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The bacterial diseases black leg and soft rot in potatoes cause heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriaceae are the causative agents of black leg and soft rot. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but bacteriophages (phages) have shown promise as potential biocontrol agents. In this study we isolated soft rot bacteria from potato tubers and plant tissue displaying soft rot or black leg symptoms collected in Danish fields. We then used the isolated bacterial strains as hosts for phage isolation. Using organic waste, we isolated phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus primarily targeting P. brasiliense; phage Ymer, Amona, Sabo, Abuela, Koroua, Taid and Pappous. TEM image of phage Ymer showed siphovirus morphotype, and the proposed Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39 kb to 43 kb. In silico host range prediction using a CRISPR-Cas spacer database suggested both P. brasiliense, P. polaris and P. versatile as natural hosts for phages within the proposed Ymer genus. A following host range experiment, using 47 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease verified the in silico host range prediction, as the genus as a group were able to infect all three Pectobacterium species. Phages did, however, primarily target P. brasiliense isolates and displayed differences in host range even within the species level. Two of the phages were able to infect two or more Pectobacterium species. Despite no nucleotide similarity with any phages in the NCBI database, the proposed Ymer genus did share some similarity at the protein level, as well as gene synteny, with currently known phages. None of the phages encoded integrases or other genes typically associated with lysogeny. Similarly, no virulence factors nor antimicrobial resistance genes were found, and combined with their ability to infect several soft rot-causing Pectobacterium species from Danish fields, demonstrates their potential as biocontrol agents against soft rot and black leg diseases in potatoes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Plant viral infections lead to accumulation of virus-derived small interfering RNAs (vsiRNAs) as a result of host defense mechanisms. High-throughput sequencing technology enables vsiRNA profiling analyses from virus infected plants, which provide important insights into virus-host interactions. Potato virus Y (PVY) is a detrimental plant pathogen that can infect a variety of solanaceous crops, e.g., potato, tobacco, tomato, and pepper. We analyzed and characterized vsiRNAs derived from Nicotiana tabacum cv. Samsun infected with two recombinant PVY strains, N-Wi and NTN. We observed that the average percentage of vsiRNAs derived from plants infected with N-Wi was higher than from plants infected with NTN, indicating that N-Wi invokes a stronger host response than NTN in tobacco. The size distribution pattern and polarity of vsiRNAs were similar between both virus strains with the 21 and 22 nucleotide (nt) vsiRNA classes as most predominant and the sense/antisense vsiRNAs ratio nearly equal in the 20-24 nt class. However, the percentage of sense vsiRNAs was significantly higher in the 25-26 nt long vsiRNAs. Distinct vsiRNA hotspots, identifying highly abundant reads of different unique vsiRNA sequences, were observed in both viral genomes. Previous studies found an A or U bias at the 5' terminal nucleotide position of 21 nt vsiRNAs; in contrast, our analysis revealed a C and U nucleotide bias. This study provides insights that will help further elucidate differential processing of vsiRNAs in plant antiviral defense., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Potato virus Y (PVY) is a member of the genus Potyvirus, family Potyviridae, is considered one of the most devastating pest affecting economically important crops, such as potato, tobacco, tomato and pepper, representing a serious threat due to high incidence and worldwide distribution. Its economic significance as well as it biological and molecular complexities have aroused great attention, thus several studies have explore it genetic characteristics. However, little is known about PVY codon usage. To shed light on the relation of codon usage among viruses and their hosts is extremely important to understand virus survival, fitness and evolution. In this study, we performed a comprehensive analysis of codon usage and composition of PVY non-recombinant strains (PVY N-NA , PVY Eu-N , PVY O , PVY O5 , PVY C ) based on 130 complete open reading frame sequences extracted from public databases. Furthermore, similarities between the synonymous codon usage of PVY and its main hosts were investigated. The results obtained in the current study suggest that the overall codon usage among PVY genotypes is similar and slightly biased. PVY codon usage is strongly influenced by mutational bias, but also by G + C compositional constraint and dinucleotide composition. Furthermore, similarities among codon usage preferences between PVY strains and analyzed hosts were observed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range and RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA3 sequences from potato and partial sequences of RNA1, 2 and 3 of isolates infecting potato and yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently described isolate from Solanum muricatum (pepino dulce), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body 'b' and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E) 410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In potato tubers showing pronounced corky ringspot symptoms, two related 'rule-breaking" tobacco rattle virus (TRV) RNA2s, named Da-2 and Db-2, were identified. Their coat protein (cp) genes are preceded on the 5' side by an additional gene for a 35 kDa protein for which no relationships with previously described TRV genes or their expression products were found. With estimated 4296 and 4247 nucleotides (nts) the two RNAs are the longest TRV RNA2s described so far. The difference in size between Da-2 and Db-2 is due to a duplication of a stretch of 49 nts in the 5' untranslated region of Da-2. An alignment of TRV coat proteins (cp) revealed that up to about amino acid (aa) 176 they form two rather uniform groups. The much shorter C-terminal parts of the cps, however, differ considerably in size and composition. With 56 aa this C-terminal part is much longer in the Da and Db sequences than in all other TRV cps. It differs in 18 positions in the two strains whereas their N-terminal 184 aa differ only in two positions. - In young potato plants developing from bud-cuttings of TRV Db-infected potato tubers which had been planted in soil free of virus and nematodes a gradual degradation of Db-2 was observed. In the newly formed rootlets already five days after planting a deletion of 80 nts was observed in the putative 2b gene which in other TRV strains encodes a protein necessary for nematode transmission. Thirty three days after planting the entire 2b gene, 119 nts at the 3'end of the cp gene and a portion of the original 3'untranslated region of Db-2 had been lost in the newly formed roots, leaves and stolons. The gene for the 35kDa protein was the only one which was not affected by deletions which seems to emphasize its importance for the virus. Fifty days after planting only TRV RNA1, but no TRV RNA2 were detectable., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the past decade recombinant strains of Potato virus Y (PVY) have overtaken the ordinary strain, PVY O , as the predominant viruses affecting the US seed potato crop. Aphids may be a contributing factor in the emergence of the recombinant strains, but studies indicate that differences in transmission efficiency of individual PVY strains either from single or mixed infections, although variable, are not generally significant. Multiple strains of PVY are present in all potato production areas and common in many potato fields. Therefore, it is likely that individual alate aphids moving through a potato field will sequentially encounter multiple strains as they "taste test" multiple potato plants while looking for a suitable host. This study examined the transmission likelihood and efficiency of three common PVY strains when acquired sequentially by individual aphids. Green peach aphids (Myzus persicae, Sulzer) were allowed a 2-3min acquisition access period (AAP) on potato leaves infected with PVY O , PVY N:O or PVY NTN , followed by another 2-3min AAP on a second potato leaf infected with a different PVY strain before being transferred to healthy potato seedlings for a 24h inoculation access period. All possible combinations of the three strains were tested. Strain-specific infection of the recipient plants was determined by TAS-ELISA and RT-PCR 3-4wk post-inoculation. The recombinant strains, PVY N:O and PVY NTN , were transmitted more efficiently than PVY O when they were sequentially acquired regardless of the order acquired. PVY N:O and PVY NTN were transmitted with similar efficiencies when they were sequentially acquired regardless of the order. The recombinant strains appear to preferentially bind to the aphid stylet over PVY O or they may be preferentially released during inoculation. This may contribute to the increased incidence of the recombinant strains over PVY O in fields or production regions where multiple PVY strains are detected., (Published by Elsevier B.V.)

Potato virus Y (PVY) is the most important viral pathogen affecting potato crops worldwide. PVY can be transmitted non-persistently by aphids that do not colonize the host plant, resulting in a rapid acquisition and transmission of the virus between plants. PVY exists as a complex of strains that can be distinguished according to their pathogenicity, serology and genomic analysis. While virus incidence remains low in Scottish seed potato crops, PVY has become the increasingly prevalent virus. The monitoring of PVY N and PVY O serotypes has revealed a recent shift towards PVY N which now accounts for more than 90% of all PVY cases. A survey of the molecular diversity of PVY N isolates indicated that 80%-90% belong to the recombinant European (EU)-NTN group, with North-American (NA)-NTN and non-recombinant EU-N variants accounting for the remainder. The shift from non-recombinant to recombinant PVY isolates is a common trend observed worldwide. Surveys of a range of PVY isolates representing the main strain and phylogenetic groups suggest that PVY has the ability to overcome hypersensitive response-mediated resistance with significant differences between isolates of the same strain group. Contrastingly, genes mediating extreme resistance (Ry adg , Ry sto ) provide efficient resistance to PVY transmission to progeny tubers. Transmission experiments in field conditions of PVY isolates representing the three main molecular groups (PVY O , PVY EU-NTN , PVY NA-NTN ) indicate that PVY EU-NTN has the highest transmission rate. Our results suggest that PVY EU-NTN isolate has a competitive advantage over PVY O and PVY NA-NTN isolates which is likely to be an important factor in shaping the evolution of viruses and the population dynamics of PVY., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses., (Published by Elsevier B.V.)

Maintenance of the rod-like structure of potato spindle tuber viroid (PSTVd), which contains over 20 loops and bulges between double-stranded helices, is important for viroid biology. To study tolerance to modifications of the stem-loop structures and PSTVd capacity for mutation repair, we have created 6 mutants carrying 3-4 nucleotides deletions or insertions at three unique restriction sites, EagI, StyI and AvaII. Differences in the infectivity of these in vitro generated PSTVd mutants can result from where the mutations map, as well as from the extent to which the secondary structure of the molecule is affected. Deletion or insertion of 4 nucleotides at the EagI and StyI sites led to loss of infectivity. However, mutants with deletion (PSTVd-Ava-del) or insertion (PSTVd-Ava-in) of 3 nucleotides (221GAC223), at the AvaII site (loop 20) were viable but not genetically stable. In all analyzed plants, reversion to the wild type PSTVd-S23 sequence was observed for the PSTVd-Ava-in mutant a few weeks after agroinfiltration. Analysis of PSTVd-Ava-del progeny allowed the identification of 10 new sequence variants carrying various modifications, some of them having retained the original three nucleotide deletion at the AvaII site. Interestingly, other variants gained three nucleotides in the deletion site but did not revert to the original wild type sequence. The genetic stability of the progeny PSTVd-Ava-del sequence variants was evaluated in tomato leaves (early infection) and in both leaves and roots (late infection), respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Apical leaf curl disease, caused by tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) is one of the most important viral diseases of potato in India. Genetic resistance source for ToLCNDV in potato is not identified so far. However, the cultivar Kufri Bahar is known to show lowest seed degeneration even under high vector levels. Hence, microarray analysis was performed to identify differentially regulated genes during ToLCNDV-[potato] infection in a resistant (Kufri Bahar) and a susceptible cultivar (Kufri Pukhraj). Under artificial inoculation conditions, in Kufri Pukhraj, symptom expressions started at 15days after inoculation (DAI) and then progressed to severe symptoms, whereas no or only very mild symptoms were observed in Kufri Bahar up to 35 DAI. Correspondingly, qPCR assay indicated a high viral load in Kufri Pukhraj and a very low viral load in Kufri Bahar. Microarray analysis showed that a total of 1111 genes and 2588 genes were differentially regulated (|log2 (Fold Change)|>2) in Kufri Bahar and Kufri Pukhraj, respectively, following ToLCNDV-[potato] infection. Gene ontology and mapman analyses revealed that these altered transcripts were involved in various biological & metabolic processes. Several genes with unknown functions were 5 to 100 fold expressed after virus infection and further experiments are necessary to ascertain their role in disease resistance or susceptibility. This study gives an insight into differentially regulated genes in response to ToLCNDV-[potato] infection in resistant and susceptible cultivars and could serve as the basis for the development of new strategies for disease management., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The ability to induce the potato tuber necrosis ringspot disease (PTNRD) is a property shared by PVY isolates belonging to different groups (e.g. PVY(N) and PVY(O)) and variants (e.g. PVY(NTN) and PVY(N)-W). The identification of viral molecular determinant(s) involved in the expression of PTNRD symptoms is essential for (i) an easier detection of tuber necrosis isolates and (ii) an improvement of our knowledge on the epidemiology of this potato disease. A reverse genetic approach associated with a biological typing of a collection of PVY chimeras and mutants indicated that residue E419 of the HC-Pro protein is linked to the ability of PVY to induce tuber necrosis on four PTNRD-susceptible potato cultivars. Indeed, the substitution of the N-type glutamic acid (E) in O-type aspartic acid (D) at position 419 in the HC-Pro cistron prevents the expression of tuber necrosis on infected tubers without reducing the virulence of the corresponding E/D419 mutant. This result opens opportunities for the future studies on potato/PVY interactions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The almost complete nucleotide (nt) sequences lacking only short primer-derived 5' and 3' terminal portions were determined for the RNA2s of two closely related tobacco rattle virus (TRV) isolates which were detected in recently infected roots of Nicotiana benthamiana bait plants (isolate HaB) or of field-grown potato plants (isolate HaW), respectively. The sequences of c. 1350 5' proximal nts in these RNA2s were almost identical to those in four previously described TRV isolates from potatoes in the Netherlands and Poland which had been propagated in tobacco leaves. The RNA2s of the earlier isolates, however, consist of only c. 2000 nts, whereas the HaW and HaB RNA2s comprise more than 4000 nts. In addition to the coat protein gene which is present in the RNA2s of all isolates, the HaW and HaB RNA2s contain two further open reading frames which in the previous isolates were apparently lost either entirely or of which small remnants only were left. The RNA2s of the previously described isolates and of our new ones have different RNA1-related 3' ends which apparently have been acquired from different supporting RNA1s. After transmission to tobacco leaves HaB RNA2 formed deletion/recombination mutants which--as in the previous isolates--consisted of only c. 2000 nts. They had acquired the 3' end from their supporting RNA1., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Potato virus X (PVX; genus Potexvirus, family Alphaflexiviridae, order Tymovirales) is one of the most widespread and intensively studied viruses of potato. However, little is known about its diversity in its likely center of radiation, the Andean region of South America. To fill this gap, the strategy of Illumina deep sequencing of small RNAs was used to obtain complete or near complete genome sequence of PVX from 5 symptomatically infected greenhouse and 3 field samples (Solanum tuberosum) from Peru. PVX sequences determined in this study were assigned into three different phylogenetic groups of isolates. Notably, a complete genome sequence of a representative of a new PVX phylogenetic lineage was obtained, which shows a high level of sequence dissimilarity to other completely sequenced isolates (∼17%). The new PVX genotype was detected in greenhouse and field samples. One of the field samples was infected with the mixture of two PVX strains, which were efficiently discriminated using small RNA sequencing approach. The study confirms the utility of small RNAs deep sequencing for successful viral strain differentiation and discovery of new viral strains and indicates a high diversity of PVX in the Andean region of South America, a pattern which may be expected also for other potato pathogens., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Deep sequencing technology has enabled the analysis of small RNA profiles of virus-infected plants and could provide insights into virus-host interactions. Potato virus Y is an economically important viral pathogen of potato worldwide. In this study, we investigated the nature and relative levels of virus-derived small interfering RNAs (vsiRNAs) in potato cv. Russet Burbank infected with three biologically distinct and economically important strains of PVY, the ordinary strain (PVY-O), tobacco veinal-necrotic strain (PVY-N) and tuber necrotic strain (PVY-NTN). The analysis showed an overall abundance of vsiRNAs of 20-24nt in PVY-infected plants. Considerable differences were present in the distribution of vsiRNAs as well as total small RNAs. The 21nt class was the most prevalent in PVY-infected plants irrespective of the virus strain, whereas in healthy potato plants, the 24nt class was the most dominant. vsiRNAs were derived from every position in the PVY genome, though certain hotspots were identified for each of the PVY strains. Among the three strains used, the population of vsiRNAs of different size classes was relatively different with PVY-NTN accumulating the highest level of vsiRNAs, while PVY-N infected plants had the least population of vsiRNAs. Unique vsiRNAs mapping to PVY genome in PVY-infected plants amounted to 3.13, 1.93 and 1.70% for NTN, N and O, respectively. There was a bias in the generation of vsiRNAs from the plus strand of the genome in comparison to the negative strand. The highest number of total vsiRNAs was from the cytoplasmic inclusion protein gene (CI) in PVY-O and PVY-NTN strains, whereas from PVY-N, the NIb gene produced maximum total vsiRNAs. These findings indicate that the three PVY strains interact differently in the same host genetic background and provided insights into virus-host interactions in an important food crop., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Four tobacco rattle virus (TRV) isolates were identified from tobacco bait seedlings planted in soil samples from Polish potato fields. Sequence analysis of the genomic RNA1 of the isolates revealed significant similarity to the isolates Ho and AL recently found in Germany. Multiple sequence alignments of the genomic RNA2 indicated that the two isolates from northern Poland (Deb57 and Slu24) are in a cluster with the isolates PSG and PLB found in the Netherlands. The remaining two isolates, from central Poland (11r21 and Mlo7), are in a distinct group with the unique isolate SYM found in England. The RNA2 sequences of the studied isolates range from 1998 nt to 2739 nt in length, and all carry deletions of the 2b and/or 2c genes. The isolate Mlo7 has an atypical RNA2 structure, having its cp gene located in its central region., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Pathogens and their vectors can interact either directly or indirectly via their shared hosts, with implications for the persistence and spread of the pathogen in host populations. For example, some plant viruses induce changes in host plants that cause the aphids that carry these viruses to settle preferentially on infected plants. Furthermore, relative preference by the vector for infected plants can change to a preference for noninfected plants after virus acquisition by the vector, as has recently been demonstrated in the wheat-Rhopalosiphum padi-Barley yellow dwarf virus pathosystem. Here we document a similar dynamic in the potato-Myzus persicae (Sulzer)-Potato leaf roll virus (PLRV) pathosystem. Specifically, in a dual choice bioassay, nonviruliferous apterous M. persicae settled preferentially on or near potato plants infected with PLRV relative to noninfected (sham-inoculated) control plants, whereas viruliferous M. persicae (carrying PLRV) preferentially settled on or near sham-inoculated potato plants relative to infected plants. The change in preference after virus acquisition also occurred in response to trapped headspace volatiles, and to synthetic mimics of headspace volatile blends from PLRV-infected and sham-inoculated potato plants. The change in preference we document should promote virus spread by increasing rates of virus acquisition and transmission by the vector., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The complete genomic RNA sequences of the tymovirus isolates Hu and Col from potato which originally had been considered to be strains of the same virus species, i.e. Andean potato latent virus (APLV), were determined by siRNA sequencing and assembly, and found to share only c. 65% nt sequence identity. This result together with those of serological tests and comparisons of the coat protein gene sequences of additional tymovirus isolates from potato suggest that the species Andean potato latent virus should be subdivided into two species, i.e. APLV and Andean potato mild mosaic virus (APMMV). Primers were designed for the broad specificity detection of both viruses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Plant virus infection can alter the suitability of host plants for their aphid vectors. Most reports indicate that virus-infected plants are superior hosts for vectors compared to virus-free plants with respect to vector growth rates, fecundity and longevity. Some aphid vectors respond preferentially to virus-infected plants compared to virus-free ones, while others avoid infected plants that are inferior hosts. Thus, it appears vectors can exploit changes in host plant quality associated with viral infection. Enhanced vector performance and preference for virus-infected plants might also be advantageous for viruses by promoting their spread and possibly enhancing their fitness. Our research has focused on two of the most important luteoviruses that infect wheat (Barley yellow dwarf virus), or potato (Potato leafroll virus), and their respective aphid vectors, the bird-cherry oat aphid, Rhopalosiphum padi, and the green peach aphid, Myzus persicae. The work has demonstrated that virus infection of host plants enhances the life history of vectors. Additionally, it has shown that virus infection alters the concentration and relative composition of volatile organic compounds in host plants, that apterae of each vector species settle preferentially on virus-infected plants, and that such responses are mediated by volatile organic compounds. The findings also indicate that plants respond heterogeneously to viral infection and as a result different plant parts change in attractiveness to vectors during infection and vector responses to virus-infected plants are dynamic. Such dynamic responses could enhance or reduce the probability of virus acquisition by individual aphids searching among plants. Finally, our work indicates that compared to non-viruliferous aphids, viruliferous ones are less or not responsive to virus-induced host plant volatiles. Changes in vector responsiveness to plants after vectors acquire virus could impact virus epidemiology by influencing virus spread. The potential implications of these findings for virus ecology and epidemiology are discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper (Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.

The symptom variations among Korean Pepper mottle virus (PepMoV) isolates infecting pepper, tomato and potato were described and the cause of variations in relation to molecular variability were investigated. In addition, the entire genome of the 13 PepMoV isolates, collected from five provinces (Kyonggi, Chungnam, Gyeongnam, Jeonbuk and Jeonnam) in Korea, were determined and compared including the previously reported Korean-Vb isolate and 2 other PepMoV isolates isolated from America (CA and FL). Our results showed that the nucleotide sequence of all Korean isolates tested were nearly identical (98-99%) and only 94% similar to American isolates. In general, the complete nucleotide sequences and deduced polyprotein sequences indicated low genetic variation among isolates showing 0.1-3% nucleotide changes per site. However, based on ratio between nucleotide diversity values in nonsynonymous and synonymous position (dN/dS ratio) surprisingly, P1 and 6K2 genes showed relatively high nucleotide substitution ratio (0.8 and 1.0 nucleotide, respectively). When the 6K2 amino acid were aligned, there were 15 amino acid substitutions found in PepMoV-infected potato and only 1 amino acid change from two isolates of PepMoV-infected bell pepper. Interestingly, three isolates including isolate numbers 731, 205135 and 205136 that possessed different aa changes at 6K2 region also showed distinct symptom differentiation in indicator hosts and cosegregated in the phylogenetic analysis. These results further proved previous studies that P1 and 6K2 genes with other proteins might have some involvement on host specificity and pathogenicity.

A novel Potato virus Y (PVY) isolate, L26, recovered from a Frontier potato line was initially typed as a PVY(NTN) strain using multiplex RT-PCR and serological assays. However, L26 induced mosaic and mild vein clearing symptoms in tobacco rather than vein necrosis characteristic of the PVY (NTN) strain. The whole genome sequence was determined for L26 and two other PVY(NTN) isolates, HR1 and N4, from Idaho that did induce vein necrosis in tobacco. The sequence of all three isolates was similar to typical European PVY(NTN) isolates that contain three recombination junctions in their genome. The sequence of the L26 genome was nearly identical to the genomes HR1, N4, and to a previously characterized PVY(NTN) isolate, 423-3, differing by only five nucleotides in the entire ca. 9.7-kb genome, only one resulting in a corresponding amino acid change, D-205 to G-205 in the central region of HC-Pro. Two "signature" amino acid residues, thought involved in induction of the vein necrosis syndrome in tobacco, K-400 and E-419, were present in the C-terminal region of HC-Pro of all three isolates. Multiple alignment of the whole genome sequences of L26 and other PVY(NTN) isolates whose phenotype in tobacco has been reported, suggests that a single nucleotide change (A-1,627 to G-1,627) resulting in the single amino acid change (D-205 to G-205) in the HC-Pro cistron of L26 correlates with the loss of the vein necrosis phenotype in tobacco. Secondary structure modeling of the HC-Pro protein predicts the G-205 residue, and the previously identified residues K-400 and E-419, would all be located on the exposed surface of the protein. Taken together, these data suggest that the vein necrosis genetic determinant of PVY in tobacco is complex and includes other element(s), in addition to the C-terminal fragment of HC-Pro.

The P1 protein of Potato leafroll virus (PLRV) is thought to play a major role in the replication cycle by promoting the maturation of the genome-linked virion protein VPg. To study the relevance of P1 and its autoproteolytic derivative P1-C25 in the viral life cycle, the V H and V L domains of monoclonal antibody mAbP1-1, raised against the C-terminus of P1, were used to develop a single chain variable fragment antibody scFvP1-1 for expression in plants. The transient expression of scFvP1-1 in tobacco (Nicotiana tabacum) strongly reduced virus accumulation, while transgenic potato (Solanum tuberosum) plants expressing scFvP1-1 showed high levels of resistance following PLRV inoculation by viruliferous aphids. This is the first report that conclusively demonstrates that a PLRV gene product is essential for the completion of the virus life cycle in vivo without genetic alteration of the viral genome. This is also the first time plantibody-mediated resistance has been demonstrated with a luteovirus.

The structure of Potato virus Y (PVY) populations causing potato tuber necrotic ringspot disease (PTNRD) was analysed. The full-length sequences of the genomic RNAs of five geographically distinct isolates from Japan were determined. Recombination and phylogenetic analyses of European, North American and Japanese isolates of PVY showed that the world PVY population has three major lineages and two sublineages. Most recombinants were interlineage, and one isolate from Europe was identified as an intralineage recombinant. No recombinants were found among Japanese PTNRD isolates, which were most closely related to PTNRD isolates previously found in North America. Comparison of the within- and between population nucleotide diversities in the N lineage sequences from Japan, Europe and North America showed that Japanese population was distinct from the European and North American populations. The nucleotide sequences of the protein 1 and coat protein genes of a further 18 isolates were determined. One Japanese clade had radiated in a star burst as shown by its deviation from the neutral equilibrium model and its small nucleotide diversity. Our results suggest that PVY PTNRD was recently introduced into Japan more than once, and has expanded throughout Japan from founder populations.

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.

Plants of three different potato cultivars/lines were transformed via Agrobacterium tumefaciens with a truncated NIb gene of a necrotic strain of Potato virus Y (PVY(N)) which had been C-terminally fused to enhanced blue-fluorescing protein. Resistance of the resulting transgenic clones was evaluated under glass house conditions using an NTN-strain of PVY. Four clones with the highest levels of resistance were chosen for further experiments. Their type of resistance was either recovery or extreme resistance. These clones and their resistance types were also characterised at the molecular level. Mechanisms other than post-transcriptional gene silencing seemed to be involved in the resistance which was not dependent on sequence homology between transgene and challenging virus. Stability of resistance was tested under field conditions. The plants usually became infected with PVY. Tubers of the clone with extreme resistance did not recover from infection whereas those from clones with the recovery type did. No influence of transgenic potatoes was apparent on aphid population numbers in test plots. Recombination events could not be detected at the RNA level between transgene and challenging virus.

Sequence analysis of RNA 2 of four Tobacco rattle virus (TRV) isolates collected from potato fields in Oregon (OR2, Umt1), Washington (BM), and Colorado (Cot2) revealed significant homologies to the ORY isolate from North America. Phylogenetic analysis based on a comparison of nucleotide (nt) and amino acid (aa) sequences with other members of the genus Tobravirus indicates that the North American isolates cluster as a distinct group. All of the RNAs are predicted to contain open reading frames (ORFs) potentially encoding the coat protein (CP, ORF 2a) and 37.6 kDa (ORF 2b) ORFs. In addition, they all contain a region of similarity to the 3' terminus of RNA 1 of ORY, including a truncated portion of the 16 kDa cistron from the 3' end of RNA 1. Three of the isolates, which are nematode transmissible, OR2, BM, and Cot2, also contain a third putative ORF (ORF 2c) which encodes a protein of 33.6 kDa. The fourth isolate, Umt1, which is not nematode transmissible, is the most divergent of the isolates as it encodes a truncated version of ORF 2c. The ORF 2c deletion in Umt1 may contribute to its inability to be transmitted by the vector. The results reported in this article indicate again that the TRV genome is flexible. Interestingly, although both isolates Umt1 and Cot2 were mechanically transmitted to tobacco from potato, only Umt1 exhibits the deletion in RNA 2. TRV Isolate Umt1, therefore, appears to be another example of rapid adaptation of the TRV genome to non-field conditions.

Viruses of the genus Potyvirus, the largest genus of plant-infecting viruses, have a messenger-polarity ssRNA genome encapsidated by approximately 2000 units of the viral coat protein (CP), resulting in filamentous virions. Only few studies have examined potyvirus virions for the presence of other structural proteins. A protein linked covalently to the 5'-end of the genome has been identified in Tobacco vein mottling virus (TVMV) and Tobacco etch virus (TEV). In TEV, it is either the viral NIa protein or only its N-terminal domain (VPg) separated autocatalytically from the C-terminal proteinase domain (NIa-Pro). Virions of TVMV carry only the VPg. We examined virions of Potato virus A (PVA) for the genome-linked protein using immunoblotting or iodination and immunoprecipitation. The VPg ( approximately 25 kDa) only, and not the unprocessed NIa, was detected. Another signal corresponding to approximately 49 kDa was detected in disrupted, RNase-treated virions with anti-VPg antibodies but not with antibodies to NIa-Pro. Since it possibly represented a dimeric form of the VPg, self-interaction of the VPg was tested using the yeast two-hybrid system, which showed that the VPg self-interacts in the absence of viral RNA.

An account is given of progress during the last 30 years in knowledge of the epidemiology of diseases caused by aphid-borne viruses in seed potato production areas of northern Europe. During this period, Potato leafroll virus (PLRV) and strains of Potato virus Y (PVY(O), PVY(N)) were consistently the most prevalent and harmful. The main factors influencing spread involve the amount of initial virus inoculum in seed crops, agricultural practices in relation to seed potato production, the status of aphids as colonising or transient vectors in the crop, variation in their life cycles and behaviour, effects of environmental factors on aphid population dynamics, and the interaction between aphid phenology and mature plant resistance. Lessons have been drawn from comparisons of the causes of outbreaks of PLRV and PVY, and various comprehensive models have been designed to predict virus spread and optimise control. The accuracy and reliability of virus detection, and chemical control of aphids have been considerably improved, but use of the latter has been severely affected by the development in Myzus persicae of resistance to most types of insecticide. It is suggested that more attention should be given to breeding virus-resistant cultivars that would lead to decreased pesticide use and thus promote more integrated environment-friendly strategies for control.

High levels of field resistance to Potato leafroll virus (PLRV; Genus: Polerovirus; Family: Luteoviridae) were achieved by expression of the unmodified, full-length PLRV replicase gene in potato plants cv. Russet Burbank. A high degree of resistance was also achieved, but less frequently, by expression of a truncated construct of the replicase gene. In limited testing, neither miss-frame nor antisense constructs of the replicase gene conferred resistance. The degree of resistance expressed among different transformant lines ranged from near immunity to full susceptibility. Resistance to the Colorado potato beetle (Leptinotarsa decemlineata Say) was combined with resistance to PLRV by expression of the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis in combination with the unmodified, full-length, viral replicase gene. Resistance was expressed as a reduced incidence of infection detectable by foliage symptoms or serological tests. Reduced incidence of infection was not associated with a decrease in virus antigen concentration in the few plants of resistant lines that became infected. Virus was not detected in the foliage of symptomless plants but was detected in progeny plants produced from the tubers of inoculated but symptomless test plants of some resistant lines. The resistance was effective under natural exposure and against plant-to-plant spread of PLRV by the aphid vector, Myzus persicae Sulzer. Three of the resistant lines selected in these studies were released and are now in commercial production.

A full-length cDNA copy of potato virus A RNA was constructed downstream from the bacteriophage T7 RNA polymerase promoter. A single extra guanosine not present in PVA RNA was added to the 5'-end of the cDNA. The capped in vitro transcripts from cloned cDNA were infectious in the mesophyll protoplasts and intact plants of Nicotiana tabacum. PVA coat protein accumulated in transcript-inoculated tobacco protoplasts and infected systematically intact plants producing high PVA titres according to the ELISA and immunoblot assay. The infected tobacco plants produced similar mild mosaic symptoms in the systematically infected leaves, irrespective of whether the RNA transcripts or parental virus were used as inoculum; the virus particles were also morphologically similar according to immunosorbent electron microscopy.