Introduction

Downy mildews (Peronosporaceae; Oomycota) are a group of obligate biotrophic fungal-like microorganisms infecting a wide range of hosts from mono- to dicotyledonous plants, and contain many economically relevant species (Thines and Choi 2016), e.g., Bremia lactucae on lettuce, Plasmopara viticola on grape, Pseudoperonospora cubensis on cucurbitaceous crops. In the family Brassicaceae, downy mildew pathogens can cause severe damage to a variety of crops, e.g., horseradish (Armoracia rusticana), mustard greens (Brassica juncea), rapeseed (B. napus), cabbage (B. oleracea), Chinese cabbage (B. rapa), arugula (Eruca vesicaria), wasabi (Eutrema japonicum), watercress (Nasturtium officinale), and radish (Raphanus sativus). Also, several ornamental plant genera, such as Aubrieta, Cheiranthus, Iberis, and Matthiola, are affected by downy mildew, as is Arabidopsis thaliana, a key model organism in the study of the flowering plants. Despite substantial efforts to resolve host ranges and the phylogeny of Brassicaceae-infecting downy mildew species (Constantinescu and Fatehi 2002; Choi et al. 2003; Göker et al. 2004, 2009), only a fraction of the known hosts could be included in these investigations so far, leaving the majority of brassicolous downy mildew (BDM) diversity unexplored.

As a consequence of the huge species diversity in downy mildews, many studies have focused on economically important downy mildew groups (Choi et al. 2009, 2011b, 2015b; Thines et al. 2009; Constantinescu and Thines 2010; Runge et al. 2011; Thines 2011; Telle and Thines 2012; Denton et al. 2015), as resolving their species boundaries has immediate importance for phytosanitary measures and quarantine (Thines and Choi 2016). The importance of understanding species boundaries and the biology of downy mildew pathogens is emphasized by the fact that several downy mildew species, such as Peronospora belbahrii (Thines et al. 2009), Pe. tabacina (Schiltz 1981), Plasmopara halstedii (Leppik 1966), and Pl. viticola (Viennot-Bourgin 1981), have been causing high economic losses outside their native range after their introduction. Due to increased global trade, the introduction of new downy mildew diseases has accelerated over the past two decades, with several new reports of downy mildew disease on a variety of crops and ornamentals, e.g., Pe. belbahrii on basil (Thines et al. 2009), Pe. salviae-officinalis on culinary sage (Choi et al. 2009), Peronospora sp. on Aquilegia (Denton et al. 2015), Pl. wilsonii on Geranium phaeum (Kruse et al. 2016), and Pl. destructor on busy Lizzie (Impatiens walleriana) (Görg et al. 2017).

Maca (or Peruvian Ginseng; Lepidium meyenii) is grown for the nutritional and health value of its root. For the last few decades, an increase in demand has occurred, promoting a quick expansion of cultivated land in both the restricted native range in the Peruvian Andes and also in other countries, especially in South-East Asia. The occurrence of downy mildew disease on maca has been reported in the native area of maca (Icochea et al. 1994), but, to our knowledge, there are no reports from outside South America.

Since November 2014, maca plants showing typical symptoms of downy mildew (Fig. 1a, b) have been found in experimental plots in the Gochang and Pyeongchang counties of South Korea, where several field trials have recently been conducted to determine the ability of the crop to grow in Korean climate and soil conditions. To our knowledge, this constitutes the first report of downy mildew on this plant in Asia. Downy mildew has to be considered as a potential threat to the cultivation of maca, but the taxonomic identity of the causal agent is uncertain. Initially, the downy mildew disease of maca has been attributed to Peronospora parasitica, which was thought to be a generalist species occurring on a broad range of the Brassicaceae and allied families. However, BDM have been generally recognized to be members of the genera Hyaloperonospora or Perofascia (Constantinescu and Fatehi 2002) and to be fairly host-specific (Göker et al. 2003, 2004, 2009; Choi et al. 2011a; Voglmayr et al. 2014). Importantly, as both Hyaloperonospora and Perofascia affect various species of Lepidium, the identification of the downy mildew pathogen of maca is uncertain even at the genus level, although from the first description of this pathogen (Icochea et al. 1994), it seems to be more likely that the species belongs to Perofascia. Given the mostly very narrow host range of BDM, e.g., with the type species H. parasitica s. str. having been confirmed only on Capsella bursa-pastoris (Gäumann 1918; Wang 1944; Chang et al. 1964; Dickinson and Greenhalgh 1977), it seemed possible that the downy mildew of maca is caused by an undescribed species of BDM. Thus, it was the aim of the present study to clarify if maca downy mildew is caused by Perofascia or Hyaloperonospora and if it can be attributed to a previously known species or represents a new species overlooked so far.

Fig. 1
figure 1

Downy mildews associated with Perofascia macaicola sp. nov. on maca (Lepidium meyenii) (KUS-F28527 – holotypus). a A typical symptom of downy mildew disease on leaves and stems; b, c close-up of dense felt-like sporulation on the upper surface; d–f conidiophores; g, h ultimate branchlets; i, j conidia. Scale bars: d–f = 100 μm; g–j = 20 μm

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Materials and methods

Oomycete samples

Six herbarium specimens of downy mildew pathogens originating from five species of Lepidium (L. coronopus, L. latifolium, L. meyenii, L. ruderale, and L. virginicum) were analyzed in this study, along with sequences of nine additional specimens from additional species (L. densiflorum, L. draba, L. ruderale, and L. virginicum) for which sequence data were previously published (Choi et al. 2003; Göker et al. 2004, 2009) and available from GenBank. Information on all specimens is shown in Table 1.

Table 1 Summary of oomycete herbarium specimens investigated in this study
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Morphological analysis

Herbarium specimens were moistened with 70% ethanol and then transferred to 60% lactic acid on a slide. The microscope preparations were warmed up, covered with coverslips, and examined using a Zeiss Imager M2 AX10 microscope (Carl Zeiss, Göttingen, Germany). Differential interference contrast (DIC) micrographs were captured with a Zeiss Axiocam MRc5 digital camera (Carl Zeiss, Göttingen, Germany) and processed using the AxioVision software (Carl Zeiss, Göttingen, Germany). Measurements were performed at 100–200× for conidiophores and at 400× for conidia and ultimate branchlets. Measurements are reported as follows; (minimum) – standard deviation towards the minimum – mean – standard deviation towards the maximum – (maximum).

DNA extraction, PCR, sequencing, and phylogenetic analysis

In total, 5–20 mg of infected plant tissue from herbarium specimens were disrupted in a mixer mill (MM2, Retsch, Haan, Germany), using three iron beads of 3 mm and 1 mm diameter per sample and shaking at 30 Hz for 3 min. Genomic DNA was extracted using the BioSprint 96 DNA Plant Kit (Qiagen, Hilden, Germany) on a KingFisher Flex (Thermo Scientific, Dreieich, Germany) robot. Polymerase chain reaction (PCR) amplification was performed with the primers ITS1-O (Bachofer 2004) and LR-0 (reverse complementary to LR-0R; Moncalvo et al. 1995) for ITS rDNA and cox2-F (Hudspeth et al. 2000) and cox2-RC4 (Choi et al. 2015a) for cox2 mtDNA. The PCR conditions for ITS and cox2 amplifications were as reported by Choi et al. (2015b). Amplicons were sequenced at the Biodiversity and Climate Research Centre (BiK-F) laboratory using primers identical to those used for amplifications.

Sequences were edited using the DNAStar software package (DNAStar Inc., Madison, WI, USA), version 5.05. An alignment of each locus was performed using MAFFT 7 (Katoh and Standley 2013) employing the Q-INS-i algorithm (Katoh and Toh 2008). We used three different tree construction methods: minimum evolution (ME), maximum likelihood (ML), and Bayesian inference (BI). ME analysis was done using MEGA 6.0 (Tamura et al. 2013), with the default settings of the program, except for using the Tamura–Nei model. For ML analyses, 1000 rounds of random addition of sequences as well as 1000 fast bootstrap replicates were performed using RAxML 7.0.3 (Stamatakis 2006), as implemented in raxmlGUI 1.3 (Silvestro and Michalak 2012) using the GTRCAT variant. BI analysis was done using siMBa (Mishra and Thines 2014), with the following parameters: GTR (substitution type), 100,000 (number of generations), 25% (fraction of samples to be discarded).

Results

Morphology

The downy mildew pathogen of maca had hyphal haustoria and appressed (parallel) branches, typical features of the monotypic genus Perofascia. However, the downy mildew pathogen of maca differs markedly from Perofascia lepidii in terms of several morphological characteristics (Table 2). The length of the conidiophores was longer in the maca downy mildew pathogen (290–350 μm) than in Pf. lepidii (180–250 μm). This difference is mostly due to the different length of trunks; 160–240 μm in maca downy mildew pathogen vs. 100–135 μm in Pf. lepidii. Branches and ultimate branchlets were mostly curved in the former species, but mostly sub-straight in the latter species. Conidia from infected maca specimens were ellipsoidal and measured, on average, 35.1 × 22.8 μm, with a length to width ratio of 1.55, but in Pf. lepidii, they were broadly ellipsoidal to ellipsoidal and measured, on average, 28.5 × 20.7 μm, with a length to width ratio of 1.44.

Table 2 Distinctive morphological characters of Perofascia macaicola and Perofascia lepidii
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Molecular phylogenetic inference

In the ITS rDNA and cox2 mtDNA regions, barcoding loci for oomycetes, the maca downy mildew pathogen and Pf. lepidii exhibited significant sequence divergences of 5.8% (46 of 790 characters are different) and 7.8% (44 of 566 characters), respectively. These divergences are higher than for closely related species of Hyaloperonospora, e.g., four species parasitic to Cardamine s.l. (in Fig. 2a), H. nasturtii-aquatici, H. cardamines-laciniatae, H. cardamines-enneaphyllos, and H. dentariae-macrophyllae, which have significantly lower mean pairwise distances of a maximum of 1.5% in ITS and 4.2% in cox2.

Fig. 2
figure 2

Minimum evolution trees based on the ITS rDNA (a) and cox2 mtDNA (b) sequences, with support values in maximum likelihood and Bayesian ineference. Support values (ME BS/ML BS/BI PP) higher than 60% are given above or below the branches. Specimens which originated from Lepidium spp. are in bold. The scale bars equal the number of nucleotide substitutions per site

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In the phylogenetic reconstructions based on ITS rDNA (Fig. 2a) and cox2 mtDNA (Fig. 2b), the maca downy mildew pathogen was placed as a sister group to Pf. lepidii with maximum support in ME, ML, and BI analyses. All specimens of Pf. lepidii originating from L. coronopus, L. densiflorum, L. latifolium, L. ruderale, and L. virginicum grouped together to form a monophyletic group with maximum support in all analyses in the ITS-based tree and high to maximum support in the cox2-based tree. Downy mildew pathogens from Lepidium ruderale belonged to either Perofascia lepidii or a presumably undescribed species of Hyaloperonospora. Downy mildew pathogens from Lepidium draba grouped together in the Hyaloperonospora clade with maximum support.

Discussion

In the first half of the 20th century, a huge amount of Peronospora species has been described (e.g., Gäumann 1918, 1923), based on morphological differences and host ranges. This view, however, had been challenged later, mainly by applied plant pathologists (e.g., Yerkes and Shaw 1959) and has been followed by most plant pathologists until the advent of molecular phylogenetics. Thus, for almost half a century, only the name “Peronospora parasitica” has been widely used for any downy mildew pathogen on Brassicaceae, instead of the about 140 other names of downy mildews occurring on Brassicaceae. Presumably, the broad species concept of Yerkes and Shaw (1959) has also influenced the initial identification of maca downy mildew as “P. parasitica” (Icochea et al. 1994), instead of considering one of the eight names available for Peronospora which have previously been described on members of the genus Lepidium (also including Cardaria and Coronopus); P. cardariae-repentis, P. coronopi, P. coronopi-procumbentis, P. lepidii, P. lepidii-perfoliati, P. lepidii-sativi, P. lepidii-virginici, and P. maublancii (Constantinescu 1991). However, molecular phylogenetic studies since the turn of the century clearly favored the narrow species circumscription of Gäumann (1918, 1923) that a species of downy mildew is usually restricted to a particular host genus or species (Riethmüller et al. 2002; Choi et al. 2003, 2011a; Göker et al. 2003, 2004, 2009; Voglmayr 2003, 2014; ; Voglmayr and Göker 2011). Based on apparent morphological differences and phylogenetic divergence, the maca downy mildew pathogen is not only distinct from H. parasitica but also from Perofascia lepidii, the sole other species of Perofascia. This is again in line with the narrow species delimitation of downy mildews. Importantly, the presence of the second species in the genus Perofascia provides a hint that several genera of downy mildews with only one species might probably contain additional species on other hosts. This is also supported by the recent finding that the genus Basidiophora with the only previously accepted species, B. entospora, does, indeed, consists of several phylogenetic lineages in need of description as a new species (Sökücü and Thines 2014).

When Constantinescu and Fatehi (2002) established the genus Perofasica, they synonymized four out of the eight species described on members of the genus Lepidium (P. coronopi, P. coronopi-procumbentis, P. lepidii-virginici, and P. maublancii) with P. lepidii. This was supported for several of these species by subsequent phylogenetic studies (Choi et al. 2003; Göker et al. 2004, 2009). Similarly, the present phylogenetic study revealed that specimens from Lepidium coronopus (type host of P. coronopi-procumbentis) and L. virginicum (type host of P. lepidii-virginici) form a well-supported group with Pf. lepidii. Although specimens from the type plants of P. coronopi and P. maublancii could not be examined in this study, the description and illustration of the two species (Gäumann 1918; Săvulescu and Rayss 1934) are reminiscent of Pf. lepidii, as previously suggested by Constantinescu and Fatehi (2002). However, these species are morphologically different from Perofascia from maca, supporting the introduction of a second species in this genus. It is notable that all previously described species and specimens investigated so far were derived from the holarctis and that the maca downy mildew pathogen is the first one investigated that originates from the southern hemisphere.

Apart from the five names of Peronospora on Lepidium which have been transferred to Perofascia, three names of Peronospora on Lepidium have either been combined into Hyaloperonospora (P. lepidii-perfoliati) or synonymized with H. parasitica (P. cardariae-repentis and P. lepidii-sativi). In the present study, two phylogenetically distant lineages of Hyaloperonospora on Lepidium were observed, specific to L. draba or L. ruderale. Including these two hosts, five species of Lepidium have been listed as hosts of P. lepidii-sativi in the original description by Gäumann (1918). Thus, a specimen from the type host plant, Lepidium sativum, should be examined in future studies to determine if either of the two lineages observed in the current study corresponds to this name. On Lepidium ruderale, both Hyaloperonospora and Perofascia were found in this study, in line with the previous morphological investigations, which reported the co-existence of the two different genera on this plant species (Constantinescu and Fatehi 2002).

Little is known about the biology and epidemiology of BDM, and this is the first report of downy mildew disease on maca outside of the high Andes of Peru. The disease has recently been introduced to South Korea, probably along with maca seeds, which would be in line with the systemic nature of the infections observed in the field and also with the fact that oospores of this pathogen have been found inside seeds (Pérez 1999). This also suggests that the pathogen is capable of spreading rapidly into other areas by seed trade. Since the cultivation of maca has recently started on a commercial scale in various countries, the downy mildew has the potential to become a serious threat to the production of this root crop. Thus, maca downy mildew should be closely monitored, and adequate quarantine and phytosanitary measures for hindering further spread should be considered.

Taxonomy

Based on differences in morphology and molecular phylogeny as well as host range, Perofascia macaicola is described here as a new species.

Perofascia macaicola Y.J. Choi, Thines, I.Y. Choi & H.D. Shin, sp. nov. Fig 1.

MycoBank no.: MB821212.

Etym.: ‘macaicola’ refers to the common name of the host plant, Lepidium meyenii.

Down hypophyllous, whitish to yellowish, dense, felt-like. Haustoria filling the host cell partly to almost completely, hyphal, branched. Conidiophores emerging through stomata, up to 20 in a fascicle, hyaline, slender, straight to slightly curved, (250–)290–350(−420) μm; trunk slightly curved, (150–)160–240(−270) μm long, (3.5–)4.0–5.5(−6.5) μm wide, more or less uniform; basal end not or somewhat swollen, callose plugs absent; branches appressed, slightly parallel, of uniform width, from simple to elaborate structured, branching mostly monopodially but rarely trichotomously (4–)5–6 times, first branching at (1/2–)2/3–3/4 of conidiophore. Ultimate branchlets in pairs or single, curved to reflexuous, (5–)12–20(−25) μm long, 1.5–2(−2.5) μm wide at the base; tip variable, from round to truncate. Conidia hyaline, ellipsoidal to broadly ellipsoidal, (25–)30–37(−40) μm long, (18–)21–24(−26) μm wide, l/w ratio (1.3–)1.41–1.62(−1.7), greatest width median, rarely sub- or supra-median, tip often apiculate in mature conidia but round in young ones, base gradually narrowing; pedicel slightly protruding, 2.5 μm wide and 2 μm long. Resting organs not seen.

Typus: South Korea: Jeollabuk-do; Gochang-gun; Gogneum-myeon, in an experimental plot, on living leaves of Lepidium meyenii affected by downy mildew disease, November 13 2014, Shin, Hyeon-Dong & Choi, In-Young (KUS-F28527 – holotypus). Ex-type sequences: KY986667 (ITS nrDNA) and KY986672 (cox2 mtDNA).

Habitat: On living leaves of Lepidium meyenii Walp. (Brassicaceae).

Distribution: Peru and South Korea.

Additional specimens examined: ditto, November 17 2014 (KUS-F28528); ditto, February 26 2015 (KUS-F28594).