Evolutionary Relationships between Rhynchosporium lolii sp. nov. and Other Rhynchosporium Species on Grasses

Kevin M King; Jonathan S West; Patrick C Brunner; Paul S Dyer; Bruce D L Fitt

doi:10.1371/journal.pone.0072536

. 2013 Oct 16;8(10):e72536. doi: 10.1371/journal.pone.0072536

Evolutionary Relationships between Rhynchosporium lolii sp. nov. and Other Rhynchosporium Species on Grasses

Kevin M King ^1,², Jonathan S West ^1,³, Patrick C Brunner ⁴, Paul S Dyer ², Bruce D L Fitt ^1,^3,^*

Editor: Michiel Vos⁵

PMCID: PMC3797698 PMID: 24146740

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.

Introduction

Leaf blotch (scald) caused by the fungal pathogen Rhynchosporium commune is an economically important disease of barley (Hordeum vulgare) crops throughout the world [1] with yield losses of 1–10% common [2]. United Kingdom (UK) survey data indicates that barley crops grown in 2005 had 0.6% area with rhynchosporium lesions on leaf two at growth stage 75 (medium milk development stage; [3]). This equates to an estimated UK national yield loss of £10.8 million per annum (at a price of £225/tonne) despite fungicide treatment [4]. The symptoms of Rhynchosporium colonisation of barley can include coalescing lesions with dark brown margins and pale green or pale brown centres [5].

Phylogeographical analyses of R. commune isolates obtained from barley across five continents led to the conclusion that the pathogen did not emerge in association with its current barley host, believed to have been first domesticated in the ‘Fertile Crescent of the Middle East about 10,000 years before the present [6]. Instead, data from phylogenetic analyses using the R. commune avirulence gene NIP1 and flanking regions [7] suggested that modern populations of R. commune originated in northern Europe approximately 2500–5000 years ago, when the pathogen switched from a wild grass species onto cultivated barley shortly after barley was introduced there, and that it subsequently spread into other barley-growing areas of the world.

The Rhynchosporium pathogen is also found on a number of other graminaceous hosts, including rye (Secale cereale), triticale (x Triticosecale), cocksfoot (Dactylis glomerata) and Italian and perennial ryegrasses (Lolium multiflorum and Lolium perenne, respectively) [8], [9], [10], [11]. Insights into the evolutionary history of the Rhynchosporium pathogen on different hosts were provided by Zaffarano et al. [12. 13], who used sequencing of several gene loci and host range testing experiments to demonstrate that the genus Rhynchosporium is comprised of at least four closely-related, but host-specialised, species. Three of the species produce conidia terminating with an oblique point, termed beak-shaped conidia [14]; (i) R. commune causing leaf blotch symptoms on barley, wall barley (Hordeum murinum), wild barley (Hordeum spontaneum), barley grass (Hordeum glaucum, Hordeum leporinum) and brome-grass (Bromus diandrus); (ii) R. agropyri on bearded couch-grass (Agropyron caninum) and couch-grass (Agropyron repens); (iii) R. secalis on rye and triticale. The remaining species R. orthosporum, which is more distantly related [13], produces cylindrically-shaped conidia [14] and is specialised on cocksfoot.

One limitation of the studies by Zaffarano et al. [12], [13] is that Rhynchosporium isolates from ryegrasses were not included. These grasses are both economically important forage grasses and commonly occurring weeds of cereal crops on most continents, and although originally native to Europe, Asia and North Africa they have now been introduced into almost all temperate countries of the world [11], [15], [16]. A previous investigation [11] had identified two types of Rhynchosporium that could cause leaf blotch on ryegrasses, with differences between them in conidial shape and host range. However, it is at present unclear how Rhynchosporium isolates from ryegrasses are related to the four described fungal species [13], none of which included ryegrass species in host range definitions Therefore, there is a need to characterise isolates from ryegrasses to determine their relatedness to the Rhynchosporium species of Zaffarano et al. [13]. Such characterisation is essential to understand any potential role for ryegrass species as a source of Rhynchosporium inoculum able to initiate leaf blotch epidemics on barley crops.

Currently, only very limited PCR (polymerase chain reaction)-based methods are available to distinguish between the four Rhynchosporium species described by Zaffarano et al. [13]. A restriction fragment length polymorphism diagnostic was developed [13] that could discriminate between isolates of R. commune and R. secalis. However, this test is time-consuming due to the requirement for an additional restriction digest step following PCR. Apart from this test, the species can be distinguished only at a molecular level on the basis of single nucleotide polymorphisms. The development of species-specific PCR diagnostic tests would provide a valuable tool to directly confirm species identity of isolates and to determine species distribution in field samples of different grass hosts.

Little is currently known about the how the four recently described Rhynchosporium species of Zaffarano et al. [13] might differ in the manner by which they colonise their plant hosts. For example, only R. commune colonisation of barley [17] and R. orthosporum colonisation of cocksfoot [10] have been the subject of detailed microscopical investigations. On both hosts, Rhynchosporium hyphae grew extracellularly (i.e. outside the plant cells) and extensive colonisation of the leaf sub-cuticular area was observed. Although Caldwell [14] made some observations about the colonisation strategy of Rhynchosporium on other grass species, no microscopic evidence is currently available to determine whether R. agropyri, R. secalis and the pathogen on ryegrasses all colonise the same sub-cuticular niche of their respective grass hosts.

This paper reports work to further investigate the different Rhynchosporium species on various grass hosts. Specifically, it uses a combination of molecular, morphological, pathological and microscopy approaches to (i) report the discovery of R. commune isolates that are pathogenic to both barley and Italian ryegrass; (ii) describe a new species that is specialised only to ryegrass species; (iii) report results of both microscopic and molecular investigations into the different host-specialised Rhynchosporium species.

Methods

Ethics Statement

Diseased plant material was collected from plots (permission obtained from IBERS-Aberystwyth University, NIAB-TAG and Rothamsted Research, all based in the UK). Diseased plant material and fungal cultures were imported into the UK under Defra plant health license number PHL174G/6192(10/2009).

Isolation of the Rhynchosporium Pathogen

Leaves of barley, couch-grass, cocksfoot, Italian ryegrass and perennial ryegrass with distinct leaf blotch lesions were collected during the period of 2009–2011 from plots, samples being collected at least ∼1 metre apart within sites. To isolate Rhynchosporium, leaf blotch lesions were cut from green leaves, rinsed first in a 70% ethanol (v/v) solution for 2 min, followed by a 10% sodium hypochlorite (v/v) solution (minimum 8% available chlorine; Fisher Scientific, UK) for 5 min, and finished by a wash in sterile distilled water for 1 min. Lesions were then dried between pieces of sterile tissue paper and incubated at 18°C on Czapek dox plates (Sigma Aldrich, UK) amended to include 0.5% mycological peptone (Oxoid, UK) and penicillin and streptomycin sulphate at a final concentration of 100 and 50 parts per million, respectively. After 3 days, mycelium growing out from lesion margins was excised and used to establish single conidial cultures on lima bean agar (LBA; Difco, UK), which were grown at 18°C for 10 days.

Fungal Isolates Obtained and Long-term Storage Procedure

A total of 44 Rhynchosporium isolates were obtained from leaves with leaf blotch lesions collected from five sites in the UK and two sites in Romania. New isolates collected included 22 isolates from Italian or perennial ryegrass in the UK, 11 isolates from couch-grass in Romania and the UK, seven isolates from cocksfoot in the UK and four isolates from barley in the UK. An additional 49 isolates, including representatives of R. commune, R. agropyri, R. secalis and R. orthosporum, obtained from international collaborators, had previously been isolated from various field sites most of which were throughout Europe. Collaborators included Dr Louise Cooke (Agri-Food and Biosciences Institute, UK), Dr James Fountaine (Scotland’s Rural College, UK), Dr Nichola Hawkins (Rothamsted Research, UK), Dr Wolfgang Knogge (Leibniz Institute of Plant Biochemistry, Germany) and Prof Bruce McDonald and Dr Tryggvi Stefansson (ETH Zürich, Switzerland). All isolates were then stored as silica stocks at −80°C [18].

DNA Extraction

All fungal isolates were revived from −80°C storage by dispensing small amounts of silica stock onto potato dextrose agar (PDA, Oxoid, UK) plates overlaid with a single cellulose disk (A.A. Packaging Ltd, UK). Plates were sealed with a double layer of parafilm (Pechiney Plastic Packaging, USA) and incubated at 18°C in the dark for 10–15 days. After this period, fungal mycelium was scraped from the surface of the overlaid cellulose disk and DNA was extracted from lyophilized tissue using a DNeasy extraction kit (Qiagen, UK), according to the manufacturer’s instructions.

RAPD-PCR and Rep-PCR Genomic Fingerprinting

Seventy-nine Rhynchosporium isolates were examined by random amplification of polymorphic DNA PCR (RAPD-PCR) fingerprinting (Table S1) as described in Murtagh et al. [19], except that a TC-512 programmable thermal controller (Techne, UK) was used for all PCR. Seven RAPD-PCR primers (Operon Technologies, UK; Table S2) were used; in preliminary testing they had been found to generate polymorphisms with a subset of Rhynchosporium isolates. No template controls were included for use with all RAPD-PCR sets and selected isolates from each of the different species were tested in duplicate to ensure that results were reproducible. PCR products (10 µl) were separated by gel electrophoresis on 1.5% agarose gels in 1×Tris-Borate-EDTA (TBE) buffer (National Diagnostics, UK) and stained with an ethidium bromide solution (200 µl of a 1 mg/ml ethidium bromide per 100 ml of 1×TBE buffer). Amplicons were viewed on a transilluminator and digital images obtained (Gene Genius Bio Imaging System, Syngene, Synoptics Ltd, UK). Unambiguous bands were chosen for scoring and their presence or absence was recorded in binary form (1 = present, 0 = absent), with data from all seven RAPD-PCR primers combined in the analyses. A neighbour joining analysis was made using Jaccard’s coefficient and a boot-strapped phylogram (based on 1,000 repeats of the tree) was produced using FreeTree software [20]. A text version of the dendrogram of the tree was exported to TreeView software [21].

Seventy-one Rhynchosporium isolates were examined with repetitive-sequence-based PCR (rep-PCR) genomic fingerprinting (Table S1) using primer pair combinations ERIC2/BOXA1R and ERICF/BOXA1R [22], [23] (Table S2). No template controls were included for use with both primer pairs and selected isolates from each of the different species were tested in duplicate to ensure that results were reproducible. Reactions were carried out in 20 µl volumes, each containing 10 µl Jumpstart RedTaq mastermix (Sigma Aldrich, UK), 7 µl sterile distilled water, 1 µl each of both primers (10 pmol µl⁻¹ stock) and 1 µl of template DNA (10 ng). For testing of five isolates (RS04ITA D-2.2, RS04ITA D-3.1, RS04ITA D-4.1, RS04ITA D-6.1, RS04ITA D-6.2), 10 µl of template DNA (total 10 ng) was added (achieved by increasing overall reaction volumes) because only dilute concentrations of template DNA were available. PCR was carried out using a PTC-100 programmable thermal controller (MJ Research, USA) and reaction conditions were an initial hold at 96°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 52°C for 1 min and 65°C for 5 min. PCR products were then analysed as described for RAPD-PCR products.

Phylogenetic Analyses

Partial sequences of the alpha-tubulin, beta-tubulin and ITS (internal transcribed spacer) gene loci were obtained for ten isolates (2lm11, 3ar10, 6ar10 and 10ar10, 59dg09, Rs04ITA D-6.2, 4lm11, 7lm11, 13lp11 and 15lp11; isolate details and accompanying GenBank accession numbers provided in Table S3) using primers (Table S2) and reaction conditions described by Zaffarano et al. [12]. No template controls were included in reactions to amplify the three gene loci. Jumpstart high fidelity mix (Roche, Germany) was used in all reactions, with reaction components selected according to the manufacturer’s instructions with the following modifications; no DMSO was included and each reaction included 10 mM of each dNTP (Fermentas, UK) and 10–25 ng of template DNA. PCR products were visualised on a 1% agarose gel to ensure the presence of a single amplicon and purified using a MinElute kit (Qiagen, UK) according to the manufacturer’s instructions. DNA was sent to Eurofin MWG Operon for bi-directional sequencing using an ABI 3730XL machine, with the exception of the beta-tubulin PCR product for which only primer BTUB21F was used.

Individual sequences were imported into the BioEdit Sequence Alignment Editor (version 7.0.9.0; [24]) and trace sequence data of poor read quality were removed. Sequences were imported into the Geneious Pro 5.5.6 software package and the partial alpha-tubulin, beta-tubulin and ITS (partial 18S rRNA, ITS1, 5.8S rRNA, partial ITS2) sequences were edited to 1538, 542 and 492 bases in length (including gaps), respectively. For the ten isolates partially sequenced in the present study, sequence data of all three gene loci were concatenated. The CLUSTAL W algorithm contained in the Geneious software package was then used to align these sequences with the concatenated haplotype sequences of R. commune, R. agropyri, R. secalis and R. orthosporum obtained by Zaffarano et al. [12].

The relationship of isolates and haplotypes was inferred using the coalescent-based Bayesian Markov Chain Monte Carlo (MCMC) method implemented in the program BEAST version 1.4.1 [25]. To allow a direct comparison with previous studies, a strict molecular clock model was applied and the phylogenetic tree was internally calibrated by assuming a time-to-most-recent-common-ancestor (TMRCA) of 2487–4791 years (mean 3625) as inferred for the cluster R commune/R. agropyri/R. secalis [12]. The MCMC analysis was run for 10⁷ generations, sampling every 1000th iteration after an initial burn-in of 10%. The performance of the MCMC process was checked for stationarity and large effective sample sizes in TRACER (available from http://beast.bio.ed.ac.uk/Tracer). A maximum clade credibility tree was constructed after discarding the first 10% of inferred trees. The mean and corresponding credibility intervals of the estimated TMRCAs were depicted using TRACER.

Microscopic Analysis of Conidial Morphology

Forty isolates, including isolates of R. commune, R. agropyri, R. secalis, R. orthosporum or Rhynchosporium isolates collected from ryegrasses (Table S1), were grown on LBA plates at 15°C in the dark. After 10 days of growth, sterile distilled water (2 ml) was added to each LBA plate and conidia were dislodged using an L-shaped sterile plastic spreader. Conidial suspensions were placed in sterile 2 ml microfuge tubes after filtration through two layers of sterile muslin to remove mycelial fragments. Conidial dimensions of 26 isolates were measured using fresh (within 8 hours of harvest) conidial suspensions maintained on ice (∼0°C), while conidial suspensions of 18 isolates (including four that were also measured fresh) were stored at −20°C and measured later. The shape of individual conidia of individual isolates was recorded as either beak-shaped or cylindrically-shaped [14]. Both the length and width of 25 mature (defined as having two cells clearly divided by a septum) conidia were measured with a digital CCD camera (Hamamatsu C8484 05G01) using HCimage software (Hamamatsu Photonics K.K., Japan).

Light microscopic images of representative conidia of 17 isolates were also made on a Zeiss axiophot light microscope; images were obtained using a QImaging monochrome camera equipped with a Retiga XEi liquid crystal RGB filter and operated using MetaMorph ver. 7.6 software. Before statistical analysis of data, isolates were separated into two different groups; those with beak-shaped conidia (R. commune including two isolates from Italian ryegrass, R. agropyri and R. secalis) and those with cylindrically-shaped conidia (R. orthosporum and Rhynchosporium isolates from ryegrasses). Data were analysed using ANOVA (GenStat version 14; [26]).

Host Range Testing

Twenty-two Rhynchosporium isolates (Table 1) were revived from −80°C silica stocks onto LBA plates as described previously in the text, with plates sealed with a double layer of parafilm and grown at 18°C in the dark for 10–12 days. Conidia were harvested from LBA plates as described previously in the text, and concentrations of suspensions were determined using a haemocytometer (Improved Neubauer haemocytometer, Weber Scientific International, UK). Aliquots (5–10 ml) of fungal inoculum (5×10⁵ conidia ml⁻¹) were prepared using sterile distilled water (containing 0.01% Tween 80; Fisher Scientific, UK) in 50 ml Falcon tubes (CellStar, USA) and frozen at −20°C until required.

Table 1. Host range experiments suggest general host-specialisation of Rhynchosporium species.

					Development of leaf blotch lesions^{a, b}
Species	Isolate	Original host	Origin	Collected	Barley^c	Cocksfoot^d	Italian ryegrass^e	Perennial ryegrass^e
R. commune	19hv09	Barley	Hertfordshire, UK	2009	+ +	− −	− −, − −	− −, − −
R. commune	53hv09	Barley	Hertfordshire, UK	2009	+ +	− −	− −, − −	− −, − −
R. commune	73hv09	Barley	Hertfordshire, UK	2009	+ +	− −	− −, − −	− −, − −
R. commune	D.1.1	Wall barley	Switzerland	2004	+ +	− −	− −, − −	− −, − −
R. commune	E.1.2	Wall barley	Switzerland	2004	+ +	− −	− −, − −	− −, − −
R. commune	2lm11	Italian ryegrass	Shropshire, UK	2011	+ +	− −	− −,++	− −, − −
R. commune	5lm11	Italian ryegrass	Shropshire, UK	2011	+ +	− −	− −, − +	− −, − −
R. orthosporum	57dg09	Cocksfoot	Aberystwyth, UK	2009	− −	+ +	− −, − −	− −, − −
R. orthosporum	59dg09	Cocksfoot	Aberystwyth, UK	2009	− −	− −	− −, − −	− −, − −
R. orthosporum	RsCH04 Bär A.1.1.3	Cocksfoot	Switzerland	2004	− −	+ +	− −, − −	− −, − −
R. orthosporum	RS04CG-BAR-A.1.1.3	Cocksfoot	Switzerland	2004	− −	+ +	− −, − −	− −, − −
R. orthosporum	RS04CG-BAR-A.1.1.4	Cocksfoot	Switzerland	2004	− −	+ +	− −, − −	− −, − −
R. lolii	6lm11	Italian ryegrass	Aberystwyth, UK	2011	− −	− −	+ −, − −	− +, − −
R. lolii	9lm11	Italian ryegrass	Aberystwyth, UK	2011	− −	− −	− −,+−	+ −, − −
R. lolii	10lm11	Italian ryegrass	Aberystwyth, UK	2011	− −	− −	− +,+−	+ −, − −
R. lolii	21lm11	Italian ryegrass	Shropshire, UK	2011	− −	− −	− +,++	− −, − −
R. lolii	22lm11	Italian ryegrass	Shropshire, UK	2011	− −	− −	+ −, − +	− +, − −
R. lolii	11lp11	Perennial ryegrass	Aberystwyth, UK	2011	− −	− −	+ −,+−	− +, − +
R. lolii	13lp11	Perennial ryegrass	Aberystwyth, UK	2011	− −	− −	− −, − −	− −,++
R. lolii	15lp11	Perennial ryegrass	Shropshire, UK	2011	− −	− −	− −, − −	− +, − −
R. lolii	16lp11	Perennial ryegrass	Surrey, UK	2011	− −	− −	− −, − −	− −,++
R. lolii	20lp11	Perennial ryegrass	Hertfordshire, UK	2011	− −	− −	− −,+−	− −, − +
n/a	Water control	n/a^f	n/a	n/a	− −	− −	− −, − −	− −, − −

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Leaf blotch disease symptoms developed at 23 days post inoculation when isolates of Rhynchosporium commune, R. orthosporum or R. lolii were cross-inoculated onto different hosts under controlled environment conditions.

Whole plants scored for presence (+) or absence (−) of leaf blotch lesions;

Scores for replicate plants are given;

Plants 14-days-old at time of inoculation;

Plants 35-days-old at time of inoculation;

Plants 35- or 40- days-old at time of inoculation (results separated by a comma);

n/a, not applicable.

All plants were grown and inoculated under controlled environment conditions, with a constant temperature of 15°C and a relative humidity of 70%. There was a 12 hour photoperiod with available light set at 700 µmol m²/sec. Seeds were sown in Rothamsted prescription mix compost (Petersfield Products, UK) at approximately 2 cm depth. Before inoculation, barley (cv. Optic) (14-day-old plants; seed supplied by the Rothamsted farm), cocksfoot (35-day-old plants) and both Italian and perennial ryegrass (35- or 40-day-old plants; seeds supplied by Herbiseed Ltd., UK) plants were grown individually in pots (4 cm×4 cm).

Immediately before inoculation, Falcon tubes containing Rhynchosporium inoculum were removed from storage at −20°C and allowed to defrost. Inoculum of each isolate was then transferred to a separate 20 ml plastic spray bottle. Conidial suspensions of each isolate were then sprayed onto two replicate whole plants of each graminaceous host, using a completely randomised design, until leaves were evenly coated with fine droplets. Plants were then maintained in a humid environment for 48 h by sealing them in 650–800 mm polyethylene bags (VWR International Ltd, UK). After this time, the top and bottom of each polyethylene bag were removed so that plants remained isolated but open to ambient relative humidity. In the experiments, sterile distilled water (containing 0.01% Tween 80) was used as a negative control. At 23 days post inoculation (dpi), whole plants were assessed for the presence or absence of typical leaf blotch lesions.

Barley and Italian ryegrass leaves that had been inoculated with two isolates of R. commune (2lm11 and 5lm11) that had produced typical leaf blotch lesions on both hosts (or with sterile distilled water, no lesions) were also examined in duplicate by scanning electron microscopy (SEM) (21 or 28 dpi as required, see Figure 4 legend).

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.

Colonisation Strategy

Plants of barley, rye, triticale, cocksfoot and Italian ryegrass were grown and inoculated under the controlled environment conditions for host range testing, except that leaves of the first three hosts were all 14-days-old at the time of inoculation and only the second fully expanded leaves were inoculated. Barley leaves (cv. Sumo) were inoculated with an isolate of R. commune (53hv09), rye leaves with an isolate of R. secalis (RS99CH1 H10B), triticale leaves with an isolate of R. secalis (I-1a), cocksfoot leaves with an isolate of R. orthosporum (RsCH04 Bär A.1.1.3) and Italian ryegrass leaves inoculated with an isolate originally collected from this host (9lm11) (see Table 2 for isolate details). Leaves of all of these graminaceous hosts with typical leaf blotch lesions were examined using SEM (14–30 dpi as required; see Figure 5 legend). In addition, couch-grass leaves colonised by R. agropyri and displaying typical leaf blotch lesions were also examined by SEM. However, it had not been possible to produce leaf blotch lesions by inoculating couch-grass with R. agropyri in controlled environments, despite the use of isolates from different geographical origins to inoculate couch grass plants of different ages. Therefore couch-grass leaf specimens colonised by R. agropyri were collected from the field in Hertfordshire (UK) in April 2010 and used for examination.

Table 2. Validation of species-specific diagnostic tests for five Rhynchosporium species.

Species	Isolate code	Host	Geographical origin	Collected	R. commune (LinA-F/R)	R. agropyri (RA6-F/R)	R. secalis (RS25-F/R)	R. orthosporum/R. lolii (2RO-F/R)	R. lolii (ERIC2/BOXAIR)
R. commune	788	Barley	France	1997	+	−	−
R. commune	K1124	Barley	UK	Unknown	+	−	−		−
R. commune	QUB 12-3	Barley	Northern Ireland, UK	Unknown	+	−	−		−
R. commune	OSA 28-2-2	Barley	Hertfordshire, UK	2002	+	−	−		−
R. commune	FI12-63	Barley	Finland	1996	+	−	−
R. commune	RS 219	Barley	UK	2004	+	−	−
R. commune	QUB 9-10	Barley	Northern Ireland, UK	Unknown	+	−	−
R. commune	R.s. 2310 4.2	Barley	France	2008	+	−	−
R. commune	R.s. 2318 4.2	Barley	France	2008	+	−	−		−
R. commune	19hv09	Barley	Hertfordshire, UK	2009	+	−	−	−	−
R. commune	53hv09	Barley	Hertfordshire, UK	2009	+	−	−	−	−
R. commune	73hv09	Barley	Hertfordshire, UK	2009	+	−	−		−
R. commune	UK7	Barley	Aberystwyth, UK	Unknown	+	−	−	−
R. commune	D.1.1	Wall barley	Switzerland	2004	+	−	−		−
R. commune	E.1.2	Wall barley	Switzerland	2004	+	−	−		−
R. commune	2lm11	Italian ryegrass	Shropshire, UK	2011	+	−	−	−	−
R. commune	5lm11	Italian ryegrass	Shropshire, UK	2011	+	−	−	−	−
R. agropyri	RS04CG-RAC-A.4.3	Couch-grass	Switzerland	2004	−	+	−		−
R. agropyri	RS04CG-RAC-A.5.2	Couch-grass	Switzerland	2004	−	+	−	−	−
R. agropyri	RS04CG-RAC-A.6.1	Couch-grass	Switzerland	2004	−	+	−	−	−
R. agropyri	1ar10	Couch-grass	Surrey, UK	2010	−	+	−		−
R. agropyri	2ar10	Couch-grass	Surrey, UK	2010	−	+	−	−	−
R. agropyri	3ar10	Couch-grass	Surrey, UK	2010	−	+	−	−
R. agropyri	6ar10	Couch-grass	Cluj-Napoca, Romania	2010	−	+	−	−	−
R. agropyri	7ar10	Couch-grass	Timisoara, Romania	2010	−	+	−	−	−
R. agropyri	8ar10	Couch-grass	Nottingham, UK	2010	−	+	−	−	−
R. agropyri	9ar10	Couch-grass	Nottingham, UK	2010	−	+	−		−
R. agropyri	10ar10	Couch-grass	Nottingham, UK	2010	−	+	−	−
R. agropyri	11ar10	Couch-grass	Nottingham, UK	2010	−	+	−	−
R. secalis	RS02CH4-2a1	Rye	Switzerland	2002	−	−	+	−	−
R. secalis	RS02CH4-4b1	Rye	Switzerland	2002	−	−	+	−	−
R. secalis	RS02CH4-5a1	Rye	Switzerland	2002	−	−	+	−	−
R. secalis	Rs02CH4-6a.1	Rye	Switzerland	2002	−	−	+		−
R. secalis	RS99CH1-H10B	Rye	Switzerland	1999	−	−	+		−
R. secalis	RS02CH4-13a1	Rye	Switzerland	2002	−	−	+	−	−
R. secalis	RS02CH4-14a1	Rye	Switzerland	2002	−	−	+	−
R. secalis	8.4	Rye	Russia	2003	−	−	+		−
R. secalis	6.2	Rye	Russia	2003	−	−	+	−	−
R. secalis	1E7a	Rye	Switzerland	1999	−	−	+	−	−
R. secalis	1B8	Rye	Switzerland	1999	−	−	+	−	−
R. secalis	1D4a	Rye	Switzerland	1999	−	−	+		−
R. secalis	I-1a	Triticale	Switzerland	2002	−	−	+	−	−
R. secalis	I-2a2	Triticale	Switzerland	2002	−	−	+	−	−
R. secalis	I-3a1	Triticale	Switzerland	2002	−	−	+		−
R. orthosporum	27dg09	Cocksfoot	Aberystwyth, UK	2009	−	−	−	+	−
R. orthosporum	51dg09	Cocksfoot	Aberystwyth, UK	2009				+	−
R. orthosporum	52dg09	Cocksfoot	Aberystwyth, UK	2009				+	−
R. orthosporum	57dg09	Cocksfoot	Aberystwyth, UK	2009	−	−	−	+
R. orthosporum	59dg09	Cocksfoot	Aberystwyth, UK	2009	−	−	−	+
R. orthosporum	RS04CG-BAR-A.1.1.3	Cocksfoot	Switzerland	2004	−	−	−	+	−
R. orthosporum	RS04CG-BAR-A.1.1.4	Cocksfoot	Switzerland	2004	−	−	−	+	−
R. orthosporum	RS04ITA D-6.1	Cocksfoot	Italy	2004	−	−	−	+	−
R. orthosporum	RS04ITA D-6.2	Cocksfoot	Italy	2004	−	−	−	+	−
R. lolii	1lm11	Italian ryegrass	Shropshire, UK	2011	−	−	−		+
R. lolii	3lm11	Italian ryegrass	Shropshire, UK	2011	−	−	−	+	+
R. lolii	4lm11	Italian ryegrass	Shropshire, UK	2011	−	−	−	+	+
R. lolii	6lm11	Italian ryegrass	Aberystwyth, UK	2011	−	−	−		+
R. lolii	7lm11	Italian ryegrass	Aberystwyth, UK	2011	−	−	−	+	+
R. lolii	8lm11	Italian ryegrass	Aberystwyth, UK	2011	−	−	−	+	+
R. lolii	9lm11	Italian ryegrass	Aberystwyth, UK	2011	−	−	−	+
R. lolii	10lm11	Italian ryegrass	Aberystwyth, UK	2011	−	−	−		+
R. lolii	21lm11	Italian ryegrass	Shropshire, UK	2011	−	−	−		+
R. lolii	22lm11	Italian ryegrass	Shropshire, UK	2011	−	−	−		+
R. lolii	11lp11	Perennial ryegrass	Aberystwyth, UK	2011	−	−	−
R. lolii	12lp11	Perennial ryegrass	Aberystwyth, UK	2011	−	−	−	+	+
R. lolii	13lp11	Perennial ryegrass	Aberystwyth, UK	2011	−	−	−	+	+
R. lolii	14lp11	Perennial ryegrass	Aberystwyth, UK	2011	−	−	−	+	+
R. lolii	15lp11	Perennial ryegrass	Shropshire, UK	2011	−	−	−	+	+
R. lolii	16lp11	Perennial ryegrass	Surrey, UK	2011	−	−	−
R. lolii	17lp11	Perennial ryegrass	Hertfordshire, UK	2011	−	−	−	+	+
R. lolii	18lp11	Perennial ryegrass	Hertfordshire, UK	2011	−	−	−	+	+
R. lolii	20lp11	Perennial ryegrass	Hertfordshire, UK	2011	−	−	−	+
Leptosphaeria maculans	LMA1	Oilseed rape	Unknown	Unknown	−	−	−	−	n/a
L. maculans	LMA5	Oilseed rape	Unknown	Unknown	−	−	−	−	n/a
Pyrenopeziza brassicae	PbCRA	Oilseed rape	UK	1988	−	−	−	−	n/a
P. brassicae	PbCRB	Oilseed rape	UK	1988	−	−	−	−	n/a
Sclerotinia sclerotiorum	SSGFRII	Oilseed rape	Unknown	Unknown	−	−	−	−	n/a
Fusarium graminearum	602.10	Wheat	Unknown	Unknown	−	−	−	−	n/a
F. culmorum	UK 99	Wheat	Unknown	Unknown	−	−	−	−	n/a
Host plant DNA^a	n/a^b	Barley	n/a	n/a	−	−	−	−	n/a
Host plant DNA^a	n/a	Rye	n/a	n/a	−	−	−	−	n/a
Host plant DNA^a	n/a	Cocksfoot	n/a	n/a				−	n/a

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All the diagnostic tests detected only the predicted Rhynchosporium 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 R. commune isolates; RA6-F/R produced an amplicon of 461-bp specific for R. agropyri isolates; RS25-F/R produced an amplicon of 1240-bp specific for R. secalis isolates; 2RO-F/R produced an amplicon of 277-bp specific for both R. orthosporum and R. lolii isolates; rep-PCR genomic fingerprinting using primers ERIC2/BOXA1R produced an amplicon of ∼400-bp specific for isolates of R. lolii.

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 Rhynchosporium DNA using the quantitative PCR assay of Fountaine et al. [30] (data not shown);

n/a = not applicable.

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.

For SEM, leaves were collected and placed in individual 9 cm diameter Petri dishes, which were lined with moist filter paper (Whatman No. 8) and sealed with a single layer of parafilm. Pieces of leaves, approximately 5 mm×5 mm, were cut out using a sterile razor blade. These were immediately mounted onto aluminium cryo-stubs using a smear of Tissue-Tek © O.C.T. compound (Sakura Finetek, USA) and plunged into pre-slushed liquid nitrogen (−280°C) to freeze them. The samples were then transferred under vacuum to the Alto 2100 (Gatan, UK) cryo-chamber stage, with the temperature maintained at −180°C. Sublimation of any contaminating ice and gold coating (10 nm thickness) was done in this chamber. Samples were then transferred to the stage of the scanning electron microscope (JSM 6360 LVSEM, Jeol, UK) with the temperature maintained at −150°C for examination and imaging using the on-board software (Jeol, UK).

Species-specific PCR Diagnostic Tests

Four PCR-based diagnostic tests, relying on either sequence alignments of known genetic loci (ITS and beta-tubulin) and non-coding nuclear RFLP loci (pRS52; [27]) or using RAPD-PCR derived sequence, were developed to both detect and distinguish between isolates of (i) R. commune (including two isolates from Italian ryegrass), (ii) R. agropyri, (iii) R. secalis, (iv) R. orthosporum and all other Rhynchosporium isolates obtained from ryegrasses. Primers (for all sequences see Table S2) were designed using Vector NTI software (Invitrogen, USA). All PCR reactions were set up on ice (∼0°C) and used a PTC-100 programmable thermal controller (MJ Research, USA); all testing using these diagnostic tests included no template controls. PCR products (10 µl) were separated by gel electrophoresis in 1×TBE buffer on 1% agarose gels. Gels were either stained with ethidium bromide solution as described previously in the text or incorporated with ethidium bromide (0.5 µg/ml) during preparation. Amplicons were viewed on a transilluminator and digital images recorded (Gene Genius Bio Imaging System, Syngene, Synoptics Ltd, UK).

R. commune-specific primers (LinA-F/R) were designed based on alignments of partial sequences of the ITS region and were predicted to produce a 145-bp amplicon specific only for template DNA of this species. PCR was carried out in 20 µl reaction volumes, each containing 10 µl RedTaq ReadyMix (2×concentrate, Sigma Aldrich, UK), 2 µl each of both primers (1 pmol µl⁻¹ stock), 5 µl of sterile distilled water and 1 µl of template DNA (10 ng). Reaction conditions were as follows; 35 cycles of 95°C for 1 min, 66°C for 1 min, 72°C for 1 min before a final elongation step of 72°C for 5 min, with a final hold at 4°C.

R. agropyri-specific primers (RA6-F/R) were designed based on alignments of pRS52 sequences [27] and were predicted to produce a 461-bp amplicon specific only for template DNA of this species. PCR was carried out in 25 µl reaction volumes, each containing 12.5 µl of RedTaq ReadyMix (2×concentrate, Sigma Aldrich, UK), 1 µl each of both primers (5 pmol µl⁻¹ stock), 9.5 µl of sterile distilled water and 1 µl of template DNA (10 ng). Reaction conditions were as follows; 35 cycles of 94°C for 1 min, 55°C for 2 min and 72°C for 2 min before a final elongation step of 72°C for 5 min, with a final hold at 4°C.

R. secalis-specific primers were developed as sequenced characterised amplified region (SCAR) markers [28]. An amplicon of ∼1240-bp was produced specifically for isolates of R. secalis in RAPD-PCR testing with primer OPW-05. This amplicon was purified from an agarose gel using a QiaQuick gel extraction kit (Qiagen, UK) and cloned using a Strataclone PCR cloning kit (Agilent Technologies, UK). Plasmids with inserts of the correct size were then purified from cultures using the Fermentas GeneJet plasmid prep kit (Fermentas, UK) and sent to Eurofins MWG Operon for bi-directional sequencing using an ABI 3730XL machine. Resulting sequence data were then used to develop SCAR-PCR primers by extending the original 10-bp RAPD primer sequence into the flanking regions. These R. secalis-specific primers (RS25-F/R) were predicted to produce a 1240-bp amplicon specific only for template DNA of this species. PCR was carried out in 25 µl reaction volumes, each containing 12.5 µl RedTaq ReadyMix (2×concentrate, Sigma Aldrich, UK), 1 µl each of both primers (1 pmol µl⁻¹ stock), 9.5 µl of sterile distilled water and 1 µl of template DNA (10 ng). Reaction conditions were as follows; 35 cycles of 94°C for 1 min, 57°C for 2 min, 72°C for 2 min before a final elongation step of 72°C for 5 min, with a final hold at 4°C.

Primers specific for R. orthosporum and most Rhynchosporium isolates from ryegrasses (2RO-F/R) were designed using alignments of partial sequences of the beta-tubulin region and were predicted to produce a 277-bp amplicon specific for template DNA of only these two species. PCR was carried out in 25 µl reaction volumes, each containing 12.5 µl RedTaq ReadyMix (2×concentrate), 1 µl each of both primers (1 pmol µl⁻¹ stock), 9.5 µl of sterile distilled water and 1 µl of template DNA (1 ng). Reaction conditions were as follows; 35 cycles of 94°C for 1 min, 52°C for 1 min, 72°C for 1 min before a final elongation step of 72°C for 5 min, with a final hold at 4°C.

The specificity of all four diagnostic tests was evaluated by screening them against template DNA from a collection of Rhynchosporium isolates (Table 2). Specificity of the four tests was further confirmed by screening against template DNA of other crop pathogens, including the closely related Pyrenopeziza brassicae [29], and different plant hosts (e.g. barley) by PCR with the addition of the appropriate DNA template (in all cases, 10 ng template DNA was added; Table 2). In addition, the sensitivities of the four diagnostic tests were evaluated by screening against a total of 25 ng of mixed template DNA, with different amounts of DNA of the respective Rhynchosporium species (10 ng, 5 ng, 1 ng, 100 pg, 1 pg, 0 pg) used in a background of corresponding healthy host plant DNA (15 ng, 20 ng, 24 ng, 24.9 ng, 24.99 ng, 25 ng, respectively; confirmed to be free of detectable Rhynchosporium DNA using the quantitative PCR assay of Fountaine et al. [30] (data not shown). However, in the 20 or 25 µl reaction volume, as appropriate, 2 µl of template DNA was included by reducing by 1 µl the volume of sterile distilled water. The R. commune test was evaluated using isolate UK7 [31] in a background of healthy barley (cv. Sumo) plant template DNA, the R. agropyri test evaluated using isolate 7ar10 in a background of healthy barley (cv. Sumo) plant template DNA, the R. secalis test evaluated using isolate I-1a in a background of healthy rye plant template DNA and the R. orthosporum/most isolates from ryegrasses test evaluated using isolate 57dg09 in a background of healthy cocksfoot plant template DNA.

Finally, a fifth PCR-based test, based on data obtained from the rep-PCR genomic fingerprinting, was developed to distinguish between confirmed (using primer pair 2RO-F/R) isolates of R. orthosporum and most isolates from ryegrasses. Rep-PCR genomic fingerprinting using primer pair ERIC2/BOXA1R against a collection of 71 Rhynchosporium isolates was carried out as described previously, with these primers predicted to produce an ∼400-bp amplicon for most isolates from ryegrasses but not for isolates of R. orthosporum.

Nomenclature

The electronic version of this article in Portable Document Format (PDF) is a work with an ISSN or ISBN number that will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, the new name contained in this work has been submitted to MycoBank (MB 803876) from where it will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/mb/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.

Results

DNA Fingerprint Analysis

Both RAPD-PCR (Fig. 1A) and rep-PCR (Fig. 1B) methodologies could discriminate between isolates of R. commune, R. agropyri, R. secalis and R. orthosporum obtained from proximate geographical origins. Furthermore, in both analyses two distinct types of Rhynchosporium isolates were found on ryegrasses. Two isolates (2lm11 and 5lm11) collected from Italian ryegrass clustered within the main R. commune grouping, forming a terminal clade. However, all other isolates collected from both Italian and perennial ryegrasses formed a separate grouping with strong bootstrap support values (>85% for both DNA fingerprinting methods) distinct from all the other previously described species of Rhynchosporium (Figs 1A and 1B), consistent with the presence of a novel species. This grouping was tentatively named Rhynchosporium lolii, subject to further evidence of speciation that would allow a formal description (see below). R. orthosporum was the closest sister species to R. lolii, the latter of which showed an intermixing of isolates from both Italian and perennial ryegrasses.

(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.

DNA Phylogeny and Times to the Most Recent Common Ancestor (TMRCA)

Bayesian phylogenetic analysis resulted in a tree topology with strongly supported clusters for all of the previously described Rhynchosporium species (Fig. 2). The phylogenetic analysis confirmed that two distinct Rhynchosporium species had been isolated from ryegrasses. Isolate 2lm11 was identified as R. commune, while isolates 4lm11, 7lm11, 13lp11 and 15lp11 grouped distinctly as the new R. lolii species. As with the previous DNA fingerprint analysis, R. orthosporum and R. lolii clustered as sister species, clearly separated from the three other species by a deep phylogenetic split. Estimates of TMRCA suggested a mean age of 736 years (160–1464 highest posterior density, HPD) for R. lolii and that it diverged from R. orthosporum ca. 5735 ybp (4335–7241). This TMRCA estimate for R. lolii overlaps largely with previous estimates for R. secalis (566–1922 ybp), R. agropyri (491–2023 ybp) and R. commune (775–1952 ybp) by Zaffarano et al. [12] (Figure S1).

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*. [12] 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).

Conidial Morphology

Isolates could be divided into two distinct groups on the basis of conidial shape. Isolates of R. commune (including isolates 2lm11 and 5lm11 from Italian ryegrass), R. agropyri and R. secalis had beak-shaped conidia, while isolates of R. orthosporum and the new R. lolii species group from ryegrass had cylindrically-shaped conidia (Fig. 3). No statistically significant differences in conidial length were observed between isolates of R. commune (mean = 16.80 µm, standard error of the mean = 0.611), R. agropyri (15.41 µm, 0.576) or R. secalis (15.64 µm, 0.773) (F _2,35 = 1.49, P = 0.239). In addition, no significant differences in conidial width were observed between isolates of R. commune (2.95 µm, 0.152), R. agropyri (3.30 µm, 0.143) or R. secalis (3.19 µm, 0.192) (F _2,35 = 1.48, P = 0.241). In contrast, statistically significant differences in conidial length were observed between isolates of R. orthosporum (16.41 µm, 0.611) and R. lolii (19.36 µm, 0.546); (F _1,35 = 12.09, P<0.01). However, no significant differences in conidial width were observed between isolates of R. orthosporum (2.92 µm, 0.152) and R. lolii (2.77 µm, 0.136) (F _1,35 = 0.53, P = 0.473). This differentiation in conidial morphology between the two groups, i.e. cylindrical vs. beak-shaped, was consistent with a deep phylogenetic split (see also Fig. 2).

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.