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

ABSTRACT: 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 information

The online version contains supplementary material available at 10.1007/s13337-021-00674-7.

SUBMITTER: Chiodi C

PROVIDER: S-EPMC7976688 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rhizomania disease, caused by the Beet necrotic yellow vein virus (BNYVV), is considered as one of the major constraints for sugar beet production, worldwide. As a result of the introgression of major resistance genes (Holly, Rz2) in commercially available sugar beet varieties, the virus has endured strong selection pressure since the 90s'. Understanding the virus response and diversity to sugar beet resistance is a key factor for a sustainable management of only few resistance genes. Here we report rhizomania surveys conducted in a rhizomania hot spot, the Pithiviers area (France) during a 4-year period and complementary to the study of Schirmer et al. (2005). The study aimed at evaluating the intra- and inter-field BNYVV diversity in response to different sources of resistance and over the growing season. To follow rhizomania development over the sugar beet growing season, extensive field samplings combined with field assays were performed in this study. The evolution of the BNYVV diversity was assessed at intra- and inter-field levels, with sugar beet cultivars containing different resistance genes (Rz1, Rz1 + Heterodera schachtii resistance and Rz1Rz2). Intra-field diversity was analyzed at the beginning and the end of the growing season of each field. From more than one thousand field samples, the simultaneous presence of the different A, B and P types of BNYVV was confirmed, with 21 variants identified at positions 67-70 of the p25 tetrad. The first variant, AYHR, was found most commonly followed by SYHG. Numerous mixed infections (9.93% of the samples), mostly of B-type with P-type, have also been evidenced. Different tetrads associated with the A- or B-type were also found with a fifth RNA-genome component known to allow more aggressiveness to BNYVV on sugar beet roots. Cultivars with Rz1+Rz2 resistant genes showed few root symptoms even if the BNYVV titre was quite high according to the BNYVV type present. The virus infectious potential in the soil at the end of the growing season with such cultivars was also lower despite a wider diversity at the BNYVV RNA3 sequence level. Rz1+Rz2 cultivars also exhibited a lower presence of Beet soil-borne virus (BSBV), a P. betae-transmitted Pomovirus. Cultivars with Rz1 and nematode (N) resistance genes cultivated in field infected with nematodes showed lower BNYVV titre than those with Rz1 or Rz1+Rz2 cultivars. Overall, the population structure of BNYVV in France is shown to be different from that previously evidenced in different world areas. Implications for long-term management of the resistance to rhizomania is discussed.

Project description:"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.

Project description:BackgroundExtracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. Several RNA isolation methods are available for the analysis of these cell-free miRNAs by RT-qPCR. Not all methods, however, are equally suitable for different biofluids. Here, we extracted total RNA from four very diverse biofluids: serum, urine, bile, and graft preservation fluid (perfusate). Four different protocols were used: a phenol-chloroform extraction and alcohol precipitation in combination with a precipitation carrier (QP) and three different column-based isolation methods, one with phenol-chloroform extraction (RN) and two without (NG and CU). For this range of clinical biofluid samples, we evaluated the potential of these different RNA isolation methods assessing recovery efficiency and the co-purification of RT-qPCR inhibiting compounds.ResultsDifferences were observed between each of the RNA isolation methods in the recovery of cel-miR-39, a synthetic miRNA spiked in during the workup procedure, and for endogenous miRNAs. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. RT-qPCR detection of synthetic miRNAs cel-miR-39, spiked in during RNA workup, cel-miR-54, spiked in during cDNA synthesis, and endogenous miRNAs was strongly improved in the presence of heparinase I for some, but not all, isolation methods. Other, co-isolated RT-qPCR inhibitors were not identified, except for biliverdin, which co-isolated from some bile samples with one of the methods. In addition, we observed that serum and urine contain compounds that enhance the binding of heparin to certain solid-phase columns.ConclusionsFor reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and in co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR inhibiting compound, but also biliverdin, co-isolated from bile, could interfere with detection.

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

Supplementary information

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