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.

Molecular and biological characterisation of two novel pomo-like viruses associated with potato (Solanum tuberosum) fields in Colombia.

Potato is the fourth most important crop worldwide that is used as a staple food, after rice, wheat and maize. The crop can be affected by a large number of pathogens, including fungi, oomycetes, bacteria and viruses. Diseases caused by viruses are among the most important factors contributing to reduced quality and yield of the crop. Potato mop-top virus (genus Pomovirus) induces necrotic flecks in the tuber flesh and skin of potato in temperate countries. Spongospora subterranea is the vector of PMTV. Both the virus and its vector cause disease in potato. In Colombia, PMTV has been detected throughout the country together with a novel pomo-like virus in the centre (Cundinamarca and Boyacá) and south west (Nariño) of the country. We studied the molecular and biological characteristics of this novel virus. Its genome resembles those of members of the genus Pomovirus, and it is closely related to PMTV. It induces mild systemic symptoms in Nicotiana benthamiana (mosaic, branch curling), but no symptoms in N. tabacum, N. debneyi and Chenopodium amaranticolor. The proposed name for the virus is "Colombian potato soil-borne virus" (CPSbV). Additionally, another pomo-like virus was identified in Nariño. This virus induces severe systemic stem declining and mild mosaic in N. benthamiana. The tentative name "soil-borne virus 2" (SbV2) is proposed for this virus. No vectors have been identified for these viruses despite several attempts. This work focused on the characterisation of CPSbV. The risk posed by these viruses if they are introduced into new territories is discussed.

Ribosomal DNA analyses reveal greater sequence variation in Polymyxa species than previously thought and indicate the possibility of new ribotype-host-virus associations.

Polymyxa species transmit viruses to many important crops. They are poorly understood obligate parasites occupying a distinct position in the Tree of Life. To better understand the potential for spread of Polymyxa-vectored diseases, ribosomal DNA was analysed from isolates covering a wide range of geographical locations, virus associations and hosts. Internal transcribed spacer 2 structure analysis indicated that Polymyxa graminis isolates could represent many species and there was more sequence variation within the known subgroups (ribotypes) than previously described. In cereal crops and soils from temperate climates Polymyxa isolates were usually ribotype I or II, but their host specificities or preferences were unclear. For the first time, there was evidence that ribotype I (in addition to ribotype II) could transmit SBWMV/SBCMV. Different ribotypes often occurred together in the same soil or plant. New hosts were identified for particular ribotypes, including the first detection of the sugar beet-infecting Polymyxa betae, in wheat. Unexpectedly, ribotype III-like sequences, usually restricted to crops in the tropics, were found in wheat from the USA. P. betae isolates showed limited variation (≤ 2%) and the recent change in susceptibility of sugar beet varieties to BNYVV in the USA is unlikely to be due to changes in P. betae.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

Beet soil-borne mosaic virus RNA-4 encodes a 32 kDa protein involved in symptom expression and in virus transmission through Polymyxa betae.

Beet soil-borne mosaic virus (BSBMV), like Beet necrotic yellow vein virus (BNYVV), is a member of the Benyvirus genus and both are transmitted by Polymyxa betae. Both viruses possess a similar genomic organization: RNA-1 and -2 are essential for infection and replication while RNA-3 and -4 play important roles in disease development and vector-mediated infection in sugar beet roots. We characterized a new species of BSBMV RNA-4 that encodes a 32 kDa protein and a chimeric form of BSBMV RNA-3 and -4. We demonstrated that BSBMV RNA-4 can be amplified by BNYVV RNA-1 and -2 in planta, is involved in symptoms expression on Chenopodium quinoa plants and can also complement BNYVV RNA-4 for virus transmission through its vector P. betae in Beta vulgaris plants. Using replicon-mediated expression, we demonstrate for the first time that a correct expression of RNAs-4 encoded proteins is essential for benyvirus transmission.

Acquisition and transmission of Peanut clump virus by Polymyxa graminis on cereal species.

The objective of this study was to investigate the specificity of the interactions between Polymyxa graminis, Peanut clump virus (PCV), and cereals, particularly the acquisition and the transmission of the virus by three P. graminis formae speciales. A new strategy has been developed: it involves using sugarcane as the common host for both the virus and its vector in order to produce the viruliferous zoospores of P. graminis f. sp. subtropicalis, temperata, and tropicalis that were then inoculated on cereal species. This experiment enabled the role of P. graminis f. sp. tropicalis and subtropicalis zoospores in PCV transmission to be demonstrated. The efficiency of this transmission was shown to vary, depending on the P. graminis special forms. Interestingly, the high transmission of the PCV isolate from Burkina Faso by an isolate of P. graminis f. sp. tropicalis from Niger on pearl millet suggests that there is a coevolution mechanism in this pathosystem. The study also provides evidence that the host plant species in which Polymyxa zoospores are produced could affect the infectivity of the vector. Finally, using Polymyxa quantitation by quantitative reverse-transcription polymerase chain reaction and in situ observations of the virus, the study demonstrates the independence of the development of PCV and its vector in the host plants.

Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil.

The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.

BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach.

Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species.