Complete Genome Sequence of Vallota Mosaic Virus Detected In A Narcissus Bulb Imported From The United States to Japan

Narcissus (Narcissus albidus) imported from the United States exhibited leaf chlorosis during post-entry quarantine. We employed next-generation sequencing (NGS) on symptomatic leaf samples and detected vallota mosaic virus (ValMV) belonging to the genus Potyvirus, family Potyviridae, as the viral agent. Sanger sequencing of PCR and rapid amplification of cDNA ends based on NGS contigs revealed that ValMV was 9,451 nucleotides (nt) in length, excluding the poly(A) tail. Nucleotide and amino acid (aa) sequences of the coat protein region had over 98% identity to previously reported ValMV isolates. At each of the 10 mature protein regions, however, sequence identity with other potyviruses was 49.5–71.9% nt and 18.3–78.9% aa, values that are below the species demarcation criteria for Potyviridae. Phylogenetic analysis revealed that our ValMV isolate is most closely related to known ValMV and is grouped within other potyviruses. Taken together, our results indicate that the newly isolated ValMV belongs to a distinct species of Potyvirus. This study provides the first report of the complete ValMV genome sequence and the first record of this virus from the narcissus.

Mealybug (Hemiptera: Pseudococcidae) Species Associated with Cacao Mild Mosaic Virus and Evidence of Virus Acquisition

Theobroma cacao is affected by viruses on every continent where the crop is cultivated, with the most well-known ones belonging to the Badnavirus genus. One of these, cacao mild mosaic virus (CaMMV), is present in the Americas, and is transmitted by several species of Pseudococcidae (mealybugs). To determine which species are associated with virus-affected cacao plants in North America, and to assess their potential as vectors, mealybugs (n = 166) were collected from infected trees in Florida, and identified using COI, ITS2, and 28S markers. The species present were Pseudococcus jackbeardsleyi (38%; n = 63), Maconellicoccus hirsutus (34.3%; n = 57), Pseudococcus comstocki (15.7%; n = 26), and Ferrisia virgata (12%; n = 20). Virus acquisition was assessed by testing mealybug DNA (0.8 ng) using a nested PCR that amplified a 500 bp fragment of the movement protein–coat protein region of CaMMV. Virus sequences were obtained from 34.6 to 43.1% of the insects tested; however, acquisition did not differ among species, X2 (3, N = 166) = 0.56, p < 0.91. This study identified two new mealybug species, P. jackbeardsleyi and M. hirsutus, as potential vectors of CaMMV. This information is essential for understanding the infection cycle of CaMMV and developing effective management strategies.

Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus

Field surveys were conducted in Greek olive orchards from 2017 to 2020 to collect information on the sanitary status of the trees. Using a high-throughput sequencing approach, viral sequences were identified in total RNA extracts from several trees and assembled to reconstruct the complete genomes of two isolates of a new viral species of the genus Tepovirus (Betaflexiviridae), for which the name olive virus T (OlVT) is proposed. A reverse transcription–polymerase chain reaction assay was developed which detected OlVT in samples collected in olive growing regions in Central and Northern Greece, showing a virus prevalence of 4.4% in the olive trees screened. Sequences of amplified fragments from the movement–coat protein region of OlVT isolates varied from 75.64% to 99.35%. Three olive varieties (Koroneiki, Arbequina and Frantoio) were infected with OlVT via grafting to confirm a graft-transmissible agent, but virus infections remained latent. In addition, cucumber mosaic virus, olive leaf yellowing-associated virus and cherry leaf roll virus were identified.

First Report of the Complete Genome Sequences of Anemone Mosaic Virus and Ranunculus Mild Mosaic Virus Isolated From Anemone Imported From the Netherlands

The anemone mosaic virus (AnMV) and ranunculus mild mosaic virus (RanMMV) infects the anemone plant with characteristic mosaic patterns on leaves. Two complete genome sequences of the two viruses imported from the Netherlands, were determined based on deep sequencing for the first time. Each of AnMV and RanMMV had 9,698 and 9,537 nucleotides (nt), excluding the poly(A) tail. They shared 80.0% nt/amino acid (aa) sequence identities or more, which are above the species demarcation value, with only AnMV and RanMMV reported previously in coat protein region, but having 68.0% nt/aa sequence identities or less with other potyviruses in each coding region of the complete sequences. Additionally, phylogenetic analysis showed that AnMV and RanMMV were included in other known potyviruses. These results suggest that both of AnMV and RanMMV were independent species belonging to the genus Potyvirus.

Cloning and Partial Characterization of Cotton Leaf Curl Burewala Virus From Khanewal

Begomoviruses are a serious threat to cotton production throughout the world. In Pakistan, enormous crop losses occur as a result of cotton leaf curl disease (CLCuD) caused by begomoviruses. Molecular characterization of begomoviruses has made possible the identification and analysis of begomoviruses prevalent in a host plant. Infected cotton leaf sample (C-59) was obtained from area around Khanewal during 2011. The total DNA was isolated from the infected sample by Cetyl trimethyl ammonium bromide (CTAB) method. An expected size band of approximately 1100bp, covering coat protein region of the virus, was amplified using universal primers. The amplified product was T/A cloned and sequenced to its entirety. DNA sequence showed 99% nucleotide sequence identity to each of Cotton leaf curl Burewala virus ((CLCuBuV; Accession No HF549Begomoviruses are a serious threat to cotton production throughout the world. In Pakistan, enormous crop losses occur as a result of cotton leaf curl disease (CLCuD) caused by begomoviruses. Molecular characterization of begomoviruses has made possible the identification and analysis of begomoviruses prevalent in a host plant. Infected cotton leaf sample (C-59) was obtained from area around Khanewal during 2011. The total DNA was isolated from the infected sample by Cetyl trimethyl ammonium bromide (CTAB) method. An expected size band of approximately 1100bp, covering coat protein region of the virus, was amplified using universal primers. The amplified product was T/A cloned and sequenced to its entirety. DNA sequence showed 99% nucleotide sequence identity to each of Cotton leaf curl Burewala virus ((CLCuBuV; Accession No HF549184)) and Cotton leaf curl Kokhran virus (CLCuKV; Accession No AJ002449)). Since CLCuBuV is a recombinant of CLCuKV and Cotton leaf curl Multan virus and the coat protein region of CLCuBuV was derived from CLCuKV that is most probable reason that the available sequence showed identity with CLCuBuV as well as CLCuKV. A complete characterization of full length virus will determine whether isolate C-59 is CLCuBuV or CLCuKV. Literature indicates that there is no existence of CLCuKV within the region and CLCuBuV is dominating within Indo-Pak184)) and Cotton leaf curl Kokhran virus (CLCuKV; Accession No AJ002449)). Since CLCuBuV is a recombinant of CLCuKV and Cotton leaf curl Multan virus and the coat protein region of CLCuBuV was derived from CLCuKV that is most probable reason that the available sequence showed identity with CLCuBuV as well as CLCuKV. A complete characterization of full length virus will determine whether isolate C-59 is CLCuBuV or CLCuKV. Literature indicates that there is no existence of CLCuKV within the region and CLCuBuV is dominating within Indo-Pak

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf patches radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in mixed-infected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90–120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf spots radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in cross-protected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90 - 120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

First Report of Cherry necrotic rusty mottle virus Infecting Sweet Cherry Trees in Korea

Cherry necrotic rusty mottle virus (CNRMV), an unassigned member in the family Betaflexiviridae, has been reported in sweet cherry in North America, Europe, New Zealand, Japan, China, and Chile. The virus causes brown, angular necrotic spots, shot holes on the leaves, gum blisters, and necrosis of the bark in several cultivars (1). During the 2012 growing season, 154 sweet cherry trees were tested for the presence of CNRMV by RT-PCR. Samples were randomly collected from 11 orchards located in Gyeonggi and Gyeongsang provinces in Korea. RNA was extracted from leaves using the NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands). The primer pair CGRMV1/2 (2) was used to amplify the coat protein region of CNRMV. Although none of the collected samples showed any notable symptoms, CNRMV PCR products of the expected size (949 bp) were obtained from three sweet cherry samples from one orchard in Gyeonggi province. The PCR products were cloned into a pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of the three Korean sequences obtained (GenBank Accession Nos. AB822635, AB822636, and AB822637) showed 97% nucleotide sequence identity with a flowering cherry isolate from Japan (EU188439), and shared 98.8 to 99.6% nucleotide and 99.6 to 100% amino acid similarities to each other. The CNRMV positive samples were also tested for Apple chlorotic leaf spot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. One of the three CNRMV-positive samples was also infected with CVA. To confirm CNRMV infection by wood indexing, Prunus serrulata cv. Kwanzan plants were graft-inoculated with chip buds from the CNRMV-positive sweet cherry trees. At 3 to 4 weeks post-inoculation, the Kwanzan plants showed quick decline with leaves wilting and dying; CNRMV infection of the indicators was confirmed by RT-PCR. To our knowledge, this is the first report of CNRMV infection of sweet cherry trees in Korea. Screening for CNRMV in propagation nurseries should minimize spread of this virus within Korea. References: (1) R. Li and R. Mock. Arch. Virol. 153:973, 2008. (2) R. Li and R. Mock. J. Virol. Methods 129:162, 2005.

First report on the association of a begomovirus with chrysanthemum indicum exhibiting yellowing of leaf vein disease characterized by molecular studies

ABSTRACT Infected leaf samples of an ornamental plant Chrysanthemum indicum showing yellowing of leaf veins were collected from gardens of New Delhi (India). An expected PCR product of size ~500 bp was amplified from total DNA extracts of symptomatic leaf samples with universal primers on the gene of coat protein region of begomovirus DNA-A component. The presence of begomoviruses was also confirmed by Southern blot analysis using control cloned DNA-A probe of Cotton leaf curl virus. Sequence analysis of the virus infecting Chrysanthemum indicum showed 99% nucleotide sequence identity with Clerodendron yellow mosaic virus (EF408037).