Validation of species-specific diagnostic tests for five <i>Rhynchosporium</i> species.

<p>All the diagnostic tests detected only the predicted <i>Rhynchosporium</i> species when tested against a range of fungal isolates and plant DNA. PCR using primer pair LinA-F/R produced an amplicon of 145-bp specific for <i>R. commune</i> isolates; RA6-F/R produced an amplicon of 461-bp specific for <i>R. agropyri</i> isolates; RS25-F/R produced an amplicon of 1240-bp specific for <i>R. secalis</i> isolates; 2RO-F/R produced an amplicon of 277-bp specific for both <i>R. orthosporum</i> and <i>R. lolii</i> isolates; rep-PCR genomic fingerprinting using primers ERIC2/BOXA1R produced an amplicon of ∼400-bp specific for isolates of <i>R. lolii</i>.</p>a<p>Barley (cv. Sumo), rye or cocksfoot DNA extracted from leaves of healthy seedlings (no leaf blotch lesions present) grown under controlled environment conditions was confirmed to be free of detectable <i>Rhynchosporium</i> DNA using the quantitative PCR assay of Fountaine <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072536#pone.0072536-Fountaine1" target="_blank">[30]</a> (data not shown);</p>b<p>n/a = not applicable.</p

Five <i>Rhynchosporium</i> species colonise the same sub-cuticular niche in their hosts.

<p>Sub-cuticular hyphal <b>(H)</b> growth of (A) <i>R. commune</i> (isolate 53hv09) at 28 days post inoculation (dpi) on a leaf of barley (cv. Sumo); (B) <i>R. agropyri</i> on a leaf of couch-grass collected from the field (Hertfordshire, UK) in April 2010; (C) <i>R. secalis</i> (RS99CH1 H10B) at 30 dpi on a leaf of rye; (D) <i>R. secalis</i> isolate (I-1a) at 28 dpi on a leaf of triticale; (E) <i>R. orthosporum</i> (RsCH04 Bär A.1.1.3) at 14 dpi on a leaf of cocksfoot; (F) <i>R. lolii</i> (9lm11) at 28 dpi on a leaf of Italian ryegrass. Scale-bars on electron micrographs are 10 <i>µm</i>.</p

PCR-based diagnostic tests to distinguish between five <i>Rhynchosporium</i> species.

<p>Five primer pairs (LinA-F/R; RA6-F/R; RS25-F/R; 2RO-F/R; ERIC2/BOXA1R) were tested with representative isolates of <i>R. commune</i> (lanes 1–3; K1124, QUB 12-3, OSA 28-2-2), <i>R. agropyri</i> (lanes 4–6; RS04CG-RAC-A.6.1, 6ar10, 10ar10), <i>R. secalis</i> (lanes 7–9; RS99CH1 H10B, E7a, I-1a), <i>R. orthosporum</i> (lanes 10–13; 27dg09, RS04CG-BAR-A.1.1.4, RS04ITA D-4.1) or <i>R. lolii</i> (lanes 13–15; 1lm11, 9lm11 and 18lp11). (A) LinA-F/R produced a 145-base pair (bp) amplicon specific for <i>R. commune</i> isolates; (B) RA6-F/R produced a 461-bp amplicon specific for <i>R. agropyri</i> isolates; (C) RS25-F/R produced a 1240-bp amplicon specific for <i>R. secalis</i> isolates; (D) 2ROR-F/R produced a 277-bp amplicon specific for both <i>R. orthosporum</i> and <i>R. lolii</i> isolates; use of rep-PCR genomic fingerprinting primers ERIC2/BOXA1R produced a ∼400-bp amplicon specific only for isolates of <i>R. lolii</i>. Different isolates are displayed in (B, C) lanes 1–3: FI12-63, QUB 9-10, 2lm11; (D) lanes 1–15∶19hv09, UK7, 2lm11, 10ar10, 6ar10, RS04CG-RAC-A.6.1, RS02CH4-4b1, RS02CH4-14a1, 6.2, 27dg09, RS04CG-BAR-A.1.1.4, RS04ITA D-4.1, 9lm11, 14lp11 and 18lp11; (E) lanes 3, 6 and 14∶2lm11, 1ar10 and 8lm11, respectively. Note that in (A) lanes 1–3 have been inserted from a different gel image. Lane labelled ‘L’ contains a 100-bp ladder (A, B, D) or a 1-kilobase ladder (C, E) (both Fermentas, UK); lane 16 is a no template control.</p

<i>R. commune</i> isolates that cause leaf blotch lesions on both Italian ryegrass and barley.

<p>Isolate 2lm11 caused lesions <b>(L)</b> when inoculated onto (A) Italian ryegrass or (B) barley (cv. Optic) leaves; scanning electron microscopic (SEM) examination of these hosts (C, D) showed sub-cuticular hyphae <b>(H)</b> and sporulation with beak-shaped conidia <b>(C)</b> on both hosts. Isolate 5lm11 also caused lesions on both Italian ryegrass (E) and barley (F); SEM examination showed that the pathogen could colonise both hosts (G, H) and sporulation with beak-shaped conidia was observed on barley. Control leaves of Italian ryegrass and barley treated with water (I, J) did not develop leaf blotch symptoms and SEM examination (K, L) found no evidence for the presence of <i>R. commune</i>. Photographs of leaf symptoms were taken at 17 (B, F, J) and 24 (A, E, I) days post inoculation (dpi); electron micrographs were taken at 21 (D, H, L) and 28 (C, G, H) dpi. All leaf pieces were c. 4 cm long; scale bars on electron micrographs are 10 <i>µm</i>.</p

Multilocus phylogeny to determine the evolutionary relationships between five <i>Rhynchosporium</i> species.

<p>Phylogenetic analysis (maximum clade credibility tree) of combined partial sequences of the alpha-tubulin, beta-tubulin and internal transcribed spacer loci show consistent differences between <i>R. commune</i>, <i>R. agropyri</i>, <i>R. secalis</i>, <i>R. orthosporum</i> and <i>R. lolii</i>. Concatenated haplotype (H) sequences sourced from Zaffarano <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072536#pone.0072536-Zaffarano1" target="_blank">[12]</a> were combined with sequence data obtained from individual isolates in the present study. Posterior probabilities are indicated for major speciation nodes. The asterisk (*) indicates the calibration point used to infer absolute times (ybp; years before present) to the most recent common ancestor (TMRCA) for these <i>Rhynchosporium</i> species (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072536#pone.0072536.s001" target="_blank">Figure S1</a>).</p

Five <i>Rhynchosporium</i> species divided into two groups by conidial shape.

<p>Isolates of <i>R. commune</i>, <i>R. agropyri</i> and <i>R. secalis</i> have beak-shaped conidia, while isolates of <i>R. orthosporum</i> and <i>R. lolii</i> have cylindrically-shaped conidia. Isolates shown are <i>R. commune</i> (collected from barley/wall barley) (A-C), <i>R. agropyri</i> (D-F), <i>R. secalis</i> (G-I), <i>R. orthosporum</i> (J-L), <i>R. lolii</i> (M-O) and <i>R. commune</i> (from Italian ryegrass) (P,Q). Isolates shown are (A-Q): 19hv09, D.1.1, E.1.2, 4ar10, 8ar10, Rs04CH Rac A.6.1, 1D4a, Rs02CH4-6a.1, I-3a1, 27dg09, 57dg09, RsCH04 Bär A.1.1.3, 12lp11, 20lp11, 22lm11, 2lm11, 5lm11. Scale bars are 20 <i>µm</i>.</p

Genomic Analysis of the Necrotrophic Fungal Pathogens <i>Sclerotinia sclerotiorum</i> and <i>Botrytis cinerea</i>

<div><p><i>Sclerotinia sclerotiorum</i> and <i>Botrytis cinerea</i> are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of <i>S. sclerotiorum</i> and two strains of <i>B. cinerea</i>. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the <i>S. sclerotiorum</i> assembly to 16 chromosomes and found large-scale co-linearity with the <i>B. cinerea</i> genomes. Seven percent of the <i>S. sclerotiorum</i> genome comprises transposable elements compared to <1% of <i>B. cinerea</i>. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of <i>B. cinerea</i>–specific secondary metabolites relative to <i>S. sclerotiorum</i>. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between <i>S. sclerotiorum</i> and <i>B. cinerea</i>. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.</p></div