌的转录间隔区ITS片段[22]。引物的序列如下:

ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4(5′-TCCTCCGCTTATTGATATGC-3′)。

反应在BIO-RAD公司的PCR扩增仪中进行,体系为50 μL,其中DNA模板(25 ng),MgCl2(2.5 mmoL/L),dNTP(200 μmol/L),ITS1、ITS4(0.25 μmol/L),Taq DNA聚合酶(1.5 U,Takara),10×PCR Buffer(100 mmol/L Tris-HCl pH值8.3,500 mmol/L KCl,0.01% gelatin)。扩增反应程序95 ℃预变性3 min后进入35个循环,94 ℃ 50 s变性,54 ℃ 50 s退火,72 ℃ 60 s延伸,72 ℃ 10 min,进入8 ℃。反应结束,1.2%琼脂糖凝胶电泳检测,于4 ℃或-20 ℃保存备用。用试剂盒进行琼脂凝胶的回收和PCR产物纯化,把纯化产物与T-载体(pMD18-TVector,Takara)连接,并按照说明书常规实验操作步骤转化到大肠杆菌DH5α中,利用蓝白斑法筛选出阳性克隆,培养并收集菌体送交Invitrogen公司测序。

从GenBank下载与形态型菌株有较高序列相似性的参考菌株的DNA序列,通过分子系统发育分析建立分子系统发育树,根据系统树反映的系统发育关系框架来推测形态型菌株的分类地位。

将不同的ITS1-5.8S-ITS4序列与GenBank数据库的序列进行比对,序列相似性程度97%以上视为同一种。确定与实验克隆亲缘关系最近的种属,构建系统发生树。

1.2.5 数据处理

分离频率(IF):分离到的某一指定类型内生真菌的菌株数量占分离的内生真菌菌株数量的百分率,用于比较和判断优势菌群。

分离率(IR):分离到的某一指定类型内生真菌的菌株数量占分离样品组织块总数的百分率。用于衡量植物组织中内生真菌的丰富程度。

通过Menhinick′s index (Dmn),计算内生真菌的物种丰度:

式中,S是同一样本(同一组织)中不同内生真菌的种类数目,N是给定样本中内生真菌的个体总数。

Fisher对数级数(α):

式中,x通过以下计算:

Shannon-Wiener多样性指数(H′):

式中,Pi是第i种的比例多度,给定为:Pi=Ni/NNi为第i种物种个体数,i=1,2,3,4,…,S。当Pi>1/S时,即为优势种属。

Simpson多样性指数(1-D):

式中,Pi是第i种的比例多度。

Margalef丰富度指数(R):

式中,S为物种数;N为个体总数。

Evenness指数(J):分析群落物种分布的均匀程度。

式中,H为Shannon-Wiener指数,S为物种总数目。

Sorenson相似性指数:

式中,j为两个样地/组织/季节共有种数或属数;a是一个样区/组织/季节的植物样本中内生真菌的种数或属数,b是另一个样区/组织/季节的植物样本中内生真菌的种数或属数;用于比较两个样区/季节之间的植物样本中内生真菌种类组成的相似程度。

2 结果与分析 2.1 茅苍术内生真菌的种类组成

共在900个叶片组织中分离得到530株内生真菌纯培养物,总分离率为58.89%。依据菌落形态和产孢结构归为不同的形态种,随后通过ITS序列测序比对,对各个形态种进行分子鉴定,最终得到28个类群(表 1)。

表 1 基于NCBI数据库BLAST比对的茅苍术内生真菌分子鉴定结果 Table 1 Molecular identification of endophytic fungal morphotypes from Atractylodes lancea based on BLAST queries in NCBI
菌株 Strains分类单元 TaxaGenBank 序列号 GenBank accession No.BLAST最接近的比对 Closest blast match (GenBank accession No.)序列相似度 Query/reference ITS length(Similarity/%)
LLX 0102Alternaria alternataKC134318Alternaria alternata (JQ416155.1)543/543 (100)
LLX 0133Alternaria tenuissimaKC202935Alternaria tenuissima (AY154712.1)571/571 (100)
LLX 0104Guignardia vaccinii KC172070Guignardia vaccinii (GU066692.1) 612/612 (100)
LLX 0105Cercospora zebrinaKC172066Cercospora zebrina (GU214656.1)534/540 (99)
LLX 0106Cercospora kikuchiiKC172078Cercospora kikuchii (HM631728.1)491/492 (99)
LLX 0107Beauveria bassianaKC172068Beauveria bassiana (JQ266220.1)538/538 (100)
LLX 0108Septoria lycopersici KC134319 Septoria lycopersici var. lycopersici (DQ841156.1) 504/505 (99)
LLX 0112Villosiclava virens (anamorph:Ustilaginoidea virens)KC172069Villosiclava virens (JF271121.1)585/587 (99)
LLX 0113Pseudocercospora marginalisKC172077Pseudocercospora marginalis (GQ852766.1)517/517 (100)
LLX 0115Pseudocercospora elaeodendriKC172073Pseudocercospora elaeodendri (GU980950.1)521/522 (99)
LLX 0116Colletotrichum jasminigenumKC172075Colletotrichum jasminigenum (JQ658871.1)558/558 (100)
LLX 0117Colletotrichum truncatumKC172074Colletotrichum truncatum (AF451899.1)561/566 (99)
LLX 0118Colletotrichum gloeosporioidesKC172072Colletotrichum gloeosporioides (GQ407097.1)575/578 (99)
LLX 0119Rhizoctonia bataticola KC172071Rhizoctonia bataticola (HQ392793.1)509/511 (99)
LLX 0120Leptospora rubellaKC172076Leptospora rubella (JQ936327.1)562/563 (99)
LLX 0121Sarocladium strictum (Acremonium strictum)KC172080Sarocladium strictum (GU595023.1)554/554 (100)
LLX 0122Acremonium alternatumKC172079Acremonium alternatum (GQ377488.1)543/544 (99)
LLX 0123Phomopsis sp. KC172081Phomopsis sp. By231 (GQ365158.1)571/572 (99)
LLX 0124Stemphylium solaniKC172065Stemphylium solani (AF203450.1)560/566 (99)
LLX 0125Cladosporium cladosporioidesKC172067Cladosporium cladosporioides (JX230994.1)528/528 (100)
LLX 0127Fusarium solaniKC202941Fusarium solani (AB470903.1)546/547 (99)
LLX 0129Nectria ipomoeaeKC202945Nectria ipomoeae (AB513849.1)553/556 (99)
LLX 0133Fusarium oxysporumKC202939Fusarium oxysporum (AY188919.1)545/551 (99)
LLX 0134Chaetomium globosumKC202936Chaetomium globosum (JN689341.1)576/578 (99)
LLX 0135Paraphoma chrysanthemicolaKC202946Paraphoma chrysanthemicola (JN585950.1)512/513 (99)
LLX 0140Nectria haematococcaKC202948Nectria haematococca (FJ441642.1)538/538 (100)
LLX 0141Pseudallescheria boydii KC202949Pseudallescheria boydii (AB369906.1)616/617 (99)
LLX 0142Edenia gomezpompaeKC202950Edenia gomezpompae (FJ839619.1)549/556 (99)

根据测序比对的结果,下载与形态型菌株有最高序列相似性的参考菌株的DNA序列,通过Invitrogen Vector NTI Advance 10 软件进行序列比对分析,最后用邻接法(Neighbor-Joining,NJ)构建系统进化树,根据系统树反映的系统发育关系框架来推测形态型菌株的分类地位(图 1)。

图 1 茅苍术内生真菌18S rDNA序列系统发育树状图 Fig. 1 Phylogenetic tree based on neighbor-joining analysis of their 18S rDNA ITS sequences of the endophytic fungal isolates obtained from Atractylodes lancea

本文在属的水平上讨论内生真菌的分离率和多样性。实验结果表明,交链孢属(Alternaria)在分离得到的内生真菌站占优势地位,其分离频率为36.04%,其次为刺盘孢属(Colletotrichum)和球座菌属(Guignardia),分离频率分别为19.43%和9.62%,其余各菌所占分离频度均较低(表 2)。

表 2 茅苍术叶片内生真菌的菌群组成 Table 2 Composition of endophytic fungi from A. lancea
内生真菌编号 Strains No.真菌属 Genus分离率 Isolation rate (IR)/%分离频率 Isolation frequency (IF)/%
ALZ-1交链孢属(Alternaria) 21.2236.04
ALZ-2球座菌属(Guignardia) 5.679.62
ALZ-3尾孢属(Cercospora)4.898.3
ALZ-4白僵菌属(Beauveria )0.110.19
ALZ-5壳针孢属(Septoria)1.783.02
ALZ-6轮枝孢属(Verticillium)0.440.75
ALZ-7假尾孢属(Pseudocercospora) 3.896.6
ALZ-8刺盘孢属(Colletotrichum) 11.4419.43
ALZ-9丝核菌属(Rhizoctonia) 1.222.08
ALZ-10Leptospora 0.330.57
ALZ-11支顶孢属(Acremonium)1.562.64
ALZ-12拟茎点霉属(Phomopsis)0.671.13
ALZ-13葡柄霉属(Stemphylium) 1.562.64
ALZ-14枝孢属(Cladosporium)0.330.57
ALZ-15镰刀菌属(Fusarium) 1.442.45
ALZ-16丛赤壳属(Nectria) 1.442.45
ALZ-17毛壳菌属(Chaetomium) 0.220.38
ALZ-18异茎点霉(Paraphoma) 0.330.57
ALZ-19假霉样真菌属(Pseudallescheria) 0.110.19
ALZ-20Edenia 0.220.38
2.2 不同化学型茅苍术内生真菌菌群的组成和多样性

MA茅苍术的内生真菌分离率要高于HA型,前者为70.22%,后者为47.53%。栽种于同一环境下的MA和HA内生真菌菌群的组成有所不同,种类多样性上呈现出明显的差异性。二者的优势菌群呈基本重合的现象,HA中的优势菌群为交链孢属、球座菌属、假尾孢属(Pseudocercospora)和刺盘孢属;MA的优势菌群为:交链孢属、球座菌属、尾孢属和刺盘孢属。以上几个属在两种化学型的茅苍术中均有分离得到,但其在菌群中所占优势有所不同(表 3)。

表 3 不同化学型苍术内生真菌菌群的组成和多样性 Table 3 Composition,isolation rate,and isolation frequency of endophytic fungi from two chemotypes of A. lancea
编号 Strains No.真菌属 Genus茅山苍术型 Maoshan chemotype A. Lance(MA) 湖北苍术型 Hubei chemotype A. lancea (HA)
分离率/% Isolation rate (IR)分离频率/% Isolation frequency (IF) 分离率/% Isolation rate (IR)分离频率/% Isolation frequency (IF)
ALZ-1交链孢属Alternaria 23.3333.2319.1140.19
ALZ-2球座菌属Guignardia 4.896.966.4413.55
ALZ-3尾孢属Cercospora4.226.015.5611.68
ALZ-4白僵菌属Beauveria 0.220.47
ALZ-5壳针孢属Septoria3.565.06
ALZ-6轮枝孢属Verticillium 0.891.87
ALZ-7假尾孢属Pseudocercospora 5.337.592.445.14
ALZ-8刺盘孢属Colletotrichum 14.4420.578.4417.76
ALZ-9丝核菌属Rhizoctonia 1.562.220.891.87
ALZ-10Leptospora 0.670.95
ALZ-11支顶孢属Acremonium3.114.43
ALZ-12拟茎点霉属Phomopsis1.111.580.220.47
ALZ-13葡柄霉属Stemphylium 2.223.160.891.87
ALZ-14枝孢属Cladosporium0.670.95
ALZ-15镰刀菌属Fusarium 1.562.221.332.80
ALZ-16丛赤壳属Nectria 2.443.480.440.93
ALZ-17毛壳菌属Chaetomium 0.440.93
ALZ-18异茎点霉Paraphoma 0.670.95
ALZ-19假霉样真菌属Pseudallescheria 0.220.47
ALZ-20Edenia 0.440.63

从二者的特异菌群差别可以明显看出菌群多样性的不同。MA的特异菌群为Leptospora属、壳针孢属(Septoria)、支顶孢属(Acremonium)、枝孢属(Cladosporium)、Edenia属;而白僵菌属(Beauveria)、轮枝霉属(Verticillium)、毛壳菌属(Chaetomium)等只在HA中分离得到。

2.3 不同季节茅苍术内生真菌组成的多样性

每个季节分别选取300个叶片组织进行内生真菌的分离。春季得到127株内生真菌,分离率为42.33%;夏季得到214株内生真菌,分离率为71.33%;秋天的结果是189株菌,分离率63.00%。由此可以看出,从春季到夏季内生真菌的定殖呈明显增加的趋势,到秋季略有降低。

从春季的样品中分离得到12个属的内生真菌,主要是交链孢属(IF=38.58%)和刺盘孢属 (IF=34.65%),其余各属真菌的分离频率在5%以下。夏季分离得到15个属的真菌,优势菌群为交链孢属 (IF=29.17%)、刺盘孢属 (IF=14.35%)、球座菌属 (IF=13.89%)、尾孢属(IF=10.65%)和假尾孢属(IF=9.72),秋季分离得到的真菌中,交链孢属的分离频率有所增加(IF=42.25%),其次为刺盘孢属(IF=14.97%)、球座菌属 (IF=11.23%)和尾孢属(IF=8.56%),其余各属真菌的分离率均较低(表 4)。

表 4 不同季节的茅苍术内生真菌菌群组成及分离率和分离频率 Table 4 Composition,isolation rate and isolation frequency of endophytic fungi in different seasons from A. lancea
种属编号 Strains No.真菌属 Genus春季Spring 夏季Summer 秋季Autumn
分离率/% Isolation rate (IR)分离频率/% Isolation frequency (IF) 分离率/% Isolation rate (IR)分离频率/% Isolation frequency (IF) 分离率/% Isolation rate (IR)分离频率/% Isolation frequency (IF)
ALZ-1交链孢属Alternaria 16.3338.5821.0029.1726.3342.25
ALZ-2球座菌属Guignardia 10.0013.897.0011.23
ALZ-3尾孢属Cercospora1.673.947.6710.655.338.56
ALZ-4白僵菌属Beauveria 0.330.79
ALZ-5壳针孢属Septoria2.676.300.330.462.333.74
ALZ-6轮枝孢属Verticillium0.330.791.001.39
ALZ-7假尾孢属Pseudocercospora 7.009.724.677.49
ALZ-8刺盘孢属Colletotrichum 14.6734.6510.3314.359.3314.97
ALZ-9丝核菌属Rhizoctonia 3.675.09
ALZ-10Leptospora 1.001.60
ALZ-11支顶孢属Acremonium0.330.792.002.782.333.74
ALZ-12拟茎点霉属Phomopsis 1.672.310.330.53
ALZ-13葡柄霉属Stemphylium 2.004.721.331.851.332.14
ALZ-14枝孢属Cladosporium0.330.790.670.93
ALZ-15镰刀菌属Fusarium 1.002.362.002.781.332.14
ALZ-16丛赤壳属Nectria 1.673.942.333.240.330.53
ALZ-17毛壳菌属Chaetomium 0.671.07
ALZ-18异茎点霉Paraphoma 1.002.36
ALZ-19假霉样真菌属Pseudallescheria 0.330.46
ALZ-20Edenia 0.670.93

交链孢属,尾孢属与刺盘孢属在3个季节中都能分离得到,且分离率远高于其他真菌。说明这3个属真菌的分布具有季节广谱性,其定殖不具有季节性差异。与此同时,某些真菌的定殖具有的明显的季节性差异,如白僵菌和异茎点霉属(Paraphoma)是春季特有的内生真菌,而丝核菌属(Rhizoctonia)、拟茎点霉属(Phomopsis)只在夏季分离得到,Leptospora属、毛壳菌(Chaetomium)、Edenia属未曾见于秋季以外的季节。

不同季节的茅苍术内生真菌多样性呈现出一定的差异性。从多样性指数来看,夏季内生真菌的Shannon-Wiener index (H′)和Simpson′s diversity index(1-D)最高(2.20,0.85),其次是秋季(1.88,0.77)和春季(1.64,0.73)。同时可以看出,春季和秋季的Margalef index (R)以及Evenness index (E)相差不大,均稍低于夏季的水平。对不同化学型的分析表明MA型的真菌多样性高于HA型。从Shannon-Wiener index (H′)和Simpson′s diversity index(1-D)可以分析得到,MA的数值(2.16,0.83)要高于HA(1.83,0.77)(表 5)。

表 5 茅苍术内生真菌菌群多样性指数 Table 5 The diversity index of endophytic fungi from A. lancea
影响因素 Influencing factors类型 Types属 Genus菌株数量 Strains number门辛尼克指数 Menhinick′s index (Dmn)费舍对数序列 Fisher′s log series index (α)香农- 威纳指数 Shannon- Wiener index (H′)辛普森 多样性指数 Simpson′s diversity index(1-D)马加利夫 多样性指数 Margalef index (R)均匀度指数 Evenness index (E)
季节春季121272.253.251.640.732.270.66
Seasons夏季162141.093.992.200.852.790.79
秋季131890.953.181.880.772.290.73
化学型茅山型163160.903.562.160.832.610.78
Chemotypes湖北型142140.963.361.830.772.420.69
2.4 茅苍术内生真菌菌群组成的相似性

不同化学型(MA和HA) 茅苍术间的内生真菌的菌群相似性系数(CS)为0.67,说明二者菌群组成具有较大的差异。对于不同季节而言,春季与夏季的相似性系数为0.71,夏秋两季的相似性系数为0.76,春季与秋季的相似性系数为0.64,说明不同季节间有一定的种类的差异,但差别不大,并且真菌的群落结构随着季节的连续变化存在一个相应的演替过程。

3 讨论

从900个茅苍术植株组织块中得到530株内生真菌,分离的菌株归为20个属,具有较高的总分离率(58.89%),这与药用植物中内生真菌多样性极其丰富的研究报道相符合。孙剑秋[23]等从北京植物园4科6种药用植物的1144个组织块中分离到内生真菌973株,有很高的分离频率(47.9%一63.1%)和分离率(70%—93%),且不同种的植物的内生真菌种类组成不同。Naik等[24]调查了南印度西高止山脉地区的药用灌木,分别于冬季、雨季和夏季从15种药用植物的9000个叶片中共分离得到了6125株内生真菌,其中交链孢属、毛壳菌属,镰刀菌属,刺盘孢属,枝孢霉属是最常见的真菌种类。在本研究所分离得到的内生菌中,交链孢属,尾孢属与刺盘孢属在不同化学型及不同季节的样本中均占有最高的分离率和分离频率。这几个属是常见的内生真菌类群,在多种生态环境下广泛存在[23, 24, 25, 26],具有很强的季节和宿主适应性,可能通过横向传播侵染不同化学型的茅苍术,并且占据了优势菌群的地位。本研究获得的茅苍术内生真菌的多样性与曹益鸣[27]等和陈佳昕[28]等的研究结果有较大的不同,这可能与实验采取的研究手段、采样材料以及植株所处的生长环境不同有关。曹益鸣等[27]在研究中采用的是单一化学型茅山野生的茅苍术,针对不同组织部位进行采样之后,通过形态学特征进行鉴定;陈佳昕等[28]选用的是江苏汤山的野生材料,未作出真菌的多样性分析。取样地环境的差异以及化学型的区别可能是造成结果差异的主要原因[23, 24, 25, 26, 29, 30, 31, 32, 33]

两种化学型茅苍术的组织中,内生真菌菌群的相似性系数为0.67,具有明显的差异性。在二者共有的内生真菌种类中,其分离率也是有所差别的。这可能是不同化学型茅苍术基因型上的差异,造成对内生真菌的定殖与侵染的的选择作用。MA的内生菌群中,除典型的优势菌群外,壳针孢属和支顶孢属的分离率仅次于优势菌群,且只在MA中分离得到,在HA中未见分离。MA是江苏地区的道地性药材,而HA在药效上低于前者。前人研究表明茅苍术道地性是多种因素共同作用的结果[16, 18, 34]。内生真菌与植物的共生作用可能影响植物次级代谢产物的积累,两种化学型的茅苍术在特异菌群上的差异可能是影响道地性的重要因素之一。

季节对内生真菌多样性的影响可能与植株的生长状态有关。曹益鸣[27]等曾对不同生长周期的茅苍术内生真菌的种类做过统计,发现内生真菌在植株不同生长期均有分布,但在苗期和花期时数量较多,种类丰富,分离率为39.6%和36.5%。另一方面,包括温度、降水等因素在内的环境因子直接作用于内生真菌,形成真菌群落的季节性演替现象。Mishra[35]等发现印度地区的心叶青牛胆(Tinospora cordifolia)内生真菌的分离频率在雨季最高,其后依次是冬季和夏季,并且季节因素对内生真菌多样性的影响作用要高于地理因素。春季是茅苍术的生长初期,尚未有大量多种类的横向传播的内生真菌定殖。而后随着季节变换,真菌在叶片内部持续生长,所以夏季内生真菌的多样性和分离率都有所提高。而秋季真菌的多样性降低,且致病性的交链孢属分离频率上升。这可能说明春夏两季某些无害的内生真菌与病原菌拮抗,抢夺叶片微环境中的生态位,从而抑制了病原菌的大规模爆发。叶片微环境内的真菌群落演替,不同种类内生真菌间竞争叶片上的生存空间,彼此之间有一定的拮抗作用,这对后续的研究工作很有启发意义。

药材的道地性受特定环境因素和生物因素的影响,其中微生物环境对药材道地性的形成有着举足轻重的作用[16, 18, 27, 34, 35]。内生菌作为微生物生态系统中的一个重要组成部分,从植物体内部对药用植物的代谢产生影响。因此,从内生菌群落的角度阐明植物次生代谢产物的形成和积累机制以及药材道地性成因将是一种新的研究思路。

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