Genome-wide Association Study Identifies New Loci for Resistance to Leptosphaeria maculans in Canola

Blackleg, caused by Leptosphaeria maculans, is a significant disease which affects the sustainable production of canola. This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to L. maculans. Genomic regions delimited with 503 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned Rlm2/LepR3 genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07 and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance and mapped within 13.2 kb from Arabidopsis R gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of R genes of A. thaliana and B. napus on the sequenced genome of B. napus cv. Darmor-bzh. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to L. maculans in canola

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Inhibition of mycelial growth of <i>L</i>. <i>maculans</i> by the stilbenes resveratrol and pterostilbene.

<p>Hyphal plugs were cultured for six days on half strength V8 agar amended with resveratrol or pterostilbene at various concentrations. Mycelial growth is expressed as the percentage (%) of growth in the treatment relative to the control. Vertical bars represent standard deviation. Chemical structures of resveratrol and pterostilbene are shown above graph.</p

Inhibition of conidia germination of five <i>L</i>. <i>maculans</i> isolates by pterostilbene.

<p>Conidia (1 x 10⁷ spores/ml) germination after 10 days on 2% water agar amended with 50 μg/ml pterostilbene (PTE) or solvent (control, CTRL).</p

Conidia germination of <i>L</i>. <i>maculans</i> on 2% water agar amended with resveratrol at various concentrations.

<p>Colonies were counted after 10 days, and germination is expressed as percentage (%) of number of colonies in the treatment relative to the control. Statistical significance in Student’s <i>t</i>-test at <i>p</i><0.05 (*) and <i>p</i><0.01 (**) with degrees of freedom = 4. Vertical bars represent standard deviation.</p

Permeabilization and cell viability of hyphae treated with 50 μg/ml pterostilbene.

<p>Uptake over time of 0.5 μM SYTOX green and 1μg/ml fluorescein diacetate (FDA) by hyphae cultures. Left axis: relative SYTOX green fluorescence (fold-change). Right axis: relative FDA fluorescence (%). Vertical bars represent standard deviation. All values were statistically significant at <i>p</i><0.01 in Student’s <i>t</i>-test with degrees of freedom = 8.</p

SYTOX green (0.5 μM) uptake into hyphae treated with 50 μg/ml pterostilbene.

<p>Bright field and fluorescent images for <b>(a)</b> control, <b>(b)</b> hyphae treated for 2 hours and <b>(c)</b> 4 hours. Black arrowheads indicate coagulation of the cytosol within the cytoplasm. White arrowheads indicate SYTOX green staining in the nuclei. Scale bar = 20 μm.</p

Sporicidal activity of pterostilbene against <i>L</i>. <i>maculans</i> spores.

<p>Growth after 10 days on 2% water agar of <b>(a)</b> 1 x 10⁷ spores/ml and <b>(b)</b> 1 x 10² spores/ml cultures. Conidia (1 x 10⁷ spores/ml) were treated with 50 μg/ml of pterostilbene (PTE) or solvent (control, CTRL) and serially diluted to 1 x 10² spores/ml.</p

Inhibition of mycelial growth of ten <i>L</i>. <i>maculans</i> isolates by pterostilbene.

<p>Hyphal plugs were cultured for six days on half strength V8 agar amended with 50 μg/ml pterostilbene. Mycelial growth is expressed as the percentage (%) of growth in the treatment relative to the control. Vertical bars represent standard deviation. All values were statistically significant at <i>p</i><0.01 in Student’s <i>t</i>-test with degrees of freedom = 4.</p

A multiplex PCR for rapid identification of Brassica species in the triangle of U

Abstract Background Within the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U’s triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here. Results A multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions. Conclusion A cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica germplasm collections in genebanks