Evolutionary relationships between Rhynchosporium lolii sp. nov. and other Rhynchosporium species on grasses

Abstract

The fungal genus Rhynchosporium (causative agent of leaf blotch) contains several host-specialised species, including R. commune (colonising barley and brome-grass), R. agropyri (couch-grass), R. secalis (rye and triticale) and the more distantly related R. orthosporum (cocksfoot). This study used molecular fingerprinting, multilocus DNA sequence data, conidial morphology, host range tests and scanning electron microscopy to investigate the relationship between Rhynchosporium species on ryegrasses, both economically important forage grasses and common wild grasses in many cereal growing areas, and other plant species. Two different types of Rhynchosporium were found on ryegrasses in the UK. Firstly, there were isolates of R. commune that were pathogenic to both barley and Italian ryegrass. Secondly, there were isolates of a new species, here named R. lolii, that were pathogenic only to ryegrass species. R. lolii was most closely related to R. orthosporum, but exhibited clear molecular, morphological and host range differences. The species was estimated to have diverged from R. orthosporum ca. 5735 years before the present. The colonisation strategy of all of the different Rhynchosporium species involved extensive hyphal growth in the sub-cuticular regions of the leaves. Finally, new species-specific PCR diagnostic tests were developed that could distinguish between these five closely related Rhynchosporium species.

Figure 1. DNA fingerprinting methods distinguish between five Rhynchosporium species.

(A) RAPD-PCR fingerprinting of 79 isolates using combined data from seven RAPD-PCR primers; (B) rep-PCR genomic fingerprinting of 71 isolates using combined data from two primer pairs (ERIC2/BOXA1R and ERICF/BOXA1R). Both unrooted trees were constructed by neighbour-joining analyses with branch lengths drawn to show genetic distance derived from Jaccard’s coefficient of band matching (scale bar: 0.1 = 10% genetic difference). Numbers at nodes indicate the percentage bootstrap support (based on 1000 re-samplings) for the groupings; only values (A) >60% and (B) >70% are shown, for clarity. Both fingerprinting methods discriminated between isolates of R. commune (blue), R. agropyri (red), R. secalis (green), R. orthosporum (yellow) and R. lolii (purple). Note that two isolates of R. commune (2lm11 and 5lm11) were collected from Italian ryegrass.

Figure 2. Multilocus phylogeny to determine the evolutionary relationships between five Rhynchosporium species.

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 R. commune, R. agropyri, R. secalis, R. orthosporum and R. lolii. Concatenated haplotype (H) sequences sourced from Zaffarano et al. 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 Rhynchosporium species (see also Figure S1).

Figure 3. Five Rhynchosporium species divided into two groups by conidial shape.

Isolates of R. commune, R. agropyri and R. secalis have beak-shaped conidia, while isolates of R. orthosporum and R. lolii have cylindrically-shaped conidia. Isolates shown are R. commune (collected from barley/wall barley) (A-C), R. agropyri (D-F), R. secalis (G-I), R. orthosporum (J-L), R. lolii (M-O) and R. commune (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 µm.

Figure 4. R. commune isolates that cause leaf blotch lesions on both Italian ryegrass and barley.

Isolate 2lm11 caused lesions (L) 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 (H) and sporulation with beak-shaped conidia (C) 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 R. commune. 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 µm.

Figure 5. Five Rhynchosporium species colonise the same sub-cuticular niche in their hosts.

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

Figure 6. PCR-based diagnostic tests to distinguish between five Rhynchosporium species.

Five primer pairs (LinA-F/R; RA6-F/R; RS25-F/R; 2RO-F/R; ERIC2/BOXA1R) were tested with representative isolates of R. commune (lanes 1–3; K1124, QUB 12-3, OSA 28-2-2), R. agropyri (lanes 4–6; RS04CG-RAC-A.6.1, 6ar10, 10ar10), R. secalis (lanes 7–9; RS99CH1 H10B, E7a, I-1a), R. orthosporum (lanes 10–13; 27dg09, RS04CG-BAR-A.1.1.4, RS04ITA D-4.1) or R. lolii (lanes 13–15; 1lm11, 9lm11 and 18lp11). (A) LinA-F/R produced a 145-base pair (bp) amplicon specific for R. commune isolates; (B) RA6-F/R produced a 461-bp amplicon specific for R. agropyri isolates; (C) RS25-F/R produced a 1240-bp amplicon specific for R. secalis isolates; (D) 2ROR-F/R produced a 277-bp amplicon specific for both R. orthosporum and R. lolii isolates; use of rep-PCR genomic fingerprinting primers ERIC2/BOXA1R produced a ∼400-bp amplicon specific only for isolates of R. lolii. 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.