First Report of Leaf Spot on Eriobotrya japonica Caused by Alternaria alternata in Southwest Korea.

Loquat (Eriobotrya japonica) is a crop cultivated in Southwest Korea, covering an area of 101 ha and yielding 120 tons at harvest (KASS, 2024). Due to its high-income potential, the cultivation area is gradually expanding. In May 2023, 30% of leaf brown spots were observed on all three trees in the Suncheonman National Garden, Suncheon (3488'57.97" N, 12750'92.83" E). As the disease progressed, the brown spot gradually enlarged, turning greyish-ivory inside and forming concentric circles. Three leaf lesions from each tree were cut into 5 x 5 mm pieces, surface-sterilized with 70% ethanol for 1 min, and washed in sterile water three times to isolate the pathogen potentially responsible for these symptoms. The samples obtained were subsequently cultured on 1.5% water agar and then incubated in the dark at 25℃. A total of nine isolates were obtained, with three isolates from each of the three trees through single-spore isolation, namely SYP-1202-1 to 3, SYP-1202-4 to 6, and SYP-1202-7 to 9. The colonies reached 90 mm in diameter after 10 days on potato dextrose agar (PDA), initially dark green, and turned sooty gray after 2 weeks. The hyphae grown on a 0.6% KCl medium for 3 days produced long chains containing three to twelve conidia. The conidia were ellipsoidal or obpyriform in shape and light brown. The conidiophores were straight or curved, measuring 12.1-75.3 x 1.6-4.8 µm (n = 100). The primary and secondary conidia measured length × width of 19.1-60.6 × 6.1-14.4 µm and 8.4-27.8 × 3.5-9.5 µm (n = 100), respectively. The conidia had 1 to 7 transverse and 0 to 3 vertical septa. The morphology of the nine isolates was identical and consistent with Alternaria species (van der Waals et al., 2011; Woudenberg et al., 2015). For molecular identification, ITS (OR844500 to OR844508), GAPDH (OR866383 to OR866391), TEF1 (OR866392 to OR866400), RPB2 (OR866401 to OR866409), Alt a1 (OR866410 to OR866418), endoPG (OR866419 to OR866427), and OPA10-2 (OR866428 to OR866436) sequences from SYP-1202-1 to 9 showed a 100% (515 bp/515 bp), 100% (579/579), 100% (240/240), 100% (753/753), 95.1% (449/472), 100% (448/448), and 100% (634/634) identity with that of type strain A. alternata CBS 115152 (KP124348, KP124202, KP125124, KP124816, KP123896, KP124049, and KP124658, respectively). A pathogenicity test was conducted on three 5-year-old E. japonica cultivar Daebang trees in pots. The surface of the five leaves per tree was sterilized with 70% ethanol for 1 min. Before inoculation, the leaves were wounded with sterile needles and sprayed with the conidial suspension (1×106 conidia/ml) produced from a 1-week-old culture grown on PDA. In contrast, control leaves were sprayed with sterile distilled water. The inoculated leaves were wrapped with black plastic bags and kept at 100% relative humidity for two days. At seven days post-inoculation, symptoms were observed on the wounded leaves, whereas the nonwounded and control leaves did not exhibit any symptoms. The experiment was performed three times in the greenhouse. For each experiment, pathogens were reisolated from the two symptomatic leaves per plant. The identity of the reisolated pathogens was then confirmed via analysis of ITS and RPB2 genes, thereby confirming adherence to Koch's postulates. To the best of our knowledge, this is the first report of E. japonica being infected by A. alternata in Korea. This report provides important information to support effective disease control strategies for E. japonica in orchards in southern Korea.

MoNOT3 Subunit Has Important Roles in Infection-Related Development and Stress Responses in Magnaporthe oryzae.

The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae.

Isolation and Identification of Alternaria alternata from Potato Plants Affected by Leaf Spot Disease in Korea: Selection of Effective Fungicides.

Brown leaf spot disease caused by Alternaria spp. is among the most common diseases of potato crops. Typical brown spot symptoms were observed in commercial potato-cultivation areas of northern Korea from June to August 2020-2021. In total, 68 isolates were collected, and based on sequence analysis of the internal transcribed spacer (ITS) region, the collected isolates were identified as Alternaria spp. (80.9%). Phylogenetic analysis revealed that a majority of these isolates clustered within a clade that included A. alternata. Additionally, the ITS region and rpb2 yielded the most informative sequences for the identification of A. alternata. Pathogenicity tests confirmed that the collected pathogens elicited symptoms identical to those observed in the field. In pathogenicity tests performed on seven commercial cultivars, the pathogens exhibited strong virulence in both wound and non-wound inoculations. Among the cultivars tested, Arirang-1ho, Arirang-2ho, and Golden Ball were resistant to the pathogens. Furthermore, among the fungicides tested in vitro, mancozeb and difenoconazole were found to be effective for inhibiting mycelial growth. In summary, our findings suggest that A. alternata plays a critical role in leaf disease in potato-growing regions and emphasise the necessity of continuous monitoring and management to protect against this disease in Korea.

First Report of Anthracnose Caused by Colletotrichum gloeosporioides on Cinnamomum camphora in Korea.

Cinnamomum camphora, known as the camphor tree, is an evergreen tree widely cultivated in Asia as an ornamental plant (Singh and Jawaid, 2012). In June 2023, several leaves on a total of 10 trees planted on a street in Suncheon, Jeonnam Province, Korea showed black spots. Disease incidence was observed in at least 15% of the 10 trees. The symptoms included circular spots with a light ash-colored center and dark brown borders. The size of lesions varied depending on the progress of the disease. The disease progressed by 30% on the tree leaves. To isolate the pathogen, we cut out the lesions on the leaf surface sterilized with 70% ethanol for one minute, washed three times with sterilized distilled water, dried, and placed on water agar. Then, it was incubated at 25°C for three days. Emerging hyphae from the samples were subcultured on potato dextrose agar (PDA), resulting in three independent isolates (SYP-F1226-1 to SYP-F1226-3) after single spore isolation from 3 independent trees. The isolates exhibited grayish fluffy mycelium in the center of the colony, while the edges were white on PDA. Conidia had rounded cylindrical shape and were 4.9 to 8.4 µm  1.4 to 3.1 µm (avg. 5.9  2.1 µm, n = 100) in size. Appressoria were round, dark gray, produced at the tip of the germ tube after a septum formed the conidium. The morphological characteristics matched those of Colletotrichum species complexes. (Damm et al., 2012; Weir et al., 2012). For molecular identification, ITS (OR647338 to 40), GAPDH (OR657042 to 44), CHS-1 (OR657045 to 47), ACT (OR657048 to 50), and CAL (OR657051 to 53) sequences from isolates SYP-F1226-1~3 showed a 99.65%, 98.56%, 99.00%, 99.28%, and 99.52% identity with that of type strain C. gloeosporioides ICMP 17821 (JX010152, JX010056, JX009818, JX009531, and JX010445, respectively). Using the MEGA X program (Kumar et al. 2018), maximum likelihood analysis based on the concatenated sequences placed the isolates within a clade comprising C. gloeosporioides. Pathogenicity of SYP-F1226-1 was tested using three leaves from a 1-year-old branch of three independent healthy C. camphora plants. The leaf surfaces were sterilized by rubbing a cotton pad soaked in 70% ethanol and then wiping them with a sterilized cotton pad. The leaves per plant were inoculated with 5 mL of a conidial suspension (1 × 105 conidia/mL), both with and without wounding. Another three control leaves were inoculated with sterile distilled water, both with and without wounding. The inoculated leaves were wrapped in a plastic bag for 48 hours under conditions of 100% relative humidity. Spot symptoms were observed on both wounded and non-wounded leaves 21 days after inoculation. No symptoms were observed in the control on either of the wounded leaves. Pathogenicity tests were performed three times. The pathogen was re-isolated from the lesion after treatment, and its identity was confirmed using the five genes and morphological characteristics. This confirms the fulfillment of Koch's postulates. C. fioriniae (Liu et al, 2022) and C. siamens (Liu et al, 2022; Khoo et al, 2023) have been reported as the causal pathogen of anthracnose in C. camphora, but C. gloeosporioides has not been reported as a pathogen in C. camphora. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on C. camphora in Korea. This study will provide symptomatic, mycological, and molecular biological information for the early detection of anthracnose disease in C. camphora plants.

The effects of fucoidan-rich polysaccharides extracted from Sargassum horneri on enhancing collagen-related skin barrier function as a potential cosmetic product.

BACKGROUND: Sargassum horneri came ashore after flowing from the South China Sea to Jeju Island a few years ago. This caused a significant environmental impact on coastal areas where S. horneri has accumulated because of decomposition and the release of toxic substances, such as hydrogen sulfide. AIMS: In this study, we evaluated a biological ingredient prepared from fucoidan-rich S. horneri and demonstrated its antiwrinkle effects on ultraviolet B (UVB)-induced fibroblast cells. MATERIALS AND METHODS: Fucoidan samples from S. horneri were prepared according to a previously published process with modifications. The compositional analysis of S. horneri fucoidan extract (SHFE) as well as its effects on antiaging were examined to determine its utility as a functional material. RESULTS: SHFE exhibited antioxidant properties using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Treatment of UVB-induced fibroblasts with SHFE significantly increased the synthesis of procollagen compared with adenosine treatment and inhibited MMP-1 and MMP-3 expression. In a clinical study, SHFE lotion improved skin barrier effects in forearms and transepidermal water loss (TEWL) values were reduced after 3 weeks of use compared with a placebo. CONCLUSION: SHFE has utility as an additive with functional antiaging effects for a range of cosmetic products as it restores skin hydration in the epidermal barrier.

Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24-36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

First Report of Sharp Eyespot of Oat (Avena sativa) Caused by Ceratobasidium cereale in Korea.

Along with barley and wheat, oats (Avena sativa) are cultivated as winter crops in Korea, and the total area for oat cultivation is 103 ha in 2021. From late March to early April 2021, sharp eyespot symptoms on oat (cv. Choyang) leaf sheaths and straws were observed in two commercial fields located in Haenam (N34°38'35.04588/E126°38'31.00668) and Gangjin (N34°38'9.46788/E126°37'19.44984), Jeollanam-do, Korea. The incidence was 5% and 7%, respectively. Small brown spots were irregular circles that began to appear on the lower sheaths, and the spots gradually enlarged in the upper part of the sheaths. The center of each lesion turned whitish-brown with dark brown margins, resulting in a blight of the sheaths. Three plants displaying typical sharp eyespot lesions were collected from each of two individual regions, Haenam and Gangjin. To isolate the causal pathogen, two infected tissues (5  5 mm) from the collected plants were surface-sterilized by treating them with 70% ethanol for 1 min and 1% NaClO for 1 min immediately after being treated with 95% ethanol for 1 min. Subsequently, the samples were rinsed three times with distilled water, dried with sterile filter paper, transferred to 1.5% water agar supplemented with 100 ppm streptomycin, and then incubated in the dark at 25°C. Hyphae emerging from the randomly selected three independent tissues from each location were subcultured on potato dextrose agar (PDA, Sparks, MD 21152, USA), resulting in three independent isolates (HNO-1, HNO-2, HNO-3) from Haenam and three (KJO1-1, KJO1-2, KJO1-3) from Ganjin after single-hypha-tip purification. Colonies on the PDA were pigmented white at first and subsequently changed to light brown after 2 weeks. All collected isolates formed globose and irregular dark brown to black sclerotia on PDA after 2 weeks. Binuclear hyphae were white to dark brown in color, branched at right angles with a septum near the branch, and multinucleate cells, suggesting that these isolates belonged to Ceratobasidium cereale (Boerema et al., 1977; Burpee, 1980; Sharon et al.,2008). For molecular identification, the ITS (GenBank accession nos. MW691851-53 for HNO-1 to HNO-3; MW691857-59 for KJO1-1 to KJO1-3), LSU (OQ397530-35), rpb2 (OQ409878-83), tef1 (OQ409884-89), and atp6 (OQ409890-95) regions of six isolates were amplified using the primer pairs ITS4/5 (White et al., 1990), LROR/LR5 (Vilgalys and Hester, 1990), bRPB2-6F/bRPB2-7.1R (Matheny, 2005; Reeb et al., 2004), TEF1-F/TEF1-R (Litvintseva et al., 2006), and ATP61/ATP62 (Kretzer and Bruns, 1999), respectively. The sequences of ITS region showed 99.7% identity with C. cereale strain WK137-56 (KY379365) and 99.8% with Ceratobasidium sp. AG-D (KP171639). Using the MEGA X program (Kumar et al. 2018), a maximum likelihood phylogenetic analysis based on the concatenated ITS-LSU, rpb2, tef1 and atp6 sequences placed the six isolates within a clade comprising C. cereale (Gónzalez et al.,2016; Ji et al., 2017; Tomioka et al., 2021; Li et al., 2014). Two representative isolate, HNO-1 and KJO1-1, were deposited in the Korean Agriculture Culture Collection (Accession No. KACC 49887 and 410268, respectively). For pathogenicity, the six isolates were cultured on sterilized ray grains at 25°C in the dark for 3 weeks as the inoculum. Five oat (cv. Choyang) seeds were sown per pot containing 80 g of the infected ray grains mixed with 150 g of composite soil and 150 ml of water (Baroker Garden Soil, Seoul Bio Co., LTD). The control was treated with 80 g of the sterilized ray grains mixed with 150 g of composite soil and 150 ml of water. All inoculated and control pots were placed in a 20°C growth chamber with a 12-h photoperiod and 65% humidity. Typical sharp eyespot symptoms were observed on the oat sheath of seedlings three weeks post-inoculation. No symptoms were observed in the control seedlings. The infection assays were repeated thrice, with similar results. The pathogen was successfully re-isolated, and its identity was confirmed via morphological and molecular analyses. In Korea, few etiological studies have been conducted on oats because they are less economical than barley and wheat. Sharp eyespot disease caused by C. cereale has already been reported in barley and wheat (Kim et al., 1991); however, this is the first report of this disease in oats in Korea.

First Report of Brown Spot Caused by Alternaria alternata on Potato (Solanum tuberosom L.) in Korea.

In June 2020, brown spot symptoms were observed in a commercial potato field located in Yeocheon, Gyeonggi Province, Korea. The symptoms were similar to those associated with early blight. Brown lesions on leaves were circular and expanded rapidly under high humidity and warm temperatures ranging 12°C at night to 30°C during daytime. Over 60% of potato (Solanum tuberosum L. cv. Superior) leaves showed the symptoms. For fungal isolation, infected leaf tissues (5 × 5 mm) from 14 infected samples were immersed in 70% ethanol for 1 min, rinsed three times in sterilized water, dried, placed on water agar amended with 100 ppm of streptomycin, and then incubated in the dark at 25°C. Hyphae emerging from the tissues were subcultured on V8-Juice agar (8% of V8-Juice, 1.5% agar, pH 7), and the obtaining cultures were subjected to single-spore isolation, resulting in 14 isolates (SYP-934~947). Three representative isolates, SYP-934 to SYP-936, were deposited in the Korean Agriculture Culture Collection (Accession Nos. KACC 410058 to KACC 410060). Conidia (n = 100) produced on the colony were brown, ellipsoid to ovoid with walls ornamented, 1 to 6 transverse and 0-3 vertical septa, and length × width of 20-45 × 7 to 24 µm (n = 100). Their morphological characteristics were consistent with Alternaria alternata (Simmons, 2007; van der Waals et al., 2011; Woundenberg et al. 2015). Sequences of the following loci in the 14 isolates were determined as described in Woundenberg et al. (2013 and 2014: the internal transcribed spacer (primer pairs VG9/ITS4, GenBank accession nos. OP581413-25), glyceraldehyde-3-phosphate dehydrogenase (gpd1/gpd2, OP588286-99), RNA polymerase second largest subunit (RPB2-5F2/fRPB2-7cR, OP588314-27), translation elongation factor 1-alpha (EF1-728F/EF1-986R, OP588300-13), Alternaria major allergen gene (Alt-For/Alt-Rev, OP588328-41), endopolygalacturonase (PG3/PG2b, OP588342-55), and an unknown gene region (OPA10-2R/OPA10-2L, OP588356-68). A neighbor-joining phylogenetic analysis based on the concatenated gene sequences, which was performed using the MEGA X program (Kumar et al., 2018), placed the 14 isolates in the clade containing A. alternata isolates. To test pathogenicity, one-month-old potato (S. tuberosum cv. Superior) plants grown in a 25°C growth chamber were sprayed with conidial suspensions (1×106 conidia/mL) prepared from 14-day-old cultures of three isolates (KACC 410058 to KACC 410060). Sterile distilled water was used as the control treatment. The inoculated pots were placed in a plastic box to maintain high humidity and incubated in the dark at 25°C for 2 days. The plants were transferred to a growth chamber (16h light with over 70% humidity at 25°C). Symptoms were first observed after 3 days post inoculation (dpi) with all three isolates, and severe brown spot symptoms were observed after 7 dpi. No symptom was observed in the control treatment. The pathogenicity assay was repeated at triplicate. Reisolated cultures from lesions were confirmed to be A. alternata based on their sequence at the rpb2 locus, thus fulfilling Koch's postulates. Alternaria alternata has been reported to cause brown spot and leaf blight on potato leaves in Israel (Dorby et al., 1984) and South Africa (van der Waals., et al. 2011). To our knowledge, this study is the first report of A. alternata causing brown spot disease in Korea.

First Report of Brown Leaf Spot Caused by Epicoccum tobaicum on Oat (Avena sativa) in Korea.

Oat (Avena sativa) is one of the Korean winter crops, and oat consumption has been increasing because it is widely perceived as a superfood. Recently, various fungal diseases have been reported likely due to climate changes during the winter season in Korea (Choi et al., 2018; Kim, 2020). During the 2020-2021 winter to spring, we surveyed new fungal diseases among cereals, including oats, in the southern region of Korea. In April 2021, brown leaf spots on oat leaves were observed in Gangjin, Jeollanam-do, Korea. These brown spots were irregular circles, ranging from 2-7 mm in diameter. Samples from three infected leaves were surface sterilized by treating them with 70% ethanol for 1 min and 1% NaOCl for 1 min. The samples were subsequently rinsed at least twice with distilled water and dried with a sterile paper towel before being placed on 1.5% water agar supplemented with 100 ppm streptomycin. Hyphal tips derived from infected tissues after incubation at 25C for 7 days were transferred to a fresh potato dextrose agar (PDA). Three isolates, labeled as KJO-AN2-S1, KJO-AN2-S2 and KJO-AN2-S3, were obtained via single hyphal tip purification. Colonies on PDA were pigmented vermilion and subsequently turned to saffron color with irregular margins after 7 days. Conidia produced on PDA were golden to dark brown, globose to subglobose, solitary, and measured 15.5-21.5 µm in diameter (n=50). Cultural and morphological characteristics suggested that these isolates belong to Epicoccum species (Chen et al. 2017). For identification by sequencing, the ITS (MW691866-68), tub2 (MW691872-74), and rpb2 (MW691869-71) regions of three isolates were amplified using the primer pairs ITS5/ITS4 (White et al., 1990), Btub2Fd/ Btub4Rd (Woudengerg et al., 2009), and RPB2-5F2 (Sung et al., 2007)/fRPB2-7cR (Liu et al., 1999), respectively. A maximum likelihood phylogenetic analysis based on the concatenated ITS, tub2, and rpb2 sequences placed the three isolates within a clade comprising E. tobaicum CBS 384.36. A mycelial plug (5 mm diameter) was inoculated onto wounded and unwounded leaves of healthy 12-day-old oat (cv. Choyang) seedlings. The control leaves were inoculated with a sterile PDA plug. All inoculated and control plants were placed in a plastic box and incubated at 20℃ in darkness with 100% humidity. After 1 day, the inoculated mycelial plug or sterile PDA plug from plants was removed; the plants in plastic boxes were then transferred to a growth chamber set at 20℃ with 12 h light and 60-70% humidity. While brown spot lesions were observed in both unwounded and wounded leaves 7 days post-inoculation, both wounded and unwounded control leaves remained asymptomatic. The pathogen was recovered from all symptomatic leaf tissues but could not be isolated from control leaves. The re-isolated pathogen was identified as E. tobaicum through morphological characterization and sequence-based identification, fulfilling Koch's postulates. This study is the first to report a causal relationship between E. tobaicum and brown leaf spot disease of oat in Korea. Identification of this newly emerging fungal disease on oats will help prepare for effectively managing this disease.

First Report of Canker Caused by Pectobacterium actinidiae on Asian Pear Trees (Pyrus pyrifolia) in South Korea.

In July 2020, pear trees (Pyrus pyrifolia cv. Niitaka) with cankers displaying dark-red bacterial ooze on the trunk and branches were found in two pear orchards located in Naju, Jeollanam-do, South Korea (34°57'50″ N, 126°43'52″ E and 34°56'14″ N, 126°33'42″ E). The incidence was 1.5% (3 out of 200 trees) and 0.83% (1 out of 120 trees), respectively. The symptoms were similar to those of the bleeding canker caused by Dickeya fangzhongdai (Choi et al. 2021), which is typically observed in October. The bacterial ooze was suspended in sterile water and streaked in Luria-Bertani (LB) medium to isolate single bacterial colonies. Two isolates (PRI-B16 and PRI-B17) from representative diseased trees were selected for investigation. Physiological and biochemical characteristics of the isolates analyzed using the BIOLOG GEN III MicroPlate™ system (Biolog, Hayward, CA, USA) were similar to the characteristics of Pectobacterium actinidiae (Portier et al. 2019). These isolates were positively utilized stachyose, L-galactonic acid-g-lactone, guanidine hydrochloride and weakly utilized (-)-D-arabitol (Portier et al. 2019). Bacterial genomic DNA was extracted from cell cultured in 5 ml LB at 28C for 2 days using G-spin DNA extraction kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol. PCR amplification was amplified as Portier et al. (2019). The generated their sequences of the small subunit ribosomal RNA (16S rRNA) using primers 27f and 1492r (Heuer et al. 1997) (Genebank accession numbers: ON951863 and ON951864) were 99.86% and 99.76% identical, respectively, to that of P. actinidiae isolate SCPJ-1 (KY307837.1) by a BLAST search against gene bank databases. The dnaX (Genebank accession nos: ON960281 and ON960282), leuS (Genebank accession nos: ON960283 and ON960284), and recA (Genebank accession nos: ON960285 and ON960286) genes of these isolates were also amplified and sequenced by previously described Stawiak et al. (2009) for dnaX and leuS, and Waleron et al. (2002) for recA. A neighbor-joining phylogenetic analysis based on the concatenated dnaX, leuS, and recA sequences placed the two isolates in a clade containing previously identified P. actinidiae isolates. A pathogenicity test was conducted using two-year-old pear (P. pyrifolia cv. Nittaka) trees grown in a greenhouse. Wounded and unwounded pear tree branches were inoculated with 10 µL of the bacterial suspension (108 CFU/ml) or sterile water as a control. The inoculated plants were maintained at 30°C without light for 2 days under 85-90% humidity. At 7 days post-inoculation, bacterial ooze was observed on the branches inoculated with a bacterial suspension, whereas branches subjected to unwounded inoculation and water inoculation exhibited no symptoms. This assay was performed three times. We reisolated two colonies from each sample showing typical bleeding symptoms and confirmed their identity by sequencing the dnaX locus. Pectobacterium actinidiae has been reported to cause canker in pear trees in Brazil (Araujo et al. 2021) as well as kiwifruit in South Korea (Koh et al. 2012). This is the first report of P. actinidiae causing canker on pear trees in South Korea and is, therefore, pathologically significant.

Comparison of projected rice blast epidemics in the Korean Peninsula between the CMIP5 and CMIP6 scenarios.

Recently, the International Panel for Climate Change released the 6th Coupled Model Intercomparison Project (CMIP6) climate change scenarios with shared socioeconomic pathways (SSPs). The SSP scenarios result in significant changes to climate variables in climate projections compared to their predecessor, the representative concentration pathways from the CMIP5. Therefore, it is necessary to examine whether the CMIP6 scenarios differentially impact plant-disease ecosystems compared to the CMIP5 scenarios. In this study, we used the EPIRICE-LB model to simulate and compare projected rice blast disease epidemics in the Korean Peninsula using five selected family global climate models (GCMs) of the CMIP5 and CMIP6 for two forcing scenarios. We found a similar decrease in rice blast epidemics in both CMIP scenarios; however, this decrease was greater in the CMIP6 scenarios. In addition, distinctive epidemic trends were found in North Korea, where the rice blast epidemics increase until the mid-2040s but decrease thereafter until 2100, with different spatial patterns of varying magnitudes. Controlling devastating rice blast diseases will remain important during the next decades in North Korea, where appropriate chemical controls are unavailable due to chronic economic and political issues. Overall, our analyses using the new CMIP6 scenarios reemphasized the importance of developing effective control measures against rice blast for specific high-risk areas and the need for a universal impact and vulnerability assessment platform for plant-disease ecosystems that can be used with new climate change scenarios in the future. Supplementary information: The online version contains supplementary material available at 10.1007/s10584-022-03410-2.

A comparative genomic analysis of lichen-forming fungi reveals new insights into fungal lifestyles.

Lichen-forming fungi are mutualistic symbionts of green algae or cyanobacteria. We report the comparative analysis of six genomes of lichen-forming fungi in classes Eurotiomycetes and Lecanoromycetes to identify genomic information related to their symbiotic lifestyle. The lichen-forming fungi exhibited genome reduction via the loss of dispensable genes encoding plant-cell-wall-degrading enzymes, sugar transporters, and transcription factors. The loss of these genes reflects the symbiotic biology of lichens, such as the absence of pectin in the algal cell wall and obtaining specific sugars from photosynthetic partners. The lichens also gained many lineage- and species-specific genes, including those encoding small secreted proteins. These genes are primarily induced during the early stage of lichen symbiosis, indicating their significant roles in the establishment of lichen symbiosis.Our findings provide comprehensive genomic information for six lichen-forming fungi and novel insights into lichen biology and the evolution of symbiosis.

Alternative splicing diversifies the transcriptome and proteome of the rice blast fungus during host infection.

Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus Magnaporthe oryzae, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-M. oryzae interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.

Time-Lapse Imaging of Root Pathogenesis and Fungal Proliferation Without Physically Disrupting Roots.

Microscopic observation of root disease onset and progression is typically performed by harvesting different plants at multiple time points. This approach prevents the monitoring of individual encounter sites over time, often mechanically damages roots, and exposes roots to unnatural conditions during observation. Here, we describe a method developed to avoid these problems and its application to study Fusarium oxysporum-Arabidopsis thaliana interactions. This method enabled three-dimensional, time-lapse imaging of both A. thaliana and F. oxysporum as they interact via the use of confocal and multi-photon microscopy and facilitated inquiries about the genetic mechanism underpinning Fusarium wilt.

Erratum: Retraction of Figure 1 in the Article "Immediate Hypersensitivity Reactions Induced by Triamcinolone in a Patient With Atopic Dermatitis".

This corrects the article on p. e87 in vol. 33, PMID: 29542298.

Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin.

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Nematicidal Activity of Grammicin Biosynthesis Pathway Intermediates in Xylaria grammica KCTC 13121BP against Meloidogyne incognita.

Grammicin, a polyketide metabolite produced by the endolichenic fungus Xylaria grammica KCTC 13121BP, shows strong nematicidal activity against Meloidogyne incognita. This study was performed to elucidate the grammicin biosynthesis pathway of X. grammica KCTC 13121BP and to examine the nematicidal activity of the biosynthesis intermediates and derivatives against M. incognita. Two grammicin biosynthesis intermediates were isolated from a T-DNA insertion transformant (strain TR-74) of X. grammica KCTC 13121BP and identified as 2-(hydroxymethyl)cyclohexa-2,5-diene-1,4-dione (compound 1) and 2,5-dihydroxybenzaldehyde (compound 2), which were also reported to be intermediates in the biosynthesis pathway of patulin, an isomer of grammicin. This indicates that the grammicin biosynthesis pathway overlaps almost with that of patulin, except for the last few steps. Among 13 grammicin biosynthesis intermediates and their derivatives (except grammicin), toluquinol caused the highest M. incognita J2 mortality, with an LC50/72 h value of 11.13 µg/mL, which is similar to grammicin with an LC50/72 h value of 15.95 µg/mL. In tomato pot experiments, the wettable powder type formulations (WP) of toluquinol (17.78 µg/mL) and grammicin (17.78 µg/mL) also effectively reduced gall formation on the roots of tomato plants with control values of 72.22% and 77.76%, respectively, which are much higher than abamectin (16.67%), but lower than fosthiazate (100%). The results suggest that toluquinol can be used directly as a biochemical nematicide or as a lead molecule for the development of new synthetic nematicides for the control of root-knot nematode diseases.

Production and Activity of Cristazarin in the Lichen-Forming Fungus Cladonia metacorallifera.

Lichens are a natural source of bioactive compounds. Cladonia metacorallifera var. reagens KoLRI002260 is a rare lichen known to produce phenolic compounds, such as rhodocladonic, thamnolic, and didymic acids. However, these metabolites have not been detected in isolated mycobionts. We investigated the effects of six carbon sources on metabolite biosynthesis in the C. metacorallifera mycobiont. Red pigments appeared only in Lilly and Barnett's media with fructose at 15 °C after 3 weeks of culture and decreased after 6 weeks. We purified these red pigments using preparative-scale high performance liquid chromatography and analyzed them via nuclear magnetic resonance. Results indicated that 1% fructose-induced cristazarin and 6-methylcristazarin production under light conditions. In total, 27 out of 30 putative polyketide synthase genes were differentially expressed after 3 weeks of culture, implying that these genes may be required for cristazarin production in C. metacorallifera. Moreover, the white collar genes Cmwc-1 and Cmwc-2 were highly upregulated at all times under light conditions, indicating a possible correlation between cristazarin production and gene expression. The cancer cell lines AGS, CT26, and B16F1 were sensitive to cristazarin, with IC50 values of 18.2, 26.1, and 30.9 µg/mL, respectively, which highlights the value of cristazarin. Overall, our results suggest that 1% fructose under light conditions is required for cristazarin production by C. metacorallifera mycobionts, and cristazarin could be a good bioactive compound.

Draft Genome Sequence of Xylaria grammica EL000614, a Strain Producing Grammicin, a Potent Nematicidal Compound.

An endolichenic fungus, Xylaria grammica strain EL000614, showed strong nematicidal effects against plant pathogenic nematode, Meloidogyne incognita by producing grammicin. We report genome assembly of X. grammica EL000614 comprised of 25 scaffolds with a total length of 54.73 Mb, N50 of 4.60 Mb, and 99.8% of BUSCO completeness. GC contents of this genome were 44.02%. Gene families associated with biosynthesis of secondary metabolites or regulatory proteins were identified out of 13,730 gene models predicted.

First Report of Anthracnose Caused by Colletotrichum fructicola on Hybrid Pear Fruit in Korea.

In August 2020, anthracnose-like symptoms was observed on pear fruit (Pyrus pyrifolia  P. communis) cultivated at 0.2 ha by the National Institute of Horticultural and Herbal Science Pear Research Institute at the Rural Development Administration (Naju, Jeonnam Province in Korea). Symptoms were observed only on fruit (112 days after full bloom (DAFB)), and disease incidences was at least 90%. Initial black specks developed into larger brown or black lesions on fruit after 3 days. Later, sunken lesions with orange conidial masses were observed. Finally, infected fruit dropped prematurely. To isolate and identify the pathogen, small pieces (5  5 mm) from the margin of lesions on fruit were surface sterilized by immersing in 70% ethanol for 1 minute, washed three times with sterile water, dried, and placed on water agar amended with 100 ppm streptomycin, then incubated in the dark at 25°C. Hyphae emerging from the three independent tissues were subcultured on Potato Dextrose Agar (PDA), resulting in three independent isolates (CP-1, CP-2, CP-3) after single spore isolation. Colonies were pale gray on PDA, but the colony edges were white. Conidia were transparent, cylindrical with rounded ends, and 13.8 to 20.1 µm  4.8 to 6.2 µm (avg. 18.3 µm  5.4 µm, n = 100) in size. Appressoria were dark brown, globose or subcylindrical, and 6.3 to 9.5 µm  5.2 to 6.9 µm in size (8.1  6.1 µm, n = 100). The morphological characteristics were similar to the descriptions of C. gloeosporioides species complex (Weir et al. 2012). Sequences of ITS (MT921589-91), GAPDH (MT921987-89), CAL (MT921990-92), ACT (MT921993-95), CHS-1 (MT921996-98), TUB2 (921999-01), and ApMAT (MT922002-04) sequences from CP-1, CP-2, and CP-3 matched with C. fruiticola strain BRIP 62871 (100%; MK298285), HXQT-2 (100%; MN52588), HXQT-2 (100%; MN52839), HXQT-2 (99.65; MN525801), ICKP18B4 (99.34%; LC494275), HB5 (100%; MH985245), and GQHZJ23 (100%; MN338294), respectively. Concatenated gene sequences were used for a phylogenetic analysis based on the maximum likelihood method. The reference gene accessions and other information are presented in Weir et al. (2012). The analysis placed the isolates within a clade comprising C. fructicola. Pathogenicity of CP-1 was tested using 120 healthy pear fruits. The fruit surfaces were sterilized with 70% ethyl alcohol for 2 min and washed twice with sterilized water. Three 120 DAFB fruits were inoculated with 10 l of a conidial suspension (1×106 conidia/ml) with and without wounding. Another three control fruits were inoculated with sterile distilled with and without wounding. The inoculated fruit were placed in a plastic box to maintain high humidity and incubated in the dark at 25°C. Symptoms were observed on both wounded fruits after 3 days post inoculation (dpi) and 5 dpi on the unwounded fruits. No symptoms were observed in the control on both the wounded fruits. Pathogenicity tests was performed in duplicate. The pathogen was re-isolated from symptomatic tissues (100%) on treatments on both the wounded and unwounded fruits, but not control. The identity of the both re-isolated pathogen from the wounded and unwounded fruits was confirmed via analysis of seven genes and morphological characteristics, thus fulfilling Koch's postulates. Although C. fructicola has been reported on apples and peaches in Korea (Kim et al. 2018; Lee et al. 2020), this is the first report of anthracnose caused by C. fructicola on pear fruit in Korea, highlighting the need for systematically investigating the diversity and incidence of pear anthracnose in Korea. This study will contribute to the development of control strategies for anthracnose disease on pear fruit in Korea.