RESUMO
As part of a cereals virome project high throughput sequencing (HTS)-based viral indexing was performed on plants with symptoms of barley yellow dwarf disease collected in June (2017-2020) in the main French cereals production areas. Total RNAs from 32 individual plants were purified (RNeasy Plant Mini Kit, Qiagen, Courtaboeuf, France) and Illumina sequenced (2x150 nt) following ribodepletion (Genewiz-Azenta, Leipzig, Germany). Following quality trimming, reads for each sample were de novo assembled (CLC Genomics Workbench 21, Qiagen) [1] and contigs annotated by BlastX analysis. In four winter barley samples collected in 2018 (18-58, 18-325 and 18-326) and 2019 (19-30A), besides contigs representing diverse viruses such as barley yellow dwarf viruses-PAV and PAS, Hordeum vulgare endornavirus, cereal yellow dwarf virus-RPV (18-326), wheat dwarf virus (18-325 and 18-326) and a novel Polerovirus (18-58 and 18-326), large contigs with high identity to barley virus G (BVG) were identified. BVG, a tentative Polerovirus, was initially reported in barley in South Korea in 2016 [2] and has so far been identified in a few other hosts including wheat, oat, maize, proso and foxtail millets as well as switchgrass. It has been reported from the USA and Australia [3] and, in Europe, from the Netherlands, Germany, Hungary and Greece [4]. Large BVG scaffolds representing near complete genomes could be reconstructed for each sample, integrating a total of 128.339, 7.188, 8.078 and 20.073 reads, for samples 19-30A, 18-58, 18325 and 18-326 respectively. Given that between 17.2 and 20.5 million reads had been obtained per sample, these values translate into between 0.04% (18-58 and 18-325) and 0.6% (19-30A) of total reads, and to average coverages of between 158x (18-58) and 2866x (19-30A) for the genomic scaffolds. The four assembled sequences (5584-5610 nt) have been deposited in GenBank (ON419453-ON419456). They are nearly identical (98.4 to 99.5% nt identity) and share between 97.7% and 98.5% nt identity with a barley reference isolate from the South Korea (NC_029906). To confirm the presence of BVG, a primer pair was designed based on available BVG sequences. Primers BVG-F(5'-CTAGCCCAACGAGTTGCGGG-3') and BVG-R(5'-GGTACAGAAGCTCTACGGTTC-3') amplifying a 394 nt were used in a two-step RT-PCR on new RNA extracts obtained from the 18-325 and 18-326 infected plants. The amplicons were directly sequenced and showed respectively 99.2% (ON419457, 18-325) and 100% (18-326) nt identity with the corresponding de novo scaffolds. The four analyzed samples have been collected respectively in 2018 (18-58, 18-325, 18-326) and 2019 (19-30A) in three different regions of France (Auvergne-Rhône-Alpes, Occitanie and Centre-Val de Loire), indicating a wide distribution and a persistence over time of BVG in France. To our knowledge, this represents the first report of a natural infection of BVG in cultivated winter barley in France. Presence of BVG may have been overlooked in a range of situation, as indicated by its retrospective discovery in a 34 years old Australian sample [3], possibly explaining its broad distribution in France. While the mixed infection status of the analyzed plants precludes any conclusion on its pathogenicity in French cereals, BVG has been reported to be associated with a range of symptoms in various hosts so that further studies to evaluate its prevalence and impact in France and to begin to understand its epidemiology are clearly warranted by the present results.
RESUMO
The European corn borer (Ostrinia nubilalis (Hübner)) is one of the most serious corn pest in Europe where it is controlled with pesticides, in particular, pyrethroids. First control failures with this chemical family occurred on the field in 2008 in the center of France, and the first resistance case was described in 2012. In the present study, we investigate resistance mechanisms involved in seven French populations of O. nubilalis collected in the field. Resistances to deltamethrin and lambda-cyhalothrin were confirmed, with a higher resistance ratio for lambda-cyhalothrin (63.79 compared to 7.67). Resistance to the two active compounds was correlated except for one population, indicating a high probability of cross-resistance. Analyses of the activity of three major families of detoxification enzymes in resistant individuals showed a significant increase of the average MFO activity in males of four populations (activity ratios of 2.76-5.73) and higher GST activity in females of two other populations (activity ratios 4.48 and 5.21). Molecular investigation of the sodium channel gene sequence showed the presence of the kdr mutation in a highly resistant individual. We designed a PCR-RFLP screening tool to search for this mutation in the field, and we found it in five populations but not in the susceptible one. The resistance of O. nubilalis to pyrethroids in France seems to result from a combination of resistance mechanisms, possibly as a consequence of a selection pressure with an exceptional duration (almost 40 yr old).