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).