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.

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.

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.