全 文 ：食用菌学报 2009.16(2):6 ～ 9
收稿日期:2009-03-25原稿;2009-04-07 修改稿
基金项目:上海市科委基金项目(编号:06DZ 22036)和农业部公益性行业科研专项项目(编号:nyhyzx07-008)的部
分研究内容
作者简介:沈　洪(1981-), 男 , 2007年毕业于南京农业大学微生物学真菌遗传与育种专业 ,发表主笔论文 2篇。
＊本文通讯作者
文章编号:1005-9873(2009)02-0006-04
采用变性梯度凝胶电泳研究羊肚菌土壤真菌群落结构
沈　洪1 , 2 , 汪　虹1 , 赵永昌3 , 冯爱萍1 , 陈明杰1＊ , 潘迎捷4
(1 农业部应用真菌资源与利用重点开放实验室, 上海市食用菌工程技术研究中心, 上海市农业遗传育种重点实验室 ,
上海市农业科学院食用菌研究所 , 上海 201106;2南京农业大学生命科学院微生物系 , 江苏南京 210095;
3云南省农业科学院生物技术资源研究所 , 云南昆明 650205;4上海海洋大学 ,上海 200090)
摘　要:采用变性梯度凝胶电泳(Denatur ing Gradient Gel Electr ophoresis)技术以土壤微生物群体的基因组
DNA 为研究对象 ,通过比较羊肚菌(Morchella sp .)出菇土壤及对照土壤中各种真菌的 28S rDNA 基因信息来
了解土壤微生物的多样性。结果发现 , 测得的 12 种真菌中 , 盘菌纲的有 10 种 , 异担子菌纲 1 种 ,还有 1 种未确
定。所取 2 份土样中仅羊肚菌生长土壤中检测出有羊肚菌;2 份样品在优势真菌种群上有一定的区别 , 盘菌
纲真菌群落存在很大的差异 , 羊肚菌出菇土壤的真菌丰度低于对照 , 可能与土壤中羊肚菌优势种群对其它种
群的抑制作用有关。
关键词:羊肚菌;土壤真菌群落;PCR-DGGE
　　高羊肚菌(Morchel la elata), 尖顶羊肚菌
(M .conica)的半人工栽培在云南等地有较大的
突破 , 但是羊肚菌发生机理还有诸多不明之
处[ 1] ,半人工栽培中需要覆土栽培 ,覆土与羊肚
菌子实体发育之间的关系有待进一步的研究[ 2] 。
覆土中的土壤微生物群系是否有助于羊肚菌子
实体的发育有待进一步的证实。
变性梯度凝胶电泳(Denaturing Gradient
Gel Elect ropho resis , DGGE)最初是 LERMAN
等人于 20世纪 80年代初期发明的[ 3] ,该技术在
一般的聚丙烯酰胺凝胶基础上 ,加入了变性剂
(尿素和甲酰胺)梯度 ,从而能够把同样长度但序
列不同的 DNA 片段区分开来。此后十几年间 ,
该技术被广泛用于微生物分子生态学研究的各
个领域 ,目前已经发展成为研究微生物群落结构
的主要分子生物学方法之一 。
本文采用 DGG E方法分析了羊肚菌形成地
土壤样品中真菌类群 ,为利用微生物分子生态研
究羊肚菌与土壤微生物的关系 ,提供参考。
1　材料与方法
1.1 样品的采集
　　2006年 4月 20日在云南省丽江市鲁甸镇东
海村(海拔 2 350 m)采集羊肚菌子实体根部 1 ～
10 cm 处土样(编号 DM),以附近菜园土作对照
(编号 DK)。样品置于 -70 ℃中速冻后于
-20 ℃保存 。
1.2 土壤样品的 DNA提取
　　按照何丽鸿等的文献[ 4]描述的方法提取两
个土壤样品的 DNA 。
1.3 28S rDNA片段的 PCR扩增及检测
　　25μL PCR 反应体系:50 ng DNA ,1×PCR
反应缓冲液 , 2 mmol/L Mg2+ , 0.2 mmol/L
dNTPs , 0.5 pmol/L 正 、反向引物 , 3U Taq 酶
(Pr omega ,中国)。PCR 扩增条件为:94 ℃预变
性 5 min;94 ℃变性 45 s , 57 ℃退火 45 s ,72 ℃
延伸 1 min ,35 个循环;72 ℃延伸 10 min 。正反
向引物的序列分别为:U1:5-GTGAA ATTGT
TGAAA GGGAA-3 和 U2:5-CGCCC GCCGC
GCGCG GCGGG CGGGG CGGGG GACTC
CTTGG TCCGT GTT-3。取 5 μL 的 PCR 产
物 ,进行 1.5%的琼脂糖凝胶电泳;电泳结束后
EB染色 , Pharmacia VDS 凝胶成像系统拍照记
录电泳结果。
DOI :10.16488/j.cnki.1005-9873.2009.02.004
第 2 期 沈　洪 , 等:采用变性梯度凝胶电泳研究羊肚菌土壤真菌群落结构
1.4 DGGE分析
　　按照赵勇等的文献[ 5] 描述的方法 ,对样品
进行 DGGE 分析 。
1.5 DGGE条带的割胶测序
　　按照 SUN 等的文献 [ 6] 描述的方法 , 对
DGGE 胶上某些条带进行割胶 、回收 、克隆 、测
序。测序工作由上海博亚生物技术有限公司
完成 。
2　结果与分析
2.1 28S rDNA PCR扩增产物的琼脂糖凝胶电泳
　　将 28S rDN A PCR扩增产物进行 1.5%琼脂
糖凝胶电泳 ,电泳结果如图 1 ,成功地扩增到约
260 bp的目的片段。电泳图谱中 1为 PCR扩增
的空白对照(在 PCR扩增时采用等体积的无菌重
蒸水替代加入样品的 DNA 模板)没有扩增出条
带 ,证明反应体系没有受到外源 DNA 的污染。
M 为 DGL 2 000分子标记 , 1为 PCR 空白对照 , 2 和 3 分别是
DK 对照土样和 DM 羊肚菌生长点土样的 PCR扩增产物
Lan e M:DGL 2 000 M ark er , lane 1:PCR negat ive cont rol ,
lanes 2 and 3:PCR amp li ficat ion resul t s for DK(cont rol soi l
sample)and DM (morel g row th soil sample)
图 1　28S rDNA PCR 产物的琼脂糖凝胶电泳图谱
Fig.1　Gel electrophoresis of 28S rDNA PCR products
2.2 28S rDNA PCR扩增产物的 DGGE图谱
　　不同真菌类群的28S rDNA PCR扩增片段 ,片
段大小基本一致 ,但其 DNA 的序列组成存在差
异 ,利用 DGGE 可以将片段大小相同但序列组成
不同的不同真菌类群的 28S rDNA PCR扩增片段
进行分离。如图 2所示 ,在 35%～ 65%的变性梯
度范围内 , 2 份取不同地点的土样样品 ,其 28S
rDNA PCR扩增产物都实现了较好的分离。
在采取的两份土样 DK和 DM 的 DGGE 带谱
比较 ,发现其中优势条带 Z1 、Z5 、Z7 、Z8 、Z9 、Z11是两
份土样中的共有条带 ,但是 Z1 、Z2 、Z3 、Z5 、Z6 、Z7 、
Z12在羊肚菌生长的土样 DM 中的丰度比较高 ,为
优势菌(较亮的条带所代表的物种 ,丰度很低的菌
种难以在 DGGE胶上检出 。条带的丰度在一定程
度上可作为起始细菌群落多样性及丰度的量度),
能辨别的条带中 ,DM 有 19条 ,DK 也有 19条 ,其
中 8条为两者共有 。在所测序分析的 12条带中 ,
Z2 、Z3 、Z4 、Z6 、Z10为土样 DM特有条带。
左边为银染照片 ,右侧为示意图 , DK 为对照土样 , DM 为羊肚菌
生长点土样 ,Z1 ～ Z12为进一步纯化测序的条带
Lef t sector:DGGE spect rogram af ter silver s taining;Right
sector:schematic repres entation of spect rogram;DK:cont rol
soil s amp le , DM :M orchella grow th soi l sample;Z1 ～ Z12 :
dominant bands selected for pu rif icat ion and sequen cing
图 2　采用 PCR 对样品 28S rDNA进行扩增后产物
的 DGGE图谱
Fig.2　DGGE spectrogram of 28S rDNA fragments
2.3 DGGE图谱中主要条带的序列测定
　　以上述 Z1 ～ Z12条带 ,进行克隆测序 ,测序结
果及 Blast比对获得的系统分类信息如表 1。
表中可以看出 ,Z2 、Z 3 、Z6 、Z7 、Z8 、Z9 、Z10都为
子 囊 菌 亚 门 (A scomycota ) 盘 菌 纲
(Pezizomyco tina)真菌 ,Z 11为子囊菌亚门真菌 ,Z 1
和 Z5 为未能培养的真菌 ,被发现存在于蘑菇堆
肥中 , 其中 Z1 分类未定 , 而 Z 5 与 uncul tured
Pezizomycot ina(DQ 273467)的相似性达到 97%,
也有可能为盘菌纲的真菌 。因此除 Z 1 不能确
定 ,Z 4 为担子菌亚门(Basidiomycota)异担子菌纲
(Heterobasidiomycetes)外 ,其余均为盘菌纲真
菌 种 类 , Z6 、 Z 9 、 Z10 为 盘 菌 纲 中 的
Sordariomycetes亚纲 , Z2 、Z8 和 Z12都属于盘菌
目(Pezizales)。
7
食　用　菌　学　报 第 16 卷
表 1　目的条带的序列比对结果
Table 1　Sequences and phylogenetic affiliations of DNA recovered from DGGE gel
样品编号
Sample
No.
相似生物种类
Blast match
organism
基因库中的登录号
GenBank
Accession No.
相似性(%)
Similar ity
分类
Taxonomy
Z1 未培养真菌
Uncultured
compost fungus
DQ365334 98
Z2 Peziza apiculata AF335129 97 Ascomycota;Pezizomycotina;Pezizomycetes;Peziz ales;
Pezizaceae;Peziza
Z3 Leptosphaerulina
australis
AY849948 99 Ascomycota; Pezizomycotina; Dothideomycetes;
Pleosporales;Pleosporaceae;Leptosphaerulina
Z4 Cryptococcus
terricolus
AJ510144 99 Basidiomycota;Hymenomyce tes;Hete robasidiomycetes;
Treme llomycetidae;F ilobasidiales;mitospor ic
Filobasidiales;Cryptococcus
Z5 未培养盘菌
Uncultured
Pezizomycotina
DQ273467 97 Ascomycota , Pezizomycotina
Z6 Lecy thophora
mutabilis
AF353604 100 Ascomycota; Pezizomycotina; Sorda riomycetes;
Sordar iomycetidae; Coniochaet ales; Coniochae taceae;
mitosporic Coniochaetaceae;Lecythophora
Z7 Leptosphaeria
calvescens
AY849944 94 Ascomycota;Pezizomycotina;Dothideomyce tes;
Pleosporales;Leptosphaer iaceae;Leptosphaeria
Z8 Ascobolus
denudatus
AY500528 98 Ascomycota;Pezizomycotina;Pezizomycetes;
Pezizales;Ascobolaceae;Ascobolus
Z9 Truncatella
laurocerasi
AF382385 98 Ascomycota;Pezizomycotina;Sordar iomyce tes;
Xylar iomyce tidae;Xylar iales;Amphisphaer iaceae;
mitosporic
Z10 Cordyceps
sinensis
AB067709 98 Ascomycota;Pezizomycotina;Sordar iomyce tes;
Hypocreomycetidae
Z11 Pyrenochae ta
nobilis
DQ678096 95 Ascomycota;Pezizomycotina;Dothideomycetes;Pleosporales;
Pleosporales incertae sedis
Z12 Morchella elata AY544665 99 Ascomycota;Pezizomycotina;Pezizomycetes;Peziz ales;
Morchellaceae;Morchella
3　讨论
　　与传统的分离 、培养法会受到所选用的培养
基以及培养条件的限制一样 ,各种新兴的分子生
态学方法也不可避免地在设计原理和技术操作
上存在一些缺陷。首先在细胞破壁及 DNA 的提
取过程中 , 由于对不同细胞的破壁效果存在差
异 ,从而导致了微生物结构分析上的偏差[ 6] ,因
此采用高效的 DNA 提取方法 ,以保证获得的
DNA 能真实地反映原始样品中的微生物组成情
况是极为重要的。
本试验采用的 DGGE 是基于 PCR技术的
核酸分析法 , 不可避免地受到 PCR扩增中一
些不可控因素的限制 , PCR 扩增的偏好性
(prefe rential amplificat ion)是一个影响实验结
果的一个重要因素 ,尤其是在对环境样品进行
的多模板扩增中该现象比较严重;而且不同微
生物的基因组大小 、28S rDNA 的拷贝数都会
对 PCR扩增产生影响 , 导致 PCR扩增的产物
量同其所反映的原始样品中相应微生物其数
8
第 2 期 沈　洪 , 等:采用变性梯度凝胶电泳研究羊肚菌土壤真菌群落结构
量间的相关性较差[ 7] 。
采用多种不同的引物同时对样品中的微生
物结构进行分析 ,有助于减少 PCR偏好性扩增对
分析结果造成的偏差;同时 ,选用专一性更高的
引物分别对各类群微生物进行研究也不失为好
的选择[ 7] 。
同时 DGGE 所能检测到的微生物数量也是
有限的:通过 DNA 的复性试验发现某土壤样品
中至少存在 104 种不同的基因组类型 ,而显然地 ,
在 DGGE电泳图谱上无法检测到这么多类群的
微生物。研究结果表明 ,只有当该类群的微生物
达到总微生物数量的 1%以上 , 才可以通过
DGGE或 SSCP 等方法被检测到[ 7 , 8] 。
从实验结果可以看出对照土样在 DGGE 中
显示的 DNA 条带明显多于羊肚菌生长的土样 ,
反映出羊肚菌生长土样中真菌的丰度低于对照
土样 ,这一结果在我们对土壤样品进行建库测序
中得到了重复(未发表数据),这可能与土壤中羊
肚菌优势种群对其它种群的抑制作用有关 。从检
测的两个试验土壤样本来看 ,在羊肚菌生长的土
壤样本中检测出了羊肚菌的 DNA 片段而在对照
样本中未检测出 ,这一结果暗示了采用 DGGE对
土壤样本的检测是可行的。
参考文献
[ 1] 朱斗锡.羊肚菌人工栽培研究进展[ J] .中国食用菌 ,
2008 , 27(4):3-5.
[ 2] 张飞翔.羊肚菌人工栽培技术[ J] .吉林农业 , 2002 ,
144:35.
[ 3] 项　丽.唐建设.变性梯度凝胶电泳(DGGE)在环境
微生物生态中的应用[ J] .安徽农学通报 , 2007 , 13
(15):28-30
[ 4] 何丽鸿 , 于荣利 , 陈明杰 , 等.采用 ARDRA 研究双孢
蘑菇培养料后发酵过程中的细菌群落结构(Ⅰ)—细
菌 16S rDNA 全长文库的建立 [ J] .食用菌学报 ,
2008 , 15(4):11-15.
[ 5] 赵　勇 , 李　武 , 周志华 , 等.秸秆还田后土壤微生
物群落结构变化的初步研究[ J] .农业环境科学学
报 , 2005 , 24(6):1114-1118.
[ 6] SUN HY , DENG SP , RA UN WR. Bacterial
community structure and div ersity in a century-old
manure-trea ted ag ro-eco sy stem [ J] .Appl Environ
M icrobio l , 2004 , 70:5868-5874.
[ 7 ] VON WINTZINGERODE F , G BEL UB ,
STACKEBRANDT E.De te rmination o f microbial
dive rsity in environmental samples:pitf alls o f PCR-
based rRNA analy sis [ J] .FEMS Microbio l Rev ,
1997 , 21(3):213-229.
[ 8] MUYZER G , SMALLA K. Application of
denaturing g radient g el electr opho resis (DGGE) and
temperature g radient gel e lectropho resis (TGGE) in
mic robial eco log y [ J] .Antonie v an Leeuwenhoek ,
1998 , 73(1):127-141.
[本文编辑] 　曹　晖
9
ACTA EDULIS FUNGI 2009.16(2):10 ～ 12
Received:March 25 , 2009;　Accepted:Apr il 7 , 2009
Suppor ted by Foundation of the Science and Technology Commission of Shanghai Municipality(No.06DZ22036)
and Project of China Agr icultural Ministr y(No.nyhyzx-07-008)
＊Corresponding author.Tel:+86-21-52630034　E-mail:mjchen@china.com
Analysis of Fungal Communities Present in Morchella
Growth Soil Using Denaturing Gradient Gel Electrophoresis
SHEN Hong
1 , 2 , WANG Hong1 , ZHAO Yongchang3 , FENG Aiping1 , CHEN Mingjie1＊ , PAN Yingjie4
(1Key Laboratory of Applied Mycological Resources and Utilization , Ministry of Agr iculture;Shanghai
Research Cen te r for Biotechnology and Engineer ing of Edible Fungi , Shanghai Key Labora tory of Agricultur al
Genetics and Breeding , I nstitute of Edible Fungi , Shanghai Academy of Agricultural Sciences , Shanghai 201106 , China;
2Depar tment of Microbiology , College of Life Sciences , Nanjing Agr icultur al University , Nanjing , Jiangsu 210095 , China;
3Yunnan Institute of Biological Technologise , Yunnan Agricultural Academy , Kunming , Yunnan 650205 , China;
4Shanghai Ocean University , Shanghai 200090 , China)
Abstract:Fungal communities inhabiting Morchella growth soil(DM) and common vegetable ga rden soil
(DK)were monitor ed using Denaturing Gradient Gel Electr ophoresis (DGGE).Sequence analysis of 12
re cover ed dominant bands r evealed the pr esence of ten fungi assigned to the Ascomycota , Pezizomycotina ,
one assigned to the Basidiomycotina , He terobasidiomyce tes , and one unidentif ied fungus.Compar ison of the
dominant fungal groups pr esent in DM and DK revealed large diff erences in the community composition with
respect to Pezizomycete fungi.Fungal communities associa ted with DM soil were much less var ied in
composition compared with those associa ted with DK soil , possibly due to inhibitory ef fe cts exer ted by the
dominant Morchella .
Key words:Morchella elata ;Morchella growth soil;f ungal community;PCR-DGGE
　　A semi-ar tificial cult ivation technique for
Morchella elata and M . conica has been
developed but the pr ocesses involved in the
formation and development of mushroom
primordia remain unclear.However , a key
step in the cul tivation procedur e is the addit ion
of casing soil.The impact of casing , and the
role(s)of the micr obial communit ies inh abit ing
the casing soil , on the development of the
Morchella fruit bodies requires fur ther study.
　　Denaturing Gradient Gel Elect rophoresis
(DGGE), developed by LERMAN et al in the
1980 s , is a useful method for studying the
st ructure of microbial communities
[ 3] .DNA
fr agments of the same size but comprised of
different sequences can be separ ated by DGGE
based on the different ial denaturing
characterist ics of the DNA as it migr ates
through a polyacrylamide gel containing
increasing concentra tions of chemical
denaturants such as ure a and formamide.
We now report data , obtained from using
DGGE to de termine the composition of the
fungal communit ies in Morchella growth soil ,
which provide a be tter understanding of the
role(s)of fungal communi ties in the promot ion
and inhibition of Morchella growth and frui t
body development.
1　Materials and Methods
1.1 Soil sampling
　　Soil samples were collected from within
one to ten centimeters of Morchel la fruit bodies
gr owing in the vicinity of Donghai V il lage
(alt itude 2 350 m), Ludian Town , Lijiang
City , Yunnan Province in mid-Apr il , 2006 ,
Contr ol soil samples were taken f rom a nearby
vege table garden.After collect ion , the samples
No.2 SHEN Hong , WANG Hong , ZHAO Yongchang , et al
were quickly fr ozen at -70 ℃ and preserved a t
-20 ℃.
1.2 DNA extraction
　 　 DNA was extracted accor ding to the
procedure described in Ref erence 4.
1.3 PCR amplification and determination of
28S rDNA
　　Reaction mixtures (25 μL) containing:
50 ng DNA , 1 ×PCR reaction buffer , 2 mmol/L
Mg
2+ ,0.2 mmol/ L dNTPs , 0.5 pmol/L each of
forward and r everse pr imers , and 3U Taq
polymer ase (Promega , China ). PCR
amplificat ion conditions were as follows:1
cycle at 94 ℃ f or 5 min;35 cycles at 94 ℃ for
45 s , 57 ℃ for 45 s and 72 ℃ for 1 min;
followed by a final extension at 72 ℃ for
10 min.The sequences of the forward (U1)
and reverse (U2) primers were 5-GTGAA
ATTGT TGAAA GGGAA-3 and 5-CGCCC
GCCGC GCGCG GCGGG CGGGG CGGGG
GACTC CTTGG TCCGT GTT-3 , respect ively.
Aliquots(5 mL)of the 28S rDNA amplificat ion
products were separated by 1.5% (w/v)
agar ose gel electrophoresis and , after staining
with e thidium br omide , the ampl ificat ion
pat terns were determined using a Pharmacia
VDS auto gel imaging analyzer.
1.4 DGGE analysis
　 　 DGGE analyses we re carr ied out as
descr ibed in Reference 5.
1.5 Cloning and sequencing of the DGGE product
　　Selected DNA bands obtained by DGGE
were recovered , cloned and sequenced as
descr ibed in Reference 6.Sequencing was
carried out by I nvit rogen Biotechnology
Co.Ltd.
2　Results and Analysis
2.1 Detection of 28S rDNA by agarose
gel electrophoresis
　　Total microbial DNA was extr acted from
two soil samples , and agarose gel
electr ophor esis revealed discrete , reproducible
bands approximately 260 bp in size (see Fig.1
in the Chinese version), indicat ing that the
extracted DNA was of high qual ity.
2.2 Analysis of 28S rDNA band patterns
generated by DGGE
　 　 Good separat ion of the 28S rDNA
amplificat ion pr oducts generated from the two
test soil samples was achieved within the 35%
～ 65% r ange of the urea/ formamide
concentr at ion gr adient (see Fig.2 in the
Chinese version).
A total of 19 different bands were de tected
in each of the two soil samples of which eight
were present in both the test and control
samples.The dominant bands Z 1 , Z5 , Z7 , Z 8 ,
Z9 and Z 11 were detected in both control and
test soils , while the dominant bands Z2 , Z 3 , Z 6
and Z12 were re corded only in the Mor chella-
associated soil sample DM.Of the 12 sequenced
bands , Z2 , Z 3 , Z 4 , Z6 and Z10 were specific to
DM.
2.3 Sequence analysis and phylogeny of the
dominant 28S rDNA bands
　　Sequencing and BLAS T results for the
bands Z 1 ～ Z12 are show n in Table 1 (see the
Chinese ver sion).
Sequence homology analysis revealed tha t
the dominant bands Z 2 , Z 3 , Z 6 , Z7 , Z8 , Z9
and Z1 0 we re members o f the A scomyco ta ,
Pezizomycot ina.Z6 , Z 9 , Z 10 were assigned to
the sub-class So rdariomycete s , while Z2 , Z8
and Z 12 were assigned to the Pezizale s.Z5
sha red 97% homology w i th uncultured
Pezizomycot ina , and Z4 exhibited high level
homolo gy w ith Cryptococcus terricolus
belonging to the Basidiomyco ta , Hetero-
basidiomycetes.
3　Discussion
　　Discrepancies in microbial community
analy sis usually result f rom dif fe rences the
microbial cell st ructure and ef ficiency o f cell w all
disrupt ion w hich , in turn , af fect the quality o f
the ex t racted DNA
[ 6] .The refore , an eff icient
and non-destructive ex traction me thod is
11
ACTA EDULIS FUNGI V ol.16
essential if the ex tracted DN A is to accurately
reflect the composition of the microbial
communi ty.Furthe r dispari ties can be avoided
by using several primer s of high specificity to
amplify the samples[ 7] .
The analyt ical potency of the DGGE
method is also limi ted because dif ferent
microbial types can be detected ef fectively only
w hen they are present in the microbial
communi ty under examinat ion in suf ficient ly
high numbers(>1%)[ 7 , 8] .
In the present study , few er bands we re
amplif ied f rom the Morchel la grow th soil than
f rom the control vegetable garden soil , possibly
indicating that the dominant Morchel la exerted
an inhibitory effect on the overall microf lora.
Few er bands w ere also obtained during the
const ruction o f a library o f fungal species
pre sent in Morchel la g row th soils , lending
fur ther support for the existence o f such
inhibi to ry effects (SHEN et al., unpublished
results).Our data show ing that Morchel la DNA
fragments w ere detectable only in Morchella
g row th soi ls indicated that i t is feasible to
analy ze the composit ion of the microbial
communi ty in such samples using the DGGE
method.
References
[ 1] ZHU DX.The progress of art if icial cultur e for
Morchella esculenta [ J] .Edible Fungi of China ,
2008 , 27(4):3-5.(in Chinese)
[ 2] ZHANG FY.Technique for ar tif icial culture for
Morchella esculenta [ J] .Jilin Agricultur e , 2002 ,
144:35.(in Chinese)
[ 3] XIANG L , TANG JS.Application of denatured
gradient gel ele ctrophoresis in environmental
microbiology [ J ] . Anhui Agr iculture Science
Bulletin , 2007 , 13 (15):28-30.(in Chinese with
English abstr act)
[ 4] HE LH , YU RL , CHEN MJ , e t al.Analysis of
bacter ial communities pr esent in Agaricus bisporus
Phase II compost using Amplified Ribosomal DNA
Restr iction Analysis(ARDRA)I-constr uction of a
bacter ial 16S rDNA libr ary[ J] .Ac ta Edulis Fungi ,
2008 , 15(4):11-19.
[ 5] ZHAO Y , LI W , ZHOU ZH , et al.Changes of
microbial community str ucture in str aw amended
soil[ J] .Journal of Agro-environmental Science ,
2005 , 24(6):1114-1118.(in Chinese with English
abstr act)
[ 6] SUN HY , DENG SP , RAUN WR. Bacter ial
community str uctur e and diversity in a century-old
manure-tr ea ted agro-ecosystem [ J] .Appl Environ
Microbiol , 2004 , 70:5868-5874.
[ 7] VON WINTZ INGERODE F , G BEL UB ,
STACKEBRANDT E.De term ination of microbial
dive rsity in environmental samples:pitf alls of PCR-
based rRNA analysis[ J] .FEMS Microbiol Rev ,
1997 , 21(3):213-229.
[ 8] MUYZER G , SMALLA K. Application of
denatur ing gradient gel electr ophoresis (DGGE)
and temperatur e gr adient gel ele ctrophoresis
(TGGE) in m icrobial ecology [ J] .Antonie van
Leeuwenhoek , 1998 , 73(1):127-141.
12