Stilbenoids as Antifungals to Counteract Rice Blast Pathogen Pyricularia oryzae

Fungi are among the greatest biotic threats to agricultural and food security. Intensive monoculture cropping and resistance to single-site fungicides in plant pathogens urge the discovery and development of novel compounds that possibly interfere with essential cellular processes in multiple ways. In this article, we describe our recent efforts addressed to the identification of natural compounds as multitarget biofungicides. A set of natural monomeric and dimeric compounds belonging to the class of stilbenoids were synthesized and tested against wild-type (WT) and strobilurin-resistant (RES) strains of Pyricularia oryzae, one of the most dangerous fungal phytopathogens. Monomers deoxyrhapontigenin, pinostilbene, and DMHS showed inhibitory activity higher than 40%, with deoxyrhapontigenin having the highest activity on mycelial growth (60–80% inhibition) on both WT and RES P. oryzae strains. Furthermore, we designed and synthesized a set of molecules having a nature-derived stilbene fragment merged with the pharmacophoric portion of strobilurins, namely, a β-methoxyacrylate moiety. We identified two molecules with activity comparable to the reference commercial fungicide azoxystrobin. However, low mycelium growth inhibition of resistant strains indicates that these compounds most likely retain the strobilurin-like mechanism of action. Overall, the results suggest that natural stilbenoids might be used as environmentally friendly biofungicides in rice blast management

Additional file 1: of Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis

Figure S1. Equine keratinocytes (EK-100) were cultured in various culture conditions. Representative phase contrast images of primary equine keratinocytes (EK-100) cultured in (a) co-culture with irradiated fibroblasts+ 10 uM Y-27632, (b) F + 10 uM Y-27632 (c,d) CNT with or without 10 uM Y-27632 and (e)KSFM+ 10 uM. All images were taken on and day7 following initial culture without passage (×10 magnification. Size bars = 400 μm). Top right images show enlarged magnification (×40 magnification, size bars = 100 μm). Figure S2. Fluorescence-activated cell sorting (FACS) analysis of human keratinocytes (HFK) and mouse fibroblasts (j2) using pan-cytokeratin antibody. HFK cells were incubated without (a) pan-CK antibody or (b) with pan-CK antibody, (c) J2 fibroblasts without pan-CK antibody, or (d) with pan-CK antibody. Figure S3. Validation of antibodies for equine tissues. Specificity and reactivity of CK-14 was tested by using diluted concentration of CK14 1:600, 1:5000, and no antibody respectively in (a) breast cancer tissue and (b) equine skin tissue. All images (×40 magnification, scale bar = 100 μm) are representative of three experimental repeats. (PPTX 5023 kb

Gene Ontology classifications of copy number variable genes in horses.

<p>Gene Ontology classifications of copy number variable genes in horses.</p

Schematic of the homozygous deletion in chr29, 28.6–28.8 Mb in two XY DSD horses.

<p><b>A</b>. chr29 ideogram showing the location of <i>AKR1C</i> genes and a control gene <i>CREM</i>; <b>B</b>. Detailed map of the CNVR showing the location of genes (black horizontal bars) and CGH signal log2 values for 47 array probes in XY DSD and control horses; <b>C</b>. FISH results with a BAC 23N13 spanning the deletion (green signal) and a control BAC 76H13 for <i>CREM</i> from a non-CNVR (red signal); <b>D</b>. PCR with CNVR-specific primers in XY DSD and control horses.</p

Chromosome-wise CNVR statistics for the horse genome.

<p>Shared – found in 2 or more individuals; private – in one horse only; novel – not reported before; the horse genome statistics was retrieved from Ensembl (<a href="http://www.ensembl.org/index.html" target="_blank">http://www.ensembl.org/index.html</a>).</p><p>Chromosome-wise CNVR statistics for the horse genome.</p

Breed- and individual-wise summary of CNV calls in horses.

<p>The number of calls per individual was not significantly different (Student's T-test <i>p</i> = 0.07) between hair and blood DNA.</p><p>Breed- and individual-wise summary of CNV calls in horses.</p

Summary statistics of all CNV studies in horses.

<p>*As reported by original studies and before consolidating overlapping and tandemly located CNVRs into a composite dataset.</p><p>**Dupuis and colleagues specified only large groups of horses (warmblood, coldblood, draft, pony) but not individual breeds.</p><p>***Results by Metzger and colleagues vary between different analysis software packages used.</p><p>Summary statistics of all CNV studies in horses.</p

Chromosome-wise distribution of genic and intergenic CNVRs in the horse genome.

<p>Chromosome-wise distribution of genic and intergenic CNVRs in the horse genome.</p

Genetic relationships of horse breeds studied for CNVs.

<p>A Maximum Likelihood tree showing genetic relationships of the horse breeds that have been studied for CNVs; * new breeds added in this study (except Swiss Warmblood);** breeds involved in 2 or more studies. Numbers denote bootstrap values.</p

A CNVR map of the horse genome.

<p>Green line – loss; red line – gain; yellow line – complex; black dots – genes involved.</p