S. D. Wyatt

The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of begomoviruses

Summary. Polymerase chain reaction (PCR) was applied to detect and establish provisional identity of begomoviruses through amplification of a ∼ 575 bp fragment of the begomoviral coat protein gene (CP), referred to as the ‘core’ region of the CP gene (core CP). The core CP fragment contains conserved and unique regions, and was hypothesized to constitute a sequence useful for begomovirus classification. Virus relationships were predicted by distance and parsimony analyses using the A component (bipartite viruses) or full genome (monopartite viruses), CP gene, core CP, or the 200 5′-nucleotides (nt) of the CP. Reconstructed trees and sequence divergence estimates yielded very similar conclusions for all sequence sets, while the CP 5′-200 nt was the best strain discriminator. Alignment of the core CP region for 52 field isolates with reference begomovirus sequences permitted provisional virus identification based on tree position and extent of sequence divergence. Geographic origin of field isolates was predictable based on phylogenetic separation of field isolates examined here. A ‘closest match’ or genus-level identification could be obtained for previously undescribed begomoviruses using the BLAST program to search a reference core CP database located at our website and/or in GenBank. Here, we describe an informative molecular marker that permits provisional begomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence.

Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses

SummaryThe 3′-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3′-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed.

NL-3 K Strain Is a Stable and Naturally Occurring Interspecific Recombinant Derived from Bean common mosaic necrosis virus and Bean common mosaic virus.

ABSTRACT A strain of Bean common mosaic necrosis virus (BCMNV) from Idaho was identified by enzyme-linked immunosorbent assay using monoclonal antibodies and determined to be similar to the NL-3 D strain (of Drifjhout) by reaction of differential bean cultivars. However, this BCMNV strain (designated NL-3 K) caused earlier and more severe symptoms on bean plants representing host groups 0, 4, and 5. The nucleotide sequence encoding the predicted polyprotein of NL-3 K was 9,893 nucleotides (nt) in length, yielding a peptide with a molecular size of 362.1 kDa compared with a 9,626-nt, 350.9-kDa polyprotein for NL-3 D. Sequence analysis of the putative P1 protein suggests that the NL-3 K strain is a recombinant between NL-3 D and the Russian strain (RU1) of Bean common mosaic virus. The P1 protein of NL-3 K consisted of 415 amino acids compared with 317 for NL-3 D. The first 114 predicted amino acids of the NL-3 K P1 region were 98% identical with RU1. The remaining 301 amino acids of the protein shared only 34% identity with RU1 but were 98% identical with NL-3 D. Primers were designed that flanked the recombination point in the P1 coding sequence of NL-3 K. An amplicon of the expected size was produced by reverse-transcriptase polymerase chain reaction of total nucleic acid extracts of bean plants inoculated with NL-3 K, but not from those with NL-3 D or RU1. The increased symptom severity on selected common bean lines induced by NL-3 K suggests that the P1 gene may play a significant role in pathogenicity and virulence.

In vitro destabilization of plant viruses and cDNA synthesis

DNA copies of a wide range of RNA viruses can be made by the direct addition of appropriately treated, purified virus particles to a reverse transcription reaction. Therefore, many problems associated with RNA isolation can be circumvented. Virus particles can be sufficiently destabilized by adjustments of salt content, buffer, pH or by the use of physical force supplied by a freeze/thaw cycle so that RNA in sufficient quantity and physical condition is available for the synthesis of in some cases, full length cDNAs. cDNAs have been made of viruses in the bromo-, poty-, carla-, ilar-, potex-, tobra and tobamovirus groups. Reported here are experiments with cowpea chlorotic mottle virus and bean common mosaic virus.

Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup

SummaryTwenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 Aff1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3′-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3′-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups.

Chickpea wilt incited by pea streak carlavirus

Pea streak carlavirus (PSV) incited a widespread wilting and yellowing disease of chickpea (Cicer arietinum) in commercial and experimental plantings in the Palouse region of eastern Washington and northern Idaho. Incidence of PSV usually ranged from 0.5 to 5%. Experimental host ranges of several Palouse PSV isolates were confined to the Fabaceae and one species of Amaranthaceae. Systemic necrosis developed in chickpea, lentil (Lens culinaris), pea (Pisum sativum), fenugreek (Trigonella foenum-graecum), and faba bean (Vicia faba), while alfalfa (Medicago sativa), white sweet clover (Melilotus alba), and hairy vetch (Vicia villosa subsp. villosa) were symptomless carriers of PSV

The complete nucleotide sequences of bean common mosaic necrosis virus strains NL-5, NL-8 and TN-1

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) belong to the genus Potyvirus and are probably the most economically important viruses affecting common bean (Phaseolus vulgaris L.). Of the two viruses, BCMNV has been the most important in common bean production in North America, East Africa and Europe due to significant yield losses incurred from infected fields [10]. BCMV and BCMNV are transmitted in a non-persistent manner by several aphid species. All known strains of each virus can be highly seed-transmitted in bean and are distributed worldwide, in large part as a result of movement of contaminated seed [4, 5]. Drijfhout [4] originally separated the two viruses and their associated strains into eight different pathogroups based on host reaction to infection under specific temperature regimes. Strains NL-5 and NL-3 from Africa were originally assigned to the BCMV pathogroup VI by Drijfhout [4], and NL-8 was assigned to pathogroup III according to host responses in 12 different common bean host groups. The TN-1 strain from Tanzania was later assigned to pathogroup VI by Silbernagel [12]. Prior to 1992, all strains of BCMNV were designated as BCMV, at which time Vetten et al. [14] designated stains NL-3, NL-5 and NL-8 as serogroup A (BCMNV). All other known strains were assigned to serogroup B (BCMV) based on serological reactions to two highly specific monoclonal antibodies, and this was further supported by molecular analysis and ultrastructural comparisons [14]. Currently, several isolates of strain NL-3 have been sequenced and are available in GenBank; however, the sequences of strains NL-5, NL-8 and TN-1 are unavailable. Therefore, we report here the complete genome sequences of these three strains of BCMNV. Common bean seeds (variety ‘Dubbele Witte’) infected with NL-5, NL-8 and TN-1, respectively, were germinated in pots containing a mixture of peat moss and perlite (Sun Gro Horticulture Canada Ltd.) in the greenhouse, and plants were observed for symptoms. Tissues from symptomatic seedlings were evaluated for infection with BCMNV by direct ELISA using monoclonal antibodies specific for serogroup A, and also to serogroup B [11] to verify that there was no contamination with any strain of BCMV. Total nucleic acids were extracted from plant tissues, and cDNA was produced by reverse transcription using MMLV reverse transcriptase (Promega Corp., Madison, WI). Thermocycling parameters were optimized, and a final profile was employed that consisted of a single cycle of 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, and 3 min at 72 C; and a final extension for 7 min at 72 C. The initial reactions included oligo-dT24 as the reverse primer and a degenerate forward primer, 5’-GAA YAG CAA TGC NAT AG-3’, that produced a fragment approximately 3425 bp in length. The degenerate primer begins at nucleotide 6206, located in the NIa protein region. Subsequent reactions to complete the genome sequence were carried out using custom primer combinations based on the partial nucleotide sequence data obtained. The 5’-proximal region of the genomic sequence R. C. Larsen (&) USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350, USA e-mail: richard.larsen@ars.usda.gov

Characterization of a New Potyvirus Naturally Infecting Chickpea

During the 1999 to 2001 growing seasons, symptoms consisting of mosaic, stunting, yellowing, wilting, shortening of internodes, and phloem discoloration were observed in chickpea (Cicer arietinum) grown in the Department of Chuquisaca in southern Bolivia. In some fields, approximately 10% of the plants exhibited viruslike symptoms and suffered greatly reduced seed yields. Lentil (Lens culinaris) was also observed to be infected but not pea (Pisum sativum) or faba bean (Vicia faba) growing in nearby fields. Infected chickpea tissue reacted positively to the potyvirus group-specific monoclonal antibody (MAb), but there was no serological reaction with antisera to the potyviruses Bean yellow mosaic virus, Clover yellow vein virus, Cowpea aphid-borne mosaic virus, Pea seedborne mosaic virus, Bean common mosaic virus, or Bean common mosaic necrosis virus. Western blots of total protein extracts probed with the potyvirus MAb revealed a single band ca. 32 kDa. Comparative sequence analysis of cDNA clones generated from the putative coat protein gene consisted of 282 amino acids (31.9 kDa) and showed moderate identities of 67, 66, 63, 63, and 61% with the coat proteins of potyviruses Pepper severe mosaic virus, Pepper yellow mosaic virus, Potato virus Y, Plum pox virus, and Pepper mottle virus, respectively. Phylogenetic analysis of the coat protein amino acid sequence revealed that this virus is a unique member of the family Potyviridae and is phylogenetically most closely related to a group of Solanaceae-infecting potyviruses rather than to other legumeinfecting potyviruses. The proposed name for the new causal agent is Chickpea yellow mosaic virus.

Comparative immunoassays of bean common mosaic virus in individual bean (Phaseolus vulgaris) seed and bulked bean seed samples.

Indirect enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) or polyclonal antiserum (PC) and dot immunoassay using a MAb were compared for the detection of bean common mosaic virus (BCMV) in seed of Phaseolus from the U.S. Department of Agriculture germ plasm collection. A small portion of seed coat, testa, and cotyledon was removed from each of 270 seeds and tested for BCMV in each of the three assays. The seeds were then planted and the resultant seedlings were tested for BCMV by ELISA:MAb (...)

The complete nucleotide sequence and genome organization of red clover vein mosaic virus (genus Carlavirus)

Red clover vein mosaic virus (RCVMV) incites disease in pea (Pisum sativum L.) crops, and was recently detected in chickpea (Cicer arietinum L.) [15] and lentil (Lens culinaris L.) [16]. Symptoms in pea, lentil and chickpea infected with RCVMV include chlorosis, stunting, rosetting of leaves, and vein clearing Early infection can cause plant death in chickpea and lentil. A lack of apical dominance and a proliferation of axillary buds frequently occurs. Pod formation is severely affected when plants are infected before flower set resulting in decreased crop yields. The virus is transmitted in a non-persistent manner by several aphid species of which the pea aphid (Acyrthosiphon pisum Harris) is the most common vector [9, 11]. RCVMV is an approved species of the genus Carlavirus in the family Flexiviridae based on serology, particle morphology and vector transmission (1). In this paper we report the complete nucleotide sequence and genome organization of an isolate of RCVMV obtained from naturally infected chickpea in Washington State. Leaf tissue from chickpea plants infected with the Washington isolate of RCVMV was triturated in 50 mM potassium phosphate buffer, pH 7.4, containing 10 mM sodium sulfite and then used to mechanically inoculate pea (cultivar ‘Puget’) as the virus propagation host. Plants were then harvested 21 days post-inoculation and the virus was purified in a sodium phosphate-sodium citrate buffer using a protocol established previously [20]. Nucleic acid was extracted from purified virions (1 mg/ml) in 300 ll resuspension buffer (16.5 mM Na2HPO4, 1.8 mM sodium citrate, pH 9.0) by the addition of 3 ll 0.1 M dithiothreitol, 10 ll proteinase K (10 mg/ml), 30 ll 10% SDS then incubated for 30 min at 37 C. Excess proteinase K was removed using an equal volume of tris-saturated phenol (pH 8.0) and chloroform. The viral nucleic acid was then concentrated by ethanol precipitation. First strand cDNA was synthesized using purified viral RNA and SuperScript II reverse transcriptase with an oligo(dT)24 primer according to the manufacture’s instructions (Invitrogen, Carlsbad, CA, USA). Second-strand synthesis was performed essentially using the procedure of Gubler and Hoffman [10]. Following ethanol precipitation, the cDNA was ligated into pBluescript SK? at the EcoRV restriction site. The recombinant plasmids were transformed into E. coli DH10-alpha, plated on selective medium containing 100 lg/ml ampicillin, and screened for inserts [19]. A clone approximately 6,000 base pairs in length originating at the poly(A) tail was selected for initial sequencing. The clone was sequenced in both directions using the dideoxy chain termination method at the Laboratory for Biotechnology and Bioanalysis, Washington State University, Pullman, WA, USA. Nucleotide sequence data were assembled using Xesee [5]. Additional clones were produced from the purified RNA as needed with custom internal primers designed using generated sequence data. The 50 terminus of the RCVMV viral genome was amplified and cloned using the 50 rapid amplification of cDNA ends (RACE) system (version 2.0; Invitrogen) using the virus-specific primers, and the Abridged Universal Amplification Primer (AUAP) according to the manufacturer’s instructions. The products were R. C. Larsen (&) USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350, USA e-mail: richard.larsen@ars.usda.gov; rclarsen@wsu.edu