Novel Naturally Occurring Bean pod mottle virus Reassortants with Mixed Heterologous RNA1 Genomes

ABSTRACT The comovirus Bean pod mottle virus (BPMV) is widespread in the soybean-growing regions in the United States. It has a bipartite genome consisting of RNA1 and RNA2, which are encapsidated separately. We previously have reported the occurrence in nature of two distinct subgroups of BPMV strains (subgroups I and II), as well as reassortants between the two subgroups. Here, we report the isolation and molecular characterization of naturally occurring partial diploid reassortant strains, which are diploid for RNA1 and haploid for RNA2. Whereas the RNA1s of the partial diploids are derived from two distinct strain subgroups (I and II), the RNA2 is derived from either subgroup I or II. The partial diploid strains induced strikingly severe symptoms on soybean, which could be explained based on the presence of two distinct RNA1s in the same plant. This conclusion was supported by the finding that pseudo-recombinants constructed with two diverse RNA1s induced very severe symptoms on soybean that mimicked those produced by the naturally occurring partial diploids. No enhancement of symptom severity was observed with pseudorecombinants constructed with closely related RNA1s. Likewise, no enhancement of symptom severity was noted with pseudo-recombinants that are diploid for RNA2 and haploid for RNA1. The potential role of genetic reassortment in the epidemiology and pathogenesis of BPMV is discussed.     

几种重要花生病毒研究新进展

 花生病毒病是影响花生生产的重要病害。近10年来,花生矮化病毒(PSV)、花生条纹病毒(PStV)和番茄斑萎病毒属(Tospovirus)病毒分子生物学研究进展,极大地丰富了对上述病毒基因组结构、遗传变异、进化的认识,以及病毒种、亚组和株系的科学划分。对PSV来说,提出了2个亚组的划分,而我国PSV株系血清学和RNA3全序列的分析,明确它们独自构成第3个新的亚组。对我国和东南亚国家PStV株系CP基因序列同源性的分析,说明在这些国家和地区PStV是单独进化的,形成不同症状类型的株系。Tospovirus属病毒分子生物学研究的深入使得该属病毒从番茄斑萎病毒(TSWV)1种增加到13种,其中侵染花生的病毒达5种,分布于不同的国家和地区。     

我国部分地区常见农作物上黄瓜花叶病毒分离物核酸多样性分析

为明确我国黄瓜花叶病毒株系分化及系统进化基本情况,从湖南、新疆、青海和海南4省区采集1 367个样品对其进行酶联免疫和RT-PCR检测,并对分离获得的15个黄瓜花叶病毒(Cucumber mosaic virus,CMV)纯化分离物CP、MP、2b核苷酸序列进行相似性和进化树分析及生物学性状比较。结果表明,辣椒、龙葵和黄瓜的CMV阳性检出率较高,分别为54.13%、29.19%和18.46%。进化树分析显示CMV-Q5与CMV亚组II的亲缘性较高;CMV-N7为新发现的重组株系,其CP、2b基因属于CMV亚组IB,MP基因却属于CMV亚组II;其余13个分离物均属于CMV亚组IB。CMV-N7和CMV-Q5在系统寄主心叶烟和枯斑寄主苋色藜上引发的症状相似,但比对照株系CMV-P3613(IB)的发病时间要晚1~2 d,系统花叶较温和,枯斑较小。表明在以上4省区常见农作物上广泛流行的CMV存在分子变异。     

Serological and biological variation between and within subgroup I and II strains of cucumber mosaic virus

Fourteen strains of cucumber mosaic virus (CMV) from Australia have been characterized by their host range and symptomatology. They were classified as subgroup I or II strains by a dot-blot molecular hybridization assay between their total viral RNAs and selected cDNAs. The strains F_NY and L_Ny, both from the USA, were used as the subgroup I- and subgroup II-type strains, respectively. A range of serological tests was used to compare these isolates. Gel immunodiffusion tests, with standard antigens homologous to the antisera prepared against glutaraldehyde-fixed virus of 11 strains, showed that they could be divided into three serogroups on the basis of spur formation in heterologous reactions. Two of the serogroups included either subgroup I or subgroup II isolates, whereas the third serogroup consisted of only one strain (Y_WA) which was homologous to all the strains tested. Use of heterologous standard antigens in this test failed to show further subgrouping of the antigens. Double-antibody sandwich (DAS) ELISA using polyclonal antibodies to distinct virus strains also placed the 14 strains in the same three serogroups. When eight different monoclonal antibodies (MAbs) were used in indirect ELISA, one of them distinguished subgroup-I strains and another distinguished subgroup-II strains; the Y_WA strain fell into subgroup II. Other MAbs showed narrower or broader specificity. Thus both molecular hybridization with total RNA and specific MAbs may be useful for separating isolates of CMV into subgroups I and II. Spur formation using heterologous standard antigens to the antisera, as well as being more difficult to interpret, was not a reliable criterion for classification.     

百合斑驳病毒云南分离物 全基因组序列分析及CP结构预测

 对云南嵩明百合上发生的百合斑驳病毒(Lily mottle virus, LMoV)进行全基因组序列测定及分析,并对LMoV嵩明分离物(LMoV-SMi1、LMoV-SMi2)和玉溪分离物(LMoV-YXi1、LMoV-YXi2)外壳蛋白(coat protein,CP)基因进行序列比较,发现云南的LMoV分为2个类群,玉溪分离物属于种群I,嵩明分离物属于种群II。2个类群间的核苷酸和氨基酸同源性分别为86.7%~89.5%、90.1%~92.7%,玉溪分离物和嵩明分离物相比,cp基因发生了3个核苷酸的缺失。对国内外LMoV所有分离物的cp基因氨基酸序列进行系统进化分析,结果表明所有LMoV分离物可划分为2个种群,种群I分离物较种群II分离物几乎均存在1个苏氨酸缺失的差异。此外,对LMoV-SMi2的CP相关特性和空间结构进行了初步预测,认为该蛋白为球状,具有较强的表面可能性,不存在跨膜区域,大多数区域能够形成主要的抗原决定簇,主要集中在aa12-22、aa31-42、aa83-99、aa179-191、aa215-223、aa249-259区段,可作为制备抗血清选择抗原的参考。LMoV-SMi2和LMoV-YXi1在二级结构和三级结构上存在一定的差异,但总体空间结构差异不大。     

Identification and characterization of peanut stunt cucumovirus from naturally infected alfalfa in Iran

A mechanically transmissible virus was isolated from a naturally infected alfalfa plant ( Medicago sativa ) in Karaj, Iran. It induced fern-leaf symptoms in Lycopersicon esculentum , general chlorosis and stunting in Arachis hypogoea , local lesions and systemic infection in Chenopodium quinoa , Phaseolus vulgaris and Vigna unguiculata but only local lesions in C. amaranticolor . In gel-immunodiffusion tests, it reacted strongly with an antiserum to peanut stunt cucumovirus (PSV), moderately with two of six antisera to cucumber mosaic cucumovirus (CMV) and with two of five antisera to tomato aspermy cucumovirus. Its spherical virions (28 nm in diameter) contained a coat protein of approximately 29 kDa and encapsidated four species of RNAs with similar electrophoretic mobilities to RNAs 1–4 of CMV strains Fny and LS and those of PSV strains J and W. Its encapsidated RNAs hybridized in slot-blot hybridization assay with the complementary DNA probe of PSV-W RNAs but not with those of CMV strains. Therefore, on the basis of biological, serological and physico-biochemical properties, the virus was identified as PSV. No satellite RNA was associated with the virus. This is the first report of PSV in Iran.     

Characterisation of black locust isolates of <Emphasis Type="Italic">Peanut stunt virus</Emphasis> (PSV) from the Pannon ecoregion show the frequent occurrence of the fourth taxonomic PSV subgroup

Biological and molecular characterisation of ten Peanut stunt virus (PSV) isolates from Robinia pseudoacacia was carried out. The host range of these isolates was similar to that of the previously described PSV strains in most cases, but on Pisum sativum and Lens culinaris latent infection was induced. Variability in systemic symptoms was observed only on Nicotiana glutinosa. The partial RNA3 sequences were determined, including the carboxyl terminal region of the movement protein gene, the intergenic region, the entire coat protein gene and the 3′ untranslated region. Nucleotide sequence comparison of the coat protein genes showed 77.6–84.2% identity with most of the known PSV strains and 96.3–98.0% identity with PSV-Rp the typical member of subgroup IV. Phylogenetic analysis indicated the presence of the ancient homologous recombination in all of the examined black locust isolates and all the isolates were members of the fourth PSV subgroup. These results showed that the isolates of the fourth subgroup are widely distributed in black locust in this region.     

Causal agent of sharka disease: new and emerging events associated with Plum pox virus characterization

Plant RNA viruses have a high genetic variation potential due to the absence of proofreading activity in their RNA replicase. In addition to mutation, recombination is generally thought to be an important source of variability. Both evolutionary processes have contributed to the diversity of Plum pox virus (PPV). There are now six recognized subgroups, strains or serotypes of PPV (D, M, Rec, EA, C and W). Isolates belonging to the PPV-Rec subgroup are derived from RNA recombination between PPV-D and PPV-M and occur frequently in various central and eastern European countries. The divergent isolate W3174 is a new and distinct strain of PPV, identified as PPV-W. It is quite conceivable that, with time, other groups will be defined and that the present classification will need revision to accommodate additional PPV variability.     

Biological and molecular characterization of a new cucurbit-infecting tobamovirus

ABSTRACT An uncharacterized virus was isolated from greenhouse-grown cucumber plants. Biological and serological data described in the present study indicated that the virus belonged in the genus Tobamovirus. The host range of the virus included several plant species within the family Cucurbitaceae. The virus designated Cucumber fruit mottle mosaic virus (CFMMV) causes severe mottling or mosaic on cucumber fruits, and its fast spread within greenhouses could lead to significant economic losses in cucumber crops. The genome of CFMMV has been completely sequenced and its genome organization was typical of a Tobamovirus. However, its sequence was distinct from other described viruses within the group of cucurbit-infecting Tobamoviruses. Comparisons of sequences and phylogenetic analysis suggested that the cucurbit-infecting Tobamoviruses be separated into two subgroups: subgroup I comprising the strains and isolates referred to in the literature as Cucumber green mottle mosaic virus (CGMMV) (CV3, CV4, CGMMV-W, CGMMV-SH, and CGMMV-Is) and subgroup II comprising CFMMV, Kyuri green mottle mosaic virus (KGMMV), and the Yodo strain of CGMMV, which is closely related to KGMMV and may be considered a strain of it.     

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.     

A varied population of cucumber mosaic virus from peppers

A varied population of Cucumber Mosaic Virus (CMV) isolates is reported from peppers ( Capsicum annuum ) in Australia. Isolates representing both CMV subgroups (serogroups) I and II have been obtained from the same field at the same sampling time. The CMV isolates were typed into subgroup I (eight isolates) and subgroup II (two isolates) using both a nucleic acid hybridization assay and an immunological assay with monoclonal antibodies. The immunological assay described allows the typing of strains in crude sap extracts, obviating the need for purified virions. The spatial and temporal coincidence of both CMV subgroups presents a situation in which pseudorecombinants with reassorted genomic components might arise.     

The Complete Nucleotide Sequence of Potato virus X Strain OS: The First Complete Sequence of a Japanese Isolate

The complete nucleotide sequence of the genomic RNA of a Japanese isolate of Potato virus X (PVX), strain OS (PVX-OS), was determined. The PVX-OS genome is 6435 nucleotides in length, excluding the 3′ poly(A) sequence, and encodes for five open reading frames (ORFs), similar to other reported PVX strains. Comparisons of the nucleotide sequence of PVX-OS with those of European and South American PVX strains revealed that the PVX-OS genomic sequence showed 95-96% homology with European strains and 77-78% with South American strains. Phylogenetic analysis of PVX-OS and other PVX strains found that PVX-OS clusters with European strains. This is the first report on the complete nucleotide sequence of a PVX strain from Japan. Received 10 May 2001/ Accepted in revised form 26 October 2001     

Identification and Subgrouping of Cucumber mosaic virus with Mouse Monoclonal Antibodies

ABSTRACT Using a mixture of isolates of Cucumber mosaic virus (CMV) from subgroups I and II as immunogens, 20 mouse hybridoma cell lines secreting monoclonal antibodies were produced. A reliable method for efficient detection and accurate subgrouping of CMV isolates has been developed. Tests with 12 well-characterized strains of CMV and other cucumoviruses demonstrated the presence of epitopes that were virus and subgroup specific. Analyses of 109 accessions of CMV isolates collected from various parts of the world revealed 70% were subgroup I, with 20% identified as subgroup II. Seven isolates (6%) did not react with group-specific antibodies but did react with antibodies that recognized all CMV isolates. Differential reactions among isolates suggested a total of 10 epi-topes were recognized. The antigenic diversity among subgroup II CMVs was greater than for the subgroup I isolates, even though fewer subgroup II isolates were tested.     

Genetic variation in Candidatus Phytoplasma australiense

This study examined whether genes that are less conserved than the 16S rRNA gene can distinguish Candidatus Phytoplasma australiense strains that are identical based on their 16S rRNA genes, with a view to providing insight into their origins and distribution, and any patterns of association with particular plant hosts. Sequence analysis of the tuf gene and rp operon showed that Ca . P. australiense strains could be differentiated into four subgroups, named 16SrXII-B ( tuf -Australia I; rp -A), 16SrXII-B ( tuf -New Zealand I; rp -B), 16SrXII-B ( tuf -New Zealand II) and 16SrXII-B ( rp -C). Strawberry lethal yellows 1, strawberry green petal, Australian grapevine yellows, pumpkin yellow leaf curl and cottonbush witches' broom phytoplasmas were designated members of the 16SrXII-B ( tuf -Australia I; rp -A) subgroup. The strawberry lethal yellows 2 and cottonbush reduced yellow leaves phytoplasmas were assigned to the 16SrXII ( tuf -New Zealand II; rp -B) subgroup. No relationship was observed between these phytoplasma subgroups and collection date, location or host plant. However, the study revealed evolutionary divergence in the 16SrXII group.