First report of walnut anthracnose caused by Colletotrichum orientalis in South Korea.

Walnut (Juglans regia L.) is known as "hodu" in South Korea. It is cultivated as a cash crop and an ornamental plant across South Korea. Walnut anthracnose is one of the main limiting factors for walnut production. In June 2022, severe anthracnose was observed in several walnut fields in Gimcheon, South Korea. The disease incidence was ~35% and symptoms on affected fruits consist of necrotic and circular lesions with or without conidial mass. Six pure isolates with similar morphological characteristics were obtained from the tissue of six affected fruits. Diseased tissues were cut into 3-to-4 mm2 pieces, surface sterilized by 1% sodium hypochlorite (NaOCl) for 2 minutes, and washed three times in sterile distilled water, then dried by blotting. Four pieces/plates were placed onto potato dextrose agar (PDA; Difco Becton Dickinson) and incubated at 25°C in the dark for 2-3 days. To obtain pure isolates, hyphal tips were transferred onto fresh PDA. The colonies of all isolates on the PDA were light gray to pinkish on the upper side and pink to vinaceous on the reverse after 7 days of incubation at 25°C in the dark. The conidia were hyaline, fusiform, aseptate, and 11.3 to 20.1 µm × 3.6 to 6.9 µm (n =100). Appressoria were single, cylendrical, smooth-walled, dark brown, and 8.1 to 14.3 × 5.4 to 10.5 µm (n =30). The morphological characteristics of the isolates were comparable with those of Colletotrichum orientalis (Chen et al. 2022). Three isolates (WN23, WN28 and WN30P) were selected based on morphological characteristics for molecular identification. The beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and histone 3 (HIS3) genes of selected isolates were amplified and sequenced using the primers Bt2a/Bt2b, ACT512F/ACT783R, CHS-79F/CHS-345R and CYLH3F + CYLH3R, respectively (Damm et al. 2012). The resulting consensus sequences of each gene were deposited in GenBank with the accession numbers (TUB2: LC742910 to LC742912; CHS-1: LC742916 to LC742918; ACT: LC742919 to LC742921, HIS3: LC742922 to LC742924). The constructed maximum likelihood tree based on TUB2, ACT, CHS-1, and HIS3 sequence data revealed that the three isolates formed a clade with C. orientalis reference strain CBS 128532 (ex-type). The pathogenicity of two representative isolates (WN28 and WN30P) was confirmed on healthy, surface-sterilized, and detached walnut fruits. Both wounded and nonwounded fruits (10/isolate) were inoculated with the conidial suspension (106 spores/ml). The wounded and unwounded control fruits were inoculated with sterile distilled water. All the fruits (inoculated and control) were incubated in containers at 25°C with a 12-h/12-h light/dark cycle. This experiment was repeated twice. Necrotic lesions were observed in all inoculated wounded fruits, while unwounded and control fruits remained asymptomatic. The pathogen was re-isolated from inoculated fruits and identified as C. orientalis by morphological characteristics and ITS and TUB2 sequences. Damm et al. (2012) has identified C. fioriniae as species belongs to two well separated clades. However, Chen et al. (2022) split the C. fioriniae species into two species, with the new species called C. orientalis, as a causal agent of apple bitter rot. In previous studies, C. fioriniae has been reported as a causal of walnut anthracnose (Luongo et al. 2021; Zhu et al. 2015; Varjas et al. 2019). To our knowledge, this is the first report of C. orientalis as the causal agent of walnut anthracnose in South Korea. Since this disease reduces the commercial value and quality of walnut fruits, effective management practices should be developed to deal with C. orientalis.

Molecular Characterization and Fungicide Sensitivity of Jujube Pathogens Colletotrichum gloeosporioides Sensu Stricto and Colletotrichum nymphaeae in South Korea.

Jujube (Ziziphus jujuba) is cultivated across South Korea because of its medicinal and economic value. It is used as a sweetener in jam, tea, and snacks and a garnish in many cuisines. Anthracnose caused by Colletotrichum spp. accounts for huge economic losses for jujube growers. In 2019 and 2020, severe anthracnose was observed in the jujube-growing areas of South Korea. The infected fruit displayed small, water-soaked, sunken, circular spots. Infected fruit were collected from different commercial orchards of Boeungun and Gyeongsan regions of South Korea, and putative causal agents were isolated on potato dextrose agar. Based on the morphological and molecular characteristics, the fungal isolates were identified as Colletotrichum gloeosporioides sensu stricto and C. nymphaeae. The pathogenicity of these isolates was confirmed by inoculating a conidial suspension (1 × 106 conidia ml-1) on healthy fruit. The in vitro sensitivity of the fungal isolates to tebuconazole, carbendazim, and azoxystrobin was also tested. All isolates showed high sensitivity to azoxystrobin in terms of mycelial growth inhibition (half maximal effective concentration value of 0.01 to 0.6 µg/ml). To the best of our knowledge, this is also the first report of jujube anthracnose caused by C. nymphaeae in South Korea.

First report of anthracnose on spotted laurel caused by Colletotrichum fructicola in South Korea.

Spotted laurel (Aucuba japonica) is a popular ornamental bush (it has two-colored leaves and red berries) and is used outdoors and indoors for decoration in South Korea. Anthracnose reduces the aesthetic value of spotted laurel leaves. In August 2022, anthracnose symptoms were observed on leaves in a park at Jeju Island, South Korea. Approximately 55% of bushes were infected by this disease. Symptoms consisted of round or irregular lesions that initially appeared as black spots and coalesced into larger, black lesions covering whole leaves and twigs. Entire leaves wither and finally die. To identify the putative causal agent, 12 affected leaves were collected, placed in a plastic box containing moist tissue, and incubated at 25 ºC in the dark to obtain conidial mass. Conidial masses were produced on leaf lesions after 2 days, and then 12 morphologically similar fungal isolates were recovered following single the spore isolation technique on solid potato dextrose agar (PDA) (Cai et al. 2009). Ten-day-old colonies were olivaceous gray with immersed perithecia on the upper side and black at the center on the reverse side. Conidia were aseptate, cylindrical with round ends and measured 14.9 - 22.7 × 5.5 - 9.4 µm (n = 80). Appressoria were brown, irregular in shape, and 7.0 - 16.1 × 5.00 - 9.9 µm (n = 50). Asci were eight-spored, banana-shaped, and measuring 60.8 - 123.1 × 13.00 - 18.9 µm (n = 30). Hyaline ascospores were single-celled, curved or straight with round ends, and ranged in size was 15.5 - 23.3 × 5.1 - 11.8 µm (n = 50). The morphological characteristics of the isolates overlapped with those of Colletotrichum species within the C. gloeosporioides complex, including Colletotrichum fructicola (Weir et al. 2012). Five genomic DNA loci of the isolates, including the partial ITS rDNA region, ACT, GAPDH, TUB, and ApMat genes, were amplified and sequenced using ITSF1/ITS4, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting consensus sequences were deposited in the GenBank and the accession numbers (ITS = LC739331- LC739334, TUB = LC739335- LC739338, GAPDH = LC739339- LC739342, ACT = LC739343 -LC739346, ApMat = LC742925 - LC742928) were obtained. A maximum phylogenetic tree was constructed based on the combined data sets of ITS, ACT, GAPDH, TUB, ApMat sequences. The isolates were clustered with reference isolates of C. fructicola (isolates ICMP18581). The pathogenicity test was performed on uninfected, healthy spotted laurel cuttings in the pot. Five leaves per seedling were selected, surface sterilized with 70% ethanol, and rinsed with sterile distilled water (SDW). A sterile pin was used to make 3 to 4 wounds on each side of the leaf from the midrib. 10 µl of spore suspension per wound spot (1 × 106 spores/ml) was applied on the wounds of one site from midrib, and SDW was placed on the wounds of other site as a control. The treated seedlings were covered with sterile plastic bag and kept in a 12-h fluorescent light/dark cycle under greenhouse conditions at 25 ± 2°C and 80% relative humidity. Two seedlings were inoculated with a single isolate, and this experiment was repeated twice. Circular or irregular lesions appeared after 5 days of inoculation, while the control remained asymptotic. Koch's postulates were fulfilled by reisolating and reidentifying the causal agent from the lesions of inoculated leaves. Colletotrichum fructicola has been reported as the causal agent of anthracnose on mango (Joa et al. 2016), apple (Kim et al. 2018), grapes (Lim et al. 2019), peaches (Lee et al. 2020), and hybrid pear (Choi et al. 2021) in South Korea. To the best of our knowledge, it is the first report of C. fructicola causing anthracnose on spotted laurel. This study will be helpful to develop effective management strategies to minimize leaf lesions.

First report of leaf anthracnose caused by Colletotrichum camelliae on tea plants (Camellia sinensis) in South Korea.

The tea plant (Camellia sinensis (L.) O. Kuntze) is a popular non-alcoholic beverage crop worldwide. The tea market in South Korea is projected to increase annually by 4.59% (Statista, 2022). Boseong, Hadong, and Jeju Island are the main tea-growing regions in South Korea. Anthracnose is one of the major diseases of tea plants and is responsible for substantial yield loss and poor tea quality. In 2021, anthracnose of tea (disease incidence of 30%) was observed in a garden (33°28'45.5"N 126°42'02.2"E) at Jeju Island, where the Yabukita cultivar has been cultivated. The typical symptoms consisted of round or irregularly shaped lesions with gray-white centers and purple-brown borders. Twelve morphologically similar isolates were recovered from 12 infected leaves using the single spore isolation method on solid potato dextrose agar (PDA) (Cai et al. 2009). Four representative isolates (GT6, GT7, GT8, and GT11) were identified based on morphology, molecular analysis, and pathogenicity tests. The upper side of seven-day-old colonies on PDA (incubated at 25 °C in the dark) was off-white with white aerial mycelia and gray-white with black zonation on their reverse side. Conidia were hyaline, aseptate, cylindrical, with both obtuse ends, and measuring 12.3 - 25.8 µm × 4.4 - 9.3 µm (n = 50). Appressoria were dark brown, irregularly shaped with a smooth edge, and measuring 7.3 -18.8 µm × 6.9 - 11.3 µm (n = 50). According to morphological characteristics, the fungal isolates were tentatively identified as the Colletotrichum gloeosporioides complex, including C. caelliae (Wang et al. 2016; Weir et al. 2012). The genomic DNA was extracted, and the ribosomal internal transcribed spacer (ITS), ß-tubulin-2 (TUB2) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, actin (ACT), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) genes were amplified and subsequently sequenced using primer sets ITS1/ITS4, BT2a/BT2b, GDF1/GDR1, ACT-512F/ACT-783R1, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting sequences were deposited in GenBank accession numbers (LC738932-LC738959). All the representative isolates were identified as C. camelliae by constructing the 50% majority rule consensus and maximum likelihood phylogenetic treebased on the combined ITS, TUB2, GAPDH, ACT, CAL, and ApMat sequences using MrBayes v. 3.2.2 and Mega X, respectively (Kumar et al., 2018; Ronquist et al. 2012). The pathogenicity of these isolates was tested on healthy leaves of 2- years-old tea seedlings (the Yabukita cultivar). Onside of unwounded or wounded leaves of seedlings were inoculated with 20 µL of conidial suspension (1 × 106 conidia or spores/ml) per spot (3-4 wounded or unwounded spots per side per leaf). Another side of the leaves received sterile distilled water and served as a control. Each treatment was replicated three times (three seedlings/isolate and four leaves per seedling) and this experiment was repeated twice. All plants were covered with plastic bags, placed in a growth chamber, and incubated at 25 °C with a 12-h photoperiod and 90% relative humidity. Typical anthracnose symptoms appeared on wounded leaves after two days of inoculation. Unwounded and controlled leaves remain asymptotic. To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaf lesions and identified as C. camelliae based on morphology and ITS sequences. Colletotrichum camelliae is a very common pathogen associated with tea anthracnose worldwide, including China (Liu et al. 2015; Wang et al. 2016).To the best of our knowledge, this is the first report of anthracnose in tea trees caused by C. camelliae in South Korea. The results of this study could help come up with better ways to keep an eye on and deal with this devastating on tea plants. Key words: Tea anthracnose, Colletotrichum camelliae, pathogenicity References Cai, L., et al. 2009. Fungal Divers. 39:183. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Liu, F. et al. 2015. Persoonia. 35: 63-86. Ronquist, F. et al. 2012. Syst. Biol. 61:539-542. Silva, D. N. et al. 2012. Mycologia. 104:396-409. Statista 2022. Statista Digital Market out Look. Available at www.statista.com. Wang, Y.-C. et al. 2016. Sci. Rep. 6: 35287. Weir, B. S., et al. 2012. Stud. Mycol. 73:115.

Morphological and molecular characteristics of fungal species associated with crown rot of strawberry in South Korea.

BACKGROUND: Crown and root rot is the most important and destructive strawberry diseases in Korea as it causes substantial economic loss. In August 2020, a severe outbreak of crown and root rot on strawberries (Fragaria × ananassa Duch.) was observed in the greenhouse at Sangju, South Korea. Infected plantlets displayed browning rot within the crown and root, stunted growth, and poor rooting. METHODS AND RESULTS: Thirty fungal isolates were obtained from the affected plantlet. Isolates were identified based on morphological characteristics and pathogenicity test as well as sequence data obtained from internal transcribed spacer, large subunit ribosomal ribonucleic acid, translation elongation factor, and RNA polymerase II-second largest subunit. Results showed that the crown and root rot of strawberry in Korea was caused by three distinct fungal species: Fusarium oxysporum f. sp. fragariae, F. solani, and Plectosphaerella cucumerina. To the best of our knowledge, F. solani, and P. cucumerina are reported for the first time as the causal agents of the crown and root rot of strawberry in South Korea. Pathogenicity tests confirmed that these isolates are pathogenic to strawberry. CONCLUSIONS: Understanding the composition and biology of the pathogen population will be helpful to provide effective control strategies for the disease.

Alternaria koreana sp. nov., a new pathogen isolated from leaf spot of ovate-leaf Atractylodes in South Korea.

BACKGROUND: A new species within the genus Alternaria was isolated from the leaf spot of Atractylodes ovata in the Mungyeong and Hwabuk-myeon districts of the Gyeongbuk province of Korea. The leaves showed disease symptoms such as circular or irregular leaf spots and brown to dark brown with gray spots at the center. The leaves also showed that concentric rings were surrounded with yellow halos. METHODS AND RESULTS: Phylogenetic analysis was conducted using the sequence dataset of the internal transcribed spacer region and part of the glyceraldehyde-3-phosphate dehydrogenase. The RNA polymerase II second largest subunit, endopolygalacturonase, Alternaria major allergen gene, anonymous gene region, and translation elongation factor 1-alpha genes were used as well. Results showed that present fungal isolates were distinct from other species of the sect. Alternaria. Morphologically, the present isolates also differed from other members of the sect. Alternaria in their production of solitary conidia or conidial chains (two units) and conidial body features. Similarly, it exhibited moderate pathogenicity in the host plant. CONCLUSIONS: This study described and illustrated A. koreana as a new species and the causal agent of the leaf-spot disease on A. ovata in Korea.

First Report of Rhizopus oryzae Causing Rhizome Soft Rot of Ovate-leaf Atractylodes in South Korea.

Ovate-leaf Atractylodes (Atractylodes ovata) (OLA) is cultivated across South Korea to fulfil domestic need as a herbal medicine. In June 2021, the rhizomes with soft rot were observed in a commercial farmer field in Mungyeong, South Korea with 25% disease incidence. Initially, watery, black and soft lesion were observed on the rhizomes. Later, abundant aerial white mycelia covered the entire rhizome resulting in rot and death. Affected rhizomes were washed thoroughly using tap water, surface sterilized with 0.5% NaOCl and rinsed with sterile distilled water. Small pieces (2 to 5 mm2) of inner diseased tissues were cut, placed on potato dextrose agar (PDA) and incubated on at 25°C in the dark. Twelve morphologically similar pure fungal isolates were obtained by hyphal tipping twice on fresh PDA. Rhizoid, sporangiophores, sporangium, and sporangiospores were observed. Rhizoid and sporangiophores were nonseptate. Sporangia were globose while columella were globose or subglobose. Sporangiospores were numerous, varied in shape and size, subglobose, or oval, and measured 9.8 ± 1.5 (6.1 to 13.5) × 7.8 ± 1.1 (5.7 to 10.1) µm (n = 50). Morphological characteristics of the isolates were analogous to Rhizopus oryzae (Kwon et al. 2012, 2015). Sequences of the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (EF-1α) gene were analyzed for molecular identification. The primer pairs ITS1F/ITS4 and MEF10/MEF4 were used to amplify ITS and EF-1α respectively (Abe et al. 2007) and sequenced (Macrogen, Inc., Seoul, Korea). The obtained sequences were lodged with accession numbers LC705530 to LC705537. The ITS and EF-1α sequences were ≥ 99.5 % and ≥ 98.5% homologous with Rhizopus oryzae (strain CBS 381.52), respectively. In the maximum likelihood phylogenetic tree based on combined ITS and EF-1α sequences, the isolates formed a distinct clade with R. oryzae references isolates. Pathogenicity tests were conducted on healthy rhizomes with isolate R4. A conidial suspension (1× 106 conidia/mL) was sprayed on the surface of fifteen surface-sterilized rhizomes while fifteen rhizomes received sterile distilled water as controls. Treated rhizomes were placed in a plastic box containing wet paper tissues and incubated at 25°C in the12/12 day/light cycle. The typical rhizomes rot symptoms as seen in the field were observed on all inoculated rhizomes after 21 days of inoculation. The noninoculated rhizomes had no obvious symptoms. The causal fungus (3 isolates) was reisolated from inoculated rhizomes and identified as R. oryzae based on morphology and EF-1α sequence. This experiment was replicated twice. To the best of our knowledge this is the first report of R. oryzae infection on OLA rhizomes in South Korea. Rhizopus oryzae contained a complex of heterothallic closely related species and has been reported to cause 'Rhizopus rot' on various plants including apple, banana, mulberry, sweet potato, and tomato (Gnanesh et al. 2020; Khokhar et al. 2019; Kwon et al. 2012, 2015; Wang et al. 2017). Rhizome soft rot poses significant threat to OLA cultivation, sustainable management practices need to be adapted to control rhizome soft rot of ovate-leaf atractylodes.

First report of Colletotrichum fioriniae causing anthracnose on the fruit of omija (schisandra) in South Korea.

Omija or Schisandra (Schisandra chinensis Baillon) is one of Korea's most unique fruits and have been used in herbal medicine. It is also used for making cold drinks during summer and hot tea during winter. Among the different diseases, anthracnose poses great threat for omija production as it reduces the yield and market quality of fruits. In September 2021, severe anthracnose symptoms were observed in some commercial farmer fields (36°36'14.9"N 127°59'31.3"E) in Mungyeong and disease incidence was approximately 30 to 35%. The symptoms on fruits were manifested as small sunken lesions. The presumed fungi were isolated from necrotic tissues on potato dextrose agar (PDA) and purified using the single spore isolation technique (Cai et al. 2009; Hassan et al.2019). A total of 20 morphological similar isolates were obtained from 18 sampled fruits. The cultures were incubated at 25°C in the dark for 5 to 7 days. The colonies were pink with white aerial mycelium on the upper side and pink with black zones on the reverse. All the colonies produced fusiform conidia measuring 12.7 to 17.5 × 4.7 to 7.4 µm (n=50). The appressoria were brown, globose to ellipsoid and measuring 6.3 to 12 × 5.9 to 9.7 µm (n=30). Morphological characteristics of present isolates were in concordance with that of Colletotrichum acutatum sensu lato (Damm et al., 2012). For molecular identification, genomic DNA of five representative isolates was extracted for sequencing of target markers and the phylogenetic tree was constructed. For amplification and sequencing of internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) chitin synthase-1 (CHS-1), histone 3 (HIS3) and beta-tubulin (TUB2), the primer pairs ITS1/ITS4, GDF1/GDR1, ACT-512F/ACT-783R, CHS1-79F/CHS-1-354R, CYLH3F /CYLH3R and BT2a /BT2b were used respectively (Damm et al., 2012). The resulting sequences were deposited in GenBank with accession numbers LC680890 to LC680914 and LC687627 to LC687631. The maximum likelihood tree based on concatenated sequences of the five genes shows that present isolates were grouped in the C. fioriniae. Pathogenicity on red schisandra fruits was confirmed using the two isolates OR1 and O59. Fruit clusters (20-30 fruits/ cluster) were surface sterilized by dipping them in 1% NaOCl for 3 minutes, rinsing in sterile distilled water, and placing them in a sterile plastic box containing moist paper tissues. All the fruits in each cluster were inoculated with 10 µl of 106 conidia/ml suspensions of each isolate by following either the wounded or un- wounded method (Hassan et al.2019). Fruit clusters treated with sterile distilled water served as a control. Three fruit clusters were inoculated per isolate for each method. With the wounding method, all the inoculated fruits showed typical anthracnose symptoms after 4 days of incubation at 25 ± 1°C in the dark, while the control and un- wounded fruits remained symptomless. This test was repeated twice. The species C. fioriniae were reisolated from the infected fruit. The morphology and ITS sequences of the reisolated fungi were consistent with the original one, fulfilling Koch's postulates. The species C. fioriniae have been described as a causal agent of anthracnose of numerous hosts worldwide including South Korea (Damm et al., 2012; Hassan et al.2019; Oo et al. 2016; Lee et al. 2020). To our knowledge, this is the first report of C. fioriniae as causal agent of omija anthracnose in South Korea. As this disease results in deterioration of fruits quality, management practices should be tailored to address C. fioriniae management. Acknowledgments This research was supported by Kyungpook National University Research Fund, 2021. References Cai, L., et al. 2009. Fungal Divers. 39:183. Damm, U., et al. 2012. Stud. Mycol. 73:37. Hassan, O., et al. 2019. Sci. Rep. 9:12089. Lee, D., et al. 2020. Mycobiology. 48: 210. Oo, M.M., et al. 2016. Mycobiology. 44: 325.

First Report on Ovate-leaf Atractylodes Damping-off Caused by Fusarium oxysporum species Complex in South Korea.

In South Korea, ovate-leaf atractylodes (OLA) (Atractylodes ovata) is cultivated for herbal medicine. During May to June 2019, a disease with damping off symptoms on OLA seedlings were observed at three farmer fields in Mungyeong, South Korea. Disease incidence was estimated as approximately 20% based on calculating the proportion of symptomatic seedlings in three randomly selected fields. Six randomly selected seedlings (two from each field) showing damping off symptoms were collected. Small pieces (1 cm2) were cut from infected roots, surface-sterilized (1 minute in 0.5% sodium hypochlorite), rinsed twice with sterile water, air-dried and then plated on potato dextrose agar (PDA, Difco, and Becton Dickinson). Hyphal tips were excised and transferred to fresh PDA. Six morphologically similar isolates were obtained from six samples. Seven-day-old colonies, incubated at 25 °C in the dark on PDA, were whitish with light purple mycelia on the upper side and white with light purple at the center on the reverse side. Macroconidia were 3-5 septate, curved, both ends were pointed, and were 19.8-36.62 × 3.3-4.7 µm (n= 30). Microconidia were cylindrical or ellipsoid and 5.5-11.6 × 2.5-3.8 µm (n=30). Chlamydospores were globose and 9.6 -16.3 × 9.4 - 15.0 µm (n=30). The morphological characteristics of present isolates were comparable with that of Fusarium species (Maryani et al. 2019). Genomic DNA was extracted from 4 days old cultures of each isolate of SRRM 4.2, SRRH3, and SRRH5, EF-1α and rpb2 region were amplified using EF792 + EF829, and RPB2-5f2 + RPB2-7cr primer sets, respectively (Carbone and Kohn, 1999; O'Donnell et al. 2010) and sequenced (GenBank accession number: LC569791- LC569793 and LC600806- LC600808). BLAST query against Fusarium loci sampled and multilocus sequence typing database revealed that 99-100% identity to corresponding sequences of the F. oxysporum species complex (strain NRRL 28395 and 26379). Maximum likelihood phylogenetic analysis with MEGA v. 6.0 using the concatenated sequencing data for EF-1α and rpb2 showed that the isolates belonged to F. oxysporum species complex. Each three healthy seedlings with similar sized (big flower sabju) were grown for 20 days in a plastic pot containing autoclaved peat soil was used for pathogenicity tests. Conidial suspensions (106 conidia mL-1) of 20 days old colonies per isolate (two isolates) were prepared in sterile water. Three pots per strain were inoculated either by pouring 50 ml of the conidial suspension or by the same quantity of sterile distilled water as control. After inoculation, all pots were incubated at 25 °C with a 16-hour light/8-hour dark cycle in a growth chamber. This experiment repeated twice. Inoculated seedlings were watered twice a week. Approximately 60% of the inoculated seedlings per strain wilted after 15 days of inoculation and control seedlings remained asymptomatic. Fusarium oxysporum was successfully isolated from infected seedling and identified based on morphology and EF-1α sequences data to confirm Koch's postulates. Fusarium oxysporum is responsible for damping-off of many plant species, including larch, tomato, melon, bean, banana, cotton, chickpea, and Arabidopsis thaliana (Fourie et al. 2011; Hassan et al.2019). To the best of our knowledge, this is the first report on damping-off of ovate-leaf atractylodes caused by F. oxysporum in South Korea. This finding provides a basis for studying the epidemic and management of the disease.

First Report of Colletotrichum aenigma Causing Anthracnose of Grape in Korea.

Grape (cv. Kyoho) is one of the most popular dessert fruits in South Korea. Anthracnose caused by Colletotrichum species is a common and very destructive disease of grape in the country. In 2019, severe outbreaks of anthracnose was observed in different grape orchards in Gimcheon (36º09´N, 128º00´ E), South Korea. The disease incidence on fruit was up to 50% in the orchards with most severe outbreaks and infected fruit displayed typical anthracnose symptoms including sunken necrotic lesions with orange-like conidial mass. For isolation of putative causal agents, nine diseased fruits were collected from three commercial orchards. A total of nineisolates were made from nine of the infected fruit by spreading spore masses (1x106 conidia mL-1) from each fruit on water agar and collecting single germinated spores after incubation at 25 ºC overnigh. The single germinated spores were transferred on to fresh potato dextrose agar (PDA) (Difco, Becton Dickinson) and incubated at 25ºC in the dark. Seven day old colonies were cottony white on the upper side and gray at the center on the reverse side. Conidia were cylindrical with round ends and measured 13.9 - 20.1 × 5.4 - 8.1 µm (mean = 16.5 × 6.6 µm, n = 30). Appressoria were brownish, sub-cylindrical with a few lobes and 10.3 -16.7 × 6.6 - 10.9 µm (mean = 13.1 × 8.1 µm, n = 30). The morphological characteristics of the solates resembled those of Colletotrichum species within the C. gloeosporioides complex (Weir et al. 2012). DNA was amplified using the following primer pairs: ITS1/ITS4, GDF / GDR, ACT-512F / ACT-783R, Bt2a/ Bt2b, and CHS79-F/CHS-354R (Weir et al. 2012). Accession numbers, LC586811 to LC586825 were obtained after depositing all the resulting sequences in GenBank. A 50% majority rules phylogenetic tree (Bayesian phylogenic analysis) was constructed based on concatenated sequences of ITS, GAPDH, ACT, TUB, and CHS using MrBayes 3.2.10. The present isolates formed a single clade with the reference isolates of C. aenigma (isolate ICMP 18608 and ICMP 18686). For a pathogenicity test, healthy grapefruits were collected from an orchards, surface sterilized by dipping in 1% sodium hypochlorite, rinsed with sterilized water and dried by blotting. A conidial suspension (1×106 conidia mL-1) in sterilized water were prepared from one week old colonies of isolates GRAP10 and GRAP12. A small wound was made on sterilized detached fruit by punching with a sterile pin. A drop of the conidial suspension was placed on the wound, while the control fruit received a drop of sterile water. Similarly, unwounded fruit were also inoculated with a single droplet of conidial suspension. For each isolate and method (wounded and unwounded), ten fruit were inoculated, and ten non-inoculated fruit were used as control. All the treated fruit were kept in a plastic box containing moist tissue and incubated at 25º C in the dark. Typical anthracnose lesions appeared on all inoculated wounded fruit while non-inoculated and inoculated unwounded fruits remained asymptotic. Koch postulates were fulfilled by re-isolating and re-identifying the causal agent from inoculated fruit. Colletotrichum aenigma has been reported as the causal agent of anthracnose on Juglans regia, Camellia sinensis and Actinidia arguta in China (Weir et al. 2012; Wang et al. 2016; Wang et al. 2018). Previous studies reported four Colletotrichum species (C. acutatum, C. gloeosporioides, C. fructicola, and C. viniferum) to cause this disease on grapes in South Korea (Oo and Oh 2017; Lim et al. 2020). To the best of our knowledge, this is the first report on grape anthracnose caused by C. aenigma in South Korea. This finding may help to take effective control measures of this disease.

Characterization of Pestalotiopsis sp. causing gray leaf spot in coconut (Cocos nucifera L.) in Bangladesh.

Coconut (Cocos nucifera L.) is one of the most important fruit trees in Bangladesh. This tree is susceptible to various pathogens. Among them, a fungus was consistently isolated from gray leaf spot symptom in coconut. This study aimed to isolate, characterize, and find the management strategies of the causal fungus of gray leaf spot disease in coconut. Both morphological and molecular characters identified the pathogen as Pestalotiopsis sp. for the first time in Bangladesh. Artificial inoculation of this fungus showed symptoms similar to those previously observed in the field. Cross-inoculation test suggests that Pestalotiopsis sp. has a wide host range. The infection process of Pestalotiopsis sp. started at 2 h after inoculation (hai) with the formation of germ tube followed by the formation of infection hyphae, which penetrated directly into the host at 6 hai. Gray leaf spot symptom was developed at 120 hai. Numerous conidia developed from the acervuli at 168 hai. These conidia acted as the source of inocula for secondary infection. The optimum temperature for the growth of Pestalotiopsis sp. was 25°C, however, the growth of Pestalotiopsis sp. ceased at 15°C and 35°C. This pathogen was completely inhibited by Autostin 50 WDG (carbendazim) at 100 ppm. Trichoderma viride (Pb-7) was found as the potential biocontrol agent against Pestalotiopsis sp. These findings could contribute to describing the disease cycle and epidemiology of Pestalotiopsis sp. that would ultimately require to undertake effective control measures against this pathogen.

First Report of Damping-off of Ovate-leaf Atractylodes Caused by Pythium aphanidermatum in South Korea.

Ovate-leaf Atractylodes (Atractylodes ovata) is a well-known medicinal plant in Korea, where the dried rhizome and the root extract are used in herbal medicines. In 2019, severe damping-off of ovate-leaf Atractylodes at the early seedling stage was observed in a commercial planting in Sangju, South Korea. Approximately 35% of young seedlings suddenly wilted and then died despite adequate soil moisture. Putative causal agents were isolated from roots of diseased plants. Roots were washed thoroughly wih tap water, cut into 1-cm-long pieces and then split in half longitudinally. Root pieces were surface disinfected in 0.1% NaOCl solution for 1 min, washed three times with sterilized distilled water, and finally blotted dry. Root tissue was placed on potato dextrose agar (PDA, Difco, and Becton Dickinson) amended with tetracycline (0.05 g/L) and incubated at 25°C in the dark. Twelve pure culture of the potential causal agent were obtained by hyphal tipping twice. Mycelia of the seven-day-cultures were white and the colony produced numerous oogonia. The oogonia were smooth, globose, terminal and rarely intercalary, and 17.3 to 27.1 µm (mean ± SD 24.3 ± 2.25 µm) in diameter. Antheridia were diclinous or monoclinous (rare) with clavate cells that were 17.9 to 31.6 × 7.1 to 8.8 µm (mean 26.8 × 7.5 µm). The stalks of the antheridia were unbranched. Oospores were plerotic or nearly plerotic and 16.3 to 25.4 µm (mean ± SD = 20.1 µm ± 3.2) in diameter. The morphological characteristics of the isolates were comparable to those of Pythium spp. including P. aphanidermatum (Van der Plaats-Niterink,1981). The internal transcribed spacers (ITS), cytochrome oxidase 1 (cox1), and cytochrome oxidase 2 (cox2) regions of genomic DNA from SRRS1, SRRS2, and SRRS4 were amplified using primer sets ITS5 + ITS4, FM52R + FM55, and FM58 + FM66 respectively (Ueta and Tojo 2016) and sequenced. Resulting sequences were deposited in GenBank under accession numbers LC569777 to LC569779 and LC569785 to LC569790. The concatenated sequence data contained 46 taxa for the ITS-cox2 phylogenetic analysis and 17 taxa for cox1. The maximum likelihood estimation and Bayesian inference consensus tree showed that the present isolates formed a clade with P. aphanidermatum (strain NBRC 100101, P36-3, and 1987-61). The pathogenicity of three isolates was tested using the procedure described by Herrero et al. (2003) with some modification. Five surface disinfected seeds were sown in a plastic pot containing autoclaved peat soil. There was a total of twelve pots. After emergence seedlings were thinned to three similar sized seedlings per pot and grown for 25 days at 25°C in the 16-h light. Seedling were then inoculated with mycelial plugs (10 mm diam.) from four day old cultures on PDA. Three plugs per pot were placed 1 cm apart from the seedlings (one plug per seedling). Three pots per isolates were inoculated and three pot with non-inoculated seedlings were used as control. All plants were grown in a growth chamber at 25°C with 16-h light and 80% relative humidity and irrigated twice per week with sterile water. All inoculated seedlings (100%) suddenly collapsed 10 days after inoculation while the control plants remained healthy and vigorous. Pythium aphanidermatum was re-isolated from the inoculated seedlings and identified following the procedures previously described thus completing Koch's postulates. This pathogen has also reported as the causal agent of damping-off of common bean, cucumber, wheat, tomato and tobacco (Al-Saadi et al. 2007; Herrero et al. 2003; and Gilardi et al. 2018). To our knowledge, this is the first report of Pythium aphanidermatum causing damping-off disease on ovate-leaf Atractylodes in South Korea, and this pathogen could threaten production. Outcomes of present study will help manage this disease with effective control measures.

Diversity, Pathogenicity, and Fungicide Sensitivity of Colletotrichum Species Associated with Apple Anthracnose in South Korea.

Apple fruits with anthracnose symptoms were collected from commercial apple orchards in different regions of the Republic of Korea, and isolations were made on potato dextrose agar to isolate the causal agents. The fungal isolates were identified based on their morphological characteristics, growth rates, and multigene sequences. Nine isolates were identified via phylogenetic analysis: three Colletotrichum fructicola, two C. fioriniae, one C. gloeosporioides sensu stricto (s.s.), two C. nymphaeae, and one C. siamense isolates. The pathogenicity of the Colletotrichum isolates was tested using detached apple fruits under laboratory conditions. This study also reidentified six Colletotrichum isolates responsible for apple anthracnose, which were deposited in the Korean Agricultural Culture Collection. Among the six isolates, three were identified as C. siamense (deposited as C. gloeosporioides s.s.), and three were C. nymphaeae (deposited as C. acutatum s.s.). All the Colletotrichum species identified in this study were highly sensitive to tebuconazole in terms of inhibition of mycelial growth (EC50 value of 0.12 to 2.1 µg/ml).

Phylogenetic and Morphological Reassessment of Mycosphaerella nawae, the Causal Agent of Circular Leaf Spot in Persimmon.

Persimmon (Diospyros kaki) fruit production is severely affected by circular leaf spot worldwide. Mycosphaerella nawae causes circular leaf spot of persimmon (CLSP) and can result in leaf spot, defoliation, early fruit maturation, and subsequent softening and abscission. The morphology and phylogenetic position of M. nawae within the family Mycosphaerellaceae is, therefore, of utmost importance given its impact on persimmon production. Based on previous morphological and molecular studies, the phylogenetic position of the anamorphic genera associated with M. nawae remain in confusion. In the present study, 15 isolates of M. nawae were collected from the tissue of living leaves exhibiting leaf spot symptoms. A subsample of three isolates was characterized phylogenetically and morphologically. Isolates were compared based on DNA sequence data for the internal transcribed spacer region (ITS1-5.8S ITS2), part of the 28S nrDNA including domains D1-D3 (LSU), actin (Act), translation elongation factor 1-alpha (EF-1α), and RNA polymerase II second largest subunit (rpb2). The anamorph and teleomorph structures, ascospore germination patterns, as well as host specificity were used to describe the isolates. The phylogenetic and morphological analyses revealed that M. nawae requires a new holomorphic genus within Mycosphaerellaceae, described herein as Plurivorosphaerella gen. nov. A host specificity test revealed that Plurivorosphaerella nawae comb. nov. (M. nawae) can superficially colonize, but not infect, apple, peach, cherry, and plum.

Molecular and Morphological Characterization of Colletotrichum Species in the Colletotrichum gloeosporioides Complex Associated with Persimmon Anthracnose in South Korea.

Anthracnose is a major disease of persimmon in the pre- and postharvest phase. Several species of Colletotrichum (Colletotrichum gloeosporioides, C. acutatum, and C. horii) have been reported as causal agents of persimmon anthracnose in South Korea. In this study, a collection of 50 isolates associated with persimmon anthracnose were collected from Sangju (n = 25) and Cheongdo-gun (n = 25), South Korea. The morphological characteristics of all 50 Colletotrichum isolates were similar, and it was difficult to identify the isolates to the species level. A subsample of eight isolates was characterized phylogenetically to ascertain species. BLAST search and phylogenetic analysis of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) genes revealed two species: C. horii as well as a previously unreported persimmon anthracnose causal agent C. siamense. C. siamense isolates were confirmed again by phylogenetic analysis of the ITS, ACT, GAPDH, calmodulin, and Apn2-Mat1-2 intergenic spacer partial mating type genes. Koch's postulates for C. horii and C. siamense were fulfilled, confirming the pathogenicity of the two species in persimmon fruit. Morphological characteristics (colony morphology and size and shape of conidia and appressoria) from two representative isolates support results of the phylogenetic analysis and match those of previous descriptions of C. horii and C. siamense.

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide Iprodione in Sclerotinia homoeocarpa Field Isolates.

The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide resistant, three multidrug resistance (MDR)-like, and five dicarboximide sensitive) were used for sequence comparison of a histidine kinase gene, Shos1, of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed nonsynonymous polymorphisms in codon 366 (isoleucine to asparagine) in Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function, using polyethylene glycol-mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N, and Shos1 deletion mutants were negatively correlated to values of iprodione-induced expression of ShHog1, the last kinase in the high-osmolarity glycerol pathway. Increased constitutive and induced expression of the ATP-binding cassette multidrug efflux transporter ShPDR1 was observed in six of seven dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance through the polymorphism in Shos1 and the overexpression of ShPDR1.

An account of Colletotrichum species associated with anthracnose of Atractylodes ovata in South Korea based on morphology and molecular data.

Ovate-leaf atractylodes (OLA) (Atractylodes ovata) is a well-known medicinal plant in Korea; its dried rhizome and root extracts are used in herbal medicine. However, anthracnose is a great challenge to the OLA cultivation in South Korea. Colletotrichum spp. is a major group of plant pathogens responsible for anthracnose on a range of economically important hosts. Its occurrence on OLA remains unresolved. To investigate the diversity, morphology, phylogeny, and biology of Colletotrichum spp., 32 fungal isolates were obtained from 30 OLA-affected leaves collected from five different farms, in two regions in South Korea, Mungyeong and Sangju. The phylogenetic analysis with four or five gene loci (ITS, TUB2, ACT, GAPDH, and CHS-1) along with morphology of 26 representative isolates delineated six previously known Colletotrichum species including C. fructicola, C. gloeosporioides sensu stricto (s.s), C. cigarro, C. plurivorum, C. siamense and C. sojae, and one new species, described here as C. ovataense. Amongst these species, C. gloeosporioides s.s. and C. plurivorum were the most prevalent species. A pathogenicity test on the detached leaves revealed that different Colletotrichum species presented a distinct degree of virulence, confirming Koch's postulates. In this study, C. fructicola, C. cigarro, C. plurivorum, C. siamense, and C. sojae were reported from A. ovata for the first time, as the causal agent of ovate-leaf atractylodes anthracnose. Understanding the diversity and biology of the Colletotrichum species population will help in managing this disease.

First Report of Damping-Off of Ovate-Leaf Atractylodes Caused by Rhizoctonia solani AG-5 in South Korea.

In May to July 2019, ovate-leaf atractylodes seedling and plant with Damping-off symptoms were observed in farmer field at Sangju and Mungyeong, Korea. Seven fungal isolates have been retrieved from diseased root tissue and identified as Rhizoctonia solani AG-5 based on morphological and molecular characteristics. To the best of our knowledge, this is the first report on damping-off of ovate-leaf atractylodes caused by R. solani AG-5 in South Korea.

Phylogenetic and Morphological Characterization of Cladosporium perangustum Associated with Flyspeck on Shine Muscat Grapes in South Korea.

The Shine Muscat is a table grape, popular in South Korea for its unique mango-flavor taste. Flyspeck is a disease that is characterized by small, black, and circular specks on the grape cuticle was first observed in several commercial orchards in Sangju, South Korea, in August 2019. Here we identified the causal agent of flyspeck based on an advanced diagnosis approach, comprised of both morphological and molecular analyses. Morphological characteristics of the cultures isolated from grape flyspeck were identical to the fungus Cladosporium perangustum. The concatenated sequences of ITS, ACT, and EF1-α were used for molecular phylogenetic analysis, BLAST searches along with Bayesian inference-based phylogeny, confirmed that the causal agent of grape flyspeck is C. perangustum. The cultured fungal isolates also produced flyspeck symptoms on healthy fruits in pathogenicity tests. To the best of my knowledge, this is the first documented evidence of any Cladosporium sp. producing flyspeck symptoms on any plant.

Identification, Characterization, and Pathogenicity of Colletotrichum Species Causing Anthracnose of Peach in Korea.

Peach (Prunus persica L.) is one of the major fruit crops in South Korea, along with apple, persimmon, and Asian pears. Peach anthracnose is a continuing threat to growers and is accountable for enormous economic loss. In July 2018, anthracnose of peach appeared at different peach orchards in Gyeongsangbuk-do region, Korea. The typical anthracnose symptoms (brown, circular, and necrotic lesions) were observed on the fruits. Anthracnose of peach was surveyed in different peach orchards of Gyeongsangbuk-do, and 20 fungal isolates from 19 diseased fruits were collected. Multigene phylogenetic analyses coupled with morphological characteristic analysis approaches were used for identifying the fungal species isolated from diseased fruits. This study confirmed three Colletotrichum species. Based on the results, Colletotrichum siamense are reported for the first time as causal agents of peach anthracnose alongside C. fructicola and C. fioriniae, which has been reported previously. Pathogenicity assays were performed for the three isolates representing all the species identified, and Koch's postulates on detached healthy peach fruits were verified. All the identified species were pathogenic on peach fruits as the typical anthracnose symptoms were reproduced. Significant variations in the virulence were observed among fungal species on peach fruit.