Evaluation of Maize Streak Virus Pathogenicity in Differentially Resistant Zea mays Genotypes

ABSTRACT We devised a rapid technique for the objective and precise assessment of both the pathogenicity of maize streak virus (MSV) isolates and the MSV resistance of maize genotypes. The technique involves the use of agroinoculation to infect maize seedlings and the objective symptom evaluation by quantification of infection rates, stunting, and chlorotic leaf areas. In assessing the MSV resistance of 19 maize genotypes, we describe how the use of differentially virulent virus isolates enables the analysis of MSV resistance phenotypes, ranging from extremely susceptible to completely immune. We further demonstrate how quantification of chlorotic leaf areas by image analysis permits differentiation between degrees of MSV resistance that are indistinguishable from one another using currently employed symptom assessment approaches. Using chlorotic area measurements, we quantify the virulence of a diverse group of 10 MSV isolates and, through agroinoculation of differentially susceptible maize genotypes, we demonstrate the use of our technique in evaluating the pathogenicity of these isolates.     

Analyses of seven new whole genome sequences of cassava brown streak viruses in Mozambique reveals two distinct clades: evidence for new species

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) is a major constraint to cassava production in Mozambique. Full genome sequences of CBSD-associated virus isolates contribute to the understanding of genetic diversity and the development of new diagnostic primers that can be used for early detection of the viruses for sustainable disease management. This study determined seven new whole CBSV genomes from total RNA isolated from cassava leaves with CBSD symptoms collected from Nampula and Zambezia in Mozambique. Phylogenetic analyses of the new genomes with published CBSV and UCBSV sequences in GenBank grouped the CBSV isolates from Mozambique into two distinct clades together with CBSV isolates from Tanzania. Clade 1 and 2 isolates shared low nucleotide (79.1–80.4%) and amino acid (86.5–88.2%) sequence identity. Further, comparisons within the seven new CBSV isolates, and between them and the single published complete CBSV sequence (CBSV_MO_83_FN434436) from Mozambique, revealed nucleotide sequence identities of 79.3–100% and 79.3–98%, respectively, and amino acid identities of 86.7–100% and 86.7–98.8%. In addition, using RDP4, a recombination analysis comprising all CBSV and UCBSV genome sequences from GenBank detect 11 recombination events. Using several comprehensive evolutionary models and statistical programs, it was confirmed that CBSV and UCBSV are distinct virus species, with an additional probable new species (clade 2).     

Viral inclusions in monocotyledons infected by maize streak and related geminiviruses

Isolates of maize streak virus (MSV) were examined by thin-section electron microscopy in plants, assessed for characteristic features of infection and compared with other related geminiviruses infecting monocotyledons from Africa, islands in the Indian Ocean, and the Pacific Island of Vanuatu. Arrays of virus particles, often crystalline, were most often seen in the nucleus. The morphology of the nuclear crystalline arrays was characteristic of certain isolates or groups of isolates (strains). Infected nuclei could be seen in cells from the phloem parenchyma, vascular bundle sheath and mesophyll tissue, and also in epidermal guard cells of plants infected with the maize strain of MSV. The particle arrays varied in morphology from regular rows of virions forming distinctive blocks, to randomly arranged aggregates in certain areas of the nucleus. We consistently failed to find viral crystalline arrays associated with infection of panicum streak virus (PSV) and sugar cane streak virus (SSV) isolates either in these hosts or in maize. Occasionally arrays of MSV particles were found outside the nuclear envelope in physiologically active cells. Accumulations or sheets of MSV particles were seen lining the walls of some phloem companion cells. Crystalline aggregates of particles were frequently observed in the cell vacuole, after lysis of the nuclear membrane of dead cells which made up the chlorotic lesions, the typical symptom of virus infection. Virus preparations from all hosts contained typical geminate particles regardless of the morphology of the virion arrays. The effect on chloroplasts appeared to vary between isolates and this is discussed in relation to lesion colour. The arrangement of virions in the nucleus as a taxonomic character is diagnostic for MSV. Inclusions with crystalline structure found in sieve elements of infected plants were not immunogold labelled when thin sections were probed using antiserum to the virus particles.     

Molecular comparisons amongst wheat bymovirus isolates from Asia, North America and Europe

To study the variation between wheat bymovirus isolates and to resolve uncertainties about the identity of the virus in some countries, leaves of infected plants were obtained from nine sites in China and from one each in Italy, Germany, USA and Canada. The German isolate was obtained from rye and the Canadian isolate was the type strain of wheat spindle streak mosaic virus (WSSMV). In RT-PCR, using primers designed from a partial sequence of a French isolate (tentatively described as WSSMV), genome fragments were obtained from the Italian and the French isolates but not from the Chinese ones. Conversely, products were consistently obtained from the Chinese isolates, but not from the Italian or French ones, when primers were designed from the sequence of a Japanese isolate of wheat yellow mosaic virus (WYMV). Nucleotide sequences were obtained from regions at or near the 3'-terminus of RNA1 of six Chinese isolates and the four from Europe and North America, usually including the coat protein. Nucleotide and amino acid sequence comparisons demonstrated that the European and North American isolates were extremely similar and were therefore WSSMV, while the Chinese isolates were close to the Japanese isolate and were thus WYMV.     

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.     

利用小RNA高通量测序技术检测玉米病毒

正玉米是我国重要的粮食作物,种植范围日趋增大,病害的发生对玉米造成极大为害,病毒病对玉米稳产高产已构成严重威胁。近年来,安徽、山东和辽宁玉米主要种植区病毒病危害较重。为了检测发病玉米的病毒种类,本研究利用小RNA高通量测序技术鉴定玉米病毒,明确种类,以期为制定抗病毒策略提供理论依据。据不完全统计,世界上有40多种玉米病毒病(http://en.wikipedia.org),在我国发生并报道的有5种,分别为玉米粗缩病、玉米矮花叶病、玉米条纹矮缩病、玉米红叶病和玉     

Genetic Diversity Among Banana streak virus Isolates from Australia

ABSTRACT Banana streak virus (BSV) is an important pathogen of bananas and plantains (Musa spp.) throughout the world. We have cloned and sequenced part of the genomes of four isolates of BSV from Australia, designated BSV-RD, BSV-Cav, BSV-Mys, and BSV-GF. These isolates originated from banana cvs. Red Dacca, Williams, Mysore, and Goldfinger, respectively. All clones contained a sequence covering part of open reading frame III and the intergenic region of the badnavirus genome. The sequences were compared with those of other badnaviruses, including BSV-Onne, a previously characterized isolate from Nigeria. The BSV-RD sequence was virtually identical to that of BSV-Onne, differing by only two nucleotides over 1,292 bp. However, BSV-Cav, -Mys, and -GF were divergent in nucleotide sequence. Phylogenetic analyses using conserved sequences in the ribonuclease H domain revealed that all BSV isolates were more closely related to each other than to any other badnavirus. BSV-Cav was most closely related to BSV-Onne, and there was 95.1% identity between the two amino acid sequences. Other relationships between the BSV isolates were less similar, with sequence identities ranging from 66.4 to 78.2%, which is a magnitude comparable to the distance between some of the recognized badnavirus species. Immunocapture-polymerase chain reaction assays have been developed, allowing specific detection and differentiation of the four isolates of BSV.     

玉米小斑病菌中一种维多利亚病毒的分离及其基因组全序列分析

 从玉米小斑病菌(Bipolaris maydis)中检测发现一种dsRNA (double-stranded RNA)病毒,暂命名为Bipolaris maydis victorivirus 2 (BmV2)。电子显微镜下观察到病毒粒子为二十面体球状,直径为40 nm左右、无包膜;病毒基因组为单条dsRNA核酸分子,长度为5 222 bp,其基因组结构与其他维多利亚病毒相似,包含两个开放阅读框(ORFs)。序列BLASTx分析发现,BmV2的基因组核酸序列与Coniothyrium minitans RNA virus Illinois isolate (CmRV-IL)具有较高的同源性(78.15%),后者CmRV-IL是单分体病毒科(Totiviridae)维多利亚病毒属(Victorivirus)的暂定种,两者的外壳蛋白(CP)和RNA依赖性RNA聚合酶(RdRp)的氨基酸序列之间的同源性分别为88.02%和89.87%,说明BmV2、CmRV-IL是同一种病毒的不同分离物。基于BmV2和选择的单分体病毒的RdRp氨基酸序列的系统发育分析表明,BmV2与单分体病毒科维多利亚病毒属中的病毒形成了一个单系进化支。     

Characterization of maize streak virus: description of strains; symptoms

Twenty-four isolates of maize streak virus (MSV) derived from maize, sugarcane and grasses were compared to a maize isolate of the virus (M(N)M) from Nigeria, using symptoms, gel diffusion and ELISA. Fourteen isolates were identified as maize strains, eight other isolates were serologically related to M(N)M but were distinct. In most cases the maize strain could be identified by the symptoms in Zea mays cv. Golden Bantam but symptom expression in grasses was not always sufficient to identify the economically important maize strain. In general, however, symptoms were similar in both grass and maize hosts. Identification by symptoms alone was further complicated by the possibility that some isolates were mixtures. There was no evidence that adaptation to grass hosts occurred, as all isolates could be transmitted to maize. It was not possible to transmit certain strains to the host species from which they were derived, even though they were transmissible to other hosts. This was assumed to be related to vector feeding behaviour. Insect toxin was responsible for certain stunting symptoms, leaf curling and vein enations often associated with MSV.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

小麦黄色花叶病毒不同分离物外壳蛋白(CP)基因序列的比较分析

 核苷酸序列分析结果表明,小麦黄色花叶病毒(W YMV)不同分离物的外壳蛋白基因存在一定的差异。邓州分离物CP基因在其31~33nt处均缺失了3个核苷酸,其余分离物与潢川分离物及日本分离物长度一致,均为882nt。不同分离物CP基因核苷酸序列同源性为97.3%~98.9%,由此推导的氨基酸序列同源性为97.6%~99.3%,外壳蛋白N末端的110个氨基酸和C末端的55个氨基酸在各个分离物间是高度保守的。潢川分离物有5个氨基酸与其它5个分离物明显不同。WYMV不同分离物外壳蛋白序列分析结果进一步确认了WYMV与WSSMV为Bymovirus属的2种不同病毒。     

Malvastrum leaf curl Guangdong virus is a distinct monopartite begomovirus

Virus isolates GD6, GD7, GD8, GD9 and GD10 were obtained from Malvastrum coromandelianum showing leaf curl symptoms in Guangdong Province of China. A specific 500 bp product was consistently detected in total DNA extracts, amplified with universal primers specific for members of the genus Begomovirus. Analysis of their partial DNA sequences revealed that they are isolates of the same begomovirus species, sharing 92·8%–97·1% nucleotide sequence identity. The complete DNA sequences of both GD6 and GD9 were found to be 2767 nucleotides, with all the characteristic features of begomovirus genome organization. The two isolates have less than 85·2% nucleotide sequence identity with other reported begomoviruses. Consequently, GD6 and GD9 are considered to be isolates of a novel begomovirus species, for which the name Malvastrum leaf curl Guangdong virus (MLCuGdV) is proposed. Sequence analyses suggest that MLCuGdV may have arisen by recombination between viruses related to Papaya leaf curl China virus , Tomato leaf curl Philippines virus and other undiscovered virus ancestors. Neither the DNA-B component nor the DNAβ molecule associated with these begomovirus isolates was found. An infectious clone of GD6 was constructed. GD6 efficiently infected Nicotiana benthamiana , N. glutinosa and Petunia hybrida by agro-inoculation, and Malvastrum coromandelianum by whitefly transmission, inducing leaf curling, vein swelling and stunting symptoms. GD6 was also infectious in N. tabacum , but did not induce observable disease symptoms.     

苹果茎沟病毒柑橘碎叶株系的分布与序列分析

为明确苹果茎沟病毒(apple stem grooving virus,ASGV)柑橘碎叶株系(citrus tatter leaf virus,CTLV)在中国柑橘产区的发生分布及其分子特性,于2017—2021年对我国12个柑橘主产区开展系统调查,并采用多重比对以及系统发育分析法对CTLV的全序列进行分析。结果表明,从12个柑橘主产区采集的2 012份疑似样品中检测出413份感CTLV阳性样品。除陕西省外,其余11个柑橘主产区样品中均检测出CTLV,并且柚类样品中CTLV的检出率最高,达到32.31%。对分离获得的22株CTLV毒株和已知的7株CTLV以及5株ASGV毒株进行全序列分析,发现所有34株毒株的核苷酸序列相似性为78.6%～99.8%,且CTLV序列的保守性较高。此外,与其他4株ASGV毒株相比,本研究中22株CTLV毒株与ASGV-MK毒株(GenBank登录号为MZ127820.1)具有更近的亲缘关系。推测CTLV中国毒株间的亲缘关系可能与其采样地和寄主品种有关。     

Asymmetrical Distribution of Barley Yellow Dwarf Virus PAV Variants Between Host Plant Species

ABSTRACT A large epidemiological study of the genetic variation of barley yellow dwarf virus (BYDV) serotype PAV involving different host plant species was conducted. French BYDV PAV isolates were collected from barley and ryegrass, and their capsid protein gene sequences characterized using restriction fragment length polymorphism, single-strand conformation polymorphism, and sequence analyses. The data show that BYDV PAV isolates from five different continents are separated into two distinct groups named cpA and cpB, which are distributed irrespective of geographical location. Amino acid identity of the capsid proteins ranged from 93 to 99.5% in group cpA and from 95 to 99.5% in group cpB, while this value was only from 82 to 88% between the groups. Moreover, isolates from each group were found preferentially (up to 98%) in one of the two plant species examined. These results show that host plant species play a role in isolate selection and maintenance and that they contribute to the genetic diversity of BYDV PAV.     

甘蔗花叶病毒2个山东分离物的全基因组序列分析

甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)是引起我国玉米矮花叶病的主要病毒。本文从山东泰安采集到2个表现矮花叶症状玉米样品的分离物(命名为DWK1和DWK2),通过RT-PCR扩增全基因组片段并测定了其序列(GenBank登录号分别为KU171814和KU171815)。序列分析结果表明,DWK1和DWK2基因组全长分别为9 575和9 576个核苷酸(nucleotides,nt),开放阅读框均为9 192 nt,编码3 063个氨基酸的多聚蛋白。DWK1和DWK2的全基因组核苷酸一致率为81.7%,DWK1与山西分离物SX(AY569692)的核苷酸一致率最高,为90.9%;DWK2与河北分离物BD8(JN021933)核苷酸一致率最高,达99.4%。二者在系统进化树中分别被聚类到Ⅰ组和Ⅳ组。重组分析发现,DWK1是HN(AF494510)、Guangdong(AJ310105)和BD8 3个分离物的重组体。选择压力分析表明,SCMV 11个蛋白的dN/dS值都小于1,均处于负选择,但在P1、P3和CP中存在正选择位点。本研究结果可为甘蔗花叶病毒株系的监测及防控提供理论指导。     

胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒

 本文报道胶体金免疫电镜技术的建立:铜网捕获病毒后,用其稀释约200倍的同源抗血清修饰处理3~5分钟,羊抗兔IgG-金复合物标记3~5分钟,经磷钨酸负染,电镜下可见病毒粒子周围金颗粒特异性吸附,而背景上非特异性吸附很少。用此技术成功地快速检测和鉴定了病汁液中线状病毒(大麦黄花叶病毒,大麦温和花叶病毒,小麦梭条斑花叶病毒,芜菁花叶病连和马铃薯Y病毒)、棒状病毒(烟草花叶病毒、长叶车前草花叶病毒和土传小麦花叶病毒)以及球状病毒(水稻矮缩病毒、黄瓜花叶病毒和大麦黄矮病毒)。用0.5M PBSpH7.0制备病汁液,可大幅度提高同源抗血清对黄瓜花叶病毒的捕获能力。