- Email: [email protected]
Three new phylogenetic lineages are the closest relatives of the widespread species Albugo candida
f u n g a l b i o l o g y 1 1 5 ( 2 0 1 1 ) 5 9 8 e6 0 7
journal homepage: www.elsevier.com/locate/funbio
Three new phylogenetic lineages are the closest relatives of the widespread species Albugo candida Young-Joon CHOIa, Hyeon-Dong SHINb, Sebastian PLOCHc, Marco THINESc,d,* a
Harvard University, Department of Organismic and Evolutionary Biology, 22 Divinity Ave., Cambridge, MA 02138, USA Korea University, Division of Environmental Science and Ecological Engineering, Seoul 136-701, Republic of Korea c Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt (Main), Germany d Johann Wolfgang Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Siesmayerstr. 70, D-60323 Frankfurt (Main), Germany b
article info
abstract
Article history:
White blister rust caused by the obligate biotroph Albugo candida (Albuginaceae; Oomycota) is
Received 8 October 2010
one of the most notorious and common diseases of Brassicaceae. During the past 5 y, A. can-
Received in revised form
dida specimens collected from about 30 host genera were phylogenetically and morpholog-
29 January 2011
ically investigated in several studies. These not only revealed that A. candida s.str. has
Accepted 8 February 2011
a broad host range, encompassing a large number of host plants belonging to Brassicales,
Available online 15 February 2011
but also the presence of previously overlooked species of Albugo with hosts in this order.
Corresponding Editor:
In this study, we examined specimens from Alyssum, Barbarea, and Rorippa, of which
Hermann Voglmayr
many species were commonly recorded as host plants of A. candida but could not be included in previous works due to the paucity of specimens available. It was revealed that
Key words:
Albugo specimens from Alyssum montanum, Barbarea vulgaris, and various Rorippa species,
Albuginales
were placed in three phylogenetically distinct clades, but closer to A. candida s.str. than
Broad species concept
any previously reported species. Oospores were observed from Albugo specimens parasitic
Host specificity
to Rorippa and could be distinguished morphologically from A. candida. Therefore, Albugo
Hyaloperonospora
rorippae sp. nov. is described and illustrated here. In addition, a key of Albugo species de-
Morphology
scribed previously from Brassicales is given. The present study reveals that a large number
Obligate biotroph
of Albugo species remain still undiscovered, and that species close to A. candida exist. This
Species key
could help elucidating the basis of the broad host range of A. candida as opposed to the nar-
White blister rust
row specialisation that is seemingly present in other species of Albugo on the Brassicaceae. ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction The Albuginaceae are obligate biotrophic oomycetes, responsible for white blister rust (WBR) disease on various angiosperms. On the Brassicaceae, WBR is probably the most common disease caused by biotrophic pathogens, along with downy mildew disease caused by members of the Peronosporaceae. During the past decades, the generalist Albugo candida (Pers.) Roussel has been
regarded as the causal agent of all incidents of WBR on Brassicaceae, mainly based on the similarity of morphological traits, which led to the application of a broad species concept, similar to downy mildews (Voglmayr 2008 and references cited therein). Recent molecular studies (Choi et al. 2006; Voglmayr € ller 2006) have supported not only its wide host & Riethmu range, but also the high degree of genetic diversity within Albugo parasitic to Brassicaceae. Subsequently, six new specialised
* Corresponding author. Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt (Main), Germany. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.02.006
Three new phylogenetic lineages close to Albugo candida
species have been introduced, Albugo koreana from Capsella bursa-pastoris (Choi et al. 2007), Albugo laibachii from Arabidopsis thaliana (Thines et al. 2009), Albugo voglmayrii from Draba nemorosa (Choi et al. 2008), Albugo hesleri, Albugo hohenheimia, and Albugo leimonios from species of the genus Cardamine (Ploch et al. 2010). In addition, Albugo lepidii (Choi et al. 2007) and Albugo resedae (Choi & Thines, in press) were confirmed as species distinct from A. candida, while Albugo chardonii from Cleomaceae was revealed to be synonymous with A. candida (Choi et al. 2007, 2009). Judging from the studies listed above, it is believed that more than a dozen distinct species so far regarded to be A. candida await discovery (Thines & Voglmayr 2009). On the other hand, the phylogenetic results also demonstrated that the host range of A. candida s.str. extends from Brassicaceae to Cleomaceae and Capparaceae (Choi et al. 2007, 2009), but possibly not to Resedaceae (Choi & Thines, in press). Only about half of about 60 brassicaceous genera which were originally recorded as host plants of A. candida (Biga 1955; Saharan & Verma 1992) have been examined during previous phylogenetic studies (Choi et al. 2006, 2007, 2008, 2009; Choi & € ller 2006; Thines et al. Thines, in press; Voglmayr & Riethmu 2009; Petkowski et al. 2010; Ploch et al. 2010), leaving Albugo on about 30 host genera unexamined. Albugo candida has previously been found deeply separated from the species recently confirmed (Choi et al. 2007; Choi & Thines, in press) and newly discovered (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010), and only recently a few specimens with close affinity to A. candida have been revealed (Ploch et al. 2010). However, based on the limited sampling from the respective host genera, Armoracia and Rapistrum, the taxonomic placement of these specimens remained unresolved. The three genera, Alyssum, Barbarea, and Rorippa, are widely distributed and of these, about 50 species have been recorded as common host plants of A. candida in monographic studies (Wilson 1907; Biga 1955; Kochman & Majewski 1970; Constantinescu & Negrean 1983; Stanyavichene 1984; Novotel’nova & Pystina 1985; Ul’yanishchev et al. 1985; Vanev et al. 1993); see also pathogenehost distribution database
599
of Farr & Rossman (2010). However, despite of their wide distribution, Albugo specimens from these genera have so far not been included in molecular phylogenetic investigations. As it was previously shown that the WBRs from the widespread genus Cardamine harbour at least three distinct species (Ploch et al. 2010), it was the aim of this study to investigate specimens from additional widespread genera for the presence of previously overlooked species.
Materials and methods Oomycete specimens Seventeen Albugo specimens on Armoracia, Alyssum, Barbarea, and Rorippa were morphologically and phylogenetically investigated; Armoracia rusticana (1), Alyssum caliacrae (1), Alyssum montanum (1), Barbarea vulgaris (3), Rorippa palustris (4), Rorippa sylvestris (3), Rorippa amphibia (2), Rorippa pyrenaica (1), and Rorippa sp. (1). For comparison, nine Albugo species described previously from the Brassicales were also included, Albugo candida, Albugo hesleri, Albugo hohenheimia, Albugo leimonios, Albugo lepidii, Albugo laibachii, Albugo koreana, Albugo resedae, and Albugo voglmayrii, with four undetermined lineages from the four host genera, Armoracia, Rapistrum, Descurainia, and Diptychocarpus. Both cox2 mtDNA and ITS rDNA sequences of several Albugo species from two previous studies (Ploch et al. 2010; Choi & Thines, in press) were retrieved from GenBank. Oomycete specimens examined in this study are listed in Table 1. Herbaria abbreviations are those from Thiers (2010). Morphological analysis was conducted as described previously (Choi et al. 2008).
Molecular analysis Genomic DNA from infected leaves of herbarium specimens was extracted using the DNeasy Plant Mini kit (Qiagen, Valencia, CA) or the Analyticjena plant DNA extraction kit as
Table 1 e Information on Albuginaceae specimens investigated in this study. Pathogen
Host
Geographic origin, collection year (Herb. no.)
GenBank Acc. No. (cox2/ITS)
Albugo sp.
Alyssum caliacrae Alyssum montanum Armoracia rusticana Barbarea vulgaris Barbarea vulgaris Barbarea vulgaris Rorippa amphibia Rorippa amphibia Rorippa palustris Rorippa palustris Rorippa palustris Rorippa palustris Rorippa pyrenaica Rorippa sylvestris Rorippa sylvestris Rorippa sylvestris Rorippa sp.
Romania, 1979 (BPI199995) Poland, 1896 (KRAM-F-000149) Germany, 1890 (BP263) USA, 1925 (BPI184667) Ireland, 1943 (BPI184670) Germany, 1992 (BPI748543) Romania, 1975 (FH HMR2610)a Romania, 1975 (BP75207) USA, 1952 (BPI185090)a USA, 1909 (BPI185091)a Australia 1992 (DAR72185) Poland, 1884 (KRAM-F-000130)a Romania, 1972 (FH HMR2197)a Poland, 1896 (KRAM-F-000138)a Poland, 1884 (KRAM-F-000134)a Romania, 1975 (FH HMR2611)a Argentina, 1987 (RD0129)b
HQ377356/HQ377366 HQ377357/ /HQ832468 HQ377358/HQ377367 HQ377359/ HQ377360/HQ377368 / HQ377361/HQ377369 / / HQ377362/HQ377370 HQ377364/HQ377371 / HQ377363/ / / HQ377365/
Albugo rorippae
a The specimens that oospores were successfully observed. b Acronym: RD, R. Delhey (private collections) in the Phytopathology Lab of Bahıa Blanca, Argentina.
600
recommended in Telle & Thines (2008). The freshly collected material was extracted using a protocol modified from May & Ristaino (2004) as described in Thines & Spring (2005). PCR amplifications of cox2 mtDNA and ITS rDNA regions were performed as outlined in Choi et al. (2006). Semi-nested reamplification of poorly amplified samples was done with the internal primer ITS1-O (Bachofer 2004) and LR0 (reverse complement to Moncalvo et al. 1995). PCR products were sequenced by two commercial sequencing companies, Macrogene (Seoul, Korea) and GATC Biotech (Konstanz, Germany) with the primers used for the PCR reactions.
Alignment and phylogenetic reconstruction The sequences were edited with the DNASTAR computer package (Lasergene, Madison, WI), version 8. Alignment of the sequences was performed using MegAlign (DNAStar) for the unambiguously alignable cox2 region and Mafft, version 6.0 (Katoh et al. 2005), was used with the G-INS-i strategy for ITS sequences. No manual “improvements” or “corrections” to the alignment were done in either case. Phylogenetic trees were obtained from the data using minimum evolution (ME), maximum likelihood (ML), and maximum parsimony (MP). An ME inference was performed in MEGA, version 4.0 (Tamura et al. 2007), with Kimura 2-parameter model. For ML inference, RAxML version 7.0.3 (Stamatakis 2006) was used with all parameters set to default values, using the GTRCAT variant. An MP heuristic search was performed with 1 K random sequence additions and branch swapping by tree bisection-reconnection (TBR), using PAUP version 4b10 (Swofford 2002). The relative robustness of the groupings and individual branches was estimated by bootstrapping (BS) using 10 K replicates, each with ten rounds of heuristic searches with TBR branch swapping on trees generated by random sequence addition. The sequence of Albugo ipomoeae-panduratae was used as outgroup, according to a study of Choi et al. (2009). The newly derived sequences were deposited in GenBank under accession numbers HQ377356eHQ377365 (cox2 mtDNA) and HQ377366eHQ377371, HQ832468 (ITS rDNA), the alignments in TreeBASE (www.treebase.org/treebase-web/home.html) under accession no. S10914, and a taxonomic novelty in MycoBank (www.MycoBank.org).
Results Molecular phylogenetic analysis The cox2 mtDNA alignment of 566 bp revealed no gaps for the genus Albugo s.str., while a gap spanning six base-pairs was observed for Albugo infecting Convolvulaceae. The ITS rDNA alignment was 857 bp in length and includes partial ITS1, complete 5.8S rDNA and partial ITS2 sequences. All unavailable data were assumed as missing from the alignment. The phylogenetic relationships between Albugo specimens were inferred from ME, ML, and MP analyses of the aligned sequences. In the cox2 mtDNA alignment, 97 characters were parsimony-informative, and the MP analysis resulted in the 44 most parsimonious trees of 277 steps, with a CI and RI of 0.7967 and 0.8862, respectively. For the ITS rDNA
Y. -J. Choi et al.
alignment, 215 characters were parsimony-informative, and the parsimony analysis produced 238 most parsimonious trees of 402 steps, with a CI and RI of 0.8383 and 0.9385, respectively. ML search in RAxML v7.0.3 using the GTRCAT variant model of sequence evolution resulted in one ML tree each with a ln L ¼ 2166.4926 and a ln L ¼ 3936.9241 for cox2 and ITS, respectively. The results of the phylogenetic reconstructions for cox2 and ITS, respectively are shown in Figs 1 and 2, respectively. A similar topology was observed in ME, ML, and MP analyses, and no highly supported, conflicting alternative topologies were inferred. Therefore, only the tree based on the ME analysis is presented, with the addition of the support values of the ML and MP analyses. Based on the cox2 mtDNA sequences (Fig 1), all phylogenetic analyses resulted in two major clades. One consists of Albugo candida s.str. from a broad range of Brassicaceae, Capparaceae, and Cleomaceae, and five closely related, yet phylogenetically distinct lineages. The second clade includes the alreadydescribed WBR specialists, Albugo hesleri, Albugo hohenheimia, Albugo koreana, Albugo laibachii, Albugo leimonios, Albugo lepidii, Albugo voglmayrii (Brassicaceae), and Albugo resedae (Resedaceae), with three undescribed species. The five phylogenetic lineages close to A. candida s.str. represent five specimens from Rorippa species, three from Barbarea vulgaris, two from Armoracia rusticana, one from Alyssum montanum, and one from Rapistrum rugosum. Among them, the Albugo specimens from Rorippa formed a monophyletic group in all three analyses (ME BS/ML BS/MP BS of 87/88/71 %), with no sequence difference among them, regardless of the different host species and geographic origins. This lineage further grouped with the specimens from Armoracia, with high to maximum BS (100/96/92 %). A well-supported group consisting of the three specimens from B. vulgaris was sister to the clade including A. candida s.str and three lineages from Armoracia, Rorippa, and Rapistrum, with high BS values of 99/90/98 %. Despite of the fact that the B. vulgaris specimens were collected in geographically distant countries, Germany, Ireland, and the USA, no sequence divergence was found. Interestingly, two specimens from Alyssum were divided into two distant branches; one from A. montanum was placed basal to A. candida s.str. and allied lineages, while the other from Alyssum caliacrae was placed in the second group including many specialised species. In the second clade, the distinctiveness of the previously described WBR specialists was confirmed, with high support in all analyses. This clade included, apart from the undescribed species specialised on A. caliacrae mentioned previously, two additional undescribed species from Descurainia and Diptychocarpus. The ITS rDNAbased analyses (Fig 2) resulted in a similar topology compared to the cox2 region, without highly supported conflicts. In contrast to the cox2 tree, a well-supported sister-group relationship of specimens from Barbarea (100/94/85 %) with those from Armoracia and Rorippa was inferred. Albugo sp. from A. caliacrae, which showed no affinity with other specialised Albugo species in cox2 analyses, clustered together with A. laibachii and Albugo sp. from Descurainia with moderate to high bootstrap support (86/95/91 %). In cox2 mtDNA, Albugo sp. on Rorippa shared a high level of sequence similarity with Albugo on A. rusticana as they differed at only two positions among 566 nucleotide characters
Three new phylogenetic lineages close to Albugo candida
601
A. candida (ex Arabis turrita) AY913803 A. candida (ex Capparis spinosa) EF655654 A. candida (ex Capsella bursa-pastoris) DQ643944 A. candida (ex Thlaspi arvense) AY913809 A. candida (ex Diplotaxis erucoides) DQ418517 64/85/63 A. candida (ex Raphanus sativus) AY927059 A. candida (ex Aubrietia deltoidea) DQ418500 A. candida (ex Biscutella laevigata) DQ418506 A. candida (ex Lunaria sp.) AY913797 A. candida (ex Iberis amara) DQ418522 A. candida (ex Berteroa incana) DQ418508 A. candida (ex Heliophila meyeri) DQ418515 A. candida (ex Erysimum cuspidatum) DQ418519 A. candida (ex Cleome anomala) AY913799 A. candida (ex Arabidopsis halleri) DQ418513 A. candida (ex Arabidopsis thaliana) FJ468361 100/96/95 A. candida (ex Brassica juncea) AY927046 A. candida (ex Draba verna) EU340140 73/87/73 A. candida (ex Sisymbrium luteum) AY913808 A. candida (ex Eruca sativa) DQ418514 Albugo sp. (ex Rapistrum rugosum) GU292101 44/58/52 89/93/83 Albugo sp. (ex Armoracia rusticana) GU292084 Albugo sp. (ex Armoracia rusticana) GU292087 100/96/92
Albugo sp. (ex Rorippa sylvestris) HQ377363 Albugo sp. (ex Rorippa palustris) HQ377364 100/90/98 Albugo sp. (ex Rorippa sp.) HQ377365 87/88/71 Albugo sp. (ex Rorippa amphibia) HQ377361 Albugo sp. (ex Rorippa palustris) HQ377362 74/-/72 Albugo sp. (ex Barbarea vulgaris) HQ377358 99/100/99 Albugo sp. (ex Barbarea vulgaris) HQ377359 Albugo sp. (ex Barbarea vulgaris) HQ377360 Albugo sp. (ex Alyssum montanum) HQ377357 A. resedae (ex Reseda alba) GU945758 99/100/98 96/93/84 A. resedae (ex Reseda alba) GU945759 Albugo sp. (ex Diptychocarpus strictus) AY927052 99/100/99 A. lepidii (ex Lepidium campestre) AY927054
A. lepidii (ex Lepidium virginicum) AY927057 Albugo sp. (ex Alyssum caliacrae) HQ377356 A. laibachii (ex Arabidopsis thaliana) FJ468371 Albugo sp. (ex Descurainia sophia) AY927051 99/100/100 A. hohenheimia(ex Cardamine hirsuta) GU292114 A. hohenheimia(ex Cardamine hirsuta) GU292118 99/100/100 59/97/60
99/100/100 99/100/100
A. voglmayrii (ex Draba nemorosa) EU240588 A. voglmayrii (ex Draba nemorosa) EU240590
A. leimonios (ex Cardamine pratensis) GU292124 A. leimonios (ex Cardamine pratensis) GU292121
A. hesleri (ex Cardamine diphylla) GU292091 A. hesleri (ex Cardamine diphylla) GU292107
99/100/100
A. koreana (ex Capsella bursa-pastoris) AY927049 A. koreana (ex Capsella bursa-pastoris) DQ643944 A. ipomoeae-pandurataeAY913804
0.02
Fig 1 e Minimum evolution tree of Albugo s.str. based on cox2 mtDNA sequences. The numbers above or below the branches indicate bootstrap support in ME, ML, and MP analyses. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitutions per site. The relevant GenBank accession numbers are indicated following the taxon names. Albugo specimens sequenced in this study are in bold.
602
Y. -J. Choi et al.
A. candida (ex Capsella bursa-pastoris) DQ643916 A. candida (ex Lunariasp.) AY929840 A. candida (ex Heliophila meyeri) DQ418493 A. candida (ex Eruca sativa) DQ418503 A. candida (ex Arabidopsis halleri) DQ418502 A. candida (ex Thlaspi arvense) AY929847 A. candida (ex Sisymbrium leteum) AY929844 A. candida (ex Iberis amara) DQ418499 A. candida (ex Arabidopsis thaliana) FJ468362 A. candida (ex Brassica juncea) AY929826 A. candida (ex Arabis turrita) AY929825 A. candida (ex Aubrietia deltoidea) DQ418500 69/80/56
A. candida (ex Berteroa incana) DQ418495 A. candida (ex Raphanus sativus) AY929841 A. candida (ex Biscutella laevigata) DQ418494
100/95/100
A. candida (ex Diplotaxis erucoides) DQ418496 A. candida (ex Erysimum cuspidatum) DQ418498
52/-/-
Albugo sp. (ex Rapistrum rugosum) GU292141 Albugo sp. (ex Barbarea vulgaris) HQ377367 Albugo sp. (ex Barbarea vulgaris) HQ377368 Albugo sp. (ex Armoracia rusticana) HQ832468 100/94/85 Albugo sp. (ex Rorippa palustris) HQ377371 97/91/71 Albugo sp. (ex Rorippa amphibia) HQ377369 93/94/91 Albugo sp. (ex Rorippa palustris) HQ377370 91/100/97
Albugo sp. (ex Alyssum caliacrae) HQ377366 A. laibachii (ex Arabidopsis thaliana) FJ468374
73/-/86/95/91 60/-/57
Albugo sp. (ex Descurainia sophia) AY929832 Albugo sp. (ex Diptychocarpus strictus) AY929833 A. voglmayrii (ex Draba nemorosa) EU240593
94/61/84 100/100/100 54/96/65 100/100/98 96/90/97
100/97/99 98/100/97 100/100/99
A. lepidii (ex Lepidium virginicum) AY929838 A. lepidii (ex Lepidium campestre) AY929835
A. hohenheimia (ex Cardamine hirsuta) GU292154 A. hohenheimia (ex Cardamine hirsuta) GU292150 A. leimonios(ex Cardamine pratensis) GU292156 A. leimonios(ex Cardamine pratensis) GU292159
A. koreana(ex Capsella bursa-pastoris) AY929831 A. koreana(ex Capsella bursa-pastoris) AY929830 A. ipomoeae-panduratae DQ643920
0.05
Fig 2 e Minimum evolution tree of Albugo s.str. based on ITS rDNA sequences. The numbers above or below the branches indicate bootstrap support in ME, ML, and MP analyses. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitutions per site. The branch of the Albugo clade from Brassicaceae is scaled down by 35 % due to over-proportional length. The relevant GenBank accession numbers are indicated following the taxon names. Albugo specimens sequenced in this study are in bold.
(Table 2). To A. candida and the three lineages on A. montanum, Barbarea, and Rapistrum sequence divergence was approximately 2 % (11 or 12 of nucleotide characters were different), 3.7 % (21 of 566), 1.6 % (9 of 566), and 2.0 % (12 of 566), respectively. In ITS rDNA, the nucleotide distances to A. candida and the three lineages on Armoracia, Barbarea, and Rapistrum were about 2.2e3.6 % (19e31 of 857 nucleotide characters were different), 0.4 % (4 of 857), 1.3 % (11 of 857), and 2.0 % (17 of 857), respectively.
Morphological analysis In sixteen herbarium specimens of Alyssum, Barbarea, and Rorippa, the sporogenous hyphae were cylindrical or clavate (Fig 3A). The sporangia were arranged in basipetal chains hyaline, globose to subglobose, with tip rounded and base rounded or sub-truncate (Fig 3B,C). Haustoria were knob-like (Fig 3D) and positioned on a stalk. Resting organs were found on specimens from the four Rorippa species, but not on Alyssum and Barbarea;
Three new phylogenetic lineages close to Albugo candida
603
Table 2 e Divergence of cox2 and ITS sequences of Albugo specimens examined. Pathogen
Albugo candida
Host (Brassicales) Albugo sp. from Rorippa (no. of characters)
cox2 mtDNA (566a) ITS rDNA (871)
Undescribed Albugo sp.
Nineteen genera
Alyssum montanum
Armoracia rusticana
Barbarea vulgaris
Rapistrum rugosum
1.9e2.1 % (11e12b) 2.2e3.6 % (19e31)
3.7 % (21) n.a.
0.3 % (2) 0.4 % (4)
1.6 % (9) 1.3 % (11)
2.0 % (12) 2.0 % (17)
a A total number of nucleotide characters. b The number of different nucleotide characters.
Fig 3 e Albugo rorippae on Rorippa palustris. (A) Sporangia produced in chains from the tips of sporogenous hyphae; (B) Primary sporangia; (C) Secondary sporangia; (D) Haustorium; (EeH) Resting organs. Scale bars: (AeC & EeH) [ 20 mm; (D) [ 5 mm. Source: KRAM-F-000130 (typus).
604
the oogonia were broadly globose or irregular, and oospores were tuberculate without blunt ridges (Fig 3EeH). Based on characteristics of sporogenous hyphae and sporangia, the Albugo specimens on Rorippa were similar to nine Albugo species so far described from Brassicales, but could be distinguished by the oospore characteristics. The surface of the oospores was irregular tuberculate without ridges and protuberances were rarely connected, but not branched (Fig 3EeH). In contrast, surface ornamentation in Albugo candida and Albugo laibachii is irregularly tuberculate but has large ridges, which are often confluent or branched, and in Albugo hohenheimia is pseudoreticulate with blunt ridges, of which the tubercles are mostly connected and often branched. The morphology of the protuberances enabled the separation of the new phylogenetic lineage from Rorippa from Albugo koreana, Albugo hesleri, Albugo lepidii, Albugo resedae, and Albugo voglmayrii. In Albugo specimens belonging to the new species, the wall ornamentation is irregularly tuberculate, while in the latter species, excluding A. koreana, it is regularly tuberculate. The larger oospores and wider warts in A. resedae and A. voglmayrii also enabled to distinguish from the present pathogen. The ornamentation observed in Albugo sp. on Rorippa is closest to A. hesleri and A. koreana, but its tubercles were more irregularly distributed, closely and densely arranged, variable in size, and often more connected than in the latter two species. Key to Albugo species parasitic to Brassicales based on oospore characteristics
Y. -J. Choi et al.
MycoBank accession MB 519654. Etym: ‘rorippae’ refers to scientific generic name of host plants examined. Hyphae sporogenae clavatae vel cylindraceae, (27.5e) 34.5e50.7(e62.5) (medio 42.6) mm longae, (10e) 10.7e14.4(e16.3) (medio 12.6) mm diam. Sporangia hyalina, globosa vel subglobosa, sporangia primaria (12e)13.6e16.3(e18) (medio 14.9) mm diam., parietibus 1.0e1.5 mm crassis, sporangia secundaria (13.5e)15.0e18.5(e21.5) (medio 16.8) mm diam., parietibus uniformibus 0.5e1.0 mm crassis. Oogonia sub-flavida, (45e)50.9e60.7(e62.5) (medio 55.8) mm diam. Oospora flavida, irregulariter tuberculata, (35e)38.8e46.0(e51) (medio 42.4) mm diam., inclusae tuberculae. Tuberculae plerumque singulares, variabiles, (1e)3e4(e5) mm longae, (1e)2e4(e6) mm diam. Typus: POLAND; Miechowice, on living leaves of Rorippa palustris affected by white blister rust disease, Oct. 1884, A. Zalewski (KRAM-F-000130 e holotypus). Sequences ex-type: HQ377364 (cox2 mtDNA) & HQ377371 (ITS rDNA). Sori distinct, round or irregular, rarely confluent, whitish or pale yellowish, 0.5e1.5(e3) mm diam, mostly on the lower surface of the leaves, rarely on upper side and stem. Mycelium intercellular, with globose haustoria with narrow and short stalk of 1e3 mm long, 2e5 mm diam, one or two per host cell. Sporogenous hyphae hyaline, clavate or cylindrical, straight to
1. Warts of wall ornamentation in oospores often connected forming ridges and often branched .............. 2 Warts tuberculated and isolated without ridges and not branched ........................... 4 2. Wall ornamentation pseudo-reticulated; warts mostly connected and branched .............. A. hohenheimia Wall ornamentation tuberculate; warts isolates or connected .............................. 3 3. Size of oospores mostly exceeding 45 mm diam; wall ornamentation mostly obvious ............... A. candida Size of oospores less than 45 mm diam; wall ornamentation often unclear .................... A. laibachii 4. Warts densely and regularly distributed, blunt or rounded; oospores often exceeding 50 mm diam ............ 5 Warts irregularly to regularly distributed, variable in shape; oospores mostly less than 50 mm in diameter ........ 6 5. Oospores yellowish to brownish; size of oospores 44e55 (av. 48.4) mm diam .................. A. voglmayrii Oospores pale yellowish; size of oospores 46e61 (av. 53.6) mm diam ....................... A. resedae 6. Warts regularly distributed, often more than 5 mm wide; tips of warts mostly blunt or rounded ............. 7 Warts irregularly distributed, mostly less than 4 mm wide; tips of warts obtuse, echinulate or truncate .......... 8 7. Warts small; 2e3.5 mm long, 2e5 mm wide ........................................ A. lepidii Warts large; 4e6 mm long, 3e6(e7) mm wide ..................................... A. hesleri 8. Warts very rarely connected, slightly curved, often elongated up to 6 mm ...................... A. koreana Warts often connected, variable in size and shape, mostly less than 4 mm ............... A. rorippae sp. nov.
Taxonomy Previously, most taxonomists and plant pathologist identified the WBR pathogen on Rorippa species as Albugo candida, which was originally described on Capsella bursa-pastoris, but Shaw (1973) considered it as Albugo cruciferarum. The latter name is now considered a synonym of A. candida (Choi et al. 2007). The results of the investigation of phylogenetic and phenetic characters in Albugo specimens from Rorippa highlight the existence of a new species of Albugo on this host, which is therefore described here. Albugo rorippae Y.J. Choi, H.D. Shin, Ploch, & Thines, sp. nov. (Fig 3)
slightly curved, (27.5e)34.5e50.7(e62.5) (av. 42.6) mm long, (10e)10.7e14.4(e16.3) (av. 12.6) mm wide (n ¼ 50), mostly grouped, thick-walled, especially towards the base up to 6 mm. Sporangia arranged in basipetal chains, hyaline, primary sporangia similar to the secondary sporangia, but the first exhibit a slightly thicker wall and somewhat smaller size; primary sporangia mostly globose or polyangular due to mutual pressure, (12e)13.6e16.3(e18) (av. 14.9) mm diam (n ¼ 80), with uniformly thin wall of ca. 1.0e1.5 mm; secondary sporangia subglobose to obovoid or globose, (13.5e)15.0e18.5(e21.5) (av. 16.8) mm diam (n ¼ 100), mostly with uniformly thin wall, but rarely slightly thickening (0.5e1.0 mm) as an equatorial
Three new phylogenetic lineages close to Albugo candida
ring, tip round, base subtruncate or rounded, pedicel mostly absent. Resting organs often present as yellowish to brown dots on both the upper and lower surfaces of the leaf spots. Oogonia irregular or broadly globose, pale yellowish, (45e) 50.9e60.7(e62.5) (av. 55.8) mm diam (n ¼ 70); wall smooth, as observed in both mature and immature oospores, 2e3 mm thick. Oospores plerotic, yellowish, globose, (32e) 35.4e42.0(e47) (av. 38.7) mm diam, but (35e)38.8e46.0(e51) (av. 42.4) mm diam (n ¼ 56) when including the height of tubercles; wall 2e3 mm thick, irregularly tuberculate, without ridges. Tubercles mostly single, rarely connected between neighbouring ones, but not branched, with a ripple-like pattern between the ridges, largely variable in shape and size, (1e)3e4(e5) mm long, (1e)2e4(e6) mm wide; apex mostly obtuse but often echinulate or truncate. Habitat: in living leaves of Rorippa amphibia, R. palustris, Rorippa pyrenaica, and Rorippa sylvestris (Brassicaceae). Additional specimens examined: see Table 1.
Discussion Before recent molecular phylogenetic analyses have proven the genetic diversity of Albugo candida (Choi et al. 2006; € ller 2006), most taxonomic and plant Voglmayr & Riethmu pathogenic studies assumed that only the generalist A. candida is responsible for WBR incidents on a large number of host plants of the Brassicaceae and allied families. Stimulated by the studies mentioned above, subsequent investigations revealed six new species, which are not closely related to A. candida, parasitising specific host plants (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010). In the present study, three additional, specialised lineages have been revealed on the host genera Alyssum, Barbarea, and Rorippa. In contrast to previously known species, these are closely related to A. candida. From these three host genera, only in specimens from Rorippa oospores were observed, which are of crucial importance for species delimitation in Albugo (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010). Oospores of Albugo from Rorippa differ from previously described species in characteristics of the oospore wall, in particular the morphology of the protuberances. The taxonomic independence of Albugo rorippae is also in agreement with a result of previous cross-inoculation experiments; Pound & Williams (1963) observed a high degree of specificity for the Albugo isolate they collected from Rorippa islandica. This isolate showed some potential to infect Armoracia, while Albugo from Armoracia was found to have limited infection potential on Rorippa. This is in line with the current study, which shows that specimens from Armoracia and Rorippa are closely related. Some differences in oospore ornamentation were observed for Albugo from Armoracia and A. rorippae (data not shown), but given the very close relationship of these lineages, several additional specimens from both host genera have to be investigated in order to evaluate, whether the lineages from Armoracia and Rorippa represent independent species. In addition to A. rorippae, we observed two phylogenetically separated lineages closely related to A. candida s.str., one
605
occurring on Alyssum montanum and another on Barbarea vulgaris, both most likely representing independent species. However these could not be morphologically described in this study, as no oospores were found in either host. A morphological form ‘Cystopus candidus (a synonym of A. candida) forma alyssi alyssoidis’ vulescu & Rayss 1930) and a host-specific form ‘C. candidus (Sa vulescu 1946) were introduced for the f. sp. alyssi-alyssoidis’ (Sa WBR agent occurring on Alyssum alyssoidis. However, as the host plant, A. alyssoidis, was not included in this study and the vulescu (1946) is too size variation of sporangia as given by Sa high for morphological delimitation, we could not determine if one of the two Albugo lineages from A. montanum and Alyssum caliacrae examined might represent this specialised form. As mentioned above, Albugo specimens from A. montanum, Armoracia, Barbarea, Rapistrum, and Rorippa are closely related to A. candida. This affinity is different from all other species of Albugo described and confirmed so far, all of which were distantly related to A. candida, with significant sequence divergences. Taking into consideration the current result and those of previous studies (Choi et al. 2006, 2007, 2008; € ller 2006; Thines et al. 2009; Thines & Voglmayr & Riethmu Voglmayr 2009; Ploch et al. 2010), it is likely that the biodiversity of Albugo on Brassicaceae is comparable to the diversity observed for members of the downy mildew genus Hyaloperonospora, which is also predominantly parasitic to € ker et al. 2004, the Brassicaceae (Choi et al. 2003, 2011; Go 2009). Future studies are thus likely to reveal additional, specialised Albugo species on Brassicaceae. The colonisation of a single host species by two distinct WBR species has been previously observed on Capsella bursapastoris (Choi et al. 2007) and Arabidopsis thaliana (Thines et al. 2009), where the presence of the generalist A. candida and apparently specialised species, Albugo koreana and Albugo laibachii, could be observed, respectively. In the present work, eleven specimens originating from four Rorippa species in different geographic regions were investigated, none of which is conspecific with A. candida. Although this does not exclude the possibility that A. candida could potentially infect these hosts, on Rorippa sp. this species seems to be less common than in A. thaliana or C. bursa-pastoris. In contrast to recently described Albugo species, which all seem to have a high degree of specialisation, possibly being restricted to only a particular host species (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010), A. rorippae might have rather a broader host range, encompassing various species of the genus Rorippa. The pattern of specialisation observed in A. rorippae is similar to a case of Albugo lepidii, for which it was shown that specimens from at least five Lepidium sp. form a monophyletic group. However, some genetic diversity was observed within A. lepidii specimens from different host plants (Ploch et al. 2010). Therefore it cannot be ruled out that some genetic isolation is already present between these lineages, which might justify classifying them as independent taxa. The genetic uniformity of A. rorippae from the hosts included in this study might reflect the close relationship of their host plants, as all Rorippa species examined presently, viz Rorippa amphibia, Rorippa palustris, Rorippa pyrenaica, and Rorippa sylvestris, belong to the section Rorippa of the genus (Bleeker et al. 2002). Similarly, the phylogenetic relationship between Albugo specimens infecting the Armoracia, Barbarea, and Rorippa might be due
606
to the fact that three genera are phylogenetically close to each other. All three genera belong to the tribe Cardamineae of Brassicaceae (Les 1994; Franzke et al. 1998; Koch et al. 2001; AlShehbaz et al. 2006). This might be the result of host clade limited colonisation and subsequent speciation, but could also represent a first hint of long-term coevolution. The host plants, Barbarea and Rorippa, are also commonly distributed in East Asian regions, including China, Japan, and Korea, but there are no records of WBR on these host genera from these countries. It can be assumed that there might be geographic barriers as proposed for A. koreana (Choi et al. 2007) and A. voglmayrii (Choi et al. 2008), which hinder the spread of the pathogens from West to East Eurasia. The finding that Armoracia is parasitised by a species distinct from A. candida points out that care should be taken not to spread this pathogen to new areas where WBR of horseradish has not yet been reported. Whether A. candida might also occur on this host needs to be clarified by comprehensive sampling from this host. Previous studies have shown a high host specificity of Albugo iso vulescu 1946; Pound & lates from Brassicaceae (Hiura 1930; Sa Williams 1963; Petrie 1988), but the phylogenetic relationships of these isolates cannot be clarified on the basis of these reports. It seems possible that distinct species were investigated, but it could also be that specialised forms of A. candida have been studied (Borhan et al. 2008). In contrast to these results, based on € rstad (1964) cross-inoculation tests, Safeeulla (1952) and Jo reported that A. candida can attack plants in the Cleomaceae, and recently Khunti et al. (2000) showed that isolates from Brassica juncea (Brassicaceae) successfully infect Cleome viscosa (Cleomaceae). The cross-inoculation results were in agreement with the molecular phylogenetic analyses of Choi et al. (2007, 2009) that Albugo specimens from C. viscosa are identical with A. candida s.str. from various plants of the Brassicaceae. However, judging from the present result, isolates of Barbarea and Rorippa, might not serve as a source of the disease on cultivated crops, although further extensive sampling from other geographic regions is required to ascertain this assumption.
Acknowledgements The authors are grateful to the curators of BP, BPI, FH and KRAM-F for providing the Albugo specimens. SP and MT are financially supported by the research funding programme “LOEWE e Landes-Offensive zur Entwicklung Wissenschaft€ konomischer Exzellenz" of Hesse’s Ministry of Higher lich-o Education, Research, and the Arts.
references
Al-Shehbaz IA, Beilstein MA, Kellogg EA, 2006. Systematics and phylogeny of the Brassicaceae (Cruciferae): an overview. Plant Systematics and Evolution 259: 89e120. Bachofer M, 2004. Molekularbiologische Populationsstudien an Plasmopara halstedii, dem Falschen Mehltau der Sonnenblume. Uni€ t Hohenheim, Germany, pp. 1e140. versita Biga MLB, 1955. Riesaminazione delle specie del genere Albugo in base alle morfologia dei conidi. Sydowia 9: 339e358.
Y. -J. Choi et al.
Bleeker W, Weber-Sparenberg C, Hurka H, 2002. Chloroplast DNA variation and biogeography in the genus Rorippa Scop. (Brassicaceae). Plant Biology 4: 104e111. € r M, Rimmer SR, Borhan MH, Gunn N, Cooper A, Gulden S, To Holub EB, 2008. WRR4 encodes a TIR-NB-LRR protein that confers broad-spectrum white rust resistance in Arabidopsis thaliana to four physiological races of Albugo candida. Molecular PlanteMicrobe Interactions 21: 757e768. Choi YJ, Hong SB, Shin HD, 2003. Diversity of the Hyaloperonospora parasitica complex from core brassicaceous hosts based on ITS rDNA sequences. Mycological Research 107: 1314e1322. Choi YJ, Hong SB, Shin HD, 2006. Genetic diversity within the Albugo candida complex (Peronosporales, Oomycota) inferred from phylogenetic analysis of ITS rDNA and COX2 mtDNA sequences. Molecular Phylogenetics and Evolution 40: 400e409. Choi YJ, Shin HD, Hong SB, Thines M, 2007. Morphological and molecular discrimination among Albugo candida materials infecting Capsella bursa-pastoris world-wide. Fungal Diversty 27: 11e34. Choi YJ, Shin HD, Ploch S, Thines M, 2008. Evidence for uncharted biodiversity in the Albugo candida complex, with description of a new species. Mycological Research 112: 1327e1334. Choi YJ, Shin HD, Thines M, 2009. The host range of Albugo candida extends from Brassicaceae through Cleomaceae to Capparaceae. Mycological Progress 8: 329e335. Choi YJ, Shin HD, Voglmayr H, 2011. Reclassification of two Peronospora species parasitic on Draba in Hyaloperonospora based on morphological and molecular phylogenetic data. Mycopathologia 171: 151e159. Choi YJ, Thines M, 2011. Morphological and molecular confirmation of Albugo resedae (Albuginaceae; Oomycota) as a distinct species from A. candida. Mycological Progress, in press, doi:10.1007/s11557-010-0683-4. Constantinescu O, Negrean G, 1983. Check-list of Romanian Peronosporales. Mycotaxon 16: 537e556. Farr DF, Rossman AY, 2010. Fungal Databases, Systematic Botany & Mycology Laboratory. ARS, USDA.http://nt.ars-grin.gov/fungaldatabases/ [continuously updated]. Franzke A, Pollmann K, Bleeker W, Kohrt R, Hurka H, 1998. Molecular systematics of Cardamine and allied genera (Brassicaceae): ITS and noncoding chloroplast DNA. Folia Geobot 33: 225e240. € ker M, Riethmu € ller A, Voglmayr H, Weiß M, Oberwinkler F, Go 2004. Phylogeny of Hyaloperonospora based on nuclear ribosomal internal transcribed spacer sequences. Mycological Progress 3: 83e94. € ker M, Voglmayr H, Bla zquez GG, Oberwinkler F, 2009. Species Go delimitation in downy mildews: the case of Hyaloperonospora in the light of nuclear ribosomal ITS and LSU sequences. Mycological Research 113: 308e325. Hiura M, 1930. Biological forms of Albugo candida (Pers.) Kuntze on some cruciferous plants. Japanese Journal of Botany 5: 1e20. € rstad I, 1964. The Phycomycetous genera Albugo, Bremia, PlasJo mopara, and Pseudoperonospora in Norway, with an appendix containing unpublished finds of Peronospora. Nytt Magasin for Botanikk 11: 47e82. Katoh K, Kuma K, Toh H, Miyata T, 2005. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: 511e518. Khunti JP, Khandar RR, Bhoraniya MF, 2000. Studies on host range of Albugo cruciferarum the incitant of white rust of mustard. Agricultural Science Digest 20: 219e221. Koch M, Haubold B, Mitchell-Olds T, 2001. Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear Chs sequences. American Journal of Botany 88: 534e544. Kochman J, Majewski T, 1970. Grzyby (Mycota). Tom IV. Glonowce stwowe Wy(Phycomycetes), Wroslikowe (Peronosporales). Pan dawnictwo Naukowe, Warszawa.
Three new phylogenetic lineages close to Albugo candida
Les DH, 1994. Molecular systematics and taxonomy of lake cress (Neobeckia aquatica; Brassicaceae), an imperiled aquatic mustard. Aquatic Botany 49: 149e165. May KJ, Ristaino JB, 2004. Identity of the mtDNA haplotype(s) of Phytophthora infestans in historical specimens from the Irish potato famine. Mycological Research 108: 471e479. Moncalvo JM, Wang HH, Hseu RS, 1995. Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87: 223e238. Novotel’nova NS, Pystina KA, 1985. Ordo Peronosporales (Fam. Pythiaceae, Phytophthoraceae, Peronosporeaceae, Cystopaceae). In: Gorlenko MV (ed.), Cryptogamic Flora of USSR. Fungi (3), vol. 11. Nauka, Leningrad. Petkowski JE, Cunnington JH, Minchinton EJ, Cahill DM, 2010. Molecular phylogenetic relationships between Albugo candida collections on the Brassicaceae in Australia. Plant Pathology 59: 282e288. Petrie GA, 1988. Races of Albugo candida (white rust and staghead) on cultivated Cruciferae in Saskatchewan. Canadian Journal of Plant Pathology 10: 142e150. Ploch S, Choi YJ, Rost C, Shin HD, Schilling E, Thines M, 2010. Evolution of diversity in Albugo is driven by high host specificity and multiple speciation events on closely related Brassicaceae. Molecular Phylogenetics and Evolution 57: 812e820. Pound GS, Williams PH, 1963. Biological races of Albugo candida. Phytopathology 63: 1146e1149. Safeeulla KM, 1952. Morphological and cytological studies of Albugo species on Ipomoea hederacea. Current Science 18: 287e288. Saharan GS, Verma PR, 1992. White Rusts: a review of economically important species. International Development Research Centre, Ottawa, Ontario. vulescu O, 1946. Studiul speciilor de Cystopus Le v.: din Europa, Sa asupra speciilor din Roma ^ nia. Teza, Stiinte cu privire speciala din Bucuresti. vulescu T, Rayss T, 1930. Contribution a la connaissance des Sa ronosporace es de Roumanie. Annales Mycologici 28: 297e320. Pe Shaw CG, 1973. Host fungus index for the Pacific Northwest e I. Hosts. Washington State University, Agricultural Experiment Station Bulletin 765: 1e121. Stamatakis E, 2006. RAxML-VI-HPC: maximum likelihood based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688e2690.
607
Stanyavichene S, 1984. Peronosporovye griby Pribltiki. ((Peronosporaceous fungi of the Baltic States)). Mokslas, Vilnius. Swofford DL, 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596e1599. Telle S, Thines M, 2008. Amplification of cox2 (w620 bp) from 2 mg of up to 129 years old herbarium specimens, comparing 19 extraction methods and 15 polymerases. PLoS ONE 3: e3584. Thiers B, 2010. Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium.http://sweetgum.nybg.org/ih/ [continuously updated]. Thines M, Choi YJ, Kemen E, Ploch S, Holub EB, Shin HD, Jones JDG, 2009. A new species of Albugo parasitic to Arabidopsis thaliana reveals new evolutionary patterns in white blister rusts (Albuginaceae). Persoonia 22: 123e128. Thines M, Spring O, 2005. A revision of Albugo (Chromista, Peronosporomycetes). Mycotaxon 92: 443e458. Thines M, Voglmayr H, 2009. Evolution, phylogeny and diversity of the white blister rusts (Albuginales). In: Lamour K, Kamoun S (eds), Oomycete Genetics and Genomics: biology, interactions with plants and animals, and research toolbox. WileyeBlackwell, pp. 77e92. Ul’yanishchev VI, Osipian LL, Kanchaveli LA, Akhundov TM, 1985. Peronosporovye Griby. ((Peronosporaceous Fungi)). In: Ul’yanishchev VI (ed.), Opredelitel’ Gribov Zakavkaz’ya. Erevan University, Erevan. Vanev SG, Dimitrova EG, Ilieva EI, 1993. Gybite v Bylgariya. 2 tom. Razred Peronosporales. ((Fungi Bulgaricae. Vol. 2. Ordo Peronosporales)). Bulgarian Academy of Sciences, Sofia. Voglmayr H, 2008. Progress and challenges in systematics of downy mildews and white blister rusts: new insights from genes and morphology. European Journal of Plant Pathology 122: 3e18. € ller A, 2006. Phylogenetic relationships of Voglmayr H, Riethmu Albugo species (white blister rusts) based on LSU rDNA sequence and oospore data. Mycological Research 110: 75e85. Wilson GW, 1907. Studies in North American Peronosporales-I. The genus Albugo. Bulletin of the Torrey Botanical Club 34: 61e84.
journal homepage: www.elsevier.com/locate/funbio
Three new phylogenetic lineages are the closest relatives of the widespread species Albugo candida Young-Joon CHOIa, Hyeon-Dong SHINb, Sebastian PLOCHc, Marco THINESc,d,* a
Harvard University, Department of Organismic and Evolutionary Biology, 22 Divinity Ave., Cambridge, MA 02138, USA Korea University, Division of Environmental Science and Ecological Engineering, Seoul 136-701, Republic of Korea c Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt (Main), Germany d Johann Wolfgang Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Siesmayerstr. 70, D-60323 Frankfurt (Main), Germany b
article info
abstract
Article history:
White blister rust caused by the obligate biotroph Albugo candida (Albuginaceae; Oomycota) is
Received 8 October 2010
one of the most notorious and common diseases of Brassicaceae. During the past 5 y, A. can-
Received in revised form
dida specimens collected from about 30 host genera were phylogenetically and morpholog-
29 January 2011
ically investigated in several studies. These not only revealed that A. candida s.str. has
Accepted 8 February 2011
a broad host range, encompassing a large number of host plants belonging to Brassicales,
Available online 15 February 2011
but also the presence of previously overlooked species of Albugo with hosts in this order.
Corresponding Editor:
In this study, we examined specimens from Alyssum, Barbarea, and Rorippa, of which
Hermann Voglmayr
many species were commonly recorded as host plants of A. candida but could not be included in previous works due to the paucity of specimens available. It was revealed that
Key words:
Albugo specimens from Alyssum montanum, Barbarea vulgaris, and various Rorippa species,
Albuginales
were placed in three phylogenetically distinct clades, but closer to A. candida s.str. than
Broad species concept
any previously reported species. Oospores were observed from Albugo specimens parasitic
Host specificity
to Rorippa and could be distinguished morphologically from A. candida. Therefore, Albugo
Hyaloperonospora
rorippae sp. nov. is described and illustrated here. In addition, a key of Albugo species de-
Morphology
scribed previously from Brassicales is given. The present study reveals that a large number
Obligate biotroph
of Albugo species remain still undiscovered, and that species close to A. candida exist. This
Species key
could help elucidating the basis of the broad host range of A. candida as opposed to the nar-
White blister rust
row specialisation that is seemingly present in other species of Albugo on the Brassicaceae. ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction The Albuginaceae are obligate biotrophic oomycetes, responsible for white blister rust (WBR) disease on various angiosperms. On the Brassicaceae, WBR is probably the most common disease caused by biotrophic pathogens, along with downy mildew disease caused by members of the Peronosporaceae. During the past decades, the generalist Albugo candida (Pers.) Roussel has been
regarded as the causal agent of all incidents of WBR on Brassicaceae, mainly based on the similarity of morphological traits, which led to the application of a broad species concept, similar to downy mildews (Voglmayr 2008 and references cited therein). Recent molecular studies (Choi et al. 2006; Voglmayr € ller 2006) have supported not only its wide host & Riethmu range, but also the high degree of genetic diversity within Albugo parasitic to Brassicaceae. Subsequently, six new specialised
* Corresponding author. Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt (Main), Germany. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.02.006
Three new phylogenetic lineages close to Albugo candida
species have been introduced, Albugo koreana from Capsella bursa-pastoris (Choi et al. 2007), Albugo laibachii from Arabidopsis thaliana (Thines et al. 2009), Albugo voglmayrii from Draba nemorosa (Choi et al. 2008), Albugo hesleri, Albugo hohenheimia, and Albugo leimonios from species of the genus Cardamine (Ploch et al. 2010). In addition, Albugo lepidii (Choi et al. 2007) and Albugo resedae (Choi & Thines, in press) were confirmed as species distinct from A. candida, while Albugo chardonii from Cleomaceae was revealed to be synonymous with A. candida (Choi et al. 2007, 2009). Judging from the studies listed above, it is believed that more than a dozen distinct species so far regarded to be A. candida await discovery (Thines & Voglmayr 2009). On the other hand, the phylogenetic results also demonstrated that the host range of A. candida s.str. extends from Brassicaceae to Cleomaceae and Capparaceae (Choi et al. 2007, 2009), but possibly not to Resedaceae (Choi & Thines, in press). Only about half of about 60 brassicaceous genera which were originally recorded as host plants of A. candida (Biga 1955; Saharan & Verma 1992) have been examined during previous phylogenetic studies (Choi et al. 2006, 2007, 2008, 2009; Choi & € ller 2006; Thines et al. Thines, in press; Voglmayr & Riethmu 2009; Petkowski et al. 2010; Ploch et al. 2010), leaving Albugo on about 30 host genera unexamined. Albugo candida has previously been found deeply separated from the species recently confirmed (Choi et al. 2007; Choi & Thines, in press) and newly discovered (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010), and only recently a few specimens with close affinity to A. candida have been revealed (Ploch et al. 2010). However, based on the limited sampling from the respective host genera, Armoracia and Rapistrum, the taxonomic placement of these specimens remained unresolved. The three genera, Alyssum, Barbarea, and Rorippa, are widely distributed and of these, about 50 species have been recorded as common host plants of A. candida in monographic studies (Wilson 1907; Biga 1955; Kochman & Majewski 1970; Constantinescu & Negrean 1983; Stanyavichene 1984; Novotel’nova & Pystina 1985; Ul’yanishchev et al. 1985; Vanev et al. 1993); see also pathogenehost distribution database
599
of Farr & Rossman (2010). However, despite of their wide distribution, Albugo specimens from these genera have so far not been included in molecular phylogenetic investigations. As it was previously shown that the WBRs from the widespread genus Cardamine harbour at least three distinct species (Ploch et al. 2010), it was the aim of this study to investigate specimens from additional widespread genera for the presence of previously overlooked species.
Materials and methods Oomycete specimens Seventeen Albugo specimens on Armoracia, Alyssum, Barbarea, and Rorippa were morphologically and phylogenetically investigated; Armoracia rusticana (1), Alyssum caliacrae (1), Alyssum montanum (1), Barbarea vulgaris (3), Rorippa palustris (4), Rorippa sylvestris (3), Rorippa amphibia (2), Rorippa pyrenaica (1), and Rorippa sp. (1). For comparison, nine Albugo species described previously from the Brassicales were also included, Albugo candida, Albugo hesleri, Albugo hohenheimia, Albugo leimonios, Albugo lepidii, Albugo laibachii, Albugo koreana, Albugo resedae, and Albugo voglmayrii, with four undetermined lineages from the four host genera, Armoracia, Rapistrum, Descurainia, and Diptychocarpus. Both cox2 mtDNA and ITS rDNA sequences of several Albugo species from two previous studies (Ploch et al. 2010; Choi & Thines, in press) were retrieved from GenBank. Oomycete specimens examined in this study are listed in Table 1. Herbaria abbreviations are those from Thiers (2010). Morphological analysis was conducted as described previously (Choi et al. 2008).
Molecular analysis Genomic DNA from infected leaves of herbarium specimens was extracted using the DNeasy Plant Mini kit (Qiagen, Valencia, CA) or the Analyticjena plant DNA extraction kit as
Table 1 e Information on Albuginaceae specimens investigated in this study. Pathogen
Host
Geographic origin, collection year (Herb. no.)
GenBank Acc. No. (cox2/ITS)
Albugo sp.
Alyssum caliacrae Alyssum montanum Armoracia rusticana Barbarea vulgaris Barbarea vulgaris Barbarea vulgaris Rorippa amphibia Rorippa amphibia Rorippa palustris Rorippa palustris Rorippa palustris Rorippa palustris Rorippa pyrenaica Rorippa sylvestris Rorippa sylvestris Rorippa sylvestris Rorippa sp.
Romania, 1979 (BPI199995) Poland, 1896 (KRAM-F-000149) Germany, 1890 (BP263) USA, 1925 (BPI184667) Ireland, 1943 (BPI184670) Germany, 1992 (BPI748543) Romania, 1975 (FH HMR2610)a Romania, 1975 (BP75207) USA, 1952 (BPI185090)a USA, 1909 (BPI185091)a Australia 1992 (DAR72185) Poland, 1884 (KRAM-F-000130)a Romania, 1972 (FH HMR2197)a Poland, 1896 (KRAM-F-000138)a Poland, 1884 (KRAM-F-000134)a Romania, 1975 (FH HMR2611)a Argentina, 1987 (RD0129)b
HQ377356/HQ377366 HQ377357/ /HQ832468 HQ377358/HQ377367 HQ377359/ HQ377360/HQ377368 / HQ377361/HQ377369 / / HQ377362/HQ377370 HQ377364/HQ377371 / HQ377363/ / / HQ377365/
Albugo rorippae
a The specimens that oospores were successfully observed. b Acronym: RD, R. Delhey (private collections) in the Phytopathology Lab of Bahıa Blanca, Argentina.
600
recommended in Telle & Thines (2008). The freshly collected material was extracted using a protocol modified from May & Ristaino (2004) as described in Thines & Spring (2005). PCR amplifications of cox2 mtDNA and ITS rDNA regions were performed as outlined in Choi et al. (2006). Semi-nested reamplification of poorly amplified samples was done with the internal primer ITS1-O (Bachofer 2004) and LR0 (reverse complement to Moncalvo et al. 1995). PCR products were sequenced by two commercial sequencing companies, Macrogene (Seoul, Korea) and GATC Biotech (Konstanz, Germany) with the primers used for the PCR reactions.
Alignment and phylogenetic reconstruction The sequences were edited with the DNASTAR computer package (Lasergene, Madison, WI), version 8. Alignment of the sequences was performed using MegAlign (DNAStar) for the unambiguously alignable cox2 region and Mafft, version 6.0 (Katoh et al. 2005), was used with the G-INS-i strategy for ITS sequences. No manual “improvements” or “corrections” to the alignment were done in either case. Phylogenetic trees were obtained from the data using minimum evolution (ME), maximum likelihood (ML), and maximum parsimony (MP). An ME inference was performed in MEGA, version 4.0 (Tamura et al. 2007), with Kimura 2-parameter model. For ML inference, RAxML version 7.0.3 (Stamatakis 2006) was used with all parameters set to default values, using the GTRCAT variant. An MP heuristic search was performed with 1 K random sequence additions and branch swapping by tree bisection-reconnection (TBR), using PAUP version 4b10 (Swofford 2002). The relative robustness of the groupings and individual branches was estimated by bootstrapping (BS) using 10 K replicates, each with ten rounds of heuristic searches with TBR branch swapping on trees generated by random sequence addition. The sequence of Albugo ipomoeae-panduratae was used as outgroup, according to a study of Choi et al. (2009). The newly derived sequences were deposited in GenBank under accession numbers HQ377356eHQ377365 (cox2 mtDNA) and HQ377366eHQ377371, HQ832468 (ITS rDNA), the alignments in TreeBASE (www.treebase.org/treebase-web/home.html) under accession no. S10914, and a taxonomic novelty in MycoBank (www.MycoBank.org).
Results Molecular phylogenetic analysis The cox2 mtDNA alignment of 566 bp revealed no gaps for the genus Albugo s.str., while a gap spanning six base-pairs was observed for Albugo infecting Convolvulaceae. The ITS rDNA alignment was 857 bp in length and includes partial ITS1, complete 5.8S rDNA and partial ITS2 sequences. All unavailable data were assumed as missing from the alignment. The phylogenetic relationships between Albugo specimens were inferred from ME, ML, and MP analyses of the aligned sequences. In the cox2 mtDNA alignment, 97 characters were parsimony-informative, and the MP analysis resulted in the 44 most parsimonious trees of 277 steps, with a CI and RI of 0.7967 and 0.8862, respectively. For the ITS rDNA
Y. -J. Choi et al.
alignment, 215 characters were parsimony-informative, and the parsimony analysis produced 238 most parsimonious trees of 402 steps, with a CI and RI of 0.8383 and 0.9385, respectively. ML search in RAxML v7.0.3 using the GTRCAT variant model of sequence evolution resulted in one ML tree each with a ln L ¼ 2166.4926 and a ln L ¼ 3936.9241 for cox2 and ITS, respectively. The results of the phylogenetic reconstructions for cox2 and ITS, respectively are shown in Figs 1 and 2, respectively. A similar topology was observed in ME, ML, and MP analyses, and no highly supported, conflicting alternative topologies were inferred. Therefore, only the tree based on the ME analysis is presented, with the addition of the support values of the ML and MP analyses. Based on the cox2 mtDNA sequences (Fig 1), all phylogenetic analyses resulted in two major clades. One consists of Albugo candida s.str. from a broad range of Brassicaceae, Capparaceae, and Cleomaceae, and five closely related, yet phylogenetically distinct lineages. The second clade includes the alreadydescribed WBR specialists, Albugo hesleri, Albugo hohenheimia, Albugo koreana, Albugo laibachii, Albugo leimonios, Albugo lepidii, Albugo voglmayrii (Brassicaceae), and Albugo resedae (Resedaceae), with three undescribed species. The five phylogenetic lineages close to A. candida s.str. represent five specimens from Rorippa species, three from Barbarea vulgaris, two from Armoracia rusticana, one from Alyssum montanum, and one from Rapistrum rugosum. Among them, the Albugo specimens from Rorippa formed a monophyletic group in all three analyses (ME BS/ML BS/MP BS of 87/88/71 %), with no sequence difference among them, regardless of the different host species and geographic origins. This lineage further grouped with the specimens from Armoracia, with high to maximum BS (100/96/92 %). A well-supported group consisting of the three specimens from B. vulgaris was sister to the clade including A. candida s.str and three lineages from Armoracia, Rorippa, and Rapistrum, with high BS values of 99/90/98 %. Despite of the fact that the B. vulgaris specimens were collected in geographically distant countries, Germany, Ireland, and the USA, no sequence divergence was found. Interestingly, two specimens from Alyssum were divided into two distant branches; one from A. montanum was placed basal to A. candida s.str. and allied lineages, while the other from Alyssum caliacrae was placed in the second group including many specialised species. In the second clade, the distinctiveness of the previously described WBR specialists was confirmed, with high support in all analyses. This clade included, apart from the undescribed species specialised on A. caliacrae mentioned previously, two additional undescribed species from Descurainia and Diptychocarpus. The ITS rDNAbased analyses (Fig 2) resulted in a similar topology compared to the cox2 region, without highly supported conflicts. In contrast to the cox2 tree, a well-supported sister-group relationship of specimens from Barbarea (100/94/85 %) with those from Armoracia and Rorippa was inferred. Albugo sp. from A. caliacrae, which showed no affinity with other specialised Albugo species in cox2 analyses, clustered together with A. laibachii and Albugo sp. from Descurainia with moderate to high bootstrap support (86/95/91 %). In cox2 mtDNA, Albugo sp. on Rorippa shared a high level of sequence similarity with Albugo on A. rusticana as they differed at only two positions among 566 nucleotide characters
Three new phylogenetic lineages close to Albugo candida
601
A. candida (ex Arabis turrita) AY913803 A. candida (ex Capparis spinosa) EF655654 A. candida (ex Capsella bursa-pastoris) DQ643944 A. candida (ex Thlaspi arvense) AY913809 A. candida (ex Diplotaxis erucoides) DQ418517 64/85/63 A. candida (ex Raphanus sativus) AY927059 A. candida (ex Aubrietia deltoidea) DQ418500 A. candida (ex Biscutella laevigata) DQ418506 A. candida (ex Lunaria sp.) AY913797 A. candida (ex Iberis amara) DQ418522 A. candida (ex Berteroa incana) DQ418508 A. candida (ex Heliophila meyeri) DQ418515 A. candida (ex Erysimum cuspidatum) DQ418519 A. candida (ex Cleome anomala) AY913799 A. candida (ex Arabidopsis halleri) DQ418513 A. candida (ex Arabidopsis thaliana) FJ468361 100/96/95 A. candida (ex Brassica juncea) AY927046 A. candida (ex Draba verna) EU340140 73/87/73 A. candida (ex Sisymbrium luteum) AY913808 A. candida (ex Eruca sativa) DQ418514 Albugo sp. (ex Rapistrum rugosum) GU292101 44/58/52 89/93/83 Albugo sp. (ex Armoracia rusticana) GU292084 Albugo sp. (ex Armoracia rusticana) GU292087 100/96/92
Albugo sp. (ex Rorippa sylvestris) HQ377363 Albugo sp. (ex Rorippa palustris) HQ377364 100/90/98 Albugo sp. (ex Rorippa sp.) HQ377365 87/88/71 Albugo sp. (ex Rorippa amphibia) HQ377361 Albugo sp. (ex Rorippa palustris) HQ377362 74/-/72 Albugo sp. (ex Barbarea vulgaris) HQ377358 99/100/99 Albugo sp. (ex Barbarea vulgaris) HQ377359 Albugo sp. (ex Barbarea vulgaris) HQ377360 Albugo sp. (ex Alyssum montanum) HQ377357 A. resedae (ex Reseda alba) GU945758 99/100/98 96/93/84 A. resedae (ex Reseda alba) GU945759 Albugo sp. (ex Diptychocarpus strictus) AY927052 99/100/99 A. lepidii (ex Lepidium campestre) AY927054
A. lepidii (ex Lepidium virginicum) AY927057 Albugo sp. (ex Alyssum caliacrae) HQ377356 A. laibachii (ex Arabidopsis thaliana) FJ468371 Albugo sp. (ex Descurainia sophia) AY927051 99/100/100 A. hohenheimia(ex Cardamine hirsuta) GU292114 A. hohenheimia(ex Cardamine hirsuta) GU292118 99/100/100 59/97/60
99/100/100 99/100/100
A. voglmayrii (ex Draba nemorosa) EU240588 A. voglmayrii (ex Draba nemorosa) EU240590
A. leimonios (ex Cardamine pratensis) GU292124 A. leimonios (ex Cardamine pratensis) GU292121
A. hesleri (ex Cardamine diphylla) GU292091 A. hesleri (ex Cardamine diphylla) GU292107
99/100/100
A. koreana (ex Capsella bursa-pastoris) AY927049 A. koreana (ex Capsella bursa-pastoris) DQ643944 A. ipomoeae-pandurataeAY913804
0.02
Fig 1 e Minimum evolution tree of Albugo s.str. based on cox2 mtDNA sequences. The numbers above or below the branches indicate bootstrap support in ME, ML, and MP analyses. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitutions per site. The relevant GenBank accession numbers are indicated following the taxon names. Albugo specimens sequenced in this study are in bold.
602
Y. -J. Choi et al.
A. candida (ex Capsella bursa-pastoris) DQ643916 A. candida (ex Lunariasp.) AY929840 A. candida (ex Heliophila meyeri) DQ418493 A. candida (ex Eruca sativa) DQ418503 A. candida (ex Arabidopsis halleri) DQ418502 A. candida (ex Thlaspi arvense) AY929847 A. candida (ex Sisymbrium leteum) AY929844 A. candida (ex Iberis amara) DQ418499 A. candida (ex Arabidopsis thaliana) FJ468362 A. candida (ex Brassica juncea) AY929826 A. candida (ex Arabis turrita) AY929825 A. candida (ex Aubrietia deltoidea) DQ418500 69/80/56
A. candida (ex Berteroa incana) DQ418495 A. candida (ex Raphanus sativus) AY929841 A. candida (ex Biscutella laevigata) DQ418494
100/95/100
A. candida (ex Diplotaxis erucoides) DQ418496 A. candida (ex Erysimum cuspidatum) DQ418498
52/-/-
Albugo sp. (ex Rapistrum rugosum) GU292141 Albugo sp. (ex Barbarea vulgaris) HQ377367 Albugo sp. (ex Barbarea vulgaris) HQ377368 Albugo sp. (ex Armoracia rusticana) HQ832468 100/94/85 Albugo sp. (ex Rorippa palustris) HQ377371 97/91/71 Albugo sp. (ex Rorippa amphibia) HQ377369 93/94/91 Albugo sp. (ex Rorippa palustris) HQ377370 91/100/97
Albugo sp. (ex Alyssum caliacrae) HQ377366 A. laibachii (ex Arabidopsis thaliana) FJ468374
73/-/86/95/91 60/-/57
Albugo sp. (ex Descurainia sophia) AY929832 Albugo sp. (ex Diptychocarpus strictus) AY929833 A. voglmayrii (ex Draba nemorosa) EU240593
94/61/84 100/100/100 54/96/65 100/100/98 96/90/97
100/97/99 98/100/97 100/100/99
A. lepidii (ex Lepidium virginicum) AY929838 A. lepidii (ex Lepidium campestre) AY929835
A. hohenheimia (ex Cardamine hirsuta) GU292154 A. hohenheimia (ex Cardamine hirsuta) GU292150 A. leimonios(ex Cardamine pratensis) GU292156 A. leimonios(ex Cardamine pratensis) GU292159
A. koreana(ex Capsella bursa-pastoris) AY929831 A. koreana(ex Capsella bursa-pastoris) AY929830 A. ipomoeae-panduratae DQ643920
0.05
Fig 2 e Minimum evolution tree of Albugo s.str. based on ITS rDNA sequences. The numbers above or below the branches indicate bootstrap support in ME, ML, and MP analyses. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitutions per site. The branch of the Albugo clade from Brassicaceae is scaled down by 35 % due to over-proportional length. The relevant GenBank accession numbers are indicated following the taxon names. Albugo specimens sequenced in this study are in bold.
(Table 2). To A. candida and the three lineages on A. montanum, Barbarea, and Rapistrum sequence divergence was approximately 2 % (11 or 12 of nucleotide characters were different), 3.7 % (21 of 566), 1.6 % (9 of 566), and 2.0 % (12 of 566), respectively. In ITS rDNA, the nucleotide distances to A. candida and the three lineages on Armoracia, Barbarea, and Rapistrum were about 2.2e3.6 % (19e31 of 857 nucleotide characters were different), 0.4 % (4 of 857), 1.3 % (11 of 857), and 2.0 % (17 of 857), respectively.
Morphological analysis In sixteen herbarium specimens of Alyssum, Barbarea, and Rorippa, the sporogenous hyphae were cylindrical or clavate (Fig 3A). The sporangia were arranged in basipetal chains hyaline, globose to subglobose, with tip rounded and base rounded or sub-truncate (Fig 3B,C). Haustoria were knob-like (Fig 3D) and positioned on a stalk. Resting organs were found on specimens from the four Rorippa species, but not on Alyssum and Barbarea;
Three new phylogenetic lineages close to Albugo candida
603
Table 2 e Divergence of cox2 and ITS sequences of Albugo specimens examined. Pathogen
Albugo candida
Host (Brassicales) Albugo sp. from Rorippa (no. of characters)
cox2 mtDNA (566a) ITS rDNA (871)
Undescribed Albugo sp.
Nineteen genera
Alyssum montanum
Armoracia rusticana
Barbarea vulgaris
Rapistrum rugosum
1.9e2.1 % (11e12b) 2.2e3.6 % (19e31)
3.7 % (21) n.a.
0.3 % (2) 0.4 % (4)
1.6 % (9) 1.3 % (11)
2.0 % (12) 2.0 % (17)
a A total number of nucleotide characters. b The number of different nucleotide characters.
Fig 3 e Albugo rorippae on Rorippa palustris. (A) Sporangia produced in chains from the tips of sporogenous hyphae; (B) Primary sporangia; (C) Secondary sporangia; (D) Haustorium; (EeH) Resting organs. Scale bars: (AeC & EeH) [ 20 mm; (D) [ 5 mm. Source: KRAM-F-000130 (typus).
604
the oogonia were broadly globose or irregular, and oospores were tuberculate without blunt ridges (Fig 3EeH). Based on characteristics of sporogenous hyphae and sporangia, the Albugo specimens on Rorippa were similar to nine Albugo species so far described from Brassicales, but could be distinguished by the oospore characteristics. The surface of the oospores was irregular tuberculate without ridges and protuberances were rarely connected, but not branched (Fig 3EeH). In contrast, surface ornamentation in Albugo candida and Albugo laibachii is irregularly tuberculate but has large ridges, which are often confluent or branched, and in Albugo hohenheimia is pseudoreticulate with blunt ridges, of which the tubercles are mostly connected and often branched. The morphology of the protuberances enabled the separation of the new phylogenetic lineage from Rorippa from Albugo koreana, Albugo hesleri, Albugo lepidii, Albugo resedae, and Albugo voglmayrii. In Albugo specimens belonging to the new species, the wall ornamentation is irregularly tuberculate, while in the latter species, excluding A. koreana, it is regularly tuberculate. The larger oospores and wider warts in A. resedae and A. voglmayrii also enabled to distinguish from the present pathogen. The ornamentation observed in Albugo sp. on Rorippa is closest to A. hesleri and A. koreana, but its tubercles were more irregularly distributed, closely and densely arranged, variable in size, and often more connected than in the latter two species. Key to Albugo species parasitic to Brassicales based on oospore characteristics
Y. -J. Choi et al.
MycoBank accession MB 519654. Etym: ‘rorippae’ refers to scientific generic name of host plants examined. Hyphae sporogenae clavatae vel cylindraceae, (27.5e) 34.5e50.7(e62.5) (medio 42.6) mm longae, (10e) 10.7e14.4(e16.3) (medio 12.6) mm diam. Sporangia hyalina, globosa vel subglobosa, sporangia primaria (12e)13.6e16.3(e18) (medio 14.9) mm diam., parietibus 1.0e1.5 mm crassis, sporangia secundaria (13.5e)15.0e18.5(e21.5) (medio 16.8) mm diam., parietibus uniformibus 0.5e1.0 mm crassis. Oogonia sub-flavida, (45e)50.9e60.7(e62.5) (medio 55.8) mm diam. Oospora flavida, irregulariter tuberculata, (35e)38.8e46.0(e51) (medio 42.4) mm diam., inclusae tuberculae. Tuberculae plerumque singulares, variabiles, (1e)3e4(e5) mm longae, (1e)2e4(e6) mm diam. Typus: POLAND; Miechowice, on living leaves of Rorippa palustris affected by white blister rust disease, Oct. 1884, A. Zalewski (KRAM-F-000130 e holotypus). Sequences ex-type: HQ377364 (cox2 mtDNA) & HQ377371 (ITS rDNA). Sori distinct, round or irregular, rarely confluent, whitish or pale yellowish, 0.5e1.5(e3) mm diam, mostly on the lower surface of the leaves, rarely on upper side and stem. Mycelium intercellular, with globose haustoria with narrow and short stalk of 1e3 mm long, 2e5 mm diam, one or two per host cell. Sporogenous hyphae hyaline, clavate or cylindrical, straight to
1. Warts of wall ornamentation in oospores often connected forming ridges and often branched .............. 2 Warts tuberculated and isolated without ridges and not branched ........................... 4 2. Wall ornamentation pseudo-reticulated; warts mostly connected and branched .............. A. hohenheimia Wall ornamentation tuberculate; warts isolates or connected .............................. 3 3. Size of oospores mostly exceeding 45 mm diam; wall ornamentation mostly obvious ............... A. candida Size of oospores less than 45 mm diam; wall ornamentation often unclear .................... A. laibachii 4. Warts densely and regularly distributed, blunt or rounded; oospores often exceeding 50 mm diam ............ 5 Warts irregularly to regularly distributed, variable in shape; oospores mostly less than 50 mm in diameter ........ 6 5. Oospores yellowish to brownish; size of oospores 44e55 (av. 48.4) mm diam .................. A. voglmayrii Oospores pale yellowish; size of oospores 46e61 (av. 53.6) mm diam ....................... A. resedae 6. Warts regularly distributed, often more than 5 mm wide; tips of warts mostly blunt or rounded ............. 7 Warts irregularly distributed, mostly less than 4 mm wide; tips of warts obtuse, echinulate or truncate .......... 8 7. Warts small; 2e3.5 mm long, 2e5 mm wide ........................................ A. lepidii Warts large; 4e6 mm long, 3e6(e7) mm wide ..................................... A. hesleri 8. Warts very rarely connected, slightly curved, often elongated up to 6 mm ...................... A. koreana Warts often connected, variable in size and shape, mostly less than 4 mm ............... A. rorippae sp. nov.
Taxonomy Previously, most taxonomists and plant pathologist identified the WBR pathogen on Rorippa species as Albugo candida, which was originally described on Capsella bursa-pastoris, but Shaw (1973) considered it as Albugo cruciferarum. The latter name is now considered a synonym of A. candida (Choi et al. 2007). The results of the investigation of phylogenetic and phenetic characters in Albugo specimens from Rorippa highlight the existence of a new species of Albugo on this host, which is therefore described here. Albugo rorippae Y.J. Choi, H.D. Shin, Ploch, & Thines, sp. nov. (Fig 3)
slightly curved, (27.5e)34.5e50.7(e62.5) (av. 42.6) mm long, (10e)10.7e14.4(e16.3) (av. 12.6) mm wide (n ¼ 50), mostly grouped, thick-walled, especially towards the base up to 6 mm. Sporangia arranged in basipetal chains, hyaline, primary sporangia similar to the secondary sporangia, but the first exhibit a slightly thicker wall and somewhat smaller size; primary sporangia mostly globose or polyangular due to mutual pressure, (12e)13.6e16.3(e18) (av. 14.9) mm diam (n ¼ 80), with uniformly thin wall of ca. 1.0e1.5 mm; secondary sporangia subglobose to obovoid or globose, (13.5e)15.0e18.5(e21.5) (av. 16.8) mm diam (n ¼ 100), mostly with uniformly thin wall, but rarely slightly thickening (0.5e1.0 mm) as an equatorial
Three new phylogenetic lineages close to Albugo candida
ring, tip round, base subtruncate or rounded, pedicel mostly absent. Resting organs often present as yellowish to brown dots on both the upper and lower surfaces of the leaf spots. Oogonia irregular or broadly globose, pale yellowish, (45e) 50.9e60.7(e62.5) (av. 55.8) mm diam (n ¼ 70); wall smooth, as observed in both mature and immature oospores, 2e3 mm thick. Oospores plerotic, yellowish, globose, (32e) 35.4e42.0(e47) (av. 38.7) mm diam, but (35e)38.8e46.0(e51) (av. 42.4) mm diam (n ¼ 56) when including the height of tubercles; wall 2e3 mm thick, irregularly tuberculate, without ridges. Tubercles mostly single, rarely connected between neighbouring ones, but not branched, with a ripple-like pattern between the ridges, largely variable in shape and size, (1e)3e4(e5) mm long, (1e)2e4(e6) mm wide; apex mostly obtuse but often echinulate or truncate. Habitat: in living leaves of Rorippa amphibia, R. palustris, Rorippa pyrenaica, and Rorippa sylvestris (Brassicaceae). Additional specimens examined: see Table 1.
Discussion Before recent molecular phylogenetic analyses have proven the genetic diversity of Albugo candida (Choi et al. 2006; € ller 2006), most taxonomic and plant Voglmayr & Riethmu pathogenic studies assumed that only the generalist A. candida is responsible for WBR incidents on a large number of host plants of the Brassicaceae and allied families. Stimulated by the studies mentioned above, subsequent investigations revealed six new species, which are not closely related to A. candida, parasitising specific host plants (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010). In the present study, three additional, specialised lineages have been revealed on the host genera Alyssum, Barbarea, and Rorippa. In contrast to previously known species, these are closely related to A. candida. From these three host genera, only in specimens from Rorippa oospores were observed, which are of crucial importance for species delimitation in Albugo (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010). Oospores of Albugo from Rorippa differ from previously described species in characteristics of the oospore wall, in particular the morphology of the protuberances. The taxonomic independence of Albugo rorippae is also in agreement with a result of previous cross-inoculation experiments; Pound & Williams (1963) observed a high degree of specificity for the Albugo isolate they collected from Rorippa islandica. This isolate showed some potential to infect Armoracia, while Albugo from Armoracia was found to have limited infection potential on Rorippa. This is in line with the current study, which shows that specimens from Armoracia and Rorippa are closely related. Some differences in oospore ornamentation were observed for Albugo from Armoracia and A. rorippae (data not shown), but given the very close relationship of these lineages, several additional specimens from both host genera have to be investigated in order to evaluate, whether the lineages from Armoracia and Rorippa represent independent species. In addition to A. rorippae, we observed two phylogenetically separated lineages closely related to A. candida s.str., one
605
occurring on Alyssum montanum and another on Barbarea vulgaris, both most likely representing independent species. However these could not be morphologically described in this study, as no oospores were found in either host. A morphological form ‘Cystopus candidus (a synonym of A. candida) forma alyssi alyssoidis’ vulescu & Rayss 1930) and a host-specific form ‘C. candidus (Sa vulescu 1946) were introduced for the f. sp. alyssi-alyssoidis’ (Sa WBR agent occurring on Alyssum alyssoidis. However, as the host plant, A. alyssoidis, was not included in this study and the vulescu (1946) is too size variation of sporangia as given by Sa high for morphological delimitation, we could not determine if one of the two Albugo lineages from A. montanum and Alyssum caliacrae examined might represent this specialised form. As mentioned above, Albugo specimens from A. montanum, Armoracia, Barbarea, Rapistrum, and Rorippa are closely related to A. candida. This affinity is different from all other species of Albugo described and confirmed so far, all of which were distantly related to A. candida, with significant sequence divergences. Taking into consideration the current result and those of previous studies (Choi et al. 2006, 2007, 2008; € ller 2006; Thines et al. 2009; Thines & Voglmayr & Riethmu Voglmayr 2009; Ploch et al. 2010), it is likely that the biodiversity of Albugo on Brassicaceae is comparable to the diversity observed for members of the downy mildew genus Hyaloperonospora, which is also predominantly parasitic to € ker et al. 2004, the Brassicaceae (Choi et al. 2003, 2011; Go 2009). Future studies are thus likely to reveal additional, specialised Albugo species on Brassicaceae. The colonisation of a single host species by two distinct WBR species has been previously observed on Capsella bursapastoris (Choi et al. 2007) and Arabidopsis thaliana (Thines et al. 2009), where the presence of the generalist A. candida and apparently specialised species, Albugo koreana and Albugo laibachii, could be observed, respectively. In the present work, eleven specimens originating from four Rorippa species in different geographic regions were investigated, none of which is conspecific with A. candida. Although this does not exclude the possibility that A. candida could potentially infect these hosts, on Rorippa sp. this species seems to be less common than in A. thaliana or C. bursa-pastoris. In contrast to recently described Albugo species, which all seem to have a high degree of specialisation, possibly being restricted to only a particular host species (Choi et al. 2007, 2008; Thines et al. 2009; Ploch et al. 2010), A. rorippae might have rather a broader host range, encompassing various species of the genus Rorippa. The pattern of specialisation observed in A. rorippae is similar to a case of Albugo lepidii, for which it was shown that specimens from at least five Lepidium sp. form a monophyletic group. However, some genetic diversity was observed within A. lepidii specimens from different host plants (Ploch et al. 2010). Therefore it cannot be ruled out that some genetic isolation is already present between these lineages, which might justify classifying them as independent taxa. The genetic uniformity of A. rorippae from the hosts included in this study might reflect the close relationship of their host plants, as all Rorippa species examined presently, viz Rorippa amphibia, Rorippa palustris, Rorippa pyrenaica, and Rorippa sylvestris, belong to the section Rorippa of the genus (Bleeker et al. 2002). Similarly, the phylogenetic relationship between Albugo specimens infecting the Armoracia, Barbarea, and Rorippa might be due
606
to the fact that three genera are phylogenetically close to each other. All three genera belong to the tribe Cardamineae of Brassicaceae (Les 1994; Franzke et al. 1998; Koch et al. 2001; AlShehbaz et al. 2006). This might be the result of host clade limited colonisation and subsequent speciation, but could also represent a first hint of long-term coevolution. The host plants, Barbarea and Rorippa, are also commonly distributed in East Asian regions, including China, Japan, and Korea, but there are no records of WBR on these host genera from these countries. It can be assumed that there might be geographic barriers as proposed for A. koreana (Choi et al. 2007) and A. voglmayrii (Choi et al. 2008), which hinder the spread of the pathogens from West to East Eurasia. The finding that Armoracia is parasitised by a species distinct from A. candida points out that care should be taken not to spread this pathogen to new areas where WBR of horseradish has not yet been reported. Whether A. candida might also occur on this host needs to be clarified by comprehensive sampling from this host. Previous studies have shown a high host specificity of Albugo iso vulescu 1946; Pound & lates from Brassicaceae (Hiura 1930; Sa Williams 1963; Petrie 1988), but the phylogenetic relationships of these isolates cannot be clarified on the basis of these reports. It seems possible that distinct species were investigated, but it could also be that specialised forms of A. candida have been studied (Borhan et al. 2008). In contrast to these results, based on € rstad (1964) cross-inoculation tests, Safeeulla (1952) and Jo reported that A. candida can attack plants in the Cleomaceae, and recently Khunti et al. (2000) showed that isolates from Brassica juncea (Brassicaceae) successfully infect Cleome viscosa (Cleomaceae). The cross-inoculation results were in agreement with the molecular phylogenetic analyses of Choi et al. (2007, 2009) that Albugo specimens from C. viscosa are identical with A. candida s.str. from various plants of the Brassicaceae. However, judging from the present result, isolates of Barbarea and Rorippa, might not serve as a source of the disease on cultivated crops, although further extensive sampling from other geographic regions is required to ascertain this assumption.
Acknowledgements The authors are grateful to the curators of BP, BPI, FH and KRAM-F for providing the Albugo specimens. SP and MT are financially supported by the research funding programme “LOEWE e Landes-Offensive zur Entwicklung Wissenschaft€ konomischer Exzellenz" of Hesse’s Ministry of Higher lich-o Education, Research, and the Arts.
references
Al-Shehbaz IA, Beilstein MA, Kellogg EA, 2006. Systematics and phylogeny of the Brassicaceae (Cruciferae): an overview. Plant Systematics and Evolution 259: 89e120. Bachofer M, 2004. Molekularbiologische Populationsstudien an Plasmopara halstedii, dem Falschen Mehltau der Sonnenblume. Uni€ t Hohenheim, Germany, pp. 1e140. versita Biga MLB, 1955. Riesaminazione delle specie del genere Albugo in base alle morfologia dei conidi. Sydowia 9: 339e358.
Y. -J. Choi et al.
Bleeker W, Weber-Sparenberg C, Hurka H, 2002. Chloroplast DNA variation and biogeography in the genus Rorippa Scop. (Brassicaceae). Plant Biology 4: 104e111. € r M, Rimmer SR, Borhan MH, Gunn N, Cooper A, Gulden S, To Holub EB, 2008. WRR4 encodes a TIR-NB-LRR protein that confers broad-spectrum white rust resistance in Arabidopsis thaliana to four physiological races of Albugo candida. Molecular PlanteMicrobe Interactions 21: 757e768. Choi YJ, Hong SB, Shin HD, 2003. Diversity of the Hyaloperonospora parasitica complex from core brassicaceous hosts based on ITS rDNA sequences. Mycological Research 107: 1314e1322. Choi YJ, Hong SB, Shin HD, 2006. Genetic diversity within the Albugo candida complex (Peronosporales, Oomycota) inferred from phylogenetic analysis of ITS rDNA and COX2 mtDNA sequences. Molecular Phylogenetics and Evolution 40: 400e409. Choi YJ, Shin HD, Hong SB, Thines M, 2007. Morphological and molecular discrimination among Albugo candida materials infecting Capsella bursa-pastoris world-wide. Fungal Diversty 27: 11e34. Choi YJ, Shin HD, Ploch S, Thines M, 2008. Evidence for uncharted biodiversity in the Albugo candida complex, with description of a new species. Mycological Research 112: 1327e1334. Choi YJ, Shin HD, Thines M, 2009. The host range of Albugo candida extends from Brassicaceae through Cleomaceae to Capparaceae. Mycological Progress 8: 329e335. Choi YJ, Shin HD, Voglmayr H, 2011. Reclassification of two Peronospora species parasitic on Draba in Hyaloperonospora based on morphological and molecular phylogenetic data. Mycopathologia 171: 151e159. Choi YJ, Thines M, 2011. Morphological and molecular confirmation of Albugo resedae (Albuginaceae; Oomycota) as a distinct species from A. candida. Mycological Progress, in press, doi:10.1007/s11557-010-0683-4. Constantinescu O, Negrean G, 1983. Check-list of Romanian Peronosporales. Mycotaxon 16: 537e556. Farr DF, Rossman AY, 2010. Fungal Databases, Systematic Botany & Mycology Laboratory. ARS, USDA.http://nt.ars-grin.gov/fungaldatabases/ [continuously updated]. Franzke A, Pollmann K, Bleeker W, Kohrt R, Hurka H, 1998. Molecular systematics of Cardamine and allied genera (Brassicaceae): ITS and noncoding chloroplast DNA. Folia Geobot 33: 225e240. € ker M, Riethmu € ller A, Voglmayr H, Weiß M, Oberwinkler F, Go 2004. Phylogeny of Hyaloperonospora based on nuclear ribosomal internal transcribed spacer sequences. Mycological Progress 3: 83e94. € ker M, Voglmayr H, Bla zquez GG, Oberwinkler F, 2009. Species Go delimitation in downy mildews: the case of Hyaloperonospora in the light of nuclear ribosomal ITS and LSU sequences. Mycological Research 113: 308e325. Hiura M, 1930. Biological forms of Albugo candida (Pers.) Kuntze on some cruciferous plants. Japanese Journal of Botany 5: 1e20. € rstad I, 1964. The Phycomycetous genera Albugo, Bremia, PlasJo mopara, and Pseudoperonospora in Norway, with an appendix containing unpublished finds of Peronospora. Nytt Magasin for Botanikk 11: 47e82. Katoh K, Kuma K, Toh H, Miyata T, 2005. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: 511e518. Khunti JP, Khandar RR, Bhoraniya MF, 2000. Studies on host range of Albugo cruciferarum the incitant of white rust of mustard. Agricultural Science Digest 20: 219e221. Koch M, Haubold B, Mitchell-Olds T, 2001. Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear Chs sequences. American Journal of Botany 88: 534e544. Kochman J, Majewski T, 1970. Grzyby (Mycota). Tom IV. Glonowce stwowe Wy(Phycomycetes), Wroslikowe (Peronosporales). Pan dawnictwo Naukowe, Warszawa.
Three new phylogenetic lineages close to Albugo candida
Les DH, 1994. Molecular systematics and taxonomy of lake cress (Neobeckia aquatica; Brassicaceae), an imperiled aquatic mustard. Aquatic Botany 49: 149e165. May KJ, Ristaino JB, 2004. Identity of the mtDNA haplotype(s) of Phytophthora infestans in historical specimens from the Irish potato famine. Mycological Research 108: 471e479. Moncalvo JM, Wang HH, Hseu RS, 1995. Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87: 223e238. Novotel’nova NS, Pystina KA, 1985. Ordo Peronosporales (Fam. Pythiaceae, Phytophthoraceae, Peronosporeaceae, Cystopaceae). In: Gorlenko MV (ed.), Cryptogamic Flora of USSR. Fungi (3), vol. 11. Nauka, Leningrad. Petkowski JE, Cunnington JH, Minchinton EJ, Cahill DM, 2010. Molecular phylogenetic relationships between Albugo candida collections on the Brassicaceae in Australia. Plant Pathology 59: 282e288. Petrie GA, 1988. Races of Albugo candida (white rust and staghead) on cultivated Cruciferae in Saskatchewan. Canadian Journal of Plant Pathology 10: 142e150. Ploch S, Choi YJ, Rost C, Shin HD, Schilling E, Thines M, 2010. Evolution of diversity in Albugo is driven by high host specificity and multiple speciation events on closely related Brassicaceae. Molecular Phylogenetics and Evolution 57: 812e820. Pound GS, Williams PH, 1963. Biological races of Albugo candida. Phytopathology 63: 1146e1149. Safeeulla KM, 1952. Morphological and cytological studies of Albugo species on Ipomoea hederacea. Current Science 18: 287e288. Saharan GS, Verma PR, 1992. White Rusts: a review of economically important species. International Development Research Centre, Ottawa, Ontario. vulescu O, 1946. Studiul speciilor de Cystopus Le v.: din Europa, Sa asupra speciilor din Roma ^ nia. Teza, Stiinte cu privire speciala din Bucuresti. vulescu T, Rayss T, 1930. Contribution a la connaissance des Sa ronosporace es de Roumanie. Annales Mycologici 28: 297e320. Pe Shaw CG, 1973. Host fungus index for the Pacific Northwest e I. Hosts. Washington State University, Agricultural Experiment Station Bulletin 765: 1e121. Stamatakis E, 2006. RAxML-VI-HPC: maximum likelihood based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688e2690.
607
Stanyavichene S, 1984. Peronosporovye griby Pribltiki. ((Peronosporaceous fungi of the Baltic States)). Mokslas, Vilnius. Swofford DL, 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596e1599. Telle S, Thines M, 2008. Amplification of cox2 (w620 bp) from 2 mg of up to 129 years old herbarium specimens, comparing 19 extraction methods and 15 polymerases. PLoS ONE 3: e3584. Thiers B, 2010. Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium.http://sweetgum.nybg.org/ih/ [continuously updated]. Thines M, Choi YJ, Kemen E, Ploch S, Holub EB, Shin HD, Jones JDG, 2009. A new species of Albugo parasitic to Arabidopsis thaliana reveals new evolutionary patterns in white blister rusts (Albuginaceae). Persoonia 22: 123e128. Thines M, Spring O, 2005. A revision of Albugo (Chromista, Peronosporomycetes). Mycotaxon 92: 443e458. Thines M, Voglmayr H, 2009. Evolution, phylogeny and diversity of the white blister rusts (Albuginales). In: Lamour K, Kamoun S (eds), Oomycete Genetics and Genomics: biology, interactions with plants and animals, and research toolbox. WileyeBlackwell, pp. 77e92. Ul’yanishchev VI, Osipian LL, Kanchaveli LA, Akhundov TM, 1985. Peronosporovye Griby. ((Peronosporaceous Fungi)). In: Ul’yanishchev VI (ed.), Opredelitel’ Gribov Zakavkaz’ya. Erevan University, Erevan. Vanev SG, Dimitrova EG, Ilieva EI, 1993. Gybite v Bylgariya. 2 tom. Razred Peronosporales. ((Fungi Bulgaricae. Vol. 2. Ordo Peronosporales)). Bulgarian Academy of Sciences, Sofia. Voglmayr H, 2008. Progress and challenges in systematics of downy mildews and white blister rusts: new insights from genes and morphology. European Journal of Plant Pathology 122: 3e18. € ller A, 2006. Phylogenetic relationships of Voglmayr H, Riethmu Albugo species (white blister rusts) based on LSU rDNA sequence and oospore data. Mycological Research 110: 75e85. Wilson GW, 1907. Studies in North American Peronosporales-I. The genus Albugo. Bulletin of the Torrey Botanical Club 34: 61e84.