Pathogenic and genetic diversity in Puccinia hordei Otth in Australasia

Two PCR-fingerprinting primers, (GACA) 4 and M13, were tested across 22 pathotypes of Puccinia hordei Otth collected from Australasia over a 30 year period, to assess their usefulness in revealing genetic variability in this pathogen. Both primers revealed polymorphisms among the pathotypes, with (GACA) 4 generating a higher level of polymorphism. Molecular analyses revealed evidence of clonality among the P. hordei pathotypes, supporting the hypothesis that some arose from mutational changes in the pathogenicity of a founding pathogen genotype. Evidence was also obtained of sexual recombination within P. hordei in Australia on the alternate host Ornithogalum umbellatum . This is the first study of genetic variation among Australasian pathotypes of P. hordei using a PCR-fingerprinting technique.


INTRODUCTION
The fungus Puccinia hordei (Ph) belongs to the genus Puccinia, the largest genus of the order Pucciniales with 3,000 to 4,000 species (Littlefield, 1981). Ph is the casual agent of barley leaf rust, an economically important disease which affects barley production in many parts of the world (Clifford, 1985). The pathogen is present in all barley growing regions of Australia , reaching epidemic levels in Queensland during 1978, 1983, 1984and 1988(Cotterill et al., 1995. A severe epidemic of leaf rust can reduce the yield of a susceptible cultivar by up to 62% , and significant yield losses have been experienced in Australia (Cotterill et al. 1995;Cotterill et al., 1992;Waterhouse, 1927), New Zealand (Arnst et al., 1979), Europe and the USA (Griffey et al., 1994;Melville et al., 1976). Ph is a macrocyclic and heteroecious rust pathogen that forms its aecial stage on various species of Ornithogalum, Leopoldia and Dipcadi in the family Liliaceae (Clifford, 1985). Different barley genotypes with resistance genes, known collectively as a differential set, were used by Levine and Cherewick (1952) and Clifford (1977) to characterise pathotypes (pts) among different isolates of Ph. The differential set used to characterise pts of Ph at the University of Sydney, Plant Breeding Institute (PBI) comprises 30 different barley genotypes with one or more resistance (Rph) genes (Park, 2003). The first assessment of pathogenic variability in Ph in Australia *Corresponding author. E-mail: karanjeet.sandhu@sydney.edu.au. Tel: +61 2 93518821. Fax: +61 2 93518875.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License was made in 1920 by Waterhouse (1927), who detected two pts, one similar to a European pt and another that differed in virulence on some genotypes compared to a pt found in North America (Waterhouse, 1952;Watson and Butler, 1947). In a later Australian study, Cotterill et al. (1995) found substantial pathogenic variation among Ph isolates collected between 1966 and 1990. This study identified 11 different pts among 154 isolates, of which pt 210P + was the most common. Up to 1995, virulence was detected for the leaf rust resistance genes Rph1, Rph2,Rph4,Rph5,Rph6,Rph8,Rph9 and Rph12, and the genes Rph3 and Rph7 remained effective (Cotterill et al., 1995). Pathotype 4610P + virulent on Rph12 was first detected in 1991 from Tasmania, after which (1996Tasmania, after which ( to 2002, more pathogenic variation was detected in Ph including the identification of two new Rph12 virulent pathotypes (pts) with added virulence for the resistance gene Rph10 (viz. pts 5610P + and 5453P -) (Park, 2003). While no virulence was detected in these studies for genes Rph3, Rph7, Rph11, Rph14, Rph15 and Rph18 (Park, 2003), virulence for Rph3 was detected in 2009 (pt 5457P + ) in northern New South Wales (NSW) (Park, 2010). This pathotype is believed to have arisen from pt 5453P -, first detected in Western Australia in 2001 (Park, 2006), via sequential single step mutations for virulence to Rph19 (pt 5453P + ) and then Rph3 (pt 5457P + ) (Park et al., 2015;Park, 2010).
While annual surveys of pathogenic variability in rust pathogens that infect cereal crops in Australia have provided evidence that variation arises via either the introduction of exotic genotypes, simple mutation, and asexual hybridisation (Wellings and McIntosh, 1990), sexual recombination is also thought to contribute to variability in the case of P. hordei (Park, 2008;Park et al., 1995). The alternate host Ornithogalum umbellatum occurs in Australia, where it is present on the Yorke Peninsula of South Australia (SA) (Wallwork et al., 1992) and in the Murrumbidgee catchment areas including Henty and Junee in NSW. While six pts of Ph were identified among uredinial isolates derived from aeciospores collected from infected plants of O. umbellatum from the Yorke Peninsula (Wallwork et al., 1992), the contribution of sexual recombination to overall genetic variability in Ph in Australia is largely unknown.
Although information on variability obtained from pathogenicity on differential genotypes is important in the genetic control of rusts, it is of limited use in assessing genetic variation in these pathogens. Both biochemical and molecular markers have been applied to evaluate genetic diversity among various plant pathogens (McDermott and McDonald, 1993). Amplified fragment length polymorphism (AFLP) analyses were used to study genetic diversity among isolates of Ph in relation to their virulence (Sun et al., 2007). This study revealed an association between molecular diversity and virulence patterns in Ph isolates collected from different geographical regions of the world. Keiper et al. (2003) studied the genetic structure of several cereal rust pathogens using various polymerase chain reaction (PCR) based tools like AFLP, selectively amplified microsatellites (SAM) and sequence-specific amplification polymorphisms (S-SAP). This study was able to discriminate fungal pathogens from five rust taxa [P. triticina (Pt), P. graminis f. sp. tritici (Pgt), P. striiformis f. sp. tritici (Pst), barley grass stripe rust caused by P. striiformis f. sp. pseudohordei (Psph) and P. graminis f. sp. avenae (Pga)], although the level of polymorphism observed within individual taxa was low. In a separate study that used AFLPs and random amplified polymorphic DNA (RAPDs), Steele et al. (2001) found no polymorphism among Australian and New Zealand isolates of Pst. However, the same AFLP primers showed five to 15% polymorphic fragments among isolates of Pst from the UK, Denmark and Colombia. These results were consistent with clonality in Australian populations of Pst. Microsatellites, or simple sequence repeats (SSRs) have also been developed and applied to study polymorphisim among different rust pathogens (Dambroski and Carson, 2008;Kolmer et al., 2011;Ordoñez et al., 2010;Mantovani et al., 2010;Keiper et al. 2006;Visser et al., 2011;Karaoglu and Park, 2014).
Another useful tool for assessing genetic diversity is "PCR-fingerprinting". This technique uses microsatellites (GACA) 4 and (GTG) 5 and the minisatellite M13 derived from the core sequence of the wild type phase M13 bacterium, as single primers in PCR to amplify hypervariable DNA sequences (Meyer et al., 2001). The PCR-fingerprinting technique has been used successfully to reveal polymorphism among various fungal and bacterial pathogens. For example, Vuyst et al. (2008) used (GTG) 5 to identify acetic acid bacteria in cocoa beans and the primers GTG, GACA and M13 were used to study population dynamics in several human pathogens (Cogliati et al., 2007;Delhaes et al., 2008;Meyer et al., 2001;Roque et al., 2006;Trilles et al., 2008). Selective amplification of the microsatellite polymorphic loci (SAMPL) markers (GACA) 4 + H-G and R1 + H-G were used to study polymorphism among 44 (25 Australasian and 19 European) isolates of Phragmidium violaceum (causal agent of blackberry rust), revealing more diversity in European isolates than in Australasian isolates, with 37 and 22% polymorphic loci, respectively (Gomez et al., 2006). In all of these studies, the primers GACA and M13 generated the most discriminating and informative DNA profiles. Efforts have been made for the first time to study genetic variation in Australasian populations of Ph using PCR-fingerprinting profiles with primers (GACA) 4 and M13.

Isolates of pathogens and DNA extraction
A total of 22 pts of Ph, comprising 20 from Australia and two from New Zealand, along with isolates of five control pathogens (Pt, Pgt, Pst, Psph and Pga) were included in this study (Table 1). All the pts used in the study were sourced from the rust collection maintained in liquid nitrogen at PBI, University of Sydney. The Ph pts used were selected to represent those identified in different regions within Australia and New Zealand in annual pathogenicity surveys conducted from 1980 to 2009. Freshly collected urediniospores were desiccated over silica for 12 h. A sample of 25 to 30 mg of urediniospores of each rust isolate was put in labelled Lysing Matrix C tubes (Impact resistant tubes with 1.0 mm silica spheres, Mp Biomedical, Ohio, USA). One milliliter of 2x Cetyl-trimethylammonium bromide (CTAB) extraction buffer [(CTAB 2% (w v -1 ), 20 mM EDTA (pH 8.0), 1.4 M NaCl, Polyvinylpyrrolidone (PVP; 40000 MW) 1% (w v -1 ), 100 mM Tris-HCl (pH 8.0) and ddH2O (double distilled autoclaved water)] was added to each sample, mixed well by inversion and tubes were submerged in ice for 2 min. Tubes were then shaken for 15 s on a FastPrep® Cell Distrupter (M.P. Biomedicals, Irvine, CA, USA) at speed 6, returned to ice for 3 min and shaken again for 20 s at the same speed. Tubes were kept in a pre-warmed water bath at 65˚C for 30 min and inverted every 10 min, after which they were removed, mixed well by inversion and the solution in each tube/sample was divided (~500 µl in each tube) into two new 1.5 ml Eppendorf tubes to generate duplicate extractions. DNA extraction was carried in a fume hood by adding ~ 250 µl of cold phenol, followed by ~ 250 µl of cold chloroform: isoamyl alcohol (24:1 v v -1 ), to each tube. Samples were mixed gently by inverting (~ 100 times) the tubes until a thick emulsion formed. Tubes were centrifuged at 13,000 rpm for 15 min and the supernatant was transferred into sterile 1.5 ml Eppendorf tubes. The process of phenol and chloroform: isoamyl alcohol extraction was repeated. About 50 µl of 3 M NaOAc and ~ 500 µl of cold isopropanol were added to each tube and tubes were then stored at -20˚C. The following day, the tubes were centrifuged at 13,000 rpm for 30 min and the DNA pellet thus formed was drained carefully. The pellets were washed with 500 µl of ethanol (70% v v -1 ), centrifuged at 13,000 rpm for 15 min, drained carefully and allowed to air dry. The dried pellet was resuspended in 100 µl ddH2O and stored overnight at 4˚C. The following day, 5 µl of Rnase-A (10 μg μl -1 ) was added to each tube and incubated at 37˚C for 2 h. All DNA samples were quantified using a Nanodrop ND-1000 spectrophotometer (Nanodrop® Technologies) and diluted to working dilution of 10 ng µl -1 using ddH2O.
PCR reactions were performed in a final volume of 50 µl which contained 3.0 µl of genomic DNA (10 ng μl -1 ), 5.0 µl of dNTPs (0.2 mM), 5.0 µl of 10x PCR buffer (NH4 Reaction buffer, Bioline), 3.0 µl of 50 mM MgCl2 (Bioline), 5.0 µl of primer (2 mM), 0.5 µl (5 u μl -1 ) of Taq DNA (Immolase DNA polymerase from Bioline) and 28.5 µl of ddH2O. PCR amplification profile comprised of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 30 s denaturation at 94°C, 60 s annealing at 47°C if M13 or at 40°C if (GACA)4 primer was used, 30 s extension at 72°C and a final extension of 7 min at 72°C. Reactions were performed in a 96-well DNA theromocycler (Eppendorf Mastercycler, Germany). PCR products were concentrated to 30 µl by placing in a fan forced oven for 45 min at 65°C and resolved on 2% high resolution agarose (MetaPhor® Agarose, Lonza, Rockland Inc.USA) gels at 80 V electrophoresis for 6 h. Five kilobite DNA marker HyperLadder™ III (Bioline) was used as reference. The separated fragments were visualised under an ultra violet light unit fitted with a GelDoc-IT UVP Camera (Bio-rad, Australia Pty. Ltd. Gladesville NSW).

Data analyses
Gel images were scored and analysed using the software GelCompar II (6 th edition, Applied Maths, Belgium). Fragment position optimisation and tolerance was set to 1 and 1.5%, respectively. Fragments were selected automatically by the GelCompar and unclear fragments were deselected manually. Based on the standard DNA ladder used, molecular weights of selected fragments were assigned automatically. Fragment scoring for the both primers ranged from 500 to 2500 bp. Genetic diversity among the Ph pts examined was evaluated using Unweighted pair group method for arithmetic averages (UPGMA) cluster analyses based on a distance matrix calculated using the Dice coefficient of similarity. The quality of similarity clusters was tested using the cluster validity index Cophenetic correlation coefficient (CPCC) using software GelCompar II. The CPCC was used to test the efficiency of the similarity clusters that resulted from the individual analyses of markers M13 and (GACA)4. The CPCC is a simple correlation coefficient between the original dissimilarity matrix and the final dissimilarity matrix (Cophenetic matrix) produced after the clustering algorithm recalculates the dissimilarities (Lessig, 1972). Dendrograms were constructed and based on similarity clusters of both primers (GACA)4 and M13, the Ph pts were clustered accordingly.

RESULTS
Both oligonucleotides (GACA) 4 and M13 amplified all pts, producing fragments in the range of 500 to 2500 bp. After deselecting unclear fragments manually, a total 27 and 28 fragments were scored automatically for markers (GACA) 4 and M13, respectively (Table 3). The UPGMA similarity dendrograms produced from the cluster analyses based on markers (GACA) 4 and M13 data grouped all 22 Ph pts and control pathogens (Figures 1  and 2). Both primers (GACA) 4 ( Figure 1) and M13 ( Figure  2) out-grouped representative control isolates of Pt, Pgt, Pst, Psph and Pga from the Ph pts examined. Both fingerprinting primers produced distinct clades for Pst and Psph, Pgt and Pga, while Pt was in a standalone group (Figures 1 and 2).
Cluster analysis based on marker M13 produced seven groups among the Ph pts with 75.9% to 100% similarities (Figure 2), while marker (GACA) 4 revealed higher variability among the Ph pts and produced 10 different groups with 70.5 to 100% similarities (Figure 1). Markers clustered pts 211P and 231P + together (Figures 1 and 2), both of which originated from New Zealand.

DISCUSSION
The evolution of new virulent pts of Ph is a significant constraint in the economical production of barley in Australia and worldwide. Understanding genetic diversity in Ph is fundamental in the efforts to develop cultivars of barley with resistance to this pathogen. For example, genetically diverse fungal pathogens may have a greater potential to evolve new pts with the ability to overcome resistance. In earlier work, six pts of Ph were identified from aeciospores collected from infected plants of O. umbellatum in SA (Wallwork et al., 1992). Furthermore, high diversities of Ph pts have been reported in SA in pathogenicity surveys, suggesting that sexual recombination is contributing to pathogen diversity (Park, 2010).  ,Rph2,Rph3,Rph4,Rph6,Rph9,Rph10,Rph12,Rph19 Isolate: Isolate ID as given in Table 1; MGP Groups of P. hordei pathotypes based on M13 analysis; GGP Groups of P. hordei pathotypes based on GACA analysis; *with respect to the resistance genes listed in Park (2003), virulence to Rph genes shown in last column is corresponding to the pathotypes shown in the previous column.

Rph1
Prior to the current study, no attempt had been made to study the genetic diversity of Ph in Australia, using PCRfingerprinting. The usefulness of the PCR-fingerprinting primers M13 and GACA in discriminating fugal pathogens has been shown in several studies (Cogliati et al., 2007;Delhaes et al., 2008;Meyer et al., 2001;Roque et al., 2006;Trilles et al., 2008). In view of this, PCRfingerprinting primers M13 and (GACA) 4 , were assessed for their utility in Ph.
Cluster analyses of marker data revealed seven to 10 clusters among the 22 Ph pts and both markers outgrouped the control pathogens. As expected, a high percentage of similarity was observed among the Ph clusters, whereas the control pathogens were more diverse. Both PCR-fingerprinting primers (GACA) 4 and M13 clearly differentiated Pt, Pgt, Pst, Psph, Pga from each other and from the pts of Ph. Markers M13 and (GACA) 4 revealed only 26.4 and 33.3% genetic similarities between Ph and the control rust pts. These findings are in accordance with earlier studies in which isolates of Pgt were clearly differentiated from isolates of Ph using AFLP markers (Sun et al., 2007).
Both markers distinguished Pst and Psph with 57.1 to 83.3% genetic similarities, which is in accordance with an earlier study of these rust pathogens by Keiper et al. (2003) in which Pst and Psph were distinct but more similar compared to other rust pathogen species. Both markers M13 and (GACA) 4 formed distinct clades of Pga and Pgt and differentiated these two from the wheat rust pathogens Pst and Pt, also consistent with earlier results of an AFLP study on these rust pathogens . The current results support the informative value and usefulness of the PCR-fingerprinting markers in differentiating species of rust pathogens.
The PCR-fingerprinting primer M13 clustered the 22 Ph pts into seven groups, while the marker (GACA) 4 resolved 10 groups among the Ph pts (Table 2) and detected more polymorphism. Interestingly, both markers grouped Ph pts 211P-and 231P + with 100% similarity ( GGP7 and MGP7 , Table 2) and differentiated them from all other Ph pts. Both pts originated from New Zealand and differ only in virulence on Rph2, Rph5 and Rph19. It is therefore possible that these two pts are simply related and their distinctiveness from the Australian pts indicates that Ph populations in the two countries are distinct. This contrasts with results from long-term surveys of pathogenic variability in wheat rust pathogens across Australia and New Zealand, which have provided Table 3. GelCompar selected fragments accross the amplifications produced by PCR-fingerprinting markers (GACA)4 and M13 where unclear fragments were deselected manually. substantial evidence of rust migration between the two land masses (Luig, 1985). These studies have also provided evidence that wheat rust movement is predominantly from west to east (Luig, 1985;. In view of this, the distinctiveness of the two pts of Ph from New Zealand from those in Australia suggests that they may have originated from a region outside Australasia and that they have remained localized to New Zealand. Based on pathogenicity, Cotterill et al. (1995) suggested that the appearance of a group of pts distinct from pt 243P and typified by pt 200P and its subsequent singlestep mutations in the form of pts 201P -, 210P and 220P in the 1980s, may have resulted from an exotic incursion. The present results support this hypothesis. Studies of pathogenic variability in all three wheat rust pathogens in Australia have provided strong evidence of clonality, with presumed clonal lineages comprising closely related pts derived by sequential single-step mutations from a common ancestor (Keiper et al., 2006). In contrast, pts of Ph detected in Australia between 1992 and 2001 did not appear to be so simply related based on pathogenicity (Park, 2003). Of the pts examined in the present study, pt 5457P + is believed to have originated from pt 5453P via step-wise mutation for virulence for Rph19 and then for Rph3 (Park, unpublished). Surprisingly, while markers (GACA) 4 and M13 grouped these two pts and separated them from all other pts, they were not identical (Figures 1 and 2, respectively). These results show that the relationship between these two pts is not as simple as thought.
The molecular analyses in the present study did, however, provide some evidence of clonal lineages in Ph in Australasia. Marker (GACA) 4 revealed pts 201P + and 201P to be 100% genetically similar ( Figure 1) and given that pt 201P + differs from 201P only in being virulent for Rph19, together these results are consistent with pt 201P + arising via a single step mutation in pt 201P with   Table 1. added virulence for Rph19. The lack of molecular variation among some of the pts studied support the hypothesis of single-step mutation being an important source of pathogenic variation in Ph, which is consistent with the results published by Steele et al. (2001) who found a similar situation among Australian isolates of Pst. Marker (GACA) 4 revealed more informative fragments compared to the M13. So PCR-fingerprinting technique using marker (GACA) 4 can be a very efficient and an effective tool to find genetic variations in Ph and other rust pts.