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菌物学报, 2021, 40(8): 1991-2007 doi: 10.13346/j.mycosystema.210010

研究论文

三种分离自冬虫夏草真菌及培养条件

孟云,1, 唐汉尧1, 施金铎2, 王敦,1,*

1.西北农林科技大学旱区作物逆境生物学国家重点实验室 陕西 杨凌 712100

2.陕西化龙山国家级自然保护区管理局 陕西 安康 725600

Three fungal strains isolated from stroma of Ophiocordyceps sinensis and their culture conditions

MENG Yun,1, TANG Han-Yao1, SHI Jin-Duo2, WANG Dun,1,*

1. State Key Laboratory of Crop Stess Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China

2. Administration of Shaanxi Hualong Mountain National Nature Reserve, Ankang, Shaanxi 725600, China

责任编辑: 韩丽

收稿日期: 2021-01-7   接受日期: 2021-02-19  

基金资助: 陕西省科技重大专项(2020zdzx03-03-02)
国家重点研发计划(2018YFD0600202-03)

Corresponding authors: *E-mail: dunwang@foxmail.com

Received: 2021-01-7   Accepted: 2021-02-19  

Fund supported: Special Funds for the Major Science of Shaanxi Province(2020zdzx03-03-02)
National Key Research and Development Program of China(2018YFD0600202-03)

作者简介 About authors

ORCID:MENGYun(0000-0002-8830-6658) 。

摘要

对采自青海玉树、青海果洛和云南迪庆的冬虫夏草鲜品进行虫生真菌的分离、纯化,获得3种生长形态不同的真菌QH 2019、GL 2019和YN 2019。通过形态学及分子鉴定,菌株QH 2019为蝙蝠蛾拟青霉Samsoniella hepiali,菌株GL 2019为粉棒束孢Isaria farinosa,菌株YN 2019为玫烟色棒束孢Isaria fumosorosea。对这3株菌的培养条件初步研究,结果表明：菌株QH 2019在1/4 SDAY培养基上菌丝日生长速率最快,为(1.94±0.55)mm/d,且菌丝致密、粗壮,含水率高达(91.90±1.22)%,虫草素和虫草酸均以PDA培养的含量最高,分别为(0.47±0.022)mg/g和(3.24±0.021)mg/g;菌株GL 2019在PDA培养基上菌丝日生长速率、菌丝含水率、虫草素含量和虫草酸含量都最高,分别为(2.37±0.20)mm/d、(88.34±2.00)%、(0.23±0.013)mg/g和(6.92±0.019)mg/g,但其菌丝在1/4 SDAY培养基上生长最为致密、粗壮;菌株YN 2019在PDA培养基上菌丝日生长速率、虫草素和虫草酸的含量最高,分别为：(2.27±0.27)mm/d、(0.50±0.012)mg/g和(11.32±0.16)mg/g,但其菌丝在1/4 SDAY培养基上含水率最高(95.23±1.65)%,且生长最为致密、粗壮。综合评价表明3株真菌中YN 2019菌丝中虫草素和虫草酸的含量高、生长速率快,有较好的药用研究价值。

关键词： 虫生真菌; 分离鉴定; 生物学特性; 发酵条件

Abstract

Three fungal strains were isolated from fresh stroma of Ophiocordyceps sinensis collected from Yushu, Guoluo of Qinghai and Diqing of Yunnan and they were identified as Samsoniella hepiali (QH 2019), Isaria farinosa (GL 2019) and Isaria fumosorosea (YN 2019) based on phylogenetic evidence. The highest mycelial growth rate of QH 2019 was observed on 1/4 SDAY medium, with quantitative value of (1.94±0.55)mm/d, and the mycelia were dense, with water content of (91.90±1.22)%. The highest content of cordycepin and cordycepic acid reaching (0.47±0.022)mg/g and (3.24±0.021)mg/g respectively was observed in culture on PDA. The mycelial growth rate, mycelial water content, cordycepin and cordycepic acid of GL 2019 were the highest in the culture growing on PDA, reaching (2.37±0.20)mm/d, (88.34±2.00)%, (0.23±0.013)mg/g and (6.92±0.019)mg/g respectively, but the mycelium grew much densely on 1/4 SDAY medium. The mycelial growth rate, cordycepin and cordycepic acid of YN 2019 were the highest in the culture growing on PDA, reaching (2.27±0.27)mm/d, (0.50±0.012)mg/g and (11.32±0.16)mg/g respectively, but the mycelium growing on 1/4 SDAY medium was much dense, with the highest mycelial water content of (95.23±1.65)%. The results suggested that Isaria fumosorosea YN 2019 had potential medicinal values.

Keywords： entomogenous fungi; isolation and identification; biological characteristic; culture condition

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本文引用格式

孟云, 唐汉尧, 施金铎, 王敦. 三种分离自冬虫夏草真菌及培养条件[J]. 菌物学报, 2021, 40(8): 1991-2007 doi:10.13346/j.mycosystema.210010

MENG Yun, TANG Han-Yao, SHI Jin-Duo, WANG Dun. Three fungal strains isolated from stroma of Ophiocordyceps sinensis and their culture conditions[J]. Mycosystema, 2021, 40(8): 1991-2007 doi:10.13346/j.mycosystema.210010

冬虫夏草是由冬虫夏草菌Ophiocordyceps sinensis (Berk.) G.H. Sung et al.侵染蝙蝠蛾幼虫所形成的子座和充满菌丝的幼虫复合体。近些年研究表明,冬虫夏草提取物和菌丝发酵液中主要的活性物质为虫草酸（钱正明等 2019）、虫草素（胡敏和程震勇 2019）、多糖（崔兵兵等 2019）等;具有诱导凋亡和抗癌（Bai et al. 2020;Qi et al. 2020）、调节机体免疫和微生物群落（Ji et al. 2020;Ying et al. 2020）、减压降脂抗糖尿（Li et al. 2020）、抗菌消炎（Li et al. 2020）、抗氧化延缓衰老（Wang et al. 2005;Wu et al. 2019）、镇静催眠和抗惊厥（丁婷等 2008）、延缓神经元细胞衰老（Cao et al. 2020）等作用。

在中国,冬虫夏草主要分布于西北西藏、青海、四川、云南3 000-5 000m的高原草甸地区（周刊社等 2018;徐梦等 2019）,由于生长环境与寄主的限制,天然冬虫夏草资源有限且呈下降趋势（杨大荣等 1996）。因此,从20世纪80年代起,研究人员对新鲜的冬虫夏草的菌种分离、人工培养开发进行了大量的研究。已有多个独立实验证明中国被毛孢Hirsutella sinensis X.J. Liu et al.是冬虫夏草菌的无性型,其发酵产物具有调节自身免疫（章水晶等 2019）、护肾（沈龙海等 2011）、抗氧化（丁婷等 2007）等功效,被制成“百令”胶囊（洪露露 2019）;从云南迪庆冬虫夏草中分离出来的蝙蝠蛾拟青霉Samsoniella hepiali (Q.T. Chen & R.Q. Dai ex R.Q. Dai et al.) H. Yu et al.（Wang et al. 2020）,其菌丝发酵产物与冬虫夏草有效成分相似（Jarmila et al. 2019）,已被开发成药物“金水宝”胶囊（赵小惠等 2020）。

为了获得具有药用价值的冬虫夏草菌及其相关菌株,本研究拟从青海玉树、果洛和云南迪庆采集的新鲜冬虫夏草中分离纯化,并对纯化的菌株进行显微形态学和分子生物学的鉴定;选择有药用价值的菌株,探究其在不同培养条件下对菌株生长和有效物质产量的影响,以期获得具有药用价值和人工培养前景的菌株资源。

1 材料与方法

1.1 材料来源

供试菌株从云南省迪庆市和青海省果洛市、玉树市采集的新鲜冬虫夏草样品中分离获得,现保存于西北农林科技大学植物保护学院昆虫与微生物资源利用实验室菌种库;样品采集于2019年5月至6月。

1.2 方法

1.2.1 培养基：1/4 SDAY选择性培养基：酵母提取物2.5g,葡萄糖10.0g,蛋白胨2.5g,琼脂20.0g,水1 000mL,121℃高压灭菌20min,备用;使用前加入氨苄西林钠0.5g和硫酸链霉素0.6g。马铃薯葡萄糖琼脂（potato dextrose agar,PDA）培养基：去皮马铃薯200g,水1 000mL,煮沸15min,过滤,取上清定容至1 000mL,再加入葡萄糖20.0g、琼脂20.0g,121℃高压灭菌20min,备用。查氏培养基（Czapek-Dox medium）：硝酸钠3.0g、磷酸二氢钾1.0g、七水合硫酸镁0.5g、氯化钾0.5g、硫酸亚铁0.01g、蔗糖30.0g、琼脂20.0g、水1 000mL,121℃高压灭菌20min,备用。

1.2.2 菌株的分离与纯化：将采集到的冬虫夏草新鲜样品用75%乙醇进行表面消毒,待表面干燥后用灭菌的手术刀切取0.5cm×0.5cm小块,接种在加入抗生素的1/4 SDAY培养基上,15℃、避光培养7d;挑取菌落边缘,进行纯化培养。

1.2.3 菌株的形态学观察：将分离、纯化的菌株接种于含1/4 SDAY培养基的培养皿和200mL组织培养瓶（培养基30mL）中,将培养皿和组培瓶在15℃恒温生化箱遮光培养30、60d,观察菌株培养皿和组培瓶中生长特征,主要包括菌落形态、菌丝颜色、渗出物颜色等。挑取少量菌丝,对菌株进行形态特征的显微观察,主要包括菌株的菌丝特征、产孢结构特征、孢子形态及大小等。参考《中国真菌志·第43卷》（梁宗琦等 2013）中的检索表及各菌株的特征描述对分离获得的菌株进行初步鉴定。

1.2.4 DNA提取、PCR扩增及测序：将分离获得的菌株接种于1/4 SDAY培养皿上,15℃、培养14d,收集培养皿表面的菌丝,根据Masoudi et al.（2018）的方法进行菌丝总DNA的提取。

以提取的总DNA为模板,进行多基因引物扩增及测序,引物序列见表1。PCR反应体系（25μL）：ddH2O 17.7µL,10×PCR Reaction Buffer 2.5µL,dNTPs（2.5mmol/L）2µL,正、反向引物（10μmol/L）各0.5µL,0.1% BSA 0.3µL,Taq酶（5U/μL）0.5µL,DNA模板1µL。

表1   本研究所用引物

Table 1  Primers used in this study for molecular identification

基因名称
Gene		引物名称
Primer		序列
Sequence (5’-3’)		参考文献
Reference
nrSSUnrSSU-CoFTCTCAAAGATTAAGCCATGCGuhr & Weig 2020
nrSSU-CoRTCACCAACGGAGACCTTG
nrLSULR5ATCCTGAGGGAAACTTCVilgalys & Sun 1994
LR0RGTACCCGCTGAACTTAAGC
TEFEF1α-EFGCYCCYGGHCAYCGTGAYTTYATBischoff et al. 2009
EF1α-ERATGACACCRACRGCRACRGTYTGSung et al. 2007
RPB1RPB1CrCCNGCDATNTCRTTRTCCATRTACastlebury et al. 2004
CRPB1ACAYCCWGGYTTYATCAAGAA
RPB2fRPB2-7cRCCCATRGCTTGTYYRCCCATLiu et al. 1999
fRPB2-5FGAYGAYMGWGATCAYTTYGG
ITSITS1TCCGTAGGTGAACCTGCGGWhite et al. 1990
ITS2GCTGCGTTCTTCATCGGATGC

注：ITS中包括核糖体ITS1、ITS2、核糖体5.8S亚基序列

Note: ITS1, ITS2 and ribosome 5.8S subunit were all included in ITS.

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ITS基因的PCR反应条件：95℃ 5min;95℃ 30s,60℃ 30s,72℃ 1min,35个循环;72℃ 10min。nrSSU基因的PCR反应条件：95℃ 4min;94℃ 50s,56℃ 50s,72℃ 2min,循环8次,每循环退火温度降低0.5℃;94℃ 50s,52℃ 50s,72℃ 2min,循环25次;72℃ 10min,12℃保存。TEF、RPB1、RPB2、nrLSU基因的PCR反应条件：95℃ 4min;94℃ 50s,56℃ 50s,72℃ 70s,循环8次,每循环退火温度降低0.5℃;94℃ 50s,52℃ 50s,72℃ 70s循环25次;72℃ 10min,12℃保存（段东娥 2019）。用1%琼脂糖胶检测PCR扩增产物,将PCR未纯化产物送至生工生物工程（上海）股份有限公司进行测序。

1.2.5 序列对比及系统发育分析：将测序结果在GenBank中进行同源序列比对;结合已报道的分离自冬虫夏草的菌株,并下载菌株的相关序列,用MEGA-X进行序列比对,在PhyloSuite v1.2.1中采用最大似然法（maximum likelihood,ML）,联合真菌ITS、nrSSU、nrLSU、TEF、RPB1、RPB2序列（表2）构建系统发育树,确定分离菌株的种属地位。

表2   用于多基因序列分析的来自GenBank中的菌株及序列登录号

Table 2  The isolates and accession number information of genes from GenBank

物种
Species		菌株号
Strain No.		基因序列登录号
GenBank accession number
nrSSUnrLSUITSTEFRPB1RPB2
Isaria farinosaGL 2019MW391721MW391777MW391716MW504644MW509836MW504642
Samsoniella hepialiQH 2019MW391720MW391779MW391718MW504645MW509835MW504643
Isaria fumosoroseaYN 2019MW391722MW391778MW391717MW509838MW509837MW509839
Samsoniella hepialiYFCC 5823MN576745MN576801-MN576971MN576861MN576915
Samsoniella hepialiYFCC 5828MN576744MN576800-MN576970MN576860MN576914
Samsoniella hepialiICMM Cs-4MN576743MN576799-MN576969MN576859MN576913
Samsoniella hepialiICMM 82-2MN576738MN576794-MN576964MN576854MN576908
Isaria farinosaMY 01338JN940994JN940901JN942620---
Isaria farinosaCBS 240.32JF415958JF415979-JF416019JN049895JF415999
Isaria farinosaNHJ 08013JN940995JN940900JN942628---
Cordyceps cf.
farinosa		OSC 111005DQ522558DQ518773-DQ522348DQ522394-
Cordyceps cf.
farinosa		OSC 111004EF468986EF468840-EF468780EF468886-
Cordyceps
farinosa		BUC 418MH879643MH879595-MH879661MH885444MH879618
Cordyceps
farinosa		BUC 386MH879641MH879593-MH879660MH885442MH879616
Cordyceps
fumosorosea		CBS 375.70AB083035AB083035AY524183MF416501MF416658MF416452
Cordyceps
fumosorosea		CBS 107.10MF416608MG665227-HM161735-MG665237
Isaria
amonen-rosea		CBS 729.73MF416604MF416551AY624169MF416495MF416652MF416446
Isaria
takamizusanensis		NHJ 3497EU369096EU369033----
Isaria
takamizusanensis		NHJ 3582EU369097EU369034--
Cordyceps cf.
takaomontana		NHJ 12623EF468984EF468838-EF468778EF468884EF468932
Cordyceps
tenuipes		BUC 394MH879642MH879594-MH879667MH885443-
Cordyceps
tenuipes		YFCC 4266MN5767774MN576830-MN577000MN576890MN576944
Cordyceps
javancia		TBRC 7260--MF140744MF140830MF140779MF140803
Cordyceps
javancia		CBS 134.22MF416610MF416558-MF416504MF416661MF416455
Cordyceps
cateniannulata		CBS 152.83AY526465MG665226AY624172JQ425687--
Cordyceps
cateniobliqua		CBS 153.83AY526466-AY624173JQ425688-MG665236
Cordyceps cf.
pruinosa		EFCC 5197EF468965EF468820-EF468760EF468868-
Cordyceps cf.
pruinosa		NHJ 10627EF468967EF468822-EF468763EF468870-
Cordyceps
cylindrica		BUN 509MH879645MH879597----
Cordyceps
cicadae		RCEF
HP090724-31		MF416605MF416552-MF416496MF416653MF416447
Beauveria
brongniartii		BCC 16585JF415951JF415967JN415967JF416009JN049885JF415991
Metacordyceps
taii		ARSEF 5714AF543763AF543787JN049829AF543775DQ522383DQ522434
Orbiocrella
petchii		NHJ 6209EU369104EU369039JN049861EU369023EU369061EU369081
Aschersonia
confluence		BCC 7961DQ372100DQ384947JN049841DQ384976DQ384998DQ452465
Virdispora
diparietispora		CBS 102797AY489703AY489735JN049838AY489630AY489668DQ522471
Ophiocordyceps
rhizoidea		NHJ 12522EF468970EF468825JN049857EF468764EF468873EF468923
Verticillium
epiphytum		CBS 384.81AF339596AF339547-DQ522361DQ522409DQ522469
Rotiferophthora
angustispora		CBS 101437AF339584AF339535AJ292412AF543776DQ522402DQ522460
Torrubiella
wallacei		CBS 101237AY184978AY184967NR111267EF469073EF469102EF469119
Hymenostilbe
aurantiaca		OSC 128578DQ522556DQ518770JN049833DQ522345DQ522391DQ522445
Metarhizium
flavoviride		ARSEF 2037AF339580AF339531AF138271DQ522353DQ522400DQ522454
Lecanicilliun
psalliotae		CBS 532.81AF339609AF339560JN049846EF469067EF469096EF469112

注：-：无

Note: -: Null.

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1.2.6 不同培养基对菌株生长速率的影响：将分离的菌株驯化培养,直至菌落为规则圆形、直径约为60.0mm时,用7.0mm打孔器沿菌落边缘打孔;再将菌碟分别接种于PDA、1/4 SDAY和查氏培养基上,15℃恒温培养;每2d测量一次菌落直径（十字交叉法）[菌丝生长速度(mm/d)=菌落半径(mm)/菌丝生长天数(d)]（Yin et al. 2009）测量至14d,各株菌每种培养基重复3次。

1.2.7 不同培养基对菌株的菌丝含水率的影响：收集各菌株同一培养基中菌丝,称量鲜重;55℃干燥至恒重、称量,计算菌丝含水率[菌丝含水率(%)=菌丝干重(g)/菌丝鲜重(g)×100]。

1.2.8 不同培养基对菌株的虫草酸和虫草素含量的影响：将干燥的菌丝粉碎,精确称取0.5g,装入10mL离心管中,以液料比20:1加入超纯水,400W、10min、常温超声提取（唐果等 2018）。将2.0、5.0、10.0、20.0、50.0μg/mL的标准品进行LC-MS检测;分别以虫草素和虫草酸浓度为横坐标、相应的峰面积为纵坐标建立标准曲线,并得出线性回归方程及R2。菌丝提取液上清过0.22μm微孔滤膜,进行LC-MS分析,根据标准曲线计算含量。

1.2.9 菌丝提取物检测条件：采用LC-MS（液质联用）的方法对菌丝提取物中的虫草酸和虫草素含量进行检测,仪器为LTQ XL mass spectrometer（Thermo Scientific,Waltham,MA,USA）;LC检测条件：色谱柱：XTerra MS C18 Column（5μm,150mm×4.6mm;Waters Corp,Milford,MA,USA）;梯度洗脱,流动相组成见表3;流速：0.5mL/min;MS检测条件：离子源：ESI（阳离子模式）,喷雾电压：4.5kV,毛细管温度：275℃,鞘气流速：20（arb）N2,虫草素和虫草酸的质谱条件见表4（张铭雅等 2019）。

表3   LC流动相组成

Table 3  Liquid chromatograph mobile phase composition

时间
Time
(min)		流动相A
Mobile
phase A (%)		流动相B
Mobile
phase B (%)		流动相C
Mobile
phase C (%)
075520
775520
808020
1308020
1475520
2175520

注：流动相A：超纯水;流动相B：乙腈;流动相C：乙酸

Note: Mobile phase A: Water; Mobile phase B: Acetonitrile; Mobile phase C: 0.5% acetic acid.

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表4   虫草素和虫草酸的质谱条件

Table 4  Mass spectrometry conditions for cordycepic acid and cordycepin

化合物名称
Compound		前体离子
Precursor ion
[M+H]+ (m/z)		质核比
Product
ion (m/z)		激发能量
Collision
energy (eV)
虫草酸
Cordycepic
acid		18369 (MS3)35
虫草素
Cordycepin		252136 (MS2)35

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1.2.10 统计学分析：本研究采用Excel软件进行数据整理,采用SPSS 22.0软件进行统计处理,采用GraphPad Prism 8系统制图。

2 结果与分析

2.1 三株菌的形态学鉴定

通过对新鲜冬虫夏草样品的分离、纯化,最终获得3株外观、形态均不相同的虫生真菌,分别命名为QH 2019、GL 2019、YN 2019。

2.1.1 菌株QH 2019：从青海省玉树市采集的新鲜冬虫夏草样品中分离、纯化,菌株在15℃培养30d,菌落直径为85.0-90.0mm,菌落正面中间呈浅黄色、周边为白色、隆起,菌丝绒毛状、致密;菌落背面中间呈黄色、周边为白色,具放射状沟纹（图1A、1D）。菌丝无色、有隔、分支,直径0.5-3.0μm;瓶梗（3.0-5.0μm）基部膨大呈椭圆形,单生或由2-4个瓶梗组成分支状分生孢子梗（5.0-10.0μm）;分生孢子(2.0-2.5)×(1.0-1.5)μm,球形至卵圆形,无色,链生（图2A-2C）。在组织培养瓶中培养60d,菌株QH 2019菌丝黄色、产生白色孢梗束（图3A、3E）,见光后菌丝逐渐变为橙黄色（图3B、3F）。

图1

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图1   三种真菌菌落形态

A、D：QH 2019正、反面;B、E：GL 2019正、反面;C、F：YN 2019正、反面

Fig. 1   The characteristic features of three fungi in petri dish.

A, D: Samsoniella hepiali QH 2019 obverse side and reverse side colony; B, E: Isaria farinosa GL 2019 obverse side and reverse side colony; C, F: Isaria fumosorosea YN 2019 obverse side and reverse side colony.


图2

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图2   三种真菌的显微形态特征

A-C：QH 2019菌株;D-F：GL 2019菌株;G-I：YN 2019菌株. 标尺=10µm

Fig. 2   The microscopic features of three fungi.

A-C: Samsoniella hepiali QH 2019 strain; D-F: Isaria farinosa GL 2019 strain; G-I: Isaria fumosorosea YN 2019 strain. Bars=10μm.


图3

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图3   三种真菌培养瓶中的形态特征

A、B、E、F：QH 2019菌株;C、G：GL 2019菌株;D、H：YN 2019菌株

Fig. 3   The characteristics of colonies of three fungi in culture bottles.

A, B, E, F: Samsoniella hepiali QH 2019 strain; C, G: Isaria farinosa GL 2019 strain; D, H: Isaria fumosorosea YN 2019 strain.


2.1.2 菌株GL 2019：从青海省果洛市采集的新鲜冬虫夏草样品中分离、纯化,菌株在15℃培养30d,菌落直径为85.0-90.0mm,菌落正面为均匀生长的白色、绒毛状菌丝,中间和边缘有孢梗束、白色至鸭黄色;菌落背面为白色、在菌碟周围呈橙黄色（图1B、1E）。菌丝无色、分隔,直径0.7-2.5μm;分生孢子梗长（100-300μm）,从基质菌丝上生长,不规则分枝,上有2-4个瓶梗组成的轮生体,瓶梗(5.0-15.0)×(1.2-2.5)µm,基部膨大呈椭圆形、上端有明显变细的颈部;分生孢子(1.8-2.5)×(1.0-1.5)µm,球形至椭圆形,长链状（图2G-2I）。在组织培养瓶中培养60d,菌株GL 2019菌丝白色、产生白色孢梗束,见光后不变色（图3D、3H）。

2.1.3 菌株YN 2019：从云南省迪庆市采集的新鲜冬虫夏草样品中分离、纯化,菌株在15℃培养30d,菌落直径为80.0-85.0mm,菌落正面中央为白色、周围为肉红色,菌丝绒毛状、面附着有肉红色液滴状渗出物;菌落背面中间为黄色、周边为白色（图1C、1F）。菌丝无色、分隔,直径1.0-3.0μm;产孢结构复杂,分生孢子梗10.0-30.0μm,上有3-6个瓶梗组成的轮生体,瓶梗（2.5-4.0μm）基部显著膨大呈卵圆或椭圆形,向上有明显的细颈;分生孢子(3.0-3.5)×(1.0-2.0)μm,透明、光滑,梭形至椭圆,长链（图2G-2I）。在组织培养瓶中培养60d,菌株YN 2019菌丝白色,上有均匀的肉红色、珊瑚状孢梗束,孢梗束上附着酒红色渗出物（图3D、3H）。

根据以上形态学观察结果和寄主种类,并结合《中国真菌志·第43卷》和Mongkolsamrit（2018）的研究结果,初步鉴定菌株QH 2019为蝙蝠蛾拟青霉Samsoniella hepiali;菌株GL 2019为粉棒束孢Isaria farinosa (Holmsk.) Fr.,异命为粉拟青霉Paecilomyces farinosa (Holmsk. ex Gray) A.H.S. Br. & G. Sm.（蒋毅和姚一建 2003;代永东等 2015）;菌株YN 2019为玫烟色棒束孢Isaria fumosorosea Wize。

2.2 两基因系统发育分析

以Aschersonia confluence BCC 7961为外群建立系统发育树,结果显示：菌株QH 2019与Samsoniella hepialid ICMM Cs-4 聚类在同一个小分支上,相似性为98%;菌株GL 2019与Isaria farinosa NHJ 08013聚类在同一个分支上,相似性为97%;菌株YN 2019与Cordyceps fumosorosea CBS 107.10聚类在同一个分支上,相似性为97%（图4）。

图4

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图4   基于PhyloSuite构建的系统发育树

Fig. 4   Phylogenetic tree based on ITS, nrSSU, nrLSU, TEF, RPB1 and RPB2 sequences by using PhyloSuite.


2.3 不同培养基对菌株生长的影响

对3株菌在不同培养基上生长的菌丝进行直径和菌落形态观察,测量和观察结果见表5、图5、图6。GL 2019在3种培养基上均能正常生长,菌落直径表现为：PDA>1/4 SDAY>查氏培养基;菌株在PDA培养基上菌丝日生长速率最快,且与其他两种培养基差异显著,在1/4 SDAY和查氏培养基上无显著差异。QH 2019在3种培养基上均能正常生长,菌落直径表现为：1/4 SDAY>PDA>查氏培养基;菌株在1/4 SDAY培养基上菌丝日生长速率最快,且与其他两种培养基差异显著,在1/4 SDAY和查氏培养基上无显著差异。YN 2019在3种不同培养基上均能正常生长,菌落直径表现为：PDA>1/4 SDAY>查氏培养基;菌株在PDA培养基上菌丝日生长速率最快,且与其他两种培养基差异显著,在1/4 SDAY和查氏培养基上无显著差异。

表5   不同菌株在不同培养基上生长状况与测量结果

Table 5  Growth characters and measurement result of three isolates grown on different media

菌株
Strain		培养基名称
Medium
type		菌丝日生长速率
Mycelial growth
rate (mm/d)		菌落形态
Colonial morphology		菌丝长势
Mycelial
growth vigor
Samsoniella
hepiali
QH 2019		PDA1.77±0.12 b菌落正面为白色;背面的中间为浅橘黄色,边缘为白色
The obverse side of colony was white; the middle of
colony reverse side was light orange with a white edge		++
1/4 SDAY1.94±0.55 a菌落正面中间为鸭黄色,边缘为白色;背面中间为黄色,
边缘为白色
The middle of the colony obverse side was yellow with a
white edge; the reverse side was yellow with a white edge		+++
查氏培养基
Czapek-Dox
medium		1.63±0.13 b菌落正面为鸭黄色,上有一层白色的气生菌丝,
背面为橘黄色,颜色较深
The obverse side of colony was yellow with a layer of white
aerial mycelium and the reverse side was dark orange		+
Isaria
farinosa
GL 2019		PDA2.37±0.20 a菌落正面中间为鸭黄色且有孢梗束,边缘为白色;
背面为乳白色
The middle of colony obverse side was yellow with coremium,
and its edge was white; the reverse side was milk white		++
1/4 SDAY2.04±0.40 b菌落正面为白色,有少量孢梗束;背面为黄色
The obverse side of colony was white with coremium
and reverse side was yellow		+++
查氏培养基
Czapek-Dox
medium		1.92±0.18 b菌落正面为白色、边缘为肉色;背面为橘黄色
The obverse side of colony was white and the edge
was flesh color; the reverse side was orange		+
Isaria
fumosorosea
YN 2019		PDA2.27±0.27 a菌落正面中间为肉红色,边缘为白色,孢梗束多;
背面为浅橘黄色,边缘为白色
The middle of colony obverse side was flesh color and its
edge was white with thick coremium; the reverse side
was light orange with a white edge		++
1/4 SDAY1.89±0.16 b菌落正面为白色,中间有浅肉红色的孢梗束,
上面有少量外渗物;背面为黄色,边缘为白色
The obverse side of colony was white, and its middle was
a light flesh color coremium covered with light exosmosis;
the reverse side was yellow with a white edge		+++
查氏培养基
Czapek-Dox
medium		1.78±0.40 b菌落正面为白色,无孢梗束;背面为橘黄色,边缘为白色
The obverse side of the colony was white without coremium;
the reverse side was orange with a white edge		+

注：“+”表示菌丝体稀疏、纤细,“++”表示菌丝较密、生长较壮,“+++”表示菌丝稠密、粗壮. 不同小写字母表示有显著差异,P<0.05. 下同

Note: “+” indicates that mycelia are sparse and slim, “++” indicates that mycelia are dense and strong, and “+++” indicates that mycelia are thick. Different lowercase letters are expressed as the significant level of 5%. The same below.

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图5

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图5   不同菌株在3种培养基上的菌落直径

Fig. 5   Colony diameter of different isolates grown on three media.


图6

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图6   不同菌株在3种培养基上生长状况

A-F：QH 2019在PDA、1/4 SDAY、查氏培养基正、反面;G-L：GL 2019在PDA、1/4 SDAY、查氏培养基正、反面;M-R：YN 2019在PDA、1/4 SDAY、查氏培养基正、反面

Fig. 6   The growth characters of different isolates grown on three media.

A-F: Obverse and reverse side of colony of QH 2019 on PDA, 1/4 SDAY, and Czapek-Dox medium; G-L: Obverse and reverse side of colony of GL 2019 on PDA, 1/4 SDAY, and Czapek-Dox medium; M-R: Obverse and reverse side of colony of YN 2019 on PDA, 1/4 SDAY, and Czapek-Dox medium.


2.4 不同培养基对菌株含水率、虫草素和虫草酸含量的影响

分别对虫草素和虫草酸标样进行LC-MS测定,可知虫草素的保留时间为：5.50min,标准曲线为：y=181744x,R2=0.9993;虫草酸的保留时间为：3.00min,标准曲线为：y=169.591x,R2=0.9936。不同菌株在3种培养基上含水率、虫草素和虫草酸含量见表6。

表6   不同菌株在不同培养基上含水率、虫草素和虫草酸含量

Table 6  Water content, cordycepin and cordycepic acid content of different isolates grown on different media

菌株
Strain		培养基种类
Medium		菌丝含水率
Mycelial water content (%)		虫草素含量
Cordycepin content (mg/g)		虫草酸含量
Cordyceps acid content (mg/g)
QH 2019PDA85.91±1.25b0.47±0.022a3.24±0.021a
1/4 SDAY91.90±1.22a0.054 ±0.014b1.27±0.060c
查氏培养基
Czapek-Dox medium		90.83±1.00a0.057±0.0016b1.75±0.026b
GL 2019PDA88.34±2.00a0.23±0.013a6.92±0.019a
1/4 SDAY86.97±0.13a0.051±0.011c2.47±0.011c
查氏培养基
Czapek-Dox medium		82.49±1.30b0.073±0.0067b5.19±0.012b
YN 2019PDA84.40±2.18b0.50±0.012a11.32±0.16a
1/4 SDAY95.23±1.65a0.11±0.011b1.25±0.011c
查氏培养基
Czapek-Dox medium		92.60±0.57a0.021±0.0006c1.70±0.010b

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QH 2019在PDA上菌丝含水率最低,虫草素和虫草酸含量最高,与其他两种培养基差异显著;菌株在1/4 SDAY培养基和查氏培养基上的菌丝含水率与虫草素含量物明显差异,但虫草酸含量差异显著。

GL 2019在查氏培养基上菌丝含水率最低,但虫草素与虫草酸含量最低,与其他两种培养基差异显著;菌株在PDA培养基上菌丝含水率最高、但与1/4 SDAY培养基无明显差异,虫草素和虫草酸含量最高,与1/4 SDAY培养基有显著差异。

YN 2019在PDA上菌丝含水率最低,虫草素和虫草酸含量最高,与其他两种培养基有显著差异;菌株在1/4 SDAY培养基和查氏培养基上的菌丝含水率差异不明显,但虫草素和虫草酸含量差异显著。

3 讨论

研究表明蝙蝠蛾拟青霉可从新鲜冬虫夏草上分离,与冬虫夏草有相似的活性物质和功效（张羽等 2019）,已被开发成药物金水宝,2000年列入《中国药典》（赵小惠等 2020）,具有很好的药用开发前景;粉棒束孢的地理分布和来源广泛,从昆虫、植物、土壤和其他真菌中均可分离获得（刘飞 2018）;玫烟色棒束孢可以从土壤（Shimizu et al. 2008）、僵虫上分离获得。本研究对冬虫夏草鲜品进行虫生真菌的分离,通过形态学及构建系统发育树鉴定发现,菌株QH 2019为蝙蝠蛾拟青霉,菌株GL 2019为粉棒束孢,YN 2019为玫烟色棒束孢。

除了蝙蝠蛾拟青霉、中国被毛孢等已经应用于药物生产外,研究表明环链棒束孢Isaria cateniannulate（唐鱼等 2020）、猫棒束孢Isaria feline（王跃凤等 2019）、细脚棒束孢Isaria tenuipes、蝉棒束孢Isaria cicadae、球孢白僵菌Beauveria bassiana、蛹虫草Cordyceps militaris等虫生真菌具有提高免疫、抗癌活性（刘建兵等 2018）;同时,改变培养基中碳氮源的种类,能提高菌株的菌丝产量和活性物质含量（洪露露 2019）。

本研究比较了从冬虫夏草鲜品上分离的3株虫生真菌的培养基类型与菌株生长状况及菌丝中活性物质虫草素、虫草酸含量的关系,筛选出虫草素和虫草酸含量高的菌株YN 2019,有望作为新的药用资源。这不仅为深入研究并获得虫草素和虫草酸高产菌株提供了新的材料,也为合理利用冬虫夏草资源、有效挽回人们对野生冬虫夏草资源及其生境的掠夺式破坏提供了可选择的途径。

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In an effort to establish a suitable alternative to the widely used 18S rRNA system for molecular systematics of fungi, we examined the nuclear gene RPB2, encoding the second largest subunit of RNA polymerase II. Because RPB2 is a single-copy gene of large size with a modest rate of evolutionary change, it provides good phylogenetic resolution of Ascomycota. While the RPB2 and 18S rDNA phylogenies were highly congruent, the RPB2 phylogeny did result in much higher bootstrap support for all the deeper branches within the orders and for several branches between orders of the Ascomycota. There are several strongly supported phylogenetic conclusions. The Ascomycota is composed of three major lineages: Archiascomycetes, Saccharomycetales, and Euascomycetes. Within the Euascomycetes, plectomycetes, and pyrenomycetes are monophyletic groups, and the Pleosporales and Dothideales are distinct sister groups within the Loculoascomycetes. We confirm the placement of Neolecta within the Archiascomycetes, suggesting that fruiting body formation and forcible discharge of ascospores were characters gained early in the evolution of the Ascomycota. These findings show that a slowly evolving protein-coding gene such as RPB2 is useful for diagnosing phylogenetic relationships among fungi.

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Fusarium asiaticum and F. graminearum are the primary causal agents of Fusarium head blight (FHB) of wheat in China. In this study, sensitivities of 159 F. asiaticum and F. graminearum isolates to a benzimidazole fungicide carbendazim (MBC) and to sterol demethylation inhibitors (DMIs) tebuconazole and prochloraz were determined. Among the 159 isolates, 9 were resistant to MBC and designated as MBC-R isolates. Three showed resistance to tebuconazole and prochloraz and designated as DMI-R isolates. There was no cross-resistance between MBC and DMI. Genetic analysis by microsatellite-primed polymerase chain reaction (PCR) showed that MBC-R or DMI-R isolates had different genotypes, which indicated that they originated from different wild-type parents. Analysis of two 14alpha-demethylase (cyp51) homologous genes (cyp51A and cyp51B) showed that the F. asiaticum isolates could be distinguished from F. graminearum isolates based on the sequence of cyp51A. Analysis of deduced amino acid sequence of cyp51A and cyp51B suggested that no mutations were associated with DMI resistance. Real-time PCR analysis showed that the DMI resistance was not related to the expression of cyp51A and cyp51B in F. asiaticum and F. graminearum, but expressions of both genes were induced greatly by the tebuconazole. Results of this study indicated that cyp51A would be an informative marker for analysis of population structure of F. asiaticum and F. graminearum, and the existence of homologous cyp51 genes in F. asiaticum and F. graminearum could provide new insights into DMI resistance in phytopathogenic fungi.

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