江苏淮安市高粱上玉米黄花叶病毒的分子检测与鉴定
Molecular Detection and Identification of Maize Yellow Mosaic Virus on Sorghum in Huai’an, Jiangsu
Wang Haitao ,, Ren Chunmei, Dong Yan, Li Shuo, Cheng Zhaobang, Ji Yinghua ,
摘要
为掌握江苏省高粱病毒病的发生情况,从江苏淮安采集高粱疑似病毒病样品20份,进行了分子检测与鉴定。结果发现,使用玉米黄花叶病毒(maize yellow mosaic virus,MaYMV)引物可扩增出与目的片段一致的条带,检出率为80%。利用MaYMV外壳蛋白(CP)基因引物,以阳性样本RNA为模板进行扩增、克隆、酶切验证和测序分析,并构建进化树发现,淮安分离物CP基因与江苏分离物CP基因的进化关系较近。这些结果表明江苏淮安高粱受到了MaYMV侵染,这是该病毒在我国高粱上的首次报道。
关键词:玉米黄花叶病毒;高粱病毒病;分子检测;进化树分析
本文引用格式
王海涛, 任春梅, 董岩, 李硕, 程兆榜, 季英华.
Wang Haitao, Ren Chunmei, Dong Yan, Li Shuo, Cheng Zhaobang, Ji Yinghua. Molecular Detection and Identification of Maize Yellow Mosaic Virus on Sorghum in Huai’an, Jiangsu. Crops, 2024, 40(1): 233-238 doi:10.16035/j.issn.1001-7283.2024.01.031
高粱是全球第五大粮食作物,全球种植面积超过4000万hm2(6亿亩),是传统酿造业、饲料加工业、再生能源产业和保健食品等领域的重要原料。高粱生育期内病虫害的发生是威胁高粱产量的关键制约因素,在我国已报道的高粱病害有30多种。有不少病毒可以感染高粱并引起典型的病毒病症状,如玉米矮花叶病毒(maize dwarf mosaic virus,MDMV)、高粱花叶病毒(sorghum mosaic virus,SrMV)、甘蔗花叶病毒(sugarcane mosaic virus,SCMV)、约翰草花叶病毒(Johnson grass mosaic virus,JGMV)、玉米条纹病毒(maize stripe virus,MStpV)和高粱褪绿叶斑病毒(sorghum chlorotic spot virus,SrCMV)等对高粱生产造成严重的威胁,然而目前植物病毒学科对高粱病毒病的研究仍相对较少。
随着高通量测序技术和小RNA深度测序的应用,越来越多的植物病毒病害被报道与发现。2016年,通过小RNA深度测序从我国云南玉米疑似病毒病样中首次鉴定到一个新病毒,命名为玉米黄花叶病毒(maize yellow mosaic virus,MaYMV),随后该病毒在我国广西、福建、四川、安徽和河南等省区被报道[1-2]。该病毒在韩国、巴西、厄瓜多尔、布基纳法索、埃塞俄比亚、肯尼亚、乌干达、卢旺达、坦桑尼亚和尼日利亚等国家和地区也陆续被报道[3⇓⇓⇓⇓⇓⇓-10]。
作者在调查江苏省淮安市玉米病毒病时发现了花叶和皱缩等疑似病毒病的症状,通过鉴定发现疑似病毒病玉米样品受到MaYMV的侵染。MaYMV是Solemoviridae病毒科马铃薯卷叶病毒属(Polerovirus)的成员,有稳定的二十面体结构,是正义单链多顺反子RNA病毒[1],由介体玉米缢管蚜(Rhopalosiphum maidis)和禾谷缢管蚜(Rhopalosiphum padi)以循环型不增殖的方式进行传播[11],侵染玉米后引起黄花叶和矮缩的症状[1,12],在一些玉米品种中可引起叶片变红的症状[13-14],除了侵染玉米外,该病毒还侵染其他禾本科植物,如小麦、高粱、小米、甘蔗和筒轴茅等[15⇓⇓⇓-19],Mlotshwa等[20]对国内不同地理分布的MaYMV的遗传变异和分子进化进行了分析,然而目前在国内尚无关于MaYMV侵染高粱的报道。
本研究对高粱疑似病毒病样进行了检测和分子鉴定,明确了MaYMV在江苏淮安高粱种植区的为害,对江苏省高粱病毒病的监测有十分重要的参考价值。
1 材料与方法
1.1 供试病样
疑似高粱病毒病害样品于2022年10月采自江苏省淮安市涟水县高沟镇(119.20°E,34.01°N),冻存于-80 ℃备用。
1.2 主要试剂
用于基因扩增的PrimeSTAR® Max DNA Polymerase(R045A)购自Takara公司;RNA提取试剂、反转录试剂、2×Rapid Taq Master Mix(P222-03)、胶回收试剂盒(DC301-01)和克隆载体(5min TA/Blunt-Zero Cloning kit,C601-01)购自南京诺唯赞生物科技股份有限公司;质粒提取试剂盒(TSP501-200)和感受态细胞(Trelief™ 5α,TSC- C01)购自北京擎科生物科技有限公司;本研究使用的其他试剂均为国产分析纯。引物委托南京金斯瑞生物科技有限公司合成。
1.3 高粱组织RNA的提取
取冻存疑似病毒病样品0.1 g,利用上海净信磨样仪将液氮冷冻的高粱叶片研磨成粉,参照RNA提取试剂说明书提取高粱样品总RNA。
1.4 RT-PCR检测
以RNA为模板,利用反转录试剂,参照说明书合成cDNA,具体步骤如下,1 μg RNA和4 μL 4×gDNA wiper mix,加无RNA酶水至16 μL。将PCR管内的样品吹打混匀后,置于42 °C孵育2 min,反应结束后,短暂离心,置于冰上冷却2 min,然后向管内加入4 μL 5×HiFiScript II qRT supermix II。将PCR管内的预混液混匀后,置于50 °C孵育15 min,85 °C反应5 s,反应结束后,短暂离心,置于冰上冷却,进行后续PCR反应。
对已报道的高粱病毒设计检测引物(表1),以反转录获得的cDNA为模板,参照2×Taq Master Mix说明书进行PCR反应,反应体系如下,10 μL 2×Taq Master Mix、2 μL cDNA模板、上下游引物各0.5 μL、7 μL无菌水。根据PCR酶试剂说明书设置反应程序如下,95 ℃预变性3 min;95 ℃ 15 s,55 ℃ 15 s,72 ℃ 15 s,35个循环;72 ℃延伸5 min;16 ℃保存。PCR反应结束后用1%琼脂糖胶进行分析。
表1 检测中所使用的病毒引物
Table 1
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1.5 江苏淮安MaYMV CP基因的克隆
根据MaYMV外壳蛋白核苷酸序列设计特异性引物如下,CP-F-Xba I:TCTAGAATGAATACG GGAGGTAGAAATGG;CP-R-Kpn I:CCATGGCTA TTTCGGGTTTTGAACATTG,下划线序列为酶切位点,以检测结果为阳性的病样cDNA为模板,检测为阴性的样品cDNA为对照,利用Prime-V STAR® Max DNA Polymerase进行PCR扩增,参考说明书设置反应体系如下,25 μL 2×PrimeSTAR® Max DNA Polymerase Mix、5 μL cDNA模板、上下游引物各1 μL、18 μL无菌水。PCR反应程序设置如下,98 ℃预变性3 min;98 ℃ 10 s,55 ℃ 15 s,72 ℃ 30 s,35个循环;72 ℃延伸8 min;16 ℃保存。PCR反应结束后用1% 琼脂糖胶进行分析,目的片段切胶后用胶回收试剂盒纯化后连接至5 min TA/Blunt-Zero克隆载体;连接产物经热激法转化入大肠杆菌Trelief™ 5α细胞后涂布于含有氨苄青霉素的LB平板上,37 ℃倒置培养12 h,挑取平板上生长的单克隆菌落进行PCR,鉴定为阳性的菌落提取质粒后进行酶切鉴定,酶切鉴定正确的样品进行序列测定。
1.6 MaYMV分离物CP的进化树分析
将测序获得的MaYMV CP氨基酸序列与已经公布的MaYMV不同分离物CP序列进行系统进化树分析。系统进化树采用Mega软件(7.0.26)的Neighbor-Joining最大复合似然法完成,进化树的可信度使用1000次复制验证。
2 结果与分析
2.1 病害田间症状
在江苏省淮安市高沟镇调查采样时发现,部分高粱植株出现新叶斑驳黄化、老叶皱缩(图1a)、植株整体矮缩(图1b)等疑似病毒病感染的症状。在调查的过程中,此类症状在田间零星发生,发病率在5%左右,调查过程中未见大面积连片或者整块田病发的情况。
图1
图1 田间采集的高粱病样症状
Fig.1 Symptoms of the virus infected sorghum in field
2.2 高粱上玉米黄化花叶病毒的检测
以采集疑似病样混合组织的RNA为模板,利用表1的引物进行RT-PCR发现,仅1种病毒引物(MaYMV)可扩增出与目的片段大小一致的条带(图2a),而其余的14种病毒未检测到条带。为了进一步明确采集的20份样品中MaYMV的侵染率,利用MaYMV引物检测,发现其中16份样品扩增到与预期大小一致的条带(图2b)。
图2
图2 采集的高粱疑似病毒样中MaYMV的RT-PCR检测
M:DNA分子量标准;(a) 不同病毒引物检测高粱样品,(b) 高粱疑似病样检测。
Fig.2 RT-PCR detection of MaYMV in suspected diseased sorghum samples
M: DNA marker; (a) Detection of sorghum samples with different virus primers; (b) Detection of suspected disease samples of sorghum.
2.3 MaYMV外壳蛋白基因CP的序列分析
为了进一步明确该病毒为MaYMV,我们设计了MaYMV外壳蛋白(coat protein,CP)基因开放阅读框(open reading fram,ORF)引物,以阳性样本为模板,扩增到与目的片段大小(594 bp)一致的特异性条带(图3a),表明该条带即为本研究要扩增的MaYMV外壳蛋白基因CP的片段。
图3
图3 淮安MaYMV CP的扩增、克隆和酶切验证
M:DNA marker;(a) 1表示MaYMV CP基因的扩增,2表示阴性;(b) CP基因克隆载体的双酶切验证。
Fig.3 The amplification, clone and verification of double digestion of Huai’an MaYMV CP
M: DNA marker; (a) 1 indicates amplification of MaYMV CP gene, 2 indicates negative; (b) Verification of double enzyme digestion of CP gene cloning vector.
为了进一步验证该结果,利用胶回收试剂盒对目的片段进行回收,参照说明书将回收片段连接至5min TA/Blunt-Zero Cloning载体,经热激法转化后将平板倒置培养12 h,挑取单菌落,利用载体通用引物(M13F、M13R)进行扩增,结果显示特异的单一条带,表明MaYMV外壳蛋白基因CP成功克隆至T载体上。
为了进一步明确克隆是否准确,挑取阳性克隆提取质粒,利用Xba I和Kpn I限制性内切酶对质粒进行酶切发现,可以切出2条完整的条带,且切出的条带与目的大小一致(图3b),该结果表明将MaYMV成功克隆至T载体。
2.4 MaYMV CP基因核苷酸序列的分析
为了保证所获得CP基因核苷酸序列的准确性,选取阳性质粒进行序列测定,结果表明,江苏淮安采集的MaYMV CP的ORF长度为594 bp(GenBank:OP947961),翻译产生197个氨基酸,编码1个大小为21.4 kDa的蛋白质。
为了进一步明确江苏淮安MaYMV的分类地位,我们利用Mega软件将本试验获得的该病毒CP基因序列与目前已公布的MaYMV、同属的玉米黄矮病毒、大麦黄矮病毒CP核苷酸序列进行聚类分析,使用Neighbour-Joining方法构建系统进化树,进化距离通过使用最大复合似然法进行计算,结果(图4)发现,江苏淮安采集到的MaYMV CP基因与同病毒不同地区分离物的进化关系较近,与江苏分离物(OP947962)进化关系最近,与同属的MYDV CP基因(MF974579.2)的亲缘关系相对较近,而与BYDV不同分离物CP(NC 043123.1、NC 021481.1、AF 218798.2、NC 003680.1)之间的进化关系远。
图4
图4 淮安MaYMV CP核苷酸序列进化树分析
Fig.4 The phylogenetic tree analysis based on nucleotide sequences of Huai’an MaYMV CP
3 讨论
自2016年在中国首次报道MaYMV的发生与为害以来[1-2],该病毒先后在亚洲、美洲和非洲等多个地区的玉米种植区发生和流行[3⇓⇓⇓⇓⇓⇓-10],对全球玉米生产造成了巨大的威胁,然而目前国内还没有关于该病毒侵染高粱的报道。江苏省高粱种植面积在7000 hm2左右,由于白酒产业发达和高粱贸易市场的优势,目前,江苏省已经成为特色高粱优势区,为此关注高粱生产过程中的病虫害具有十分重要的意义。本研究于2022年对江苏省淮安市的高粱病毒病进行了调查,并在疑似病毒病样中检测到了MaYMV侵染,而未检测到其他已报道的可侵染高粱的病毒,这是该病毒在我国高粱上的首次报道,关于该病毒在江苏省乃至全国高粱种植区上的发病率以及对产量的影响还没有相关报道。
本研究采集的MaYMV高粱病样具有花叶、植株矮缩等典型的病毒病症状,与前人[1-2]报道的玉米上的典型症状相似,即皱缩、花叶和植株矮缩。有报道[13-14,20]发现,MaYMV侵染玉米后可引起叶片变红,目前尚未在高粱上发现这一现象。最近,Mlotshwa等[20]构建了MaYMV的侵染性克隆,并对其侵染引起玉米红叶的原因进行了解析。MaYMV除了侵染玉米还可以侵染双子叶植物本生烟[20],表明该病毒可能还有一种至多种双子叶寄主植物。
水稻、小麦、玉米和高粱等禾本科作物在苗期受到病毒侵染后,随着适宜作物生长的季节到来,病毒在寄主植物内的增殖增多,导致植物出现典型的病毒病症状,使作物减产,甚至绝产。MaYMV主要由蚜虫进行传播,带毒蚜虫从冬小麦迁移至春玉米进而侵染高粱,实现病毒在不同作物间的传播。防治上应通过控制蚜虫数量达到预防病毒侵染的目的。目前,已知马铃薯卷叶病毒属成员病毒以网格蛋白介导的内吞方式突破介体蚜虫的肠道和唾液腺屏障[21],然而关于玉米缢管蚜和禾谷缢管蚜传播该病毒的机制及其如何突破介体蚜虫各种组织和膜屏障的机制仍不清楚。
本研究利用RT-PCR对采集到的20份高粱疑似病毒病样品进行了检测,发现MaYMV的检出率高达80%,表明该病毒对于江苏省高粱和玉米等禾本科作物的生产具有十分重要的威胁,为此当下研究该病毒的任务较为迫切。除了普通的RT-PCR检测方法外,Li等[22]开发了RT-LAMP方法,对MaYMV的检测手段进行了丰富。本研究利用Neighbour-Joining方法和最大复合似然法构建了MaYMV各地区分离物CP基因的系统进化树(图4),表明MaYMV已从最初的玉米蔓延扩散至高粱上。
MaYMV除了编码CP蛋白外,还编码P0和MP等蛋白,然而目前关于这些蛋白在植物和昆虫细胞内的亚细胞定位及具体的功能仍不清楚。马铃薯卷叶病毒属代表种马铃薯卷叶病毒(potato leafroll virus,PLRV)的P0、P1和P7蛋白通过抑制因介体蚜虫取食引起的寄主植物激素变化,进而促进介体蚜虫的生殖[23]。然而,目前关于MaYMV各蛋白的功能以及与寄主植物和介体蚜虫之间互作的研究还没有报道。为了预防MaYMV在江苏省乃至长江流域的大面积暴发与流行,当前亟需对该病毒及其介体蚜虫以及寄主玉米之间的互作进行深入研究。
4 结论
本研究利用15对引物对采集到的高粱疑似病毒病样本进行检测,发现样本受到了MaYMV侵染。随后利用MaYMV引物对20份样本单独检测发现,MaYMV的感染率高达80%,利用分子生物学和生物信息学手段进一步明确了高粱疑似病样中MaYMV的侵染。
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A maize-infecting polerovirus variously named maize yellow dwarf virus RMV2 (MYDV-RMV2) and maize yellow mosaic virus (MaYMV) has been discovered and previously described in East Africa, Asia, and South America. It was identified in virus surveys in these locations instigated by outbreaks of maize lethal necrosis (MLN), known to be caused by coinfections of unrelated maize chlorotic mottle virus (MCMV) and any of several maize-infecting potyviruses, and was often found in coinfections with MLN viruses. Although sequenced in many locations globally and named for symptoms of related or coinfecting viruses, and with an infectious clone reported that experimentally infects, rudimentary biological characterization of MaYMV in maize, including insect vector(s) and symptoms in single infections, has not been reported until now. We report isolation from other viruses and leaf tip reddening symptoms in several maize genotypes, along with transmission by two aphids, and. This is important information distinguishing this virus and demonstrating that in single infections it causes symptoms distinct from those of potyviruses or MCMV in maize, and identification of vectors provides an important framework for determination of potential disease impact and management.
[12]
Wang F, Zhou B G, Gao Z L, et al.
A new species of the genus Polerovirus causing symptoms similar to maize yellow dwarf virus-RMV of maize in China
Plant Disease, 2016, 100(7):1508.DOI:10.1094/PDIS-11-15-1259-PDN [本文引用: 1]
[13]
Shi Y J, Han X Y, Li Q L, et al.
First report of maize yellow mosaic virus causing maize reddening in Henan, China
Plant Disease, 2022, 106(12):3220.[本文引用: 2]
[14]
Sun S R, Chen J S, Yang J, et al.
First report of maize yellow mosaic virus infecting sugarcane in China
Plant Disease, 2019, 103(9):2482-2483.[本文引用: 2]
[15]
Lim S, Yoon Y, Jang Y W, et al.
First report of maize yellow mosaic virus infecting Panicum miliaceum and Sorghum bicolor in South Korea
Plant Disease, 2018, 102(3):689.[本文引用: 1]
[16]
Yahaya A, Rwahnih M A, Dangora D B, et al.
First report of maize yellow mosaic virus infecting sugarcane (Saccharum spp.) and itch grass (Rottboellia cochinchinensis) in Nigeria
Plant Disease, 2017, 101(7):1335.[本文引用: 1]
[17]
Nithya K, VishnuVardhan J, Balasaravanan S, et al.
First report of maize yellow mosaic virus (MaYMV) infecting sugarcane in India and its molecular characterization
Australasian Plant Pathology, 2021, 50:633-638.[本文引用: 1]
[18]
Guo M Y, Yuan X Y, Song Y L, et al.
First report of maize yellow mosaic virus (MaYMV) naturally infecting wheat in China
Plant Disease, 2022, 106(10):2763.[本文引用: 1]
[19]
Sun S R, Chen J S, He E Q, et al.
Genetic variability and molecular evolution of maize yellow mosaic virus populations from different geographic origins
Plant Disease, 2021, 105(4):896-903.DOI:10.1094/PDIS-05-20-1013-RE URL [本文引用: 1]
Maize yellow mosaic virus (MaYMV) hosted in various gramineous plants was assigned to the genus Polerovirus (family Luteoviridae) in 2018. However, little is known about its genetic diversity and population structure. In this study, 509 sugarcane leaf samples with mosaic symptoms were collected in 2017 to 2019 from eight sugarcane-growing provinces in China. Reverse-transcription PCR results revealed that four positive-sense RNA viruses were found to infect sugarcane, and the incidence of MaYMV among samples from Fujian, Sichuan, and Guangxi Provinces was 52.1, 9.8, and 2.5%, respectively. Based on 82 partial MaYMV sequences and 46 whole-genome sequences from different host plants, phylogenetic analysis revealed that MaYMV populations are very closely associated with their source geographical regions (China, Africa, and South America). Pairwise identity analysis showed significant variability in genome sequences among MaYMV isolates with genomic nucleotide identities of 91.1 to 99.9%. In addition to codon mutations, insertions or deletions also contributed to genetic variability in individual coding regions, especially in the readthrough protein (P3-P5 fusion protein). Low gene flow and significant genetic differentiation of MaYMV were observed among the three geographical populations, suggesting that environmental adaptation is an important evolutionary force that shapes the genetic structure of MaYMV. Genes in the MaYMV genome were subject to strong negative or purification selection during evolution, except for the movement protein (MP), which was under positive selection pressure. This finding suggests that the MP may play an important role in MaYMV evolution. Taken together, our findings provide basic information for the development of an integrated disease management strategy against MaYMV.
[20]
Mlotshwa S, Khatri N, Todd J, et al.
First report of cDNA clone- launched infection of maize plants with the polerovirus maize yellow mosaic virus (MaYMV)
Virus Research, 2021, 295:198297.[本文引用: 4]
[21]
Gray S, Gildow F E.
Luteovirus-aphid interactions
Annual Review of Phytopathology, 2003, 41:539-566.PMID:12730400 [本文引用: 1]
Members of the Luteoviridae are transmitted by aphids in a circulative, nonpropagative manner that requires the virus to be acquired through gut tissue into the aphid hemocoel and then exit through salivary tissues. This process is aphid species-specific and involves specific recognition of the virus by unidentified components on the membranes of gut and salivary tissues. Transport through the tissues is an endocytosis/exocytosis process. Both structural proteins of the virus are involved in the transmission process, with multiple protein domains regulating the movement and survival of the virus in the aphid and plant. Here we review what is known about the genetic, cellular, and molecular mechanisms regulating these complex and specific virus-aphid interactions.
[22]
Li X Q, Hu W L, Li Y, et al.
Development of an RT-LAMP assay for the detection of maize yellow mosaic virus in maize
Journal of Virological Methods, 2022, 300:114384.DOI:10.1016/j.jviromet.2021.114384 URL [本文引用: 1]
[23]
Patton M F, Bak A, Sayre J M, et al.
A polerovirus, potato leafroll virus, alters plant-vector interactions using three viral proteins
Plant Cell and Environment, 2020, 43(2);387-399.DOI:10.1111/pce.v43.2 URL [本文引用: 1]
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