scholarly journals Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 459
Author(s):  
Ahmed R. Sofy ◽  
Mahmoud R. Sofy ◽  
Ahmed A. Hmed ◽  
Rehab A. Dawoud ◽  
Ehab E. Refaey ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Solanum Melongena ◽  
Carotenoid Content ◽  
Leaf Biomass ◽  
Parsimony Method ◽  
Cp Gene ◽  
Morphological Criteria ◽  
Das Elisa

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

scholarly journals First Report of China Rose (Hibiscus rosa-sinensis) as a Host of Alfalfa mosaic virus in Spain

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 462-462 ◽  
Author(s):  
G. Parrella ◽  
E. Fiallo-Olivé ◽  
J. Navas-Castillo
Keyword(s):  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Nucleotide Identity ◽  
Rt Pcr ◽  
Cp Gene ◽  
Hibiscus Rosa Sinensis ◽  
Bright Yellow ◽  
Spanish Isolate ◽  
China Rose ◽  
Das Elisa

China rose (Hibiscus rosa-sinensis L.) is an ornamental plant grown throughout the tropics and subtropics. In June 2011, a China rose plant (sample CV-1) showing bright yellow “aucuba”-type mosaic, mainly at the center of the leaves, was found in a public garden in Caleta de Vélez (Málaga Province, southern Spain). Electron microscope examination of negatively stained preparations from the symptomatic plant revealed the presence of semispherical to bacilliform virus-like particles of 30 to 56 × 16 nm. Sap extracts also reacted positively in double-antibody sandwich (DAS)-ELISA to antiserum against Alfalfa mosaic virus (AMV) (Bioreba AG, Reinach, Switzerland). RNA of this sample was extracted with the RNeasy Kit (Qiagen, Valencia, CA) and tested by reverse-transcription (RT)-PCR with AMV specific primers (2), using AMV and GoTaq Master Mixes (Promega, Madison, WI) for cDNA synthesis and amplification, respectively. After cloning and sequencing, the ~750-bp DNA fragment was confirmed as the coat protein (CP) gene of AMV (GenBank Accession No. HE591387) with the highest nucleotide identity of 96% to AMV isolates belonging to subgroup IIA (e.g., GenBank Accession No. AJ130707). Sap from affected leaves of sample CV-1 was mechanically inoculated onto herbaceous indicator plants (Chenopodium amaranticolor, C. quinoa, and Ocimum basilicum). Both Chenopodium species developed chlorotic local lesions followed by mosaic within 3 days after inoculation, and O. basilicum showed bright yellow mosaic of calico type 2 weeks postinoculation. These symptoms are consistent with those reported for AMV in these hosts (1). Virus infection in the inoculated plants was confirmed by DAS-ELISA and RT-PCR. To gain insight on the prevalence and genetic variability of AMV in China rose, a survey was carried out in nearby locations in the provinces of Málaga (14 samples from Torre del Mar and 5 samples from Rincón de la Victoria) and Granada (12 samples from La Herradura). Leaf samples were analyzed by tissue blot hybridization with an AMV-specific digoxigenin-labeled RNA probe obtained from the RNA 1 of the Spanish isolate Tec1 (3), and only two samples from Torre del Mar tested positive. One of these samples (TM-2) was used to amplify by RT-PCR the AMV CP gene that was cloned and sequenced (GenBank Accession No. HE591386). The highest nucleotide identity of the TM-2 CP gene (98%) was with the subgroup IIB Spanish isolate Tec1, whereas identity with the CV-1 isolate was 95%. Nevertheless, phylogenetic analysis (neighbor-joining method) showed that both CV-1 and TM-2 isolates belong to the recently proposed AMV subgroup IIB (3), which includes the Tec1 isolate and two other isolates from ornamental plants, Phlox paniculata from the United States (GenBank Accession No. DQ124429) and Viburnum lucidum from Spain (GenBank Accession No. EF427449). These results show that AMV subgroup IIB is emerging as a complex cluster of virus isolates that currently are reported to infect only ornamentals. To our knowledge, this is the first report of AMV naturally occurring in China rose. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Arch. Virol. 156:1049, 2011.

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

2006 ◽  
Vol 96 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
H. Xu ◽  
J. Nie
Keyword(s):  
Mosaic Virus ◽  
Gene Sequence ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Simple Method ◽  
Cp Gene ◽  
Nucleotide Sequence Alignment

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.

scholarly journals Detection and molecular characterization of Pepper mild mottle virus in Serbia

Genetika ◽  
2015 ◽  
Vol 47 (2) ◽  
pp. 651-663 ◽  
Author(s):  
Dragana Milosevic ◽  
Ivana Stankovic ◽  
Aleksandra Bulajic ◽  
Maja Ignjatov ◽  
Zorica Nikolic ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Molecular Characterization ◽  
Great Influence ◽  
Alfalfa Mosaic Virus ◽  
Resistance Breeding ◽  
Elisa Test ◽  
Mottle Virus ◽  
Pepper Mild Mottle Virus ◽  
Das Elisa

During 2009 and 2010, a survey was conducted in pepper crops to detect the possible presence of Pepper mild mottle virus (PMMoV) in Serbia. A total of 239 pepper samples from 39 crops at 26 localities were collected and analyzed for the presence of PMMoV, Cucumber mosaic virus (CMV), Potato virus Y (PVY), and Alfalfa mosaic virus (AMV), using DAS-ELISA test. Although it was detected in a small percentage, PMMoV could pose a threat to pepper production in Serbia due to its rapid seed-borne spread. Presence of PMMoV was confirmed by serological and biological detection, followed by conventional reverse transcription RT-PCR, using primers specific for the RNA-dependent RNA polymerase (RdRp) and the coat protein (CP) genes. Molecular identification confirmed that the Serbian isolates belong to PMMoV pathotypes P1,2 which do not break the resistance gene L3. Reconstructed phylogenetic tree confirmed the allocation of the Serbian isolates together with the majority of PMMoV isolates which belong to pathotypes P1,2. This study represents the first serological and molecular characterization of PMMoV infection of pepper in Serbia, and provides important data on the population structure. The obtained data could have great influence on pepper production in Serbia as well as future pepper resistance breeding in the country.

scholarly journals Natural Infection of Field-Grown Borage (Borago officinalis) by Alfalfa mosaic virus in Spain

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 698-698 ◽  
Author(s):  
C. Mallor ◽  
M. Luis-Arteaga ◽  
M. A. Cambra ◽  
S. Fernández-Cavada
Keyword(s):  
Mosaic Virus ◽  
Indicator Species ◽  
Natural Infection ◽  
Alfalfa Mosaic Virus ◽  
Solanum Melongena ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Consumption ◽  
Vegetable Crops ◽  
Borago Officinalis ◽  
Local Lesions

Alfalfa mosaic virus (AMV) has a wide host range and is distributed throughout the world. It causes disease in several vegetable crops, including bean, celery, lettuce, pea, pepper, and tomato (1). In Spain, it has been found naturally infecting alfalfa, pepper, and tomato. During the autumn of 1999, in the area of Zaragoza (northeastern Spain), several plants expressing foliar yellow mosaic symptoms were observed in borage grown for human consumption in open field plots. The commercial value of the symptomatic plants was greatly reduced. The symptoms were similar to those previously obtained in greenhouse-grown borage plants mechanically inoculated with three tomato isolates of AMV (2). The following indicator species, including virus-free borage plants, were mechanically inoculated with sap from leaves of symptomatic borage plants, and reactions were recorded: chlorotic and necrotic local lesions on Tetragonia expansa and Vigna unguiculata; chlorotic local lesions and systemic mosaic on Chenopodium quinoa, C. amaranticolor, Cucumis sativus ‘Marketmore’, Gomphrena globosa, and Nicotiana glutinosa; systemic mosaic, sometimes associated with localized reactions, on Ocimum basilicum, Capsicum annuum ‘Doux des Landes’ and ‘Yolo Wonder’, N. benthamiana, N. clevelandii, N. rustica, N. sylvestris, N. tabacum ‘Paraguay’ and ‘Xanthi nc’, Petunia hybrida, Physalis floridana, and Solanum melongena ‘Cerna krazavitska’. The reactions are in agreement with the indicator host reactions described for AMV (1). Symptoms on virus-free borage plants mechanically inoculated with sap from symptomatic borage and from inoculated C. amaranticolor and O. basilicum experimental hosts were similar to those observed in naturally infected borage. Positive serological reactions (A405 values more than three times greater than those of the negative controls) in double-antibody sandwich enzyme-linked immunosorbent assay using commercially prepared antiserum against AMV (Agdia, Inc., Elkhart, IN) were obtained with extracts of naturally infected borage leaves and with systemically infected indicator species. Alfalfa plots located in the vicinity of the symptomatic borage plants could be the source of virus for borage infections. To our knowledge, this is the first report of natural AMV infection in Borago spp. References: (1) E. M. J. Jaspars and L. Bos. CMI/AAB. No. 229, 1980. (2) M. Luis-Arteaga and J. M. Alvarez. Inf. Téc. Econ. Agr. 92:70, 1996.

scholarly journals First Report of Cucumber mosaic virus Infecting Peperomia tuisana in Serbia

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 1004-1004 ◽  
Author(s):  
K. Milojević ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Ristić ◽  
D. Milošević ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Disease Incidence ◽  
Impatiens Necrotic Spot Virus ◽  
Rt Pcr ◽  
Test Species ◽  
First Report ◽  
Cp Gene ◽  
Negative Controls ◽  
Das Elisa

Peperomia tuisana C.DC. ex Pittier (family Piperaceae) is an attractive succulent grown as an ornamental. Despite its tropical origins, it can be successfully grown indoors in any climate. In March 2012, three samples of P. tuisana showing virus-like symptoms were collected from a commercial greenhouse in Zemun (District of Belgrade, Serbia) in which estimated disease incidence was 80%. Infected plants showed symptoms including necrotic ringspots and line patterns that enlarged and caused necrosis of leaves. A serious leaf drop led to growth reduction and even death of the plant. Leaves from three symptomatic P. tuisana plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against the most common viral pathogens of ornamentals: Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV) (1,2). Commercial positive and negative controls were included in each ELISA. Serological analyses showed that all plants were positive for CMV and negative for TSWV and INSV. The ELISA-positive sample (isolate 1-12) was mechanically inoculated onto five plants each of three test species as well as of healthy young P. tuisana using 0.01 M phosphate buffer (pH 7). Chlorotic local lesions on Chenopodium quinoa and severe mosaic and leaf malformations were observed on all inoculated Nicotiana tabacum ‘Samsun’ and N. glutinosa. Also, the virus was successfully mechanically transmitted to P. tuisana that reacted with symptoms identical to those observed on the original host plants. All mechanically inoculated plants were positive for CMV in DAS-ELISA. For further confirmation of CMV infection, reverse transcription (RT)-PCR was performed on extracts made from symptomatic P. tuisana, N. tabacum ‘Samsun,’ and N. glutinosa leaf materials. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was carried out using One-Step RT-PCR Kit (Qiagen). A CMV-specific primer pair, CMVCPfwd and CMVCPrev (3), which amplifies an 871-bp fragment of the entire coat protein (CP) gene and part of 3′- and 5′-UTRs, were used for both amplification and sequencing. Total RNAs obtained from the Serbian CMV isolate (HM065510) and healthy P. tuisana were used as positive and negative controls, respectively. A product of the correct predicted size was obtained in all naturally and mechanically infected plants, as well as positive control. No amplicon was recorded in the healthy control. The amplified product derived from isolate 1-12 was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions, deposited in GenBank (KC505441), and analyzed by MEGA5 software (4). Sequence comparison of the complete CP gene (657 nt) revealed that the Serbian isolate 1-12 shared the highest nucleotide identity of 99.1% (99.5% amino acid identity) with the Japanese isolate (AB006813). To our knowledge, this is the first report on the occurrence of CMV in P. tuisana in Serbia. This is also an important discovery since P. tuisana is commonly grown together with other ornamental hosts of CMV, and thus could represent a serious threat for future expansion of CMV in the greenhouse floriculture industry in Serbia. References: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (2) S. Flasinski et al. Plant Dis. 79:843, 1995. (3) K. Milojevic et al. Plant Dis. 96:1706, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

scholarly journals First Report of Alfalfa mosaic virus and Soybean dwarf virus on Soybean in North Dakota

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1829-1829 ◽  
Author(s):  
H. A. Hobbs ◽  
L. L. Domier ◽  
B. D. Nelson
Keyword(s):  
North Dakota ◽  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Dwarf Virus ◽  
Field Sample ◽  
Total Rna ◽  
Qrt Pcr ◽  
Field Samples ◽  
Soybean Dwarf Virus ◽  
Das Elisa

Soybean (Glycine max L.) is the major oilseed crop in North Dakota, with production concentrated in the eastern half of the state. Only one virus, Soybean mosaic virus, has been reported from soybean in North Dakota (4). In July and August of 2010, 200 soybean fields from 25 counties were surveyed for Alfalfa mosaic virus (AMV) and Soybean dwarf virus (SbDV). AMV and SbDV have been detected infecting soybean in multiple Midwestern states and are reported to reduce yields in soybean (1,3). Each field was sampled with a grid pattern across the area with at least 8 km between fields. From each field, leaves were collected from 20 plants without regard for symptoms along a transect of approximately 170 m. Leaves from each field were bulked and sap was extracted in phosphate buffer and stored at –80°C until tested using double-antibody sandwich (DAS)-ELISA with positive controls and reagents and protocols from Agdia Inc. (Elkhart, IN). Using DAS-ELISA, AMV was detected in eight of the 200 soybean fields. For sequence-based virus detection, total RNA was extracted from all field samples using a Qiagen RNeasy Plant Mini Kit (Germantown, MD), pooled, depleted of ribosomal RNA (RiboZero Epicentre, Madison, WI), reverse transcribed, sequenced using an Illumina HiSeq2000 (San Diego, CA), and compared to all available viral amino acid and nucleotide sequences. The analysis detected AMV and SbDV sequences in the pool of 200 fields. The presence of AMV and SbDV was confirmed by quantitative real-time reverse transcription (qRT)-PCR (1,3). For AMV, total RNA extracted from bulked leaves from each of the 200 fields was tested using AMVspecific primers (5′-ATGCTACCCAGGCATGTATATTT-3′ and 5′-GCTGCATCTTTCGCCAGAA-3′) and a FAM-labeled minor-groove binding TaqMan probe (5′-TGGACGTTACCCCCGGA-3′). One field sample from Cass county positive for AMV by ELISA was also positive for AMV by qRT-PCR, confirming the presence of AMV in the field sample. For SbDV, an RNA pool representing all 200 fields, subpools, and individual field samples was analyzed by qRT-PCR (1) and DAS-ELISA. One field sample from Grand Forks County tested positive for SbDV by qRT-PCR and DAS-ELISA, confirming the presence of SbDV in the field sample. Because leaf samples were collected and pooled prior to analysis, the symptom phenotypes of individual field plants could not be correlated with positive ELISA or qRT-PCR results. AMV was reported by the American Phytopathological Society Virus Working Group (2007 to 2008) to be widely prevalent in North Dakota, but we found no peer-reviewed reports of verified AMV identification on any crop in the state. To our knowledge, this is the first confirmed report of AMV and SbDV infecting soybean in North Dakota. Serious infestations by the soybean aphid, Aphis glycines, requiring chemical control, have occurred in recent years in North Dakota. Because A. glycines is a vector for both viruses (1,2), the distribution, incidence, and agronomic impact of AMV and SbDV could be affected in years when A. glycines infestations are high. In addition, AMV is seedborne in soybean and may cause seed mottling, a concern for the food-grade soybean industry where production is primarily for export. References: (1) V. D. Damsteegt et al. Plant Dis. 95:945, 2011 (2) J. H. Hill et al. Plant Dis. 85:561, 2001. (3) H. A. Hobbs et al. Plant Health Progress doi:10.1094/PHP-2010-0827-01-BR, 2010. (4) B. D. Nelson and L. L. Domier. Plant Dis. 93:760, 2009.

scholarly journals First Report of a Mexican Isolate of Papaya mosaic virus in Papaya (Carica papaya) and Pumpkin (Cucurbita pepo)

Plant Disease ◽  
2001 ◽  
Vol 85 (5) ◽  
pp. 558-558 ◽  
Author(s):  
J. C. Noa-Carrazana ◽  
L. Silva-Rosales
Keyword(s):  
Mosaic Virus ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reactions ◽  
Detection Techniques ◽  
Polymerase Chain Reactions ◽  
Cp Gene ◽  
Pcr Products ◽  
Papaya Mosaic Virus ◽  
Das Elisa

Papaya mosaic virus (PMV) is a member of the Potexvirus group and has filamentous particles of 530 nm with a positive sense single-stranded RNA of 6.6Kb. PMV was detected in Mexico in diseased papaya plants growing alone and in mixed plantations with pumpkin. Reverse transcription polymerase chain reactions (RT-PCR) and standard double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) procedures were used on 45 leaf samples from single plants in seven locations in southeast Mexico (States of Yucatan, Campeche, and Quintana Roo). PCR primer design was based on a GenBank sequence with accession number D13957 (1). Amplified PCR products were cloned using a TOPO TA Cloning Kit and sequenced by the dideoxy chain termination method. Twenty-six samples tested positive for PMV using one or both detection techniques: 23 of 41 from papaya and three of four from pumpkin. The two sequences reported here, YY-15 and YY-22 (from papaya and pumpkin respectively, with accession numbers AYO17186 and AYO17187), were 1180 nucleotides long and contained a fragment of ORF3, the complete ORF4 and the putative CP gene, including the 3′ end untranslatable region. Within the CP gene sequence, the amino acid sequence derived had a similarity of 88% with that of D13957 from the GenBank. The similarity of the CP between the two Mexican isolates (from papaya and pumpkin) was 94% and would therefore represent two variants of PMV. A healthy papaya plant in the greenhouse, inoculated with tissue from an infected papaya plant from the field, tested positive for PMV in DAS-ELISA. (PVX was used as a negative control). These results confirmed the identity of the isolate as PMV. Reference: (1) T. L. Sit, M. G. AbouHaidar, and S. Holi. J. Gen. Virol. 70:2335-2331, 1989.

scholarly journals First Record and Complete Nucleotide Sequence of Alfalfa mosaic virus from Lavandula stoechas in Italy

Plant Disease ◽  
2010 ◽  
Vol 94 (7) ◽  
pp. 924-924 ◽  
Author(s):  
G. Parrella ◽  
N. Acanfora ◽  
M. G. Bellardi
Keyword(s):  
Nucleotide Sequence ◽  
Mosaic Virus ◽  
Complete Genome ◽  
Alfalfa Mosaic Virus ◽  
Virus Like Particles ◽  
Lavandula Stoechas ◽  
Cp Gene ◽  
Bright Yellow ◽  
Italian Isolate ◽  
Local Lesions

During spring 2009, lavender plants (Lavandula stoechas L.) showing a bright yellow mosaic of calico type and light stunting were observed in a commercial nursery in Liguria Province in northern Italy. Of 300 plants inspected, ~2% were symptomatic. Preliminary observations of leaf sap with the transmission electron microscope revealed bacilliform virus-like particles in three symptomatic plants, whereas no virus-like particles were observed in asymptomatic plants. The same symptomatic plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, and Alfalfa mosaic virus (AMV). All three plants reacted positively against AMV antibodies, but not the other antibodies. A crude sap extract obtained from a single symptomatic plant, hereafter referred to as the Lst isolate, was prepared by macerating 1 g of fresh leaves in 4 ml of sodium phosphate 0.03 M, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Sap extract was mechanically inoculated onto a set of herbaceous hosts. Inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in bean (Phaseolus vulgaris L., cv. Borlotto) and cowpea (Vigna unguiculata L., cv. Black eye); necrotic local lesions followed by systemic necrosis in broad bean (Vicia faba L., cv. Super Simonia) and tomato (Solanum lycopersicum L., cv. San Marzano); and bright yellow mosaic (calico type) in basil (Ocimum basilicum L., cv. Gigante). To sequence the entire genome of the Lst isolate, total RNA was extracted from infected samples with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to AMV-specific reverse transcription-PCR by using four primer pairs for each genomic RNA of overlapping oligonucleotides according to the complete genome sequence of AMV 425L isolate (GenBank No. L00163 for RNA1, X01572 for RNA2, and K03542 for RNA3). The 5′- and 3′-terminals regions of each RNA were amplified with the strategy described by Lozano et al. (1) and specific AMV oligonucleotides designed for the corresponding viral RNA. The complete genome of the AMV-Lst isolate comprised 3,643 nucleotides for RNA1 (No. FN667965), 2,593 nucleotides for RNA2 (No. FN667966), and 2,038 nucleotides for RNA3 (No. FN667967). Comparative sequence analyses revealed that the AMV-Lst isolate from Italy shared overall nucleotide sequence identities with the AMV isolate 425L of 97.1, 95.5, and 94.7% for RNA1, 2, and 3, respectively. P1 and P2 replicase genes and the movement protein and coat protein (CP) genes of AMV-Lst isolate showed, respectively, 97.2, 95.1, 96.2, and 97.8% identity with those from the 425L isolate. The AMV-Lst CP gene was shorter by nine nucleotides compared with the CP gene of 425L. A phylogenetic tree, obtained with neighbor-joining and maximum likelihood methods, showed that the Lst isolate grouped within subgroup I of AMV isolates confirmed that the differences between subgroups I and II correlate mainly with the geographic origin of isolates (2). Lst represents the first Italian isolate of AMV completely sequenced, and to our knowledge, this is the first report of this virus in L. stoechas. References: (1) G. Lozano et al. Arch. Virol. 151:581, 2006. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000.

scholarly journals The spreading of Alfalfa mosaic virus in lavandin in Croatia

2014 ◽  
Vol 29 (2) ◽  
pp. 115-122 ◽  
Author(s):  
Ivana Stankovic ◽  
Karolina Vrandecic ◽  
Jasenka Cosic ◽  
Katarina Milojevic ◽  
Aleksandra Bulajic ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Chenopodium Quinoa ◽  
Alfalfa Mosaic Virus ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
Lavandula Stoechas ◽  
Pcr Products ◽  
Test Plants ◽  
Das Elisa

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.

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