Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

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.

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1.

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses.

"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 subgenus Alphanecrovirus (genus Necrovirus; family Tombusviridae). 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 of Chenopodium 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.

Genome composition analysis of multipartite BNYVV reveals the occurrence of genetic re-assortment in the isolates of Asia Minor and Thrace.

Beet necrotic yellow vein virus (BNYVV) is the cause of rhizomania, an important disease of sugar beet around the world. The multipartite genome of the BNYVV contains four or five single-stranded RNA that has been used to characterize the virus. Understanding genome composition of the virus not only determines the degree of pathogenicity but also is required to development of resistant varieties of sugar beet. Resistance to rhizomania has been conferred to sugar beet varieties by conventional breeding methods or modern genome engineering tools. However, over time, viruses undergo genetic alterations and develop new variants to break crop resistance. Here, we report the occurrence of genetic reassortment and emergence of new variants of BNYVV among the isolates of Thrace and Asia Minor (modern-day Turkey). Our findings indicate that the isolates harbor European A-type RNA-2 and RNA-3, nevertheless, RNA-5 is closely related to East Asian J-type. Furthermore, RNA-1 and RNA-4 are either derived from A, B, and P-types or a mixture of them. The RNA-5 factor which enhance the pathogenicity, is rarely found in the isolates studied (20%). The creation of new variants of the virus emphasizes the necessity to develop new generation of resistant crops. We anticipate that these findings will be useful for future genetic characterization and evolutionary studies of BNYVV, as well as for developing sustainable strategies for the control of this destructive disease.

Comparative Transcriptome Analysis Provides Molecular Insights into the Interaction of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus with Their Host Sugar Beet.

Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) are closely related species, but disease development induced in their host sugar beet displays striking differences. Beet necrotic yellow vein virus induces excessive lateral root (LR) formation, whereas BSBMV-infected roots appear asymptomatic. A comparative transcriptome analysis was performed to elucidate transcriptomic changes associated with disease development. Many differentially expressed genes (DEGs) were specific either to BNYVV or BSBMV, although both viruses shared a high number of DEGs. Auxin biosynthesis pathways displayed a stronger activation by BNYVV compared to BSBMV-infected plants. Several genes regulated by auxin signalling and required for LR formation were exclusively altered by BNYVV. Both viruses reprogrammed the transcriptional network, but a large number of transcription factors involved in plant defence were upregulated in BNYVV-infected plants. A strong activation of pathogenesis-related proteins by both viruses suggests a salicylic acid or jasmonic acid mediated-defence response, but the data also indicate that both viruses counteract the SA-mediated defence. The ethylene signal transduction pathway was strongly downregulated which probably increases the susceptibility of sugar beet to Benyvirus infection. Our study provides a deeper insight into the interaction of BNYVV and BSBMV with the economically important crop sugar beet.

Prevalence and Distribution of Beet Necrotic Yellow Vein Virus Strains in North Dakota and Minnesota.

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.

Genomes of the wild beets Beta patula and Beta vulgaris ssp. maritima.

We present draft genome assemblies of Beta patula, a critically endangered wild beet endemic to the Madeira archipelago, and of the closely related Beta vulgaris ssp. maritima (sea beet). Evidence-based reference gene sets for B. patula and sea beet were generated, consisting of 25 127 and 27 662 genes, respectively. The genomes and gene sets of the two wild beets were compared with their cultivated sister taxon B. vulgaris ssp. vulgaris (sugar beet). Large syntenic regions were identified, and a display tool for automatic genome-wide synteny image generation was developed. Phylogenetic analysis based on 9861 genes showing 1:1:1 orthology supported the close relationship of B. patula to sea beet and sugar beet. A comparative analysis of the Rz2 locus, responsible for rhizomania resistance, suggested that the sequenced B. patula accession was rhizomania susceptible. Reference karyotypes for the two wild beets were established, and genomic rearrangements were detected. We consider our data as highly valuable and comprehensive resources for wild beet studies, B. patula conservation management, and sugar beet breeding research.

Positive selection and recombination shaped the large genetic differentiation of Beet black scorch virus population.

Beet black scorch virus (BBSV) is a species in the Betanecrovirus genus, in family Tombusviridae. BBSV infection is of considerable importance, causing economic losses to sugar beet (Beta vulgaris) field crops worldwide. Phylogenetic analyses using 3'UTR sequences divided most BBSV isolates into two main groups. Group I is composed of Iranian isolates from all Iranian provinces that have been sampled. Chinese, European, one North American and some other Iranian isolates from North-Western Iran are in Group II. The division of Iranian BBSV isolates into two groups suggests numerous independent infection events have occurred in Iran, possibly from isolated sources from unknown host(s) linked through the viral vector Olpidium. The between-group diversity was higher than the within-group diversity, indicating the role of a founder effect in the diversification of BBSV isolates. The high FST among BBSV populations differentiates BBSV groups. We found no indication of frequent gene flow between populations in Mid-Eurasia, East-Asia and Europe countries. Recombination analysis indicated an intra-recombination event in the Chinese Xinjiang/m81 isolate and an inter-recombination breakpoint in the viral 3'UTR of Iranian isolates in subgroup IranA in Group I. The ω ratios (dNS/dS) were used for detecting positive selection at individual codon sites. Amino acid sequences were conserved with ω from 0.040 to 0.229 in various proteins. In addition, a small fraction of amino acids in proteins RT-ORF1 (p82), ORF4 (p7b) and ORF6 (p24) are positively selected with ω > 1. This analysis could increase the understanding of protein structure and function and Betanecrovirus epidemiology. The recombination analysis shows that genomic exchanges are associated with the emergence of new BBSV strains. Such recombinational exchange analysis may provide new information about the evolution of Betanecrovirus diversity.

Beta vulgaris mitovirus 1 in diverse cultivars of beet and chard.

Recent results indicate that mitoviruses, which replicate persistently in host mitochondria, are not restricted to fungi, but instead are found also in plants. Beta vulgaris mitovirus 1 (BevuMV1) is an example first discovered in sugar beet cultivars. For the current study, complete coding sequences of 42 BevuMV1 strains were newly determined, derived from not only sugar beet but also fodder beet, table beet, and Swiss chard cultivars of Beta vulgaris, as well as wild sea beet. BevuMV1 is thus a common phytobiome component of this valuable crop species. Most of the new BevuMV1 sequences originated from RNA extracted from B. vulgaris seed clusters, consistent with vertical transmission of this virus. Results suggest that BevuMV1 entered the B. vulgaris lineage prior to human cultivation and also provides a marker for tracing the maternal ancestry of B. vulgaris cultivars. Especially notable is the monophyletic relationship and limited sequence divergence among BevuMV1 strains from cultivars that are thought or shown to share the "Owen" trait for cytoplasmic male sterility, which is transmitted by maternal mitochondria and has been broadly established in commercial breeding lines of B. vulgaris since the mid-20th century.

Virus-mediated export of chromosomal DNA in plants.

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species.

Newly Distinguished Cell Punctures Associated with Transmission of the Semipersistent Phloem-Limited Beet Yellows Virus.

Here we report on plant penetration activities (probing) by the aphid Myzus persicae (Sulzer, 1776) in association with the transmission, acquisition, and inoculation of the semipersistent Beet yellows virus (BYV; Closterovirus) in sugar beet. During electrical penetration graph (EPG) recording of stylet pathways, standard intracellular stylet punctures occur which are called potential drop (pd) waveforms. In addition to the standard pd, there also appeared to be a unique type of intracellular stylet puncture that always preceded the phloem salivation phase (waveform E1). This type of pd, the phloem-pd, showed properties distinct from those of the standard pds and has never been described before. We manually ended EPG recordings during the acquisition and inoculation tests by removing aphids from the source or test plant after specific waveforms were recorded. Inoculation of BYV occurred at the highest rate when probing was interrupted just after a single or various phloem-pds. In contrast, BYV acquisition showed an intimate association with sustained phloem sap ingestion from phloem sieve elements (SEs) (E2 waveform). Our work shows for the first time that the inoculation of a phloem-limited virus occurs during specific intracellular stylet punctures and before phloem salivation (waveform E1). Further studies are needed to establish in what cells this novel phloem-pd occurs: phloem parenchyma, companion, or SE cells. The role of the different stylet activities in the acquisition and inoculation of BYV by M. persicae is discussed.IMPORTANCE We discovered the specific feeding activities of Myzus persicae (Sulzer, 1776) associated with the transmission of Beet yellows virus (BYV; Closterovirus). Our work strongly suggests that aphids can insert their stylets into the membranes of phloem cells-visualized as a unique type of waveform that is associated with the inoculation of BYV. This intracellular puncture (3 to 5 s) occurs just before the phloem salivation phase and can be distinguished from other nonvascular stylet cell punctures. This is the first time that the transmission of a phloem-limited semipersistent virus has been shown to be associated with a unique type of intracellular puncture. Our work offers novel information and strongly contributes to the existing literature on the transmission of plant viruses. Here we describe a new kind of aphid behavioral pattern that could be key in further works, such as studying the transmission of other phloem-limited viruses (e.g., luteoviruses).

Massive up-regulation of LBD transcription factors and EXPANSINs highlights the regulatory programs of rhizomania disease.

Rhizomania of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is characterized by excessive lateral root (LR) formation leading to dramatic reduction of taproot weight and massive yield losses. LR formation represents a developmental process tightly controlled by auxin signaling through AUX/IAA-ARF responsive module and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcriptional network. Several LBD transcription factors play central roles in auxin-regulated LR development and act upstream of EXPANSINS (EXPs), cell wall (CW)-loosening proteins involved in plant development via disruption of the extracellular matrix for CW relaxation and expansion. Here, we present evidence that BNYVV hijacks these auxin-regulated pathways resulting in formation LR and root hairs (RH). We identified an AUX/IAA protein (BvAUX28) as interacting with P25, a viral virulence factor. Mutational analysis indicated that P25 interacts with domains I and II of BvAUX28. Subcellular localization of co-expressed P25 and BvAUX28 showed that P25 inhibits BvAUX28 nuclear localization. Moreover, root-specific LBDs and EXPs were greatly upregulated during rhizomania development. Based on these data, we present a model in which BNYVV P25 protein mimics action of auxin by removing BvAUX28 transcriptional repressor, leading to activation of LBDs and EXPs. Thus, the evidence highlights two pathways operating in parallel and leading to uncontrolled formation of LRs and RHs, the main manifestation of the rhizomania syndrome.

Comparison of RNA extraction methods for the detection of BNYVV rhizomania virus from roots of sugar beet.

Rhizomania is one of serious threat to sugar beet production in Morocco and in several parts of the world. This disease led to a statistically significant decrease in the quality and yield of sugar beet plantations. Therefore, this study aimed at comparing the efficacy of six commonly used RNA extraction methods for the detection, recovery of RNA of beet necrotic yellow vein virus (BNYVV) and removal of amplification inhibitors by reverse transcription-polymerase chain reaction (RT-PCR). The efficiency of these extraction methods was then compared to that of a commercial isolation kit with high content of phenolic compounds. The results showed that the extraction with the lithium chloride technique, the commercial kit, and direct and membrane spotting crude extract methods were found effective in yielding a higher purity and a higher concentration of RNA when compared to the other tested methods. Extraction with the lithium chloride technique and the Qiagen kit (RNeasy Plant Mini Kit) allowed the most intense band, whereas the CTAB method has generated the least intense band. Furthermore, the silica capture extraction method did not yield any RNA after extraction and electrophoresis. Consequently, it was concluded that, of these six methods, the lithium chloride technique and the Qiagen kit are the most appropriate for the extraction of viral RNA from sugar beet samples prior to RT-PCR for detecting BNYVV.

Biological properties of Beet soil-borne mosaic virus and Beet necrotic yellow vein virus cDNA clones produced by isothermal in vitro recombination: Insights for reassortant appearance.

Two members of the Benyviridae family and genus Benyvirus, Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa. These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants.

An Iranian genomic sequence of Beet mosaic virus provides insights into diversity and evolution of the world population.

Beet mosaic virus (BtMV), the only Potyvirus known to infect sugar beet, occurs worldwide in beet crops. The full genome sequencing of a BtMV isolate from Iran (Ir-VRU), enabled us to better understand the evolutionary history of this virus. Selection analysis suggested that BtMV evolution is mainly under negative selection but its strength varies in different proteins with the multifunctional proteins under strongest selection. Recombination has played a major role in the evolution of the BtMVs; only the Ir-VRU and USA isolates show no evidence of recombination. The ML phylogenies of BtMVs from coat protein and full sequences were completely congruent. The primary divergence of the BtMV phylogeny is into USA and Eurasian lineages, and the latter then divides to form a cluster only found in Iran, and a sister cluster that includes all the European and Chinese isolates. A simple patristic dating method estimated that the primary divergence of the BtMV population was only 360 (range 260-490) years ago, suggesting an emergence during the development of sugar beet as a crop over the past three centuries rather than with the use of leaf beet as a vegetable for at least 2000 years.

Influence of Beet necrotic yellow vein virus and Freezing Temperatures on Sugar Beet Roots in Storage.

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is a yield-limiting sugar beet disease that was observed to influence root resistance to freezing in storage. Thus, studies were conducted to gain a better understanding of the influence of BNYVV and freezing on sugar beet roots to improve pile management decisions. Roots from five commercial sugar beet cultivars (one susceptible and four resistant to BNYVV) were produced in fields under high and trace levels of rhizomania pressure and subjected to storage using five temperature regimes ranging from 0 to -4.4°C for 24 h. After cold treatment, eight-root samples were stored in a commercial indoor storage building (set point 1.1°C) for 50 days in 2014 and 57 days in 2015. Internal root temperature, frozen and discolored tissue, and moisture and sucrose loss were evaluated. The air temperature at 0, -1.1, and -2.2°C matched internal root temperature but internal root remained near -2.2°C when air temperature was dropped to -3.3 and -4.4°C. In a susceptible cultivar produced under high rhizomania pressure, the percentage of frozen tissue increased (P < 0.0001) from an average of 0 to 7% at 0, -1.1, and -2.2°C up to 16 to 63% at -3.3°C and 63 to 90% at -4.4°C, depending on year. Roots from the susceptible cultivar produced under low rhizomania pressure and those from the resistant cultivars from both fields only had elevated (P ≤ 0.05) frozen tissue at -4.4°C in 15 of 18 cultivar-year combinations. Frozen tissue was related to discolored tissue (r2 = 0.91), weight loss (r2 = 0.12 to 0.28), and sucrose reduction (r2 = 0.69 to 0.74). Consequently, BNYVV will not only lead to yield and sucrose loss in susceptible sugar beet cultivars but also to more frozen root tissue as temperatures drop below -2.2°C. Based on these observations, the air used to cool roots in nonfrozen sugar beet piles throughout the winter should not drop below -2.2°C to maximize sucrose retention.

Seed Transmission of Beet Curly Top Virus and Beet Curly Top Iran Virus in a Local Cultivar of Petunia in Iran.

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2-78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8-18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses.

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

Beet curly top virus Strains Associated with Sugar Beet in Idaho, Oregon, and a Western U.S. Collection.

Curly top of sugar beet is a serious, yield-limiting disease in semiarid production areas caused by Beet curly top virus (BCTV) and transmitted by the beet leafhopper. One of the primary means of control for BCTV in sugar beet is host resistance but effectiveness of resistance can vary among BCTV strains. Strain prevalence among BCTV populations was last investigated in Idaho and Oregon during a 2006-to-2007 collection but changes in disease severity suggested a need for reevaluation. Therefore, 406 leaf samples symptomatic for curly top were collected from sugar beet plants in commercial sugar beet fields in Idaho and Oregon from 2012 to 2015. DNA was isolated and BCTV strain composition was investigated based on polymerase chain reaction assays with strain-specific primers for the Severe (Svr) and California/Logan (CA/Logan) strains and primers that amplified a group of Worland (Wor)-like strains. The BCTV strain distribution averaged 2% Svr, 30% CA/Logan, and 87% Wor-like (16% had mixed infections), which differed from the previously published 2006-to-2007 collection (87% Svr, 7% CA/Logan, and 60% Wor-like; 59% mixed infections) based on a contingency test (P < 0.0001). Whole-genome sequencing (GenBank accessions KT276895 to KT276920 and KX867015 to KX867057) with overlapping primers found that the Wor-like strains included Wor, Colorado and a previously undescribed strain designated Kimberly1. Results confirm a shift from Svr being one of the dominant BCTV strains in commercial sugar beet fields in 2006 to 2007 to becoming undetectable at times during recent years.

Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for detection of beet necrotic yellow vein virus.

Sugar beet can be infected by many different viruses that can reduce yield; beet necrotic yellow vein virus (BNYVV) is one of the most economically important viruses of this crop plant. This report describes a new reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for identification of BNYVV. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection (without RNA extraction) of BNYVV was developed here and compared with DAS-ELISA and RT-LAMP assays. Our results show that the IC-RT-LAMP assay is a highly reliable alternative assay for identification of BNYVV.