Unraveling the Life Cycle of Nyssopsora cedrelae: A Study of Rust Diseases on Aralia elata and Toona sinensis.

Rust disease poses a major threat to global agriculture and forestry. It is caused by types of Pucciniales, which often require alternate hosts for their life cycles. Nyssopsora cedrelae was previously identified as a rust pathogen on Toona sinensis in East and Southeast Asia. Although this species had been reported to be autoecious, completing its life cycle solely on T. sinensis, we hypothesized that it has a heteroecious life cycle, requiring an alternate host, since the spermogonial and aecial stages on Aralia elata, a plant native to East Asia, are frequently observed around the same area where N. cedrelae causes rust disease on T. sinensis. Upon collecting rust samples from both A. elata and T. sinensis, we confirmed that the rust species from both tree species exhibited matching internal transcribed spacer (ITS), large subunit (LSU) rDNA, and cytochrome oxidase subunit III (CO3) mtDNA sequences. Through cross-inoculations, we verified that aeciospores from A. elata produced a uredinial stage on T. sinensis. This study is the first report to clarify A. elata as an alternate host for N. cedrelae, thus providing initial evidence that the Nyssopsora species exhibits a heteroecious life cycle.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Hosta longipes in Korea.

Hosta longipes (Franch. & Sav.) Matsum. (Asparagaceae) is a perennial, herbaceous plant, native to Japan and Korea (Lee et al. 2021). In Korea, the plant is used as an edible vegetable and ornamental (Kang and Ju 2015). During 2021-2022, anthracnose symptoms were observed on leaves of H. longipes with over 70% disease incidence in Wanju-gun (35°38'47''N; 127°31'16''E) and Jangsu-gun (35°35'31''N; 127°30'03''E) in Jeollabuk-do, Korea. The disease initially appeared on old leaves, gradually spreading to young ones. The symptoms were characterized as yellow to white discoloration on the upper leaf surface with black necrotic tissue in the center of the lesion. Three H. longipes samples with anthracnose symptoms were collected. From each, a monoconidial isolate was obtained and then deposited in the Korea Agricultural Culture Collection (accession Nos. KACC 410038, 410391, and 410443). The dried specimens were housed at the herbarium of Jeonbuk National University (JBNU0129, 0137) and Korea University (KUS-F33379). Conidiomata was acervular, 65 to 80 × 56 to 70 µm in diam. Setae were dark brown, 2 to 4-septate, 63 to 161 µm long, being formed on a pale brown cushion. Conidia were hyaline, smooth-walled, aseptate, slightly curved, base truncate, 3.9 to 5.1 × 17 to 23 µm. The appressoria were solitary, olivaceous-brown, ovoid or irregularly shaped. Two-week-old colonies grown on PDA at 25 ℃ were 20-25 mm in diameter, initially white, then turned gray with age, with cottony aerial mycelium. The morphological and cultural characteristics of the fungus were consistent with those of Colletotrichum spaethianum (Allesch.) Damm, P.F. Cannon & Crous (Damm et al. 2012). To confirm morphology-based identification, the nucleotide sequences of the internal transcribed spacer (ITS) rDNA region, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin (actA), chitin synthase (CHS1), histone (HIS3) and tubulin (TUB2) genes were determined for KACC410443, as outlined by Cannon et al. (2012) and Damm et al. (2009). The resulting sequences were submitted into GenBank (PP000829 for ITS, PP133094 for GAPDH, PP083418 for actA, PP133091 for CHS1, PP133097 for HIS3, and PP133099 for TUB2) and compared with reference sequences in GenBank using BLASTn search tool. The results showed a 100% match with C. spaethianum (MT611068), C. incanum (MN880260) and C. truncatum (EF016303) for ITS, and 100% with C. spaethianum for GAPDH (MH370513), actA (MH045677), CHS1 (MH370520), HIS3 (MH985161), and TUB (MH456884). Pathogenicity was tested by inoculating conidial suspension (1 ×104 cfu/ml) of three-week-old fungal colonies of the isolate KACC410443 onto leaves of three healthy potted plants. Prior to inoculation, leaves were deliberately wounded by pinpricking with a sterilized needle. Two wounded but non-inoculated plants served as controls. Plants were maintained in a greenhouse at 25 to 30 °C. Inoculated plants developed anthracnose symptoms after eight days, while the control plants remained symptomless. The fungus isolated from the inoculated plants was morphologically identical to that observed initially, fulfilling Koch's postulates. To our knowledge, there is no previous record of C. spaethianum on H. longipes, although C. spaethianum has been reported to infect another species, H. plantaginea (Cheon and Jeon 2016). This is the first report of this fungus on H. longipes in Korea (KSPP 2024) and globally (Farr and Rossman 2024). The anthracnose on this ornamental plant can be considered a new severe threat to planting strategies in gardens.

First Report of Powdery Mildew Caused by Golovinomyces adenophorae on Adenophora triphylla var. japonica in Korea.

Adenophora triphylla var. japonica (Campanulaceae), known as Japanese lady bell, is native to East Asia. It has been used as a medicinal plant but is widely cultivated in Korea as an indigenous vegetable (Park et al. 2011). In the summer of 2020, about 100 plants in an experimental plot at the National Institute of Forest Science, Seoul, Korea, showed powdery mildew symptoms with a 100% disease incidence. Signs first appeared as white colonies, subsequently expanding over the leaves, stems, and inflorescences. Infected young shoots were elongated and became slender. Chasmothecia were found in late October. Voucher specimens were deposited in the Korea University Herbarium (KUS-F). Conidiophores arising from the lateral part of the hyphae were upright, 100 to 220 × 10 to 12 µm, and produced 2 to 5 immature conidia in chains with sinuate edge lines. Basal parts of foot-cells in conidiophores were curved. Conidia were barrel-shaped to ellipsoid, 26 to 40 × 14 to 20 µm, and produced germ tubes on the perihilar position of the conidia. Chasmothecia with short mycelioid appendages were gregarious, 144 to176 µm in diam., and contained 8 to 22 asci. Asci were clavate-saccate with short stalks, 60 to 82 × 28 to 42 µm, and contained two spores. Ascospores were broadly ellipsoid, cytoplasm-dense without vacuoles, colorless, and 22 to 28 × 12 to 18 µm. The structures and measurements were consistent with those of Golovinomyces adenophorae (R.Y. Zheng & G.Q. Chen) Heluta (Braun & Cook, 2012). To confirm the morphology-based identification, two herbarium specimens (KUS-F29252 and F31898) were sequenced for the internal transcribed spacer (ITS) and large subunit (LSU) regions with PM10/ITS4 and PM3/TW14 primers, respectively (Bradshaw and Tobin, 2020). A Blastn search revealed high similarities in the ITS and LSU sequences, with 99.81% (538/539 bp) and 99.86% (697/698 bp) to G. adenophorae sequences (AB077633 and AB077632), respectively. All resulting sequences were deposited in GenBank under accession numbers OR841069-70 for ITS and OR841071 for LSU. A pathogenicity test was performed through inoculation by gently dusting the conidia from a detached symptomatic leaf onto the leaves of five healthy plants. Five non-inoculated plants served as controls. Following inoculation, plants were covered with plastic film and maintained in a greenhouse (24 to 32°C) until symptoms developed. Powdery mildew colonies developed on the inoculated plants after twelve days, whereas the control plants remained symptomless. The inoculated pathogen was confirmed morphologically and molecularly by the sequence comparison aforementioned, fulfilling Koch's postulates. Based on morphological characteristics and the sequencing data, the powdery mildew was identified as G. adenophorae. The association of G. adenophorae and Adenophora spp. has been known in China, Japan, Kazakhstan, and the Far East of Russia (Farr and Rossman, 2023). This is the first report of powdery mildew caused by G. adenophorae on A. triphylla var. japonica in Korea. Since the commercial cultivation of this plant aims to harvest young shoots as one of the most popular vegetables in Korea, appropriate control measures for the powdery mildew should be considered.

First Report of Leaf Spot Caused by Arthrinium arundinis on Amaranthus hybridus in Korea.

Amaranthus hybridus (=A. patulus), often called green amaranth, is an annual herbaceous plant of the Amaranthaceae. This plant is considered a harmful weed in the agricultural context of North America and has expanded its distribution to Asia and Europe. In Korea, it has become a problematic invasive issue, leading to economic losses due to reduced crop yields and rising weed management costs (Park et al., 2014), although its seeds and young leaves are edible and frequently consumed. In October 2020, we observed leaf spot symptoms on A. hybridus plants that were growing within perilla farms (Perilla frutescens var. japonica) located in Damyang (35°14'07"N, 126°59'40"E), Korea, with a disease incidence of 20 to 30% of the inspected plants. The initial signs appeared as grey to brown dots on the leaves, which gradually expanded into irregular, brown patches with a diameter of 2-3 cm. A single spore was isolated from the diseased leaf under a dissecting microscope, placed onto a 2% water agar plate, and incubated in darkness at 25°C for three days. Pure cultures were obtained by transferring single hyphal tips onto potato dextrose agar (PDA) plates. Five single-spore isolates were the same in the cultural and morphological examination, and a representative isolate (P309) was preserved at the Korean Agricultural Culture Collection (KACC49813), Korea. Colonies appeared light gray to white with regular margins and reached 4 to 5 cm in diameter after a week. After two weeks, black patches of spores were often visible in the aerial mycelia. Conidiophores were brown to pale brown, often branched, thick-walled, and measured 6.8 × 2.7 µm (n = 30). Conidia were single-celled, dark brown, globose to ellipsoid, and measured 6.8 × 5.0 µm (n = 50), with a ratio of length/width of 1.1 to 1.6 (n = 50). These morphological characteristics matched those of Arthrinium arundinis (Crous et al., 2013). For molecular identification, genomic DNA was extracted from conidia and mycelia on two-week-old PDA culture of the KACC49813. PCR was performed for the internal transcribed spacer (ITS) (primers ITS1/ITS4, White et al. 1990), the large subunit (LSU) rDNA (primers LROR/LR5, Vilgalys et al. 1990), the beta-tubulin gene (TUB) (primers T1/Bt-2b, O'Donnell and Cigelnik 1997), and the translation elongation factor 1-alpha (TEF) (primers EF1-728F/EF-2, Crous et al. 2013). A BLASTn search of the resulting sequences of ITS (560 bp; OL744431), LSU (881 bp; OL744432), TUB (790 bp; PP084934), and TEF (445 bp; PP084935) revealed 100 % similarity (e-value=0.0, coverage=100%) to previously reported sequences of Arthrinium arundinis (e.g. MF627422 for ITS, KF144930 for LSU, KF144973 for TUB, and KY705146 for TEF), confirming the identity of the Korean isolate. Pathogenicity assays were performed twice by spraying 1 ml of a conidial suspension (1.1 × 104 conidia per mL) onto the leaf surface of sixteen healthy A. hybridus plants. Sixteen control plants were sprayed with sterile water. All plants were kept in a growth chamber at 80% relative humidity and 23 °C with a 12-h light/dark cycle. Three weeks after the inoculation, initial symptoms mirroring the aforementioned ones appeared, while the control plants remained symptomless. Fungal isolates were successfully re-isolated from the inoculated leaves, and their identity as A. arundinis was confirmed by DNA sequencing, thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot caused by A. arundinis on Amaranthus hybridus, not only in Korea but globally. Arthrinium arundinis has also been reported as a plant pathogen on some agricultural crops (Ji et al. 2020; Liao et al. 2022; Farr and Rossman 2023), suggesting its polyphagous behavior. Then, this pathogen could represent a potential risk to the cultivation of edible amaranth in Korea and other crops where Amaranthus species are spread as weeds. For this reason, continuous monitoring is necessary to assess the impact of this fungus on Amaranthus and other crops.

Development and Validation of a Multiplex TaqMan Probe qPCR Assay Specific for Three Rust Fungi Infecting Meliosma myriantha.

Rust fungi are the largest group of obligate plant pathogens and cause severe damage to global forests and agricultural security. Meliosma myriantha, a tree species native to East Asia (China, Japan, and Korea), is vulnerable to three rust species: Neophysopella meliosmae, N. meliosmae-myrianthae, and N. vitis. The early symptoms of infection are indistinguishable between these species, making accurate and rapid diagnosis challenging. The urediniospores of N. meliosmae-myrianthae and N. vitis are also known to infect economically relevant grapevines (Vitis spp.) and ivies (Parthenocissus spp.), respectively, rendering early detection and identification even more important. To address this issue, we developed a multiplex quantitative PCR (qPCR) assay equipped with TaqMan probes targeting the internal transcribed spacer rDNA sequences specific to the three rust pathogens. This assay successfully detected minute quantities (5 fg for N. meliosmae-myrianthae and 50 fg for N. meliosmae and N. vitis) of DNA from the three Neophysopella species and demonstrated consistent reliability when applied to fresh and herbarium samples collected from M. myriantha, grapevines, and ivies. In conclusion, this novel assay is a rapid and robust diagnostic tool for the three rust pathogens, N. meliosmae, N. meliosmae-myrianthae, and N. vitis, and offers the potential to identify and detect their global movement and spread to grapevines, ivies, and trees.

Morphology, Molecular Phylogeny, and Fungicide Sensitivity of Phytophthora nagaii and P. tentaculata in Korea.

Phytophthora species, classified under Oomycota, cause significant damage to various crops and trees. The present study introduced Phytophthora species, P. nagaii and P. tentaculata, new to Korea, which pose notable risks to their respective host plants. Our research provided a comprehensive description of these species taking into account their cultural features, morphological characteristics, and molecular phylogenetic analysis using the internal transcribed spacer rDNA region and cytochrome c oxidase subunit mtDNA genes (cox1 and cox2) sequences. In addition, this study first evaluated the sensitivity of P. nagaii and P. tentaculata to five anti-oomycete fungicides, finding both species most responsive to picarbutrazox and P. tentaculata resistant to fluazinam. The data can guide targeted treatment strategies and offer insights into effective control methods. The findings expand our understanding of the diversity, distribution, and management of Phytophthora species in Korea.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Xanthium orientale in Korea.

Xanthium orientale L. (syn. Xanthium canadense Mill., Asteraceae), known as cocklebur, is an annual weed native to North America, which is now a neophyte distributed throughout the world. This plant was accidentally introduced to Korea in the late 1970s ( So et al. 2008) and is considered a problematic exotic weed in orchards, for which many herbicides are ineffective (Kim et al. 2020). In September 2018, powdery mildew was observed on X. orientale in Jeju, Korea. The disease incidence ranged from 40 to 60%. Voucher specimens were deposited in the Korea University Herbarium (Accession No. KUS-F30795) and Kunsan National University Herbarium (KSNUH1988). Symptoms appeared as round to irregular white patches with abundant hyphal growth on the leaf surface. Hyphal appressoria were nipple-shaped, and 3 to 6 µm diam. Conidiophores (n = 30) were 145 to 206 × 9 to 11.6 µm and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot-cells of the conidiophores were straight, cylindrical, and 43 to 100.9 µm long. Conidia (n = 30) were ellipsoid-ovoid, doliiform to somewhat limoniform, 25.2 to 31.8 × 13.6 to 16.8 µm (l/w 1.6 to 2.1), and devoid of distinct fibrosin bodies. The morphological characteristics corresponded to those of Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, under Golovinomyces spadiceus (Beck. & M.A. Curtis) U. Braun; Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), large subunit (LSU) (Bradshaw and Tobin 2020), the intergenic spacer (IGS) of rDNA, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Bradshaw et al. 2022) were amplified for a herbarium specimen (KUS-F30795). A BLASTn search of these sequences revealed 100% identity with reference sequences of G. ambrosiae on diverse Asteraceae plants (AB077644 for ITS, AB077643 for LSU, ON361171 for IGS, and ON075648 for GAPDH). However, there was a single nucleotide difference on both the IGS and GAPDH sequences when compared to the closely related species Golovinomyces latisporus. The sequences were deposited in GenBank (Accession No. OQ165157 (ITS), OQ165164 (LSU), OR050524 (IGS), and OR086076 (GAPDH)). Phylogenetic analyses of ITS, LSU, IGS, and GAPDH sequences revealed the Korean sample formed a well-supported group with other G. ambrosiae sequences, confirming its identity. A pathogenicity test was performed through inoculation by gently pressing diseased leaves onto the leaves of five healthy plants. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25±2°C. Powdery mildew colonies developed on the inoculated plants after ten days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the initially diseased leaves, fulfilling Koch's postulates. Powdery mildew on X. orientale has previously been reported as Golovinomyces cichoracearum (≡ Erysiphe cichoracearum) sensu lato in the USA, G. ambrosiae (= G. spadiceus) throughout all continents, and Podosphaera fusca sensu lato (now P. xanthii) in Korea (Braun and Cook 2012; Farr and Rossman 2023). To date, powdery mildew in Korea has been reported only on Xanthium strumarium as G. cichoracearum s. lat. and Podosphaera xanthii (KSPP 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on X. orientale in Korea.

Organic Farming Allows Balanced Fungal and Oomycetes Communities.

Conventional and organic farming systems affect soils differently, thereby influencing microbial diversity and composition. Organic farming, which relies on natural processes, biodiversity, and cycles adapted to local conditions, is generally known to improve soil texture and alleviate microbial diversity loss compared with that of conventional farming, which uses synthetic inputs such as chemical fertilisers, pesticides, and herbicides. Although they affect the health and productivity of host plants, the community dynamics of fungi and fungi-like oomycetes (under Chromista) in organic farmland are poorly understood. The present study aimed to determine the differences in the diversity and composition of fungi and oomycetes inhabiting organic and conventional farm soils using culture-based DNA barcoding and culture-independent environmental DNA (eDNA) metabarcoding. Four tomato farms with different farming practices were selected and investigated: mature pure organic (MPO) via non-pesticide and organic fertiliser, mature integrated organic (MIO) via non-pesticide and chemical fertiliser, mature conventional chemical (MCC) via both pesticide and chemical fertiliser, and young conventional chemical (YCC). Culture-based analysis revealed that different genera were dominant on the four farms: Linnemannia in MPO, Mucor in MIO, and Globisporangium in MCC and YCC. eDNA metabarcoding demonstrated that the fungal richness and diversity on the MPO farm were higher than that on other farms. Both conventional farms exhibited simpler fungal and oomycete network structures with lower phylogenetic diversity. Interestingly, a high richness of oomycetes was shown in YCC; in which, Globisporangium, a potential pathogenic group on tomato plants, was abundantly observed. Our findings indicate that organic farming enhances fungal and oomycete diversity, which may provide robust support for maintaining healthy and sustainable agricultural practices. This study contributes to our knowledge on the positive effects of organic farming on crop microbiomes and provides essential information for maintaining biological diversity.

Regulation of c-SMAC formation and AKT-mTOR signaling by the TSG101-IFT20 axis in CD4+ T cells.

CD4+ T cells play major roles in the adaptive immune system, which requires antigen recognition, costimulation, and cytokines for its elaborate orchestration. Recent studies have provided new insight into the importance of the supramolecular activation cluster (SMAC), which comprises concentric circles and is involved in the amplification of CD4+ T cell activation. However, the underlying mechanism of SMAC formation remains poorly understood. Here, we performed single-cell RNA sequencing of CD4+ T cells left unstimulated and stimulated with anti-CD3 and anti-CD28 antibodies to identify novel proteins involved in their regulation. We found that intraflagellar transport 20 (IFT20), previously known as cilia-forming protein, was upregulated in antibody-stimulated CD4+ T cells compared to unstimulated CD4+ T cells. We also found that IFT20 interacted with tumor susceptibility gene 101 (TSG101), a protein that endocytoses ubiquitinated T-cell receptors. The interaction between IFT20 and TSG101 promoted SMAC formation, which led to amplification of AKT-mTOR signaling. However, IFT20-deficient CD4+ T cells showed SMAC malformation, resulting in reduced CD4+ T cell proliferation, aerobic glycolysis, and cellular respiration. Finally, mice with T-cell-specific IFT20 deficiency exhibited reduced allergen-induced airway inflammation. Thus, our data suggest that the IFT20-TSG101 axis regulates AKT-mTOR signaling via SMAC formation.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Erigeron annuus in Korea.

Erigeron annuus (L.) Pers., known as annual fleabane or eastern daisy fleabane, is native to North America and was unintentionally introduced to Korea in the 1910s (Park, 1995). It is now widely naturalized throughout Korea and was designated as one of the ten major introduced plants in Korea by the Korea National Arboretum. In September 2012, several dozen annual fleabanes were found to be heavily infected with powdery mildew. Symptoms first appeared as circular to irregular white patches, which subsequently showed abundant hyphal growth on both sides of the leaves. The same symptoms have continuously been found on annual fleabane throughout the country, where the disease incidence was often higher than 80%. Five voucher specimens were deposited in the Korea University Herbarium (KUS-F30208, 31414, 31774, 31784 and 32003). Hyphae were septate, branched, and 4.5 to 6.7 µm wide. Appressoria on the mycelium were lobed. Conidiophores (n = 30), measured 154 to 215 × 9 to 12.5 µm, were simple and produced 2 to 4 immature conidia in chains with a sinuate outline, followed by 2 to 3 cells. Foot-cells of conidiophores were straight, cylindrical, and 40 to 98 µm long. Conidia (n = 30) were hyaline, ellipsoid to barrel-shaped, measured 25.3 to 35.8 × 13 to 17 µm (length/width ratio = 1.62 to 2.31), lacked distinct fibrosin bodies, and showed reticulate wrinkling of the outer walls. Primary conidia were apically rounded and basally truncated and generally smaller than the secondary conidia. Germ tubes were produced on the subterminal position of conidia. No chasmothecia were observed. The structures described above were typical of the Euoidium anamorph of the genus Golovinomyces, and the fungus measurements were compatible with those of G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Qiu et al., 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA from the five herbarium specimens were amplified with primers PM10/ITS4 for ITS and PM3/TW14 for LSU (Bradshaw and Tobin, 2020; Mori et al., 2000; White et al., 1990) and sequenced directly. The resulting sequence was deposited in GenBank (Accession No. OP788040-4 for ITS and OP788045-9 for LSU). Comparison with the sequences available in the GenBank database revealed that the isolates showed 100% sequence similarity with those of G. ambrosiae from the family Asteraceae (e.g., MT355557, MF612182, etc.). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto the leaves of five healthy potted plants. Five non-inoculated plants served as controls. Plants were maintained in a greenhouse at 22 to 28°C. Inoculated plants developed signs and symptoms after seven days, whereas the control plants remained healthy. The fungus present on the inoculated plants was morphologically identical to that observed initially on diseased plants, fulfilling Koch's postulates. Powdery mildew infections of Erigeron spp. associated with Golovinomyces species have been known in the United States, France, and China (Farr and Rossman, 2022). To our knowledge, this is the first report of powdery mildew disease caused by G. ambrosiae on E. annuus outside of North America as well as in Korea. According to our field observation, powdery mildew infections were found only on annual fleabanes growing in shady areas, not in sunny places.

Uncharted Diversity and Ecology of Saprolegniaceae (Oomycota) in Freshwater Environments.

The fungal-like family Saprolegniaceae (Oomycota), also called "water mold," includes mostly aquatic saprophytes as well as notorious aquatic animal pathogens. Most studies on Saprolegniaceae have been biased toward pathogenic species that are important to aquaculture rather than saprotrophic species, despite the latter's crucial roles in carbon cycling of freshwater ecosystems. Few attempts have been made to study the diversity and ecology of Saprolegniaceae; thus, their ecological role is not well-known. During a survey of oomycetes between 2016 and 2021, we investigated the diversity and distribution of culturable Saprolegniaceae species in freshwater ecosystems of Korea. In the present study, members of Saprolegniaceae were isolated and identified at species level based on their cultural, morphological, and molecular phylogenetic analyses. Furthermore, substrate preference and seasonal dynamics for each were examined. Most of the species were previously reported as animal pathogens; however, in the present study, they were often isolated from other freshwater substrates, such as plant debris, algae, water, and soil sediment. The relative abundance of Saprolegniaceae was higher in the cold to cool season than that in the warm to hot season of Korea. This study enhances our understanding of the diversity and ecological attributes of Saprolegniaceae in freshwater ecosystems.

Multiplex TaqMan qPCR Assay for Detection, Identification, and Quantification of Three Sclerotinia Species.

White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.

Activation of ADRB2/PKA Signaling Pathway Facilitates Lipid Synthesis in Meibocytes, and Beta-Blocker Glaucoma Drug Impedes PKA-Induced Lipid Synthesis by Inhibiting ADRB2.

Meibomian gland dysfunction is one of the main causes of dry eye disease and has limited therapeutic options. In this study, we investigated the biological function of the beta 2-adrenergic receptor (ADRB2)/protein kinase A (PKA) pathway in lipid synthesis and its underlying mechanisms in human meibomian gland epithelial cells (HMGECs). HMGECs were cultured in differentiation media with or without forskolin (an activator of adenylate cyclase), salbutamol (an ADRB2 agonist), or timolol (an ADRB2 antagonist) for up to 4 days. The phosphorylation of the cAMP-response element-binding protein (CREB) and the expression of peroxisome proliferator activator receptor (PPAR)γ and sterol regulatory element-binding protein (SREBP)-1 were measured by immunoblotting and quantitative PCR. Lipid synthesis was examined by LipidTOX immunostaining, AdipoRed assay, and Oil Red O staining. PKA pathway activation enhanced PPARγ expression and lipid synthesis in differentiated HMGECs. When treated with agonists of ADBR2 (upstream of the PKA signaling system), PPARγ expression and lipid synthesis were enhanced in HMGECs. The ADRB2 antagonist timolol showed the opposite effect. The activation of the ADRB2/PKA signaling pathway enhances lipid synthesis in HMGECs. These results provide a potential mechanism and therapeutic target for meibomian gland dysfunction, particularly in cases induced by beta-blocker glaucoma drugs.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on New York Aster (Symphyotrichum novi-belgii) in Korea.

Symphyotrichum novi-belgii (L.) G.L. Nesom (syn. Aster novi-belgii L.), known as New York aster, is a perennial herb used in gardens and as a potted plant. The plant is native to North America but has been developed into various horticultural varieties. In Korea, it is one of the most common plants used for autumn bloom. In September 2011, New York asters (variety unknown) showing typical signs of powdery mildew were observed in a public garden in Seoul, Korea. Since then, the disease on New York asters has been continuously found in parks and flower markets in different regions of Korea. Voucher specimens (n=3) were deposited in the Korea University Herbarium (KUS-F 30752, 31865, and 32103). On leaves, circular to irregular white patches appeared which subsequently showed abundant hyphal growth on both sides of the leaves and on young stems and inflorescences, reducing the aesthetic value and vigor of the plants affected. Hyphae were septate, branched, and 4 to 8 µm wide. Appressoria on the mycelium were nipple-shaped. Conidiophores measured 110 to 200 × 9 to 11.5 µm, were simple, and produced 2 to 5 immature conidia in chains with a sinuate outline, followed by 2 to 3 cells. Foot-cells of conidiophores were straight, cylindric, and 55 to 125 µm long. Conidia were hyaline, ellipsoid to barrel-shaped, measured 22 to 52 × 15 to 20 µm (length/width ratio = 1.5-2.5), lacked distinct fibrosin bodies, and produced germ tubes on the subterminal position, with reticulate wrinkling of the outer walls. No chasmothecia were observed. The structures described above were typical of the Oidium subgenus Euoidium anamorph of the genus Golovinomyces, and the fungus measurements were consistent with those of G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA were amplified with primers PM10/ITS4 for ITS and PM3/TW14 for LSU (Mori et al. 2000, Bradshaw and Tobin 2020). The resulting sequences were deposited in GenBank (Accession No. OP028065-7 for ITS and OP028053-5 for LSU). A GenBank BLAST search of these sequences revealed 100% identity with sequences of G. ambrosiae on many asteraceous plants, including S. novi-belgii from China (MK452575-9 for ITS and MK452648-52 for LSU). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto leaves of five healthy potted New York aster plants. Five non-inoculated plants served as controls. Plants were maintained in an incubator at 24°C. Inoculated plants developed signs and symptoms after three weeks, whereas the control plants remained symptomless. The fungus present on the inoculated plants was morphologically identical to that observed initially on diseased plants, fulfilling Koch's postulates. The powdery mildew infections of S. novi-belgii associated with G. ambrosiae have been widely known in Europe and North America but only recently in China (Qiu et al. 2020, Farr and Rossman 2022). In Japan, Podosphaera fuliginea was known to be associated with powdery mildew of S. novi-belgii (Farr and Rossman 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on S. novi-belgii in Korea. The powdery mildew on this ornamental plant can be considered a severe threat.

CD5 Suppresses IL-15-Induced Proliferation of Human Memory CD8+ T Cells by Inhibiting mTOR Pathways.

IL-15 induces the proliferation of memory CD8+ T cells as well as NK cells. The expression of CD5 inversely correlates with the IL-15 responsiveness of human memory CD8+ T cells. However, whether CD5 directly regulates IL-15-induced proliferation of human memory CD8+ T cells is unknown. In the current study, we demonstrate that human memory CD8+ T cells in advanced stages of differentiation respond to IL-15 better than human memory CD8+ T cells in stages of less differentiation. We also found that the expression level of CD5 is the best correlate for IL-15 hyporesponsiveness among human memory CD8+ T cells. Importantly, we found that IL-15-induced proliferation of human memory CD8+ T cells is significantly enhanced by blocking CD5 with Abs or knocking down CD5 expression using small interfering RNA, indicating that CD5 directly suppresses the IL-15-induced proliferation of human memory CD8+ T cells. We also found that CD5 inhibits activation of the mTOR pathway, which is required for IL-15-induced proliferation of human memory CD8+ T cells. Taken together, the results indicate that CD5 is not just a correlative marker for IL-15 hyporesponsiveness, but it also directly suppresses IL-15-induced proliferation of human memory CD8+ T cells by inhibiting mTOR pathways.

First Report of Puccinia modiolae Causing Rust Disease on Malva verticillata in Korea.

Malva verticillata (Malvaceae), commonly called Chinese mallow or whorled mallow, is an annual herb native to East Asia and is currently distributed worldwide. In Korea, this plant is cultivated as a leafy vegetable and cooked like spinach or used in soups and also as a medicine material. In March 2022, typical symptoms of rust disease were observed on M. verticillata in a plastic house (37°22'12″ N, 127°34'30" E) in Yeoju, Korea. Yellow or light green round chlorotic spots appeared on the upper surface of infected leaves, while reddish-brown or dark brown rust pustules formed on its lower surface. Infection occurred in 10% of M. verticillata plants surveyed, and disease severity ranged between 30-90%. A representative sample was deposited in the Kunsan National University Herbarium (KSNUH1762). Telia were mostly hypophyllous, reddish-brown to dark brown, round, mostly grouped, and 0.3-0.7 mm in diameter. Teliospores were mostly two-celled, but rarely one or three-celled, yellowish to light brown, fusoid, and 42.9-101 × 10.8- to 18.8 µm (average 72.7 ± 12.3 × 14.2 ± 1.92 µm [mean ± SD]; n = 50), with a smooth, hyaline to yellowish wall of 1.0-2.5 µm thickness. The morphological characteristics were similar to those reported for Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). To confirm the morphological identification, genomic DNA was extracted from the teliospores of an infected leaf. The internal transcribed spacer (ITS) with primers ITS5-u and ITS4rust (Pfunder and Schürch 2001) and the large subunit (LSU) rDNA with primers LRust1R and LRust3 (Beenken et al. 2012) were amplified for sequencing. The resulting sequences were deposited in GenBank with accession numbers ON631218 for ITS and ON631226 for LSU. BLASTn search showed that the Korean sample was identical to the ITS sequences of P. modiolae from Modiola caroliniana (MK458693-MK458697) and the LSU sequences from M. caroliniana, Malva sylvestris, and Alcea rosea (MH742976, MH742977, and MH742978). In the phylogenetic trees of the ITS and LSU sequences, the Korean sample was grouped with the reference sequences of P. modiolae, with the maximum supporting value. For the pathogenicity test, rust-infected leaf discs were placed on the upper or lower surfaces of leaves of three healthy M. verticillata. Three non-inoculated plants served as controls. Inoculated and non-inoculated plants were maintained in a growth chamber at 22°C, a 16/8 h light cycle, and 80% humidity. After three weeks, all inoculated plants developed evident rust symptoms on the upper and lower surfaces of the leaves on which the leaf discs were placed, whereas the control plants remained symptomless. The pathogen present on the inoculated plants was confirmed to be the same pathogen as that observed in the field, fulfilling Koch's postulates. Based on the morphological investigation, sequence analysis, and pathogenicity tests, P. modiolae was identified as the causal agent of rust disease on M. verticillata. To date, this pathogen has been reported on seven Malvaceae plants, including Alcea rosea, Althaea officinalis, Lavatera arborea, Malva parviflora, Malva sylvestris, Modiola caroliniana, and Modiola sp., in North and South America (Farr and Rossman 2022). However, it has not been reported in Asia or Korea. This study is the first report of rust disease on M. verticillata worldwide. Considering its high incidence rate and severe damage, this pathogen is a potential concern for the cultivation of M. verticillata in Korea. This finding could contribute to developing phytosanitary and control treatments for this disease.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Leucanthemum vulgare in Korea.

Leucanthemum vulgare Lam. (Asteraceae), known as ox-eye daisy, is a perennial herb native to Europe and western Asia (Clements et al. 2004, McDougall et al. 2018). In Korea, this plant was introduced for ornamental purposes but has been naturalized as a widespread invasive species. In June 2015, symptoms of a powdery mildew disease were observed on L. vulgare in a public garden in Goseong (38°14'18"N, 128°32'56"E), Korea. Since then, its findings have continued throughout the country, including Mokpo and Seogwipo (in 2018), Hongcheon and Seoul (in 2020), Boeun, Gunsan, and Namwon (in 2022), where the disease incidence was often higher than 80%. Symptoms first appeared as circular to irregular white powdery patches covering leaves and stems. Affected plants became distorted, eventually losing their aesthetic and ornamental value. A total of sixteen samples were deposited in the herbarium of Korea University (KUS-F), Korea. Microscopic observations showed that hyphal appressoria were nipple-shaped. Conidiophores were cylindrical, 98 to 157 × 9 to 12 µm, and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot cells were cylindrical, straight, and 37 to 65 µm long. Conidia were ellipsoid to barrel-shaped, 23 to 39 × 12 to 19 µm, with a length/width ratio of 1.4 to 2.3 and devoid of fibrosin bodies. Germ tubes were produced in the perihilar position of the conidia. Primary conidia were apically rounded and basally subtruncated. No chasmothecia were found until the plants died in winter. The morphological characteristics were typical for anamorph of the genus Golovinomyces. To identify the fungus, genomic DNA was extracted from the four herbarium specimens (KUS-F 28650, 30839, 31728, and 31787). PCR products were amplified using the primer sets PM10/ITS4 for internal transcribed spacer (ITS) and PM3/TW14 for the large subunit (LSU) of the rDNA (Mori et al. 2000, Bradshaw and Tobin 2020). Sequences obtained in the present study were deposited at GenBank (accession numbers ON834488-91 for ITS and ON834494-7 for LSU). A BLASTn search of the Korean specimens showed 100% identity with reference sequences of G. ambrosiae in GenBank (KX98730, MK452580, and MK452588 for ITS and MF612182, MK452653, and MK452661 for LSU). In phylogenetic trees of a concatenated dataset of the ITS and LSU sequences, the Korean specimens formed a well-supported clade with the reference sequences of G. ambrosiae. Pathogenicity tests were carried out by touching and dusting an infected leaf (KUS-F 31787) onto the upper leaf surface of five healthy plants. Five non-inoculated plants served as controls. After two weeks, all inoculated plants formed white patches on the surface of leaves and stems, whereas the control plants remained symptomless. The fungus on the inoculated plants was identical to that observed on the initially diseased plant, fulfilling Koch's postulates. As a result, the causal agent of the powdery mildew on L. vulgare was confirmed as G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook, based on the current taxonomy and nomenclature of this species by Qiu et al. (2020).. Previously powdery mildew collections on L. vulgare have been reported as Golovinomyces cichoracearum (≡ Erysiphe cichoracearum) s. lat. in Estonia, Finland, Germany, and Switzerland, Golovinomyces biocellatus in Spain, and Podosphaera fusca (probably P. xanthii according to the current taxonomy) in the former Soviet Union (now Russia and adjacent countries) (Farr and Rossman 2022). This study is the first report of powdery mildew disease caused by G. ambrosiae on L. vulgare in Korea. Qiu et al. (2020) confirmed the occurrence of G. ambrosiae on L. maximum, another species of the genus Leucanthemum. As powdery mildew causes damage to the cultivation of L. vulgare by loss of ornamental value, appropriate control measures should be developed.

The Role of Autophagy in the Function of CD4+ T Cells and the Development of Chronic Inflammatory Diseases.

Uncontrolled acute inflammation progresses to persistent inflammation that leads to various chronic inflammatory diseases, including asthma, Crohn's disease, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. CD4+ T cells are key immune cells that determine the development of these chronic inflammatory diseases. CD4+ T cells orchestrate adaptive immune responses by producing cytokines and effector molecules. These functional roles of T cells vary depending on the surrounding inflammatory or anatomical environment. Autophagy is an important process that can regulate the function of CD4+ T cells. By lysosomal degradation of cytoplasmic materials, autophagy mediates CD4+ T cell-mediated immune responses, including cytokine production, proliferation, and differentiation. Furthermore, through canonical processes involving autophagy machinery, autophagy also contributes to the development of chronic inflammatory diseases. Therefore, a targeted intervention of autophagy processes could be used to treat chronic inflammatory diseases. This review focuses on the role of autophagy via CD4+ T cells in the pathogenesis and treatment of such diseases. In particular, we explore the underlying mechanisms of autophagy in the regulation of CD4+ T cell metabolism, survival, development, proliferation, differentiation, and aging. Furthermore, we suggest that autophagy-mediated modulation of CD4+ T cells is a promising therapeutic target for treating chronic inflammatory diseases.

Chemokine Receptor Profiles of T Cells in Patients with Age-Related Macular Degeneration.

PURPOSE: To evaluate the expression of multiple chemokine receptors in peripheral blood T cells from patients with age-related macular degeneration (AMD). MATERIALS AND METHODS: Peripheral blood mononuclear cells and/or aqueous humor were obtained from 24 AMD patients and 24 age- and sex-matched healthy controls. Chemokine receptor expression on T cells from peripheral blood was determined by multicolor flow cytometry. The levels of chemokines and cytokines in the aqueous humor from 12 AMD patients and six healthy controls were assessed. RESULTS: AMD patients had increased expressions of CCR4 in CD4+ T cells (p=0.007) and CRTh2 in CD8+ T cells (p=0.002), and decreased expressions of CXCR3 in CD4+ T cells (p=0.029) and CXCR3, CCR5, and CX3CR1 in CD8+ T cells (p=0.005, 0.019, and 0.007, respectively). Monocyte chemoattractant protein-1 levels were increased in the aqueous humor from AMD patients (p=0.018), while the levels of interleukin (IL)-4 and IL-22 were significantly decreased compared to controls (p=0.018 and 0.041, respectively). CONCLUSION: The chemokine receptor profiles of T cells are altered in AMD patients compared to healthy controls without noticeable associations with chemokine levels in the aqueous humor. Further evaluation is needed to clarify the role of these alterations in AMD pathogenesis.

Early Detection of the Spinach Downy Mildew Pathogen in Leaves by Recombinase Polymerase Amplification.

Downy mildew of spinach, caused by Peronospora effusa, is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic because symptoms can develop postharvest. Therefore, early detection methods for P. effusa could help producers identify infection before visible symptoms appear. Recombinase polymerase amplification (RPA) provides sensitive and specific detection of pathogen DNA and is a rapid, field-applicable method that does not require advanced technical knowledge or equipment-heavy DNA extraction. Here, we used comparative genomics to identify a unique region of the P. effusa mitochondrial genome to develop an RPA assay for the early detection of P. effusa in spinach leaves. In tandem, we established a TaqMan quantitative PCR (qPCR) assay and used this assay to validate the P. effusa specificity of the locus across Peronospora spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, P. schachtii. TaqMan qPCR and RPA have detection thresholds of 100 and 900 fg of DNA, respectively. Both assays could detect P. effusa in presymptomatic leaves, with RPA-based detection occurring as early as 5 days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24 h of plant exposure to airborne spores. Implementation of the RPA detection method could provide real-time information for point-of-care management strategies at field sites.