Discovery and characterization of two highly divergent variants of a novel potyvirus species infecting Madagascar periwinkle (Catharanthus roseus L.).

During summer 2022, a cluster of Madagascar periwinkle plants with white and mauve flowers were observed with foliar mild yellow mosaic symptoms on a private property in Harlingen, Cameron County, Texas. The symptoms were reproduced on mechanically inoculated periwinkle and Nicotiana benthamiana plants. Virions of 776 to 849 nm in length and 11.7 to 14.8 nm in width were observed in transmission electron microscopy of leaf dip preparations made from symptomatic periwinkle leaves. Highthroughput sequencing (HTS) analysis of total RNA extracts from symptomatic leaves revealed the occurrence of two highly divergent variants of a novel Potyvirus species as the only virus-like sequences present in the sample. The complete genomes of both variants were independently amplified via RT-PCR, cloned, and Sanger sequenced. The 5' and 3' of the genomes were acquired using RACE methodology. The assembled virus genomes were 9,936 and 9,944 nucleotides (nt) long and they shared 99.9-100% identities with the respective HTS-derived genomes. Each genome encoded hypothetical polyprotein of 3,171 amino acids (aa) (362.6 kDa) and 3,173 aa (362.7 kDa), respectively, and they shared 77.3%/84.4% nt/aa polyproteins identities, indicating that they represent highly divergent variants of the same Potyvirus species. Both genomes also shared below species threshold polyprotein identity levels with the most closely phylogenetically related known potyviruses thus indicating that they belong to a novel species. The name periwinkle mild yellow mosaic virus (PwMYMV) is given to the potyvirus with complete genomes of 9,936 nt for variant 1 (PwMYMV-1) and 9,944 nt for variant 2 (PwMYMV-2). We propose that PwMYMV be assigned into the genus Potyvirus (family Potyviridae).

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

Evidence of occurring alfalfa mosaic virus in potato plants in Assam, India.

Alfalfa mosaic virus (AMV, family Bromoviridae, genus Alfamovirus) has an extensive host range. The reports of AMV available in India were dated far back as 1979 and 1981 found in alfalfa and brinjal crops respectively. In January 2019, field surveys were conducted for viral diseases infecting potato in Sonitpur and Jorhat districts of Assam state of India. Severe yellow mosaic or calico pattern symptom, consistent with infection with AMV were observed with an incidence of approximately 25% of the plants found in farmer's fields. Sixty different symptomatic leaf samples including those associated with AMV observed were collected at random and were analysed to detect the presence of AMV. Leaf samples were frozen in liquid nitrogen and total RNA extracted from them were analyzed by one step polymerase chain reaction to detect the presence of AMV reported in potato inducing similar symptoms using a specific pair of primers for coat protein gene. An expected amplicon size of 351 bp was observed in 70% of the symptomatic leaf samples when the PCR products were analyzed on a 1.2% agarose gel. The PCR product for one sample each from the surveyed districts was eluted, purified and sequenced. The sequence results obtained were compared with those deposited in GenBank database. Blastn analysis of the sequenced isolates submitted to GenBank revealed nucleotides similar to AMV Iran isolate sequences. To our knowledge, this is the first report of AMV infecting potato in India.