Search

MN55 is an apple (Malus ×domestica Borkh.) cultivar recently released by the University of Minnesota apple breeding program, with fruit marketed in the U.S. as Rave®. When stored for 4 months at 0 to 4 °C, MN55 fruit can develop several storage disorders, including skin dimpling. Skin dimpling incidence was greater for fruit harvested 1 week later than those harvested earlier. Dimpling was not alleviated by prestorage treatments of 1-methylcyclopropene or diphenylamine or by holding fruit at room temperature for 1 day before long-term cold storage. However, dimpling incidence was very low when fruit were stored at 6 to 7 °C. Because viruses have been implicated in other fruit dimpling disorders, the presence of viruses in MN55 leaves and fruit was studied. Apple stem pitting virus (ASPV) was detected by microscopy, reverse transcriptase polymerase chain reaction (RT-PCR) methodology, and high throughput sequencing (HTS) in peel of fruit from MN55 trees that exhibited skin dimpling after 4 months of storage at 0 to 1 °C. ASPV was also detected in supermarket-purchased fruit of other cultivars with noticeable skin dimpling. Although ASPV was not conclusively demonstrated to cause skin dimpling in our work, its prevalence indicates that further investigations are warranted to determine the relationship between viruses and skin deformities in stored apples.

Cereal chlorotic mottle virus (CCMoV) is a cicadellid-transmitted plant rhabdovirus associated with chlorotic and necrotic streaks on several gramineous hosts and weeds. The virus was initially described in 1979 in Australia, but its genome has never been sequenced. In this study, the complete genome sequence of a Moroccan isolate of CCMoV was generated by high-throughput sequencing from infected oat leaves (Avena sativa). The genome is 13,800 nt long, containing seven open reading frames (ORFs) arranged in the canonical organization of rhabdoviruses: 3'-nucleocapsid (N), phosphoprotein (P), unknown protein (p3), unknown protein (p4), matrix (M), glycoprotein (G), viral polymerase (L)-5'. Pairwise analysis showed that maize fine streak virus (MFSV, genus Gammanucleorhabdovirus) was the closest relative. The amino acid identity values between homologous proteins from CCMoV and MFSV are as follows: 59.27% (N), 36.7% (P), 24% (P3), 62% (P4), 43.70% (M), 49.15% (G), 60.93% (L). Based on its phylogenetic relationship and analogous genome architecture, CCMoV should be assigned as member of the genus Gammanucleorhabdovirus. The low sequence similarity observed between CCMoV and MFSV suggests that CCMoV is a member of a distinct virus species.

Filamentous structures were observed in purified extracts from chrysanthemum, gerbera, sunflower and zinnia. When purified filament proteins were subjected to SDS-PAGE, the major protein associated with filaments from all three species has an apparent molecular mass of ≈25 kDa. Protein bands from chrysanthemum, gerbera, and zinnia were subjected to N-terminal protein sequencing while proteins from sunflower were sequenced by CID MS/MS. All of the sequences shared highest similarity to the kunitz trypsin inhibitor family. The sequencing results indicated that the proteins lacked the signal sequences. We tested the gerbera filament protein for glycosylation and found that it was a glycoprotein. Together these results indicate that the filaments are composed of mature KTI protein. This is the first report of a KTI assembling into filaments and the first report of a filament forming Asteraceae enzyme.

The term “anthropocene” (Crutzen, 2002) refers to our current geological epoch and illustrates the manifold influences of human existence and actions on geology and evolution. Ornamentals are a true anthropogenic product solely manufactured to please the eye of the beholder. They are produced and traded all over the globe thus opening gateways for viruses. We investigated consequences of anthropogenic impact on virus spread and diversity in ornamentals. We focused on Tobamoviruses that are mechanically transmitted and are lacking a true natural vector. Our study spanned the period from 2000 to 2016 and identified Tobamovirus infections in host plants belonging to 15 different plant families. The ten identified virus species belonged mostly to Tobamovirus subgroup 1. In Solanaceae as well as in Cactaceae members of both Tobamovirus subgroup 1 and 3 were present. Different patterns of virus-host plant associations seem to exist when comparing members of both virus subgroups. Phylogenetic analysis of a partially sequenced Tobamovirus isolated from Hoya, Asclepiadaceae, in 2014 indicates the emergence of a new Tobamovirus species positioned separately from known viruses of the subgroup 3 cluster. Similar evidence has been reported independently from Florida (Schubert and Davison, 2012; Adkins et al., 2016) indicating manmediated global spread of this virus.

A disease of Rudbeckia hirta (Black-eyed Susan), characterized by severe flower deformation, was observed in Minnesota during 2010–2016. A previously undescribed virus species, named Rudbeckia flower distortion virus (RuFDV, family Caulimoviridae, genus unassigned), was determined to be the causal agent of the disease. Symptoms induced by RuFDV infection resemble those characteristic of phytoplasma-induced diseases, but no phytoplasmas were detected in RuFDV-infected R. hirta. The virus, and the disease were transmitted readily by mechanical inoculation and by the aphid Myzus persicae, but only to R. hirta. Virions of RuFDV are icosahedral, 42–45 nm in diameter, and contain a circular 8222 bp dsDNA genome containing seven open reading frames (ORFs). The ORFs 2–6 have 28–52% amino acid sequence identity to the movement protein (MP), coat protein (CP), aspartic protease (AP), reverse transcriptase (RT) and RNase H, and translational transactivator (TA) domains of known caulimoviruses. The two remaining ORFs (1 and 7) have no significant amino acid sequence similarity to known viruses. Although the RuFDV ORF 6 is significantly truncated relative to those of other known caulimoviruses, neither this nor the concomitant absence of characteristic virus-encoded cytoplasmic inclusion bodies appears to adversely affect aphid transmission of this virus. Phylogenetic analysis based on the sequence of the RT region revealed no close relationship to known members of the family Caulimoviridae. Based on sequence similarity, genome organization and phylogenetic relatedness, RuFDV appears to be distinct from any currently recognized taxonomic grouping in the family Caulimoviridae.

Prune dwarf virus (PDV) is one of the most dangerous pathogens of fruit trees worldwide. One of the most important proteins required for PDV infection is replicase. (P1 protein) which anchored viral RNA and builds replication complex along with RNA depended polymerase. Despite the importance of PDV as a pathogen, our knowledge regarding tissue/cellular localization and structure of PDV P1 protein is still incomplete. The aim of this work was to localize replicase distribution in leaf tissues and cells by immunofluorescent and immunogold labeling of Nicotiana tobaccum cv Samsun and development of a 3D model of PDV replicase. In this paper we demonstrate that PDV replication, is similar to that of Alfalfa mosaic virus and is strongly connected with tonoplasts. In addition, PDV replicase and coat protein (CP) were also found to be strongly associated with membranes of endoplasmic reticulum and, indicating the potential involvement of these membrane structures in the processes related to viral infection. Bioinformatic analyzes based on 3D modeling and structure prediction revealed that P1 protein has a potential transmembrane domain which enables protein anchoring to tonoplast during replication complex assembly.

Epimediums (Epimedium grandiflorum) are popular perennial groundcovers and are known to produce a wide range of medicinal effects. Extracts have long been used in traditional Chinese medicine. Carnation ringspot virus (CRSV) has a wide host range. In 2018, symptoms were observed on epimedium in three different landscape settings across St. Paul, MN. Leaf symptoms consisted of conspicuous mottling, chlorotic ringspots, and distortion of leaf margins. CRSV was confirmed by reverse transcription polymerase chain reaction. The CRSV host range now includes epimedium, which is the first species of the Berberidaceae family shown to be susceptible to CRSV. The potential risk of infection in other ornamental plant species in the family should be evaluated.

Virions of Kalanchoë top-spotting virus (KTSV) were purified from infected leaf tissue of Kalanchoë blossfeldiana using a procedure that prevented loss of virus in the initial extraction step. The double-stranded DNA viral genome was cloned and sequenced. The KTSV genome was 7,591 bp in size and contained three open reading frames capable of encoding proteins of 21, 14, and 223 kDa, respectively. The size and organization of the KTSV genome were similar to those of other mealybug-transmitted badnaviruses. Several oligonucleotide primer pairs, based on the KTSV genomic sequence, were used to efficiently detect the virus in plants, thereby removing a major constraint to reliable screening of kalanchoë propagating stock and breeding lines for KTSV infection. Two KTSV sequences, one symptom-inducing and the other not, were identified and differentiated by polymerase chain reaction (PCR) amplification and digestion of the resulting amplicon with restriction endonucleases. Preliminary results from graft-transmission tests and PCR indexing suggest that the nonsymptomatic form of KTSV may represent an integrated viral element. The occurrence of such integrated pararetroviral elements poses practical problems for routine PCR indexing of breeding and propagating stock, and also raises the possibility of symptomatic episomal infections arising from these viral integrants.

A disease of penstemon (Penstemon digitalis) occurring in commercial nurseries in Minnesota in 2004 to 2006 and characterized by red foliar ringspots, leaf deformation, and plant stunting was found to be caused by a strain of Turnip vein-clearing virus (TVCV) that was named Penstemon ringspot virus (PenRSV). This is the first report of a viral disease of penstemon. The genome organization of PenRSV was similar to that of the crucifer-infecting tobamoviruses. The nucleotide sequence of PenRSV was almost identical (99%) to that of TVCV, but the two viruses differed importantly in host range and symptoms induced. The only sequence difference between PenRSV and TVCV occurred at the 3′ end of open reading frame I, where the amino acid sequence FRDSNL in TVCV is replaced by FRGQQL in PenRSV. The experimental host range of PenRSV included species in the families Brassicaceae (Cruciferae), Cactaceae, Cucurbitaceae, Leguminosae, Malvaceae, and Solanaceae. This virus poses a potential threat to commercial nursery and bedding plant production because of its wide host range and because it will escape detection by immunoenzymatic screening procedures for tobamoviruses based on use of antibodies to Tobacco mosaic virus (TMV).

A previously uncharacterized virus with flexuous filamentous particles 660 nm in length was identified in the United States in florists' hydrangea (Hydrangea macrophylla), in which it caused chlorotic mottling, leaf deformation, and discoloration. The virus, tentatively named Hydrangea chlorotic mottle virus (HdCMV), was transmitted readily by mechanical inoculation and by Myzus persicae, but infected only H. macrophylla. The amino acid sequence of a 1.7-kb amplicon comprising the 3′ terminus of the HdCMV genome contained one partial and three complete putative open reading frames (ORFs) most similar in size, arrangement, and sequence to the homologous regions of the genomes of known carlaviruses. Based on virion morphology, genome properties, and current criteria for species demarcation, it was concluded that HdCMV represented a new species in the genus Carlavirus. Hydrangea ringspot virus (HdRSV, genus Potexvirus) occurred in mixed infections with HdCMV, but the two viruses could be distinguished readily by serological tests.

Sedums (Sedum spp.; Crassulaceae) are perennial landscape plants that are grown widely because they are drought tolerant and winter hardy. Plants of Sedum ‘Matrona’ showing faint foliar ringspot symptoms were collected at a nursery retail outlet in St. Paul, MN in July 2008 and tested for possible viral infection by transmission electron microscopic (TEM) examination of negatively stained, partially purified leaf tissue extracts (1). The only virus-like particles observed were rigid, rod-shaped particles similar to those of Tobacco rattle virus (TRV) and other tobraviruses. A random sample of 100 measurements showed particles 20 nm in diameter with two modal lengths of 115 nm and 175 nm. These virus-like particles were confirmed to be those of TRV by immunosorbent electron microscopy (1) using antiserum to TRV (ATCC PVAS 75) and by reverse transcription (RT)-PCR using total RNA extracted with the RNeasy Kit (Qiagen, Valencia, CA) and primers that yield a 462-bp amplicon from TRV RNA 1 (4). An amplicon of the expected size was obtained by RT-PCR and its nucleotide sequence (GenBank Accession No. GQ268817) had 95 to 99% identity to published TRV sequences (AAW13192 and AAB48382). Two additional amplicons generated by RT-PCR from separate plants were identical in size and nucleotide sequence to the first. On the basis of virion morphology, serological relatedness, and sequence identity, the virus associated with mild ringspot symptoms in sedum was identified as an isolate of TRV. To our knowledge, this represents the first report of TRV incidence in sedum. Although Arabis mosaic virus is the only other virus reported to occur in sedum (2), we have observed numerous, flexuous filamentous 750 to 800 nm virus-like particles in partially purified extracts of a range of sedums showing mild mosaic and/or vein-clearing symptoms in Minnesota. Similar virus-like particles were not observed by TEM in partially purified extracts from TRV-infected ‘Matrona’ plants, suggesting that they did not contribute to the symptoms observed. We have reported previously (3) the occurrence of TRV in a variety of widely grown perennial ornamentals that provide potential sources of inoculum for spread of this virus by nematode vectors (Trichodorus and Paratrichodorus spp.) that occur commonly in garden soil, and Sedum is now added to the list of potential TRV reservoir plants. References: (1) Y. S. Ahlawat et al. Plant Dis. 80:590, 1996. (2) A. Gera et al. Acta Hortic. 722:175, 2006. (3) B. E. Lockhart et al. Plant Dis. 79:1249, 1995. (4) D. J. Robinson. J. Virol. Methods 40:57, 1992.

Pachysandra terminalis Siebold & Zucc. (Japanese pachysandra, spurge) is widely used as a groundcover. In early 2012, Japanese pachysandra plants from Missouri, which originated in Pennsylvania, showed symptoms of light and dark green mosaic, leaf deformation, concentric ringspots, and stunting. Initial screening of symptomatic leaf tissue by transmission electron microscopy (TEM) using partially purified extracts confirmed the presence of spherical (~28 nm) and bacilliform (18-nm diameter, 35- to 58-nm length) virus particles. Immunosorbent electron microscopy (ISEM) using antisera to a clover isolate of Alfalfa mosaic virus (AMV) (PVAS 92) and to Cucumber mosaic virus (CMV) (ATCC PVAS-30) obtained from the American Type Culture Collection, Manassas, VA, confirmed the presence of AMV and CMV. No other type of virus-like particles were observed by TEM. After 6 months, nearly 20% of the 4,000 pachysandra cuttings exhibited the described symptoms. However, it is possible that more than 20% of the cuttings were infected with both viruses and not yet exhibiting symptoms. Reverse-transcription PCR (RT-PCR) was done using total RNA extracted with a Qiagen RNeasy kit and Ready-To-Go RT-PCR beads (GE Healthcare, UK Limited, UK). The primer pair CMV-1 (5′-GCCGTAAGCTGGATGGACCA) and CMV-2 (5′-TATGATAAGAAGCTTGTTTTCGCG) were used (3) to obtain a 502-bp amplicon from the coat protein (CP) region of CMV RNA 3. The product was ligated and cloned (pGEM-T Easy Vector System; Promega, USA). Three clones were sequenced (UMGC, USA), and the consensus sequence (Sequencher 5.1, Gene Codes Corp., USA) was deposited in GenBank (Accession No. JX227938). The sequence obtained had 100% identity with a homologous CP CMV sequence (AFQ94058) and 99% identity with several other homologous CP CMV sequences (CAX62443, CCK24369, and 15 others). It also contained an EcoRI site at nucleotides 332 to 337, characteristic of CMV Type II isolates (3). The primer pair AMV1F (5′-ATCCACCGATGCCAGCCTTA) and AMV1R (5′-TTCCGCCTCACTGCTGCTG) generated a 1,047-bp product from AMV RNA1 that was deposited in GenBank (JX227937). This product had 100% identity with a homologous AMV sequence (AFQ94057), and 99% identity with several other homologous AMV sequences (AGV15824, ADO85715, CBX36144). From the data presented here, it was concluded that the pachysandra had a mixed infection of AMV and a Type II isolate of CMV. Occurrence of AMV in pachysandra was first reported in New Jersey in 1982 (2) and reported for the first time in France and Germany in 2000 (1). The presence of CMV infection in pachysandra has not been reported in the present literature. Some of the symptoms associated with AMV infection in pachysandra in New Jersey (2) and Europe (1) were similar to the symptoms produced by pachysandra plants infected with both viruses (ring spots, mosaic, and line patterns). However, some symptoms were unique to the mixed infection in pachysandra by AMV and CMV (leaf deformation, stunting). A potential source of this co-infection could occur when plants are grown near alfalfa fields (AMV infection by aphids) and undergo vegetative propagation (CMV infection by contaminated tools). This is the first report of pachysandra co-infected by AMV and CMV in the United States. References: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000. (2) D. E. Hershman and E. H. Varney. Plant Dis. 66:1195, 1982. (3) S. Wylie et al. Aust. J. Agric. Res. 44:41, 1993.

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 gmax for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla ‘Sunmandeho’ Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5′-GGN AAY AAY AGY GGN CAR CC-3′) and PV1 (5′-20(T)V-3′) (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3′ end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla ‘Sunmandeho’ Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla. References: (1) J. Chen et al. Arch Virol. 146:757, 2001. (2) A. Gibbs and K. Ohshima. Ann. Rev. Phytopathol. 48:205, 2010. (3) R. L. Jordan et al. Acta Hortic. 901:159, 2011. (4) S. C. Maciell et al. Sci. Agric. Piracicaba, Brazil. 68:687, 2011. (5) D. Mollov et al. Arch Virol. 158:1917, 2013.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

Icosahedral virus-like particles were isolated from Petunia hybrida cuttings with interveinal chlorotic mottling. The virus was transmitted by mechanical inoculation from infected to healthy P. hybrida, and was found to contain a bipartite RNA genome of 7.6 and 3.8 kilobases. Full genomic sequence was obtained by high-throughput sequencing combined with RACE amplification of the 5′-termini of RNAs 1 and 2, and reverse-transcription PCR amplification of the 3′-termini with oligo-dT and sequence specific primers. Based on particle morphology, genome organization, and phylogenetic analyses, it was concluded that the new virus is a member of the genus Nepovirus, subgroup A. This new virus causing a leaf mottling disease of petunia was provisionally named Petunia chlorotic mottle virus (PCMoV).

Blueberry shock virus (BlShV), an Ilarvirus sp. reported only on blueberry, was associated with scarring, disfigurement, and premature reddening of cranberry fruit. BlShV was detected by triple-antibody sandwich enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction, and isometric virions of 25 to 28 nm were observed in cranberry sap. The virus was systemic, although unevenly distributed in plants. The coat protein of BlShV from cranberry shared 90% identity compared with BlShV accessions from blueberry on GenBank. Phylogenetic analysis of isolates of BlShV from cranberry collected from Wisconsin and Massachusetts did not indicate grouping by state. BlShV was detected in cranberry pollen, and seed transmission of up to 91% was observed. Artificial inoculation of cranberry flowers by pollination did not cause virus transmission. In some Nicotiana spp., rub inoculation of leaves with homogenized BlShV-positive cranberry flowers resulted in systemic infection. Cranberry plants recovered from symptoms the year after berry scarring occurred but continued to test positive for BlShV. The virus caused significant reduction in the average number of marketable fruit and average berry weight in symptomatic cranberry plants but recovered plants yielded comparably with healthy plants. Although recovery may limit the immediate economic consequences of BlShV, long-term implications of single- or mixed-virus infection in cranberry is unknown.

The lady’s slipper orchids are a subfamily encompassing over 160 species, including the state flower of Minnesota, Cypripedium reginae. Hydrangea is a genus of about 75 species of shrubs and trees that are popular in perennial gardens. Chlorotic and necrotic foliar symptoms were observed in lady’s slipper orchid and Hydrangea arborescens on plants in St. Paul, Minnesota. From partially purified extracts, virus particles resembling those of tobacco rattle virus (TRV) were observed. TRV-specific primers amplified products from both hydrangea and lady’s slipper and were then sequenced. The sequences matched published TRV sequences with 99% identity, confirming the presence of the virus. TRV has a broad host range including ornamental, vegetable, and weed hosts. This is the first report of TRV infection in both lady’s slipper and hydrangea in Minnesota and the United States.

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

The cell wall provides the structure of the plant, and also acts as a barier against biotic stress. The vein necrosis strain of Potato virus Y (PVYNTN) induces necrotic disease symptoms that affect both plant growth and yield. Virus infection triggers a number of inducible basal defense responses, including defense proteins, especially those involved in cell wall metabolism. This study investigates the comparison of cell wall host dynamics induced in a compatible (potato cv. Irys) and incompatible (potato cv. Sarpo Mira with hypersensitive reaction gene Ny-Smira) PVYNTN–host–plant interaction. Ultrastructural analyses revealed numerous cell wall changes induced by virus infection. Furthermore, the localization of essential defensive wall-associated proteins in susceptible and resistant potato host to PVYNTN infection were investigated. The data revealed a higher level of detection of pathogenesis-related protein 2 (PR-2) in a compatible compared to an incompatible (HR) interaction. Immunofluorescence analyses indicated that hydroxyproline-rich glycoproteins (HRGP) (extensin) synthesis was induced, whereas that of cellulose synthase catalytic subunits (CesA4) decreased as a result of PVYNTN infection. The highest level of extensin localization was found in HR potato plants. Proteins involved in cell wall metabolism play a crucial role in the interaction because they affect the spread of the virus. Analysis of CesA4, PR-2 and HRGP deposition within the apoplast and symplast confirmed the active trafficking of these proteins as a step-in potato cell wall remodeling in response to PVYNTN infection. Therefore, cell wall reorganization may be regarded as an element of “signWALLing”—involving apoplast and symplast activation as a specific response to viruses.

The purification of viruses from bananas is a complex and laborious process due to the large quantities of polysaccharides and secondary metabolites in the plant tissue. The banana streak viruses (BSVs) are generally difficult to purify to a level that allows generation of high quality antisera. This study presents an improved method of purification of Banana streak MY virus (BSMYV), which allowed us to generate highly specific chicken and rabbit antisera and to detect the capsid proteins for the first time. Finally, we demonstrate further enrichment of BSMYV by isoelectric focusing.

In sub-Saharan Africa, maize is a staple food and key determinant of food security for smallholder farming communities. Pest and disease outbreaks are key constraints to maize productivity. In September 2011, a serious disease outbreak, later diagnosed as maize lethal necrosis (MLN), was reported on maize in Kenya. The disease has since been confirmed in Rwanda and the Democratic Republic of Congo, and similar symptoms have been reported in Tanzania, Uganda, South Sudan, and Ethiopia. In 2012, yield losses of up to 90% resulted in an estimated grain loss of 126,000 metric tons valued at $52 million in Kenya alone. In eastern Africa, MLN was found to result from coinfection of maize with Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), although MCMV alone appears to cause significant crop losses. We summarize here the results of collaborative research undertaken to understand the biology and epidemiology of MLN in East Africa and to develop disease management strategies, including identification of MLN-tolerant maize germplasm. We discuss recent progress, identify major issues requiring further research, and discuss the possible next steps for effective management of MLN.

International audience; Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae, and fungi (1, 2). This broad distribution suggests the ancient origin(s) of these viruses, possibly [...]

The results also suggest one-step RT-PCR from Phalaenopsis plants and Orchid fleck virus (OFV) isolates can give false negatives for asymptomatic plants. Further work on detection methods for OFV is under way. This is the first confirmation of OFV infecting orchids in the United States. Accepted for publication 24 September 2015. Published 30 September 2015.

Two sugarcane cultivars (R570 and SP71-6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf-free environment (Montpellier, France). Plants were grown as plant cane for 5-6 months and the experiment was repeated for three consecutive years (2003-2005) in a greenhouse. Several sugarcane-growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71-6163 were infected by SCYLV genotypes BRA-PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71-6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions. (Resume d'auteur)

Ustilago maydis virus H1(Umv-H1) is a mycovirus that infectsUstilago maydis, a fungal pathogen of maize. AsZea mayswas domesticated, it carried with it many associated symbionts, such that the subsequent range expansion and cultivation of maize should have affected maize symbionts' evolutionary history dramatically. Because transmission of Umv-H1 takes place only through cytoplasmic fusion during mating ofU. maydisindividuals, the population dynamics ofU. maydisand maize are expected to affect the population structure of the viral symbiont strongly. Here, the impact of changes in the evolutionary history ofU. maydison that of Umv-H1 was investigated. The high mutation rate of this virus allows inferences to be made about the evolution and divergence of Umv-H1 lineages as a result of the recent changes inU. maydisgeographical and genetic structure. The phylogeographical history and genetic structure of Umv-H1 populations in the USA and Mexico were determined by using analyses of viral nucleotide sequence variation. Infection and recombination frequencies, genetic diversity and rates of neutral evolution were also assessed, to make inferences regarding evolutionary processes underlying the population genetic structure of ancestral and descendent populations. The results suggest that Mexico represents the ancestral population of Umv-H1, from which the virus has been carried withU. maydispopulations into the USA. Thus, the population dynamics of one symbiont represent a major evolutionary force on the co-evolutionary dynamics of symbiotic partners.

Banana streak disease is caused by several distinct badnavirus species, one of which is Banana streak Obino l'Ewai virus. Banana streak Obino l'Ewai virus has severely hindered international banana (Musa spp.) breeding programmes, as new hybrids are frequently infected with this virus, curtailing any further exploitation. This infection is thought to arise from viral DNA integrated in the nuclear genome of Musa balbisiana (B genome), one of the wild species contributing to many of the banana cultivars currently grown. In order to determine whether the DNA of other badnavirus species is integrated in the Musa genome, PCR-amplified DNA fragments from Musa acuminata, M. balbisiana and Musa schizocarpa, as well as cultivars ‘Obino l'Ewai’ and ‘Klue Tiparot’, were cloned. In total, 103 clones were sequenced and all had similarity to open reading frame III in the badnavirus genome, although there was remarkable variation, with 36 distinct sequences being recognized with less than 85 % nucleotide identity to each other. There was no commonality in the sequences amplified from M. acuminata and M. balbisiana, suggesting that integration occurred following the separation of these species. Analysis of rates of non-synonymous and synonymous substitution suggested that the integrated sequences evolved under a high degree of selective constraint as might be expected for a living badnavirus, and that each distinct sequence resulted from an independent integration event.

A Rosa hybrida plant was identified with rose rosette disease symptoms and was positive for Rose rosette virus (RRV) by reverse transcription PCR. It is important to monitor routinely roses for RRV symptoms and to test and rogue symptomatic plants. This is the first report of RRV infecting roses in Minnesota.

Recently-introduced inter-specific #Musa# hybrids, bred for improved yield and resistance to diseases, have been found to be widely infected with banana streak virus (BSV), the causal agent of banana streak disease (BSD). One hypothesis suggests: (1) that BSD occurrence in these inter-specific hybrids results from activation of BSV-01 endogenous pararetrovirus sequences (EPRV) integrated into the #Musa# genome rather than from external sources of infection, and (2) that the process of genetic hybridisation may be one factor involved in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid (#Musa# AAB) population produced by inter-specific hybridisation between virus and disease-free diploid #Musa balbisiana# (BB) and tetraploid #Musa acuminata# (AAAA) parents. Half of the F1 progeny of this cross expressed BSV particles. Using PCR amplification to determine the presence or absence of BSV-01 EPRVs, it was determined that this endogenous sequence was specific to the #M. balbisiana# genome and occurred in a homozygous state. Using bulk segregant analysis, ten AFLP markers co-segregating with the absence and/or presence of BSV infection were identified in the #M. balbisiana# genome, but were absent from the #M. acuminata# genome. Seven of these markers segregated with the presence of a BSV particle and three with the absence of BSV particles. Analysis of the segregation of these markers using a test-cross configuration allowed the construction of a genetic map of the linkage group containing the locus associated with BSV infection in the F1 hybrid population. These data indicate that a genetic mechanism is involved in BSV appearance, and suggest that a monogenic allelic system confers the role of carrier to the #M. balbisiana# parent.

Sugarcane yellow leaf virus (SCYLV) was first detected in sugarcane of Reunion Island in 1997. A field experiment was undertaken to assess the potential impact of this virus on sugarcane production. The agronomic characteristics of SCYLV-infected plants were compared to those of virus-free plants of three sugarcane cultivars (R570, R577 and R579) which occupy more than 90% of the cultivated sugarcane area on Reunion Island. In the plant crop, significant losses in stalk weight (28%) and in sugar content (11%) were detected for cultivar R577, but not for either of the two other cultivars. In the first ratoon crop, yield reduction was detected for cultivar R577 (37%), but also for cultivar R579 (19%). Cultivar R577 also showed significant losses in sugar content (12%) due to reduced amount and quality of extracted cane juice. No yield reduction was found for cultivar R570, although stalk height and diameter were reduced in SCYLV-infected canes of this cultivar in the first ratoon crop. Leaf yellowing was observed at harvest of plant and ratoon crops when sugarcane was no longer irrigated, and 10–59% of symptomatic stalks could be attributed to the presence of SCYLV. The most severe yellowing symptoms were related to infection of sugarcane by the virus.

Summary Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l’Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l’Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the ‘dead’ virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants.

Phlox are herbaceous perennial ornamentals native to North America grown for their flower color, range in flowering time, scent, and differing forms. Candidatus Phytoplasma asteris, first found to occur in Chinese asters, is spread by aster leafhoppers and in 2001 was reported to be a serious threat to phlox. There have been several reports of Ca. P. asteris in garlic and small grains in Minnesota. This is the first report of Candidatus Phytoplasma asteris in phlox in Minnesota and the United States. Accepted for publication 28 June 2016. Published 6 September 2016.

Tubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV). The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein. Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV-infected C. diffusa. These tubular structures traversed the cell wall at points where this was thickened or protruded. No similar structures were observed in healthy C. diffusa. These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.

Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.

Commelinayellow mottle virus (CoYMV) is the type member of the badnaviruses, a genus of plant pararetroviruses. The N-terminus of the polyprotein encoded by ORF III has limited similarity to known cell-to-cell movement proteins. To test the hypothesis that the N-terminus is required for viral movement, the phenotypes caused by mutations constructed in this region were determined. Similar to mutants affected in the reverse transcriptase, mutants affected in the putative movement protein were unable to cause a systemic infection. However, when the abilities of the mutated viral genomes to direct virion assembly and replication were tested using anin vitrostem-culture system, the mutants affected in the putative movement protein were found to assemble virions, whereas the reverse transcriptase mutants were unable to do so. Moreover, the putative movement protein mutants were shown to be replication competent by detection and mapping of one of the genomic discontinuities that are the hallmark of replication by reverse transcription. Thus the N-terminal region of ORF III is required for the systemic movement but not for the replication of CoYMV.

A previously undescribed badnavirus was found to be a causal agent of a disease of black pepper (Piper nigrum) in Malaysia, the Philippines, Sri Lanka and Thailand, and was also associated with a disease of betelvine (P. betle) in Thailand. Disease symptoms included chlorotic mottling, chlorosis, vein-clearing, leaf distortion, reduced plant vigor and poor fruit set. The virus, named Piper yellow mottle virus (PYMV), had non-enveloped bacilliform virions averaging 30 × 125 nm in size and containing a double-stranded DNA genome. An isolate of PYMV from Thailand was transmitted by mechanical inoculation and by the citrus mealybug, Planococcus citri, from infected P. nigrum and P. betle to healthy P. nigrum seedlings, which developed symptoms similar to those observed in naturally-infected plants. A serological relationship between PYMV and isolates of banana streak (BSV) and sugarcane bacilliform (ScBV) viruses, but not six other badnaviruses, was detected by immunosorbent electron microscopy (ISEM). Genomic PYMV sequences were amplified by polymerase chain reaction (PCR) using badnavirus-specific oligonucleotide primers, and sequence analysis comparisons of the putative reverse transcriptase (RT) domain showed PYMV to be closely related to other mealybug-transmitted badnaviruses. Black pepper infected with PYMV sometimes contained one or more isometric virus-like particles, and PYMV may therefore be only one component of a virus complex infecting black pepper in Southeast Asia.

Antibodies were prepared against bacterially expressedCommelinayellow mottle badnavirus (CoYMV) proteins. Antiserum against purified virions and antiserum against the C-terminus of the putative coat protein-encoding region of ORF III detected the same virus-specific proteins, indicating that the CoYMV coat protein is encoded in ORF III. In addition to the two major forms of the coat protein (37 and 39 kDa), several high molecular weight virus-specific proteins were detected when virions were isolated without chloroform treatment. These proteins are possible ORF III polyprotein processing intermediates and might be associated with “immature” virions which are eliminated by chloroform treatment. As predicted by the genomic sequence, a 20-kDa virus-specific protein was detected by an antiserum raised against the C-terminus of the putative ORF I protein. Results of filtration experiments suggest that the ORF I protein is equally associated with virions and with plant component(s). The association between the ORF I protein and the virions was further confirmed using immunosorbent electron microscopy and immunogold labeling. The ORF I protein was not detected in virus preparations treated with chloroform, and colocalized with virions containing immature coat protein on sucrose–cesium sulfate density gradients, suggesting that it is associated with immature virions. An antiserum raised against the putative ORF II gene product detected a 15-kDa virus-specific protein whose association with the virions was unaffected by chloroform treatment. The ORF II protein was found to be sensitive to some protease(s) that copurified with the virions, and protease inhibitors preventing this degradation have been identified.

The verified presence of TuMV in symptomatic plants fulfils Koch's postulates establishing its role in the etiology of the disease. Infection of garlic mustard by TuMV has been reported previously from Central Europe and Ontario. The only other report of natural viral infection in garlic mustard in the US is the occurrence of Cucumber mosaic virus in this host in Ohio. Accepted for publication 29 July 2012. Published 24 August 2012.