Alfalfa Mosaic Virus Strain Research Articles

Host-specific loss of sequences of an alfalfa mosaic virus isolate during systemic infection

Infectivity of an alfalfa mosaic virus (AMV) isolate from Leonotis nepetaefolia in different tomato cultivars was analyzed. Symptoms typical of AMV infection were observed in indicator plants, but not in Flora Dade and Rio Grande tomato cultivars; however, mild symptoms were observed in cv. Rutgers. Furthermore, at least 1 kb of the 3´ segment of RNA 2 and the coat protein gene were missing in systemic leaves of inoculated Rio Grande and Flora Dade plants, while in cv. Rutgers infected with this AMV strain all genomic components were detected. Northern blot analysis of plants infected with the aforementioned AMV isolate confirmed the absence of the CP gene, but suggested rearrangements in both RNA 2 and 3. Factors that may affect differential movement or systemic accumulation of genomic components in multipartite viruses in plants are discussed.

Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt.

Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.

DETECTION OF ALFALFA MOSAIC VIRUS IN LAURUSTINUS IN APULIA

Laurustinus (Viburnum tinus L.), an evergreen shrub of the Mediterranean scrub, is widely used in Italy as an ornamental in private and public gardens. In the spring of 2014, conspicuous interveinal yellow spots and mottling were observed on the foliage of a V. tinus plant cv. Eve Price growing in the Campus of the University of Bari (Italy). Since Alfalfa mosaic virus (AMV) has been reported from V. tinus and related species (V. opulus and V. lucidum) in several countries, and in northern Italy (De Sanctis and Quacquarelli, 1985; Parrella et al., 2011), the symptomatic plant was tested by RT-PCR for the presence of this virus. The specific primer set CPAMV1/CPAMV2, designed in the viral RNA3 sequence (Finetti-Sialer et al., 1997) amplified the expected 754 bp fragment, spanning the whole coat protein gene and the 3’-UTR. There was no amplification from symptomless plants. The amplified fragment was cloned into pSC-A-amp/kan, custom-sequenced (Macrogen Europe, The Netherlands) and the sequence was deposited in GenBank as accession No. KP233749. The cloned sequences were BLAST compared with those of a Chilean strain of AMV from V. tinus and a Spanish isolate from V. lucidum, with which an identity at the nucleotide level of 99% and 94%, respectively, was found. These results demonstrate the presence of an AMV strain in laurustinus in southern Italy.

OCCURRENCE OF ALFALFA MOSAIC VIRUS (AMV) DISEASES ON POTATO CROPS IN NORTHERN CYPRUS

Alfalfa mosaic virus (AMV) was for the first time detected in potato, peppers and alfalfa crops growing in North Cyprus. This virus was frequently found on the hosts nearby alfalfa plantation areas. The detection and characterization of AMV was made by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The collected potato (154), alfalfa (81) and pepper (63) samples were proceeded to test by DAS ELISA and RT-PCR. RT-PCR tests using primers, specific for the coat protein gene of AMV, resulted in an amplicon of the expected size (351 bp). The amplicons were then direct sequenced and deposited in GenBank (JN104033, JN104034). Direct sequencing analyses showed that potato, alfalfa and pepper AMV virus isolates were highly similar to each other. Sequence analysis (BioEdit v 7.0.5) and a comparison with other AMV isolates showed about 96% (nt) identities with FN667767 and HQ185596. Comparison of the isolates of AMV to each other and type of symptoms expression of viruses on three host plants revealed that potato, pepper and alfalfa plants were infected by the same AMV strains. The results of serological and molecular assays clearly suggested that alfalfa plants is an over wintering host and source of inoculums of AMV. The virus from the infection source (the alfalfa fields) was transmitted by the migration of aphids at the nearby potato and pepper fields. Latter on, AMV has been completed the life cycle on potato and pepper plants at the end of growing seasons.

Diagnostic Potential of Polyclonal Antibodies Against Bacterially Expressed Recombinant Coat Protein of Alfalfa mosaic virus.

Alfalfa mosaic virus (AMV), a pathogen of a wide range of plant species, including Glycine max (soybean), is poorly immunogenic. Polyclonal antibodies were generated against bacterially expressed recombinant coat proteins (rCPs) of two biologically distinct AMV strains in rabbits and compared with those raised against native and glutaraldehyde-treated virions of the same strains. Analyses showed that sera against rCPs had comparable antibody titers in indirect enzyme-linked immunosorbent assay with those raised against virions when soybean sap containing homologous viruses served as antigens. Polyclonal antibodies against rCPs were specific, sensitive, and detected all AMV isolates that originated from soybean fields from geographically different regions of the United States. Comparison of CP genes of these isolates showed 96 to 99 and 96 to 100% nucleotide and amino acid sequence identities, respectively, suggesting that they are all closely related. This was further confirmed by phylogenetic analysis where they were all clustered together along with representative AMV strains belonging to group I. Collectively, our data demonstrate that, despite poor immunogenicity of AMV, polyclonal antibodies against rCP are effective probes for detection and diagnosis of the virus.

Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

ABSTRACT Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

A Short Insert in the Leader Sequence of RNA 3L, A Long Variant of Alfalfa mosaic virus RNA3, Introduces Two Unidentified Reading Frames

N20-RNA 3L, a large form of RNA 3 associated with Alfalfa mosaic virus (AMV) strain N20 comprises 2281 nt and has approximately 97% overall sequence similarity to the longest previously described RNA 3 of AMV strain YSMV (YSMV-RNA 3; 2188 nt). Compared with YSMV-RNA 3, N20-RNA 3L contains an additional 97 nt in the 5' leader upstream of the open reading frames for movement protein (MP) and coat protein (CP). Two overlapping unidentified reading frames (URF1 and URF2) result from this modification, each of which code for putative translation products of 21 amino acids. The URF1 putative peptide has a hydrophilic N-terminus and a hydrophobic C-terminus, indicating a possible association with both host cell membrane and cytosol whereas the putative URF2 product is predominantly hydrophobic. A further structural modification found in N20-RNA 3L is a new tandem repeat of 243 nts which overlaps with the MP open reading frame.

The Am Gene Controlling Resistance to Alfalfa mosaic virus in Tomato Is Located in the Cluster of Dominant Resistance Genes on Chromosome 6

ABSTRACT The dominant gene Am from Lycopersicon hirsutum f. sp. glabratum PI134417 confers resistance to most strains of Alfalfa mosaic virus, including the recently identified necrotic strains. The phenotypic response includes a lack of symptom development following mechanical inoculation of leaves. To study the resistance mechanism controlled by Am, biological (back-inoculation to susceptible hosts), serological (double-antibody sandwich, enzyme-linked immunosorbent assay), and molecular (reverse transcription-polymerase chain reaction and hybridization with specific riboprobes) methods of virus detection have been conducted on mechanically inoculated PI134417 leaves. The virus was never recovered, indicating that Am acts by an inhibition of viral accumulation during the early events of the virus life cycle. Am has been mapped genetically to the short arm of tomato chromosome 6 in the resistance hotspot, which includes the R-genes Mi and Cf-2/Cf-5 and the quantitative resistance factors Ty-1, Ol-1, and Bw-5.

First Report of Alfalfa mosaic virus in Physostegia virginiana.

Physostegia virginiana Benth. (false dragon head) is a perennial plant from the family Lamiaceae cultivated as an ornamental in gardens and for cut-flower production. In 2000, stunting of plants and yellow-to-brown ringspots on leaves were observed in cut-flower production in the Alpes Maritimes Department (southeast France). These symptoms greatly decreased the commercial value of the stems. The disease was attributed to Alfalfa mosaic virus (AMV) because extracts of infected plant tissues revealed typical bacilliform particles by electron microscopy, produced symptoms typical of AMV after inoculation of a range of previously described test plants (1), and reacted positively in enzyme-linked immunosorbent assays (ELISA) with antibodies raised to a tomato strain of AMV (from G. Marchoux, INRA, France). After isolation from single local lesions on Vigna unguiculata, the AMV isolate was multiplied in cv. Xanthinc tobacco, where it induced local and systemic ringspot symptoms. Infected Xanthinc plants served as sources of inocula for subsequent mechanical- and aphid (Myzus persicae)-transmission tests to healthy seedlings of P. virginiana (seeds from the botanic garden of Nancy, France; 36 plants for each inoculation procedure). Chlorotic and necrotic local lesions were observed in 25% of mechanically inoculated plants. Three months after inoculation, uninoculated leaves of all mechanically inoculated plants and 30.5% of aphid-inoculated plants tested positive for AMV based on ELISA. During the first year after inoculation, less than 10% of infected plants showed typical systemic symptoms. This proportion reached 40% during the second year. Recently, we observed similar symptoms in P. virginiana plants cultivated in public gardens in Intercourse (Pennsylvania), Toronto (Ontario, Canada) and Montreal (Quebec, Canada). Using ELISA, AMV was detected in symptomatic plants from these three additional locations. Reference: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000.

Partial Resistance of Transgenic Peas to Alfalfa Mosaic Virus under Greenhouse and Field Conditions

Transformed pea (Pisum sativum L.) lines were produced with two chimeric gene constructs encoding the coat protein (CP) of alfalfa mosaic virus (AMV) strain NZ1. To determine whether transformed lines have improved AMV resistance, progeny of independently derived transgenic lines were tested in the greenhouse and five lines with improved virus resistance were identified. Resistance was observed only in individual plants that accumulated detectable CP product from the transgene, suggesting that resistance is CP mediated. Plants that accumulated detectable amounts of transgene CP product yet were susceptible to AMV were found among the progeny of most lines, indicating that these lines are only partially resistant to the challenging AMV strains, 425 and NZ1(Lincoln). A field test was conducted with the progeny of four independently derived transgenic lines. To test for improved virus resistance under field conditions, plots of transgenic lines and nontransgenic controls were inoculated with two AMV strains, NZ1(Lincoln) and NZ34. Analysis of disease severity in seedlings in inoculated plots confirmed that partial virus resistance can be produced by genetically modifying peas with AMV CP sequences.

Resistance to Alfalfa mosaic virus in transgenic barrel medic lines containing the virus coat protein gene

Medicago truncatula is used as a pasture legume and a source of nitrogen for grain crops in southern Australia. Alfalfa mosaic virus (AMV) infection reduces herbage production and nodulation. The coat protein gene of a South Australian strain of AMV (AMV N20) has been transferred to Medicago truncatula cv. Jemalong 2HA using Agrobacterium-mediated transformation. The most detailed investigations were carried out with the coat protein gene in the sense orientation (CP+). Progeny (T1, T2, T3) raised from self-pollinated primary transformants (T0) containing the coat protein CP+ gene were resistant to AMV. Based on Southern analysis and segregation, the transformants contained a single gene copy. In the T3 generation, one line was immune and one line showed resistance to AMV N20. The immune line contained no detectable virus when plant sap from either inoculated or systemic leaves was bioassayed on Phaseolus vulgaris. This line was also immune to the heterologous AMV S40 isolate. A line with the coat protein gene in antisense orientation (CP–) showed delayed systemic infection but was not immune. We conclude that coat protein mediated protection (CPMP) is an effective strategy for controlling AMV infection and should be further evaluated in the field.

First Report of Alfalfa mosaic virus in Pachysandra terminalis in Europe.

Pachysandra terminalis (Buxaceae) was introduced from Japan to Europe in 1882. This ornamental plant is grown in northern Europe as a ground cover in shaded sites. Line patterns, more or less necrotic ringspots, and mosaic symptoms on leaves of pachysandra plants have been seen in public gardens in France (Strasbourg, Colmar, Mulhouse, and Nantes) and Germany (Freiburg im Breisgaü). Extracts of plant tissues obtained from these five sites were used for mechanical- and aphid-transmission experiments, enzyme-linked immunosorbent assays (ELISA) with antibodies directed toward a tomato strain of Alfalfa mosaic virus (AMV) (from G. Marchoux, INRA, France), and electron microscopy. All inoculations produced symptoms typical for AMV in Nicotiana tabacum cv. Xanthi-nc tobacco, Chenopodium amaranticolor, C. quinoa, Vigna unguiculata, Phaseolus vulgaris, Vicia faba, Pisum sativum, and Ocimum basilicum (2). Moreover, Medicago arborea, a new host for AMV, showed systemic mosaic on leaflets. On these and the original pachysandras, AMV was readily detected by ELISA. After isolation from three to four local lesions on Vigna unguiculata and further multiplication in tobacco, one isolate was purified. Bacilliform particles of three sizes, typical for AMV, were seen by electron microscopy. Transmissions of the strain to ELISA-negative pachysandras was achieved by mechanical inoculations (7 of 20 inoculated plants were ELISA positive) and by the aphid species Myzus persicae and Aphis craccivora (14 of 17 inoculated plants were infected). Symptoms were observed 2 months after inoculation; some of the plants remained symptomless but were AMV positive in ELISA. As early as 1970, similar symptoms were reported in pachysandra in New Jersey, and AMV was isolated from affected plants (1). However, inoculations of healthy pachysandra plants with AMV was not performed. Our results show the need for an AMV indexing protocol in the propagation of pachysandra to control its spread.

Amino acids of alfalfa mosaic virus coat protein that direct formation of unusually long virus particles.

In contrast to most alfalfa mosaic virus (AMV) strains (YSMV, S, M and 425), AMV strains VRU and 1 5/64 can form abnormally long virus particles, an ability which has been linked to the coat protein (CP). In order to study this phenomenon, the CP-encoding RNAs 3 of AMV strains VRU and 1 5/64 were cloned and fully sequenced. Comparative sequence analyses of AMV RNA 3 sequences derived from different strains revealed two non-conservative amino acid substitutions, Ser65 and Leu175, which occur exclusively in the closely related VRU- and 15/64-CPs. When these amino acid alterations were introduced into the CP of AMV strain 425 unusually long virus particles were assembled. This confirms that amino acids Ser66 and Leu175 of the CPs of AMV strains VRU and 15/64 are involved in the formation of tubular virus particles.

Resistance to Alfalfa Mosaic Virus in Transgenic Burley Tobaccos Expressing the AMV Coat Protein Gene

There are no known genetic sources of resistance to alfalfa mosaic virus (AMV) in the genus Nicotiana. In this communication, we describe how we genetically engineered an AMV (strain 425) coat protein (CP) gene into three commercial burley tobacco (N. tabacum L.) genotypes, BY 21, TN 86, and KY 8959, and the evaluation of the transgenic lines in greenhouse and field experiments. In a replicated greenhouse trial, 11 AMV‐CP transgenic lines in a BY 21 background were resistant to two AMV isolates, AMV‐425 and AMV‐KY, but the lines were less resistant to another isolate, AMV‐NC. In a 1994 field experiment, we found that nine BY 21 AMV‐CP transgenic lines were resistant to AMV‐KY when inoculated both before and after transplanting. The level of resistance varied among the nine lines. In 1995, 10 randomly selected AMV‐CP lines in each of the three genetic backgrounds were evaluated in a replicated field trial. Mean infection scores of each set of lines were significantly less than those of their non‐transgenic counterparts, but there were no significant differences among these three mean infection scores. However, there were significant differences among lines within a genetic background for their response to AMV infection. High sensitivity to AMV infection, found in genotypes TN 86 and KY 8959 that possess an endogenous gene for resistance to potyviruses, was not evident in their AMV‐CtrPa nsgenic lines. The CP mediated resistance to AMV was effective in reducing viral symptoms in the field and it may provide a valuable source for AMV resistance in tobacco.

The sense and antisense coat protein gene of alfalfa mosaic virus strain N20 confers protection in transgenic tobacco plants

The coat protein gene of a South Australian strain of alfalfa mosaic virus (AMV-N20 [NcS]) has been cloned, sequenced, and transferred into Nicotiana tabacum L. cv. Xanthi via Agrobacterium tumefaciens under the control of the CaMV 35S promoter. A number of lines (T0 generation) were selected with the coat protein gene either in sense orientation (CP+) or in antisense orientation (CP–). The T0 plants were tested for their gene expression and susceptibility to the homologous AMV strain. A significant delay in the onset of symptoms and a reduction in virus accumulation was observed in CP+ plants mechanically inoculated with AMV. CP– plants were also significantly protected but less so than the CP+ plants. Plants transformed with the expression vector only (CP0) showed a minor resistance to local infection on inoculated leaves compared with untransformed plants. The strategy of coat protein mediated protection (CPMP) using the CP gene in either messenger sense or antisense would therefore be appropriate for testing on economically important pasture legumes.

Analysis ofCis-Acting Elements in the 5′ Leader Sequence of Alfalfa Mosaic Virus RNA 3

The leader sequence of RNA 3 of the Leiden isolate of alfalfa mosaic virus strain 425 consists of 345 nucleotides (nt) and contains four putative stem–loop structures each with a motif in the loop that resembles the internal control region 2 (ICR2) of tRNA genes. The sequence of the 5′ terminal 112 nt of this leader contains one of these stem–loop structures and is sufficient for a reduced accumulation of RNA 3 in protoplasts and a delayed accumulation in plants (E. A. G. van der Vossenet al., Nucleic Acids Res.21, 1361–1367 (1993). A number of mutations were made in this 112-nt leader sequence to investigate its role in RNA 3 accumulation. Deletion of nucleotides 23–43, 44–90, or 55–112 and inversion or duplication of nucleotides 44–90 all abolished RNA 3 accumulation. Similarly, two base substitutions in the ICR2 motif (nucleotides 60–77) abolished RNA 3 accumulation. Mutations in the stem sections of the putative stem–loop structure had various effects on RNA 3 accumulation and supported the notion that this structure is important for plus-strand promoter activity.

トマト黄斑モザイク病を起こすアルファルファモザイクウイルスの性状

A new disease of tomato with yellow mosaic symptoms was found in Japan in 1983. A strain of alfalfa mosaic virus (AMV-T), proved to be the causal agent of the disease, was characterized comparing with another isolate of AMV from red clover (AMV-R). The two isolates varied in symptomatology in tomato, pea and petunia. Neither of the isolates affected soybean which is susceptible to many strains of AMV. AMV-T and AMV-R were slightly different from each other in polyacrylamide gel electrophoresis of double-stranded RNAs extracted from Nicotiana glutinosa plants infected with the two isolates, indicating that they had distinct electrophero-types. In agar gel diffusion using anti-AMV-T serum, a spur was formed, which was not formed in a test using anti-AMV-R serum, indicating that these two isolates were serologically related but antigenically different.

Serological Differentiation of Some Strains of Alfalfa Mosaic Virus with Polyclonal Antibodies

Five biologically distinct but antigenically similar alfalfa mosaic virus (AMV) strains were tested for minor antigenic differences by immunodiffusion tests and indirect enzyme-linked immunosorbent assay (ELISA) by using antisera elicited to native and glutaraldehyde-fixed AMV particles of each of the virus strains. In addition, antisera to coat protein preparations of two of the strains were also used. In immunodiffusion tests with antisera to fixed virus particles and either fixed or native virus preparations as test antigens, no antigenic differences were detected among any of the strains (...)

Differentiation and Antigenic Characterization of Closely Related Alfalfa Mosaic Virus Strains with Monoclonal Antibodies

A panel of 15 mouse monoclonal antibodies (MAbs) was raised against five strains of alfalfa mosaic virus (AMV) which were closely related antigenically but biologically distinct. A wide diversity of MAb specificity was revealed by screening them in three formats of indirect ELISA, using native and glutaraldehyde-fixed AMV particles as well as isolated coat protein preparations. Of these MAbs, seven reacted specifically with only one AMV strain in at least one ELISA format and at least one MAb was capable of identifying each of the strains. One of the MAbs reacted with a cryptotope, whereas the other recognized different subtypes of either metatopes or neotopes, indicating that the AMV particle has a complex antigenic structure. Only two of the MAbs precipitated AMV in agarose gels. Another two, which recognized epitopes on coat protein subunits, also reacted well in immunoblots. One of the precipitating MAbs recognized an epitope which appears to be common to AMV and cucumber mosaic virus.

Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance

Agrobacterium-mediated transfer from a binary vector was used to produce transgenic Nicotiana tabacum plants that expressed coat protein of the plant virus, alfalfa mosaic virus (AMV). Expression levels of the chimeric gene, which was under the control of the cauliflower mosaic virus 19S promoter, were determined in primary transformed plants, in the progeny from self-fertilization and in the progeny from crosses to normal tobacco. RNA transcripts that were of the expected size as well as a protein of the M(r) and antigenicity of AMV coat protein accumulated in the transgenic plants. Plants that expressed the highest levels of coat protein developed fewer primary infections following inoculation with two strains of AMV and developed systemic infection slower than did plants that did not express coat protein. Resistance was specifically against virions of the AMV strains. AMV RNA and the unrelated virus, tobacco mosaic virus, were as infectious on progeny that expressed coat protein as they were on progeny that did not. The relationship between the virus resistance expressed by these transgenic plants and that observed in virus cross-protection is discussed.