Alfalfa Mosaic Virus Isolate Research Articles

Molecular characterization and evolution of the resident population of some alfalfa mosaic virus (AMV) isolates in Egypt

BackgroundAlfalfa mosaic virus (AMV) is an important virus affecting many vegetable crops in Egypt. In this study, virus isolates were collected from naturally infected potato, tomato, alfalfa and clover plants that showed suspected symptoms of AMV in different locations of Beheira and Alexandria governorates during the 2019–2020 growing season. The relative incidence of the virus ranged from 11–25% based on visual observations of symptoms and ELISA testing. A total of 41 samples were tested by ELISA using polyclonal antisera for AMV. Four AMV isolates collected from different host plants, named AM1 from potato, AM2 from tomato, AM3 from alfalfa and AM4 from alfalfa, were maintained on Nicotiana glutinosa plants for further characterization of AMV.ResultsElectron micrographs of the purified viral preparation showed spheroidal particles with a diameter of 18 nm and three bacilliform particles with lengths of roughly 55, 68, and 110 nm and diameters identical to those of the spheroidal particles. The CP gene sequence comparisons of four AMV isolates (AM1, AM2, AM3 and AM4) showed the highest nucleotide identity of 99.7% with the Gomchi isolate from South Korea infecting Gomchi (Ligularia fischeri) plants. Phylogenetic analysis showed that the present isolates were grouped together into a distinct separate clade (GPI) along with the Gomchi isolate from South Korea. Similarly, the deduced amino acid sequence comparisons of Egyptian AMV isolates revealed that amino acids Q29, S30, T34, V92 and V175 were conserved among the Egyptian isolates in GPI.ConclusionThe present study found strong evolutionary evidence for the genetic diversity of AMV isolates by the identification of potential recombination events involving parents from GPI and GPII lineages. Additionally, the study found that Egyptian AMV isolates are genetically stable with low nucleotide diversity. Genetic analysis of the AMV population suggested that the AMV populations differ geographically, and AMV CP gene is under mild purifying selection. Furthermore, the study proposed that the Egyptian AMV population had common evolutionary ancestors with the Asian AMV population. Antioxidant enzymes activity was assessed on N. glutinosa plants in response to infection with each AMV isolate studied, and the results revealed that the enzyme activity varied.

Serologic and molecular determination and phylogenetic analysis of some viruses infecting pepper in Turkey

Viruses are among the major agents causing yield loss in pepper production. During the 2016-2017, a survey was performed in pepperfields to determine of some viruses in pepper plants showing virus-like symptoms in different provinces of Turkey. Plant samples wereassayed using DAS-ELISA with specific antisera for cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV), tomato yellow leafcurl virus (TYLCV), alfalfa mosaic virus (AMV), and pepper mild mottle virus (PMMoV). The DAS-ELISA results indicated that 42.2, 17.5,17.5, and 10.6 of the 421 samples were infected with CMV, TSWV, AMV, and PMMoV, respectively. The positive samples were furtheranalysed by RT-PCR and sequencing. Based on sequence analyses, a phylogenetic tree was drawn for each of CMV, PMMoV and AMV.Phylogenetic analysis showed that CMV isolates were placed in subgroup IA within the major group I. The AMV isolates were classifiedwithin four clades and showed a high variability. PMMoV isolates were grouped with Serbia, Brazil, and Japan isolates within the group I.One AMW isolates determined possible recombinant isolates. Further studies needed for more information about recombinant isolatesand evolutions. The analyses of the molecular structure of viruses is important for the accurate diagnosis of the pathogen and to applythe right method of management strategies for controlling plant viruses in agriculture.

Evolutionary history and global spatiotemporal pattern of alfalfa mosaic virus.

Alfalfa mosaic virus (AMV) is an important plant virus causing considerable economic loss to alfalfa production. Knowledge of the evolutionary and demographic history of the pathogen is limited but essential to the development of effective and sustainable pathogen management schemes. In this study, we performed worldwide phylodynamic analyses of AMV based on 154 nucleotide sequences of the coat protein gene, sampled from 1985 to 2020, to understand the epidemiology of this pathogen. Bayesian phylogenetic reconstruction estimates that the crown group of AMV dates back to 1840 (95% credibility interval, 1687-1955). We revealed that AMV continuously evolves at a rate of 4.14 × 10-4 substitutions/site/year (95% credibility interval, 1.04 × 10-4 - 6.68 × 10-4). Our phylogeographic analyses identified multiple migration links between Europe and other regions, implying that Europe played a key role in spreading the virus worldwide. Further analyses showed that the clustering pattern of AMV isolates is significantly correlated to geographic regions, indicating that geography-driven adaptation may be a factor that affects the evolution of AMV. Our findings may be potentially used in the development of effective control strategies for AMV.

Molecular characterisation of Alfalfa mosaic virus isolates in potato from the Tokat province, Türkiye

The Alfalfa mosaic virus (AMV) was detected in potato fields in the Tokat province. The coat protein (CP) sequences of AMV isolates from the Tokat province were determined and compared with sequences of reference AMV isolates from GenBank. Total nucleic acid (TNA) was extracted from plants with positive results according to serological test results. Then, reverse-transcription polymerase chain reaction (RT-PCR) was performed using primer pair specific to partial the coat protein region, and positive PCR products were sent for sequence analysis in both directions. Two Turkish AMV isolates (AMV-PN3-5 and AMV-PN3-6) had a 96-99% nt homology amongst themselves, according to nucleotides (nt) sequence analysis. Based on the phylogenetic tree obtained from 24 AMV isolates from GenBank for both sequences, the two Turkish AMV isolates were clustered in subgroup I containing Iranian, Canadian, Turkish, Korean, and Serbian isolates, at the nucleotide level. Sequence comparison showed that these two isolates of AMV shared 96% to 99.7% sequence similarity with the twenty-six reported isolates of AMV obtained from GenBank. This is the first report on the genetic variability of AMV isolates infecting potato crops in the Tokat province.

Phylogenetic Relationship of Alfalfa Mosaic Virus (AMV) Isolate Identified in Bingöl Province of Turkey

Alfalfa mosaic virus (AMV) is one of the most important viral diseases of alfalfa plant among the forage crop, causing significant annual economic losses. The agent is also of potential importance to other cultivars such as tomatoes, potatoes, and peppers in most cases. The identification and phylogenetic relationships of AMV were carried out by reverse-transcription polymerase chain reaction (RT-PCR), following by bacterial cloning. The cDNA of alfalfa samples (12) were subjected to RT-PCR tests using primer pairs, specific for the capsid protein gene (CP) of AMV, resulting in a DNA fragment of approximately 700 bp as expected. The amplicons were directly cloned and then resulting sequences were deposited in GenBank (Acc. No: MW962977, MW962976). The BLASTn analysis of both sequences demonstrated that AMV virus isolates from alfalfa were highly similar to other AMV isolates from various crops in the world, with nucleotide identity ranging from 97 to 99.37%. The results of phylogenetic dendrogram based on CP gene sequences clearly suggested that our isolates are closely related to four AMV isolates from alfalfa in Turkey. To our knowledge, this study is the first report of molecular phylogeny and AMV presence in alfalfa exhibiting yellowing, mottling, and leaves abnormalities in Bingöl province, Turkey.

Whole-Genome Characterization of Alfalfa Mosaic Virus Obtained from Metagenomic Analysis of Vinca minor and Wisteria sinensis in Iran: with Implications for the Genetic Structure of the Virus.

Alfalfa mosaic virus (AMV), an economically important pathogen, is present worldwide with a very wide host range. This work reports for the first time the infection of Vinca minor and Wisteria sinensis with AMV using RNA sequencing and reverse transcription polymerase chain reaction confirmation. De novo assembly and annotating of contigs revealed that RNA1, RNA2, and RNA3 genomic fragments consist of 3,690, 2,636, and 2,057 nucleotides (nt) for IR-VM and 3,690, 2,594, and 2,057 nt for IR-WS. RNA1 and RNA3 segments of IR-VM and IR-WS closely resembled those of the Chinese isolate HZ, with 99.23–99.26% and 98.04–98.09% nt identity, respectively. Their RNA2 resembled that of Canadian isolate CaM and American isolate OH-2-2017, with 97.96–98.07% nt identity. The P2 gene revealed more nucleotide diversity compared with other genes. Genes in the AMV genome were under dominant negative selection during evolution, and the P1 and coat protein (CP) proteins were subject to the strongest and weakest purifying selection, respectively. In the population genetic analysis based on the CP gene sequences, all 107 AMV isolates fell into two main clades (A, B) and isolates of clade A were further divided into three groups with significant subpopulation differentiation. The results indicated moderate genetic variation within and no clear geographic or genetic structure between the studied populations, implying moderate gene flow can play an important role in differentiation and distribution of genetic diversity among populations. Several factors have shaped the genetic structure and diversity of AMV: selection, recombination/reassortment, gene flow, and random processes such as founder effects.

Occurrence and molecular characterization of alfalfa mosaic virus in eggplant in Serbia

In the 2018-19 growing season, a total of 51 leaves of eggplant plants grown under field conditions were collected randomly from nine private gardens at four different localities in the Province of Vojvodina. Eggplants with nearly 40% of plants showing bright yellow to white mosaic or mottling of leaves were found throughout the inspected fields (gardens). The collected samples were analyzed for the presence of alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV) and potato virus Y (PVY) using commercial double-antibody sandwich (DAS)-ELISA kits. Serological analysis of eggplant samples revealed the presence of AMV in 80.39% collected samples. None of the analyzed samples was positive for CMV and PVY. The virus was successfully mechanically transmitted to test plants including Nicotiana benthamiana, Chenopodium quinoa, C. amaranticolor, as well as eggplant seedlings, confirming the infectious nature of the disease. The presence of AMV in eggplants was further verified by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, using the primers CP AMV1 and CP AMV2 that amplify part of the coat protein (CP) gene. The phylogenetic analysis showed that Serbian AMV isolates grouped into a separate well-supported group together with AMV isolates from Italy, Croatia and previously characterized isolates from Serbia. To our knowledge, this is the first report of AMV infection of eggplant in Serbia.

First Report of Alfalfa mosaic virus in Chayote in Italy.

Chayote (Sechium edule (Jacq.) Sw.) is a vigorous perennial and climbing cucurbits, native to Mesoamerica, and cultivated for alimentary purposes in the American continent, Australia, New Zealand, South Europe, Asia and Africa. During spring 2019, some chayote plants showing bright yellow vein banding rings and lines were observed in a private garden in South Italy (Campania region). Symptoms coalesced in some leaves, covering almost the whole foliar area. Double-stranded RNAs were extracted from symptomatic leaves of a single chayote plant and reverse-transcribed, randomly amplified, and submitted to Illumina sequencing (Marais et al., 2018). Reads were assembled using CLC Genomics Workbench 11.1 (http://www.clcbio.com). Contigs were then annotated by Blastn and Blastx comparison with the Genbank database, which allowed the identification of eight contigs of between 380 and 980 nucleotides sharing significant identity with alfalfa mosaic virus (AMV) genomic RNAs. No other viral contigs were identified. Mapping of reads on AMV genomic RNAs identified 4,209 AMV reads (1.26% of total reads) and allowed the scaffolding of the contigs into three scaffolds corresponding to the three AMV genomic RNAs. To complete the sequence of the AMV chayote isolate genome (named See-1), primers were designed from the contig sequences and used to amplify RACE PCR products spanning the 5' and 3' terminal regions of the three genomic RNAs using the SMARTer™ RACE cDNA Amplification Kit (Clontech, China). All amplicons were cloned into the pGEM-T vector (Promega, USA) and sequenced (three clones for each amplicon) by Microsynth Seqlab (Microsynth AG, Switzerland). Finally, the complete genomic sequences of the three RNAs were assembled by MacVector 17.5 (MacVector Inc., USA). The RNA1, RNA2 and RNA3 of See-1 are 3,643, 2,593 and 2,037 nt respectively (GenBank accession Nos. MT093209 to MT093211), and share the highest nt sequence identity with the RNA1 and RNA3 of AMV isolate (HZ) from tobacco (99.5% for RNA1, HQ316635; 98.7% for RNA3, HQ316637) and with the RNA2 of isolate AMV-Gym from Gynostemma pentaphyllum (98.1%, MH332898), both from China. AMV isolate See-1 was classified as belonging to subgroup I based on the presence of a BamH I and two AvaII sites in the CP ORF (Parrella et al., 2000). Reverse transcription polymerase chain reaction, using primers targeting the CP gene (Parrella et al., 2000), confirmed AMV infection in three symptomatic cayote plants including that used for Illumina sequencing, with 100% of nt sequence identity of amplicons. Three plants each of Chenopodium amaranticolor, Nicotiana benthamiana and Solanum lycopersicon were mechanically inoculated with sap from isolate See-1 infected plant, leading to the appearance of typical AMV symptoms in all three hosts ten days post-inoculation (Jaspars & Bos, 1980). This note describes the first detection of AMV in cayote in Italy and, to the best of our knowledge, in the world. In some areas of Southern Italy, climatic conditions are favorable enough to allow chayote development in the wild. Further studies would be desirable to determine the distribution and incidence of AMV in chayote and to understand the possibility that this species may play a role in AMV epidemiology, representing a threat to other susceptible crops.

Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus.

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa.

Molecular characterization of Alfalfa mosaic virus (AMV) isolates in alfalfa and other plant species in different regions in Saudi Arabia

In a survey conducted in 2012 and 2013, 1166 samples from alfalfa plants and 202 samples from symptomatic weeds and cultivated plants growing adjacent to alfalfa fields were collected. Using DAS-ELISA, Alfalfa mosaic virus (AMV) was detected in 58.4% of the alfalfa samples and in 63.9% of the weeds and cultivated plants samples. ELISA detection of AMV was confirmed by testing representative samples from all regions by RT-PCR using a pair of primers (AMV/F and AMV/R) specific to the AMV coat protein (CP) gene. The size of the major product obtained from AMV-infected plants was identical to the 700 bp size expected from the CP gene of AMV. The amino acids and the nucleotide sequences of 17 Saudi AMV isolates from alfalfa, and 16 from other cultivated plants and weeds detected in different regions in Saudi Arabia showed a percentage of similarity that ranged between 87.9%–100% among them and 86.2% - 100% when compared with sequences of 15 different AMV isolates reported in the GenBank. This is the first time AMV was detected in Vigna unguiculata and in the following weed plant species: Chenopodium quinoa, Convolvulus arvensis, Malva parviflora, Hibiscus spp., Hippuris vulgaris, Cichorium intybus, and Flaveria trinervia in Saudi Arabia.

Potato Tuber Necrosis Induced by Alfalfa Mosaic Virus Depends on Potato Cultivar Rather Than on Virus Strain.

Alfalfa mosaic virus (AMV) was identified as the causal agent of internal tuber necrosis in the potato cultivar Innovator in New Brunswick, Canada. Further pathological characterization of the isolate (designated as isolate CaM) was performed on six potato cultivars and one breeding clone. Upon mechanical inoculation, four cultivars (Innovator, Yukon Gold, Rochdale Gold-Dorée, and Shepody) showed needle-sized necrotic spots and increasing calico symptoms on new leaves, whereas the remaining cultivars only developed calico symptoms on new leaves. All tubers of CaM-infected Innovator and Shepody plants developed sporadic internal necrotic spots, as did ca. 23 and 8% tubers of CaM-infected Yukon Gold and Rochdale Gold-Dorée, respectively. Sequence analysis of the CP gene of CaM with AMV isolates from potato, all presumed belonging to the "non-necrotic" strain and retrieved from GenBank, indicated that CaM shared >97.1% sequence identity with all but four Egyptian isolates. At the complete genome level, phylogenetic analysis of all available sequences demonstrated that RNA 1 and RNA 3 can be grouped into three major clades each, whereas RNA 2 can be clustered into two clades. CaM and Ca175-1, an AMV isolate that was deemed non-necrotic in a previous study, had different phylogenetic clade patterns, indicating different RNA 1-RNA 2-RNA 3 haplotypes: IA-I-IB (CaM) versus Ca175-1 (IB-II-IA). Despite the difference in haplotype composition, CaM and Ca175-1 induced similar levels of internal necrosis in tubers of Innovator and its parent Shepody. The results suggest that the internal necrosis in AMV-infected tubers depends on potato cultivar rather than on AMV strain/haplotype, and CaM is just a "regular" isolate of AMV.

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.

Molecular and partial biological characterization of the coat protein sequences of Iranian alfalfa mosaic virus isolates

Alfalfa mosaic virus (AMV) is one of the most important viruses that cause significant losses to alfalfa worldwide. A total of 1066 symptomatic alfalfa, 143 weeds and other secondary hosts were collected and assayed for AMV infection. The virus was detected in 360 samples (33.8% incidence) of alfalfa by plate-trapped antigen (PTA)-ELISA. Among the weed species and the other plants tested, potato, pepper and seven weed species were infected with AMV. The coat protein (CP) gene sequences from 59 Iranian isolates were PCR amplified, cloned, sequenced, and compared with the sequences available in GenBank. Sequence analysis of the amplified regions showed that 55 of the Iranian CP sequences share >98% pairwise identity with each other. The remaining four share between 89 and 96% identity. Our host studies indicated that the Iranian AMV isolates were classified into two groups. Only four Iranian isolates were clustered in the symptom-group B and caused relatively milder symptoms compared to those found in symptom-group A.

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.

Development and validation of PCR assays for detection of alfalfa dwarf disease-associated viruses in Australian lucerne pastures

In 2010, a severe disease of lucerne (Medicago sativa L.) named alfalfa dwarf disease (ADD) was reported in Argentina. High throughput sequencing of diseased plants indicated the presence of five viruses, the (−) ssRNA virus, alfalfa dwarf virus (ADV), the (+) RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus (BLRV) and alfalfa enamovirus 1 (AEV-1) and the ssDNA virus, alfalfa leaf curl virus (ALCV). In this study, we determined which ADD-associated viruses are present in Australian lucerne that showed diverse virus-like symptoms. A duplex RT-PCR was developed for simultaneous detection of ADV and AMV using a cloned non-infectious ADV RNA fragment as positive control. Similarly, the presence of BLRV and AEV-1 was determined by duplex RT-PCR, and ALCV by PCR. Only AMV and BLRV that are endemic to Australia were detected. None of the novel exotic viruses ADV, AEV-1 and ALCV were detected in lucerne samples collected between 2015 and 2017. However, AMV and BLRV were detected in 78% and 70% of tested samples, respectively. Based on analysis of coat protein (CP) nucleotide sequences, Australian BLRV isolates are closely related to each other and to the Argentine Manfredi isolate. Phylogenetic analyses based on CP gene nucleotide sequences confirmed separation of AMV isolates into two subgroups. The majority of AMV isolates, including all those from Argentina and Australia, clustered in subgroup I with isolates from various hosts and geographic origins. All Australian AMV isolates were closely related to AMV isolated from ADD-affected lucerne in Argentina.

Molecular detection and identification of alfalfa mosaic virus (AMV) on pepper cultivated in open fields in R. Macedonia

Alfalfa mosaic virus (AMV) is one of the most distributed and economically important plant viruses in pepper in R. Macedonia. Serological detection of AMV in eight important pepper production regions in R. Macedonia and molecular identification of a representative isolate were performed. The virus detection of AMV was conducted using DAS-ELISA method. In order to make molecular detection, RT-PCR was performed. Phylogenetic analysis was conducted, based on the partial sequences of the coat protein gene. A genetic relationship of the Macedonian isolate KUA7-2013 gained in this study was compared with 29 AMV isolates from other parts of the world. High level of nucleotide (92-94.4%) and amino acid identities (91.9-97.1%) were determined. In the constructed phylogenetic tree, the Macedonian AMV isolate was clustered in group II together with isolates from France and Great Britain. In this study, for the first time in R. Macedonia, an isolate of AMV was identified at the molecular level.

Biological and Molecular Characterization of Alfalfa Mosaic Virus Infecting Trumpet Creeper (Campsis radicans) in Iran

AbstractSamples of trumpet creeper (Campsis radicans) leaves showing mottling and mosaic were collected from plants growing in a private garden in Tehran province, Iran, in 2012. Symptomatic leaf samples were tested for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) infection in enzyme‐linked immunosorbent assay (ELISA), using specific antibodies. None of the samples were positive for CMV and PSV; however, all reacted positively with that of AMV antiserum. In biological assay, systemic infection was found on Datura stramonium, Nicotiana tabacum cvs., White Burley, and Xanthi, 21 days postinoculation (DPI), while necrotic local lesions were obtained following inoculation of Phaseolus vulgaris and Vigna unguiculata within three to four DPI. Using a pair of primers specific for AMV, a DNA fragment of 880 bp was RT‐PCR‐amplified. Analysis of the sequences revealed the presence of 657 nucleotides of AMV complete coat protein (CP) gene (translating 218 amino acid residues). Phylogenetic analysis using neighbour‐joining (NJ) method clustered AMV isolates into two main types and the IRN‐Tru (GenBank Accession No. JX865593) isolate fell into type I. Pairwise nucleotide distances also confirmed two main types with the highest and lowest similarities for type I and II, respectively. The association of AMV with mosaic disease of C. radicans represents the first record from the world.

First Report of Cucumber mosaic virus Infection in Pachysandra in the United States.

Pachysandra terminalis Siebold & Zucc. (Japanese pachysandra, spurge) is widely used as a groundcover. In early 2012, Japanese pachysandra plants from Missouri, which originated in Pennsylvania, showed symptoms of light and dark green mosaic, leaf deformation, concentric ringspots, and stunting. Initial screening of symptomatic leaf tissue by transmission electron microscopy (TEM) using partially purified extracts confirmed the presence of spherical (~28 nm) and bacilliform (18-nm diameter, 35- to 58-nm length) virus particles. Immunosorbent electron microscopy (ISEM) using antisera to a clover isolate of Alfalfa mosaic virus (AMV) (PVAS 92) and to Cucumber mosaic virus (CMV) (ATCC PVAS-30) obtained from the American Type Culture Collection, Manassas, VA, confirmed the presence of AMV and CMV. No other type of virus-like particles were observed by TEM. After 6 months, nearly 20% of the 4,000 pachysandra cuttings exhibited the described symptoms. However, it is possible that more than 20% of the cuttings were infected with both viruses and not yet exhibiting symptoms. Reverse-transcription PCR (RT-PCR) was done using total RNA extracted with a Qiagen RNeasy kit and Ready-To-Go RT-PCR beads (GE Healthcare, UK Limited, UK). The primer pair CMV-1 (5'-GCCGTAAGCTGGATGGACCA) and CMV-2 (5'-TATGATAAGAAGCTTGTTTTCGCG) were used (3) to obtain a 502-bp amplicon from the coat protein (CP) region of CMV RNA 3. The product was ligated and cloned (pGEM-T Easy Vector System; Promega, USA). Three clones were sequenced (UMGC, USA), and the consensus sequence (Sequencher 5.1, Gene Codes Corp., USA) was deposited in GenBank (Accession No. JX227938). The sequence obtained had 100% identity with a homologous CP CMV sequence (AFQ94058) and 99% identity with several other homologous CP CMV sequences (CAX62443, CCK24369, and 15 others). It also contained an EcoRI site at nucleotides 332 to 337, characteristic of CMV Type II isolates (3). The primer pair AMV1F (5'-ATCCACCGATGCCAGCCTTA) and AMV1R (5'-TTCCGCCTCACTGCTGCTG) generated a 1,047-bp product from AMV RNA1 that was deposited in GenBank (JX227937). This product had 100% identity with a homologous AMV sequence (AFQ94057), and 99% identity with several other homologous AMV sequences (AGV15824, ADO85715, CBX36144). From the data presented here, it was concluded that the pachysandra had a mixed infection of AMV and a Type II isolate of CMV. Occurrence of AMV in pachysandra was first reported in New Jersey in 1982 (2) and reported for the first time in France and Germany in 2000 (1). The presence of CMV infection in pachysandra has not been reported in the present literature. Some of the symptoms associated with AMV infection in pachysandra in New Jersey (2) and Europe (1) were similar to the symptoms produced by pachysandra plants infected with both viruses (ring spots, mosaic, and line patterns). However, some symptoms were unique to the mixed infection in pachysandra by AMV and CMV (leaf deformation, stunting). A potential source of this co-infection could occur when plants are grown near alfalfa fields (AMV infection by aphids) and undergo vegetative propagation (CMV infection by contaminated tools). This is the first report of pachysandra co-infected by AMV and CMV in the United States.

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

The spreading of Alfalfa mosaic virus in lavandin in Croatia

A survey was conducted in 2012 and 2013 to detect the presence and distribution of Alfalfa mosaic virus (AMV) in lavandin crops growing in continental parts of Croatia. A total of 73 lavandin samples from six crops in different localities were collected and analyzed for the presence of AMV and Cucumber mosaic virus (CMV) using commercial double-antibody sandwich (DAS)-ELISA kits. AMV was detected serologically in 62 samples collected at three different localities, and none of the samples tested positive for CMV. For further analyses, six selected samples of naturally infected lavandin plants originating from different localities were mechanically transmitted to test plants: Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana and Ocimum basilicum, confirming the infectious nature of the disease. Molecular detection was performed by amplification of a 751 bp fragment in all tested samples, using the specific primers CP AMV1/CP AMV2 that amplify the part of the coat protein (CP) gene and 3?-UTR. The RT-PCR products derived from the isolates 371-13 and 373-13 were sequenced (KJ504107 and KJ504108, respectively) and compared with the AMV sequences available in GenBank. CP sequence analysis, conducted using the MEGA5 software, revealed that the isolate 371-13 had the highest nucleotide identity of 99.5% (100% amino acid identity) with an isolate from Argentina originating from Medicago sativa (KC881010), while the sequence of isolate 373-13 had the highest identity with an Italian AMV isolate from Lavandula stoechas (FN667967) of 98.6% (99% amino acid identity). Phylogenetic analysis revealed the clustering of selected isolates into four molecular groups and the lavandin AMV isolates from Croatia grouped into two distinct groups, implying a significant variability within the AMV lavandin population.