【作者】 Hesham Mohamed Ahmed Abouelnasr；

【导师】 韩成贵；

【作者基本信息】 中国农业大学 ， 植物病理学， 2014， 博士

【摘要】 中国是世界最大的马铃薯生产国,1980至2012年间,马铃薯已经成为中国第四大粮食作物,产量增加显著,每公顷从9.7增加到15.2t。而美国、德国和埃及的平均单产分别为34,31和21t/ha,与他们相比相比,我国马铃薯单产还是比较低。马铃薯病害被认为是限制我国马铃薯产量的一个主要因素,由马铃薯病毒病引起马铃薯退化,造成损失可达15%至90%。其中,侵染马铃薯的香石竹潜隐病毒属病毒(carlaviruses)中PVS和新发现的马铃薯H病毒(Potato virus H, PVH)发生频率很高,已知PVS与其他病毒复合侵染能导致更严重的症状和产量损失。多数研究人员主要关注于实用检测方法和防控技术的研究,而有关PVS不同株系致病性,PVH的寄主范围和全长侵染性cDNA克隆构建等研究工作尚未见报道。因此,本论文开展并取得了以下四方面的研究结果。本论文检测并统计了2010-2014年间马铃薯病毒病在中国部分省份的发生和分布情况。从不同省份的马铃薯田块采集了具有病毒病症状的马铃薯植株,通过RT-PCR和Western杂交方法对这些样品中可能含有的7种病毒进行检测。检测结果表明这些马铃薯样品的带毒率为51.7%。检出率最高的三种病毒为：马铃薯X病毒,马铃薯S病毒和马铃薯H病毒,分别是32.7,36.6和12.1%。马铃薯Y病毒,马铃薯卷叶病毒,马铃薯A病毒和马铃薯M病毒检出率分别为7.9,6.8,3.0和1.8%。此外,还发现PVS发生分布广泛,其在单独侵染,双侵染和复合侵染的马铃薯样品中检出率分别为7.5,20.4和7.2%。根据PVS在藜科植物(Chenopodium spp.)上的症状反应,PVS被分为两个株系：不能系统侵染藜的株系(Ordinary:PVSO)和能系统侵染藜的株系(Andean:PVSA或PVSCS)本论文的研究中,通过鉴别寄主反应,RT-PCR以及Western blotting检测,从采集到的马铃薯样品中发现三个PVS分离物：PVSo云南分离物(PVSO-YN), PVSCS山东分离物(PVSCS-SD)和PVSA黑龙江分离物(PVSCS-HL). PVSCS-HL与PvSCS-SD能够局部和系统侵染藜,而PVSO-YN只能局部侵染藜。这些分离物中只有PVSCS-SD能系统侵染番茄。随后,分别获得了PVSO-YN分离物的基因组全长序列和PVSCS-HL分离物从TGB1(ORF3)至3’poly A末端的序列(共2573nts)。除去3’poly (A)尾巴,PVSO-YN基因组全长为8488nt,含有6个重叠的开放阅读框(ORF)。PVSO-YN与3个PVSI株系分离物在所有的ORFs中核苷酸水平的同源性为93-97%；而和另外两个pygCS株系分离物相比,除ORF2以外(95-97%)一致性均较低(77-90%)。此外,PVSCS-HL的部分序列与PVSO-YN分离物在核苷酸和氨基酸水平的同源性为83-97%和88-97%。基于核苷酸和氨基酸序列的进化树分析显示了类似的结果,PVSO-YN与3个Ordinary株系分离物聚为一支(亲缘关系较近),而PVSCCS-HL分离物归属于Andean分离物,但形成单独的一支。马铃薯H病毒(Potato virus H, PVH)是Betaflexiviridae科Carlavirus属的新成员,在马铃薯上被首次检测到,其广泛分布于云南,广西,河北,辽宁,黑龙江,新疆省和内蒙古自治区。本论文中,通过2条或者3条对应于基因组的cDNA片段亚克隆和拼接的方法成功构建了基于pCass-4Rz载体由35S启动子驱动的PVH的全长侵染性cDNA克隆(pCa-PVH-3和pCa-PVH-2)a农杆菌浸润接种后,侵染性克隆在本生烟和马铃薯植株上的侵染性通过RT-PCR检测得到验证。PVH的侵染性cDNA克隆的构建,为今后利用反向遗传学手段研究病毒基因的表达和功能、病毒复制和病毒-寄主-介体互作等提供了强有力的工具。此外,本论文在中国首次报道了马铃薯H病毒能侵染人参果(Solanum muricatum)。在一次关于PVH和PVM的血清学实验中,偶然从来自北京无症的组培人参果苗中检测到了PVH。为确认该结果,于2012-2013年间,在甘肃、云南和广西省进行了采样调查。随机采集无明显症状的人参果果实和叶片,通过RT-PCR结合测序和血清学的方法进行检测。从果实和叶片中提取RNA,并利用引物对PVHCPF/PVHCPR进行RT-PCR检测,同时利用PVH CP的特异性抗体对样品进行Western blotting检测。在50棵随机取样的样品中,有9棵的果实和叶片为PVH阳性。随后,利用特异引物扩增得到相应的RT-PCR产物(2.6kb,包含编码三联基因区、外壳蛋白和富半胱氨酸蛋白的基因序列),克隆到pMD19-T载体上并进行序列测定(即PVH-Pepino, GenBank登录号KF546312)。序列比对表明PVH-Pepino与已报道的马铃薯分离物PVH-Ho和PVH-YN的核苷酸同源性为91-98%。PVH-Pepino与PVH-Ho和PVH-YN的CP同源性分别为98%和99%,但与其它已报道的carlaviruses的CP同源性只有57-67%。因此,本论文首次发现PVH能无症地侵染人参果。就目前所知,这是S. muricatum作为PVH自然寄主的首次报道。我们的结果表明,Carlavirus属的新种PVH可能拥有比最初预想还要广的寄主谱。马铃薯和人参果都是在中国广泛种植的作物,但PVH在人参果上表现为无症(无症携带),在马铃薯病株上仅有弱症状的特性使得PVH的检测和防控变得更加困难。更多还原

【Abstract】 China is the first largest potato producer in the world. Between1980and2012, potato ranks fourth among the most important food crops in China, potato yields have a significant increase from9.7to15.2t/ha. However, potato yields in China are low relative to world levels, comparing with USA, Germany and Egypt average34,31and21t/ha, respectively. And potato viral diseases were considered as a major factor for potato production, for viruses infections have led to potato degeneration with yield reductions of from15%to90%. Regarding to potato carlaviruses, the incidence of PVS and PVH infecting potatoes is very high in China, as we know; mixed infections between PVS with other viruses can dramatically increase the severity of symptoms which further reduce potato yields. Most researchers only focus on development of appropriate diagnostic and control measures. However, studies on characterization of PVS strains, new host and construction of PVH infectious cDNA clone have not been reported.During2010-2014, incidence and distribution of potato viruses in some provinces of China was conducted. Virus disease-like symptoms in potato plants were sampled from different provinces of potato fields and were detected for seven viruses by RT-PCR and western blotting assays. Investigation of the incidence of the viral infections counts for51.7%in potato plants. Potato virus X, S and H are the most prevalent potato viruses sharing32.4,36.6and12.1%, respectively. Potato virus Y, Potato leaf roll virus, Potato virus A and Potato virus M were found in7.9,6.8,3.0and1.8%, respectively. In addition, PVS was widespread during survey in single, double and mixed infections sharing7.5,20.4and7.2%, respectively.Potato virus S (PVS, Carlavirus) has been divided into two strains:Chenopodium non-systemic (Ordinary:PVSo) and Chenopodium systemic (Andean:PVSA or PVSCS) according to the reaction on Chenopodium spp. In my study, we showed that there are three PVS isolates:PVS ordinary isolate collected from Yunnan (PVSo-YN), and two Andean isolates collected from Shandong (PVSCS-SD) and from Heilongjiang (PVSCS-HL) were identified by host reactions, RT-PCR and western blotting. In Chenopodium spp. phenotype screening of the Shandong (PVSCS-SD) and Heilongjiang (PVSCS-HL) isolates revealed localized infections followed by systemic infections, but the Yunnan isolate (PVSo-YN) only exhibited with localized infection. Of these isolates, only PVSCS-SD infected tomato plants systemically. Subsequently, complete genome sequence of PVSo-YN isolate and partial sequences of PVSCS-HL covering TGB1(ORF3) to the3’ poly A terminus (2573nts) were determined. The PVSo-YN genome contains six overlapping ORFs and consists of8,488nucleotides (nt), excluding the3’poly (A) tail. PVSo-YN shares nt sequence identities of93-97%in all the ORFs of three PVSo strain isolates, whereas this isolate has lower (77-90%) identities with the two PVScsstrains, except for ORF2where they share95-97%identity. On the other hand, PVSCS-HL partial sequence shared nt and aa sequence identities of83-97%and88-97%with PVSo-YN strain. Amino acid alignments showed that PVSCS/O strains with different positions could be characterized the relationship between systemic and localized infections on Chenopodium. Phylogenetic tree analyses based on nucleotide and amino acid sequences also showed that PVSO-YN clusters with the three Ordinary strain isolates, and that the PVSCS-HL isolate form a separated cluster and also belongs to Andean isolates.Potato virus H(PVH), a proposed new member in the genus Carlavirus in the family Betaflexiviridae, was first discovered in potatoes and occurred widely in Yunnan, Guangxi, Inner Mongolia, Hebei, Liaoning, Heilongjiang and Xinjiang. Herein, the full length infectious cDNA clones of PVH (pCa-PVH-3and pCa-PVH-2) have been generated successfully by sub-cloning three or two corresponding genomic cDNA fragments into pCass-Rz vector containing double35S promoter. The clones can be inoculated to N.benthamiana and Potato plants by agro-infiltration inoculation; the infectivity was confirmed by RT-PCR detection. The PVH infectious clone will be an important tool in the study of PVH by using reverse genetic approach, such as viral gene expression and their functions, virus replication and virus-host or vector interactions in the future.In addition, Potato virus H infecting pepino (Solanum muricaturn) was identified for the first time in China. During a study of serological relationships between PVH and PVM on potatoes, potato virus H (PVH) was detected serendipitously in symptomless pepino plantlets in Beijing, grown from tissue culture stocks. To confirm the presence of PVH on S. muricatum, surveys were conducted in2012and2013in Gansu, Yunnan and Guangxi provinces and Beijing. Fruits and leaves were collected randomly from pepino plants displaying no obvious symptoms. For PVH detection, a combination of RT-PCR, genome sequencing and serological assays were used. RNAs extracted from fruits and leaves were amplified using RT-PCR with primer pairs PVHCPF and PVHCPR, and extracted samples were reacted by Western blotting with the specific polyclonal antiserum against PVH. Among the fifty plants randomly collected, fruits and leaves of nine plants tested positive for PVH. Subsequently, an RT-PCR product of the expected size (2.6kb) encompassing the triple-gene block, the capsid protein gene, and the cysteine-rich protein gene, was amplified with a specific primer pair, and then cloned into pMD19-T and sequenced(PVH-Pepino with GenBank Accession no. KF546312). Further sequence comparison showed that PVH-Pepin shared91-98%nucleotide sequence identity in the genes mentioned above with those of the reported potato isolates PVH-Ho and PVH-YN.PVH-Pepino shared deduced amino acid identity of98-99%in CP gene with PVH-Ho and PVH-YN, respectively, but only shared57-67%amino acid identities with other reported carlaviruses. Thus, latent infection of PVH on S. muricaturm was confirmed. To our knowledge, this is the first report of S. muricaturm as a natural host of PVH. Our results suggest that PVH, as a new member of the genus Carlavirus, has a wider host range than originally expected. Potatoes and pepino are crops widely grown in China. The fact that no symptoms were expressed by PVH in pepino plants (symptomless carrier) and only mild symptoms expressed by PVH in diseased potatoes makes detection and remediation of this disease more difficult.更多还原