First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 µm long and 3.1 to 4.5 µm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and ß-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

The complete mitochondrial genome of a wood-decaying fungus Vanderbylia fraxinea (Polyporaceae, Polyporales).

Vanderbylia fraxinea (Bull.) D.A. Reid, 1973 is an important wood-inhabiting fungus that plays a significant role in nutrient recycling in most forest ecosystems. In this study, the complete mitochondrial genome of V. fraxinea was characterized through de novo assembly using Illumina sequencing data and genome annotation. The mitochondrial genome is a circular molecule of 115,473 bp with a GC content of 28.66%. It comprises a total of 62 genes. Among these, 36 are protein-coding genes including 21 free-standing open reading frames (ORFs), 24 transfer RNA genes, and two ribosomal RNA genes. Core gene set commonly found in fungal mitochondrial genomes is also present in this genome, such as the apocytochrome b (cob), three subunits of the cytochrome c oxidase (cox1, cox2, and cox3), seven subunits of the NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), and three subunits of the ATP synthase (atp6, atp8, and atp9), as well as ribosomal RNA subunits (rns and rnl) and a set of transfer RNA genes. Phylogenetic analysis of the protein-coding sequences from the mitochondrial genome revealed a close relationship between V. fraxinea and the Ganoderma species within the Polyporaceae family.

Haplotype diversity of Heterodera koreana (Tylenchida: Heteroderidae), affecting bamboo in Korea.

In a survey of plant-parasitic nematodes in agricultural fields, cyst-forming nematodes were found in soil planted bamboo in Korea. The aim of this study was to identify the cyst nematodes based on morphological and molecular characteristics. As the results, the morphology and morphometrics of cysts and second-stage juveniles (J2s) were consistent with those of previous descriptions of Heterodera koreana. In phylogenetic analyses based on DNA sequences, these cyst nematodes were clustered together with clade of H. koreana in internal transcribed spacer (ITS) region, and large subunit D2-D3 segments (LSU D2-D3). These nematodes were clustered together with clade of H. koreana in cytochrome c oxidase subunit I (COI) gene, but a haplotype was different when compared with previous reported haplotypes (haplotype A-C) in Japan. This study showed these cyst nematodes were identified as H. koreana, and a new haplotype of H. koreana is distributed in Korea. We suggest that the new haplotype of H. koreana name as haplotype D.

First Report of Stunt Nematode, Tylenchorhynchus zeae, on Corn in Republic of Korea.

Corn (Zea mays), one of the major food sources it contains rich in fiber and many vitamins, is one of the most widely consumed cereal grains in Republic of Korea. A survey of plant-parasitic nematodes (PPNs) was carried out in corn fields in Goesan, Republic of Korea from August in 2021. PPNs were extracted from the corn roots and soil using modified Baermann funnel methods and were identified using morphological and molecular analyses. Among the roots and soil samples of 21 fields, 5 fields (23.8%) were infected with stunt nematodes. Tylenchorhynchus zeae was originally described in India from soil around corn and is reported to dwarf plants, yellow leaves (Sethi and Swarup, 1968). Morphologically, characteristics of females were very similar to T. zeae with cylindrical body and slightly ventrally arcuated after fixation. Lip region slightly offset from body with four annuli. Stylet with anteriorly flattened knobs, the vulva was located in the center of the body, didelphic-amphidelphic reproductive system and tail conoid, tail terminus with obtuse smooth, with four incisures areolated throughout body. Bodies of males were similar to females but with shaper tails, with relatively strong bursa and spicules (Fig. S1). The morphology of Korean populations was in agreement with the described populations of India and China (Alvani et al., 2017; Xu et al., 2020). Measurements and micrographs with the light-microscope (DM5000; Leica[Germany]) and camera (DFC450; Leica[Germany]) were taken from females (n=10) for mean, standard deviation and range of body length: 553.2 ± 41.2 (492.7-643.6) µm, maximum body width: 19.4 ± 1.0 (17.6-21.0) µm, stylet length: 18.1 ± 0.4 (17.5-18.7) µm, percent of distance from anterior end to vulva / body length: 58.5 ± 1.3 (56.1-60.9), tail length: 31.7 ± 1.2 (30.3-34.0) µm, and distance of anterior to excretory pore: 96.5 ± 1.8 (94.1-99.4) µm. In addition, PCR was performed for the 28S rDNA D2-D3 segments using the primers D2A and D3B, and ITS region with the primers TW81 and AB28. The newly obtained sequences were submitted to GenBank database under accession numbers ON909086, ON909087 and ON909088 of 28S rDNA D2-D3 segments, and ON909123, ON909124 and ON909125 of ITS region. The resulting 28S rDNA D2-D3 segment sequences were 100% identical to KJ461565 and the BLASTn search of the ITS region sequences was most similar to T. zeae (KJ461599), which is the species isolated from corn in Spain. The identities of ITS region sequences on these populations were 99.89% (893/894), with no insertions/deletions. The phylogenetic relationships of the population strongly support T. zeae (Fig. S2). Phylogenetic relation analysis based on the two genes was constructed using PAUP version 4.0 and MrBayes 3.1.2 programs. To confirm pathogenicity, a modified version of Koch's postulates was conducted in the greenhouse by inoculating 100 females and males onto each of five pots of seedling corn (cv. Daehakchal) filled with the sterilized sandy soil and maintained for 60 days at 25℃ under the conditions. Tylenchorhynchus zeae reproduction factor was 2.21 ± 0.37 was observed at the end of the trial in soil on pots. The stunted and swollen roots and dwarfed and yellowing leaf shoots symptoms in the greenhouse pots trial were confirmed the same as those typical damage symptoms. To the best of our knowledge, this is the first report of T. zeae in Republic of Korea. The host range of T. zeae includes some economic crops such as cabbage, cauliflower, grapevine, and olive (Chen at al., 2007; Handoo et al., 2014). It is necessary to investigate the damage to economic crops in the Republic of Korea to this nematode.

Characterization of Heterodera sojae Virulence Phenotypes in Korea.

The white soybean cyst nematode Heterodera sojae, isolated from the roots of soybean in Korea, is widespread in most provinces of the country and has the potential to be as harmful to soybean as H. glycines. Determining the virulence phenotypes of H. sojae is essential to devising management strategies that use resistant cultivars. Consequently, virulence phenotypes of 15 H. sojae populations from Korea were determined on seven soybean lines and one susceptible check variety. Two different HS types were found to be present in Korea; the more common HS type 2.5.7, comprising 73.3% of the H. sojae populations and the less common HS type 0, constituting only 26.7% of the tested populations. Considering the high frequency of H. sojae adaptation to soybean indicator lines, the PI 88788 group may not be a possible source of resistance while PI 548402, PI 90763, PI 437654, and PI 89772 can be used as resistance sources for soybean breeding programs aimed at developing H. sojae-resistant soybean cultivars in Korea.

First Report of Pratylenchoides leiocauda parasitic on Perilla frutescenes in the Republic of Korea.

Perilla (Perilla frutescens (L.) Britton var. japonica) is an annual herbaceous crop belonging to Lamiaceae. It is one of the most important crops in Korea. A survey of plant parasitic nematodes was carried out in 70 perilla fields in Geumsan and Miryang, Korea in 2020. Migratory endo-parasitic nematodes were extracted from the perilla roots using modified root incubation methods (Mountain and Patrick, 1959). False root-lesion nematodes (Pratylenchoides) were recovered from 41.4% of the fields. They were identified using morphological and molecular analyses. Among the Pratylenchoides species, Pratylenchoides leiocauda was described in France from soil around non-identified grass and weeds. P. leiocauda is reported to weaken the root systems of tomato, potato and strawberry (Sher, 1970; Li et al., 2020). The morphological characteristics of males and females from the 2020 survey were very similar to P. leiocauda. The lateral field contained six incisures and the pharyngeal glands overlapped the intestine dorsally for 1-2 times the body width. Additionally, they had a second nucleus near the pharyngeal intestine valve, female tails with incisures, and a smooth conical tail with 18-23 annules. Measurements and micrographs with the light-microscope (DM5000, Leica, Germany) and camera (DFC450, Leica, Germany) were taken from females (n=23) for mean, standard deviation and range of body length: 824.5 ± 71.0 (657.7-993.8) µm, maximum body width: 28.4 ± 2.7 (24.8-36.2) µm, stylet length: 22.8 ± 0.7 (21.4-24.0) µm and percent of distance from anterior end to vulva / body length: 57.7 ± 1.4 (54.0-60.3). Males (n=5) were characterized by a body length: 653.5 ± 64.3 (555.3-714.7) µm, a stylet length: 20.9 ± 1.0 (19.7-22.0) µm and a spicule length: 26.1 ± 2.5 (22.3-28.5) µm. Morphological measurements and characteristics (Fig. 1A-F) were compared to previous descriptions of P. leiocauda. As a result, the Korean population was larger than that of the originally described population from France, and was more similar to the Chinese population (Sher, 1970; Azizi et al., 2016; Li et al., 2020). In addition, PCR was performed for the COⅠ, LSU rRNA D2-D3 and ITS region using the primers (Mwesige et al., 2020). The sequenced COⅠ, LSU rRNA D2-D3 segments and ITS region are 447, 776 and 777 bp, respectively. The newly obtained sequences were submitted to GenBank database under accession numbers MZ418130, MZ414163 and MZ418133 of ITS region. A BLASTn search of the LSU rRNA D2-D3 sequence was most similar to P. leiocauda (MN539650), which is the species isolated from tomato in China (Li et al., 2020). The identities of these two sequences were 99.9% (775/776), with no insertions/deletions. The phylogenetic relationships strongly support P. leiocauda (Fig. 1G). Symptoms of perilla associated with P. leiocauda under field conditions included small and curled leaves, chlorosis of leaf edges, dark brown root epidermis and root necrosis (Fig. 2A, B). Koch's postulates were completed by inoculating 100 P. leiocauda into ten 45-cm-in-diameter pots filled with sterilized sandy loam soil and planted with perilla cultivar 'Ipdeulkkae 1-ho'. The potted plants were maintained for 50 days at 25℃ under greenhouse conditions. A P. leiocauda reproduction factor was 1.45 ± 0.20 was observed at the end of the trial, with 106 ± 21.6 recovered from soil and 39 ± 6.7 from root tissue. The root and shoot symptoms in the greenhouse trial were the same as those observed under field conditions (Fig. 2C, D). This, therefore, is the first record of P. leiocauda associated with perilla in Korea and management is warranted.

Occurrence of the White Soybean Cyst Nematode, Heterodera sojae, and the Soybean Cyst Nematode, H. glycines, in Korea.

Soybean cyst nematode (Heterodera glycines, SCN) is the most harmful pathogen of soybean (Glycine max (L.) Merr.) worldwide. In 2016, a new soybean-parasitic cyst nematode, Heterodera sojae (the white soybean cyst nematode) was found parasitizing the roots of soybean plants in Korea. To investigate the distribution and population density of H. sojae, 943 soil samples were collected from soybean fields in all nine provinces in Korea in 2017 to 2018. Cyst nematodes were detected in 343 samples (36.4%) from eight of the nine provinces, except the island of Jeju province. Among the 343 samples, H. glycines was found in 227 samples (66.2%), H. sojae in 95 samples (27.7%), and 21 samples (6.1%) were infested with both H. sojae and H. glycines. Wide distribution of H. sojae in soybean fields indicates that H. sojae is an important cyst nematode species parasitizing soybean together with H. glycines.

Differential Morphological, Structural and Biological Characteristics of Cysts in Heterodera Species in Korea.

Morphological (cyst shape, color, and sizes [length (L), maximum width (W), volume and "a" (L/W)]), structural (vulvar cone slope angle [VCSA], surface wrinkle [VCSW], cyst wall thickness, composition, and texture) and biological characteristics (fecundity, hatching, and emergence [number of second-stage juveniles (J2) from a cyst]) in preceding Heterodera glycines (Hg), currently-recorded H. sojae (Hs) and H. trifolii (Ht) were examined by microscopy. Cysts were lemon-shaped, indicating the genus is Heterodera except for Hs that formed frequently globular cysts with significantly flatter VCSA (102.2°) with smooth VCSW than Hg (50.6°) and Ht (82.0°), but not genus Globodera because of the presence of vulvar cone in Hs. Ht was significantly larger in all morphological characteristics than Hg and Hs, suggesting Ht may be diagnosed differentially by cyst sizes and also host plant preferences. Hs showed smaller "a" value with more globular shape and stronger structures with more thickened and strengthened collagen-like texture of cyst wall than Hg and Ht. This suggests Hs may be diagnosed differently by structural characteristics from the others, especially Hg with similar cyst sizes. There were no significant differences in emergence (inoculum potential) among cyst nematodes due to the offset of fecundity and hatching rate; however, the inoculum potential of Hs may be not so persistent as Hg and Ht in fields because of its lower fecundity and higher hatching rate (causing rapid inoculum loss) than the others. These characteristics of cysts provide information useful for simple and differential diagnoses and reliable management of cyst nematodes.

Identification of Heterodera glycines (Tylenchida; Heteroderidae) Using qPCR.

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R 2 = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

New Cyst Nematode, Heterodera sojae n. sp. (Nematoda: Heteroderidae) from Soybean in Korea.

A new soybean cyst nematode Heterodera sojae n. sp. was found from the roots of soybean plants in Korea. Cysts of H. sojae n. sp. appeared more round, shining, and darker than that of H. glycines. Morphologically, H. sojae n. sp. differed from H. glycines by fenestra length (23.5-54.2 µm vs. 30-70 µm), vulval silt length (9.0-24.4 µm vs. 43-60 µm), tail length of J2 (54.3-74.8 µm vs. 40-61 µm), and hyaline part of J2 (32.6-46.3 µm vs. 20-30 µm). It is distinguished from H. elachista by larger cyst (513.4-778.3 µm × 343.4-567.1 µm vs. 350-560 µm × 250-450 µm) and longer stylet length of J2 (23.8-25.3 µm vs. 17-19 µm). Molecular analysis of rRNA large subunit (LSU) D2-D3 segments and ITS gene sequence shows that H. sojae n. sp. is more close to rice cyst nematode H. elachista than H. glycines. Heterodera sojae n. sp. was widely distributed in Korea. It was found from soybean fields of all three provinces sampled.