Bean Yellow Mosaic Virus Isolates Research Articles

Diversity of the Polish isolates of bean yellow mosaic virus (BYMV) isolated from broad bean

Bean yellow mosaic virus (BYMV) is one of the most widespread and damaging viruses of cultivated legumes in the world. In order to characterise BYMV isolates detected in ten broad bean plants in Poland, total nucleic acids were isolated, a fragment of the gene encoding the polyprotein was amplified by RT-PCR and then sequenced. The sequences of the coat protein (CP) gene of the three Polish isolates (E1, E2, E3) were compared with 33 sequences retrieved from the GenBank database. The sequences of the tested isolates showed 81,8–99,8% identity at the nucleotide level and 84,9–99,6% at the amino acid level with other BYMV isolates. The analysis performer based on CP sequences confirmed the genetic diversity of the BYMV population and revealed the presence of three phylogenetic groups. The Polish isolates were classified into I (E1 and E2) and III (E3) group.

ФІЛОГЕНЕТИЧНИЙ АНАЛІЗ ІЗОЛЯТІВ ВІРУСУ ЖОВТОЇ МОЗАЇКИ КВАСОЛІ ІЗГЛАДІОЛУСІВВУКРАЇНІ

Bean yellow mosaic virus (BYMV) is the one of most prevalent and harmful viruses infecting gladiolus plants worldwide. The aim of the study was to perform phylogenetic analysis of two Ukrainian BYMV isolates from gladioli grown in different regions of Ukraine and determine phylogenetic relationships of the isolates to other BYMVs. Visual diagnostics, enzyme-linked immunosorbent assay in DAS-ELISA modification, total RNA extraction, RT-PCR, phylogenetic analysis and statistical data analysis were used in this research. The results of the studies showed that gladioli of variety Pamyat’ (Poltava region, 2018) and var. Galyna Zelenobirska (Kyiv region, 2020) with symptoms of chlorotic stripes and mosaics on the leaves, flower color breaks are infected by BYMV. Fragments of the CP gene sequence of Ukrainian gladiolus isolates of bean yellow mosaic virus, named GlMP-18 (MK416160) and BYMV-Gl-SV-20 (MZ286966) were sequenced and deposited to the NCBI GenBank. Nucleotide sequences of these isolates corresponding to 578 nt of the coat protein gene (CP) located at the position 8727–9305 of the viral genome and amino acid sequences were compared with 40 known BYMV isolates/strains. Phylogenetic analysis demonstrated that GlMP-18 and BYMV-Gl-SV-20 have identity of nucleotide sequence 100% and amino acid sequence 100% with each other. Both Ukrainian isolates clustered with the Monocot group. The identity of the СР gene sequences of the two gladiolus isolates GlMP-18 and BYMV-Gl-SV-20 from geographically remote regions of the country and in different years of selection indicates a common origin of isolates and probable their distribution of planting material.

Co-infection of bean yellow mosaic virus and cucumber mosaic virus in Gladiolus sp. plants: phylogenetic analysis of Ukrainian isolates

Co-infection of bean yellow mosaic virus (BYMV) and cucumber mosaic virus (CMV) was confirmed in gladioli var. Lehenda Kyieva, Pamyat’, Victoria Skvyrska by TEM, DAS-ELISA, and RT-PCR. The symptoms of co-infection were chlorotic streaks and mosaics on the leaves, flower colour breaking. The infectious nature of the symptoms was proven by biotesting method (Koch’s postulates). Partial CP gene sequences of three gladiolus BYMV isolates BYMV-Gl-LeK-19 (MZ286969), GlMP-18 (MK416160), BYMV-Gl-Skv-20 (MZ286967) and three CMV isolates CMV-Gl-LeK-19 (MW847707), CMV-Gl-MP-19 (MW847708), CMV-Gl-Skv-20 (MW847710) were deposited to the NCBI GenBank. BYMV isolates are clustered with Monocot group, having identity with the isolates from this group of 94.8–99.4% nt and 95.3–100% aa. CMV isolates belong to subgroup IA and share with them identity of 81.2–99.3% nt and 82.5–97.6% aa. Analysis revealed high level of CP nucleotide sequence diversity for BYMV-Gl-LeK-19, BYMV-Gl-Skv-20 and CMV-Gl-Skv-20 as some identified substitutions were nonsynonymous.

Phylogenetic analysis of a Bean yellow mosaic virus isolate from Iran and selecting the phylogenetic marker by comparing the individual genes and complete genome trees of BYMV isolates

Bean yellow mosaic virus (BYMV) is an economic potyvirus with worldwide distribution and a broad host range. Recently, phylogenetic studies of BYMV isolates have indicated the importance of comparing the full genome sequences to obtain a reliable classification. However, most available isolates in public databases are partially sequenced, and virologists continue to prefer partial genomes or individual genes for phylogenetic studies. As such, to avoid unbiased classification, phylogenetic studies should be based on either phylogenetic gene markers or a multigene dataset instead of just one random gene. In the present study, we sequenced the full genome of a BYMV isolate collected from an Iranian faba bean field using the Illumina Novaseq 6000 platform. The phylogenetic analysis revealed that the Iranian isolate is closely related to three isolates originating from faba bean fields in Japan, Australia and Germany. To identify the phylogenetic markers of BYMV, the topology distance between reconstructed trees based on individual genes and the whole genome was calculated using topology congruence tests considering only the topology of the compared trees and a character congruence test taking into account the characters, tree topology and estimated bootstrap support value. Accordingly, nuclear inclusion proteins a and b (NIa and NIb) and viral protein genome linked (VPg) were selected as evolutionary markers which can be used as appropriate representations of the full genome in phylogenetic studies. Furthermore, supermatrix approach was used to compare all BYMV isolates that have been fully or partially sequenced. The results demonstrate that all submitted BYMV strains can be divided into 10 main monophyletic clades with high bootstrap values; these are named BYM-1 through BYM-10 to avoid confusion with previously introduced nomenclature. Moreover, the results reveal that clustering based on phylogenetic analysis and their original host is not completely consistent, which supports recent phylogenetic studies based on available whole genome sequences.

УРАЖЕНІСТЬ РОСЛИН ГЛАДІОЛУСІВ ВІРУСОМ ЖОВТОЇ МОЗАЇКИ КВАСОЛІ, ВІРУСОМ ОГІРКОВОЇ МОЗАЇКИ ТА ВІРУСОМ КІЛЬЦЕВОЇ ПЛЯМИСТОСТІ ТЮТЮНУ НА ТЕРИТОРІЇ ДЕЯКИХ ПІВНІЧНИХ І ЦЕНТРАЛЬНИХ ОБЛАСТЕЙ УКРАЇНИ

Gladioli can be affected with 15 species of viruses, which cause significant economic losses to both floriculture and agriculture. The most prevalent and harmful for gladioli are Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV), circulating on the territory of Ukraine on vegetable, legume and other crops, and also Tobacco ringspot virus (TRSV), which is included into the List of regulated pests of Ukraine and is the subject to strict control. The aim of the work was to conduct testing of gladiolus plants for the presence of viral infection symptoms and to test them for the affection with the most widespread and dangerous viruses, namely: BYMV, CMV, TRSV. Visual diagnostics, enzyme-linked immunosorbent assay in DAS-ELISA modification, transmission electron microscopy method and statistical data analysis were used in this research. The results of the studies showed absence of TRSV in all tested samples. For the first time in Ukraine, it has been established that gladioli are infected by Bean yellow mosaic virus. Its circulation on gladioli has been registered in Poltava, Kyiv and Sumy regions. Gladioli were also found to be affected by CMV or mixed infection of these pathogens. Gladioli infection by BYMV and CMV in Kyiv region is 88.2% and 93.8%, in Poltava – 69.2% and 55.5%, in Sumy – 66.6% and 0%, respectively. Typical symptoms on gladiolus plants caused by CMV and BYMV isolates are leaf chlorotic stripes and flower color break, less often – spotting on the leaves and plant stunting. It has been revealed that affection of gladioli by CMV and BYMV can be asymptomatic. The diversity, nature and course of viral infections in gladioli demonstrate the relevance of further research and their monitoring in Ukraine.

First Report of Bean Yellow Mosaic Virus Infecting Nasturtium (Tropaeolum majus) in Hawaii

Garden nasturtium (Tropaeolum majus L.) is an herbaceous annual plant commonly used as a garden ornamental, culinary herb, or a medicinal plant. It has become naturalized in many locations and is currently considered an alien invader in New Zealand, Lord Howe Island, and Hawaii (U.S. Forest Service 2012). Bean yellow mosaic virus (BYMV) is an aphid-transmitted nonpersistent potyvirus with a wide host range (Parrella and Lanave 2009). In February 2018, we observed nasturtium plants exhibiting severe chlorotic mosaic symptoms at the Kula Botanical Gardens on Maui, HI (20°44′40.01″ N, 156°19′03.00″ W). We tested six samples that had severe yellow mosaic and leaf distortion symptoms and three leaves from nonsymptomatic plants using potyvirus-specific ELISA (Agdia, Elkhart, IN) and universal potyvirus-specific reverse transcription polymerase chain reaction (RT-PCR) with potyvirus nuclear inclusion body (NIb) primers NIb2F and NIb3R (Zheng et al. 2010). All of the symptomatic nasturtium samples tested positive for potyvirus infection in both tests, whereas three nonsymptomatic nasturtium leaves tested negative in both tests. To identify the specific potyvirus involved, amplicons (309 bp) of the NIb region that were generated by RT-PCR using universal potyvirus-specific primers were sequenced (accession MH422546). BLASTn analysis of the sequences showed that this virus shared 93% nucleotide identity with a BYMV isolate from Australia (HG970862), and BLASTx analysis showed it shared 93% amino acid identity with a polyprotein of BYMV isolate from Australia (CDO67686). Two additional sets of PCR primers were used to further characterize the isolate: BYMV NIb-specific primers BYMV-NIb-3 (5′-CCACTGTTATTCCCTTTAAAC-3′) and BYMV-NIb-5 (5′-AATAAGTTCTACAGTTTGCACC-3′), and BYMV coat protein (CP)-specific primers BYMV-CP-3 and BYMV-CP-5 (Wang et al. 2017). BLASTn analysis of the 257-bp NIb amplicon (accession MH422547) shared 94% identity to an Australian isolate of BYMV (HG970847), and the 595-bp CP amplicon (accession MH422548) shared 98% identity to another Australian isolate of BYMV (HG970860). BLASTx analysis of the 257-bp NIb amplicon shared 93% identity to an Australian isolate polyprotein of BYMV (AHQ95554), and the 595-bp CP amplicon shared 98% identity to another Japan isolate polyprotein of BYMV (BAA93683). All six symptomatic samples also tested positive for BYMV by triple-antibody sandwich ELISA using a BYMV-specific antibody (DSMZ, Braunschweig, Germany). From March to June 2018, 24 additional nasturtium samples were collected from two different locations on Maui, and all tested positive for BYMV infection by RT-PCR and ELISA. All of the symptomatic nasturtium samples tested positive for BYMV infection in both tests, while three nonsymptomatic nasturtium leaves from other plants tested negative in both tests. To our knowledge, this is the first report of BYMV infecting nasturtium plants in Hawaii. Nasturtium is a widely grown herbaceous ornamental in Hawaii. It could be harboring BYMV in the off-season and moving it to cultivated legume plants. Therefore, further study is needed to determine whether legume aphids can transmit BYMV from nasturtium to healthy legume plants. In addition, further studies on the distribution of BYMV-infected nasturtium and identification of other BYMV-infected plants in the Hawaiian Islands are needed.

Production of polyclonal antibodies against the BYMV isolate infecting gladiolus crop naturally and development of polyclonal antibodies based serological diagnostic system

In this study a bean yellow mosaic virus isolate infecting gladiolus crop with leaf mosaic and color breaking symptoms on flower was characterized based on mechanical transmission, symptomatology on hosts, and viron particle structure and capsid protein molecular weight determination. The virus isolate was successfully purified from the propagating host and purified viron particles were used for the production of polyclonal antibodies for the development of a cost effective and sensitive serological diagnostic system. The produced polyclonal antibodies would be useful for the assessment of BYMV infection in gladiolus crop and seed materials.

Molecular characterization of viruses infecting canna in Russia

Canna yellow streak virus (CaYSV), Canna yellow mottle virus (CaYMV), Bean yellow mosaic virus (BYMV), and Cucumber mosaic virus (CMV) were detected by reverse transcription-polymerase chain reaction in 16 local and introduced canna cultivars grown in the collection of the Nikita Botanical Gardens, Crimea. CaYSV was detected in all the cultivars tested, and most of the cultivars had mixed infection with two or three viruses. Analysis of the 3′-terminal genomic sequences revealed a high degree of diversity among isolates of CaYSV and BYMV suggesting their multiple introductions in the Gardens. Phylogenetic analysis of the coat protein (CP) gene showed that all the Crimean BYMV isolates together with the Chinese and the Indian ones from canna formed a separate cluster of canna-adapted isolates. Unique amino acid substitutions at positions 58, 70, 106, 168 and 215 of the CP were found in all isolates from canna that may be relevant to the adaptation of BYMV to this host. The sequences of the CaYMV isolates were highly conserved. The sequence identities of their ORF3 genomic segment ranged from 98.7–100% and from 97.7–100% at the nucleotide and amino acid levels, respectively. The CMV isolate was most closely related to the isolate Fny (D10538) from muskmelon. This is the first report of CaYSV, CaYMV, BYMV and CMV on canna in Russia confirming their worldwide distribution on this culture.

Plant virology and next generation sequencing: experiences with a Potyvirus.

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.

Bean Yellow Mosaic Virus in Soybean from Argentina

AbstractAn association of Bean yellow mosaic virus (BYMV), (genus Potyvirus), with yellow mosaic leaf symptoms on soybean in Argentina was determined by enzyme‐linked immunosorbent assay, a result confirmed by transmission electron microscopy and reverse transcription‐polymerase chain reaction using a degenerate primer targeting the C‐terminal region of BYMV′s NIb. The sequence analysis showed that the isolate had 80.6–91.6% identities with other BYMV isolates from different strain groups, indicating that it is a strain of BYMV.

New Record of Association of Bean yellow mosaic virus with Mosaic Disease of Vicia faba in India

Faba bean (Vicia faba L.) is a species of bean belonging to the family Fabaceae extensively cultivated in Africa and Asia for its high nutritional values. In India and Nepal, it is called as “Bakulla”, its young pods are consumed as a green vegetable. It is used as human food in developing countries and as animal feed in industrialized countries. It is rich in l-dopa and used in medicine to treat Parkinson’s disease and to control hypertension (http://en.wikipedia.org/wiki/Vicia_faba). During a survey in February of 2011 severe mosaic symptoms were observed on about 35 % (38/109) V. faba plants growing in kitchen gardens at Lucknow. The symptoms were mild to severe (Fig. 1a) and infected plants were dwarf as compared to healthy plants. Since infection of Bean yellow mosaic virus (BYMV) is reported on V. faba [6], therefore, infection of potyvirus was suspected. Fig. 1 aBean yellow mosaic virus (BYMV) infected V. faba leaf showing mosaic symptoms. b Phylogenetic analysis of nucleotide sequence of Bean yellow mosaic virus isolate-1 ({type:entrez-nucleotide,attrs:{text:JF798580,term_id:341650451,term_text:JF798580}} ... For the detection of BYMV, total RNA was isolated from leaves of naturally infected V. faba plants from two locations using the RaFlex total RNA isolation kit (GeNei, India) and Reverse transcription-polymerase chain reaction (RT-PCR) were performed using a pair of degenerate primers (Pot 1 and Pot 2) capable of amplifying a 1.6–2.1 Kb fragment from 3′ end of the genome (virion protein gene and part of the NIb gene) of 17 species of the Potyviridae [4]. RT-PCR amplification resulted in the expected size (~1.6 Kb) amplicons from symptomatic V. faba (4/5) similar to a positive control (potyvirus infected gladiolus) but not from the healthy one. An additional specific band of ~0.8 Kb was also obtained in all the symptomatic samples. These results indicated presence of potyvirus infection in V. faba. For identification of potyvirus associated with mosaic symptoms of V. faba, the amplicons of ~1.6 Kb obtained from two symptomatic samples were sequenced directly and deposited in GenBank under Accession numbers: {type:entrez-nucleotide,attrs:{text:JF798580,term_id:341650451,term_text:JF798580}}JF798580 (isolate-1) and {type:entrez-nucleotide,attrs:{text:JN692500,term_id:779787813,term_text:JN692500}}JN692500 (isolate-2). BLASTn analysis of isolate-1 from V. faba ({type:entrez-nucleotide,attrs:{text:JF798580,term_id:341650451,term_text:JF798580}}JF798580) revealed highest sequence identities 87 % with polyprotein gene of various BYMV strains: from Japan ({type:entrez-nucleotide,attrs:{text:AB097089,term_id:40645273,term_text:AB097089}}AB097089, {type:entrez-nucleotide,attrs:{text:AB079782,term_id:32351753,term_text:AB079782}}AB079782, {type:entrez-nucleotide,attrs:{text:AB097090,term_id:40645275,term_text:AB097090}}AB097090); China ({type:entrez-nucleotide,attrs:{text:AJ311371,term_id:29134762,term_text:AJ311371}}AJ311371) and Taiwan ({type:entrez-nucleotide,attrs:{text:AM884180,term_id:198446713,term_text:AM884180}}AM884180). It also showed 86 % sequence identities with strains of BYMV: M11 ({type:entrez-nucleotide,attrs:{text:AB079886,term_id:109638255,term_text:AB079886}}AB079886) and IbG ({type:entrez-nucleotide,attrs:{text:AB079887,term_id:109638257,term_text:AB079887}}AB079887) from Japan and Vanilla ({type:entrez-nucleotide,attrs:{text:AY845011,term_id:61740434,term_text:AY845011}}AY845011, {type:entrez-nucleotide,attrs:{text:AY845012,term_id:61740436,term_text:AY845012}}AY845012) from India. The sequence identity of isolate-2 ({type:entrez-nucleotide,attrs:{text:JN692500,term_id:779787813,term_text:JN692500}}JN692500) was highest 86 % with isolates-1 ({type:entrez-nucleotide,attrs:{text:JF798580,term_id:341650451,term_text:JF798580}}JF798580) and 85–84 % with BYMV strains ({type:entrez-nucleotide,attrs:{text:AB079782,term_id:32351753,term_text:AB079782}}AB079782, {type:entrez-nucleotide,attrs:{text:AB097089,term_id:40645273,term_text:AB097089}}AB097089, {type:entrez-nucleotide,attrs:{text:AY845012,term_id:61740436,term_text:AY845012}}AY845012, {type:entrez-nucleotide,attrs:{text:AM884180,term_id:198446713,term_text:AM884180}}AM884180, {type:entrez-nucleotide,attrs:{text:AJ311371,term_id:29134762,term_text:AJ311371}}AJ311371, {type:entrez-nucleotide,attrs:{text:AY845011,term_id:61740434,term_text:AY845011}}AY845011, {type:entrez-nucleotide,attrs:{text:AB097090,term_id:40645275,term_text:AB097090}}AB097090, {type:entrez-nucleotide,attrs:{text:AB079886,term_id:109638255,term_text:AB079886}}AB079886, {type:entrez-nucleotide,attrs:{text:AB079887,term_id:109638257,term_text:AB079887}}AB079887). The 86–87 % sequence identities of isolates under study with other BYMV strains reported all over the world and in accordance with the species demarcation criteria and identification guidelines for potyviruses [1] suggested the virus isolates of V. faba under study as two isolates of BYMV. Phylogenetic analysis of nucleotide sequence of the BYMV isolates-1 and 2 of V. faba ({type:entrez-nucleotide,attrs:{text:JF798580,term_id:341650451,term_text:JF798580}}JF798580 and {type:entrez-nucleotide,attrs:{text:JN692500,term_id:779787813,term_text:JN692500}}JN692500) with the various BYMV strains of V. faba and other host species reported all over the world was performed using MEGA 4.0 tool. The BYMV strains clustered into two clusters, where the isolates under study clustered in cluster 1 along with Indian BYMV strains of Vanilla fragrans ({type:entrez-nucleotide,attrs:{text:AY845011,term_id:61740434,term_text:AY845011}}AY845011, {type:entrez-nucleotide,attrs:{text:AY845012,term_id:61740436,term_text:AY845012}}AY845012). The analysis of the BYMV isolate-1 ({type:entrez-nucleotide,attrs:{text:JF798580,term_id:341650451,term_text:JF798580}}JF798580) and isolate-2 ({type:entrez-nucleotide,attrs:{text:JN692500,term_id:779787813,term_text:JN692500}}JN692500) also revealed closest relationships with BYMV strains of Gentiana scabra ({type:entrez-nucleotide,attrs:{text:AB097089,term_id:40645273,term_text:AB097089}}AB097089, {type:entrez-nucleotide,attrs:{text:AB079782,term_id:32351753,term_text:AB079782}}AB079782, {type:entrez-nucleotide,attrs:{text:AB097090,term_id:40645275,term_text:AB097090}}AB097090) from Japan; close relationships with BYMV strains of G. scabra ({type:entrez-nucleotide,attrs:{text:AB079886,term_id:109638255,term_text:AB079886}}AB079886, {type:entrez-nucleotide,attrs:{text:AB079887,term_id:109638257,term_text:AB079887}}AB079887) from Japan, Eustoma russellianum ({type:entrez-nucleotide,attrs:{text:AM884180,term_id:198446713,term_text:AM884180}}AM884180) from Taiwan, {type:entrez-nucleotide,attrs:{text:AJ311371,term_id:29134762,term_text:AJ311371}}AJ311371 from China, V. fragrans ({type:entrez-nucleotide,attrs:{text:AY845011,term_id:61740434,term_text:AY845011}}AY845011, {type:entrez-nucleotide,attrs:{text:AY845012,term_id:61740436,term_text:AY845012}}AY845012) from India and distinct relationship with other BYMV strains of V. faba reported from Australia and Japan (Fig. 1b). Based on nucleotide sequence identities and phylogenetic relationships of the virus isolates of V. faba with various strains of BYMV, the virus isolates of V. faba under study were identified as two BYMV isolates. These isolates did not differ in symptoms on V. faba plant. The occurrence of BYMV on gladiolus in India [5], Japan [6], V. fragrans in India [2], V. faba in Australia [3], Egypt [7] and Iran [8] were reported previously. However, to our knowledge this is the first report of the natural occurrence of BYMV on V. faba in India.

First Report of Bean yellow mosaic virus in Alaska from Clover (Trifolium spp.)

In mid-June 2008, distinct mosaic leaves were observed on a cluster of clover (Trifolium spp.) with light pink and white flowers growing at the edge of a lawn in Palmer, AK. Virus minipurification from leaves of affected clover and protein extractions on a polyacrylamide electrophoresis implicated a ~35-kDa putative coat protein (CP). Subsequent western blots and ELISA with a universal potyvirus antiserum (Agdia Inc., Elkhart, IN) confirmed potyvirus identity. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA) from the same plant were used for reverse transcription (RT)-PCR. Three sets of degenerate primers that targeted potyvirus-specific genes, HC-Pro (helper component protease) and CI (cylindrical inclusion protein) and the genomic 3'-terminus that included a partial NIb (nuclear inclusion), CP (coat protein), and UTR (untranslated region), produced the expected PCR segments (~0.7, ~0.7, and ~1.6 kbp, respectively) on 1% agarose gels (1). Direct sequencing of the HC-Pro (GenBank No. GQ181115), CI (GQ181116), and CP (GU126690) segments revealed 98, 97, and 99% nucleotide identities (no gaps), respectively, to Bean yellow mosaic virus (BYMV)-chlorotic spot (CS) strain, GenBank No. AB373203. The next closest BYMV percent identity comparisons decreased to 79% for HC-Pro (GenBank No. DQ641248; BYMV-W), 79% for CI (U47033; BYMV-S) partial genes, and 96% for CP (AB041971; BYMV-P242). Mechanical inoculations of purified virus preparations produced local lesions on Chenopodium amaranticolor Coste & A. Reyn. (2 of 5) and C. quinoa Willd. (6 of 7), and mosaic on Nicotiana benthamiana Domin (5 of 5). BYMV was specifically confirmed on tester plants using a double-antibody sandwich (DAS)-ELISA BYMV (strain 204 and B25) kit (AC Diagnostics, Inc., Fayetteville, AR) as directed. The absence of another potyvirus commonly found in clover, Clover yellow vein virus (ClYVV), was verified in parallel DAS-ELISA ClYVV assays (AC Diagnostics, Inc). The BYMV isolate was maintained in N. benthamiana, and virion or sap extracts inoculated to the following host range (number of infected/total inoculated plants [verified by BYMV ELISA]): Cucumis sativus L. 'Straight Eight' (0/5), Gomphrena globosa L. (1/4), Nicotiana clevelandii A. Gray (4/7), Phaseolus vulgaris L. 'Bountiful' (1/3), Pisum sativum L. (Germplasm Resources Information Network Accession Nos. -PI 508092 (8/12), -W6 17525 (13/13), -W6 17529 (0/13), -W6 17530 (13/14), -W6 17537 (0/12), -W6 17538 (0/12), and -W6 17539 (0/21), Tetragonia tetragoniodes (2/2), Trifolium pretense L. 'Altaswede' (6/10), T. repens L. 'Pilgrim' (0/8), and Vicia faba L. (1/3). All infected plants had symptoms ranging from systemic mosaic (T. pretense, P. sativum) to leaf distortions (N. clevelandii, Tetragonia tetragoniodes). Interestingly, the host range and genomic sequences of the BYMV Alaskan strain resemble the BYMV-CS (chlorotic spot) strain that was originally isolated from a diseased red clover (T. pretense) plant in Japan more than 40 years ago (2). Although BYMV occurs worldwide and has a wide host range in dictoyledonous and monocotyledonous plants (3), to our knowledge, this is the first report of a natural occurrence of BYMV in Alaska. The incidence and distribution of BYMV in clover and other plant species are not known in Alaska.

Variability of Bean yellow mosaic virus isolates in the Czech Republic

Three isolates of Bean yellow mosaic virus (BYMV) from the Czech Republic originating from gladiolus plants were examined according to their biological and molecular characteristics. Partial sequence of coat protein-nuclear inclusion protein b (CP-NIb) coding region (768 bp) of these isolates were determined and compared with the corresponding sequences of different BYMV isolates obtained from GeneBank. Phylogenetic analysis showed that the Czech BYMV isolates were distributed across the three groups of phylogenetic tree. Their placement was not dependent on the geography or host plant.

Cross-Protection Against Bean yellow mosaic virus (BYMV) and Clover yellow vein virus by Attenuated BYMV Isolate M11

Attenuated isolate M11 of Bean yellow mosaic virus (BYMV), obtained after exposing BYMV-infected plants to low temperature, and its efficacy in cross-protecting against infection by BYMV isolates from gladiolus, broad bean (Vicia faba) and white clover (Trifolium repens) was assessed with western blotting and reverse transcription-polymerase chain reaction. The level of cross-protection varied depending on the challenge virus isolates. Cross-protection was complete against BYMV isolates from gladiolus, but incomplete against BYMV isolates from other hosts. M11 also partially cross-protected against an isolate of Clover yellow vein virus. A comparison of the nucleotide sequence of M11 and those of BYMV isolates from gladiolus and from other hosts showed higher homology among gladiolus isolates than the homology between gladiolus isolates and nongladiolus isolates. In the phylogenetic trees, constructed using the nucleotide sequences of an overall polyprotein of the genomes, five gladiolus isolates clustered together, completely separated from the three BYMV isolates from other hosts. A comparison of the amino acid sequences between M11 and its parental isolate IbG, and analysis of recombinant infectious clones between M11 and IbG revealed that an amino acid at position 314 was involved in the attenuation of BYMV.

First Report of Bean yellow mosaic virus from Diseased Lupinus luteus in Eastern Washington.

Lupine accessions from the Cool Season Food Legume Seed Collection are grown for seed regenerations in Pullman, WA by the Agricultural Research Service, Western Regional Plant Introduction Station. Selected seed was germinated in the greenhouse and assayed by indirect ELISA using antiserum for potyvirus group detection (Agdia, Inc., Elkhart, IN). Healthy transplants were grown for seed collection on outside plots. In July of 2005, more than 90% of 307 Lupinus luteus L. transplants developed severe yellowing, necrosis, and stunting with an estimated 5% plant death. Plants were heavily infested with aphids and leaf sap was serologically positive for potyvirus. Partially purified virus preparations from infected plants contained filamentous particles and a 35-kDa protein that reacted with universal potyvirus antiserum on western blots. Reverse transcription (RT)-PCR using potyvirus universal primers (2) and cDNA derived from virion RNA generated a ~1.7-kbp product that was cloned and sequenced. The sequenced portion of the genomic RNA contained 1,610 nucleotides (nt) on its 3'-terminus (GenBank Accession No. EU144223) that included a partial nuclear inclusion protein, NIb, (1 to 637 nt) with the conserved amino acid (aa) replicase motif GDD (131 to 139 nt), the coat protein (CP) gene of 821 nt (638 to 1,459 nt), and a 171-nt untranslated region (1,460 to 1,630 nt) attached to a poly(A)tail. The CP sequence contained a NAG motif instead of the DAG motif commonly associated with aphid transmission. Searches in the NCBI GenBank database revealed that the CP aa and nt sequences contained conserved domains with isolates of Bean yellow mosaic virus (BYMV). A pairwise alignment (ClustalX) (4) of the CP aa from 20 BYMV isolates with the BYMV-Pullman isolate revealed identities from 96% (BYMV-S, U47033) to 88% (BYMV-MI [X81124)] -MI-NAT [AF434661]). This meets the species demarcation criteria of more than ~80% identity for inclusion with BYMV (1). Virion mechanical inoculations resulted in local lesions on Chenopodium amaranticolor Coste et Reyn and C. quinoa Willd., necrotic blotches on Phaseolus vulgaris L., and yellow spots and systemic movement in L. succulentus Douglas ex. K. Koch, L. texensis 'Bluebonnet', and L. texensis 'Maroon'; BYMV was confirmed by western blots and ELISA. The experimental inoculations represent the first documented report of BYMV in the annual L. succulentus and biennial L. texensis species. Since BYMV is seedborne and transmitted by many aphid species (3), it is possible that several lupine transplants escaped potyvirus detection, and secondary transmission of BYMV to plants occurred by aphids. During the 1950s, BYMV was confirmed in several annual lupines grown as crops in the southeastern United States (3). To our knowledge, this is the first report of BYMV occurring naturally in a lupine species in Washington. BYMV is a destructive virus to lupine species worldwide and has a wide host range in Fabaceae. This research directly contributes toward the maintenance of virus-free lupine seed for distribution to scientists focusing on lupine research.

Molecular characterization of a Bean yellow mosaic virus isolate from Syria

Bean yellow mosaic virus (BYMV, genus Potyvirus, family Potyviridae ) was studied by comparing sequences from the coat protein (CP) gene of a Syrian isolate with sequences of six other isolates from the NCBI database. A homology tree of the CP sequences was developed using DNAMAN Software. BYMV isolates were grouped into two clusters of which the first comprised the Syrian isolate together with the Indian, Australian and Japanese isolates, and the second the BYMV isolates from China, the Netherlands and the USA. Moreover, the homology tree showed that the Syrian isolate was very close to the Indian one, with 99% homology.

Three Previously Unrecorded Viral Diseases of Astilbe, Fuschia, and Thermopsis Species in Minnesota

Interest in virus diseases of perennial ornamentals has been increasing because of their increasing monetary value, because wholesale producers perceive an advantage in marketing disease-free stock, and because widespread international movement of these plants carries the risk of introduction of exotic viruses. In an ongoing study to identify and document viral diseases of perennial ornamentals used in the United States commercial horticultural industry, three virus-like diseases of astilbe (Astilbe chinensis), fuschia (Fuschia cv. Gartenmeister) and false lupine (Thermopsis caroliniana) occurring in Minnesota were investigated. Symptomatic plants were selected from lots in commercial greenhouses and garden centers in several locations in Minnesota. Astilbe with systemic chlorosis was found to be infected with Tobacco ringspot virus (TRSV). Fuschia with leaf mottling and leaf deformation was found to be infected with Cucumber mosaic virus (CMV), and false lupine with mosaic and leaf deformation symptoms was found to be infected with Bean yellow mosaic virus (BYMV). Identification of the three viruses was based on: 1) virion presence and morphology in partially purified leaf extracts using electron microscopy (EM) and immunosorbent electron microscopy (1); 2) enzyme-linked immunosorbent assay using crude leaf extracts; and 3) biological properties, including symptoms produced in indicator plants. Antisera to BYMV (ATCC PVAS-368), CMV (ATCC PVAS-30), and TRSV (ATCC PVAS-157) were obtained from the American Type Culture Collection, Manassas, VA. No other virus-like particles were observed with EM in partially purified leaf extracts of the three plants, no virus-like particles were observed in similar preparations from asymptomatic plants, and indicator plant tests did not indicate the presence of any other mechanically transmissible viruses. The TRSV isolate from astilbe and the BYMV isolate from false lupine produced typical symptoms on indicator plants susceptible to known isolates of these two viruses. The CMV isolate from fuschia was similar to previously described isolates of CMV (2) in most respects and was readily transmitted in a nonpersistent manner by Myzus persicae, but was unusual in that it did not infect Nicotiania benthamiana, N. glutinosa, and tomato, which are normally highly susceptible to infection by CMV. The identity of the fuschia CMV isolate was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR) amplification with CMV-specific oligonucleotide primers (3). The PCR product was of the predicted size (500 bp) and was cleaved by restriction digestion with EcoRI, suggesting that the fuschia virus is a Type II CMV isolate (3). To my knowledge, this is the first report of TRSV infection in astilbe, CMV infection in fuschia, and of a viral disease of false lupine.

Selection of an attenuated isolate of Bean yellow mosaic virus for protection of dwarf gentian plants from viral infection in the field

An attenuated isolate of Bean yellow mosaic virus (BYMV), designated B-33, that caused barely any mottling on the leaves of gentian plants (Gentiana scabra) was isolated from dwarf gentian plants. Infection with this isolate did not cause severe symptoms in several leguminous plants or in prairie gentian (Eustoma grandiflorum) plants. Dwarf gentian plants that had been infected with an inoculum of partially purified B-33 were propagated vegetatively from cuttings without difficulty. The B-33 isolate could be distinguished from other, more virulent, isolates by comparing symptoms on prairie gentian plants and by examining restriction fragment length polymorphism after reverse transcription and amplification by the polymerase chain reaction. In cross-protection tests, dwarf gentian plants that had been infected with B-33 were protected from infection with the virulent BYMV isolate 35-1. Potted plants that had been inoculated with B-33 grew larger and produced more flowers than unprotected plants. Thus, B-33 is an effective agent for controling mosaic and atrophic diseases caused by BYMV in clonal dwarf gentian plants.

Comparison of Pathogenicity and Nucleotide Sequences of 3′-terminal Regions of Bean yellow mosaic virus Isolates from Gladiolus

Sixty-four isolates of Bean yellow mosaic virus (BYMV) from cultivated and naturalized gladioli were divided into two pathogenic groups, necrotic spot (NS) and chlorotic spot (CS) groups on Chenopodium quinoa. NS-type isolates (S-22N and E-24N), CS-type isolates (S-22C and E-92C), and broad bean isolates (Sb-50C and Sb-12C) differed in their pathogenicity on Antirrhinum majus, Nicotiana benthamiana, Phaseolus vulgaris, Spinacia oleracea and Vigna unguiculata. The four gladiolus isolates were different from BYMV-B, -P, -O and C1YVV-N in their pathogenicity on these plants, while the two broad bean isolates were similar to BYMV-B, originally from broad bean. The nucleotide (nt) sequences of the 3′-terminal region of the BYMV RNA genome of the two NS-type isolates, the two CS-type isolates, the two broad bean isolates and BYMV-B, -P and -O were determined. In a phylogenetic tree based on the CP amino acid (aa) sequences, the two NS-type isolates clustered together (identity 98.4% and 98.2% at the nt and aa level, respectively). The two CS-type isolates clustered with BYMV-O (93.2 to 99.3% nt identity and 95.6 to 98.5% aa identity). The two broad bean isolates clustered with BYMV-B (99.0 to 99.5% nt identity and 98.9 to 99.6% aa identity). BYMV-P clustered with BYMV-CS (identity 97.7% and 99.3% at the nt and aa level, respectively). The obtained sequences were compared with those of the 3′-terminal regions of seven published BYMV isolates. In a phylogenetic tree based on deduced aa sequences, BYMV isolates were divided into four clusters.

Bean yellow mosaic potyvirus infection of alternative annual pasture, forage, and cool season crop legumes: susceptibility, sensitivity, and seed transmission

Seven field and 5 glasshouse experiments were done during 1994–98 to determine the relative susceptibilities and sensitivities of a wide range of alternative annual pasture, forage, and crop legumes to infection with isolate MI of bean yellow mosaic virus (BYMV). Seed harvested from some species was also tested for seed transmission of the virus. Seven of 18 genotypes belonging to 17 species of annual pasture and forage legumes evaluated in 2 replicated field experiments were ranked as highly susceptible to BYMV, 7 as susceptible, 2 as moderately resistant, 1 as resistant, and 1 as highly resistant. The most susceptible and sensitive were Biserrula pelecinus, Trifolium cherleri, T. incarnatum, and T. spumosum. Ornithopus sativus was resistant but sensitive, whereas Hedysarum coronarium was highly resistant. H. coronarium was not infected when manually inoculated repeatedly with 3 different BYMV isolates. Seventy-three of the 94 genotypes of 7 crop legume species tested in the same replicated field experiments were ranked as highly susceptible, including 58/68 of Lens culinaris. Of the remaining genotypes, 6 were susceptible, 5 moderately resistant, 9 resistant, and 1 highly resistant. Five other crop legumes were included in other field experiments in which these species were ranked as highly susceptible (1) or resistant (4). Overall, the most susceptible and sensitive crop legume species were Lens culinaris (most genotypes), Lathyrus cicera, L. ochrus, and Vicia narbonensis. Lathyrus sativus (3 genotypes only), V. sativa (4 genotypes), Cicer arietinum, Pisum sativum, and V. faba were resistant to isolate MI, and Lens culinaris ILL7163 was highly resistant. When infected, C. arietinum was ranked as highly sensitive but symptoms within the other resistant crop species varied in sensitivity between genotypes. Extreme resistance was confirmed in Lens culinaris ILL7163 when it was manually and aphid-inoculated repeatedly with 3 different BYMV isolates. When testing seedlings for seed transmission of BYMV, germination on moist paper towels before testing usually proved more effective than growing in soil in the glasshouse. Low rates of seed transmission of BYMV (0.03–1%) were detected in 9 alternative pasture or forage and 3 alternative crop legume species. This is the first report of seed transmission of BYMV in these species. The pasture or forage species with the highest seed transmission rates were T. clypeatum and T. spumosum (both 1%). The crop legume species in which seed transmission was found were L. cicera (0.1%), L. sativus (0.2%), and V. sativa (0.5%). The high susceptibility and sensitivity to BYMV in some alternative annual pasture, forage, and crop legumes is a cause for concern, especially when they are intended for sowing in BYMV-prone high rainfall zones. Seed transmission of BYMV also leads to inadvertent introduction of the virus to new sites.