Plasmodiophorid Vector Research Articles

Quantification of rhizomania virus by automated RNA isolation and PCR based methods in sugar beet.

Rhizomania is a grave disease affecting sugar beet (Beta vulgaris L.). It is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), an RNA virus transmitted by the plasmodiophorid vector Polymyxa betae. Genetic resistance to the virus has been accomplished mostly using phenotype-genotype association studies. As yet, the most convenient method to ascertain plant resistance has been the quantification of viral titer in roots through the ELISA test. This method is particularly time-consuming and clashes with the necessities of modern plant breeding. Here, we propose an alternative and successful phenotyping method based on the automatic extraction of the viral RNA from sugar beet roots and its relative and absolute quantification by quantitative real-time PCR (qRT-PCR) and digital PCR (dPCR), respectively. Such a method enables an improved standardization of the study, as well as an accurate quantification of the virus also in those samples presenting low virus titer, with respect to the ELISA test.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13337-021-00674-7.

Pest categorisation of beet necrotic yellow vein virus.

Following a request from the EU Commission, the Panel on Plant Health performed a categorisation of beet necrotic yellow vein virus (BNYVV), the causal agent of the sugar beet rhizomania disease. The virus is currently listed in Annex III as a protected zone (PZ) quarantine pest of the Commission Implementing Regulation (EU) 2019/2072. The identity of the BNYVV is well established. BNYVV is a soil‐borne virus transmitted by the obligate root plasmodiophorid endoparasite Polymyxa betae. BNYVV is widely distributed in the EU, but is not reported in the following EU PZs: Ireland, France (Brittany), Portugal (Azores), Finland and Northern Ireland. The virus may enter, become established and spread in the PZs via P. betae resting spores with soil and growing media as such or attached to machinery and with roots and tubercles of species other than B. vulgaris and with plants for planting. Introduction of BNYVV would have a negative impact on sugar beet and other beet crops in PZs, because of yield and sugar content reduction. Phytosanitary measures are available to reduce the likelihood of entry and spread in the PZs. Once the virus and its plasmodiophorid vector have entered a PZ, their eradication would be difficult due to the persistence of viruliferous resting spores in the soil. The main knowledge gaps or uncertainties identified concerning the presence of BNYVV in the PZs and the incidence and distribution of BNYVV in Switzerland, a country to which a range of specific requirements do not apply. BNYVV meets all the criteria that are within the remit of EFSA to qualify as a potential protected zone union quarantine pest. Plants for planting are not considered as a main means of spread, and therefore BNYVV does not satisfy all the criteria evaluated by EFSA to qualify as potential Union regulated non‐quarantine pest.

Field evaluation for rhizomania resistance of transgenic sugar beet events based on gene silencing

Rhizomania is one of the most damaging and widespread diseases in the major sugar beet (Beta vulgaris L. subsp. vulgaris) growing regions worldwide. The causal agent, Beet necrotic yellow vein virus, is transmitted by a plasmodiophorid vector, Polymyxa betae Keskin. In this study, the inhibition of BNYVV multiplication was evaluated, under field conditions, in six sugar beet transgenic events generated based on gene silencing of the major coat protein gene (CP21). Phenotypic criteria, including scoring for disease symptoms and an immunoassay for viral accumulation, indicated that the transgenic events were less susceptible to rhizomania and had lower virus titre rates than non-transgenic plants of the susceptible cultivar ‘9597’. Disease severity and virus titres were comparable to the resistant cultivar ‘Dorothea’, which carries the Rz1-resistance gene and was bred through traditional means. This study provides a proof of concept for the effectiveness of the gene-silencing mechanism for introducing rhizomania resistance in sugar beet at the field level.

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.

Identification of strain types of some Beet necrotic yellow vein virus isolates determined in Northern and Central Parts of Turkey

Beet necrotic yellow vein virus (BNYVV), the agent of rhizomania disease, causes severe economic losses in sugar beet fields in all over the world. The virus is transmitted by a plasmodiophorid vector, Polymyxa betae Keskin. Twenty soil samples, collected from sugar beet fields in northern and central parts of Turkey during surveys in 2004 and 2005 and known to be infested with viruliferous cultures of P. betae carrying BNYVV, were selected and used in this study. Sample selection was made according to symptom expression of beet seedlings in preliminary bait plant tests and locations of the soil samples that accurately represent the region from which they were taken. Total RNAs were extracted from sugar beet plants grown in these soils and used to amplify RNA-2 (nt. 19-1088) and RNA-3 (nt. 50-1268) of BNYVV by reverse transcription polymerase chain reaction (RT-PCR) method. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified products showed that most of BNYVV isolates studied were A-type strain, however, two isolates did not exactly match the band profile of A-type strain. Additionally, the presence of BNYVV RNA-5 component was investigated by RT-PCR using the primers specific for P26 coding region. Four samples belonging to three provinces were found to be involving RNA-5 segment (20%).

Agroinoculation of Beet necrotic yellow vein virus cDNA clones results in plant systemic infection and efficient Polymyxa betae transmission

Agroinoculation is a quick and easy method for the infection of plants with viruses. This method involves the infiltration of tissue with a suspension of Agrobacterium tumefaciens carrying binary plasmids harbouring full-length cDNA copies of viral genome components. When transferred into host cells, transcription of the cDNA produces RNA copies of the viral genome that initiate infection. We produced full-length cDNA corresponding to Beet necrotic yellow vein virus (BNYVV) RNAs and derived replicon vectors expressing viral and fluorescent proteins in pJL89 binary plasmid under the control of the Cauliflower mosaic virus 35S promoter. We infected Nicotiana benthamiana and Beta macrocarpa plants with BNYVV by leaf agroinfiltration of combinations of agrobacteria carrying full-length cDNA clones of BNYVV RNAs. We validated the ability of agroclones to reproduce a complete viral cycle, from replication to cell-to-cell and systemic movement and, finally, plant-to-plant transmission by its plasmodiophorid vector. We also showed successful root agroinfection of B. vulgaris, a new tool for the assay of resistance to rhizomania, the sugar beet disease caused by BNYVV.

A BRIEF OUTLINE OF THE DEVELOPMENT OF PLANT VIROLOGY IN THE 20TH CENTURY

The development of plant virology as a science is, almost totally, a 20th century phenomenon. Early papers on plant viruses emphasize pathogenicity and ultra-filterability and report transmission by leafhopper and aphid vectors, and from soil or through seed. Successive advances were largely driven by access to new techniques. For example, biochemical approaches, and local- lesion and serological assays, led to purification and crystallization of virus particles, and their identification as nucleoproteins; electron microscopy and X-ray crystallography revealed their detailed structure. Biological tests revealed mite, nematode, chytrid and plasmodiophorid vectors. Viroids were discovered. Proof of the infectivity of viral RNA and DNA, followed by sequencing of complete viral genomes, allowed their genes and control elements to be defined. Although some viral genomes were monopartite and others multipartite, each typically encoded proteins involved in replication, movement within the plant, particle structure and, commonly, transmission by vectors. The processes involved in vector transmission, viral replication, disease induction and resistance, and the role of gene silencing, were then studied intensively. Genetic variability was analyzed and short-term evolution explored. A soundlybased virus taxonomy was devised and now includes over 70 plant virus genera containing some 800 species. Virus ecological and epidemiological research established patterns of virus spread and aided the development of control measures. These include schemes for producing virus-tested stocks of vegetatively propagated plants, quarantine regulations, application of vectorcontrolling chemicals to crops and use of virus-resistant cultivars that contain conventional or man-made resistance genes. These and other changes in agricultural practices have resulted in substantial increases in crop yields and food security. Since about 1950, results of research on viruses infecting the whole spectrum of host Corresponding author: B.D. Harrison Fax: +44.1382562426 E-mail: bryan.harrison@scri.ac.uk taxa have been integrated to constitute virology, with plant virology contributing many seminal discoveries. The biological phase of plant virology has progressed to molecular biological and molecular genetic phases, and plant viruses have become tools in molecular biology, cell biology and biotechnology.

First report of Barley yellow mosaic virus infecting barley in Poland

Barley yellow mosaic virus (BaYMV) is a dangerous pathogen of winter barley in Europe and Japan, transmitted by the plasmodiophorid vector Polymyxa graminis (Adams, 1996). No data on the occurrence of barley yellow mosaic viruses were recorded in Poland until spring 2008. First disease symptoms appeared in at least 50% of winter barley fields by late March, affecting mainly cv. Vanessa in Lower Silesia region. These were associated with mosaic and leaf yellowing, including longitudinal chlorotic flecks on younger leaves of some plants, decreased growth, and irregular yellow patches in the fields. Leaves of barley plants with symptoms were collected during April and May, and tested by ELISA using antibodies against BaYMV (Loewe), yielding positive results with 135 out of 352 samples. The OD values ranged from three to 10 times higher than the negative controls. Bymovirus-like flexuous particles, about 700 nm long, were observed under the electron microscope (Philips EM-201) in leaf extracts only from samples from symptom-bearing leaves. Fourteen plant samples, found to be ELISA positive for BaYMV were indexed by reverse transcription‐polymerase chain reaction (RT-PCR). Total RNA was isolated from 100‐120 mg fresh plant tissue using RNeasy Mini Kit (Qiagen), reverse transcribed and then amplified by PCR using a one step RT-PCR kit (Qiagen) with primers BaYMV-F and BaYMV-R (Vaianopoulos et al ., 2003). PCR products of the expected size (346bp) observed in nine samples were separated electrophoretically in a 1% agarose gel. The consensus sequence from two amplicons was deposited in GenBank (FJ436987). The BaYMV partial coat protein sequence showed the highest identity (100%) with that of BaYMV from UK (AJ515485). The virus was easily propagated in glasshouse conditions by soil-transmission experiments. The identification of BaYMV in Poland is an important indication for barley growers to cultivate only resistant winter barley cultivars in the regions of the disease incidence.

Another quarter century of great progress in understanding the biological properties of plant viruses

The last quarter century saw a massive application of new molecular and cell biological, and molecular genetical, techniques in research on plant viruses. Hundreds of complete virus genomic nucleotide sequences were analysed, their constituent genes and control elements identified, and similarities and differences revealed in genome organisation, so justifying the modern taxonomic classification of the viruses. Numerous virus replication systems were described and some viral replicases isolated. Especially good progress was made in understanding cell-to-cell and long-distance virus transport within plants; in cloning dominant and recessive plant genes controlling virus resistances, and identifying the cognate viral avirulence factors; in unraveling mechanisms of virus transmission by invertebrate and plasmodiophorid vectors; and in showing through these advances how viruses utilise and subvert endogenous eukaryotic processes. The discovery of gene silencing, and of viral silencing-suppressor proteins, transformed ideas on how virus replication is controlled, and explained the phenomena of recovery from disease, cross-protection between virus strains and synergy between unrelated viruses. Transgenic, virus-resistant plants were created, tested successfully in field conditions and a few commercialised. Factors underlying the appearance of new disease epidemics were identified. Genetic recombination was reported and found to make an important contribution to generating virus variation, and some major virus evolutionary pathways were recognised by comparative genome analysis. The scene is set for further major advances in the coming decades.

Molecular aspects of plant virus transmission by olpidium and plasmodiophorid vectors.

The genome structures of a large number of viruses transmitted by olpidium and plasmodiophorid vectors have been determined. The viruses are highly diverse, belonging to 12 genera in at least 4 families. Plasmodiophorids are now classified as protists rather than true fungi. This finding, along with the recognition of the great variety of viruses transmitted by olpidium and plasmodiophorid vectors, will likely lead to an elaboration of the details of in vitro and in vivo transmission mechanisms. Recent progress in elucidating the interaction between Cucumber necrosis virus (CNV) and its zoospore vector suggests that specific sites on the capsid as well as on the zoospore are involved in transmission. Moreover, some features of CNV/zoospore attachment are similar to poliovirus/host cell interactions, suggesting evolutionary conservation of functional features of plant and animal virus capsids.

Response of Hard Red Winter Wheat to Soilborne wheat mosaic virus Using Novel Inoculation Methods

ABSTRACT Soilborne wheat mosaic virus (SBWMV) is an agronomically important pathogen of wheat that is transmitted by the soilborne plasmodiophorid vector Polymyxa graminis. In the laboratory, attempts to generate SBWMV-infected plants are often hampered by poor infectivity of the virus. To analyze the mechanism for virus resistance in wheat cultivars, we developed novel inoculation techniques. A new technique for foliar inoculation of SBWMV was developed that eliminated wound-induced necrosis normally associated with rub inoculating virus to wheat leaves. This new technique is important because we can now uniformly inoculate plants in the laboratory for studies of host resistance mechanisms in the inoculated leaf. Additionally, wheat plants were grown hydroponically in seed germination pouches and their roots were inoculated with SBWMV either by placing P. graminis-infested root material in the pouch or by mechanically inoculating the roots with purified virus. The susceptibility of one SBWMV susceptible and three field resistant wheat cultivars were analyzed following inoculation of plants using these novel inoculation techniques or the conventional inoculation technique of growing plants in P. graminis-infested soil. The results presented in this study suggest that virus resistance in wheat likely functions in the roots to block virus infection.

Evidence that soilborne wheat mosaic virus moves long distance through the xylem in wheat.

Soilborne wheat mosaic virus (SBWMV) is a member of the genus Furovirus of plant viruses. SBWMV is transmitted to wheat roots by the plasmodiophorid vector Polymyxa graminis. Experiments were conducted to determine the path for SBWMV transport from roots to leaves. The results of immunogold labeling suggest that SBWMV enters and moves long distance through the xylem. SBWMV may enter primary xylem elements before cell death occurs and then move upward in the plant after the xylem has matured into hollow vessels. There is also evidence for lateral movement between adjacent xylem vessels.