Smilax auriculata: A new host for Orchid fleck dichorhavirus identified in Florida, USA.

In June 2020, Orchid fleck virus (OFV) was detected in a species of Liriope in Leon and Alachua County, Florida (Fife et al; 2021). In October of the same year, four adjacent dune/ear-leaf greenbrier vines, Smilax auriculata (Smilaceae: Liliales), showed yellowing and mottling symptoms (Figure 1). Infected and healthy S. auriculata leaves samples were collected in Alachua County by the Florida Department of Agriculture and Consumer Services, Gainesville, Florida. OFV primers successfully detected in four Smilax samples by conventional RT-PCR assay. Amplicon sequences (Acc. No. MZ645935 and MZ645938) shared 99% nucleotide identity with OFV infecting orchids (LC222629) and citrus (MK522804). The OFV subgroup I (OFV-Orc1) and subgroup II (OFV-Orc2) specific primers (Kondo et al 2017) were utilized to confirm the presence of OFV type strains infecting Smilax. Sanger sequencing of subgroup I specific amplicons (MZ645934) shared 99% nucleotide identity with OFV-Orc1 (LC222629) whereas subgroup II specific amplicon sequence (MZ645930) shared 98-99 % nucleotide identity with OFV-Orc2 (AB244417). Further confirmation was done by USDA-APHIS-PPQ-Plant Pathogen Confirmatory Diagnostics Laboratory utilizing optimized conventional RT-PCR protocols (Roy et al. 2020) and deep sequencing on a on a NextSeq550 Illumina platform. Assembled reads identified seven non-overlapping viral contigs. Five RNA1 and two RNA2 contigs covered more than 97% of the bipartite OFV genome with average coverage depth of 5297.61 and 5186.04, respectively. Contigs of RNA1 and RNA2 shared 98-99% nt identity to OFV-Orc2-RNA1 (AB244417) and OFV-Orc-RNA2 (AB244418 and LC222630). No other pathogen sequences were identified. This is the first time the genus Smilax has been identified as a natural host of OFV. Very recent findings of OFV-Orc in Florida in Liriope, Aspidistra, and Ophiopogon among the Asparagaceae family members (Fife et al; 2021) and now in the Smilacaceae suggest a broader host range of the virus than previously known; further research should be conducted to better characterize the potential risk of introduction into citrus in Florida.

First Report of Cucurbit Chlorotic Yellows Virus affecting Watermelon in USA.

Watermelon (Citrullus lanatus) is a high nutrient crop, high in vitamins and very popular in the U.S and globally. The crop was harvested from 101,800 acres with a value of $560 million in the U.S (USDA-NASS, 2020). California, Florida, Georgia and Texas are the four-leading watermelon-producing states in the U.S. During the fall season of 2020, plants in two North Florida watermelon fields, one in Levy County (~20 acres) and one in Suwannee County (~80 acres) with varieties Talca and Troubadour, respectively, exhibited viral-like symptoms. The fields had 100% disease incidence that led to fruit quality issues and yield losses of 80% and above. Symptoms observed in the watermelon samples included leaf crumpling, yellowing and curling, and vein yellowing similar to that of single/and or mixed infection of cucurbit leaf crumple virus (CuLCrV; genus: Begomovirus, family: Geminiviridae), cucurbit yellow stunting disorder virus (CYSDV; genus: Crinivirus, family: Closteroviridae) and squash vein yellowing virus (SqVYV; genus: Ipomovirus, family: Potyviridae), although the vine decline symptoms often associated with SqVYV infection of watermelon were not observed. All three viruses are vectored by whiteflies and previously described in Florida (Akad et al., 2008; Polston et al., 2008; Adkins et al., 2009). To confirm the presence of these viruses, RNA was isolated from 20 symptomatic samples using the RNeasy Plant Mini Kit (Qiagen, USA) as per protocol. This was followed by RT-PCR (NEB, USA) using gene-specific primers described for CuLCrV, CYSDV and SqVYV (Adkins et al., 2009). Amplicons of expected sizes were obtained for all the viruses with the infection of CuLCrV in 17/20, CYSDV in 16/20, and SqVYV in 8/20 samples. In addition, the presence of cucurbit chlorotic yellows virus (CCYV; genus: Crinivirus, family: Closteroviridae) in mixed infection was confirmed in 4/20 samples (3 leaves and 1 fruit) by RT-PCR with primers specific to the CCYV coat protein (CP), heat shock protein 70 homolog (HSP70h) and RNA dependent RNA polymerase (RdRp) designed based on the available CCYV sequences (Sup Table. 1). The RT-PCR amplification was performed using a symptomatic watermelon sample and the amplicons of RdRp, HSP70h and CP were directly sequenced by Sanger method, and the sequences of the amplicons were deposited in GenBank under the accession number: MW527462 (RdRp, 952 bp), MW527461 (HSP70h, 583 bp) and MW527460 (CP, 852 bp). BLASTn analysis demonstrated that the sequences exhibited an identity of 99% to 100% (RdRp and HSP70h, 100%; and CP, 99%) with the corresponding regions of the CCYV isolate Shanghai from China (accession number: KY400636 and KY400633). The presence of CCYV was further confirmed in the watermelon samples by ELISA (Loewe, Germany) using crude sap extracted from the RT-PCR-positive, symptomatic watermelon samples. CCYV was first identified in Kumamoto, Japan in 2004 on melon plants (Gyoutoku et al. 2009). The CCYV was previously reported on melon from Imperial Valley, California (Wintermantel et al., 2019), and more recently on squash in Tifton, Georgia (Kavalappara et al., 2021) and cantaloupe in Cameron, Texas (Hernandez et al., 2021). To our knowledge, this is the first report of CCYV on field watermelon production in the U.S. Continued monitoring of the CCYV in spring and fall watermelon crop, and cucurbit volunteers and weeds will be critical toward understanding the spread of this virus and its potential risk to watermelon in Florida and other regions of the U.S.

Air potato (Dioscorea bulbifera) plants displaying virus-like symptoms are co-infected with a novel potyvirus and a novel ampelovirus.

Air potato (Dioscorea bulbifera) plants being grown at the Florida Department of Agriculture and Consumer Services Division of Plant Industry Biological Control Laboratory II in Alachua County, Florida were observed exhibiting foliar mosaic symptoms characteristic of virus infection. A double-stranded RNA library generated from a symptomatic plant underwent high-throughput sequencing to determine if viral pathogens were present. Sequence data revealed the presence of two viral genomes, one with properties congruent with members of the genus Potyvirus (family Potyviridae), and the other with members of the genus Ampelovirus (family Closteroviridae). Sequence comparisons and phylogenetic placement indicate that both viruses represent novel species. The names "dioscorea mosaic virus" and "air potato virus 1" are proposed for the potyvirus and ampelovirus, respectively.

A Survey of Declining Palms (Arecaceae) With 16SrIV-D Phytoplasma to Evaluate the Distribution and Host Range in Florida.

The 16SrIV-D phytoplasma was first identified in Florida in 2006. Since its discovery, it has spread throughout most of the state. It is most prevalent in the central part of Florida, from Hillsborough County on the west coast to St. Lucie County on the east coast. The 16SrIV-D phytoplasma is the causal agent of lethal bronzing disease (LBD), which is also known as Texas Phoenix palm decline (TPPD). It affects a variety of common and economically important ornamental palm species as well as the native and ecologically important species, Sabal palmetto. It has spread into the southern portions of Florida, where the palm species diversity is higher. The aims of this survey were to document the spread of disease in terms of geographic and host range one decade after its introduction into Florida, and to assess the risk that LBD poses to the nursery and landscaping industries. The survey included samples received from stakeholders throughout the state, covering 18 counties, as well as a systematic sampling of palms at the Fort Lauderdale Research and Education Center (FLREC), where the disease is spreading actively. The findings of this survey resulted in the detection of LBD in eight new counties, including Collier, Hernando, Jefferson, Martin, Miami-Dade, Monroe, Seminole, and St. Johns, and the expansion of LBD into four new host species, Cocos nucifera, Livistona chinensis, Butia capitata, and Carpentaria acuminata. These findings are crucial for stakeholders because they highlight new hosts of 16SrIV-D phytoplasma and the geographic expansion of the disease, indicating that vigilance is needed when surveying declining palms.

Detection of Jasmine virus H and characterization of a second pelarspovirus infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants displaying virus-like symptoms.

Star jasmine (Jasminum multiflorum) plants growing in Hawaii expressing a diverse array of virus-like foliar symptoms were examined for the presence of a causal agent. Symptomatic tissues collected from three locations on the island of Oahu, Hawaii consistently harbored double-stranded (ds)RNAs approximately 4.2 and 1.7 kbp in size. Sanger and high-throughput sequencing approaches revealed these dsRNAs were from two distinct virus species co-infecting the same host plant. One of these two viruses was the recently characterized Jasmine virus H (JaVH), and the second we designated as Jasmine mosaic-associated virus (JMaV). Both viruses were subsequently found, by high-throughput sequencing, in a single angelwing jasmine (J. nitidum) plant exhibiting similar ringspot symptoms and growing at the U.S. National Arboretum in Washington, DC. Phylogenetic placement, genome organization, and sequence comparisons indicate these two viruses are classifiable as members of the genus Pelarspovirus (family Tombusviridae). To determine if either of these viruses were associated with the observed symptoms, a PCR-based detection assay was developed to detect and distinguish these two viruses in several Hawaii-grown plants. All 32 samples collected from four Oahu locations displayed symptoms. All 32 samples were positive for JaVH, and 16 were positive for JMaV. An asymptomatic star jasmine plant from the island of Hawaii was negative for both JaVH and JMaV. Both viruses were also found in a symptomatic J. sambac sample from Maryland while only JMaV was detected in a symptomatic Jasminum sp. sample from California.

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii.

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.

Deep sequencing of banana bract mosaic virus from flowering ginger (Alpinia purpurata) and development of an immunocapture RT-LAMP detection assay.

Banana bract mosaic virus (BBrMV) has never been reported in banana plants in Hawaii. In 2010, however, it was detected in a new host, flowering ginger (Alpinia purpurata). In this study, we characterize the A. purpurata isolate and study its spread in flowering ginger in Hawaii. A laboratory study demonstrated that BBrMV could be transmitted from flowering ginger to its natural host, banana, therefore raising a serious concern about the potential risk to the rapidly growing banana industry of Hawaii. To quickly monitor this virus in the field, we developed a robust immunocapture reverse transcription loop-mediated isothermal amplification (IC-RT-LAMP) assay. Deep sequencing of the BBrMV isolate from A. purpurata revealed a single-stranded RNA virus with a genome of 9,713 nt potentially encoding a polyprotein of 3,124 aa, and another predicted protein, PIPO, in the +2 reading-frame shift. Most of the functional motifs in the Hawaiian isolate were conserved among the genomes of isolates from one found in the Philippines and India. However, the A. purpurata isolate had an amino acid deletion in the Pl protein that was most similar to the Philippine isolate. Phylogenetic analysis of an eastern Pacific subpopulation that included A. purpurata was closest in genetic distance to a Southeast Asian subpopulation, suggesting frequent gene flow and supporting the hypothesis that the A. purpurata isolate arrived in Hawaii from Southeast Asia.

Polymorphic landscape of SARS-CoV-2 genomes isolated from Indian population in 2020 demonstrates rapid evolution in ORF3a, ORF8, nucleocapsid phosphoprotein and spike glycoprotein.

India, with around 15 million COVID-19 cases, recently became the second worst-hit nation by the SARS-CoV-2 pandemic. In this study, we analyzed the mutation and selection landscape of 516 unique and complete genomes of SARS-CoV-2 isolates from India in a 12-month span (from Jan to Dec 2020) to understand how the virus is evolving in this geographical region. We identified 953 genome-wide loci displaying single nucleotide polymorphism (SNP) and the Principal Component Analysis and mutation plots of the datasets indicate an increase in genetic variance with time. The 42% of the polymorphic sites display substitutions in the third nucleotide position of codons indicating that non-synonymous substitutions are more prevalent. These isolates displayed strong evidence of purifying selection in ORF1ab, spike, nucleocapsid, and membrane glycoprotein. We also find some evidence of localized positive selections ORF1ab, spike glycoprotein, and nucleocapsid. The CDSs for ORF3a, ORF8, nucleocapsid phosphoprotein, and spike glycoprotein were found to evolve at rapid rate. This study will be helpful in understanding the dynamics of rapidly evolving SARS-CoV-2.

Clg2p interacts with Clf and ClUrase to regulate appressorium formation, pathogenicity and conidial morphology in Curvularia lunata.

Ras is a small GTPase that regulates numerous processes in the cellular development and morphogenesis of many organisms. In this study, we identified and functionally characterized the Clg2p gene of Curvularia lunata, which is homologous with the Ras protein. The Clg2p deletion mutant (ΔClg2p) had altered appressorium formation and conidial morphology and produced fewer, smaller lesions compared with the wild-type strain. When a dominant Clg2p allele was introduced into the mutant, all of these defective phenotypes were completely restored. To further understand the regulation of Clg2p in appressorium formation and conidial morphology, and its role in pathogenicity, seven Clg2p-interacting proteins were screened using a yeast two-hybrid assay. Two of these proteins, Clf, a homologue of Mst11, which corresponds to MAP kinase kinase kinase in Magnaporthe oryzae, and urate oxidase (designated ClUrase) were functionally characterized. Clg2p specifically interacted with Clf through its RA domain to regulate appressorium formation and pathogenicity, whereas the Clg2p-ClUrase interaction regulated conidial morphology without affecting fungal pathogenicity. This report is the first to elucidate the regulatory mechanism of the key Ras protein Clg2p in C. lunata.

Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.