Molecular studies on a complex of potyviruses infecting solanaceous crops, and some specific virus-host interactions

This thesis constitutes a comprehensive analysis of the molecular and biological characteristics of three potyviruses (genus Potyvirus, family Potyviridae) naturally occurring in cultivated and wild species of family Solanaceae: Peru tomato virus (PTV), Potato virus V (PVV) and Wild potato mosaic virus (WPMV). In addition, the studies presented in this thesis focus on the genetic variability of isolates of PTV and PVV and on the role of the Potato virus A (PVA) 6K2 protein as a host-specific determinant of virus movement and symptom induction. Determination of the complete genomic sequences of PVV, PTV and WPMV demonstrated that these viruses are typical members of the genus Potyvirus. Furthermore, comparison of the polyprotein amino acid sequences and the biological and serological characteristics of these three viruses supported their current taxonomic position as independent species of the genus Potyvirus. The nucleotide sequences of the P1 protein, coat protein and non-translated regions of European and South American PVV isolates were determined and compared. Results showed limited genetic variability among the European isolates, in contrast to the higher variability found among the South American isolates of PVV. Phylogenetic analysis defined two distinct clusters, grouping the European isolates together but placing two South American isolates to a different group; these two isolates of PVV did not induce a hypersensitive response in an Nv gene-carrying potato cultivar in contrast to the European PVV isolates. Thus, European and South American PVV isolates belong to different strain groups. In addition, great genetic variability was detected among PTV isolates. Analysis of phylogenetic relationships among PTV, PVV, WPMV and other members of the genus Potyvirus commonly found infecting solanaceous crop plants showed that PTV, PVV and WPMV are the most closely related viruses which together with Potato virus Y, Pepper mottle virus, Pepper severe mosaic virus and Pepper yellow mosaic virus constitute a group distinguishable from other potyviruses. Thus, members of this group seem to share a common ancestor. The 6K2 protein of PVA was modified by deleting various portions or by introducing six histidine residues (6xHis) into various positions of this protein. These modifications disturbed functions required for viral infection in Nicotiana tabacum. Furthermore, inoculation of the insertion constructs to N. benthamiana plants did not result in systemic infection with the exception of one plant. This plant lacked typical PVA symptoms but had virus titers similar to the plants infected with the wild type virus: a single point mutation (Gly2 ® Cys2) in the 6xHis-containing 6K2 had restored the viral movement functions. However, partial deletion of the 6xHis-tag to gain the original size of the 6K2 protein was required to restore the induction of symptoms in N. benthamiana and to enable systemic infection of N. tabacum. Taken together, these results indicate the 6K2 is a host-specific determinant for long-distance movement and exemplify that mutations that arise during viral propagation represent a mechanisms by which viruses can evolve and adapt to different hosts

Virus i jordbær 2017. Resultater fra OK-programmet.

Det ble sommeren 2017 gjennomført et kartleggingsprogram for å undersøke om importert plantemateriale av jordbær kunne være infisert av virus. Det ble tatt ut stikkprøver i jordbærfeltene hos utvalgte bærprodusenter som hadde baser sin produksjon på importerte planter, til sammen 150 prøver. Disse prøvene ble testet for fire bladlusoverførte virus som er relativt vanlige i Europa, men ikke i Norden: jordbær-nervebåndvirus (strawberry vein banding virus), jordbær-mildmosaikkvirus (strawberry mottle virus), jordbær-bladgulningvirus (strawberry mild yellow edge virus) og jordbærbladkrøllevirus (strawberry crinkle virus). Det ble påvist virus i prøver fra tre lokaliteter: strawberry crinkle virus ble funnet på tre steder, mens strawberry mild yellow edge virus ble funnet på ett sted i en prøve som hadde dobbeltinfeksjon med strawberry mild yellow edge virus og strawberry crinkle virus. Prøvene hadde ikke synlige symptomer.publishedVersio

Kartlegging av virus i jordbær 2017 og 2018. Resultater fra OK-programmet.

Det ble sommeren 2017 og 2018 gjennomført et artleggingsprogram for å undersøke om plantemateriale av jordbær som var nylig importert, kunne være infisert av virus. Det ble tatt ut stikkprøver i jordbærfeltene hos utvalgte bærprodusenter som hadde baser sin produksjon på importerte planter, i 2017 til sammen 150 prøver fra 20 bruk i fylkene Hedmark, Oppland, Vestfold, Buskerud, Agder, Rogaland og Sogn og Fjordane. I 2018 ble det tatt ut 156 prøver fra 14 bruk i fylkene Østfold, Akershus, Møre og Romsdal og Trøndelag. Disse prøvene ble testet for fire bladlusoverførte virus som er relativt vanlige i Europa, men ikke i Norden: jordbær-nervebåndvirus (strawberry vein banding virus, SVBV), jordbær-mildmosaikkvirus (strawberry mottle virus, SMoV), jordbær-bladgulningvirus (strawberry mild yellow edge virus, SMYEV) og jordbær-bladkrøllevirus (strawberry crinkle virus, SCV). Det ble i 2017 påvist virus i prøver fra tre lokaliteter: SCV ble funnet på tre steder, mens SMYEV ble funnet på ett sted i en prøve som hadde dobbeltinfeksjon med SMYEV og SCV. Prøvene hadde ikke synlige symptomer. I 2018 ble det blant 156 prøver påvist virus i én prøve – det var SVBV i en prøve av ‘Sonata’ i Akershus. Denne prøven hadde heller ikke synlige symptomer.publishedVersio

Begrenset kartlegging av virusoverførende bladlus i potet i Norge i 2011

Bladlusoverført virus er et problem i potet i Norge. Hvert år er det settepotetpartier i den sertifiserte avlen som ikke kan godkjennes fordi innholdet av PVY/PVA er for høye. For høyt virusinnhold i potetprodusentenes egen oppformering er også et problem. For årene 2008, 2009, 2010 og 2011 har det blitt sendt inn virusprøver til NAK (Nederland) fra egne oppformerte settepoteter hos potetdyrkerne. Resultatene viste høye innhold av både PVA og PVY. Dette kan fort gi store avlingstap. I Norge har vi ikke god nok kunnskap om hvilke lusarter som herjer i potetåkre. Med bakgrunn i problemstillingen skissert over ønsket Norsk Landbruksrådgiving at Bioforsk Plantehelse skulle opparbeider seg mer kunnskap om følgende: Hvilke bladlusarter er det i norske potetåkre? Prosjekt: Kartlegging av bladlusarter i potetåkre. Hvor aktive er de i overføring av virus? Hvilken skade gjør disse bladlusartene i potetåkre i Norge? Prosjekt: Forsøk i potetåker med bladlusproblemer. Hvordan kan lus i potet overvåkes, for dermed å sette inn riktig tiltak til rett tid, slik at skader ikke oppstår i avlinga. For eksempel få til en ”indeks” (terskel) som ut fra bladluspopulasjonen angir når riset skal fjernes for å redusere virusmengden i settepotetene. Prosjekt: Vurderes når kartlegging og resultater fra forsøk er ”på plass”publishedVersio

Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2–3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated

Next generation sequencing as a method to verify virus elimination using heat treatment and meristem tip culture in the five most widely used sweet potato varieties in Ethiopia

Sweet potato (Ipomoea batatas L. Lam) has become one of the staple crops in Africa in the last 20 years. In Ethiopia, sweet potato is the second most widely grown root crop and is the first regarding the production per hectare. Thus, there is a great demand of planting material throughout the country. Currently, planting material is usually obtained from own previous season harvest, local markets or from the neighboring fields since no certified clean planting material production scheme has been established in Ethiopia yet. Unfortunately, this practice has contributed to the spread of viral diseases throughout the country. Elimination of viruses from infected plants is a tedious job, which requires efficient methods to eliminate the virus and also to verify that the plants are indeed virus-free. In the case of sweet potato, it was observed that heat treatment, combined with meristem tip culture is an efficient method for virus elimination. Previous findings indicate that reverse transcription (RT) PCR is more efficient than ELISA to verify the efficiency of virus elimination. In this study, the use of next generation sequencing (NGS) was explored as a verification method and compared with RT-PCR. The results show that NGS seems to be more efficient than RT-PCR, although also prone to inconclusive results.publishedVersio

Comparative transcriptome analysis in Triticum aestivum infecting wheat dwarf virus reveals the effects of viral infection on phytohormone and photosynthesis metabolism pathways

Wheat dwarf virus (WDV), a mastrevirus transmitted by the leafhopper Psammotettix alienus, causes a severe disease in cereal crops. Typical symptoms of wheat plants infected by WDV are yellowing and severe dwarfing. In this present study, RNA-Seq was used to perform gene expression analysis in wheat plants in response to WDV infection. Comparative transcriptome analysis indicated that a total of 1042 differentially expressed genes (DEGs) were identified in the comparison between mock and WDV-inoculated wheat plants. Genomes ontology (GO) annotation revealed a number of DEGs associated with different biological processes, such as phytohormone metabolism, photosynthesis, DNA metabolic process, response to biotic stimulus and defense response. Among these, DEGs involved in phytohormone and photosynthesis metabolism and response pathways were further enriched and analyzed, which indicated that hormone biosynthesis, signaling and chloroplast photosynthesis-related genes might play an important role in symptom development after WDV infection. These results illustrate the dynamic nature of the wheat-WDV interaction at the transcriptome level and confirm that symptom development is a complex process, providing a solid foundation to elucidate the pathogenesis of WDV.publishedVersio

Virus i jordbær 2017. Resultater fra OK-programmet.

Det ble sommeren 2017 gjennomført et kartleggingsprogram for å undersøke om importert plantemateriale av jordbær kunne være infisert av virus. Det ble tatt ut stikkprøver i jordbærfeltene hos utvalgte bærprodusenter som hadde baser sin produksjon på importerte planter, til sammen 150 prøver. Disse prøvene ble testet for fire bladlusoverførte virus som er relativt vanlige i Europa, men ikke i Norden: jordbær-nervebåndvirus (strawberry vein banding virus), jordbær-mildmosaikkvirus (strawberry mottle virus), jordbær-bladgulningvirus (strawberry mild yellow edge virus) og jordbærbladkrøllevirus (strawberry crinkle virus). Det ble påvist virus i prøver fra tre lokaliteter: strawberry crinkle virus ble funnet på tre steder, mens strawberry mild yellow edge virus ble funnet på ett sted i en prøve som hadde dobbeltinfeksjon med strawberry mild yellow edge virus og strawberry crinkle virus. Prøvene hadde ikke synlige symptomer

Kartlegging av virus i jordbær 2017 og 2018. Resultater fra OK-programmet.

Det ble sommeren 2017 og 2018 gjennomført et artleggingsprogram for å undersøke om plantemateriale av jordbær som var nylig importert, kunne være infisert av virus. Det ble tatt ut stikkprøver i jordbærfeltene hos utvalgte bærprodusenter som hadde baser sin produksjon på importerte planter, i 2017 til sammen 150 prøver fra 20 bruk i fylkene Hedmark, Oppland, Vestfold, Buskerud, Agder, Rogaland og Sogn og Fjordane. I 2018 ble det tatt ut 156 prøver fra 14 bruk i fylkene Østfold, Akershus, Møre og Romsdal og Trøndelag. Disse prøvene ble testet for fire bladlusoverførte virus som er relativt vanlige i Europa, men ikke i Norden: jordbær-nervebåndvirus (strawberry vein banding virus, SVBV), jordbær-mildmosaikkvirus (strawberry mottle virus, SMoV), jordbær-bladgulningvirus (strawberry mild yellow edge virus, SMYEV) og jordbær-bladkrøllevirus (strawberry crinkle virus, SCV). Det ble i 2017 påvist virus i prøver fra tre lokaliteter: SCV ble funnet på tre steder, mens SMYEV ble funnet på ett sted i en prøve som hadde dobbeltinfeksjon med SMYEV og SCV. Prøvene hadde ikke synlige symptomer. I 2018 ble det blant 156 prøver påvist virus i én prøve – det var SVBV i en prøve av ‘Sonata’ i Akershus. Denne prøven hadde heller ikke synlige symptomer.publishedVersio

An update of sweet potato viral disease incidence and spread in Ethiopia

Sweet potato (Ipomoea batatas (L.) Lam.) is an important root crop for poor farmers in developing countries. Since the late 1980s, viral diseases have increasingly become a threat to sweet potato production in Ethiopia. This review paper presents the role of sweet potato production for ensuring food security, the level of sweet potato virus research, including the types of viral species identified and their current level of incidences in Ethiopia. Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus 2 (SPV2), Sweet potato virus G (SPVG), and Cucumber mosaic virus (CMV) were reported in Ethiopia, where the first two are the most common and exist at high incidences. In addition, this paper discusses the virus vectors, virus transmission methods to new farms, factors exacerbating the rate of viral incidence and the methods used to reduce the incidences. Moreover, it highlights methods of sweet potato viruses’ detection and cleaning of infected materials in use and the challenges encountered towards the efficient utilization of the methods. Finally, we suggest major intervention techniques that will integrate all key players in managing the impact of the virus on sweet potato production to improve productivity and ensuring food security in Ethiopia. The findings obtained from this review could be an input for the current research on sweet potato improvement (both planting materials and routines) in Ethiopia.publishedVersio