Albugo S.Str. (Albuginales; Oomycota) Is Not Restricted to Brassicales but Also Present on Fabales

Org Divers Evol (2011) 11:193–199 DOI 10.1007/s13127-011-0043-5

ORIGINAL ARTICLE

A new perspective on the evolution of white blister rusts: Albugo s.str. (Albuginales; Oomycota) is not restricted to Brassicales but also present on Fabales

Young-Joon Choi & Marco Thines & Hyeon-Dong Shin

Received: 21 July 2010 /Accepted: 28 February 2011 /Published online: 13 March 2011 # Gesellschaft für Biologische Systematik 2011

Abstract For almost all groups of pathogens, unusual and Resedaceae in the order Brassicales. In the present study, rare host species have been reported. Often, such associa- molecular phylogenetic analysis of cox2 mtDNA sequences tions are based on single or few collections only, which are and morphological investigations on an original specimen frequently hard to access. Many of them later prove to be confirmed the occurrence of a member of Albugo on due to misidentification of the host, the pathogen, or both. Fabaceae hosts, with the characteristic thin wall of the Therefore, such reports are often disregarded, or treated secondary sporangia, which is almost uniform in thickness. anecdotally in taxonomic and phylogenetic studies, regard- In phylogenetic analyses the species results as embedded less of their potential importance to unravelling the within Albugo s.str. Therefore, it is concluded that the evolution of the entire group. Concerning oomycete natural host range of Albugo s.str. extends from Brassicales biotrophs there are several reports of unusual and rare to Fabales via host jumping. Our results underscore that hosts for hardly known pathogens. In the order Fabales, for unrevised reports of pathogens from unusual hosts should example, a single species of Albugo, A. mauginii, was be reconsidered carefully to obtain a more complete picture described as parasitic to Onobrychis crista-galli about of pathogen diversity and evolution. 80 years ago, but not recorded again. All other confirmed members of Albugo s.str. are parasitic to representatives of Keywords Ancient DNA . cox2 mtDNA . Host jumping . the families Brassicaceae, Capparaceae, Cleomaceae, and Pathogen evolution . Unusual hosts

Y.-J. Choi : H.-D. Shin (*) Division of Environmental Science and Ecological Engineering, Introduction Korea University, – Seoul 136 701, South Korea Within almost all groups of plant pathogenic fungal or e-mail: [email protected] fungus-like organisms, such as rusts, smuts, mildews, and Y.-J. Choi oomycetes, some species have been reported from unusual Department of Organismic and Evolutionary Biology, hosts that do not fit the host range of the specific subgroup. Harvard University, For example, Ustilaginaceae Tul. & C. Tul. are well-known 22 Divinity Ave., Cambridge( MA 02138, USA parasites of grasses (Poaceae Barnhart), and most other Ustilaginales also are parasites of Poales s.l. However, M. Thines members of Melanopsichium Beck are parasitic to Polyg- Biodiversity and Climate Research Centre (BiK-F), onaceae Juss., even though the genus belongs to the Senckenberganlage 25, 60325 Frankfurt Main, Germany Ustilaginaceae (Begerow et al. 2006), despite its deviating morphology. Within the Microbotryaceae R.T. Moore, M. Thines which are predominantly parasites of dicotyledonous Institute of Ecology, Evolution and Diversity, Department plants, diverging hosts can be observed in Bauerago of Biological Sciences, Johann Wolfgang Goethe University, Siesmayerstr. 70, Vánky, which is parasitic to Juncaceae Juss. (Kemler et 60323 Frankfurt Main, Germany al. 2006). In downy mildews (Peronosporales) it was 194 Y.-J. Choi et al. recently discovered that Pseudoperonospora Rostovzev is scope study were heated, covered with coverslips, and not restricted to Cucurbitaceae Juss. and Rosales, but examined with brightfield and DIC light microscopy, using extends to Balsaminaceae A. Rich. (Voglmayr et al. an Olympus BX51 microscope (Olympus; Tokyo, Japan) 2009). The order Albuginales is one of two groups of for measurements and a Zeiss AX10 microscope (Carl obligate plant parasites within Oomycetes (Hudspeth et al. Zeiss; Göttingen, Germany) mainly for photographs. 2003; Riethmüller et al. 2002; Thines et al. 2008; Thines Measurements were performed at 1000× magnification for and Spring 2005). Unexpectedly, the Albuginales are a sporangia, and at 200–400× for other organs; the results are highly divergent lineage with several derived character reported in the following format: (minimum value)— states unique to oomycetes, which are distantly related to standard deviation from the minimum-mean-standard devi- the downy mildews, as well as to the other culturable ation from maximum—(maximum). To test whether the genera Phytophthora and Pythium. Over 50 species have host plant belongs to Fabaceae, morphological investiga- been described as causal agents for white blister rust tions and ITS rDNA sequence analyses were performed. disease. One family in Monocotyledoneae (Walker and Specimen examined: Albugo mauginii (Parisi) Cif. & Priest 2007) and 23 families of 12 orders in Dicotyledoneae Biga (1955), labelled ‘Cystopus candidus var. mauginii’— (Biga 1955; Choi and Priest 1995), including economically LIBYA, Cyrenaica, on Onobrychis crista-galli, 24 March important agricultural crops and common weeds, are 1925, Rosa Parisi (BPI 185277), sequences ex-type affected by these pathogens. About 80 years ago, Albugo FJ820994 (GenBank accession number) for cox2 mtDNA. mauginii (Parisi) Cif. & Biga, 1955 was first described on Onobrychis crista-galli Lam., 1779, which belongs to DNA extraction, amplification, and sequencing Fabaceae Lindl. (Parisi 1926). To our knowledge, A. mauginii has not been recorded or collected since. The DNA was extracted from sporogenous hyphae and sporan- family Albuginaceae J. Schröt. was long regarded as gia formed on the lower surface of the infected leaves for comprising a single genus, Albugo (Pers.) Roussel, but Albugo mauginii, and from uninfected leaves for the host recently it was revealed that four distinct lineages exist in plant. DNA extraction was performed according to the this family (Voglmayr and Riethmüller 2006). These methodology described in Lee and Taylor (1990). For cox2 lineages represent specific host ranges, with Albugo s.str. amplification and sequencing of the pathogen, the being parasitic to Brassicaceae Burnett, Albugo s.l. parasitic Oomycota-specific primers and the PCR conditions to Convolvulaceae Juss., Pustula Thines parasitic to described by Hudspeth et al. (2000)wereemployed, Asteridae, and Wilsoniana Thines parasitic to Caryophylli- whereas primers ITS1 and ITS4 (White et al. 1990) were dae (Thines and Spring 2005). The phylogenetic affinities used for the amplification of the nrITS region of the host to either Pustula or Wilsoniana of some species affecting plant, with an annealing temperature set to 58°C instead of Caryophyllidae were not resolved unequivocally. The 50°C. PCR products were purified using a QIAquick gel Fabales are hosts very divergent from the host families so extraction kit (Qiagen; Hilden, Germany), and sequenced far included in phylogenetic studies, although, along with on an automatic sequencer (ABI Prism TM 377 DNA Brassicales, it belongs to the rosids sensu APG III (APG Sequencer; Applied Biosystems, Foster City, CA, USA), 2009). Thus, the phylogenetic position of Albugo mauginii using the BigDye™ (Applied Biosystems; Foster City, CA, is of great interest for elucidating the overall evolution of USA) cycle sequencing kit, version 3.1. the white blister rusts. Therefore, the present study aimed at confirming the identity of the host as a member of the Phylogenetic analysis Fabales, and at investigating the phylogenetic relationships between A. mauginii and other species, based on morpho- The newly obtained sequences were edited using the logical and molecular phylogenetic analyses. DNASTAR computer package (Lasergene; Madison, WI), version 5.05. For comparison to other Albugo species, an alignment of cox2 mtDNA sequences used by Choi et al. Material and methods (2008) was obtained from TreeBASE (accession no. S2020). Alignment of the sequences was performed using Oomycete isolate CLUSTAL X (Thompson et al. 1997), which was feasible as the alignment contained only few gaps. Phylogenetic An original specimen of A. mauginii was found to be analyses were done on the resulting alignment using present in the U.S. National Fungus Collections (BPI), Maximum Likelihood (ML) and Maximum Parsimony Beltsville, MD, USA. The specimen was in good condition, (MP) methods. For ML inference, RAxML version 7.0.3 andpermissionwasgrantedforDNAextractionand (Stamatakis 2006) was used with all parameters set to sequencing of the cox2 mtDNA. Preparations for micro- default values, using the GTRMIX variant. A MP heuristic A new perspective on the evolution of white blister rusts 195 search was performed with 1000 random sequence addi- arranged according to the ‘total score’, and all of these tions and branch swapping by tree bisection-reconnection belonged to the family Fabaceae. Among them, the top 100 (TBR), using PAUP* version 4b10 (Swofford 2002). For hits included Onobrychis (97), Hedysarum (2), and Ever- both analyses, the relative robustness of the individual smannia (1). The sequence of the host plant showed the branches was estimated by bootstrapping (BS; Felsenstein highest sequence similarity (99 and 98%) to O. crista- 1985) using 1000 replicates. The resulting trees were edited galli (accession nos. GQ246076 and AB329700) sequen- with TreeView version 1.6.6 (Page 1996). The final ces obtained by Ahangarian et al. (2007). The total score alignment and trees obtained have been deposited in and BLAST E-value of these hits were 502 and 4E-139, TreeBASE (www.treebase.org/treebase-web/home.html) and 471 and 7E-130, respectively. Therefore, we deter- and are available under accession no. S11201. The ITS mine the host plant as O. crista-galli or a closely related rDNA sequence of the host plant was compared against the species belonging to the genus Onobrychis, confirming GenBank nr database (www.ncbi.nlm.nih.gov/blast/) using that this fabaceous plant is a natural host plant of the megablast algorithm and default search parameters Albuginales. (Altschul et al. 1990). Morphological analysis of the pathogen

Results In Onobrychis crista-galli infected by A. mauginii, the leaf surface had sparse yellow lesions, with pustule-like sori on Identification of the host the opposite surface. Pustules were found mostly on the lower surface, rarely on the upper surface. The sori were The vegetative morphology of the host plant matched that formed subepidermally, usually white in colour but occa- of Onobrychis Mill., 1754, but the species could not be sionally light yellow, of elongate and irregular shape, rarely determined. In the comparison with other ITS sequences confluent (Fig. 1a). Haustoria were stalked, knob-like in deposited in the NCBI database, the 500 best hits were shape, and unbranched. The grouped sporogenous hyphae

Fig. 1 Albugo mauginii on Onobrychis crista-galli.(a) Sori on infected leaves. (b, c) Sporogenous hyphae. (d) Primary sporangia. e Secondary sporangia. Scale bars=20 μm 196 Y.-J. Choi et al. were hyaline, cylindrical or clavate, straight to slightly wall in the basal and lateral portion. However, the absence curved, (21−)23.5–27.8–31.9(−37) μm long, (9−)10.8– of resting organs in the original specimen limited a 12.5–14.1(−15) μm wide (n=68), and thick-walled, espe- comparison at species level. cially towards the base (up to 4.5 μm; Fig. 1b, c). Sporangia were arranged in basipetal chains, hyaline, Phylogenetic analysis globose to subglobose, (10.5−)13.5–15.3–17.0(−20) μmin diameter (n=85); the primary sporangia were similar to the The phylogenetic relationship among the Albuginales secondary ones, although the former exhibited a slightly species was inferred from the ML and MP analyses of thicker wall (1.0–1.5 μm) than the latter (0.5–1.0 μm) partial cox2 mtDNA. Out of a total of 561 characters, 130 (Fig. 1d, e, respectively). Resting organs were not seen. were parsimony-informative; parsimony analysis resulted in The morphological characteristics of A. mauginii are 2 most parsimonious trees of 428 steps with a consistency similar to those of A. candida (Pers.) Roussel, A. hesleri Y. index (Kluge and Farris 1969) of 0.6308 and a retention J. Choi, S. Ploch & Thines, A. hohenheimia Thines, S. index (Farris 1989) of 0.8076. Since no differences were Ploch & Y.J. Choi, A. koreana Y.J. Choi, Thines & H.D. found between the tree topologies of the ML and MP Shin, A. laibachii Thines & Y.J. Choi, A. leimonios S. analyses, only the ML tree is shown in Fig. 2.The Ploch, Thines, C. Rost & Y.J. Choi, A. lepidii A.N.S. Rao, phylogenetic inference is that A. mauginii is nested within A. resedae (Jacz.) Cif. & Biga, and A. voglmayrii Y.J. Choi, Albugo s.str. from Brassicales, with significant support in both Thines & H.D. Shin within Albugo s.str. from Brassicales, analyses (99 ML BS, 100 MP BS). The species is placed as the secondary sporangia could not be clearly differenti- among the specialised species of Albugo, a large subgroup ated from primary ones and had only a slightly thickened containing A. hesleri, A. hohenheimia, A. koreana, A.

Fig. 2 Phylogenetic tree of Albuginales species inferred from ML analysis using partial cox2 mtDNA. Number terms at nodes: ML/MP bootstrap support values >50%. Branch length reflects number of nucleotide changes between respective taxa; scale bar represents number of nucleotide substitutions per site A new perspective on the evolution of white blister rusts 197 laibachii, A. leimonios, A. lepidii, A. resedae, and A. realized only recently that a high degree of genetic diversity voglmayrii, but distant from A. candida.Inthecox2-based is present among the species parasitic to Brassicaceae, and tree, A. mauginii wasplacedassistertoA. resedae from that a variety of distinct, specialised species is present on Reseda alba, with significant support in both analyses (94 ML Brassicales (Choi et al. 2006, 2007, 2008, 2011; Choi and BS, 92 MP BS). These two species clustered with Albugo sp. Thines 2010; Ploch et al. 2010; Thines et al. 2009b). It was from Diptychocarpus strictus (86 ML BS, 89 MP BS). also demonstrated that A. candida s.str. has a broad host range, infesting representatives of about 20 genera of the Brassicaceae and also some Cleomaceae Bercht. & J. Presl Discussion and Capparaceae Juss. (Choi et al. 2009). The host range of species with a confirmed placement in Albugo s.str. was From the family Fabaceae, white blister rust has been previously restricted to the four families of the Brassicales, reported only on Onobrychis crista-galli in the Libyan i.e. Brassicaceae, Capparaceae, Cleomaceae, and Reseda- province of Cyrenaica (Kranz 1965). Later, the name ceae Martinov. The present study reveals it to extend to Cystopus candida var. mauginii was introduced for the Fabales as well. It is noteworthy that A. mauginii and A. pathogen by Parisi (1926). Albugo mauginii has been given resedae are found to be sister species, as both do not occur species rank by Biga (1955), but the species was described on Brassicaceae s.l. (including Cleomaceae and Cappar- only on the basis of host preference and sporangial size, aceae). Potentially, this group has evolved effectors target- without further morphological differences to other Albugo ing conserved pathways, and is currently in the process of species. Although two plant disease lists from Libya establishing itself on the new hosts as a consequence of the (El-Buni and Rattan 1981; Kranz 1965) included the white host jump. Future studies willshow whether additional rare blister rust on Onobrychis, they only cited previous reports. species of Albugo might also belong to this group. Therefore, the BPI specimen investigated here is part of the Like other obligate biotrophic fungi (Begerow et al. sole collection of A. mauginii. Based on high ITS sequence 2006; Kemler et al. 2006), the white blister rusts evolve similarity (99%) as well as on leaf morphology, the host with their plant hosts, and eventually the two groups’ could be identified as a member of the genus Onobrychis, phylogenies are linked to some extent, showing host clade thereby confirming the parasitic relation between a species limited colonisation (Thines and Spring 2005; Voglmayr of Albuginales and a member of Fabaceae. and Riethmüller 2006). So far, three genera have been Interestingly, in our molecular phylogenetic analyses described in the Albuginales: Albugo, Pustula,and Albugo mauginii was nested within Albugo s.str., a clade Wilsoniana. The members of the latter two are parasitic to otherwise containing parasites of Brassicales. The morphol- Asteridae and Caryophyllidae, respectively, while the ogy of the species is in agreement with this, as it has only a species remaining in Albugo can be further separated into slightly thicker wall in the basal and lateral parts than in the three subgroups: Albugo s.str. parasitic to Brassicales apical part of the sporangial wall, and only slightly (Brassicaceae, Cleomaceae, Capparaceae, Resedaceae), thickened walls in the secondary sporangia (Constantinescu Albugo s.l. parasitic to Convolvulaceae, and some white and Thines 2006). With these morphological characteristics blister rust species parasitic to Caryophyllales, with it differs distinctly from two other genera of the Albugina- unresolved affinity to either Wilsoniana (Thines and Spring ceae, Pustula and Wilsoniana, and also from Albugo s.l., 2005)orPustula (Voglmayr and Riethmüller 2006). Based parasitic to Convolvulaceae, which exhibit an equatorial or on the close relationship of A. mauginii from Fabales to almost evenly distributed wall thickening, respectively. The Albugo species from Brassicales, a host-jumping event wall ornamentation of the resting organs appears to emerge from Brassicales to Fabales is the most likely explanation; as the most important character to distinguish closely codivergence with the respective host would be a less related species in the Albuginales (Choi et al. 2007, 2008; parsimonious assumption. Host jumping is not rare in Ploch et al. 2010; Thines et al. 2009b; Voglmayr and obligate parasites. It has been reported from a downy mildew, Riethmüller 2006). Unfortunately, no oospores could be Pseudoperonospora cubensis s.l. (including the possibly observed on the specimen available for study. However, in conspecific P. humuli), which is parasitic to hosts in three the phylogenetic analyses using cox2 sequences A. maugi- distantly related families: Cucurbitaceae, Cannabaceae, and nii was grouped together with the subgroup of Albugo s.str., Balsaminaceae (Choi et al. 2005; Runge et al. 2011; which includes the generalist species A. candida and the Voglmayr et al. 2009). Host jumping has also been reported specialised species A. hesleri, A. hohenheimia, A. koreana, in powdery mildews (Inuma et al. 2007; Matsuda and A. laibachii, A. leimonios, A. lepidii, A. resedae, and A. Takamatsu 2003) and rusts (van der Merwe et al. 2008; voglmayrii. On the other hand, A. mauginii was found to be Savile 1979). It can be assumed that speciation of the white distant from A. candida, which had long been regarded as blister rusts can take four different evolutionary paths. (1) the sole member of Albugo parasitic to Brassicaceae. It was Various degrees of coevolution with their plant hosts, eventu- 198 Y.-J. Choi et al. ally resulting in the pathogen’s dependence on a specific host families of flowering plants: APG III. Botanical Journal of the – genus or even a single species. This is the case in all Albugo Linnean Society, 161, 105 121. Begerow, D., Stoll, M., & Bauer, R. (2006). A phylogenetic species investigated so far, with the exception of A. candida, hypothesis of Ustilaginomycotina based on multiple gene leading to clade-limited speciation. (2) Allopatric speciation analyses and morphological data. Mycologia, 98, 906–916. by geographic isolation as in A. koreana (Choi et al. 2007) Biga, M. L. B. (1955). Review of the species of the genus Albugo – and possibly A. voglmayrii (Choi et al. 2008). (3) Sympatric based on the morphology of the conidia. Sydowia, 9, 339 358. Birch, P. R., Rehmany, A. P., Pritchard, L., Kamoun, S., & Beynon, J. speciation due to unknown niche adaptation factors, as in A. L. 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