Hosta Virus X Research Articles

Establishment of meristem culture for virus-free and genetically stable production of the endangered plant Hosta capitata

Hosta species endemic to Korea are currently suffering from infection with Hosta virus X (HVX), and it has been difficult to produce virus-free plants when using infected plants as a source of material for in vitro and vegetative propagation in continuous production processes. Genetic variation in in vitro-regenerated plants has also been detected in some Hosta cultivars. Therefore, we aimed to develop an in vitro propagation method for the production of virus-free and genetically stable plants. First, we collected Hosta capitata showing symptoms of viral infection and confirmed the presence of an HVX virus gene using reverse transcription (RT)–PCR, followed by optimization of the sterilization of in vivo bulbs. Meristems derived from sterilized bulbs were cultured in media containing different concentrations of 1-naphthaleneacetic acid (NAA) with either benzyl aminopurine (BA) or thidiazuron (TDZ). The combination of 0.1 mg/l NAA and 3.0 mg/l BA generated the highest number of shoots per explant, and these shoots had a higher fresh weight. The addition of nano-silver (NAg) particles to the regeneration medium distinctly stimulated plant growth. According to the results of RAPD analysis, the meristem-derived plants were genetically stable compared with donor plants grown in a greenhouse, and absence of the HVX gene was also observed, indicating freedom from HVX infection. Therefore, they would be highly valuable for use in the landscape industry. We expect that the method proposed in this study will also prove helpful for generating virus-free, genetically stable plants in the commercial production of other Hosta species.

Development of reverse transcription loop‐mediated isothermal amplification assay for sensitive and rapid detection of Hosta virus X

AbstractThe one‐step real‐time turbidity loop‐mediated isothermal amplification assay (RealAmp) was developed to detect Hosta virus X (HVX), the most devastating threat to hosta industry. The reaction was performed in a single tube at 63°C for 15 min, and real‐time turbidimetry was used to monitor the amplification results. Specificity and sensitivity analyses demonstrated that this RealAmp method was sensitive as real‐time TaqMan RT‐PCR and about 100‐fold higher than conventional RT‐PCR with no cross‐reaction with other viral pathogens. Field samples detection showed that HVX could be identified effectively with this method. Overall, this RealAmp assay for HVX detection was simple, specific, sensitive, convenient and time‐saving and could assist in the quarantine measures for prevention and control of the disease caused by HVX.

First report of Hosta virus X infecting hosta plants in Ukraine.

In September 2011, the leaf samples of hosta cultivar 'Sum and substance' were collected from the collection of Gryshko' National Botanical Garden in Kyiv. The leaves showed dark green streaking and puckering along the leaf veins. Transmission electron microscopy revealed the presence of filamentous viral particles 13 nm in diameter and 470-580 nm in length. Reverse transcription PCR (RT-PCR) analysis confirmed the presence of Hosta virus X (HVX). The sequencing of the complete genome revealed 99% identity to HVX-37 and 97.5% identity to HVX-Kr. Notably, ORF4 initiation codon presented a non-conventional start codon (UUG) like it was previously identified in HVX-37.

Sensitive detection and discrimination method for studying multiple infections of five major plant viruses infecting ornamental plants in nursery environments

Tobacco mosaic virus (TMV), Hosta virus X (HVX), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are a few of the major viruses that infect ornamental and nursery plants. These viruses cause significant losses that impact growers and the ornamental industry. Often, a single ornamental plant is co-infected by more than one virus, which makes identification and discrimination of these viruses a difficult task, thus creating delays and limiting regulatory measures for effective quarantine. The aim of this study is to develop a sensitive, rapid, economic, and reliable multiplex Reverse Transcription PCR (RT-PCR) for simultaneous detection and discrimination of these five viruses. Specific PCR primers were designed using the consensus sequences of corresponding capsid protein (CP) genes of HVX and CMV, the nucleocapsid protein (NP) genes of TSWV and INSV, and the movement protein (MP) and CP genes of TMV. The primers were validated in vitro using single and multiplex RT-PCR assays. The detection limit of each primer set in multiplex RT-PCR was 100 fg (TMV), 1 fg (HVX), 10 fg (CMV), 10 pg (TSWV) and 10 pg (INSV). Forty-six infected nursery samples collected from different locations in the USA were screened for virus infections using this multiplex RT-PCR. The multiplex RT-PCR has a potential for its application in routine diagnostics, quarantine, and epidemiological studies. The developed method is reliable, sensitive, and economic for testing a wide range of ornamental and nursery plants for detection of these viruses.

First Report of Hosta virus X Infecting Hosta Plants in China.

Hosta (Hosta spp.) plants showing leaf deformation, puckering, and ink-bleed symptoms were collected in July 2012 from a park at Dongcheng district, Beijing, China. Three out of six samples tested positive for Hosta virus X (HVX) by immunostrip and double-antibody sandwich (DAS)-ELISA with HVX-specific serological reagents from Agdia Inc. (Elkhart, IN, USA). Filamentous viral particles were trapped and observed from the infected hosta leaf sap by immuno-serological electron microscopy (ISEM) (antibodies from Agdia). To confirm HVX infection, three ELISA-positive samples were analyzed by reverse transcription-PCR assay, using virus-specific primers HVXf (5'-ATCCGTTATCTACAGGGGACCAG-3') and HVXr (5'-TAAGTTAGTGGAACGGTTAGCCCGAT-3') that amplified a 1,067-bp fragment including the coat protein (CP) coding region. The CP nucleotide sequence comparisons showed 99% to 100% homology among the three isolates named HVXBJ4, HVXBJ5, and HVXBJ6 (GenBank Accession No. JX535292, JX535293, and JX535294) and with the HVX sequences previously reported in GenBank. HVX has been reported from the United States, Korea, the Netherlands, Poland, France, the Czech Republic, and New Zealand (1,2). To our knowledge, this is the first report of HVX infecting hosta plants in China. As an ornamental and medicinal plant, hosta has been cultivated in China for more than 2,000 years. The presence of HVX in Beijing is a potential threat to the landscape in the city. HVX can be spread by vegetative propagation material or mechanical contact (3). Hence, to cultivate HVX-free hosta and restrict the movement of HVX-infected hosta is vitally important in the future. HVX has become economically important in the world more recently. Globalization of trade in hosta plants has increased the risk of movement of HVX. The national plant protection organization should establish effective quarantine strategy and the growers take proper planting measures to avoid further spreading of this virus.

Biological and Molecular Characterization of a U.S. Isolate of Hosta virus X

Hosta virus X (HVX) is rapidly becoming a serious pathogen of commercially important hosta plants worldwide. We report here biological and molecular characterization of a U.S. isolate of HVX, HVX-37. HVX-37 infectivity was tested in 21 hosta cultivars over three growth seasons, and three types of responses were defined based upon the ability of the virus to cause local and/or systemic infections. Four cultivars resistant to systemic HVX infection were identified. The full-length sequence of the HVX-37 genome was determined, the first complete sequence of a U.S. HVX isolate. Comparison with the previously sequenced HVX-Korea (Kr) genome revealed a high level of sequence similarity, as well as some differences. Notably, a 105-nucleotide long, near-perfect direct repeat in the Kr isolate is absent in HVX-37. The accuracy of the HVX-37 genome sequence was confirmed by infectivity of in vitro transcripts synthesized from a full-length HVX-37 cDNA on Nicotiana benthamiana and hosta plants.

Phylogeny, coat protein genetic variability, and transmission via seeds ofHosta Virus X

The complete genome of Hosta Virus X (HVX), which is thought to be a distinct species of Potexvirus, was sequenced. Nucleotide sequences of HVX were compared with those of other members of the genus Potexvirus and phylogenetic tree was constructed. The range of identities of viral replicase open reading frame 1 (ORF1) between HVX and other potexviruses were 43.1%-55.1% and 35.9%-46.6% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis was performed according to the amino acid sequence of the replicase to determine the position of HVX in the genus Potexvirus. Results from the phylogenetic analysis demonstrated that HVX was in the same group as Cassava common mosaic virus (CsCMV), Plantago asiatica mosaic virus (PlAMV), Tulip virus X (TVX), and Hydrangea ring spot virus (HdRSV). In particular, coat protein (CP) sequences among viruses from different Hosta cultivars were revealed to be less variable than those from different isolates of Potato virus X (PVX), a Potexvirus type species. In the present study, HVX was transmissible by seeds of the Hosta "Blue Cadet" cultivar. Moreover, HVX was detected in the embryo but not in the seed coat or endosperm of the seed.

Presence of Hosta virus X in New Zealand

Leaf mosaic and deformation symptoms were observed on hosta plants (Hosta spp.) in a nursery in Kihikihi in the North Island of New Zealand. The virus associated with these symptoms has been identified as Hosta virus X (HVX), using electron microscopy, mechanical transmission, ELISA and RT-PCR techniques. This is the first report of HVX in New Zealand although this virus is believed to have been present and widespread in the country for more than 10 years.

First report of Hosta virus X infecting Hosta in the Czech Republic

Hostas (Hosta spp.) are popular herbaceous perennial plants represented by over 7000 varieties, and widely cultivated due to their diversity in leaf shape and colour patterns, shade tolerance and pest resistance. In the Czech Republic, hostas are grown as outdoor ornamental plants. At least four viruses in hosta have been reported in the world, but Hosta virus X (HVX) is the most economically important virus infecting hostas (Ryu et al., 2006). HVX is a member of genus Potexvirus (family Flexiviridae), and was first identified and described in Minnesota, USA in 1996 (Currier & Lockhart, 1996). Since then, HVX was reported in hosta in Tennessee and Ohio, USA (Fajolu et al., 2009; De la Torre, 2009) and from Korea and Poland (Park & Ryu, 2003; Cajza & Zielińska, 2007). Leaves infected with HVX commonly show symptoms of mosaic, mottling, interveinal chlorosis between secondary veins and leaf desiccation, and deformation. Infected plants often exhibit reduced growth and dieback. HVX can be transmitted from infected to healthy plants by cutting practices used for propagation and breeding, as well as in HVX-contaminated soils (Ryu et al. 2006). During 2010, a total of twenty five hosta plants of five different species and twenty different cultivars showing various virus-like symptoms including mosaic, mottling (Fig. 1), leaf chlorosis and leaf distortion were collected from six locations throughout the Czech Republic. Presence of HVX was also tested on plants without symptoms, but occurrence of HVX was not detected in these plants. Plants were mostly obtained from the collection of hosta cultivars at Lednice (Faculty of Horticulture, Mendel University Brno), Botanical Garden and Arboretum (Mendel University Brno), horticulture in Prague, and gardens in Brno. Other samples were purchased at garden centres in South Moravia and Central Bohemia. Total RNA was isolated from 100 mg of fresh leaf tissue from all hosta plants collected using the RNeasy Plant Mini Kit (Qiagen, Germany). The presence of HVX was confirmed by RT-PCR using a specific primer pair PHVXCP5 and PHVXCP3 that amplifies the HVX coat protein (CP) gene (Park & Ryu, 2003). HVX amplification products of the expected size (663 bp) were obtained for 19 out of 25 hosta plants analysed. Seven isolates of HVX were sequenced and resulting sequences were aligned and showed nucleotide identities ranging from 99 to 100%, when compared with each other. BLAST analysis yielded identities from 98 to 100% when HVX sequences were compared with those available in GenBank (HVX-Kr, Accession No. AJ620114 and HVX-U, AJ517352). Comparison of deduced amino acid sequence of CP gene from all the Czech isolates and HVX-Kr and HVX-U showed identities from 99 to 100%. The nucleotide sequence data of CP genes reported in this article have been deposited in GenBank under Accession Nos. JF301948-JF301954. In Europe, HVX has been previously reported only from Poland (Cajza & Zielińska, 2007). Therefore this is the first report of the occurrence of HVX in the Czech Republic. HVX is easily mechanically transmitted and can survive in the infected plants without showing symptoms for years. More than 56 hosta cultivars have been found to be susceptible to this virus in USA (De la Torre, 2009). To minimise HVX spread, the planting of HVX-free hostas and destroying of HVX-infected plants are recommended. The combination of RT-PCR and sequencing proved the best approach for a reliable and specific diagnostic of HVX in the Czech Republic. This project was supported by IGA AF MENDELU Brno No. IP 6/2010 and IGA AF MENDELU BRNO No. TP 4/2010.

Genetic variability and phylogenetic analysis of hosta virus X

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3-100% and 98.2-100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4-100% nucleotide and 97-100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.

Hosta virus X Detected in Kansas

Hosta virus X (HVX) is a mechanically-spread Potexvirus that infects several species in the genus Hosta. The virus causes a variety of symptoms including color-streaking, mosaic, distortion, and necrosis, though some infected plants remain symptomless. To our knowledge, these are the first occurrences of HVX in Kansas. Accepted for publication 16 April 2007. Published 26 September 2007.

Occurrence of Arabis mosaic virus in Hostas in the United States.

Hostas (Hosta spp.) are one of the most widely grown and economically important landscape perennials in the nursery industry in North America. Several viruses including Hosta virus X (HVX), Tobacco rattle virus (TRV), Tobacco ringspot virus (ToRSV), Tomato ringspot virus (TomRSV), Impatiens necrotic spot virus (INSV), and Tomato spotted wilt virus (TSWV) are known to occur in hostas (4). This report confirms the occurrence of an additional virus, Arabis mosaic virus (ArMV), in hostas in North America. This virus was first identified during the summer of 2004 in Hosta fortunei 'Sharmon' in several garden centers in Minneapolis and St. Paul, MN. Entire lots of this variety, numbering several dozen plants, showed symptoms consisting of blanching of the foliage similar to those caused by ToRSV and TomRSV infection (4). Symptoms persisted throughout the growing season. Virus-like particles, 28 to 30 nm in diameter, were observed by electron microscopy in partially purified extracts of symptomatic leaf tissue following fixation with 5% glutaraldehyde and negative staining with 2% sodium phosphotungstate, pH 7.0. Particles had an angular outline and some were penetrated by stain. No other virus-like particles were observed in these extracts. The particles were identified as those of ArMV. Identification was made using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) with antiserum to ArMV (PVAS-587) obtained from the American Type Culture Collection, Manassas, VA. In the spring and summer of 2005, ArMV was again identified as described above in 'Sharmon', H. undulata 'Albomarginata' samples from Minnesota, Michigan, and Nebraska, and H. 'Marion Bachman' and H. 'Touch of Class' from two wholesale nurseries in Minnesota. Symptoms in these hosta cultivars were similar to those observed in 'Sharmon' and were accompanied by stunting and leaf deformation. A portion of the coat protein (CP) gene of the ArMV isolate from 'Sharmon', designated ArMV-H, was amplified using reverse transcription-polymerase chain reaction (RT-PCR) with ArMV-specific CP primers (3) and total RNA extracted with a RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Amplicons of the expected size (220 bp) were cloned and five clones were sequenced. Nucleotide sequence identities of the ArMV-H CP sequence to corresponding ArMV databank entries varied from 94 to 88% (Genbank Accession Nos. AY017339 and D10086 and X55460 and X81815, respectively). Interestingly, the hosta ArMV isolate was not transmitted by mechanical inoculation to diagnostically susceptible indicator plants (cucumber, tobacco, and petunia) (2) or to hosta (H. undulata 'Albormarginata', H. 'Honeybells', and H. 'Royal Standard'). Testing by using ELISA and ISEM showed that 'Sharmon' source plants contained high levels of ArMV antigen and virions, and a high percentage of virions were not penetrated by negative stain, indicating that they were not empty (i.e., devoid of RNA). It appears that ArMV-H may be transmitted only vertically, (i.e., clonal propagation) and this raises some interesting questions about the molecular basis of this anomaly. An isolate of ArMV from hops was similarly reported to have a very restricted host range (1) suggesting a possibility of a common mechanism of host range restriction.

Virus infection of ornamental plants in Hungary

Recently the economic importance of the ornamental plant production has shown remarkable growth. Following the country's joint to the EU increased competition has become characteristic of the Hungarian market. More than 20 viruses have been isolated in the various vegetatively propagated Petunia species and varieties [Richert (1992), Lesemann and Dalchow (1995), Bellardi et al. (1996), Mavric et al. (1996) Salamon (1996), Richert-POggeler and Shepherd (1997), Feldhof et al. (1998), Boonham et al. (1999), Alexandre et al. (2000)]. Metelik et al. (1994, 1996), Mokrd and Gfitzovd (1994), Lesemann and Winter (2002) as well as Pham et al., (2002) examined the health condition of Zanthedeschia aetiopica and other Zanthedeschia species. The presence of Bean yellow mosaic virus (BYMV), Dasheen mosaic virus (DsMV), Cucumber mosaic virus (CMV), Konjac mosaic virus (KoMV) and Tomato spotted wilt vims (TSVW) were detected. Hosta virus X (HVX) and Impatiens necrotic spot vims (INSV) were isolated in the examined Hosta species (Lockhart 2002). Lockhart et al. (2002) verified the presence of Turnip mosaic virus (TuMV) in Heuchera samples and that of Alfalfa mosaic vims (AIMV) in Pulmonaria species. The goal of our work was to make a virological survey of herbaceous ornamental plants in Hungary.

Characterization of a Potexvirus InfectingHostaspp.

A previously undescribed potexvirus, named Hosta virus X (HVX), was found in 17 naturally infected hosta (Hosta spp.) cultivars from Minnesota, Indiana, Illinois, Iowa, and Michigan. HVX was readily transmitted mechanically but infected only Nicotiana benthamiana and Hosta spp., in which symptoms ranged from severe mosaic and leaf necrosis to latency. Particles of HVX averaged 530 nm in length, had a buoyant density in cesium chloride of 1.28 gm/cm 3 , and contained a single genomic species of ssRNA approximately 3 kb in size. The capsid protein of HVX had a molecular mass of approximately 27 kDa. In indirect enzyme immunoassays, HVX reacted with an antiserum to clover yellow mosaic virus (C1YMV), and less strongly with antiserum to hydrangea ringspot virus (HRSV), but not with antisera to any of 14 other potexviruses tested. However, in reciprocal tests, CIYMV reacted only very weakly with HVX antiserum. HVX did not infect any of three cultivars of pea that were susceptible to infection by CIYMV, and CIYMV did not infect any of three hosta cultivars susceptible to infection by HVX. Spread of HVX infection in hosta appears to occur by vegetative propagation and accidental mechanical transmission, and management of the disease can be achieved by virus indexing and cultural practices that minimize the risk of virus spread to susceptible cultivars.