The effect of mature plant resistance in sugar beet (Beta vulgaris spp. vulgaris) on survival, fecundity and behaviour of green peach aphids (Myzus persicae).

Several studies have shown the negative effects of mature plant resistance (MPR) on aphids in sugar beet, which is correlated to the formation of black deposits in their stomach. However, the underlying mechanism of MPR still needs to be elucidated, by understanding the toxicity effects of MPR on aphids and the role of the plant phenological stage and the environment. Here, we report that MPR in sugar beet does not only affect Myzus persicae mortality rate and the formation of a black deposit in the aphid stomach, but also aphid fecundity and behaviour. In addition, experiments in climate-controlled and field settings showed quantitative variation in MPR to M. persicae between six genotypes of sugar beet. Our results indicate that environmental effects, such as temperature, play a major role in MPR and underscore the importance of proper climate-controlled experiments for investigating MPR. In climate-controlled experiments, 83.3% of aphids on old leaves developed a black deposit, in contrast to only 16.8% of aphids on young leaves. This shows that not only plant age, but also leaf age plays a major role in the intensity of MPR. Further research will be needed to identify the underlying mechanism, before MPR can be used as a viable and sustainable solution to aphid pests in sugar beet.

Creation of a new genus in the family Secoviridae substantiated by sequence variation of newly identified strawberry latent ringspot virus isolates.

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named "Stralarivirus". Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV.

Efficiency of insect-proof net tunnels in reducing virus-related seed degeneration in sweet potato.

Virus-related degeneration constrains production of quality sweet potato seed, especially under open field conditions. Once in the open, virus-indexed seed is prone to virus infection leading to decline in performance. Insect-proof net tunnels have been proven to reduce virus infection under researcher management. However, their effectiveness under farmer-multiplier management is not known. This study investigated the ability of net tunnels to reduce degeneration in sweet potato under farmer-multiplier management. Infection and degeneration were assessed for two cultivars, Kabode and Polista, grown in net tunnels and open fields at two sites with varying virus pressures. There was zero virus incidence at both sites during the first five generations. Sweet potato feathery mottle virus and sweet potato chlorotic stunt virus were present in the last three generations, occurring singly or in combination to form sweet potato virus disease. Virus infection increased successively, with higher incidences recorded at the high virus pressure site. Seed degeneration modelling illustrated that for both varieties, degeneration was reduced by the maintenance of vines under net tunnel conditions. The time series of likely degeneration based on a generic model of yield loss suggested that, under the conditions experienced during the experimental period, infection and losses within the net tunnels would be limited. By comparison, in the open field most of the yield could be lost after a small number of generations without the input of seed with lower disease incidence. Adopting the technology at the farmer-multiplier level can increase availability of clean seed, particularly in high virus pressure areas.

Alstroemeria yellow spot virus (AYSV): a new orthotospovirus species within a growing Eurasian clade.

An orthotospovirus distinct from all other orthotospoviruses was isolated from naturally infected alstroemeria plants. Disease symptoms caused by this virus mainly consisted of yellow spots on the leaves based on which the name alstroemeria yellow spot virus (AYSV) was coined. A host range analysis was performed and a polyclonal antiserum was produced against purified AYSV ribonucleoproteins which only reacted with the homologous antigen and not with any other (established or tentative) orthotospovirus from a selection of American and Asian species. Upon thrips transmission assays the virus was successfully transmitted by a population of Thrips tabaci. The entire nucleotide sequence of the M and S RNA segments was elucidated by a conventional cloning and sequencing strategy, and contained 4797 respectively 2734 nucleotides (nt). Simultaneously, a next generation sequencing (NGS) approach (RNAseq) was employed and generated contigs covering the entire viral tripartite RNA genome. In addition to the M and S RNA nucleotide sequences, the L RNA (8865 nt) was obtained. The nucleocapsid (N) gene encoded by the S RNA of this virus consisted of 819 nucleotides with a deduced N protein of 272 amino acids and by comparative sequence alignments to other established orthotospovirus species showed highest homology (69.5% identity) to the N protein of polygonum ringspot virus. The data altogether support the proposal of AYSV as a new orthotospovirus species within a growing clade of orthotospoviruses that seem to share the Middle East basin as a region of origin.

The complete nucleotide sequence of chrysanthemum stem necrosis virus.

The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331 kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99 % identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates.

A bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors.

Bead-based suspension array systems enable simultaneous fluorescence-based identification of multiple nucleic acid targets in a single reaction. This study describes the development of a novel approach to plant virus and vector diagnostics, a multiplexed 7-plex array that comprises a hierarchical set of assays for the simultaneous detection of begomoviruses and Bemisia tabaci, from both plant and whitefly samples. The multiplexed array incorporates genus, species and strain-specific assays, offering a unique approach for identifying both known and unknown viruses and B. tabaci species. When tested against a large panel of sequence-characterized begomovirus and whitefly samples, the array was shown to be 100% specific to the homologous target. Additionally, the multiplexed array was highly sensitive, efficiently and concurrently determining both virus and whitefly identity from single viruliferous whitefly samples. The detection limit for one assay within the multiplexed array that specifically detects Tomato yellow leaf curl virus-Israel (TYLCV-IL) was quantified as 200fg of TYLCV-IL DNA, directly equivalent to that of TYLCV-specific qPCR. Highly reproducible results were obtained over multiple tests. The flexible multiplexed array described in this study has great potential for use in plant quarantine, biosecurity and disease management programs worldwide.

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses.

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.

Tomato marchitez virus, a new plant picorna-like virus from tomato related to tomato torrado virus.

A new virus was isolated from a tomato plant from the state of Sinaloa in Mexico. This plant showed symptoms locally known as 'marchitez disease': severe leaf necrosis, beginning at the base of the leaflets, and necrotic rings on the fruits. A virus was isolated from the infected plant consisting of isometric particles with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7221 (RNA1) and 4898 nts (RNA2). The viral capsid contains three coat proteins of 35, 26 and 24 kDa, respectively. The abovementioned characteristics: symptoms, morphology, number and size of coat proteins, and number of RNAs are similar to those of the previously described tomato torrado virus (ToTV). Sequence analysis of the entire viral genome shows that this new virus is related to, but distinct from, ToTV and that these members of two obviously new virus species belong to the recently proposed plant virus genus Torradovirus. For this new virus, the name tomato marchitez virus (ToMarV) is proposed.

Identification and characterisation of tomato torrado virus, a new plant picorna-like virus from tomato.

A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of 'torrado disease'; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.

First Report of Tomato infectious chlorosis virus in Tomato in Indonesia.

In 2002, a breeding company submitted several samples of tomato (Lycopersicon esculentum) for diagnosis. Samples originated in Indonesia and were taken from protected and nonprotected crops. Plants exhibited severe chlorosis on fully expanded leaves, while young leaves were symptomless. Symptoms resembled those of the criniviruses Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV). Moreover, large numbers of whiteflies, potential vectors of these viruses, had been observed at the plots with symptomatic plants. A reverse transcription-polymerase chain reaction (RT-PCR) with specific primers for TICV (1) yielded amplicons of the expected size of approximately 500 bp for all samples. One of the amplicons was sequenced (Genbank Accession No. AY221097) and revealed more than 98.9% identity to six isolates of TICV in NCBI Genbank. cDNA synthesis using the universal crinivirus primer HSP_M2-DW (5' -TCRAARGTWCCKCCNCCRAA-3') followed by PCR with a ToCV specific primerset (ToCV-UP 5'-TCATTAAAACTCAATGGGACCGAG-3' and ToCV-DW 5'-GCGACGT AAATTGAAACCC-3') was negative in all cases. Grafting of symptomatic shoots onto healthy tomato seedlings of cv. Money-maker showed transmission of the virus, as chlorosis appeared on fully expanded leaves of lateral shoots after 6 weeks. The presence of TICV in the graft-inoculated plants was confirmed by RT-PCR. Furthermore, mechanical inoculation to a range of herbaceous test plants did not evoke any virus symptoms, indicating the absence of mechanically transmissible viruses. Although other nonmechanically transmissible viruses cannot be fully excluded, the results together with the symptoms observed, indicate that TICV is the cause of the disease. TICV has been reported from Greece, Italy, Japan, Spain, and the United States, but to our knowledge, this is the first report of TICV in Indonesia. Reference: (1) A. M. Vaira et al. Phytoparasitica 30:290, 2002.

Pepper yellow mosaic virus, a new potyvirus in sweetpepper, Capsicum annuum.

A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3' terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3'-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3'-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested.

Natural Infection of Alstroemeria brasiliensis with Lily Mottle Virus.

During a survey for a European Union-funded project on viruses of Alstroemeria, two A. brasiliensis plants were found expressing virus-like symptoms, including leaf chlorosis with deep-green oval spots and flower color breaking. In enzyme-linked immunosorbent assays (ELISA), no positive reaction was obtained with antisera to Alstroemeria mosaic, Alstroemeria carla, Cucumber mosaic, Freesia mosaic, or Tobacco rattle virus or potyvirus-specific monoclonal antibodies (Agdia, Elkhart, IN). ELISA reactions were positive with antisera to Lily mottle (LMoV) and Rembrandt tulip breaking viruses (1). In electron microscopy preparations of A. brasiliensis, potyvirus-like particles were observed. Using sap-inoculation, the virus was transferred to a range of host species. Chenopodium quinoa, Nicotiana occidentalis accession 37B, and N. occidentalis subsp. obliqua (P1) expressed local lesions; N. clevelandii expressed local and systemic mottle; and N. benthamiana expressed local lesions, systemic vein yellowing, and leaf crinkling. Isolated total RNA from infected N. benthamiana was used for initial cDNA synthesis and polymerase chain reaction amplification with a potyvirus-specific primer set (2). The amplicon (≈670 bp) was cloned and sequenced. The sequence showed 92% homology with the corresponding region of LMoV RNA (GenBank accession no. S44147). The results confirm the infection of A. brasiliensis with LMoV. This is the first report of natural infection of Alstroemeria by LMoV. References: (1) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993. (2) R. A. A. van der Vlugt et al. Phytopathology 89:148, 1999.

First Report of Pepino Mosaic Virus on Tomato.

Early in 1999 a new viral disease occurred in protected tomato (Lycopersicon esculentum) crops in the Netherlands. Infected plants showed yellow leaf spots and mosaic. Transmission electron microscopic analysis revealed particles typical of potexviruses. Only three potexviruses have been reported to infect solanaceous crops: Pepino mosaic virus (PepMV), Potato aucuba mosaic virus (PAMV), and Potato virus X (PVX). Inoculation of test plants and serological tests showed that the new virus clearly differed from PAMV and PVX. Immuno-electron microscopy with antiserum to PepMV (1), the original PepMV isolate, and the virus from tomato showed decoration titers of 1:800 (homologous) and 1:400, respectively. Neither virus reacted with antiserum to PVX, nor did PVX react with antiserum to PepMV. Results of host plant analysis with 17 plant species mostly resembled those expected for PepMV. Nucleotide sequence alignment of DNA fragments obtained by reverse-transcriptase polymerase chain reaction with a specific primer set for potexviruses, directed against the RNA polymerase region, showed 93% identity between PepMV and the virus from tomato, while homologies with PVX, PAMV, and other potexviruses were <60%. Results indicate that the potexvirus in tomato is PepMV. PepMV was first found in pepino (Solanum muricatum) in Peru in 1974 and described by Jones et al. in 1980 (1). This is the first report of a natural infection of tomato by PepMV. Reference: (1) R. Jones et al. Ann. Appl. Biol. 94:61, 1980.

Natural Infection of Alstroemeria caryophyllea with Ornithogalum mosaic virus.

During a survey for a European Union-funded project on the viruses of Alstroemeria, an A. caryophyllea plant was found expressing virus-like symptoms, including dark green vein banding, necrotic spots, and flower color breaking. In enzyme-linked immunosorbent assays (ELISA), no positive reaction was obtained with antisera to Alstroemeria mosaic, Alstroemeria carla, Cucumber mosaic, Freesia mosaic, or Tobacco rattle virus. A positive ELISA reaction was obtained with potyvirus-specific monoclonal antibodies (Agdia, Elkhart, IN) and antiserum to Ornithogalum mosaic virus (OrMV) (1). In electron microscopy leaf dip preparations of A. caryophyllea, potyvirus-like particles were observed. Using sapinoculation, the virus was transferred to Chenopodium amaranticolor and C. quinoa, resulting in local lesions 6 days postinoculation. The presence of OrMV in both Chenopodium spp. was confirmed by electron microscopy and ELISA with antiserum to OrMV. Sequence alignment of DNA fragments (740 bp) obtained in immunocapture-reverse transcription-polymerase chain reaction on RNA isolated from the suspect virus, using a potyvirus-specific primer set (2), showed 91% homology with the corresponding region of OrMV RNA (GenBank accession no. D00615). The results confirm the infection of A. caryophyllea by OrMV. This is the first report of natural infection of Alstroemeria by OrMV. References: (1) J. T. Burger and M. B. von Wechmar. Phytopathology 79:385, 1989. (2) R. A. A. van der Vlugt et al. Phytopathology 89:148, 1999.

Nucleotide sequence of the 3' terminal region of the genome of four lettuce mosaic virus isolates from Greece and Yemen.

Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3' terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3' non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1(1) and mo1(2) and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR.

Further Evidence that Shallot Yellow Stripe Virus (SYSV) Is a Distinct Potyvirus and Reidentification of Welsh Onion Yellow Stripe Virus as a SYSV Strain.

ABSTRACT An antiserum to shallot yellow stripe virus (SYSV) was raised and used in combination with a range of other antisera to potyviruses of Allium spp. in electron microscopic decoration experiments. The serological results corroborated an earlier finding that the type isolates of SYSV and Welsh onion yellow stripe virus (WoYSV) are closely related to each other and only distantly related to onion yellow dwarf (OYDV) and leek yellow stripe (LYSV) viruses, the two other major potyviruses infecting Allium spp. Moreover, the decoration results indicated that Japanese potyviruses named OYDV and Wakegi yellow dwarf virus are isolates of SYSV. Sequence analysis of the 3'-terminal regions of the SYSV and WoYSV ge-nomes revealed coat protein (CP) amino acid and 3'-nontranslated region (3'-NTR) nucleotide sequence identities of 95 and 89%, respectively. The CP amino acid and 3'-NTR nucleotide sequences of these viruses differed from those of OYDV and LYSV by >25 and >67%, respectively. The serological and molecular studies showed that SYSV and WoYSV are different strains of a potyvirus distinct from OYDV and LYSV. For priority reasons, we propose that these strains together with the Wakegi-type isolates of OYDV described in Japan be referred to as SYSV and that SYSV isolates from Allium spp. other than shallot be designated as the Welsh onion strain of SYSV (SYSV-Wo).

Complex formation determines the activity of ribozymes directed against potato virus YN genomic RNA sequences.

A ribozyme was synthesized against a conserved region in the RNA-dependent RNA-polymerase encoding cistron of the important plant pathogen potato virus Y (PVY). This ribozyme was shown to cleave PVY-specific RNA-transcripts efficiently in vitro, with up to 95% of the substrate RNA being cleaved within 2 h incubation at 37 degrees C. A second ribozyme, designed with much shorter viral complementary arms in an attempt to optimize the efficiency of the cleavage reaction, surprisingly failed to cleave the substrates previously cleaved by the longer ribozyme. A much shorter PVY specific RNA-transcript of only 37 nucleotides (nt), however, was cleaved by this short ribozyme proving its ribozymic activity and indicating that the cleavage activity of the ribozyme is, in part, determined by the substrate involved. Analysis of cleavage reactions on non-denaturing polyacrylamide (PAA) gels indicated that incorrect basepairing, interfering with correct formation of the hammerhead structure, was likely to be responsible for the absence of detectable cleavage of the larger substrates by the short ribozyme.

Taxonomic relationships between distinct potato virus Y isolates based on detailed comparisons of the viral coat proteins and 3'-nontranslated regions.

Detailed comparisons were made of the sequences of the coat protein (CP) cistrons and 3'-nontranslated regions (3'-NTR) of 21 (geographically) distinct isolates of potato virus Y (PVY) and a virus isolate initially described as pepper mottle virus (PepMoV). Multiple sequence alignments and phylogenetic relationships based on these alignments resulted into a subgrouping of virus isolates which largely corresponded with the historical strain differentiation based on biological criteria as host range, symptomatology and serology. Virus isolates belonging to the same subgroup shared a number of characteristic CP amino acid and 3'-NTR nucleotide residues indicating that, by using sequences from the 3'-terminal region of the potyvirus genome, a distinction could be made between different isolates of one virus species as well as between different virus species. RNA secondary structure analysis of the 3'-NTR of twelve PVY isolates revealed four major stem-loop structures of which, surprisingly, the loop sequences gave a similar clustering of isolates as resulting from the overall comparisons of CP and 3'-NTR sequences. This implies a biological significance of these structural elements.

Evidence for sense RNA-mediated protection to PVYN in tobacco plants transformed with the viral coat protein cistron.

The coat protein (CP) cistron of the tobacco veinal necrosis strain of potato virus Y (PVYN), supplemented with translational start signals, was cloned into an Agrobacterium tumefaciens Ti transformation vector. Transformation of tobacco leaf discs resulted in 99 transgenic lines which were subsequently analysed for the presence and expression, at both the transcriptional and translational level, of the CP-gene. Although CP-specific RNA transcripts were produced in all plants no CP could be detected by several sensitive immunological techniques. Upon mechanical inoculation of progeny lines of self-pollinated original transformants (S1) with PVYN, protection levels of 20 and 95%, respectively, could be observed in two out of ten lines tested. This level of protection increased to 100% in the S2 progeny obtained from self-pollination of virus-protected S1 plants. Transformation of tobacco leaf discs with a PVYN CP construct from which the ATG start codon had been removed by site-directed mutagenesis resulted in 57 transgenic lines that all produced CP-specific transcripts. Mechanical inoculation with PVYN of S1 progeny plants of several of these lines resulted in resistance to a similar level and extent as in the S1 progeny of plants transformed with the intact CP cistron. The results obtained strongly suggest that the resistance observed in the transgenic plants is principally based on the presence of PVYN CP RNA sequences rather than on the accumulation of viral coat protein.