Aphid Transmission Research Articles

Analysis of the biological and molecular variability of Watermelon mosaic virus isolates from Iran

Watermelon mosaic virus (WMV) is one of the most important viruses that causes different symptoms in Cucurbitaceae. WMV is a potyvirus with a worldwide distribution, but occurs most commonly in temperate and Mediterranean regions. Cucurbit species grown in Yazd, Esfahan, West Azerbaijan, Hormozgan, and Kerman provinces were surveyed for the relative incidence of WMV in 2004-2005. A total of 757 symptomatic cucurbit and 31 weed species were collected and assayed for infection with WMV. Of 788 leaf samples from cucurbit and weed plants, 190 samples were positive by double antibody sandwich ELISA (DAS-ELISA) using specific polyclonal antibody. Among the weed species tested only colocynth (Citrullus colocynthis) was found to be infected with WMV. The coat protein (CP) gene from 18 representative isolates was PCR amplified, cloned, sequenced, and compared with the sequences available in GeneBank. Phylogenetic analysis using 778 nucleotide long sequences of the coat protein gene showed that these isolates fell into two; groups I and II. Only one isolates (KER.JI.1) was classified in the group II. This isolate had a wider host range and infected Nicotiana debneyii and Datura metel. None of the other 17 isolates could infect these two species. Members of group I were divided into three subgroups; A, B, and C. The subgroup I(B) appears to be a new subgroup comprising only of the Iranian isolates. Phylogenetic analysis based on 200 nucleotides coding for the N-terminal segment of the CP showed that all Iranian isolates except KER.JI.1 clustered with the previously reported WMV strains. All Iranian isolates had a DAG amino acid triplet which is involved in aphid transmissibility. This is the first report on sequence analysis of the nearly full-length CP cDNA clones of WMV isolates from Iran.

Small deletions in the potato leafroll virus readthrough protein affect particle morphology, aphid transmission, virus movement and accumulation

Potato leafroll virus (PLRV) capsid comprises 180 coat protein (CP) subunits, with some percentage containing a readthrough domain (RTD) extension located on the particle's surface. The RTD N terminus is highly conserved in luteovirids and this study sought to identify biologically active sites within this region of the PLRV RTD. Fourteen three-amino-acid-deletion mutants were generated from a cloned infectious PLRV cDNA and delivered to plants by Agrobacterium inoculations. All mutant viruses accumulated locally in infiltrated tissues and expressed the readthrough protein (RTP) containing the CP and RTD sequences in plant tissues; however, when purified, only three mutant viruses incorporated the RTP into the virion. None of the mutant viruses were aphid transmissible, but the viruses persisted in aphids for a period sufficient to allow for virus transmission. Several mutant viruses were examined further for systemic infection in four host species. All mutant viruses, regardless of RTP incorporation, moved systemically in each host, although they accumulated at different rates in systemically infected tissues. The biological properties of the RTP are sensitive to modifications in both the RTD conserved and variable regions.

Aphid Transmission of Plant Viruses

A majority of plant viruses are transmitted between hosts by insect vectors, and it is often important to use insect transmission in the laboratory to maintain virus isolates or to study virus-vector-plant interactions. Although many of these viruses can also be mechanically transmitted in the laboratory using infected sap, maintenance by mechanical transmission can often lead to changes in the virus, either minor changes in gene sequences or, in some cases, major deletions of genome sequences. These can affect both virus-vector and virus-host interactions. This unit describes some simple and practical methods for conducting virus transmission experiments using sap-sucking insects.

Molecular, serological and transmission electron microscopic analysis of the barley yellow dwarf virus-PAVand the cereal yellow dwarf virus-RPV in canary seed (Phalaris canariensisL.)

Canary seed (Phalaris canariensis L.) is a cereal crop belonging to the tribe Phalarideae of the Poaceae family. A prevailing virus infection, which causes dwarfing of plants and yellowing of leaves, was observed in canary seed fields in the Tekirdag province of Turkey. The aim of this study was to identify, clone and sequence the cereal viruses naturally occurring on P. canariensis by employing serological tests as DAS-ELISA and TAS-ELISA tests combined with transmission electron microcopy (TEM) and molecular analysis. One hundred and one plant samples showing symptoms were collected and tested serologically using polyclonal antisera against Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV). The results of both immunoassays showed that 48% of the samples were infected with BYDV-PAV, 2% with CYDV-RPV and 14% were mixed infection. 36% of the samples were uninfected or infected at level below detection. Aphid transmission experiments revealed that barley (cv. Rubina) exhibited characteristic of CYDV-RPV. Investigations of infected canary grass seedlings using transmission electron microscopy (TEM) supported the findings of serological tests and revealed the presence of isometric particles of approximately 25 nm in diameter. These results were also confirmed by using BYDV-PAV and CYDV-RPV specific primers. Sequence analysis of cDNA and probable translation products revealed a high level of homology to BYDV-PAV and CYDV-RPV isolates found in other plant species. The sequence data obtained from this research were deposited in the EMBL/GenBank Data Libraries under the accession nos. EGO19056 and EF372272.

SEROLOGICAL AND MOLECULAR TYPING OF PLUM POX VIRUS ISOLATES IN THE NORTH OF ROMANIA

Plum pox virus (PPV) is considered as the most dangerous viral pathogen of stone fruits. Although PPV is widespread in Romania and causes serious yield losses, little is known about the variability of its isolates. To secure this information we investigated 43 PPV isolates collected from five different plum orchards in the North of Romania in the Bistrita plum-growing area. PPV strains were serologically tested by TAS-ELISA using PPV-D and PPV-M specific monoclonal antibodies. Molecular strain typing was done by IC-RT-PCR targeting three genomic regions corresponding to (Cter)CP, (Cter)NIb/(Nter)CP and CI. RFLP analysis was used to distinguish the two major strains, D and M based on a RsaI polymorphism located in (Cter)CP. All PCR products targeting (Cter)CP and 8 PCR products spanning the (Cter)NIb/(Nter)CP cistrons were sequenced. All PPV isolates typed as PPV-M by serological analysis and by molecular differentiation in the genomic region corresponding to (C-ter)CP were confirmed by nucleotide sequencing to be homologous to PPV recombinant (PPV-Rec) previously reported. All these recombinant isolates share the same recombination breakpoint and conserve the DAG motif, which is considered essential for aphid transmission. This genetic similarity confirms that PPV-Rec may represent an ancestral group with a common evolutionary origin. Overall results provided evidence for endemic distribution of PPV-Rec in plum trees grown in the North of Romania.

Identification and Partial Characterization of a New Luteovirus Associated with Rose Spring Dwarf Disease.

A number of viruses in the genera Ilarvirus and Nepovirus have been shown to be associated with specific diseases in rose, but many graft-transmissible rose diseases still have unknown etiologies. One of these diseases originally was detected by grafting from nonsymptomatic roses to Rosa multiflora indicator plants. Double-stranded RNAs (dsRNAs) were recovered and used as templates for cDNA synthesis and generating a cDNA library. Analysis of deduced amino acid sequences clearly positioned this virus as a member of the family Luteoviridae. The name rose spring dwarf associated virus (RSDaV) is tentatively proposed for the novel virus because the symptoms of this virus on R. multiflora are consistent with previous descriptions of rose spring dwarf disease (RSD). Phylogenetic analysis revealed a close relationship of RSDaV with members of the genus Luteovirus. Aphid transmission studies identified the rose-grass aphid (Metapolophium dirhodum) and yellow rose aphid (Rhodobium porosum) as vectors for this new virus. Host range data showed that RSDaV has a host range including both monocots and dicots. A specific reverse-transcription polymerase chain reaction assay was developed and revealed the presence of the RSDaV in several rose cultivars. RSDaV-inoculated rose plants developed RSD symptoms, confirming its role in the etiology of the disease.

Note: Characterization of aPeanut mottle virus isolate infecting peanut in Israel

Peanut mottle virus (PeMoV) was identified for the first time in Israel in peanut plants expressing mottle symptoms. Particle morphology, biological properties and serology suggested that this virus belongs to the genusPotyvirus. The characteristics of the Israeli (IL) PeMoV were compared with those of previously reported isolates. Using RT-PCR, a 1393-bp fragment consisting of the helper component — proteinase (HC-Pro) and a 988-bp product containing the coat protein (CP) were amplified, cloned and sequenced. Comparison of the HC-Pro sequences for PeMoV-IL and PeMoV-M (reported previously), showed 98% homology at the amino acid (aa) level. The aa sequence homology of the entire CP of isolate IL and six other PeMoV isolates ranged from 92% to 98%. A phylogenetic analysis carried out using the CP nucleotide sequence data indicated close similarity between PeMoV-IL and an Australian isolate and between two M isolates. The conserved KITC and CCC motifs in the HC-Pro were replaced by KVSC and ASC, respectively, in PeMoV-IL as in strain M. The DAG motif in the CP was replaced by DAA in the PeMoV isolates including IL. These results prompted the examination of aphid transmissibility of PeMoV-IL which were low and variable among experiments. These results differ from a previous report showing high aphid transmission of PeMoV.

Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids

Hairpin RNAs have been used to confer resistance to viruses in plants through RNA silencing. However, it has not been demonstrated that RNA silencing was effective against inoculation by aphids of non-persistently transmitted viruses, the major route of plant virus spread in nature. As a proof-of-principle strategy, we made use of Agrobacterium tumefaciens to transiently express a hairpin RNA homologous to Potato virus Y (PVY) in plant tissues. A complete and specific interference with aphid transmission of PVY was achieved by inducers of RNA silencing, as demonstrated by specific siRNAs accumulation in agroinfiltrated tissues. To our knowledge, this is the first report of successful interference with non-persistent transmission of a plant virus using RNA interference.

Genome structure and organization of a member of a novel and distinct species of the genus Caulimovirus associated with dahlia mosaic

The genome structure and organization of a new and distinct caulimovirus that is widespread in dahlia (Dahlia variabilis) was determined. The double-stranded DNA genome was ca. 7.0 kb in size and shared many of the features of the members of the genus Caulimovirus, such as the presence of genes potentially coding for the movement protein, the inclusion body protein, and the reverse transcriptase (RT), and an intergenic region consisting of a potential 35S promoter. However, the virus differed from the previously described dahlia mosaic caulimovirus and other known caulimoviruses in that the aphid transmission factor (ATF) was absent and the putative coat protein contained a C-terminal deletion and was fused in-frame with the RT. Sequence identity at the amino acid level with known caulimoviruses including a previously reported caulimovirus from dahlia was low and ranged from 32 to 72%. The absence of an ATF and the highly divergent nature of the genomic sequence are characteristics of this new caulimovirus that is widely associated with dahlia.

High expression of foreign proteins from a biosafe viral vector derived from Turnip mosaic virus

A vector derived from an original infectious clone of Turnip mosaic potyvirus (TuMV) has been developed. The vector (p35Tunos-vec01) was made through the creation of a new unique insertion site for foreign genes between the NIb and the CP genes of the viral genome. The jellyfish green fluorescent protein (GFP) and E. coli beta-glucuronidase (GUS) were expressed in Arabidopsis thaliana plants infected with chimeric vectors carrying their corresponding genes. In the case of GUS, expression levels that were 15-50 fold higher than nuclear transgenic A. thaliana lines carrying the same gene were attained. Acceptable levels of gene stability were found in the infected plants, compatible with the use of the vector for protein production in plants. A non-aphid transmissible version of the vector was also made and its lack of aphid transmission was extensively shown. The vector was not transmitted through seeds or by contact between plants.

Establishing a testing method for the aphid transmission of SPFMV and evaluating viral resistance in transgenic sweet potatoes

The sweet potato, Ipomoea batatas (L.) Lam., is the third most important root crop in the world after the potato and cassava. In particular, the sweet potato is not only an important crop in tropical, subtropical and temperate regions, but also an efficient biomass-producing plant for starch. The sweet potato is widely recognised as being prone to infection by sweet potato feathery mottle virus (SPFMV). Generally, SPFMV is transmitted by aphids (Myzus persicae), and the infected tuberous roots are used for vegetative propagation. However, neither the transmission of SPFMV using the aphid transmission test nor the aphid transmit theory itself has been established for sweet potato. The present study establishes a testing method for the aphid transmission of SPFMV and evaluates viral resistance in transgenic sweet potatoes. As a result of some examinations, we establish a testing method for resistance to SPFMV by aphid transmission in sweet potato. Furthermore, we evaluated the resistance to SPFMV in transgenic sweet potato using the improved aphid transmission method, and determined higher levels of resistance to SPFMVs in transgenic sweet potatoes. These results suggest that transgenic sweet potatoes show resistance to SPFMVs in the field.

Effect of Hairy Nightshade (Solanum Sarrachoides) Presence on Potato Nematodes, Diseases, and Insect Pests

Hairy nightshade is a common weed in potato rotations in the western United States. As a close relative of potato, hairy nightshade can host numerous potato nematodes, diseases, and insect pests. Hairy nightshade hosts three common parasitic nematodes of potato, Columbia and northern root-knot nematodes, and stubby root nematode. Tubers of a potato breeding line with roots that are resistant to Columbia root-knot nematode—race 1, were damaged when grown in the presence of hairy nightshade. The weed provided an alternate host for the nematode, which then allowed the nematode to infect susceptible tubers. Stubby root nematodes transmit tobacco rattle virus (TRV), the causal agent for corky ringspot disease (CRS) of potato. CRS disease was maintained in soil when hairy nightshade was present in rotation crops of alfalfa or Scotch spearmint that otherwise eliminated the disease. Hairy nightshade also is a host of potato leaf roll virus (PLRV), which is transmitted by green peach aphids (GPA). GPA preferentially land and readily reproduce on hairy nightshade. Aphid transmission of PLRV from hairy nightshade to potato was four times greater than the virus transmission rate from potato to potato. Integrated management of these potato nematodes, diseases, and insect pests also should include strategies to control hairy nightshade in potato and rotation crops.

Nucleotide Sequence Analysis of the Coat Protein Genes of Two Isolates of Sweet potato feathery mottle virus from Central Taiwan

Two potyvirus-like isolates, CY1 and CY2, were collected from sweet potato displaying leaf symptoms of mosaic or vein mottling at Chia-Yi area. Taiwan, by single-lesion isolation on Chenpodium quinoa plants. Using the degenerate primers for potyviruses, a 1.2 kb and a 1.3 kb DNA fragments were amplified from CY1-and CY2-infected tissues of C. quinoa, respectively, by reverse-transcription polymerase chain reaction (RT-PCR). After clotting and sequencing, the two cDNA fragments were found to be of 1205 and 1351 bp. and corresponding to a part of the 3' end of nuclear inclusion (NIb) region and the 5' end of the coat protein (CP) region of potyviruses. For amplification of the region corresponding to the 3' end of CP region, the 3' untranslated region (3'-UTR), and the poly A tail, RT-PCR was conducted with the oligo (dT) primer and the specific Primers designed from the known sequence. The assembled cDNA sequences of 1249 and 1383 bp, respectively, from CY1 and CY2 were elucidated to reflect the 3'-terminal region of nuclear inclusion b (NIb) protein gene [85 nt (28 aa) / 213 nt (71 aa)], the complete CP gene [939 nts (313 aa) /945 nts (315 aa)], and 3'-uTR (both 225 nt) and a poly (A) tail. Sequence analysis indicated that the two viruses were isolates of Sweet potato feathery mottle virus (SPFMV). The two isolates showed 80.6% nucleotide identity and 86.3% amino acid identity in their CP genes. A putative proteolytic cleavage site Q/S was predicted between NIb protein and CP. A DAG triplet for aphid transmissibility of potyviruses was found at the 9-11 residues from the N-terminus of both CP genes. Phylogenetic analysis of CP sequences revealed that SPFMV-CY1 belonged to the group C and is as closely related to the isolates 115/1S, 5, Arua 10a, O and TZ4. The sequence relationships between the two isolates and potyviruses revealed that SPFMV-CY1, SPFMV-CY2 were closely to the sweet potato infecting potyviruses, SPVY, SPVG and SPLV, reflecting a more recent evolutionary relationship.

Variation in the pathogenicity of two Turnip mosaic virus isolates in wild UK Brassica rapa provenances

Brassica rapa can be infected with Turnip mosaic virus (TuMV) as a result of manual inoculation or aphid transmission, but infected plants have not been found in the field. In this study, B. rapa plants grown from seed collected from two field sites in southern England were mechanically inoculated with one of two distinct isolates (pathotypes) of TuMV under glasshouse conditions. These had either been isolated from Brassica oleracea growing wild in Wales, (GBR 83, pathotype 3) or Dorset (GBR 98, pathotype 1). Use of ELISA as an index of infection in manually inoculated B. rapa showed that although seed provenance had a small effect on the proportion of plants infected, the biggest factor was the virus isolate. Both virus isolates infected both lines of B. rapa, but invaded at different rates, although both resulted in easily discernible symptoms. The severity of symptoms was not related to amounts of virus in the infected plants. A significantly greater proportion of plants were infected with GBR 83 at 45 days post-inoculation (d.p.i.) than GBR 98. but GBR 98 caused significantly more severe and obvious symptoms as well as greater mortality at 119 d.p.i., in plants from both sites than GBR 83.

Estimation of the number of virus particles transmitted by an insect vector

Plant viruses are submitted to narrow population bottlenecks both during infection of their hosts and during horizontal transmission between host individuals. The size of bottlenecks exerted on virus populations during plant invasion has been estimated in a few pathosystems but is not addressed yet for horizontal transmission. Using competition for aphid transmission between two Potato virus Y variants, one of them being noninfectious but equally transmissible, we obtained estimates of the size of bottlenecks exerted on an insect-borne virus during its horizontal transmission. We found that an aphid transmitted on average 0.5-3.2 virus particles, which is extremely low compared with the census viral population into a plant. Such narrow bottlenecks emphasize the strength of stochastic events acting on virus populations, and we illustrate, in modeling virus emergence, why estimating this parameter is important.

Electron-lucent inclusion bodies are structures specialized for aphid transmission of cauliflower mosaic virus

Cauliflower mosaic virus (CaMV) is transmitted by aphids. For acquisition by the vector, a transmissible complex must form, composed of the virus particle, the viral coat-associated protein P3 and the helper protein P2. However, the components of the transmissible complex are largely separated in infected plant cells: most P3 virions are confined in electron-dense inclusion bodies, whereas P2 is sequestered in electron-lucent inclusion bodies (elIBs). This spatial separation controls virus acquisition by favouring the binding of virus-free P2 to the vector first, rendering the vector competent for later uptake of P3 virions. Consequently, sequential acquisition of virus from different cells or tissues is possible, with important implications for the biology of CaMV transmission. CaMV strains Campbell and CM1841 contain a single amino acid mutation (G94R) in the helper protein P2, rendering them non-transmissible from plant to plant. However, the mutant P2-94 protein supports aphid transmission when expressed heterologously and supplied to P3-CaMV complexes in vitro. The non-transmissibility of P2-94 was re-examined in vivo and it is shown here that the non-transmissibility of this P2 mutant is not due to low accumulation levels in infected plants, as suggested previously, but more specifically to the failure to form elIBs within infected plant cells. This demonstrates that elIBs are complex viral structures specialized for aphid transmission and suggests that viral inclusion bodies other than viral factories, most often considered as 'garbage cans', can in fact exhibit specific functions.

Biological properties and relative fitness of inter-subgroup cucumber mosaic virus RNA 3 recombinants produced in vitro

In vitro reverse transcription of a mixture of total RNA from plants infected with the I17F or R strains of cucumber mosaic virus (CMV), representative of subgroups IA and II, respectively, results in viral cDNA populations including rare recombinant RNA 3 molecules, some of which also have point mutations. The biological properties of 17 recombinants in the capsid gene or the 3' non-coding region of RNA 3 were evaluated when associated with I17F RNAs 1 and 2. Six viruses displayed deficiencies (non-viability, deficiencies for movement and/or replication, delayed infection, loss of aphid transmissibility). Nine induced symptoms close to those of I17F-CMV on tobacco and pepper plants. All recombinants bearing the movement protein (MP) of R-CMV and part or most of the capsid protein (CP) of I17F-CMV, as well as the recombinant created in vitro by exchanging the corresponding open reading frames, also induced filiformism on tobacco, but induced only faint symptoms on melon. Two recombinants induced atypically severe symptoms on both tobacco and pepper. Most of the recombinants generally accumulated to lower levels than the wild-type I17F strain in tobacco. Three recombinants, however, including one responsible for severe symptoms, accumulated to generally higher levels than I17F-CMV. When two of these were tested in co-infection experiments with I17F RNA 3, they proved to be poorly competitive, suggesting that they would be unlikely to emerge in the field.

Complete nucleotide sequence of a Japanese isolate of Chrysanthemum virus B (genus Carlavirus)

The complete nucleotide sequence of a Chrysanthemum virus B isolate from Japan (CVB-S) has been determined. The genomic RNA of CVB-S is 8,990 nucleotides long, excluding the poly(A) tail and, like that of other carlaviruses, contains six open reading frames (ORFs). Multiple alignment and phylogenetic analyses indicated that the phylogenetic relationship among members of the genus Carlavirus is very diverse, with phlox virus S being the closest relative of CVB. In aphid transmission tests, CVB-S was transmitted at a very low rate by Aphis gossypii, a new vector of the virus.

Cucumber mosaic virus isolated from Aconitum spp. in Japan

Viruses were isolated from leaves of plants of Aconitum species with symptoms such as mottling and yellowing in Hokkaido and Gunma prefectures in Japan. These viruses were identified as Cucumber mosaic virus (subgroup II) based on particle morphology, host range, aphid transmission, and serology.

Role of <I>Myzus persicae</I> (Hemiptera: Aphididae) and Its Secondary Hosts in Plum Pox Virus Propagation

Plum pox virus (family Potyviridae, genus Potyvirus, PPV) is one of the most important viral pathogens of plants in the genus Prunus, particularly Prunus persica L. The role of the Myzus persicae (Sulzer) (Hemiptera: Aphididae) as a vector of PPV-M, and its role in spreading PPV-M, was investigated. PPV-M-infected peach trees were used as inoculum sources, and transmission to 15 herbaceous species commonly present in and around peach orchards was evaluated. The presence of PPV-M in secondary hosts after aphid transmission was verified by reverse transcription-polymerase chain reaction tests. The results indicate that Saponaria ocymoides L., Pisum sativum L., Trifolium repens L., Trifolium pratense L., Lepidium sativum L., Matricaria chamomilla L., Centaurea cyanus L., Bellis perennis L., Papaver rhoeas L., and Zinnia elegans L. became infected. Although Lupinus polyphyllus Lindley, Taraxacum officinale L., Achillea millefolium L., Amaranthus retroflexus L., and Linum rubrum L. did not become infected, they are hosts of M. persicae. Among the 10 positive species that were infected, the species most common in peach orchards, T. pratense, T. repens, B. perennis, and M. chamomilla, were used as source plants for the transmission studies to the peach tree. Our study reveals the ability of M. persicae to transmit PPV-M from herbaceous hosts to peach trees, describes PPV-M symptoms in herbaceous species, and discusses the role of M. persicae and its hosts as a source of PPV-M in peach orchards.