CN114561300B - 一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株wh504-50 - Google Patents

一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株wh504-50 Download PDF

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CN114561300B
CN114561300B CN202210444189.8A CN202210444189A CN114561300B CN 114561300 B CN114561300 B CN 114561300B CN 202210444189 A CN202210444189 A CN 202210444189A CN 114561300 B CN114561300 B CN 114561300B
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张晓婷
高飞
谢源
李洪连
周琳
杜鹏强
潘鑫
刘东伟
代君丽
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Abstract

本发明属于微生物领域，涉及一种假禾谷镰孢菌株，特别是指一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株WH504‑50。所述菌株的保藏号为CCTCC No：M20211108，保藏日期为2021年8月30日。所述假禾谷镰孢巨型Toti病毒（Fusarium pseudograminearum megatotivirus 1，FpgMTV1）为两条dsRNA序列的病毒，含有FpgMTV1的假禾谷镰孢菌菌株WH504‑50在镰孢菌病害防治方面的应用。

Description

一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷 镰孢菌株WH504-50
技术领域
本发明属于微生物领域，涉及一种假禾谷镰孢菌株，特别是指一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株WH504-50。
背景技术
假禾谷镰孢菌(Fusarium pseudograminearum)是引起小麦茎基腐病的优势病原菌，目前广泛存在于河南、河北、山西、安徽、江苏、山东、陕西等黄淮麦区多个省份，发生蔓延呈现不断扩大和加重的趋势，严重危害小麦生产。本课题组前期也发现一株假禾谷镰孢菌FC136-2A，该病原菌内含有的Fusarium pseudograminearum megabirnavirus 1（FpgMBV1）对病原菌的危害主要体现在对寄主致病力的抑制。
利用弱毒相关病毒对植物病原真菌致病力的抑制作用进行生物防治的应用中，栗疫菌(Cryphonectria parasitica) 中的 CHV1 最早引起人们重视。近年来，我国真菌病毒学家证实，核盘菌(Sclerotinia sclerotiorum) 中的SsHADV-1 可以在寄主真菌的营养体不亲和型菌株之间扩散和复制，并引起寄主真菌的致病力衰退，具有良好的生防潜能。
本课题组一直致力于对小麦致病菌病原菌的研究，在前期科研方法的基础上，一直希望筛选到用于特异性减弱假禾谷镰孢菌产孢能力和分生孢子生长发育的菌株。
发明内容
为解决上述技术问题，本发明筛选到一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株WH504-50。
本发明的技术方案是这样实现的：
用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株WH504-50，所述菌株的拉丁文名称为Fusarium pseudograminearum，其保藏单位为中国典型培养物保藏中心，保藏号为CCTCC No：M20211108，保藏日期为2021年8月30日，保藏地址为湖北省武汉市武昌区八一路299号。
进一步，所述假禾谷镰孢菌株WH504-50上寄生有病毒FpgMTV1。
进一步，所述megatotivirus病毒FpgMTV1为两条dsRNA序列的病毒，两条dsRNA序列分别如SEQ ID No.1和SEQ ID No.2所示。
上述的假禾谷镰孢菌株WH504-50在抑制镰孢菌产孢能力中的应用。
上述的假禾谷镰孢菌株WH504-50在抑制分生孢子生长发育中的应用。
上述的假禾谷镰孢菌株WH504-50在抑制镰孢菌生长发育中的应用。
本发明具有以下有益效果：
（1）发明人对不同小麦产区的假禾谷镰孢菌进行了真菌病毒筛查和菌株生物学性状观察，发现菌株WH504-50中存在一种dsRNA病毒；该dsRNA病毒的基因组由2条dsRNA组成，分别长为12300bp（L1-dsRNA）和3818bp（L2-dsRNA）；基因组测序和分析结果表明，该病毒是一种新的病毒，属于Megatotiviridae，将它命名为Fusarium pseudograminearummagatotivirus 1，缩写为FpgMTV1。
（2）本发明要解决的技术问题是提供一种在减弱假禾谷镰孢菌产孢能力和分生孢子生长发育方面具有显著效果的假禾谷镰孢菌病毒FpgMTV1。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为假禾谷镰孢菌株WH504-50的菌落形态图，a为菌落正面，b为菌落反面。
图2为特异性引物对Fp1-1/Fp1-2的PCR扩增结果图，其中M-DNA ladder； 1：假禾谷镰孢对照菌株FC136-2A、2：WH504-50、3：WH504-50-V-、CK：清水对照。
图3为菌株分生孢子形态观察和产孢量测定，其中A为孢子形态；B为产孢量和分生孢子平均长度测定。
图4为菌株玻璃纸穿透试验，其中a为菌株在玻璃纸上生长3 d时菌落形态，b为去掉玻璃纸后2 d时菌落形态。
图5为菌丝尖端形态显微观察。
图6为WH504-50中的dsRNA电泳图谱。
图7为FpgMTV1的基因组结构示意图。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
一、假禾谷镰孢菌株WH504-50的分离鉴定和保存
假禾谷镰孢菌株WH504-50的菌落形态如图1所示。
利用特异性引物对Fp1-1/Fp1-2对假禾谷镰孢菌株WH504-50的菌落进行PCR其中假禾谷镰孢的特异性引物为：
Fp1-1 5’-CGGGGTAGTTTCACATTTCCG-3’
Fp1-2 5’-GAGAATGTGATGACGACAATA-3’
PCR扩增反应体系为：在25 μL体系中，2×Taq PCR Master Mix 12.5 μL；Fp1-1（10 μM）1 μL ，引物 Fp1-2（10 μM）1 μL；模板DNA（50 ng·μL-1）体积为1 μL，ddH2O补足到25 µl。反应程序：94℃ 3 min，94℃ 30 s，57℃ 30 s，72℃ 45 s，40个循环；72℃终延伸7min；16℃保存。
扩增结果如图2所示，可以确定菌株WH504-50为假禾谷镰孢菌。
二、假禾谷镰孢菌株WH504-50产孢量测定和分生孢子显微观察
用直径5 mm的打孔器在刚纯化的菌落边缘内侧打菌饼，并用无菌挑针将WH504-50、WH504-50-V-和对照菌株FC136-2A接种到含有50 mL CMC的150 mL锥形瓶中，每个菌株做三个重复，放在空气恒温振荡器中进行摇动培养，25℃，180 r/min培养5 d，用Microcloth过滤，然后用血球计数板在显微镜下观察，测定菌株产孢量。用移液枪吸取20 μL过滤后的孢子悬浮液至载玻片上，用正置显微镜（ZEISS Axio Imager2）对分生孢子进行形态观察并拍照记录，如图3所示。
结果显示，通过测定菌株产孢量和孢子大小，携带FpgMTV1病毒菌株WH504-50相比于脱毒菌株WH504-50-V-和对照菌株FC136-2A的产孢能力显著减弱，FpgMTV1还显著降低菌株分生孢子长度，说明FpgMTV1能显著抑制宿主菌株产孢能力和分生孢子生长发育。
三、假禾谷镰孢菌株WH504-50玻璃纸穿透力测定
用直径5 mm的打孔器在菌落边缘内侧打菌饼，用无菌挑针将菌饼分别接种到铺有玻璃纸的PDA培养基中间位置，菌丝面朝下，25℃，倒置培养2 d，在超净工作台中小心打开培养皿盖子，用酒精灯烧过的镊子在玻璃纸边缘将玻璃纸轻轻揭掉，避免菌丝飘落至PDA培养基上，用新的培养皿盖子盖住去掉玻璃纸的培养皿，在培养箱中25℃培养3 d，观察培养皿中是否存在菌落，如图4所示。
结果显示，携带FpgMTV1病毒菌株WH504-50菌丝不能穿透玻璃纸到PDA培养基上，脱毒菌株WH504-50-V-对玻璃纸的穿透能力不受影响，说明FpgMTV1能抑制宿主菌株生长和降低对玻璃纸的穿透力。
四、假禾谷镰孢菌株WH504-50菌丝尖端形态观察
用直径5 mm的打孔器在刚纯化的菌落边缘内侧打菌饼，并用无菌挑针接种到PDA平板培养基的中央位置，菌丝面朝下，在距离菌饼1 cm 处放置无菌盖玻片，25℃，黑暗培养，直到菌株生长到培养皿中放置的盖破片上0.5 cm时，用无菌镊子将盖玻片表明的气生菌丝轻轻刮去，并将带菌丝的盖玻片一面倒置在滴有无菌水的载玻片上，用正置显微镜（ZEISS Axio Imager2）对菌丝尖端进行形态观察并拍照，如图5所示。
结果显示，携带FpgMTV1病毒菌株WH504-50菌丝尖端相比于脱毒菌株出现了菌丝呈波浪弯曲现象，而脱毒菌株WH504-50-V-菌丝尖端正常笔直生长，说明FpgMTV1能影响宿主菌丝尖端的正常生长。
五、假禾谷镰孢菌株WH504-50中发现真菌病毒FpgMTV1的基因组dsRNA
收集PDA培养基上培养5天的假禾谷镰孢菌株WH504-50菌丝，用液氮将菌丝在研体中碾成粉末，装入灭菌离心管中。每0.2 g样品中加入400 μL 2×STE，酚：氯仿：戊醇=25:24:1溶液400 μL、10%SDS 92 μL，室温震荡混匀10 min。12000 rpm离心10 min，收集上清，移至新管。每600 μL上清液加入114 μL ETOH混匀后再加入0.04 g无离子纤维素粉（CF-11），震荡混匀，冰浴30 min。12000 rpm离心1 min，弃上清液，加入600 μL 1×STE with17%ETOH，混匀，静置3 min。重复步骤4三次，12000 r离心2 min后弃上清液，加入600 μL 1×STE，混匀震荡5 min，12000 rpm离心5 min，将上清液移至新管。加入等体积预冷的异丙醇，-20℃下沉淀30 min以上。4℃下，12000 rpm离心2.5 min，弃上清液，用200 μL 75%ETOH洗沉淀。12000 rpm离心1 min倒去残液，风干或37℃温箱内干燥10 min。用25 μL DEPC-H2O溶解沉淀。加入2 μL DNase I， 4 μL 10×DNase I buffer和1 μL RNA酶抑制剂，用DEPC-H2O定容至40 μL，小心混匀后于37℃酶解60 min。取5 μL酶解后的dsRNA进行1％琼脂糖凝胶电泳。
结果显示在大约12 kb和大于4 kb处存在两条dsRNA片段（图6），将其分别命名为L1-dsRNA和L2-dsRNA。
六、真菌病毒FpgMTV1的基因组dsRNA序列测定和分析
以菌株WH504-50中所得dsRNA为模板，用随机引物Random Primer(26-mer) (5’-GACGTCCAGATCGCGAATTCNNNNNN-3’)进行反转录，反转录酶为Promega的M-MLV。 利用引物Random Primer(20-mer) (5’-GACGTCCAGATCGCGAATTC-3’)进行PCR扩增，所用的酶是康润诚业生物科技有限公司（北京）的2×SuperStar Plus Mix with Loading Dye。PCR产物经1%琼脂糖凝胶电泳后回收500bp以上的条带，按照OMEGA公司的DNA凝胶回收试剂盒说明进行。
回收后将目的片段连接到T克隆载体pMD19-T(TaKaRa)，挑选阳性克隆送上海生工生物工程（上海）股份有限公司进行测序。根据已获得随机片段克隆序列，用primerpremier 5.0设计引物(包括正向、反向互补)进行PCR扩增。扩增产物与T载体的连接，转化大肠杆菌并测序。反应程序：94℃ 3 min，94℃ 30 s，（Tm-5）℃ 30 s，72℃ 3min，40个循环；72℃终延伸7 min；16℃保存。5’末端序列扩增采用经典的5’RACE克隆方法，利用末端脱氧核苷酸转移酶(TdT)在反转录产物cDNA的3’端加上poly A尾巴，然后利用接头引物及序列特异引物进行巢式PCR扩增。PCR产物与T载体连接，转化大肠杆菌并测序。3’末端序列扩增采用oligo RNA末端连接RACE(RLM-RACE)，在T4 RNA连接酶的作用下，将一断具有特殊修饰的寡核苷酸链(5’端磷酸化修饰，3’端氨基修饰)连接到RNA链的3’羟基末端。根据寡核苷酸链序列设计特异引物，与3’端序列特异引物一同将3’末端序列扩增出来。PCR产物与T载体连接，转化及验证测序。
使用DNAMAN软件的序列拼接功能将随机引物cDNA克隆序列、特异引物cDNA克隆序列、RACE克隆序列拼接起来，构成完整的病毒基因组序列。获得新病毒全基因组序列后，从NCBI Genbank 数据库下载+ssRNA或dsRNA 病毒各科病毒的核酸序列，利用 CLUSTAL_ X和MEGA 7.0和NCBI数据库（https://www.ncbi.nlm.nih.gov/）上的相关软件进行分析。
通过序列测定和拼接，获得了长为12300bp（L1-dsRNA）碱基序列如SEQ ID NO：1所示和3818bp（L2-dsRNA）碱基序列如SEQ ID NO：2所示的2条dsRNA序列（参见sequencelist），分析表明，L1-dsRNA和L2-dsRNA的GC含量分别是48.4%和45.6%，L1-dsRNA包含两个开放阅读框（Open Reading Frame, ORF）, L2-dsRNA只包含一个ORF（图7）。L1-dsRNA中ORF1长为8334nt,编码蛋白质(2787aa)，ORF2长为3666nt,编码蛋白质(1221aa)。L2-dsRNA中ORF3长为3666nt,编码蛋白质(1221aa)。根据基因序列和进化树分析，申请人明确了这两条dsRNA是病毒科Megatotiviridae的一种新病毒的基因组，申请人将其命名为Fusariumpseudograminearum megatotivirus 1 （FpgMTV1）。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
<110> 河南农业大学
<120> 一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株WH504-50
<141> 2022-04-26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12300
<212> RNA
<213> 病毒(virus )
<400> 1
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ugcgucucga acuggaugca guuguucagu aggauaggug auccauccaa gguugccaag 11400
cuugaacaau accuguauuc agacguggac aauauuuuaa agcauuauaa uguuagagcu 11460
gaucaccgaa uacuacacac cccaagaucc gagggcgguu ucgguuuguc gcguuuacaa 11520
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aaauugucgu augacaggac uuucgcaagc acgcugauau cggacgaaga ccgauuauca 11700
acauucaagg uggcuaagac cacacaaccg gcuccgaagc gccuguacaa ugucaaauac 11760
gucaacaaga agcucacaca guuuggcgau gagcugauug agaaacuaaa cucgcccacu 11820
agagugacgg cugacuuguu guugucuaag ggugauuaca uaccagaggu gagagaugga 11880
cugcuagcaa gugggauguu uucaucguua uucgaacaaa caccggauaa ucaaaagaga 11940
uacuuguuca ccagaccgau cguguacgau caauugguag agagguucgg caaggguaga 12000
gcugcggcug ucaucuuuaa aggucuagau uggguucagu uuagaucuug ggaguuugac 12060
guugauuucu ucaaccuuau ugcugcacag uauguguuau ucugguuauc aacaaaucuu 12120
aacaaauaua ugacguugau ucaacuucaa cuguuuaugg aaacggagau gcaucgauua 12180
auguccaaac uacacuauca aaucagaaac uaaaacgagu ugagaggguu gagggaaaga 12240
ccacucacaa cggaccaccu gucgaggugg ggcgcucuua ugguccaaga ccaaaucguc 12300
<210> 2
<211> 3818
<212> RNA
<213> 病毒(virus )
<400> 2
aagagcagga aucucacaaa acuuccaaau augagaaagu aaaacauuca caguuuuucu 60
cucuaaugga gagauuuacc gggcgugaag guacuuccuc acuucaauca uuucuugauu 120
cugaggugag ggacaacccu cacgucaaca gaauaaagaa caaguuguuu ucugagacua 180
gugcugacau ugacaagguc cuugauaaaa cgggacaggu cagagucaac auacaggcua 240
aagaaaaaca agacaucauc aacauguuaa agaagcgcau agcugaugac aacaugucuc 300
gaccaaucgu aguagcgcau cgcaacuaug guaagucuuu caucacugcg agaucaccga 360
cagcgcugga cauagaugac uucacaccga gugaaguguu caguggugaa ccgaaccagu 420
uacuggcgag gggucaaaac ccguaucgug ccaucuuaau ggcaucgaga augggcaaga 480
caaagcuguc acucaacaac cccgggaagg uuguagaugu cgaugacauu augaaacgcc 540
uuucugccga uauuaaaaac aaugacagua aauuguuguu gaacgauaaa ccugaggugu 600
uaauccauaa acuggaggga aacuucauca ccaaaggaau caugcaacua aucaaagaca 660
acuuacaucu auacguugac gggaccaaga uagugcucuu acauggacca ucggaagucc 720
cggaaggcau ggauguccug gauuguuuaa guuucccuua cuacaagcac acgggagaga 780
gcgaugauug gcauaaaguu cagacuuaug acaauacggu acgagauugu cugucauaca 840
ggcaacccgu cacgucgugg uuacagguca ggaccagauu guugcgucau gaucucagaa 900
ugcccgauga cuuuaaaaca accggugcuc cguucggucg cucaacucac guaccacuca 960
agcaguggaa uuuugagcga caggaugguu ggauacuuuu caauaaaaag aaacacgacu 1020
aucagagaca agcucuaaac aacgccuacc ccgaugggug ggcgaccaga ggcagaggag 1080
ccuuguuaua uacgcauucg aucgaagacg ucaaacgccu gggagguaug uuacucgcag 1140
uaguugaaua uaccgguaau gacgggaugg aucuuauggc agugcauaac aggcacacaa 1200
cucguaaagc agcuggacag ugcggccaga cgaucauaag acucagugac aagaagauga 1260
accugacuca gcacgagaga uuguggagua uguacgcucg caaauucucu ucgacagcuu 1320
gguuugaaca guuuaaguua gacaagaucg aacaaggagu uaucaagacu ugggacgaac 1380
aaacaaucaa guacuuacac uccacacagg gugaguggug ugcguuccag cuuaauaacg 1440
acgucuuauu ccagaauugg uucauggaca augagaccua caagcucaag cgauucagga 1500
cuaaggagug gaauuaucac cuggcauaca ucucggacug cgcuaugcuc uugaagcagu 1560
ucaacccaca uguguucuca uucuuguugg ccgugugugg gaccuuuaau cucacauauc 1620
agucauauuc gaaaauaaug aaauugaugu cgaacauggu uaaggcauuc aacaauucgu 1680
gguguccggg uuggauggac ugggucgacc uggcccgcuu agccggauuc aguaugaagu 1740
uugaugcuaa auuguacgcc gaagacauua agaccuggcu uuuagagaac gagaagucga 1800
ggcauuaugu cgaaggcagu gagcucaagu uucuguccga auucgaaaaa ucuguggaau 1860
uauuguuagc aaaugaugcg cccaagaacg cacugacaau caaacaauuc uugaacgacc 1920
cggccaagug ggccguuucg ggcagugcca agggaguaga auugccaaga uuagauaacg 1980
gcaggaagcu gcgcaaaacc aaacgugcag cggguuuccu ucuucaauau gaagaacuca 2040
gagcagcauu ugaucgugac acuuccgaca auuauguuuu ugacaagaaa gaaccaaaca 2100
ggaacagacc ugugaucaac ucugaucuag gcauguaucu aaaaaugagc uaucucgacg 2160
accucgucua caaaauggug ucagaggagu auagucagug uucucccauc uuuaagaacu 2220
uugacuauau ugcugauaag acauaccaag ugaaacacau ugguaggaag aaucgagucu 2280
gcuuaccacu agaccaaucu gguuuugagc gacagaccag cuuugcuaug cuggacaaga 2340
uccucggcgu cauugaacgu cgguuagcau acgacgacgu cgagagucga cagacacuca 2400
aauugaucag gagugguauc uucaauucua cacuccacuu ugaugaugca ccggaaauug 2460
auggacaacg gauuacgaau gguuuguccu ccgguuggaa auggacggca cugaucaaca 2520
cgaucaucaa ccuugccgaa uucaucaccu gcgccagauu acuugacuug caauaccaca 2580
accugugcgc acugggagac gauaccagag uacaacucaa gucauauucu gaugcccgcg 2640
cagugcucga cugguacaaa ucugccagua uagauaucaa cgagaaacua gcuaucauga 2700
gccacaccug ugacgaguuc cuacgcaagg uaacacagua cgacccagug accaagcgcc 2760
guugggugag uggcuacuac aaucguaugc uaguugccau cauguacaag aaccccaaau 2820
ccgcggaccc ugcggauguc aacgaagcca uagagacaug cgucucgaac uggaugcagu 2880
uguucaguag gauaggugau ccauccaagg uugccaagcu ugaacaauac cuguauucag 2940
acguggacaa uauuuuaaag cauuauaaug uuagagcuga ucaccgaaua cuacacaccc 3000
caagauccga gggcgguuuc gguuugucgc guuuacaacc cggugggcca gcagucauca 3060
uggauuauga ggcgaucaau caccaggugc cuuugagcgc caggagacaa gcgcagaucc 3120
aagacaacuu guugacuacc uaucgguuac cgaaugugaa auugucguau gacaggacuu 3180
ucgcaagcac gcugauaucg gacgaagacc gauuaucaac auucaaggug gcuaagacca 3240
cacaaccggc uccgaagcgc cuguacaaug ucaaauacgu caacaagaag cucacacagu 3300
uuggcgauga gcugauugag aaacuaaacu cgcccacuag agugacggcu gacuuguugu 3360
ugucuaaggg ugauuacaua ccagagguga gagauggacu gcuagcaagu gggauguuuu 3420
caucguuauu cgaacaaaca ccggauaauc aaaagagaua cuuguucacc agaccgaucg 3480
uguacgauca auugguagag agguucggca aggguagagc ugcggcuguc aucuuuaaag 3540
gucuagauug gguucaguuu agaucuuggg aguuugacgu ugauuucuuc aaccuuauug 3600
cugcacagua uguguuauuc ugguuaucaa caaaucuuaa caaauauaug acguugauuc 3660
aacuucaacu guuuauggaa acggagaugc aucgauuaau guccaaacua cacuaucaaa 3720
ucagaaacua aaacgaguug agaggguuga gggaaagacc acucacaacg gaccaccugu 3780
cgaggugggg cgcucuuaug guccaagacc aaaucguc 3818

Claims (3)

1.用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株WH504-50，其特征在于：所述菌株的拉丁文名称为Fusarium pseudograminearum，其保藏单位为中国典型培养物保藏中心，保藏号为CCTCC No：M20211108，保藏日期为2021年8月30日，保藏地址为湖北省武汉市武昌区八一路299号。
2.权利要求1所述的假禾谷镰孢菌株WH504-50在抑制镰孢菌产孢能力中的应用。
3.权利要求1所述的假禾谷镰孢菌株WH504-50在抑制镰孢菌分生孢子生长发育中的应用。
CN202210444189.8A 2022-04-26 2022-04-26 一株用于抑制镰孢菌产孢能力和分生孢子生长发育的假禾谷镰孢菌株wh504-50 Active CN114561300B (zh)

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