A new chrysanthemum potyvirus: molecular evidence

Abstract

A number of viruses are known to infect chrysanthemum plants, however in the present study a previously unknown potyvirus was detected using techniques such as ELISA, RT-PCR and hybridization. The ELISA-positive samples were amplified using a potyvirus group-specific primer which gave an amplification of ～850 bp. The amplified product was cloned and sequenced, and shows 72 – 73% homology with known potyviruses that infect chrysanthemums such as Potato virus Y potyvirus, Soyabean mosaic virus and Turnip mosaic potyvirus when compared to the sequence available in the database. However, present potyvirus isolates show 93% homology with Chilli veinal mottle virus and Pepper vein banding virus. The results were further confirmed by Northern hybridization. This is the first report of a potyvirus similar to Chilli veinal mottle virus, and Pepper vein banding virus infecting chrysanthemums.     

Resistance to Vanilla Necrosis Potyvirus in Transgenic Nicotiana benthamiana Plants Containing the Virus Coat Protein Gene

The full-length vanilla necrosis potyvirus (VNV) coat protein (CP) gene was introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens-mediated transformation. Four constructs contained either: sense (+) CP sequence, antisense (-) CP sequence, sense CP sequence with a Kozak's consensus ATG resulting in a change in the first amino acid, or antisense CP sequence with the Kozak's modification. When mechanically inoculated with a high concentration of VNV, one of the plant lines containing the full-length sense CP gene was highly resistant to virus infection. Plants from the resistant lines expressed the CP at a relatively low level compared to susceptible lines containing the same construct. Plants containing the other three constructs were either susceptible or showed delayed symptom expression.     

Genome comparison and primer design for detection of Tamarillo leaf malformation virus (TaLMV)

Tamarillo (Solanum betaceum Bosh) is a plant from the highland tropics of great importance in the fruit agroindustry of Colombia. In recent years, there has been a dramatic decrease in the local tamarillo fruit production as a result of an emerging disease caused by the potyvirus Tamarillo leaf malformation virus (TaLMV). Symptoms of TaLMV infection include rugose mosaics, severe leaf deformation, defoliation and reduction in plant longevity. In this work, we present a second genome sequence of TaLMV obtained from a tamarillo-growing region in northern Antioquia (Colombia). This genome was compared with a previously sequenced isolate from La Union in eastern Antioquia; this information was used to design specific primers targeting the CP and CI regions of the TaLMV. These primer sets will be useful in future epidemiological studies, seed certification schemes and genetic improvement programmes using real-time RT-PCR (RT-qPCR) and/or RT-PCR.     

Complete nucleotide sequence analysis of <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> Indian isolate: further evidence for natural recombination among potexviruses

The complete nucleotide sequence of an Indian strain of Cymbidium mosaic virus (CymMV) was determined and compared with other potexviruses. Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV) and Potato aucuba mosaic virus (PAMV). Different sets of primers were used for the amplification of different regions of the genome through RT-PCR and the amplified genes were cloned in a suitable vector. The full genome of the Indian isolate of CymMV from Phaius tankervilliae shares 96–97% similarity with isolates reported from other countries. It was found that the CP gene of CymMV shares a high similarity with each other and other potexviruses. One of the Indian isolates seems to be a recombinant formed by the intermolecular recombination of two other CymMV isolates. The phylogenetic analyses, Recombination Detection Program (RDP2) analyses and sequence alignment survey provided evidence for the occurrence of a recombination between an Indian isolate (AM055720) as the major parent, and a Korean type-2 isolate (AF016914) as the minor parent. Recombination was also observed between a Singapore isolate (U62963) as the major parent, and a Taiwan CymMV (AY571289) as the minor parent.     

Konjac mosaic virus Naturally Infecting Three Aroid Plant Species in Andhra Pradesh,India

The genomes of three potyvirus isolates from, respectively, naturally infected Colocasia esculenta, Caladium spp. and Dieffenbachia spp. in Andhra Pradesh, India, were amplified by RT‐PCR using degenerate potyvirus primers. Sequence analysis of RT‐PCR amplicons (1599 nucleotides) showed maximum identity of 97% with the KoMV‐Zan isolate of Konjac mosaic virus (KoMV) from Taiwan (A/C AF332872). The three isolates had a maximum identity of 99.4%. The length of coat protein (CP) gene of three isolates was 846 nucleotides encoding 282 amino acids with a deduced size of 32.25 kDa. The CP gene of the isolates had, respectively, 78.1–95.7% and 88.2–96.4% identity at nucleotide and amino acid levels with KoMV isolates. The CP gene of the three isolates had 93.1–100% (nucleotide) and 98.2–100% (amino acid) identity. The 3′‐UTR of the three isolates showed maximum identity of 91.1–100% identity between and with other KoMV isolates. In the CP amino acid–based phylogenetic analyses, the isolates branched as a distinct cluster along with known KoMV isolates. The three potyvirus isolates associated with mosaic, chlorotic feathery mottling, chlorotic spots, leaf deformation and chlorotic ring spots on three aroids were identified as isolates of KoMV for the first time from Andhra Pradesh, India.     

Nucleotide sequence of coat protein gene for GPV isolate of barley yellow dwarf virus and construction of expression plasmid for plant

GPV is a Chinese serotype isolate of barley yellow dwarf virus (BYDV) that has no reactionwith antiserum of MAV, PAV, SGV, RPV and RMV. The sequence of the coat protein (CP) of GPV isolate of BYDV was identified and its amino acid sequence was deduced. The coding region for the putative GPV CP is 603 bases nucleotides and encodes a Mr 22218 (22 ku) protein. The same as MAV, PAV and RPV, GPV contained a second ORF within the coat protein coding region. This protein of 17024 Mr (17 ku) is thought to correspond to the Virion protein genome linked (Vpg). Sequence comparisons of the CP coding region between the GPV isolate of BYDV and other isolates of BYDV have been done. The nucleotide and ammo acid sequence homology of GPV has a greater identity to the sequence of RPV than those of PAV and MAV. The GPV CP sequence shared 83.7% of nucleotide similarity and 77.5% of deduced amino add similarity, whereas that of the PAV and MAV shared 56.9%. 53.2% and 44.1%. 43.8% respectively. According to BYDV-GPV CP seque     

Bean common mosaic virus and cucumber mosaic virus naturally infecting Aristolochia spp. plants in Brazil

In April 2022, Aristolochia plants with symptoms of mosaic were observed in a garden at Jardim Botânico Plantarum, Nova Odessa, São Paulo State, Brazil. Potyviridae-like particles were observed by transmission electron microscopy in leaf extracts. Total RNA extracted from symptomatic plants used in RT-PCR with universal and BCMV-specific primers detected the potyvirus bean common mosaic virus (BCMV). The cucumovirus cucumber mosaic virus (CMV) was identified only in Aristolochia littoralis plants that tested negative by RT-PCR for BCMV. Phylogenetic analysis grouped samples of Aristolochia in a different clade among samples of Phaseolus vulgaris. Phylogenetic analysis indicated that the CMV isolate from Aristolochia belongs to the CMV group IA. BCMV was mechanically transmitted to healthy plants of A. fimbriata, Chenopodium quinoa, P. vulgaris cv. Jalo and Macroptilium lathyroides. CMV was mechanically transmitted to plants of A. fimbriata and C. quinoa. The BCMV and CMV were aphid transmitted only by Aphis gossypii to Aristolochia plants. This is the first report of BCMV and CMV infecting Aristolochia plants in Brazil.     

Calanthe Mild Mosaic Virus, a New Potyvirus Causing a Mild Mosaic Disease of Calanthe Orchid in Japan

Calanthe mild mosaic potyvirus (CalMMV), a previously undescribed virus found in several locations in Japan, causes mild leaf mosaic and flower colour breaking of Calanthe plants. CalMMV was mechanically transmitted only to Calanthe sp., Phalaenopsis sp. and Tetragonia expansa of 50 plant species tested and was transmitted by the aphid Myzus persicae in a nonpersistent manner. The virus has flexuotis particles about 764 nm long and induced the formation of intracellular cytoplasmic cylindrical inclusions. The virus particles contain a single poly-peptide of 32.0 kDa and a single RNA of mol. weight 3.1 × 10⁶. As determined by immuno-electron microscopy, CalMMV is distantly related to the Japanese isolate of dendrobium mosaic potyvirus (DeMV-J), but it showed no serological relationship to any of seven other potyviruses. The sequence of the 3'-terminal 1306 nucleo-tides of the viral genome was determined. The coat protein (CP) coding sequence is predicted to be 804 nucleotides in length, encoding a protein of 268 amino acids with a calculated mol. weight of 30 389. The 3' noncoding region is 169 nucleotides long and is followed by a polyadenylate tract. The amino acid sequence of the CP of CalMMV was 73% homologous to that of DeMV-J, but less than 66% to other potyviruses.     

Complete Nucleotide Sequence of the RNA 3 of Bacopa chlorosis virus and Production of Polyclonal Antibodies to a Recombinant Coat Protein

The complete sequence of the RNA 3 of a virus causing chlorosis in Impatiens in Germany was determined and identified as an isolate of Bacopa chlorosis virus (BaCV, genus Ilarvirus). BaCV has previously only been reported from bacopa in the USA, but no coat protein (CP) sequence has been previously available. Both RNA 3 encoded proteins, CP and movement protein, showed highest sequence identity to Parietaria mottle virus, a subgroup 1 ilarvirus. Attempts to purify BaCV failed, so an antiserum was raised against a recombinant CP. The polyclonal antiserum so produced allowed specific detection of BaCV but showed no serological cross‐reaction with other ilarviruses and was unsuitable for immunoelectron microscopy. The host range includes many important flowering plant species, highlighting the potential threat BaCV might pose for the horticultural industry. This is the first report of BaCV occurring in Germany and outside the US.     

An Unusual Feature at the N-terminal end of the Coat Protein of Maize dwarf mosaic virus isolated in Hungary

Maize dwarf mosaic is the most widespread virus disease affecting corn production in Hungary. In attempts to identify the causal virus by test plant reactions, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), only Maize dwarf mosaic virus (MDMV) was detected. To further characterize Hungarian isolates of MDMV, one isolate from each of the sweet corn varieties Dallas, Royalty and GH23‐85 was selected for sequence analysis of its coat protein (CP) gene. The three Hungarian isolates shared CP amino acid sequence similarities of 95–98% not only with one another but also with MDMV isolates from other countries. However, the N‐terminus of the CP of the ‘Dallas’ isolate was unusual in containing a stretch of 13 additional amino acids. This is the first report of variation in the size of the N‐terminus of the MDMV CP.     

Molecular characterisation of papaya ringspot potyvirus isolates from India

Papaya ringspot potyvirus (PRSV) causes major diseases of papaya and cucurbits in the Indian subcontinent. Based on biological properties, PRSV isolates are classified as either papaya infecting (P), or non-papaya infecting (W) types. To characterise the P and W isolates from India at the molecular level, c. 1.7 Kb of the 3′-terminal regions comprising a part of the nuclear inclusion b (Nib) gene, the complete capsid protein (CP) gene and the untranslated region (UTR) of both the P and W isolates were cloned and sequenced. Comparative sequence analyses showed that the 3′-UTRs in isolates P and W were 209 nucleotides in length excluding the poly (A) tail, and shared 96% identity. The CP genes of the two isolates were also similar, with 87% nucleotide identity and 93% amino acid identity. The amino acid differences between the CP genes were mostly confined to the amino terminus. The DAG triplet associated with aphid transmissibility was present in the CP of isolate W, but it was replaced by DAD in the P isolate. The partially sequenced Nib genes were also 90% identical, but isolate W contained an additional amino acid (threonine) just upstream of the cleavage site (Q/S) between Nib and CP. This is the first reported comparison of the molecular characterisation of PRSV-P and W isolates from the Indian subcontinent.     

The Natural Occurrence of Turnip Mosaic Potyvirus in Allium ampeloprasum

An isolate of turnip mosaic potyvirus (TuMV) was obtained from Allium ampeloprasum grown in commercial greenhouses in Israel. Symptoms on infected plants include systemic chlorosis and yellow stripes, accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically, and in a non-persistent manner by aphids, among Allium, Chenopodium. Gomphrena and some Nicotiana spp. Purified preparations contained numerous filamentous particles similar to those observed in crude extracts of infected leaves. Particles from crude plant extracts had a normal length of 806 nm. Cells of infected plants contained cylindrical cytoplasmic inclusions with pinwheel, scrolls and laminated aggregates which indicated the presence of a potyvirus of Edwardson's subgroup III. and which resemble those of turnip mosaic virus (TuMV), The virus reacted strongly with antiserum to typical isolates of TuMV in immunoelectron microscopy and western blotting but not with antisera to several other potyviruses. Based on serological reactivity, electron microscopy, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV.     

Plum Pox Virus in Japanese Plum from Argentina: Serological Detection and Molecular Characterization of an Isolate from cv. Red Beauty

A Plum pox virus (PPV) isolate detected in a Japanese plum orchard in Pocito (San Juan, Argentina) was transmitted mechanically to Prunus tomentosa and Nicotiana benthamiana. DAS‐ELISA and DASI‐ELISA indicated the virus presence and serological relationship with D‐strain isolates; IC‐RT‐PCR amplified a 1.2‐kb fragment of the virus genome encoding the CP‐3′ nc region. The analysis of the sequence showed the presence of the DAG motif at the 5′ end of the capsid protein and the Rsa I and Alu I sites at the 3′ end. The phylogenetic relationships and multiple alignment with PPV isolates from NCBI database indicated greatest (+98%) homology with the D strain and close identity with MNAT1 ( AF360579 ) USA peach isolate. The sequence analysed showed two amino acid mutations towards the 5′ N‐terminus of CP (the most variable region) with respect to a consensus of PPV D‐strain isolates. This is the first molecular characterization of 3′terminal genome region of PPV isolate to confirm D strain in a Japanese plum from Argentina.     

Analysis of the Coat Protein Gene of Indian Strain of Apple Stem Grooving Virus

A survey was undertaken in the temperate fruit growing regions of Himachal Pradesh (HP) and Jammu & Kashmir (J&K). Apple stem grooving virus (ASGV), a Capillovirus, was detected in different cultivars of apple, nectarines, plum, cherry, quince and apricot by double antibody sandwich ELISA (DAS-ELISA). The coat protein (CP) gene sequence of an amplicon produced by RT-PCR, confirmed the association of ASGV in apple cultivar Starkrimson, collected from Himachal Pradesh. The CP of Indian ASGV isolate shared 100 % sequence identity with a Brazilian isolate (AF438409). Sequence analysis by Recombination Detection Program (RDP2) indicated no recombination event for the Indian isolate. However, recombination was detected in Chinese, Korean and Citrus tatter leaf virus-Taiwan (CTLV) strains of ASGV. The study describes first report of ASGV infection in India and characterization of its CP gene.     

Complete Genome Sequence of a Novel Wild tomato mosaic virus Isolate Infecting Nicotiana tabacum in China

Virus particles of approximately 740–760 nm in length and 13 nm in diameter were observed from a diseased Nicotiana tabacum (tobacco) plant in Sichuan Province, China. The complete genomic sequence of the virus isolate XC1 was determined to contain 9659 nucleotides without 3′ terminal poly(A) tail. XC1 has a genome typical of members of the genus Potyvirus, encoding a large polyprotein of 3075 amino acids. Putative proteolytic cleavage sites and a number of well characterized functional motifs were identified by sequence comparisons with those of known potyviruses. Sequence comparison revealed that XC1 shared the highest level of nucleotide sequence identity (76.5%) with Wild tomato mosaic virus (WTMV). Phylogenetic analysis showed that XC1 was closely related to the WTMV Guangdong isolate with an identity of 94.3% between CP gene sequence of the two viruses. We thus named XC1 WTMV‐XC‐1 as a novel isolate of WTMV. The full sequence of WTMV‐XC‐1 may serve as a basis for future investigations on the gene diversity of WTMV.