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.

First Report of Fruit Scab Caused by Alternaria alternata on Actinidia chinensis in Korea.

Kiwi (Actinidia chinesis) is an economically important fruit in Korea, with 1,300 ha cultivated and a production of approximately 25,000 tons per year (Kim and Koh, 2018; Kim and Choi, 2023). In late June 2020, fruit scab symptoms were observed on A. chinensis var. rufopulpa in an orchard in Suncheon, Korea. The incidence of scab symptoms among 20-year-old trees was over 75%, primarily superficial, but rendered the fruit less marketable. In the initial stages of the disease, small, light-brown, circular, and oval spots were formed. As the superficial spots expanded, they became cracked scabs measuring 1 to 7 cm with light edges at the later stages. To isolate the causal pathogen, two lesions were cut from two sections of symptomatic tissue, from each of seven fruits from seven trees. Lesions were surface-sterilized with 70% ethanol for 1 min and washed three times with sterilized distilled water (SDW). The sterilized pieces were placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for one week. After subculturing on PDA, single-spore isolation produced 14 isolates: SYP-410 to 423). All 14 colonies appeared greyish-green and cottony on PDA after 7 d. Conidia were pale brown, ellipsoid to obclavate, with ornamented walls, 1 to 6 transverse and 0 to 3 vertical septa, and length × width of 21.5 to 53.4 × 7.3 to 19.2 µm (avg. 33.0 × 12.0 µm, n = 100). Their morphological characteristics were consistent with Alternaria spp. (van der Waals et al. 2011; Woudenberg et al. 2015). We randomly selected three isolates from the morphologically similar cultures and named them SYP-412 to 414 for further investigation. The ITS (GenBank accession nos.: OR901850 to 52), gapdh (OR924309 to 11), tef1 (OR924312 to 14), rpb2 (OR924315 to 17), Alt a1 (OR924318 to 20), endoPG (OR924321 to 23), and OPA10-2 (OR924324 to 26) sequences from SYP-412 to 414 had a 100% (515 bp/515 bp), 100% (578/578), 100% (240/240), 100% (724/724), 95.55% (451/472), 99.33% (445/448), and 100% (634/634) identity with that of type strain A. alternata CBS 918.96 (AF347032, AY278809, KC584693, KC584435, AY563302, KP124026, and KP124633), respectively. Results from the maximum likelihood phylogenetic analysis, based on the seven concatenated gene sequences, placed the representative isolates in a clade with A. alternata. Pathogenicity of SYP-412 was tested using 12 surface-sterilized two-month-old kiwifruits on a 20-year-old trees. Six kiwifruits were spray-inoculated with 5 mL of a conidial suspension (1 × 106 conidia/ml) generated after culturing in PDA medium for 7 d, with or without wounding. Another six control fruits were inoculated with SDW with and without wounding. The inoculated kiwifruits were enclosed in plastic bags to maintain high humidity for one day. Scab symptoms were observed in both wounded and unwounded fruits six weeks after inoculation, but not in the control. The pathogenicity test was performed on a total of three separate trees twice. To satisfy Koch's postulates, A. alternata was re-isolated from all the symptomatic tissues and confirmed by analyzing the ITS and rpb2 genes. Although scab disease caused by A. tenuissima (now A. alternata) has been previously reported in kiwifruit of A. chinensis var. rufopulpa in China (Woudenberg et al. 2015; Ma et al., 2019), this is the first report of its occurrence on kiwifruit in Korea and will help in future detection and control.

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.

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 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.

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 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.

First Report of Bleeding Canker of Pear Tree Trunks Caused by Dickeya fangzhongdai in Korea.

Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53'50.54″ N, 126°39'00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 µl and 2 µl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.

High Contiguity Whole Genome Sequence and Gene Annotation Resource for Two Venturia nashicola Isolates.

Venturia nashicola is a fungal pathogen that causes Asian pear scab disease. This pathogen is of particular importance in Northeast Asian countries, where Asian pears are grown industrially. Scab disease in Asian pear is currently controlled by fungicide spraying and this situation calls for developing scab resistant cultivars. High-quality genome data are therefore required for in-depth comparative genome analysis of different isolates of V. nashicola and V. pyrina, a closely related species, which only infects European pear plants. Here, we report the high-contiguity whole genome assembly of two V. nashicola isolates, which is expected to enable genome comparisons for identification of the genes involved in host range determination of V. nashicola.

An Alternative Method to Evaluate Resistance to Pear Scab (Venturia nashicola).

Two pear cultivars with different degrees of resistance to Venturia nashicola were evaluated on the basis of a disease severity rating for pear scab resistance under controlled environmental condition. Two inoculation techniques were tested: the procedure for inoculation by dropping conidia suspension of V. nashicola; the procedure by deposition of agar plug on the abaxial surface of pear leaves. All tested cultivars resulted in blight symptoms on the inoculated leaves and became spread to uninoculated region or other leaves. Although both methods provide satisfactory infection of V. nashicola on pear leaves, the mycelial plug method of inoculation was more reliable than the spray inoculation method for the evaluation of pear scab disease resistance. The incubation period of V. nashicola in the resistant pear cultivar, Greensis was longer than that in the susceptible cultivar, Hwasan.

Draft Genome Sequence of Streptomyces Strain SJ1-7, a Soil Bacterial Isolate.

The draft genome sequence of Streptomyces strain SJ1-7, a bacterial strain isolated from the rhizosphere of a Pinus densiflora plant, is reported. The whole-genome assembly comprised 7.9 Mbp, with a GC content of 71.80% and 4,262 predicted protein-coding genes.

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.

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.

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.

Retrospective Study on the Seasonal Forecast-Based Disease Intervention of the Wheat Blast Outbreaks in Bangladesh.

Seasonal disease risk prediction using disease epidemiological models and seasonal forecasts has been actively sought over the last decades, as it has been believed to be a key component in the disease early warning system for the pre-season planning of local or national level disease control. We conducted a retrospective study using the wheat blast outbreaks in Bangladesh, which occurred for the first time in Asia in 2016, to study a what-if scenario that if there was seasonal disease risk prediction at that time, the epidemics could be prevented or reduced through prediction-based interventions. Two factors govern the answer: the seasonal disease risk prediction is accurate enough to use, and there are effective and realistic control measures to be used upon the prediction. In this study, we focused on the former. To simulate the wheat blast risk and wheat yield in the target region, a high-resolution climate reanalysis product and spatiotemporally downscaled seasonal climate forecasts from eight global climate models were used as inputs for both models. The calibrated wheat blast model successfully simulated the spatial pattern of disease epidemics during the 2014-2018 seasons and was subsequently used to generate seasonal wheat blast risk prediction before each winter season starts. The predictability of the resulting predictions was evaluated against observation-based model simulations. The potential value of utilizing the seasonal wheat blast risk prediction was examined by comparing actual yields resulting from the risk-averse (proactive) and risk-disregarding (conservative) decisions. Overall, our results from this retrospective study showed the feasibility of seasonal forecast-based early warning system for the pre-season strategic interventions of forecasted wheat blast in Bangladesh.

Genetic Diversity of the Pear Scab Fungus Venturia nashicola in Korea.

Scab disease caused by Venturia nashicola is of agroeconomic importance in cultivation of Asian pear. However, little is known about the degree of genetic diversity in the populations of this pathogen. In this study, we collected 55 isolates from pear scab lesions in 13 major cultivation areas in Korea and examined the diversity using sequences of internal transcribed spacer (ITS) region, ß-tubulin (TUB2), and translation elongation factor-1α (TEF-1α) genes as molecular markers. Despite a low level of overall sequence variation, we found three distinctive subgroups from phylogenetic analysis of combined ITS, TUB2, and TEF-1α sequences. Among the three subgroups, subgroup 1 (60% of isolates collected) was predominant compared to subgroup 2 (23.6%) or subgroup 3 (16.4%) and was distributed throughout Korea. To understand the genetic diversity among the subgroups, RAPD analysis was performed. The isolates yielded highly diverse amplicon patterns and none of the defined subgroups within the dendrogram were supported by bootstrap values greater than 30%. Moreover, there is no significant correlation between the geographical distribution and the subgroups defined by molecular phylogeny. Our data suggest a low level of genetic diversification among the populations of V. nashicola in Korea.

Development of Carrot Medium Suitable for Conidia Production of Venturia nashicola.

The causal fungus of pear scab, Venturia nashicola, grows slowly and rarely produces conidia on artificial media in the laboratory, but it produced conidia on the Cheongah medium containing Cheongah powder. V. nashicola grew too slow to produce conidia until 15 days after cultivation but produced conidia with 4 × 104 conidia/plate 30 days after cultivation on the Cheongah medium containing 1% Cheongah powder. V. nashicola showed a peak production of conidia with 4.5 × 105 conidia/plate 60 days after cultivation on the carrot medium containing 2% carrot powder, one of the constituents of Cheongah powder. The carrot medium is considered to be the best medium to obtain conidia of V. nashicola in the laboratory until now. This is the first report on the development of a suitable medium for conidia production of V. nashicola, as far as we know.

Outbreak and Spread of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae pv. actinidiae Biovar 3 in Korea.

A bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa), is a causal agent of kiwifruit bacterial canker worldwide. Psa biovar 3 (Psa3) was first detected in 2011 at an orchard in Dodeok-myeon, Goheunggun, Jeonnam Province in Korea. In this study, we present the results of an epidemiological study regarding Psa3 occurrence on kiwifruit orchards in Korea for the period of 2013 to 2015. Since the first detection of Psa3 in 2011, there was no further case reported by 2013. However, Psa3 was rapidly spreading to 33 orchards in 2014; except for three orchards in Sacheonsi, Gyeongnam Province, most cases were reported in Jeju Island. Entering 2015, bacterial canker by Psa3 became a pandemic in Korea, spreading to 72 orchards in Jeju Island, Jeonnam, and Gyeongnam Provinces. Our epidemiological study indicated that the first Psa3 incidence in 2011 might result from an introduction of Psa3 through imported seedlings from China in 2006. Apart from this, it was estimated that most Psa3 outbreaks from 2014 to 2015 were caused by pollens imported from New Zealand and China for artificial pollination. Most kiwifruit cultivars growing in Korea were infected with Psa3; yellow-fleshed cultivars (Yellow-king, Hort16A, Enza-gold, Zecy-gold, and Haegeum), red-fleshed cultivars (Hongyang and Enza-Red), green-fleshed cultivars (Hayward and Daeheung), and even a kiwiberry (Skinny-green). However, susceptibility to canker differed among cultivars; yellow- and red-fleshed cultivars showed much more severe symptoms compared to the green-fleshed cultivars of kiwifruit and a kiwiberry.