Pseudoperonospora cubensis Causing Downy Mildew on Luffa aegyptiaca in Korea
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Abstract
In November 2021, sponge gourd (Luffa aegyptiaca) plants with typical downy mildew symptom were observed in Korea. Based on morphological characteristics of sporangiophores and sporangia, combined with molecular phylogenetic analysis of the internal transcribed spacer rDNA and cox2 mtDNA regions, the causal pathogen was identified as Pseudoperonospora cubensis (Oomycota). This is the first report of downy mildew caused by P. cubensis on L. aegyptiaca in Korea. Given that P. cubensis exhibits high disease incidence and severity on many Cucurbitaceae crops, it could pose a significant threat to sponge gourd cultivation.
The genus Pseudoperonospora, which belongs to Oomycota, is an obligate biotrophic group responsible for downy mildew disease (Thines, 2014). Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev is a well-known species causing cucumber downy mildew (CDM) on many Cucurbitaceae crops worldwide (Choi et al., 2005; Cohen et al., 2015; Lebeda and Cohen, 2011; Ojiambo et al., 2015; Savory et al., 2011; Sun et al., 2017; Thines and Choi, 2016). CDM appears as mosaic-like spots on leaves and limits photosynthesis, resulting in stunted growth, immature fruit formation, and premature defoliation (Lebeda et al., 2011; Rhouma et al., 2022; Zhang et al., 2019). Asexual structures, including sporangiophores and sporangia, are developed mainly on the lower leaf surface but also on the upper surface in severe cases. Economic losses due to CDM are increasing annually in many countries (Lebeda et al., 2011; Savory et al., 2011; Sun et al., 2022). In Korea, P. cubensis has been reported to affect various Cucurbitaceae plants, including cucumber, gourd, melon, pump-kin and watermelon (Choi et al., 2005; Choi and Shin, 2008; Kwon and Park, 2006; Lee et al., 2021; Shin and Choi, 2006; The Korean Society of Plant Pathology, 2022).
In November 2021, downy mildew disease was observed at a farm of Luffa aegyptiaca in Iksan-si, Korea. The affected leaves exhibited yellow to light green, vein-limited, mosaic-like spots (Fig. 1A, B). A representative sample was deposited in the Kunsan National University Herbarium (KSNUH) with an accession number of KSNUH 1753. Morphological characteristics of the causal pathogen were observed using a stereo microscope (M205C microscope; Leica, Wetzlar, Germany) equipped with a Dhyana 400DC camera (Tucsen, Fuzhou, China) and a DIC microscope (Axio Imager M2 AX10 microscope) with an Axio Cam 512 camera (Carl Zeiss, Jena, Germany). Sporangiophores were tree-like, hyaline, straight or slightly curved, protruded through stomata, and measured (107.8–) 120.04–146.71 (–163) (average, 133.38) μm in height and (3.4–) 3.97–5.17 (–5.9) (average, 4.57; n=50) μm in width (Fig. 1C-I). Trunks were (67.2–) 81.46–104.16 (–120.9) (average, 92.81; n=50) μm in length from the bottom to the first branch. Ultimate branchlets were straight to slightly curved and paired, with the longer branchlets of (5–) 7.15–13.17 (–16.3) (average, 10.16) × (1.6–) 2.04–2.89 (–3.8) (average, 2.47) μm (n=50) and the shorter ones of (3.4–) 4.5–9.58 (–12.9) (average, 7.04) × (1.6–) 1.92–2.55 (–3.3) (average, 2.23) μm (n=50), with a blunt tip (Fig. 1F-I). Sporangia were pale brown-ish, ovoidal or lemon-shaped, and measured (24.4–) 26.84–31.09 (–32.6) (average, 28.97) × (16.7–) 18.19–20.97 (–22.8) (average, 19.58) μm (n=50), with a length/width ratio of (1.27–) 1.38–1.57 (–1.68) (average, 1.48) (n=50). The sporangia had an operculum at the apex and a pedicel at the base (Fig. 1J-M). The morphological characteristics of sporangiophores and sporangia were consistent with those of Pseudoperonospora cubensis (Waterhouse and Brothers, 1981), except for the relatively shorter sporangiophores, compared with those (approximately 150–600 μm) of P. cubensis samples originated from different host plants (Runge and Thines, 2011).
Downy mildew symptoms and morphological characteristics of Pseudoperonospora cubensis occurring on Luffa aegyptiaca. (A, B) Vein-limited, mosaic-like spots on the upper (A) and lower (B) surfaces of an L. aegyptiaca leaf. (C-E) Sporangiophores and sporangia of P. cubensis on an infected leaf observed under a stereo microscope (scale bars: 150 μm). (F-I) Sporangiophores of P. cubensis observed under a DIC microscope. (J-M) Sporangia with an operculum at the apex and a pedicel at the base (scale bars: 50 μm for sporangiophores and 20 μm for sporangia).
To perform molecular phylogenetic analysis, G-DNA was extracted from the herbarium specimen (KSNUH 1753) using the MagListo 5M plant genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). Polymerase chain reaction (PCR) was performed with primers DC6/LR0 (Cooke et al., 2000; Thines, 2007) for the internal transcribed spacer (ITS) region of ribosomal DNA and cox2-F/cox2-RC4 (Choi et al., 2015; Hudspeth et al., 2000) for cytochrome c oxidase II (cox2) of mitochondrial DNA. The PCR mixture included 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), making a total volume of 25 μl by filling with nuclease-free water (Sigma-Aldrich, Merck, St. Louis, MO, USA). The amplification protocols were as follows: for the ITS region, an initial denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 54.5°C for 40 sec, elongation at 72°C for 1 min, and a final elongation at 72°C for 4 min. For the cox2 region, initial denaturation at 95°C for 4 min, followed by 35 cycles of 95°C for 40 sec, 49.5°C for 40 sec, 72°C for 1 min, and a final elongation at 72°C for 7 min. 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 (1,138 bp for ITS region and 554 bp for cox2 gene) were edited using the DNAStar software package version 5.05 (DNAStar, Inc., Madison, WI, USA) and deposited with The National Center for Biotechnology In-formation (NCBI) GenBank (accession numbers: PQ047487 for ITS and PQ059186 for cox2). Sequence comparison using NCBI BLAST+ version 2.15.0 (Camacho et al., 2009) revealed a 100% identity with reference sequences of P. cubensis (OP142404.1 for ITS and HM988996.1 for cox2). To perform phylogenetic analysis, the sequences were aligned using the MAFFT online service version 7 (Katoh et al., 2019). Minimum evolution and maximum likelihood trees were reconstructed in MEGA version 11 (Tamura et al., 2021) by applying the Tamura-Nei model method with 1,000 rounds of bootstrapping for tree reliability. In a phylogenetic tree inferred from cox2 sequences (Fig. 2), the pathogen associated with L. aegyptiaca placed within the Clade 1 group of P. cubensis with high supporting values of 98% in minimum evolution and 97% in maximum likelihood analyses. Similar to the cox2 tree, the ITS tree showed that the Korean sample grouped with P. cubensis, but there was no distinction between Clades 1 and 2 (data not shown).
Phylogenetic analysis of Pseudoperonospora species inferred from minimum evolution analysis using cox2 mtDNA sequences. Boot-strapping values (minimum evolution/maximum likelihood bootstrapping) above 60% are shown above the branches (1,000 replicates). The scale bar equals the number of nucleotide substitutions per site. Two coloured boxes indicate two different phylogenetic clades of Pseudoperonospora cubensis: the blue box for Clade I and the green box for Clade II. The Koran sample is highlighted in bold within Clade I.
As many Cucurbitaceae plants are globally cultivated for food and medicinal purposes, the increasing damage caused by CDM presents a significant concern worldwide (Cohen et al., 2013; de Moraes et al., 2023; Kitner et al., 2015; Lebeda and Cohen, 2011; Quesada-Ocampo et al., 2012; Runge et al., 2011). The CDM is notably severe in greenhouse environments due to high humidity and moderate temperatures (Lebeda and Cohen, 2011). To manage P. cubensis, various methods are employed: physical strategies such as regulating the temperature and humidity of the cultivation environments and chemical approaches by applying fungicides (Jones et al., 2021; Miao et al., 2018; Ojiambo et al., 2015). Recently, a biological control system has been established by interacting with other fungi and enhancing the immunity of plants (Gabriel-Ortega et al., 2020; Sun et al., 2022). The present study is the first report on P. cubensis on L. aegyptiaca in Korea, providing detailed morphological and phylogenetic data on P. cubensis, a generalist of Cucurbitaceae crops.
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Conflicts of Interest
No potential conflict of interest relevant to this article was reported.