Asparagus Virus Research Articles

A novel ilarvirus protein CP-RT is expressed via stop codon readthrough and suppresses RDR6-dependent RNA silencing.

Ilarviruses are a relatively understudied but important group of plant RNA viruses that includes a number of crop pathogens. Their genomes comprise three RNA segments encoding two replicase subunits, movement protein, coat protein (CP), and (in some ilarvirus subgroups) a protein that suppresses RNA silencing. Here we report that, in many ilarviruses, RNA3 encodes an additional protein (termed CP-RT) as a result of ribosomal readthrough of the CP stop codon into a short downstream readthrough (RT) ORF. Using asparagus virus 2 as a model, we find that CP-RT is expressed in planta where it functions as a weak suppressor of RNA silencing. CP-RT expression is essential for persistent systemic infection in leaves and shoot apical meristem. CP-RT function is dependent on a putative zinc-finger motif within RT. Replacing the asparagus virus 2 RT with the RT of an ilarvirus from a different subgroup restored the ability to establish persistent infection. These findings open up a new avenue for research on ilarvirus silencing suppression, persistent meristem invasion and vertical transmission.

Host range and molecular and ultrastructural analyses of Asparagus virus 1 pathotypes isolated from garden asparagus Asparagus officinalis L.

Asparagus samples were examined from growing areas of Germany and selected European as well as North, Central and South American countries. Overall, 474 samples were analyzed for Asparagus virus 1 (AV1) using DAS-ELISA. In our survey, 19 AV1 isolates were further characterized. Experimental transmission to 11 species belonging to Aizoaceae, Amarantaceae, Asparagaceae, and Solanaceae succeeded. The ultrastructure of AV1 infection in asparagus has been revealed and has been compared with the one in indicator plants. The cylindrical inclusion (CI) protein, a core factor in viral replication, localized within the cytoplasm and in systemic infections adjacent to the plasmodesmata. The majority of isolates referred to pathotype I (PI). These triggered a hypersensitive resistance in inoculated leaves of Chenopodium spp. and were incapable of infecting Nicotiana spp. Only pathotype II (PII) and pathotype III (PIII) infected Nicotiana benthamiana systemically but differed in their virulence when transmitted to Chenopodium spp. The newly identified PIII generated amorphous inclusion bodies and degraded chloroplasts during systemic infection but not in local lesions of infected Chenopodium spp. PIII probably evolved via recombination in asparagus carrying a mixed infection by PI and PII. Phylogeny of the coat protein region recognized two clusters, which did not overlap with the CI-associated grouping of pathotypes. These results provide evidence for ongoing modular evolution of AV1.

Asparagus virus 1. [Distribution map

Abstract A new distribution map is provided for Asparagus virus 1 (Potyviridae: Potyvirus). Host: asparagus ( Asparagus officinalis ). Information is given on the geographical distribution in Asia (China, Yunnan, Japan, Hokkaido), Europe (Germany, Italy, Sicily, Poland, UK) and North America (USA, California, Connecticut, Delaware, Kansas, Michigan, New Jersey, Washington).

Identification and pathogenicity of plant viruses infecting Valeriana fauriei in Japan

Samples from Valeriana fauriei Briq. plants were collected from six prefectures to survey viral pathogens. By next-generation sequencing and RT-PCR analysis, asparagus virus 2 (AV-2), broad bean wilt virus 2 (BBWV-2), cucumber mosaic virus (CMV), gaillardia latent virus (GaILV), ligustrum necrotic ringspot virus (LNRSV), and tomato spotted wilt orthotospovirus (TSWV) were detected. Many collected samples were coinfected with 2–4 viruses. V. officinalis was also back-inoculated with one or a mixture of isolated viruses to confirm pathogenicity of isolated viruses. This report is the first of GaILV and LNRSV in Japan and the first of AV-2 on V. fauriei.

Feeding behavior of Myzus persicae on asparagus species susceptible and resistant to Asparagus virus 1

AbstractAsparagus virus 1 (AV‐1) infects Asparagus officinalis L. (Asparagaceae) in the field worldwide. However, various wild relatives of A. officinalis are resistant to AV‐1. Here we study the behavior of the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), on 19 AV‐1‐resistant wild relatives of A. officinalis. We focus on behavior that is associated with regular cell penetration, relevant for inoculation of AV‐1, and sieve element penetration to check for vector resistance and its potential influence on AV‐1 transmission. Parameters, relevant for the transmission of non‐persistent viruses and host plant acceptance, were obtained by the electrical penetration graph technique. Furthermore, phylloclade architecture of A. officinalis and its wild relatives was examined to study its influence on aphid behavior. Behavior of M. persicae displays many cell penetrations and long ingestion periods on A. officinalis, compared to the generally shorter cell penetrations (reduced potential for virus transmission) and reduced or no ingestion (phloem‐located aphid resistance) on wild relatives. Because effects on aphid behavior are not consistent throughout the group of the tested wild relatives of A. officinalis, with some wild relatives being susceptible to M. persicae, a common genetic background for AV‐1 and aphid resistance appears to be unlikely. However, the reduced potential of virus transmission as well as aphid resistance shown by some wild relatives may be useful for future breeding programs.

Progress in the development of garden asparagus resistant toAsparagus virus1 (AV-1)

A genetically determined resistance to the Asparagus virus 1 was identified within the Mediterranean wild relative Asparagus amarus. Field experiments under climatic conditions in Germany showed for A. amarus a relative low yield potential (~20%) compared with various cultivars of A. officinalis. Analyses of volatile organic compounds revealed a number of significant differences. Furthermore, the wild relative is hexaploid (2n=6x=60), expresses a strong bitter taste and is frost tolerant up to -15°C. A backcross programme was started for introgression the AV-1 resistance from the wild relative into the genome of garden asparagus. Interspecific hybrids were obtained by reciprocal crosses between A. amarus and tetraploid A. officinalis via embryo rescue approach. Four F1 progenies were cloned in vitro and transplanted in the field. Two progenies showed resistance to AV-1, while two progenies were susceptible.

Somatic hybridization for transfer of resistance against Asparagus virus 1 (AV-1) from Asparagus amarus into A. officinalis

Asparagus is a valued vegetable in people's diet in many countries of the world. The production of asparagus increased remarkably in the last few years. Comprehensive evaluations have shown that there exist no cultivars of A. officinalis with resistance against Asparagus virus 1 (AV-1) causing a high loss of yield and quality. Resistance was found in wild species of the genus Asparagus such as in A. amarus. Somatic hybridization could be a tool to overcome crossing barriers between species. The sophisticated technology requires plant species adapted protocols. Here, a method starting with suspension cultures of A. officinalis and A. amarus was presented as source for protoplasts. Fusion was performed using either electromanipulation or polyethylene glycol solution. Fused protoplasts were cultivated in liquid protoplast cultivation medium PPM1 supplemented with 1 g L-1 L-glutamine. First cell divisions were noticed already after eight to ten days. About five weeks after fusion microcolonies were plated on solid Murashige and Skoog medium (MS) + 1 mg L-1 2,4-dichlorophenoxyacetic acid. After several subcultures, developing plants were transferred to MS + 0.2 mg L-1 α-naphthalene acetic acid for further growth. Regenerated plants were acclimatized to greenhouse conditions. By fluorescence flow cytometry the DNA amount was estimated. RAPD analyses revealed regenerated plants with DNA profiles with fragments from only one protoplast donor but also with fragments from both donors as it is expected for somatic hybrids. Resistance to AV-1 was tested by DAS-ELISA.

Effects ofAsparagus virus 1(AV-1) on yield and quality components on asparagus

Effects of<i>Asparagus virus 1</i>(AV-1) on yield and quality components on asparagus

Breeding programs to transmitAsparagus virus 1resistance

Breeding programs to transmit<i>Asparagus virus 1</i>resistance

Evaluation of genetic resources in the genus Asparagus for resistance to Asparagus virus 1 (AV-1)

Forty-four Asparagus officinalis cultivars, gene bank accessions and breeding lines as well as thirty-four accessions of wild relatives of Asparagus were evaluated for resistance to Asparagus virus 1. Three different test strategies were developed for the assessment of individual plants: (1) natural infection under field conditions, or two vector-mediated infection assays using the green peach aphid Myzus persicae (2) in an insect-proof gauze cage or (3) in a climate chamber. The AV-1 infections were verified by DAS-ELISA and RT-PCR approaches. All tested 660 individual plants of A. officinalis germplasm were susceptible to AV-1 infection. In contrast, in 276 plants of 29 Asparagus wild accessions no virus infection could be detected. These resistant accessions comprised of nineteen diploid, tetraploid and hexaploid species of both the Eurasian clade and the African clade of the asparagus germplasm. Data of the AV-1 resistance evaluation are discussed in relation to the genetic distance of the resistance carrier and potential application in breeding.

Molecular identification and biological characterization of a new potyvirus in lettuce.

A potyvirus causing necrosis and leaf distortion on lettuce was found in the Lazio region of Italy. Host range analysis showed its ability to infect only Chenopodium quinoa and C. amaranticolor in addition to some lettuce cultivars. The virus could be transmitted by aphids of the species Myzus persicae. The complete 9829-nt genome was characterized. BLAST analysis of sequence of the complete encoded polyprotein showed that the most closely related virus is asparagus virus 1, with 52% amino acid sequence identity. These results suggest that this virus should be considered a member of a new species in the genus Potyvirus.

Seed and pollen transmission of asparagus virus 2

Asparagus virus 2 (AV2) is widespread in asparagus crops worldwide and is known to be seed-transmitted. To determine the effect of AV2- infected parental source on development of infected seed, male and female asparagus plants which were AV2-infected or AV2-free were cross-pollinated and allowed to develop seed in an insect-proof greenhouse. ELISA tests of shoots from the resulting seedlings showed that AV2 was transmitted into 17 % of germinated seedlings when only the male parent was infected, into 47 % when only the female was infected and to 95 % when both parents were infected. Infection of parent plants resulted in reductions in successful pollination (21–29 %), weight (11–18 %) and number of seed (12–46 %), as well as germination (8–23 %) and seedling viability (4–15 %). Most of the AV2-free female plants pollinated with infected pollen became infected; ELISA tests showed that 83 % of plants and 36 % of shoots tested positive the following year, and by the second year RT-PCR testing showed that 75 % of plants and 55 % of shoots were infected. The significance of vertical transmission via infected seed and horizontal transmission by pollination is discussed with respect to control strategies.

Detection of plant viruses in mixed infection by a macroarray-assisted method.

The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and "microtube hybridization" (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect asparagus viruses (Asparagus virus 1 and Asparagus virus 2) from latently infected asparagus plants.

Complete genome sequences of two biologically distinct isolates of Asparagus virus 1.

The complete genome sequences of two asparagus virus 1 (AV-1) isolates differing in their ability to cause systemic infection in Nicotiana benthamiana were determined. Their genomes had 9,741 nucleotides excluding the 3'-terminal poly(A) tail, encoded a polyprotein of 3,112 amino acids, and shared 99.6% nucleotide sequence identity. They differed at 37 nucleotide and 15 amino acid sequence positions (99.5% identity) scattered over the polyprotein. The closest relatives of AV-1 in amino acid sequence identity were plum pox virus (54%) and turnip mosaic virus (53%), corroborating the classification of AV-1 as a member of a distinct species in the genus Potyvirus.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

The global trade in fresh produce and the vagility of plant viruses: a case study in garlic.

As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic (Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses (Onion yellow dwarf virus, Leek yellow stripe virus), carlaviruses (Shallot latent virus, Garlic common latent virus) and allexiviruses (Garlic virus A, B, C, D, and X), for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic (A. vineale L.), a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.

Incidence and genetic variability of Asparagus virus 1 in naturally infected asparagus.

A study to determine the incidence of viral diseases in green asparagus ( Asparagus officinalis L.) was carried out in Italy, sampling spears, young stems and ferns from com- mercial fields in several regions. Asparagus bunches from supermarkets originating from Italy as well as from sev- eral other countries, were also examined. Molecular assays (RT-PCR) were developed for the simultaneous detection of Asparagus virus 1 (AV-1) and Asparagus virus 2 (AV-2). The incidence of the two viruses was quite different. AV-2 was restricted to a few Italian cultivation areas and was never detected in samples from Spain, Peru, Mexico, Ec- uador, Argentina, or the USA. In contrast, AV-1 was widely spread in Italian asparagus crops with an average infection rate of about 50.7% and was detected in imported aspara- gus at different rates. Partial sequences of AV-1 isolates from different geographical areas were obtained. Their 3’ terminal genomic region comprising the partial coat protein gene and the 3’ untranslated region of 18 isolates were compared to each other and to species of the genus Potyvirus . Results revealed that AV-1 has become the most widespread virus in commercial asparagus crops of Italy, and presumably in other asparagus producing countries. Further, our study provides, for the first time, valuable information on the molecular variability of AV-1 isolates from different parts of the world.

Resistance to Asparagus virus 1 in the Wild Relative Asparagus amarus

AbstractFive asparagus cultivars, three breeding lines and the wild relative Asparagus amarus were tested for natural infection by Asparagus virus 1 (AV‐1) in experimental fields at two locations over 3 and 4 years, respectively. In the first year after re‐planting the annual crowns in the field, more than 90% of tested plants of cultivars were infected by AV‐1. In the third and fourth year, 100% of tested plants of cultivars were AV‐1 infected. In comparison, all plants of the wild relative A. amarus were completely free of AV‐1, suggesting a high level of resistance. Additionally, 1‐year‐old glasshouse‐cultivated plants of A. officinalis and A. amarus were placed in an AV‐1 provocation cabin under field conditions. Seven months later, 100% of the A. officinalis plants showed a high virus concentration in ELISA, whereas no AV‐1 was detectable in the A. amarus plants. This result was confirmed by highly sensitive AV‐1‐specific RT‐PCR. To exclude vector resistance, the feeding behaviour of green peach aphid Myzus persicae was tested over 12 h using the electrical penetration graph method. Both asparagus genotypes were accepted by the aphids as potential hosts, but the feeding time was significantly longer on A. amarus. A genetic distance analysis of the various cultivars of Asparagus officinalis and selected wild relatives of the JKI collection was carried out, resulting in a clear discrimination of cultivars and wild relatives, especially A. amarus. The potential breeding value of the putative resistance carrier is discussed.

The 2b protein of Asparagus virus 2 functions as an RNA silencing suppressor against systemic silencing to prove functional synteny with related cucumoviruses

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1–3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.

The nucleotide sequences of the RNA 1 and RNA 2 of asparagus virus 2 show a close relationship to citrus variegation virus

Asparagus virus-2 (AV-2) is a member of the genus Ilarvirus and infects the vegetable Asparagus officinalis L. The virus is transmitted through seed. Even though the presence of AV-2 causes reductions in vigor and productivity, plants grown from infected seed show no obvious symptoms. The virus has been reported from Europe, North America, and Japan. However, because of the ease with which the virus is seed borne, it has probably been moved to wherever seed lots of asparagus have been distributed [20]. Other ilarviruses are also seed borne and, in addition, have been shown to be transmitted naturally through pollen and the feeding activity of thrips [11]. Ilarviruses are members of the family Bromoviridae. They share traits at the molecular level with the extensively studied cucumoviruses and bromoviruses and have a particularly close relationship to the alfamovirus alfalfa mosaic virus [1]. Ilarviruses possess a tripartite genome of positive-sense ssRNAs. The RNA 1 is monocistronic, coding for a viral replicase. The RNA 2 is bicistronic in some members of the genus but monocistronic in others. The open reading frame (ORF) proximal to the 5 0 UTR codes for the viral polymerase. The second, smaller ORF found in some members of the genus is located toward the 3 0 terminus of the molecule and expressed through a subgenomic RNA that codes for a 2b protein. The initial ORF of RNA 3 codes for the movement protein (MP), and the second ORF of RNA 3 codes for the coat protein (CP). The CP gene is expressed via a subgenomic RNA 4 [7]. AV-2 is a member of subgroup 2 of the ilarviruses, having been shown to be related to the other members of this subgroup: citrus leaf rugose virus (CiLRV), citrus variegation virus (CVV), elm mottle virus (EMoV), spinach latent virus (SpLV), and tulare apple mosaic virus (TAMV), both serologically and at the molecular level [19]. The complete sequence for the RNA 3 of AV-2 has been described [17], as have fragments of sequence for the RNA 1 and RNA 2 [GenBank accessions U93601, U93602, U93603]. Here, we present the complete sequences for the RNA 1 and RNA 2 of AV-2 and discuss the close relationship between this virus and another member of subgroup 2, citrus variegation virus.