Identification of <i>Golovinomyces ambrosiae</i> Causing Powdery Mildew on <i>Eupatorium</i> spp. in Korea

Jun Hyuk Park; Hyeon-Dong Shin; Young-Joon Choi

doi:10.5423/RPD.2024.30.4.448

ABSTRACT

Powdery mildew is a common fungal disease affecting Eupatorium species, including E. japonicum, E. lindleyanum, E. makinoi var. oppositifolium, and E. tripartitum, in Korea, posing challenges for both ornamental and medicinal uses. Through morphological examination and molecular phylogenetic analyses of the internal transcribed spacer, large subunit, intergenic space rDNA regions and the glyceraldehyde-3-phosphate dehydrogenase gene, the causal pathogen was identified as Golovinomyces ambrosiae, distinct from G. circumfusus, which infects Eupatorium cannabinum in Europe. This study represents the first report of E. tripartitum infected by G. ambrosiae in Korea. The distinct separation between G. ambrosiae and G. circumfusus on Eupatorium species reflects their different geographic distributions between East Asia and Europe, likely resulting from a recent host expansion of G. ambrosiae following the introduction of Eupatorium into East Asia rather than a co-introduction of both the plant and pathogen.

The genus Eupatorium (Asteraceae) comprises approximately 40 species of perennial herbaceous plants distributed across North America, Europe, and East Asia (Schmidt and Schilling, 2000). These plants are traditionally used in herbal medicine due to their bioactive compounds (Hensel et al., 2011). Studies have shown the antibacterial and antioxidant properties of Eupatorium, aiding the development of related commercial products (Liu, et al. 2015). Additionally, several species are valued for ornamental purposes, which enhances their economic significance (Putri, et al. 2022).

Powdery mildew fungi (order Erysiphales) are obligate plant pathogens that require a living host to grow and reproduce, making them impossible to culture in artificial media. These fungi are widely distributed on Eupatorium species across the Americas, East Asia, and Europe. They have been classified into the genera Golovinomyces, Neoerysiphe, and Podosphaera (formerly Sphaerotheca) (Amano, 1986; Braun and Cook, 2012; Qiu, et al. 2020). Recent phylogenetic studies have identified two species of Golovinomyces on Eupatorium: G. circumfusus, which infects E. cannabinum in Europe, and G. ambrosiae, which infects E. japonicum and E. makinoi in Japan (Qiu, et al. 2020). Distinguishing between these species is crucial for understanding their geographic spread, host range, and the impact of powdery mildew on different Eupatorium species.

Powdery mildew fungi (Erysiphales) are obligate plant pathogens that require a living host plant to grow and re-produce, making them unculturable in artificial media. These fungi are widely distributed on Eupatorium spp. across the Americas, East Asia, and Europe (Farr and Rossman, 2024). They have been classified under Golovinomyces (Braun and Cook, 2012; Qiu et al., 2020), Neoerysiphe (Braun and Cook, 2012), and Podosphaera (formerly Sphaerotheca) (Amano, 1986; Braun, 1995). Recent phylogenetic studies have identified two species of Golovinomyces on Eupatorium spp.: G. circumfusus infecting E. cannabinum in Europe, whereas G. ambrosiae infecting E. japonicum and E. makinoi in Japan (Qiu, et al., 2020). Distinguishing between these species is crucial for understanding their spread, host range, and the impact of powdery mildew on different Eupatorium species in varied geographic regions. In Korea, powdery mildew has been reported on four Eupatorium spp., namely, E. chinense var. simplicifolium (synonym of E. makinoi var. oppositifolium), E. fortunei (synonym of E. japonicum), E. lindleyanum (Shin, 1994), and E. rugosum (Shin, 2000). The causal agent was previously identified solely based on morphological characteristics as Golovinomyces cichoracearum sensu lato, other than G. circumfusus and G. ambrosiae. Thus, taxonomic re-examination is needed to clarify the previous records in Korea. The present study aimed to identify the powdery mildew agent on Eupatorium species in Korea using morphological and molecular phylogenetic analyses.

From 1978 to 2024, fourteen powdery mildew samples from four Eupatorium species plants (E. japonicum, E. lindleyanum, E. makinoi var. oppositifolium, and E. tripartitum) were collected across various regions of Korea. The disease initially appeared as white powdery patches on leaves, stems, and inflorescences, later coalescing into larger colonies. (Fig. 1A-F). All herbarium specimens were preserved in the Korea University Herbarium (KUS-F) (Table 1).

Fig. 1.

Powdery mildew symptoms (A-F) and morphological characteristics (G-L) of Golovinomyces ambrosiae on Eupatorium species. (A) Eupatorium japonicum, (B, C) Eupatorium makinoi var. oppositifolium, (D, E) Eupatorium tripartitum, (F) A herbarium specimen of Eupatorium lindleyanum, (G, H) Conidiophore, (I) Primary conidium, (J) Secondary conidium, (K) Conidium with a germ tube, (L) Nipple-shaped appressorium. Scale bar=10 µm.

Table 1.

Powdery mildew specimens from Eupatorium spp. in Korea

Host plant	Herbarium no.	Date	Geographic location	GenBank accession numbers
Host plant	Herbarium no.	Date	Geographic location	ITS	LSU	IGS	GAPDH
Eupatorium japonicum	KUS-F11067	Sep. 1991	Gangneung	-	-	-	-
E. lindleyanum	KUS-F25432	Oct. 2010	Chungju	PQ644049	PQ643305	PQ649836	PQ649845
E. lindleyanum	KUS-F10070	Oct. 1987	Chuncheon	-	-	-	-
E. makinoi var. oppositifolium	KUS-F29337	Jul. 2016	Haenam	PQ644046	PQ643306	PQ649838	PQ649841
E. makinoi var. oppositifolium	KUS-F27506	Aug. 2013	Pocheon	PQ644047	PQ643308	PQ649837	PQ649840
E. makinoi var. oppositifolium	KUS-F32978	Jun. 2022	Buan	-	-	PQ649835	-
E. makinoi var. oppositifolium	KUS-F33418	Oct. 2022	Imsil	-	-	PQ649834	PQ649844
E. makinoi var. oppositifolium	KUS-F11872	Aug. 1992	Suwon	-	-	-	-
E. makinoi var. oppositifolium	KUS-F16617	Sep. 1999	Seoul	-	-	-	-
E. makinoi var. oppositifolium	KUS-F18693	Oct. 2001	Samcheok	-	-	-	-
E. tripartitum^a	KUS-F32169	Nov. 2020	Seoul	PQ644048	PQ643307	PQ649839	PQ649842
E. tripartitum^a	KUS-F34419	Nov. 2024	Seoul	-	PQ643309	PQ649833	PQ649843

ITS, internal transcribed spacer; LUS, large subunit; IGS, intergenic space; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

^a New host plant of powdery mildew in Korea.

The morphological characteristics of the causal pathogen were examined using a DIC microscope (Zeiss Imager M2 AX10; Carl Zeiss, Jena, Germany) equipped with an AxioCam 512 microscope camera (Carl Zeiss). Hyphae were straight to sinuous, measuring 5-6.2 μm in diameter. Conidiophores (n=20) were 122-190 (av. 139)×9.4-12 (av. 10.1) µm, producing 2-4 immature conidia in chains with a sinuate outline. Foot-cells were straight at the basal cell, followed by 2-3 straight cells and 41 to 60 (av. 44.6) µm (Fig. 1G,H). Conidia (n=30) were ellipsoid to oval, 28-37 (av. 32.3)×14-17 (av. 16) µm with a length/breadth ratio of 1.6-2.3, devoid of distinct fibrosin bodies, producing a germ tube on the perihilar position (Fig. 1I). Primary conidia were apically conical to rounded, basally sub-truncate (Fig. 1J). Germ tubes were typically subterminal, indicative of the Euoidium type (Fig. 1K). Hyphal appressoria were nipple-shaped and 3-5 μm in diameter (Fig. 1L). Chasmothecia were not observed. These characteristics matched those of the anamorph of Golovinomyces ambrosiae (Braun and Cook, 2012).

For molecular phylogenetic analysis, genomic DNA (G-DNA) was extracted from the herbarium specimens using the MagListo 5M Plant Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). Polymerase chain reaction (PCR) was performed to amplify the internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions of rDNA, and the glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) gene of nDNA, using primer sets PM10/ITS4 (Bradshaw and Tobin, 2020; White, et al., 1990), PM3/TW14 (Bradshaw and Tobin, 2020), IGS-12a/NS1R (Carbone and Kohn, 1999), and GoGPD-F/GoGPD-R (Park and Choi, 2024), respectively. The PCR mixture consisted of 400 nM of each primer, 1 μl of G-DNA, and 0.8 μg/μl of bovine serum albumin (Biosesang, Seongnam, Korea) in the AccuPower PCR Premix (Bioneer). The total volume was adjusted to 25 μl using nuclease-free water (Sigma-Aldrich; Merck, St. Louis, MO, USA). The PCR conditions followed the methods described by Bradshaw and Tobin for ITS and LSU (Bradshaw and Tobin, 2020), Qiu et al. (2020) for IGS and Park and Choi (2024) for GAPDH. The PCR amplicons were visualized by electrophoresis on a 1.5% agarose gel, purified using the AccuPrep PCR Purification Kit (Bioneer), and sequenced by Macrogen (Daejeon, Korea).

The resulting sequences (563-621 bp for ITS, 746-897 bp for LSU, 254-304 bp for IGS region rDNA and 178-190 bp for GAPDH gene) were edited using the DNAStar software package version 5.05 (DNAStar, Inc., Madison, WI, USA) and registered in the National Center for Biotechnology Information GenBank (Table 1). Sequence alignment of each marker was performed using MAFFT online service version 7 (Katoh and Standley, 2013). A concatenated dataset of ITS, LSU, IGS, and GAPDH sequences was used to construct a phylogenetic tree. Maximum likelihood tree was reconstructed in MEGA version 11 (Tamura et al., 2021) using the Tamura-Nei models, with 1,000 bootstrap replicates to assess tree reliability. The multi-loci phylogenetic tree (Fig. 2) revealed that seven Korean samples formed a well-supported group with reference sequences of G. ambrosiae, clearly distinct from G. circumfusus. The present study provides reliable evidence for distinguishing between G. ambrosiae and G. circumfusus. The identification of Eupatorium specimens from Korea and Japan as G. ambrosiae supports previous findings that this species infects Eupatorium spp. in East Asia (Qiu et al., 2020).

Fig. 2.

Phylogenetic analysis of Golovinomyces species inferred from maximum likelihood analysis based on a concatenated dataset of the internal transcribed spacer, large subunit, intergenic space rDNA and glyceraldehyde-3-phosphate dehydrogenase gene sequences. Boot-strap values above 60% are indicated above the branches. The blue box represents Golovinomyces ambrosiae. The Korean specimens sequenced in the present study are shown in bold. The scale bar represents the number of substitutions per site.

To our knowledge, this is the first report of G. ambrosiae infecting Eupatorium tripartitum in Korea. Morphological and molecular analyses confirm that G. ambrosiae is the causal agent of powdery mildew on E. japonicum, E. lindleyanum, E. makinoi var. oppositifolium, and E. tripartitum in Korea. Although we were unable to obtain sequence data from a powdery mildew specimen (KUS-F11067) collected from E. japonicum, its morphology matched G. ambrosiae. Additionally, a careful re-examination of the Eupatorium rugosum sample (KUS-F16617), previously identified as infected by G. cichoracearum s. lat. (Shin, 2000), revealed that the host plant is indeed E. japonicum, with G. ambrosiae as the causal agent.

The distinction between G. ambrosiae and G. circumfusus on Eupatorium species reflects their different geographic origins. Since Eupatorium spp. spread from North America over Europe to Asia (Schmidt and Schilling, 2000), and G. ambrosiae is a plurivorous species capable of infecting a wide range of host plants across multiple families (Bradshaw et al., 2022; Qiu et al., 2020), the presence of G. ambrosiae on Eupatorium spp. in East Asia likely results from a recent host expansion of G. ambrosiae, after the introduction of Eupatorium spp., rather than a co-introduction along with its host. Similar host expansion events have been observed in other powdery mildew species, such as Erysiphe alphitoides, E. quercicola, Golovinomyces orontii, and Podosphaera xanthii (Kiss et al., 2020; Vagi et al., 2007). Therefore, the present findings provide additional evidence of the host expansion dynamics of powdery mildew fungi.

Identification of Golovinomyces ambrosiae Causing Powdery Mildew on Eupatorium spp. in Korea

ABSTRACT

Fig. 1.

Table 1.

Fig. 2.

NOTES

REFERENCES