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Fluorescent staining of septa and nuclei in Ophiocordyceps sinensis and Cordyceps militaris

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冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色 胡晓棣
冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色 胡晓棣
冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色 胡晓棣
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Abstract

As important medicinal fungi, both Ophiocordyceps sinensis and Cordyceps militaris have gained significant scientific attention in recent decades. However, very little work on mycelial staining method, which is the basis of cytological research, has been reported. Fluorescent staining is a simple and fast staining technique. In the present study, four dyes were tested, singly
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
菌物学报
jwxt@im.ac.cn22September2016,35(9):1099‐1105
Http://journals.im.ac.cnMycosystemaISSN1672‐6472CN11‐5180/Q
Tel : +86‐10‐64807521Copyright©2016InstituteofMicrobiology,CAS, all rights reserved.
研究论文 ResearchpaperDOI:10.13346/j.mycosystema.150139

基金项目：国家自然科学基金（31170017，31400018）；青海省科技计划（2014‐NS‐524，2014‐NS‐525）；国家科技支撑计
划（2013BAD16B013）
SupportedbytheNationalNaturalScienceFoundationofChina(31170017,31400018);QinghaiScience&Technology
Department(2014‐NS‐524,2014‐NS‐525)andKeyTechnologiesResearch&DevelopmentProgramofChina(2013BAD16B013).
*Correspondingauthor.Tel : +86‐10‐64807518;Fax:+86‐10‐64807468;E‐mail:yaoyj@im.ac.cn
Received:2015‐06‐04,accepted:2015‐10‐12
冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色
胡晓棣 1,3李熠 1,2任蜀豫 1姚一建 1*
1中国科学院微生物研究所真菌学国家重点实验室北京100101
2福建农林大学植物保护学院福建福州350002
3中国科学院大学北京100049
摘要：冬虫夏草和蛹虫草作为重要的药用真菌，得到广泛的重视，迄今已有大量的研究报道。然而，作为细胞学研究
的基础处理方法，其菌丝隔膜和细胞核染色却缺乏必要的研究。荧光染色是一种程序简便、快速灵敏的染色方法。本研
究选用 DAPI、PI、CalcoflourWhite 和刚果红等 4种染料，对冬虫夏草、蛹虫草菌丝的隔膜和细胞核进行单独与组合染色
实验，通过显微观察比较得出较好的染色方法。结果表明 DAPI 对冬虫夏草和蛹虫草菌丝的细胞核染色效果都较好，而
CalcoflourWhite 对两者的细胞壁染色效果较好且隔膜清晰。DAPI 与CalcoflourWhite 两者进行冬虫夏草菌丝组合染色的效
果为最佳，但在蛹虫草菌丝染色中效果不太稳定。对蛹虫草菌丝较好的组合染色是 DAPI 与刚果红的组合，但其染色结果
需要在激光共聚焦显微镜下观察。
关键词：荧光染料，4’,6‐二脒基‐2‐苯基吲哚，碘化丙啶，荧光增白剂，刚果红
FluorescentstainingofseptaandnucleiinOphiocordycepssinensisand
Cordycepsmilitaris
HUXiao‐Di1,3LIYi1,2RENShu‐Yu1YAOYi‐Jian1*
1StateKeyLaboratoryofMycology,InstituteofMicrobiology,ChineseAcademyofSciences,Beijing100101,China
2CollegeofPlantProtection,FujianAgricultureandForestryUnive rsity,Fuzhou,Fujian350002,China
3UniversityofChineseAcademyofSciences,Beijing100049,China
Abstract:Asimportantmedicinalfungi,bothOphiocordycepssinensisandCordycepsmilitarishavegainedsignificantscientific
attentioninrecentdecades.However,verylittleworkonmycelialstainingmethod,whichisthebasisofcytologicalresearch,has
beenreported.Fluorescentstainingisasimpleandfaststainingtechnique.Inthepresentstudy,fourdyesweretested,singly
ISSN1672‐6472CN11‐5180/QMycosystemaSeptember22,2016Vol. 35No.9
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andcombinedly,foruseonOphiocordycepssinensisandCordycepsmilitarisaspossiblefluorescentstainsoffungalnucleiand
septa.TheresultsshowedthatDAPIfluorescentstainedbetterthanPIforthenuclei,andtheCalcoflourWhiteisbetterthan
CongoRedinstainingthesepta.Inthecombinedstainings,DAPIandCalcoflourWhitestainednucleiandsepta,respectively,
moreeffectivelyinO.sinensisthaninC.militaris,whereasthecombinationofDAPIandCongoRedismoresuitableforthelatter
byusingthelaserconfocalmicroscope.
Keywords:fluorescentdye,DAPI,PI,CalcoflourWhite,CongoRed
冬虫夏草 Ophiocordycepssinensis(Berk.)G.H.
Sung,J.M.Sung,Hywel‐Jones&Spatafora 和蛹虫草
Cordycepsmilitaris(L.)Fr. 都是广义虫草属
Cordycepss.l.的成员。虫草是一类具有重要药用和
开发价值的真菌，其中冬虫夏草为我国传统名贵滋
补中药材，而蛹虫草则经常被作为冬虫夏草替代品
来使用，两者具有广阔的市场前景。对这两种虫草
的生物学、基因组学、资源、活性物质、菌丝体培
养和人工栽培等方面都有比较深入的研究（Huang
2003；Dong&Yao2011；Lietal.2013），但在细胞
学研究方面尚有待进一步深入。
目前在冬虫夏草和蛹虫草的研究中，大部分采
用的是光学显微镜直接观察的方法，如观察菌落、
菌丝体、分生孢子和子囊孢子等（曾纬等2006；
何苏琴等2011）。偶尔也见使用真菌观察中最常
见的棉兰等染色方法（杨振芳2009）。近年来荧
光染料染色也逐渐得到使用，如 Ren&Yao（2013）
采用 Calcoflour对冬虫夏草分生孢子细胞壁进行
染色观察；Huetal.（2013）使用 4’,6‐二脒基‐2‐苯
基吲哚（DAPI）和荧光增白剂（CalcoflourWhite）
进行组合染色观察到冬虫夏草菌丝体、分生孢子和
子囊孢子细胞内都呈单核状态。此外还有使用
Hoechst33258 荧光染料对粉被虫草 Cordyceps
pruinosaPetch 进行染色观察的报道（梁宗琦&Fox
1997）。然而，为了更深入地进行虫草菌细胞学研
究，荧光染色方法的使用需要更进一步的探讨。
真菌染色研究已至少有 100 多年的历史。早在
上个世纪 20 年代，Feulgren&Rossenbeck（1924）
发明的福尔根反应法就得到了广泛的应用。之后，
以碱性品红为代表的染色剂开始应用于真菌观察，
比早期的染色剂更为敏感，但是特异性不太强
（Delamater1948；Colemanetal.1981）。到 1969
年，以激光辐射为基础的荧光染色方法开始出现，
并能用于快速测定单细胞群体中每个细胞的 DNA
含量（VanDillaetal.1969）。从上个世纪 70 年代
至今，荧光染色方法得到了大批学者的关注并应用
于各类生物类群，使染色方法向前迈进了一大步，
同时也促进了大量荧光染料的开发，如 Holmquist
（1975）使用 Hoechst33258 染色观察了果蝇的染
色体，而Andersonetal.（2010）则用 PontamineFast
Scarlet4B（P4B）染色观察了植物根部细胞壁扩张
时细胞膜的重新定位。在真菌研究中，荧光染色方
法同样得到大量使用，促进了真菌细胞学等研究的
发展。Bagoetal.（1998）采用碘化丙啶（PI）染
色的方法对丛枝菌根真菌 Gigasporarosea 菌丝进
行活体观察并总结出细胞核行为。Jany&
Pawlowska（2010）使 用 DAPI 染色，并通过激光共
聚焦显微镜进行 Glomeromycota 的活体观察，探
讨多核孢子与物种古老性的关系。同时，组合荧
光染色的方法也得到使用，如针对植物病原真菌
的细胞核和隔膜的组合染色（康振生等 1993）；
使用 Hoechst33258 和CalcoflourWhite 组合染色
观察多核菌丝中的细胞核行为（Maheshwari
2005）等。
鉴于对广义虫草属菌丝染色观察的研究报道
不多，其细胞学研究亟需深入，本研究初步探讨了
胡晓棣 等 /冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色
菌物学报
1101
不同荧光染色方法和不同染色剂对菌丝隔膜和细
胞核的染色效果，通过显微观察比较得出对冬虫夏
草和蛹虫草菌丝染色效果较好的方法，为广义虫草
属菌丝染色与细胞学研究提供技术支持。
1材料与方法
1.1供试菌株
冬虫夏草菌使用本实验室保存的菌株 762、
1224、1227、1229、2238、2241、2243，保 存 于 4℃
的培养基中。主要供试菌株为 1229，该菌株是由
本课题组从新鲜标本分离得到的单子囊孢子菌株。
蛹虫草菌使用本实验室保存的菌株 1018、1033、
1035、1065、1809、2087，保存于 4℃的 PDA 培
养基中，主要供试菌株为 1065 菌株。
1.2菌丝样品制备
在无菌超净台中，用打孔器从冬虫夏草固体平
板中取出 3个直径约为 6mm 的菌块，接入装有
50mLPDA 麦麸液体培养基的 250mL 三角瓶中，在
18℃下 100r/min 避光培养 14d 做为接种液。取
0.5mL 接种液接至直径 9cm 的固体平板上，涂布均
匀，将无菌盖玻片（10mm×10mm）以 45°角度插
入培养基中，每个平板均匀插入 10 片盖玻片。正
置培养 1d，待液体被吸收后再倒置于 18℃恒温培
养箱中避光培养 60d。待菌丝爬到盖玻片约 2/3 处时
取出备用，染色之前先在 10%福尔马林中固定 1h。
用无菌水吹打蛹虫草固体平板培养物，洗下成
熟孢子，制成孢子悬液。取 0.5mL 孢子悬液接至直
径9cm的固体平板上，涂布均匀，将无菌盖玻片
（10mm×10mm）以 45°角度插入培养基中，每个
平板均匀插入 10 片盖玻片。正置培养 1d，待液体
被吸收后再倒置于 20℃恒温培养箱中避光培养
3–5d。待菌丝爬到盖玻片约 2/3 处时取出备用，染
色之前先在 10%福尔马林中固定 1h。
1.3荧光染料及配制
称取荧光染料 5mgDAPI （4,6‐diamidino‐2‐
phenylindole，Biotopped 公司），用少量无菌水溶
解后定容成 50μg/mL的母液，于‐20℃下保存。使
用时用 PBS 缓冲液（pH6.8，0.1mol/L）稀释成
5μg/mL 的染色液（Liescheetal.2013）。激发光与
发射光波长为 340/488nm，激发显蓝色荧光。
荧光染料 FluorescentBrightener28（Calcoflour
White，荧光增白剂，Sigma 公司）。配制方法同上，
稀释终浓度为 2.5μg/mL（Thomasetal.2013）。激 发
光与发射光波长为 345/430nm，激发显蓝色荧光。
荧光染料 Propidiumiodide（PI，碘化丙啶，Sigma
公司）。按上述方法配制，使用时稀释成 20μg/mL
的染色液（Bagoetal.2013）。激发光与发射光波
长为 535/615nm，激发显红色荧光。
染料刚果红（CongoRed，上海试剂三厂）。配
制的染色液浓度为 1%（M/V），即将 1g 刚果红溶
于蒸馏水，至终体积 100mL。
1.4样品的荧光染色处理
单独荧光染色方法使用细胞核染料 DAPI、PI
和细胞壁染料 CalcoflourWhite、刚果红分别对冬
虫夏草和蛹虫草菌丝进行单独染色。用镊子取出布
有菌丝的盖玻片，擦去反面的菌丝。向不同盖玻片
着生菌落的位置滴加染料，刚果红 200mL、DAPI
200mL、PI200mL、CalcoflourWhite2滴，除前者
染色 12h，其他各种染料均染 30min。染色后，PBS
缓冲液冲洗 1min。将带有菌落一侧盖玻片朝下，
盖到滴有 PBS 缓冲液的载玻片上，置于荧光显微
镜下观察。
组合荧光染色将两类染料两两配对，对冬虫夏
草和蛹虫草菌丝的细胞核与细胞壁进行染色，共有
DAPI 与CalcoflourWhite、DAPI 与刚果红、PI 与
CalcoflourWhite、PI 与刚果红等 4种组合。染色时，
除了刚果红先采用上述方法单独对细胞壁进行染
色，用 PBS 缓冲液冲洗约 1min 后再滴加细胞核染
料，再用 PBS 缓冲液冲洗约 1min。其他组合的两
种染料同时进行混合染色，30min 后用 PBS 缓冲液
冲洗约 1min。显微镜观察方法同上。
ISSN1672‐6472CN11‐5180/QMycosystemaSeptember22,2016Vol. 35No.9
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1.5样品的观察及照相
本研究使用的显微设备为 ZeissIMAGERA2 型
正置荧光显微镜和 LeicaSP8 双扫描模式激光共聚
焦显微镜。前者一般在 400 倍下进行显微观察，
并采用蔡司显微镜数码摄像系统 AxiocamMRC 以
及配套的 DAPI、Tex a s R e d 荧光模块，通过 Axiovision
Rel.4.6 软件拍照成像。后者观察倍数通常为 630
倍，通过 LeicaLASAF 软件采集图像。除了特别提
到使用激光共聚焦显微镜，本文中涉及的观察均使
用荧光显微镜。
2结果与分析
2.1冬虫夏草单独染色与组合染色
2.1.1冬虫夏草单独染色结果：DAPI 对冬虫夏草细
胞核的染色特异性很高，可以清晰地观察到菌丝中
蓝色圆形的细胞核（图 1A）。PI 也使细胞核染色，
但其特异性和稳定性都比前者差一些（图 1B）。细
胞壁染料 CalcoflourWhite 的染色作用明显，菌丝
的细胞壁及其隔膜均为蓝色，且隔膜尤为清晰，呈
光亮蓝色（图 1C）。刚果红染色的细胞壁和隔膜也
很明显（图 1D），但染色效果经常不稳定。
2.1.2冬虫夏草组合染色结果：DAPI 与Calcoflour
White 的组合对冬虫夏草菌丝的染色结果，经镜检
观察显示隔膜和细胞核清晰可见，效果明显。观察
中可见菌丝细胞多为单核，偶见双核存在（图 1E）。
刚果红与 DAPI 组合的染色，由于在荧光显微镜下
隔膜的亮度较低，需要通过激光共聚焦显微镜进行
观察。结果显示的细胞核呈弥散状（图 1F）。PI 与
细胞壁染色剂组合进行染色时，特异性都很低，无
法观察到细胞核。
2.2蛹虫草单独染色与组合染色
2.2.1蛹虫草单独染色结果：使用 DAPI 对蛹虫草
细胞核进行染色，结果清晰且特异性高，细胞核明
显（图 2A）；而 PI 对细胞核染色的特异性差，整
个菌丝往往都呈红色，通过升高或降低其浓度进行
的染色实验也还是表现出同样的效果（图 2B）。
图1冬虫夏草菌丝荧光染色A：DAPI 对细胞核的染色；
B：PI 对细胞核的染色；C：CalcoflourWhite 对细胞壁的染
色；D：刚果红对细胞壁的染色；E：DAPI 和CalcoflourWhite
的组合染色；F：DAPI 和刚果红的组合染色（激光共聚焦显
微镜）.图中三角形示细胞核，箭头示细胞壁.
Fig.1Fluorescencestainingofseptaandnucleiin
Ophiocordycepssinensis.A:NucleistainingwithDAPI;B:
NucleistainingwithPI;C:SeptastainingwithCalcoflour
White;D:SeptastainingwithCongoRed;E:Combined
stainingwithDAPIandCalcoflourWhite;F:Combined
stainingwithDAPIandCongoRed(laserscanningconfocal
microscopy).Thetrianglesindicatenucleiandthearrows
indicatesepta.
CalcoflourWhite 对细胞壁的染色使隔膜呈现亮蓝
色，灵敏度较高（图 2C）。刚果红对细胞壁具有一定
特异性，但在荧光显微镜下隔膜的亮度较低（图 2D）。
2.2.2蛹虫草组合染色结果：DAPI 与Calcoflour
White 组合对蛹虫草菌丝的染色效果不太稳定，有
胡晓棣 等 /冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色
菌物学报
1103
时可以观察到部分菌丝的细胞核和隔膜（图 2E）。
DAPI 与刚果红组合进行的染色结果有一定的特异
性，但是由于刚果红的染色较弱，需要在激光共聚
焦显微镜进行观察（图 2F）。PI 与细胞壁染色剂组
合进行的染色无法观察到细胞核。
图2蛹虫草菌丝荧光染色A：DAPI 对细胞核的染色；B：
PI 对细胞核的染色；C：CalcoflourWhite 对细胞壁的染色；
D：刚果红对细胞壁的染色；E：DAPI 和CalcoflourWhite
的组合染色；F：DAPI 和刚果红的组合染色（激光共聚焦显
微镜）.图中三角形示细胞核，箭头示细胞壁.
Fig.2FluorescencestainingofseptaandnucleiinCordyceps
militaris.A:NucleistainingwithDAPI;B:Nucleistainingwith
PI;C:SeptastainingwithCalcoflourWhite;D:Septastaining
withCongoRed;E:CombinedstainingwithDAPIand
CalcoflourWhite;F:CombinedstainingwithDAPIandCongo
Red(laserscanningconfocalmicroscopy).Thetriangles
indicatenucleiandthearrowsindicatesepta.
2.3荧光染色效果总结
冬虫夏草和蛹虫草菌丝的单独与组合荧光染
色的结果见表 1。
3讨论
在广义虫草属菌丝隔膜和细胞核染色方面，国
内外已有少量报道，但缺少较为系统的研究。本研
究针对冬虫夏草和蛹虫草菌丝的荧光染色方法作
了初步的探究，分别对菌丝隔膜和细胞核进行单独
染色和组合荧光染色，通过观察比较得出了灵敏度
较高、特异性较强的染色方法。
荧光染色的结果表明，DAPI 与CalcoflourWhite
分别对冬虫夏草和蛹虫草菌丝的细胞核与隔膜具
有较好的染色效果（图 1A，1C，2A，2C），这两 者
也是对冬虫夏草进行混合染色的最佳染色剂组合
（图 1E）。而对蛹虫草菌丝较好的组合染色是 DAPI
和刚果红，但其染色结果需要在激光共聚焦显微镜
下观察（图 2F）。
通过对菌丝进行组合染色，能够同时观察到菌
丝中隔膜和细胞核的数目与位置，以及菌丝分支情
况和菌丝间的联络等，从而可以对冬虫夏草和蛹虫
草菌丝体进行系统全面的观察与细胞学研究。实际
上许多真菌的细胞核行为研究，特别是食药用菌杂
交育种以及菌丝交配核迁移等方面都可以使用荧
光染色方法来进行。
有报道认为冬虫夏草菌丝细胞具单个细胞核
（Huetal.2013），但其双核的菌丝细胞也在研究
中发现（Bushleyetal.2013）。本研究通过荧光染
色观察，发现冬虫夏草菌丝中确实存在双核（图
1E）甚至多核的现象。这种双核或多核现象对冬虫
夏草菌的细胞核行为、基因组或者物种进化是否具
有重要意义，还需要进一步研究。
本研究通过显微观察比较得出了较好的荧光
染色方法，但是存在一定的局限性。使用 DAPI 与
CalcoflourWhite 对冬虫夏草菌丝进行组合染色时，
由于两种染料的激发与发射波长很相近，在荧光显
ISSN1672‐6472CN11‐5180/QMycosystemaSeptember22,2016Vol. 35No.9

http://journals‐myco.im.ac.cn
1104
表1冬虫夏草和蛹虫草菌丝的单独与组合荧光染色结果
Table1TheresultsoffluorescentstaininginOphiocordycepssinensisandCordycepsmilitaris
荧光染料
Fluorescentdye
冬虫夏草
Ophiocordycepssinensis
蛹虫草
Cordycepsmilitaris
亮度
Brightness
特异性
Specificity
亮度
Brightness
特异性
Specificity
DAPI++ √√√ ++ √√√
PI++√√ ++√
CalcoflourWhite++ √√√ ++ √√√
CongoRed+√√ +√√
DAPI&CalcoflourWhite++ √√√ ++√√
DAPI&CongoRed+√√ +√√
PI&CalcoflourWhite++√ ++√
PI&CongoRed+√ +√
注：符号“+”、“++”分别指染色效果较暗和明亮；符号“√”、“√√”、“√√√”均表明着色，且分别指细胞结构不清晰、可
见以及清晰可见.
Note:“+”,“++”indicatethecellularstructureswerestainedfaintlyandbrightly;“√”, “√√”, “√√√”meanthestainingresultswere
poor,fineandexcellent.
微镜下都呈现蓝紫色荧光，只能通过形状的不同来
辨别隔膜与细胞核，因此还需要继续探究不同颜色
的双染方法，增强染色结果的直观性。而蛹虫草菌
丝的组合荧光染色方法也还不完善，还有待进一步
提高。
[REFERENCES]
AndersonCT,CarrollA,AkhmetovaL,SomervilleC,2010.
Real‐timeimagingofcellulosereorientationduringcell
wallexpansioninArabidopsisroots.PlantPhysiology,
152(2):787‐796
BagoB,ZipfelW,W illiamsRM,ChamberlandH,LafontaineJG,
WebbWW,PichéY,1998.Invivostudiesonthenuclear
behaviorofthearbuscularmycorrhizalfungusGigaspora
roseagrownunderaxenicconditions.Protoplasma,
203(1‐2):1‐15
BushleyKE,LiY, WangWW,WangXL,JiaoL,SpataforaJW,
Yao YJ,2013.Isolationofthemat1‐1matingtype
idiomorphandevidenceforselfingintheChinese
medicinalfungusOphiocordycepssinensis.Fungal
Biology,117(9):599‐610
ColemanAW,MaguireMJ,ColemanJR,1981.Mithramycin‐
and4’‐6‐diamidino‐2‐phenylindole(DAPI)‐DNAstaining
forfluorescencemicrospectrophotometricmeasurement
ofDNAinnuclei,plastids,andvirusparticles.Journalof
Histochemistry&Cytochemistry,29(8):959‐968
DeLamaterED,1948.Basicfuchsinasanuclearstainforfungi.
Mycologia,40(4):423‐429
DongCH,Yao YJ ,2011.Onthereliabilityoffungalmaterials
usedinstudiesonOphiocordycepssinensis.Journalof
IndustrialMicrobiology&Biotechnology,38(8):
1027‐1035
FeulgrenR,RossenbeckH,1924.Mikroskopisch‐chemischer
NachweiseinerNucleinsäurevomTypus der
Thymonucleinsäureunddie‐daraufberuhendeelektive
FärbungvonZellkerneninmikroskopischenPräparaten.
Hoppe‐SeylersZeitschriftfürPhysiologischeChemie,
135(5‐6):203‐248
胡晓棣 等 /冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色
菌物学报
1105
HeSQ,Wa ngSX,LuoJC,LiFQ,JinXL,WenCH,MaFQ ,Zhou
ZX,Tan g DZ,2011.There‐studyformorphologyof
OphiocordycepssinensisandHirsutellasinensis.Journal
ofMicrobiology,38(11):1730‐1738(inChinese)
HolmquistG,1975.Hoechst33258fluorescentstainingof
Drosophilachromosomes.Chromosoma,49(4):333‐356
HuX,ZhangYJ,XiaoGH,ZhengP, XiaYL,ZhangXY,StLegerRJ,
LiuXZ,WangCS,2013.Genomesurveyuncoversthe
secretsofsexandlifestyleincaterpillarfungus.Chinese
ScienceBulletin,58(23):2846‐2854
HuangL,LiangY, GuoF,ZhouZ,ChengB,2003.Simultaneous
separationanddeterminationofactivecomponentsin
CordycepssinensisandCordycepsmilitarrisbylc/esi‐ms.
JournalofPharmaceutical&BiomedicalAnalysis,33(5):
1155‐1162
JanyJL,PawlowskaTE,2010.Multinucleatesporescontribute
toevolutionarylongevityofasexualGlomeromycota.
TheAmericanNaturalist,175(4):424‐435
KangZS,LiZQ,ShangHS,MaQ,Wang HW,1993.On
techniqueofdoubleflourescencestainingfornucleiand
septaofplantpathogenicfungi.ActaMycologicaSinica,
12(2):174‐176(inChinese)
LiY,JiaoL,Yao Y, 2013.Non‐concertedITSevolutioninfungi,
asrevealedfromtheimportantmedicinalfungus
Ophiocordycepssinensis.MolecularPhylogenetics&
Evolution,68(2):373‐379
LiangZQ,FoxRTV,1997.Vegetative compatibilityof
inter‐andintra‐monoconidiumstrainsofanamorphof
Cordycepspruinosa.Mycosystema,16(3):216‐223(in
Chinese)
LiescheJ,ZiomkiewiczI,SchulzA,2013.Super‐resolution
imagingwithpontaminefastscarlet4bsenablesdirect
visualizationofcelluloseorientationandcellconnection
architectureinonionepidermiscells.BMCPlantBiology,
13(2):285‐288
MaheshwariR,2005.Nuclearbehaviorinfungalhyphae.
FEMSMicrobiologyLetters,249(1):7‐14
RenSY,YaoYJ,2013.Evaluationofnutritionalandphysical
stressconditionsduringvegetativegrowthonconidial
productionandgerminationinOphiocordycepssinensis.
FEMSMicrobiologyLetters,346(1):29‐35
ThomasJ,IngerfeldM,NairH,ChauhanSS,CollingsDA,2013.
Pontaminefastscarlet4b:anewfluorescentdyefor
visualisingcellwallorganisationinradiatapinetracheids.
WoodScience&Tech n o l o g y ,47(1):59‐75
VanDillaMA,TruiulloTT,MullaneyPF,CoulterJR,1969.Cell
microfluorometry:amethodforrapid fluorescence
measurement.Science,163(3872):1213‐1214
Yan gZF,2009.Studyonbiologicalcharacteristicsandsome
activecomponentofCordycepsbassiana.MasterThesis,
OceanUniversityofChina,Qingdao.1‐72(inChinese)
ZengW, YinDH,LiQS,LiL,2006.ThegrowthofCordyceps
sinensis(Berk.)Sacc.intheinfectionandparasitic
phases.Mycosystema,25(4):646‐650(inChinese)
[附中文参考文献]
何苏琴，王三喜，罗进仓，李风庆，金秀琳，文朝慧，马
福全，周昭旭，唐德志，2011.冬虫夏草和中国被毛
孢形态学再研究.微生物学通报，38(11):1730‐1738
康振生，李振岐，商鸿生，马青，王惠文，1993.植物病
原真菌细胞核和隔膜的双重荧光染色技术.真菌学
报，12(2):174‐176
梁宗琦，FoxRTV，1997.粉被虫草无性型单孢子株间和单
孢子株内的营养亲和性.菌物系统，16(3):216‐223
杨振芳，2009.球孢虫草（Cordycepsbassiana）生物学特
性及活性成分研究.中国海洋大学硕士毕业论文，青
岛.1‐72
曾纬，尹定华，李泉森，李黎，2006.冬虫夏草菌侵染及
寄生阶段的生长发育研究.菌物学报，25(4):646‐650
（本文责编：王敏）
... The PI fluorescent stain can pass through the damaged cell membrane into the cells and combine with the nucleic acid material to emit red fluorescence when the cell membrane of A. carbonarius H9 spores is damaged [42]. No cell-emitted red fluorescence was observed in the control spore cells without polypeptides treatment, but a certain number of cells were observed to emit red fluorescence in spore cells treated by polypeptides with concentrations of 1.6, 6.4 mg/L, as shown in Figure 6. ...
... It is generally believed that the inhibition of polypeptide on the fungal spore survival rate is mainly manifested through the destruction of spore cell membranes (Qian et al., 2015;Cai et al., 2020). The polypeptides with antifungal effects such as Bacillomycin D (a polypeptide derived from B. subtilis) [42], PAF26 [15], etc., were found to have significant inhibitory and destructive effects on fungal spore cell membranes. It is possible that antifungal peptides may destroy the spore cell membrane, leading to the leakage of nucleic acids and proteins, and to the production of reactive oxygen species that cause apoptosis of spore cells, which eventually inhibits germination and the survival rate of fungal spore [26,29]. ...
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