Sugarbeet provides an important source of sucrose, a stable, environmentally safe, and low-cost staple in the human diet. Viral diseases arising in sugarbeet ultimately impact sugar content, which translates to financial losses for growers. To manage diseases and prevent such losses from occurring, it is essential to characterize viruses responsible for disease. Recently, our laboratory identified a tobacco necrosis virus A variant named Beta vulgaris alphanecrovirus-1 (BvANV-1) in sugarbeet roots. We validated the infectivity of BvANV-1 using two DNA-based clones: one sequence referred to as the wild type and another as mutant #7 that harbors one nonsynonymous and one synonymous mutation in the viral replicase gene (p23) and one synonymous mutation in the p8 movement protein gene relative to the wild type. The host range of the virus was determined through inoculating a series of local weed species and agriculturally relevant hosts, which showed that soybean and pinto bean are potentially important hosts of this virus. Using both clones, viral transmission to sugarbeet by Olpidium virulentus was verified. Using site-directed mutagenesis of mutant #7, we demonstrated that the amino acid change in the p23 gene alone restored the phenotype similar to the wild type on Nicotiana benthamiana-inoculated leaves. Additionally, this latter change and the synonymous mutation in the p8 gene were both required to re-establish systemic infection in N. benthamiana similar to the wild type. Our analysis reveals differences among both variants and lays the groundwork for characterizing the role of BvANV-1 proteins during infection. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Sugarbeet (Beta vulgaris L.), grown worldwide for sugar production, is susceptible to various viral diseases. Identifying the causal viral pathogen can be difficult due to mixed infections that might contribute to the disease symptoms. In the fall of 2022, widespread yellowing, chlorosis, vein clearing, and curling leaf symptoms were observed on sugarbeet plants across ~40 acres of commercial fields in southeast Parma, Idaho, with a disease incidence of 10 to 15%. Leaf symptoms resembled virus yellows, a known sugarbeet disease caused by a complex of viruses, Beet yellows virus, Beet western yellows virus, and Beet chlorosis virus (Wintermantel, 2005). To identify the causal viral pathogens responsible for the symptoms, total RNA was isolated from a pool of symptomatic leaves from four plants using the RNeasy Plant Mini Kit (Qiagen, CA). After depleting the ribosomal RNA, sequencing libraries were constructed and subjected to high throughput sequencing (HTS) using the Illumina NovaSeq 6000 with a 150-bp paired-end platform (Novogene, CA). After adapter trimming, a total of 71.67 million high-quality reads (Q>30) were obtained. Following the removal of reads mapped to host genome, the remaining 14.24 million reads were assembled into contigs using the SPAdes assembler (Bankevich et al. 2012; Prjibelski et al. 2020). The resulting 101513 contigs were aligned with the NCBI nonredundant database to identify contigs matching known viruses. The assembled contigs generated the complete genome of L, M, and S segments of Orthotospovirus iridimaculaflavi (Iris yellow spot virus, IYSV) with 98.08%, 98.98% and 96.65% identity with the corresponding reference sequences NC_029799, FJ361359 and OR526471 with 99 to 100% coverage. The full-length sequences of L (8863 nt), M (4823 nt), and S (3121 nt) segments of IYSV found in Idaho were deposited in GenBank with accession numbers PQ246270, PQ246271 and PQ246272, respectively. Additionally, a single 2860 nt contig exhibiting 98.57% identity and 100% coverage to Becurtovirus spinaciae (Spinach curly top Arizona virus, SpCTAV) HQ443515 identified in sugarbeet was deposited in GenBank with PQ246273. The SpCTAV was initially identified in spinach in AZ (Hernandez-Zepeda et al. 2013) and has recently been found in red table beet being grown in Idaho (Ramachandran et al. 2023) and in beet leafhopper (Circulifer tenellus) from Idaho (Strausbaugh et al. 2024). Accordingly, the current study identified SpCTAV in sugarbeet from Idaho. The IYSV incidence in the southeast Parma region of Idaho was further examined in additional sugarbeet samples collected in 2024. Total RNA was extracted from the leaves of 15 individual plants, and RT-PCR produced the expected amplicons of the L, M, and S segments of IYSV in all 15 plants. Sanger sequencing confirmed all the amplicons from 15 plants with 97.35% to 99.4% identity to the corresponding HTS of IYSV. To verify the presence of SpCTAV, genomic DNA was isolated and Sanger sequencing of the PCR product confirmed SpCTAV in 4 of 15 samples with 98.37% identity to the HTS sequence. Although virus yellows disease was suspected based on foliar symptoms, the HTS analysis revealed no known viruses in the analyzed sugarbeet samples. This is the first report of IYSV and SpCTAV in sugarbeet grown in Idaho. Monitoring the prevalence of IYSV, an RNA virus, and SpCTAV, a DNA virus, and their coexistence will clarify their role in the recently observed virus yellows-like disease symptoms in sugarbeet from Idaho.

RNA viruses have evolved numerous strategies to overcome host resistance and immunity, including the use of multifunctional proteases that not only cleave viral polyproteins during virus replication but also deubiquitinate cellular proteins to suppress ubiquitin (Ub)-mediated antiviral mechanisms. Here, we report an approach to attenuate the infection of Arabidopsis thaliana by Turnip Yellow Mosaic Virus (TYMV) by suppressing the polyprotein cleavage and deubiquitination activities of the TYMV protease (PRO). Performing selections using a library of phage-displayed Ub variants (UbVs) for binding to recombinant PRO yielded several UbVs that bound the viral protease with nanomolar affinities and blocked its function. The strongest binding UbV (UbV3) candidate had a EC50 of 0.3 nM and inhibited both polyprotein cleavage and DUB activity of PRO in vitro. X-ray crystal structures of UbV3 alone and in complex with PRO reveal that the inhibitor exists as a dimer that binds two copies of PRO. Consistent with our biochemical and structural findings, transgenic expression of UbV3 in the cytosol of A. thaliana suppressed TYMV replication in planta, with the reduction in viral load being correlated to UbV3 expression level. Our results demonstrate the potential of using UbVs to protect plants from tymovirus infection, a family of viruses that contain numerous members of significant agricultural concern, as well as other plant viruses that express functionally related proteases with deubiquitinating activity.

In this study, meta-transcriptome sequencing was conducted on a total of 18 sugarbeet (Beta vulgaris L. subsp. vulgaris) sample libraries to profile the virome of field-grown sugarbeet to identify the occurrence and distribution of known and potentially new viruses from five different states in the United States. Sugarbeet roots with symptoms resembling rhizomania caused by beet necrotic yellow vein virus (BNYVV), or leaves exhibiting leaf-curling, yellowing to browning, or green mosaic were collected from the sugarbeet growing areas of California, Colorado, Idaho, Minnesota, and North Dakota. In silico analysis of de novo assembled contigs revealed the presence of nearly full-length genomes of BNYVV, beet soil-borne virus (BSBV), and beet soil-borne mosaic virus (BSBMV), which represent known sugarbeet-infecting viruses. Among those, BNYVV was widespread across the locations, whereas BSBV was prevalent in Minnesota and Idaho, and BSBMV was only detected in Minnesota. In addition, two recently reported Beta vulgaris satellite virus isoforms (BvSatV-1A and BvSatV-1B) were detected in new locations, indicating the geographical expansion of this known virus. Besides these known sugarbeet-infecting viruses, the bioinformatic analysis identified the widespread occurrence of a new uncharacterized Erysiphe necator-associated abispo virus (En_abispoV), a fungus-related virus that was identified in all 14 libraries. En_abispoV contains two RNA components, and nearly complete sequences of both RNA1 and RNA2 were obtained from RNASeq and were further confirmed by primer-walking RT-PCR and Sanger sequencing. Phylogenetic comparison of En_abispoV isolates obtained in this study showed varying levels of genetic diversity within RNA1 and RNA2 compared to previously reported isolates. The undertaken meta-transcriptomic approach revealed the widespread nature of coexisting viruses associated with field-grown sugarbeet exhibiting virus disease-like symptoms in the United States.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

In the fall 2021, red table beet plants (Beta vulgaris L. cv 'Eagle') exhibiting stunted growth with shorter petioles were observed at an incidence of 10 to 15 percent in a production field in Payette County, Idaho, United States. In addition to stunting, beet leaves displayed yellowing and mild curling and crumpling, and the roots exhibited hairy root symptoms (sFig.1). To identify potential causal viruses, total RNA was isolated from the leaf and root tissue using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to high-throughput sequencing (HTS). Two libraries were prepared, one for the leaf sample and another for the root sample using a ribo-minus TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA). HTS was performed with 150 bp paired-end sequencing on a NovaSeq 6000 (Novogene, Sacramento, CA). Following adapter trimming and removal of host transcripts, 5.9 and 16.2 million reads were obtained from the leaf and root samples, respectively. These reads were de novo assembled using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The assembled leaf sample contigs were aligned to the NCBI non-redundant database to identify contigs matching known viruses. A single contig of 2845 nts that shared 96% coverage and 95.6% sequence identity to the pepper yellow dwarf strain of beet curly top virus (BCTV-PeYD, EU921828; Varsani et al., 2014), and 98% coverage and 98.39% identity with an isolate of BCTV-PeYD (KX529650) from Mexico, was identified in the leaf sample (GenBank Accession OP477336). To validate the HTS detection of BCTV-PeYD, total DNA was isolated from the leaf sample and a 454 bp fragment of the C1 gene (replication-associate protein) was PCR amplified and Sanger sequencing of the amplicon revealed 99.7% identity to the HTS assembled BCTV-PeYD sequence. In addition to the PeYD strain of BCTV, the Worland strain of BCTV (BCTV-Wor) was detected as a single 2930 nt contig with 100% coverage and 97.3% identity to the BCTV-Wor isolate CTS14-015 (KX867045) known to infect sugar beet in Idaho. Of note, there are 11 strains of BCTV and among those, the BCTV-Wor strain induces mild symptoms in sugar beet (Strausbaugh et al., 2017), whereas BCTV-PeYD was found only in pepper from New Mexico. Further, two contigs of 2201 nts and 523 nts were assembled generating a nearly complete genome of spinach curly top Arizona virus (SpCTAV) in the leaf sample with 99% coverage and 99.3% identity (GenBank Accession OQ703946) to the reference genome of SpCTAV (HQ443515; Hernandez-Zepeda et al., 2013). To validate the HTS results, total DNA was isolated from the leaf tissue and PCR amplified a 442 bp fragment that overlaps the V1, V2, and V3 ORFs and its sequence revealed 100% identity with the HTS assembled SpCTAV. The roots sample also showed HTS reads corresponding to BCTV-PeYD and SpCTAV. In addition, beet necrotic yellow vein virus (BNYVV) was detected in the root sample with 30% coverage, but no sequence reads matching to BNYVV was detected in the leaf sample. BNYVV is known to infect sugar beet causing rhizomania (Tamada et al., 1973; Schirmer et al., 2005). To further confirm the BNYVV HTS results, total RNA was extracted separately from the root and leaf tissue, and RT-PCR was performed with primers that were designed to amplify portions of BNYVV RNAs (Weiland et al., 2020). RT-PCR analysis generated the appropriate amplicons with expected sequences corresponding to the RNA-1, RNA-2, RNA-3, and RNA-4 of BNYVV as determined by Sanger sequencing implying BNYVV the causal agent of hairy root symptoms. Similar to observations seen for BNYVV infection in conventional sugar beet varieties, no amplification was detected for BNYVV in the RNA extracted from leaf tissue, indicating that the RT-PCR results are consistent with the HTS analysis. This is the first report of BCTV-PeYD and SpCTAV observed naturally infecting red table beet in Idaho suggesting the geographical expansion of these viruses. The co-existence of BCTV-PeYD and SpCTAV with limited host range needs to be investigated to determine the actual cause of the observed foliar symptoms. This report provides the basis for further research to understand the pathogenic nature of these viruses and their potential threat to red table beet and sugar beet production in Idaho.

Sugar beet (Beta vulgaris L.) is an important crop grown for its sucrose content used in sugar production around the world. Tomato bushy stunt virus (TBSV) is an RNA virus that belongs to the Tombusvirus genus of the family Tombusviridae (Hearne et al., 1990). The virus was first isolated from tomato, and it is known to infect a wide range of plants (Smith, 1935; Martelli et al., 1988; Hafez et al., 2010). In 1980, a natural infection of TBSV was reported in sugar beet leaves with chlorotic and necrotic ring spots and line pattern symptoms based on serological affinity to TBSV anti-sera in Czechoslovakia (Novak and Lanzova, 1980). In March 2021, sugarbeet plants showing stunted and bushy growth with yellowing and necrotic leaves were observed in a production field in the Imperial Valley of California. Harvested roots exhibited stunted and abnormal growth compared to roots from healthy plants (sFig. 1A). These symptoms prompted a screen for potential infection by TBSV. Root-tissue harvested from the symptomatic sugar beet was initially screened using a TBSV double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Inc., Elkhart, IN), which reacted positive for TBSV. To obtain the full-length sequence of TBSV and potentially other viruses in the sample, total RNA isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) from the root-tissue was subjected to high-throughput sequencing (HTS). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA) and sequenced using Illumina NovoSeq 6000 paired-end platform (Novogene, Sacramento, CA). A total of 52 million reads were obtained after removing the adapters and reads mapping to the host genome. These high-quality reads were de novo assembled into 75,891 contigs that are larger than 500 base pairs using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The resulting contigs were searched for matching sequences to known viruses using the NCBI non-redundant database. A single contig of 4770 nts representing the full-length genome of TBSV was generated (Accession number OP477335), which showed 100% coverage to previously reported TBSV isolates 'statice' (AJ249740.1) and 'nipplefruit' (AY579432.1) with 92.19% and 91.25% nucleotide sequence identities, respectively, and thus confirming the presence of TBSV in sugar beet root-tissue. However, it showed 74% coverage with only 87% nucleotide identity to a previously reported Lettuce necrotic stunt virus (LNSV) from sugar beet, a tombusvirus that was re-classified as Moroccan pepper virus (MPV) due to high degree (>97%) of sequence identity (Obermeier et al., 2001; Wintermantel and Anchieta, 2012; Wintermantel and Hladky, 2013). The coat protein is conserved within species in tombusvirus, and it plays a significant role by providing serological relationships to tombusvirus taxonomy. The coat protein of TBSV-isolate of this study shared 98.45% and 96.91% identities at amino acid level with TBSV 'nipplefruit' (AY579432.1) and TBSV 'statice' (AJ249740.1) isolates, respectively. In contrast, it showed only 61.56% identity with the coat protein of MPV as shown in the phylogenetic tree indicating that the TBSV-isolate reported here is different from MPV (sFig. 2). To confirm the presence of TBSV, reverse-transcription (RT)-PCR was performed using the total RNA isolated from the root-tissue with primers (VR306: 5'-CGCTCACGAGCCCAGCATCCTTGA-3' and VR297: 5'-ACACCGCCACAGGAGCCATGATTG-3') designed based on the HTS data to amplify a portion of the TBSV genome. Sequencing of the RT-PCR product confirmed the presence of TBSV sequence with 99.1% identity to the TBSV-isolate identified in this study. Further, mechanical inoculation of total RNA isolated from the symptomatic sugar beet roots produced local lesions and systemic necrosis symptoms on the leaves of Chenopodium quinoa (sFig. 1B). Sequencing of the amplicon obtained using RT-PCR with primers VR306 and VR297 confirmed the presence of TBSV in C. quinoa. In addition to TBSV, several viral contigs representing Beet necrotic yellow vein virus were identified in the root-tissue indicating mixed infection in the field. To our knowledge, this is the first report that documents the occurrence of TBSV in sugar beet in the United States. Since TBSV is a soil-borne virus, our findings indicate the need for further studies focused on the frequency and coexistence of the TBSV with BNYVV in sugar beet production fields to understand the disease complexity resulting from potential mixed infections.

Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

"Rhizomania" of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector,Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenusAlphanecrovirus(genusNecrovirus; familyTombusviridae). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves ofChenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, a disease of global importance to the sugar beet industry. The most widely implemented resistance gene to rhizomania to date is Rz1, but resistance has been circumvented by resistance-breaking (RB) isolates worldwide. In an effort to gain greater understanding of the distribution of BNYVV and the nature of RB isolates in Minnesota and eastern North Dakota, sugar beet plants were grown in 594 soil samples obtained from production fields and subsequently were analyzed for the presence of BNYVV as well as coding variability in the viral P25 gene, the gene previously implicated in the RB pathotype. Baiting of virus from the soil with sugar beet varieties possessing no known resistance to rhizomania resulted in a disease incidence level of 10.6% in the region examined. Parallel baiting analysis of sugar beet genotypes possessing Rz1, the more recently introgressed Rz2, and with the combination of Rz1 + Rz2 resulted in a disease incidence level of 4.2, 1.0, and 0.8%, respectively. Virus sequences recovered from sugar beet bait plants possessing resistance genes Rz1 and/or Rz2 exhibited reduced genetic diversity in the P25 gene relative to those recovered from the susceptible genotype while confirming the hypervariable nature of the coding for amino acids (AAs) at position 67 and 68 in the P25 protein. In contrast to previous reports, we did not find an association between any one specific AA signature at these positions and the ability to circumvent Rz1-mediated resistance. The data document ongoing virulence development in BNYVV populations to previously resistant varieties and provide a baseline for the analysis of genetic change in the virus population that may accompany the implementation of new resistance genes to manage rhizomania.

Aphanomyces cochlioides is an oomycete organism, distantly related to members in the genera Pythium and Phytophthora. It is considered to be a water mold that affects sugarbeets in both the seedling and adult stages of the plant. During its life cycle, A. cochlioides produces two very different types of spores – motile zoospores and resting oospores. Zoospores are considered to be the infectious entity produced by the pathogen, whereas oospores are the stable, overwintering form of the organism, capable of surviving many years in the infested soil.

Maize rayado fino virus (MRFV) possesses an open reading frame (ORF43) predicted to encode a 43 kDa protein (p43) that has been postulated to be a viral movement protein. Using a clone of MRFV (pMRFV-US) from which infectious RNA can be produced, point mutations were introduced to either prevent initiation from three potential AUG initiation codons near the 5'-end of ORF43 or prematurely terminate translation of ORF43. Inoculation of maize seed via vascular puncture inoculation (VPI) resulted in plants exhibiting symptoms typical of MRFV infection for all mutants tested. Furthermore, corn leafhoppers (Dalbulus maidis) transmitted the virus mutants to healthy plants at a frequency similar to that for wild-type MRFV-US. Viral RNA recovered from plants infected with mutants both prior to and after leafhopper transmission retained mutations blocking ORF43 expression. The results indicate that ORF43 of MRFV is dispensable for both systemic infection of maize and transmission by leafhoppers.

A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

Oat blue dwarf virus (OBDV) is a member of the genus Marafivirus whose genome encodes a 227 kDa polyprotein (p227) ostensibly processed post-translationally into its functional components. Encoded near the 3' terminus and coterminal with the p227 ORF are ORFs specifying major and minor capsid proteins (CP). Since the CP expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the OBDV CP encoding gene and examined expression in protoplasts. Results support a model in which the 21 kDa major CP is the product of direct translation of a sgRNA, while the 24 kDa minor CP is a cleavage product derived from both the polyprotein and a larger ~26 kDa precursor translated directly from the sgRNA. Cleavage occurs at an LXG[G/A] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system.

Cercospora leaf spot, caused by the hemibiotrophic fungal pathogen Cercospora beticola, is the most economically damaging foliar disease of sugarbeet worldwide. Although most C. beticola populations display characteristics reminiscent of sexual recombination, no teleomorph has been described. To assess whether populations in northern United States have characteristics consistent with sexual reproduction, 1024 isolates collected over a 3-y period were analyzed for frequency and distribution of mating type genes. After clone correction, an approximately equal distribution of mating types was found for each sampling year. Mating type frequency was also assessed in individual lesions. Lesions always consisted of isolates with a single mating type and microsatellite haplotype, but both mating types and up to five microsatellite haplotypes could be found on an individual leaf. The MAT1-1-1 and MAT1-2-1 genes were sequenced from 28 MAT1-1 and 28 MAT1-2 isolates, respectively. Three MAT1-1-1 nucleotide haplotypes were identified that encoded a single amino acid sequence. For MAT1-2-1, five nucleotide haplotypes were identified that encoded four protein variants. MAT1-1-1 and MAT1-2-1 gene expression analyses were conducted on plants inoculated with either or both mating types. MAT1-1-1 expression remained low, but MAT1-2-1 spiked during late stages of colonization. A segment of the MAT1-2-1 coding sequence was also found in MAT1-1 isolates. Taken together, these results suggest that C. beticola has the potential for sexual reproduction.

Vascular puncture inoculation (VPI) of plant viruses previously has been conducted either manually or by use of a commercial engraving tool and laboratory-fabricated needle arrays. In an effort to improve this technique, a linear-motion tattoo machine driving industry-standard needle arrays was tested as a means of delivering plant viruses into maize and small grain seed embryos. The new method was applied in the successful transmission of maize rayado fino virus (MRFV), the type member of the genus Marafivirus, from an archived sample to maize. Subsequent transfer of MRFV from the sap of an infected plant using the method produced an average infection rate in maize of 70% (range 39–93%). Maize, oat, and triticale were successfully infected with oat blue dwarf virus (OBDV) using the method; similar infection rates were observed between maize seeds inoculated with the tattoo machine and those inoculated with the engraving machine when using prefabricated needle arrays. No infection was obtained in repeated tests with barley yellow dwarf virus (BYDV-PAV) or cereal yellow dwarf virus (CYDV-RPV) using either sap or RNA from infectious cloned cDNA. Replacement of the original engraving-tool with a linear-motion tattoo machine in VPI provides greater flexibility and convenience in a quiet, readily-available instrument, while improving reproducibility through the use of prefabricated needle arrays.

Maize rayado fino virus (MRFV) is distinct from other marafiviruses in that its genome reportedly lacks a poly(A) tail at the 3' terminus. We now show that the MRFV genome is indeed polyadenylated.

Oat blue dwarf virus (OBDV) is a small, phloem-limited marafivirus that replicates in its leafhopper vector. We have developed complete cDNA clones of OBDV from which infectious transcripts may be derived––the first such clones for any propagatively transmitted plant virus. Prior to clone construction, the reported sequences of the 5′ and 3′ ends were confirmed using 5′ RACE, primer extension, and ligation-anchored PCR. Using vascular puncture of maize seeds with capped transcripts, multiple clones were shown to be infectious at an average rate of 24.3% (range 14–36%). Aster leafhoppers successfully transmitted OBDV to oats and barley after feeding on detached, infected maize leaves. Proteins and RNAs consistent in size with those expected in OBDV infection were detected in young leaves via western and northern blotting, respectively. One construct, pOBDV-2r, was designated as the reference clone. An infectious clone of OBDV will be valuable in examining the interaction of this virus with both its insect and plant hosts.

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.