Diversity, Phylogeny, and Host-Specialization of Hyaloperonospora Species in Korea.

The genus Hyaloperonospora (Peronosporaceae; Oomycota) is an obligate biotrophic group that causes downy mildew disease on the Brassicaceae and allied families of Brassicales, including many economically relevant crops, such as broccoli, cabbage, radish, rape, and wasabi. To investigate the diversity of Hyaloperonospora species in northeast Asia, we performed a morphological analysis for the dried herbarium specimens collected in Korea, along with molecular phylogenetic inferences based on internal transcribed spacer rDNA and cox2 mtDNA sequences. It was confirmed that 14 species of Hyaloperonospora exist in Korea. Of these, three species, previously classified under the genus Peronospora, were combined to Hyaloperonospora: H. arabidis-glabrae comb. nov. (ex Arabis glabra), H. nasturtii-montani comb. nov. (ex Rorippa indica), and H. nasturtii-palustris comb. nov. (ex Rorippa palustris). In addition, finding two potentially new species specific to northeast Asian plants is noteworthy in support of the view that the species abundance of Hyaloperonospora has been underestimated hitherto.

The Peronosporaceae is the largest obligate biotrophic family of the phylum Oomycota. They cause downy mildew disease on a wide range of mono-and dicotyledonous plants [1], among which the Brassicaceae and allied families of Brassicales are the most common host plants. A total of 140 names have been introduced to accommodate the Peronospora species infecting the brassicaceous plants [2], more than half of which have been described by Gäumann [34], who discriminated them mainly using subtle morphological difference and host specialization. In addition to the establishment of the new genus Hyaloperonospora for the brassicolous downy mildews (BDMs) [5], recent phylogenetic studies revealed a high level of species diversity and host-specialization of this genus [6789101112]. The collective results support the view that the narrow species circumscription [34] better reflects the natural taxonomic system for BDMs. The results refute the broad species concept of Yerkes and Shaw [13], who attributed the disease to a single species, H. parasitica, although for the last 50 years this concept has been more widely applied to plant pathology and related fields.

Downy mildew is an important disease for many economically relevant brassicaceous crops, particularly species or varieties of Brassica, e.g., the flowerhead types (cauliflower, broccoli), the leaf brassicas (cabbage, Brussels sprouts), the root brassicas (turnips, swedes), oil brassicas (rape), and the horticultural variety (B. oleracea var. acephala). In Korea, commercial crops, including brassicas and radishes, have experienced ongoing severe economic losses [14]. Furthermore, the active importation and cultivation of various overseas crops have led to the emergence of new diseases affecting broccoli [15], tatsoi [16], and spider flower [17]. Previously, Choi et al. [18] described a high phylogenetic diversity for Hyaloperonospora specimens collected in Korea, but the discovered lineages could not be assigned at the species level because of the lack of morphological data and the broad species concept, which prevailed at that time in the taxonomy of downy mildews. The concept has continued to the first monographic studies for the Peronosporaceae in Korea [19]. However, in keeping with the aforementioned taxonomic system for downy mildews, recent studies have certainly reflected the narrow species concept for Hyaloperonospora species parasitic to Cardamine [12] and Draba [11] in Korea (Table 1).

Table 1

List of brassicolous downy mildew genera, Hyaloperonospora and Perofascia, in Korea

aNewly reported in Korea by the present work.

This study evaluated the species diversity of BDMs in Eastern Asia using the herbarium specimens collected in Korea, for which mycological characteristics, phylogeny, and host-specialization of Hyaloperonospora species were investigated. We report the presence of eight interesting species, including three new combinations, two new species candidates, and three species that have been previously unrecorded in Korea (Table 1).

MATERIALS AND METHODS

Oomycete samples

Brassicaceous plants with downy mildew infections were collected from different sites of Korea for the past 15 years. Information on the dried herbarium samples selected for morphological and molecular phylogenetic analyses is provided in Table 2.

Table 2

Information of Hyaloperonospora specimens used in the present study

Morphological analysis

Conidiophores and conidia formed from the underneath of infected leaves or stems were transferred to a drop of lactic acid on a slide glass, covered with a cover slip, and briefly heated using an alcohol lamp. Slides were examined and photographed using a model BX53F microscope (Olympus, Tokyo, Japan) equipped with a DigiRetina 16M digital camera (Tucsen, Fuzhou, China). Measurements are reported as maxima and minima in parentheses and the mean plus and minus the standard deviation of a number of measurements given in parentheses.

DNA extraction, PCR, and sequencing

Genomic DNA was extracted from the infected dry host tissue using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany). The ribosomal internal transcribed spacer (ITS) regions were amplified using primers ITS1-O [24] and LR0 [25]. For the mitochondrial cytochrome c oxidase subunit II (cox2) sequences, primers cox2-F [26] and cox2-RC4 [27] were used for amplification and sequencing. PCR conditions for ITS and cox2 amplifications were identical as outlined in Choi et al. [28]. The PCR products were purified and sequenced by a DNA sequencing service (Macrogen Inc., Seoul, Korea), with the same primers used for amplification.

Phylogenetic analysis

The ITS rDNA and the cox2 mtDNA sequences were edited using the DNAStar software package ver. 5.05 (DNAStar, Inc., Madison, WI, USA). Alignments of each locus were performed using MAFFT 7 [29] and the Q-INS-i algorithm [30], in addition to the previously published reference sequences of Hyaloperonospora and an outgroup taxon, Perofascia lepidii. Minimum evolution (ME) and maximum likelihood (ML) methods were used to construct two different trees. ME analysis was done using MEGA 7.0 [31] with the default settings of the program, except for replacement with the Tamura-Nei model. For ML analysis, 1,000 rounds of random addition of sequences as well as 500 fast bootstrap replicates were performed with RAxML 7.0.3 [32] using the GTRCAT model.

RESULTS

Phylogenetic analyses

After the exclusion of excessive gap regions and large insertions present in the ITS rDNA regions of some Hyaloperonospora species, e.g., H. parasitica s.s. ex Capsella bursa-pastoris, 1,222 characters were included in analyses, of which 379 were parsimony informative. The cox2 matrix contained 509 characters, from which 150 were parsimony informative. As tree topologies of ME and ML inferences were fully compatible (data not shown), only each ME tree is shown for each locus (Fig. 1 for ITS, Fig. 2 for cox2), with ME and ML bootstrap values above 60% at first and second position above/below the branches.

Fig. 1

Minimum evolution trees based on the internal transcribed spacer rDNA sequences, with support values in maximum likelihood inference. Bootstrapping support values (minimum evolution/maximum likelihood) higher than 60% are given above/below the branches. The specimens collected in Korea are shown in bold, and previously unknown species in Korea were highlighted in the colored boxes. The scale bar equals the number of nucleotide substitutions per site.

Fig. 2

Minimum evolution trees based on the cox2 mtDNA sequences, with support values in Bayesian inference. Bootstrapping support values (minimum evolution/maximum likelihood) higher than 60% are given above/below the branches. The specimens collected in Korea are shown in bold, and previously unknown species in Korea were highlighted in the colored boxes. The scale bar equals the number of nucleotide substitutions per site.

In phylogenetic investigations (Figs. 1 and 2), the accessions from different species of Arabis were divided into four distant lineages: one representing H. arabidis-alpinae parasitic on Arabis alpina, a second lineage of H. arabidis-turritae on Arabis turrita, a third lineage on Arabis soyeri previously described by Göker et al. [7], and a fourth clade with two Korean accessions on Arabis glabra, representing a new combination, H. arabidis-glabrae. An unknown lineage of Hyaloperonospora originating from Catolobus pendulus (= Arabis pendula) was supported by high supporting values in both ME/ML analyses of ITS and cox2. It further clustered together with H. arabidis-turritae, H. mayli, and H. thlaspeos-perfoliati. There has been no prior report of downy mildew on this plant, but, considering the phylogenetic divergence, it could be an unknown species.

The accessions from Rorippa species were contained in two major clades (Fig. 1). The first monophyletic clade represented H. nasturtii-montani. The second clade consisted of H. nasturtii-islandicae and H. nasturtii-palustris. The grouping of the latter two species, which are parasitic to only a single plant Rorippa palustris, was highly supported with the values of 91/98%, but each combination harbored only a distinct Hyaloperonospora species.

In the ITS tree, the accessions of the H. brassicae complex were contained in four closely related clades, as suggested by Göker et al. [7]: a first highly supported monophyletic clade consisting of the specimens parasitic on B. campestris, B. narinosa, and B. oleracea, a second on Sinapis alba, a third on Sinapis arvense, and a fourth on Raphanus sativus and R. raphanistrum. Interestingly, the accessions from spider flower, Tarenaya hassleriana (= Cleome spinosa), were placed within the first clade of H. brassicae (Figs. 1 and 2), with no nucleotide differences in the ITS sequences and only one difference in the cox2 sequences, hinting at their close genetic relationship.

The Korean accessions collected from Cardamine impatiens fell within the H. dentariae clade, with weak supporting values in ITS dataset and the maximum value in cox2 mtDNA. In the cox2 dataset, they further formed their own well-supported subclade with the maximum supporting value in both analyses, as shown by Voglmayr et al. [12], who observed the presence of host-specific substructures within the H. dentariae clade. Interestingly, there was a phylogenetic divergence between European and Korean accessions originating from the same plant, C. impatiens. An unknown lineage of Hyaloperonospora originated from Cardamine scutata. It was closest to H. sp. ex Arabis soyeri, but could be a hitherto overlooked species. The Korean accessions collected from Thlaspi arvense grouped with the GenBank sequences of H. thlaspeos-arvensis, with the maximum support in the ITS sequences. This is the first report of H. dentariae and H. thlaspeos-arvensis in Korea.

Taxonomy

Based on the molecular phylogenetic and morphological data, we combined three names of Peronospora to Hyaloperonospora: H. arabidis-glabrae comb. nov., H. nasturtii-montani comb. nov., and H. nasturtii-palustris comb. nov. Along with H. dentariae, H. thlaspeos-arvensis, and Hyaloperonospora sp. [Peronospora cf. cleomes], they were previously unknown to Korea.

Hyaloperonospora arabidis-glabrae (Gäum.) Jae S. Lee & Y. J. Choi, comb. nov. (MB#821809)

Basionym: Peronospora arabidis-glabrae Gäum., Beih. Bot. Zbl. 35: 520 (1918) (MB#231470).

Description: Down hypophyllous, whitish to yellowish, dense, felt-like. Haustoria filling the host cell partly to almost completely, lobate. Conidiophores emerging through stomata, up to 25 in a fascicle, hyaline, somewhat stout, straight to slightly curved, (220–)250–450(–550) µm; trunk straight, (150–)200–300(–350) µm long, (7–)9–12(–14) µm wide (n = 30). Ultimate branchlets mostly in pairs, but rarely single, reflexuous to sigmoid, (14–)16–30(–47) µm long, (1.0–)1.6–2.3(–2.5) µm wide at the base; tip obtuse or subtruncate (n = 30). Conidia hyaline, ellipsoidal to subglobose, (19–)20–23(–26) µm long (av. 21.14), (17–) 18–21(–24) µm wide (av. 19.14), a length to width ratio of (1.00–)1.04–1.18(–1.22) (av. 1.11, n = 30), the greatest width median, rarely sub- or supra-median, tip and base rounded; pedicel mostly present as a scar. Resting organs not seen.

Habitat: On living leaves and stems of Arabis glabra (Brassicaceae).

Specimen examined: KZITFG0000000009 (KUS-F25778), Korea, Yangpyeong, Jungmisan Recreational Forest (37°34′53″ N, 127°27′29″ E), 10 Jun 2011, H. D. Shin & Y. J. Choi.

Note: On Arabis glabra, two species of Peronospora, Peronospora arabidis-glabrae and P. arabidis-turritae (now under Hyaloperonospora), were introduced by Gäumann [3], who discriminated between them by the height of conidiophores; 300–400 µm in the former species and 200–250 µm in the latter. The Korean sample was mostly more than 300 µm, well consistent with P. arabidis-glabrae. The conidia of the Korean specimen were slightly smaller than P. arabidis-glabrae [3] (av. 21.14 × 19.14 µm vs. 23.8 × 21.9 µm), but still larger than P. arabidis-turritae (av. 18.9 × 16.4 µm). In addition, these two species were phylogenetically distant (Fig. 1).

Hyaloperonospora nasturtii-montani (Gäum.) Jae S. Lee & Y. J. Choi, comb. nov. (MB#821810)

Basionym: Peronospora nasturtii-montani Gäum., Beih. Bot. Zbl. 35: 528 (1918) (MB#150102).

Description: Down hypophyllous, whitish, dense, felt-like. Haustoria filling the host cell partly to almost completely, lobate. Conidiophores emerging through stomata, 20–30 in a fascicle, hyaline, somewhat stout, straight, (250–)330–600(–780) µm (av. 530 µm); trunk straight, (180–)220–500(–580) µm (av. 390 µm) long, (8–)10–16(–20) µm wide (n = 30). Ultimate branchlets mostly in pairs, curved to sigmoid, (8–)10–16(–21) µm long, (1.1–)1.2–1.7(–1.9) µm wide at the base; tip obtuse or subtruncate (n = 30). Conidia hyaline, subglobose, (19–)24.0–30.0(–36) µm long (av. 27), (16–)21–27(–31) µm wide (av. 24), a length to width ratio of (1.05–)1.09–1.19(–1.22) (av. 1.14, n = 30), the greatest width at median, with a rounded tip and base; pedicel mostly absent, but rarely present as a scar. Resting organs not seen.

Habitat: On living leaves of Rorippa indica (= Nasturtium montanum) (Brassicaceae).

Specimen examined: KZITFG0000000024 (KUS-F22396), Korea, Pyeongchang, Jinbu-myeon (35°08′59″ N, 128°57′19″ E), 28 Oct 2006, H. D. Shin & Y. J. Choi.

Hyaloperonospora nasturtii-palustris (Gäum.) Jae S. Lee & Y. J. Choi, comb. nov. (MB#821812)

Basionym: Peronospora nasturtii-palustris S. Ito & Tokun., Transactions of the Sapporo Natural History Society 14: 31 (1935) (MB#272042).

Description: Down hypophyllous, whitish, dense, felt-like. Haustoria filling the host cell partly to almost completely, lobate. Conidiophores emerging through stomata, 20–30 in a fascicle, hyaline, somewhat stout, straight, (200–)250–350(–400) µm; trunk straight, (100–)150–250(–300) µm long, (9–)11–15(–16) µm wide (n = 30). Ultimate branchlets mostly in pairs, curved to sigmoid, (3–)6–13(–17) µm long, (1.3–)1.5–2.5(–2.7) µm wide at the base; tip obtuse or subtruncate (n = 30). Conidia hyaline, broadly ellipsoidal to subglobose, (15–)17–20(–22) µm long (av. 18.82), (12–) 13–15(–16) µm wide (av. 14.07), a length to width ratio of (1.07–)1.23–1.45(–1.54) (av. 1.34, n = 30). Resting organs not seen.

Habitat: On living leaves and stems of Rorippa palustris (Brassicaceae).

Specimen examined: KZITFG0000000006 (KUS-F23085), Korea, Namyangju, Deokso Experiment Farm of Korea University (37°35′03″ N, 127°14′11″ E), 22 Oct 2007, H. D. Shin & Y. J. Choi.

Note: Peronospora rorippae-islandicae (now, under Hyaloperonospora) has been introduced to accommodate the downy mildew pathogen on Rorippa palustris [3]. From the same host plant, the second species, P. nasturtii-palustris, was described by Ito and Tokunaga [33]. As these two species slightly differed by the size of conidia (av. 16.62 × 13.79 µm vs. av. 18.05 × 14.71 µm), Gustavsson [34] synonymized the latter species under the former species. The Korean accession (av. 18.82 × 14.07 µm), with a typical morphology of Hyaloperonospora, is closer to P. nasturtii-palustris.

Hyaloperonospora sp. [Peronospora cf. cleomes]

Reference: Peronospora cleomes Hansf., E. African Agric. J. Kenia 8: 250 (1943) (Nom. inval. Article 39).

Description: Down hypophyllous, whitish to yellowish, dense, felt-like. Haustoria filling the host cell partly to almost completely, lobate. Conidiophores emerging through stomata, up to 20 in a fascicle, hyaline, somewhat stout, straight, (250–)300–400(–450) µm; trunk straight, (150–) 200–300(–350) µm long, (7–)9–13(–16) µm wide (n = 30). Ultimate branchlets mostly in pairs, flexuous to sigmoid, (10–)13–23(–31) µm long, (1.4–)1.6–2.5(–3.3) µm wide at the base; tip obtuse or subtruncate (n = 30). Conidia hyaline, broadly ellipsoidal to subglobose, (22–)23–25(–27) µm long (av. 24.50), (20–)22–23(–25) µm wide (av. 21.29), a length to width ratio of (1.04–)1.09–1.21(–1.30) (av. 1.15, n = 30), the greatest width median, rarely sub- or supramedian, tip and base rounded; pedicel mostly present as a scar. Resting organs not seen.

Habitat: On living leaves of Tarenaya hassleriana (Chodat) H. H. Iltis (= Cleome hassleriana; C. spinosa) (Cleomaceae).

Specimen examined: KZITFG0000000010 (KUS-F25462), Korea, Ganghwa, Ganghwa-ri (37°44′38″ N, 126°28′05″ E), 11 Oct 2010, H. D. Shin & Y. J. Choi.

Note: Spider flower is popular in public parks and private gardens worldwide. For the downy mildew pathogen on the plant, two names, Peronospora cleomes [35] and Plasmopara venezuelana [36], were invalidly published, not being accompanied by a Latin description (ICBN Article 39). They are typical of the genus Hyaloperonospora, as also recognized by Constantinescu and Fatehi [5]. Although the latter name has priority, several morphological characteristics of the Korean sample differed from the type specimen CUP-VZ-002477 of Pl. venezuelana (data not shown), and unfortunately, no authentic specimen available for P. cleomes remained. In addition to the close phylogenetic affinity between the Korean sample and H. brassicae s.l., and due to the nomenclature issues mentioned, we leave behind the combination of this species under Hyaloperonospora.

Hyaloperonospora dentariae (Rabenhorst) Voglmayr, Mycol. Prog. 13: 140 (2014) (MB#803661)

Description: Down hypophyllous, whitish, dense, felt-like. Haustoria filling the host cell partly to almost completely, lobate. Conidiophores emerging through stomata, up to 30 in a fascicle, hyaline, somewhat stout, straight, (250–)300–450(–590) µm; trunk straight, (100–)200–350(–400) µm long, (9–)10–13(–17) µm wide (n = 30). Ultimate branchlets mostly in pairs, curved to sigmoid, (5–)7–13(–17) µm long, (1.2–)1.5–2.2(–2.5) µm wide at the base; tip obtuse or subtruncate (n = 30). Conidia hyaline, broadly ellipsoidal to subglobose, (12.5–)14.0–18.0(–19) µm long (av. 16.0), (10–)11.3–15.5(–17) µm wide (av. 12.4), a length to width ratio of (1.05–)1.11–1.29(–1.35) (av. 1.2, n = 30), the greatest width median, rarely sub- or supra-median, tip and base rounded; pedicel mostly present as a scar. Resting organs rarely present and visible as yellow to brown spot on leaves. Oogonia irregular in shape, 36–57 µm diameter (n = 30); wall wrinkled, yellowish, 2–5(–7) µm thick. Oospores plerotic to aplerotic, globose, (22–)25–30 µm diameter, yellowish, 2–3.5 µm thick wall (n = 30).

Habitat: On living leaves and stems of Cardamine impatiens (Brassicaceae).

Specimen examined: KZITFG0000000011 (KUS-F23320), Korea, Cheorwon, Bokjusan Natural Recreation Forest (38°08′40′ N, 127°28′23′ E), 16 May 2008, H. D. Shin & Y. J. Choi.

Note: This cosmopolitan species is parasitic to four different species of Cardamine, including C. impatiens. The morphology fits well with the description of H. dentariae collected from C. heptaphylla [312], except for minor differences in the conidial size. In the cox2 tree, as suggested by Voglmayr et al. [12], there were a few subclades within “H. dentariae complex”, among which the Korean samples formed a well-supported group (Fig. 3), somewhat apart from other isolates of the complex. Further study is necessary to determine whether it may actually represent taxonomically separable entity at the species or the subspecific level.

Fig. 3

Morphological characteristics of four Hyaloperonospora species; H. arabidis-glabrae ex Arabis glabra (in the first column or the left), H. nasturtii-montani ex Rorippa indica (second), H. nasturtii-palustris ex Rorippa palustris (third), and Hyaloperonospora sp. [Peronospora cf. cleomes] ex Tarenaya hassleriana (fourth). A-D, Conidiophores; E–H, Ultimate branchlets; I–P, Conidia. Sources: KZITFG0000000009 (KUS-F25778) for H. arabidis-glabrae, KZITFG0000000024 (KUS-F22396) for H. nasturtii-montani, KZITFG0000000006 (KUS-F23085) for H. nasturtii-palustris, and KZITFG0000000010 (KUS-F25462) for H. sp. [Peronospora cf. cleomes] (scale bars: A–D = 100 µm, E–P = 10 µm).

Hyaloperonospora thlaspeos-arvensis (Gäum.) Göker, Riethm., Voglmayr, M. Weiss & Oberw., Mycol. Prog. 3: 89 (2004) (MB#371143)

Description: Down hypophyllous, whitish, dense, felt-like. Haustoria filling the host cell partly to almost completely, mostly only a haustorium but rarely two in a host cell, vesicle-like or lobate, variable in size (up to 30 µm), with stalk surrounded by thick sheath at the part of entry into the host cell. Conidiophores emerging through stomata, 10–30 in a fascicle, hyaline, somewhat stout, straight, (250–) 300–450(–550) µm; trunk straight, (180–)200–350(–450) µm long, (10–)13–16(–18) µm wide (n = 30). Ultimate branchlets mostly in pairs, curved to sigmoid, (12–)18–28(–33) µm long, (1.5–)1.8–2.4(–2.6) µm wide at the base; tip obtuse or subtruncate (n = 30). Conidia hyaline, broadly ellipsoidal to subglobose, (25–)26.5–30.5(–33) µm long (av. 28.04), (18–)20.5–24.0(–25) µm wide (av. 22.1), a length to width ratio of (1.1–)1.28–1.35(–1.5) (av. 1.31, n = 30), the greatest width median, rarely sub- or supra-median, tip and base rounded; pedicel mostly present as a scar. Resting organs not seen.

Habitat: On living leaves and stems of Thlaspi arvense (Brassicaceae).

Specimen examined: KZITFG0000000012 (KUS-F24060), Korea, Chuncheon, Hoengseong Natural Recreation Forest (37°32′19′ N, 128°06′46′ E), 10 May 2009, H. D. Shin & Y. J. Choi.

DISCUSSION

The species abundance of the genus Hyaloperonospora remains underestimated in northeast Asia. Two monographs have been published for the family Peronosporaceae in China [37] and Korea [19]. Both studies regarded BDMs as only a species, H. parasitica (= Peronospora parasitica), or a few species, undoubtedly according to the “one host family-one pathogen” concept of Yerkes and Shaw [13]. The recent phylogenetic investigations, however, clearly favor the narrow species circumscription of Gäumann [34] that a species of Hyaloperonospora parasitize a particular genus or species of plants of Brassicaceae [678910111238]. The studies highlight the necessity of re-evaluating the species diversity of Hyaloperonospora in this region. In the present study, we confirm the presence of 14 species of this genus, of which six species have been unknown in Korea: H. arabidis-glabrae, H. dentariae, H. nasturtii-montani, H. nasturtii-palustris, H. thlaspeos-arvensis, and H. sp. [Peronospora cf. cleomes]. Although the number of species still seems lower, compared with other European countries, interestingly we discovered two unknown lineages of Hyaloperonospora, specific to two Asian indigenous plants, Cardamine scutata Thunb. and Catolobus pendulus L. (= Arabis pendula). Cardamine scutata is a common herb distributed in northeast Asia, including China [39], Japan [40], Korea [41], and Far Eastern Russia [42]. Similarly, C. pendulus is mainly distributed in northeast Asia, but has also been rarely reported in Europe [4344]. To date, there has been no report of downy mildew on these two plants worldwide. However, considering the phylogenetic divergence and the narrow host specialization previously known for Hyaloperonospora species, they are considered two new species. Finding two potentially new species on northeast Asian plants is noteworthy in support of our argument that the diversity of Hyaloperonospora species present in Asia has been underestimated.