First Report of Anthracnose on Euonymus japonicus Thunb. Caused by a Provisionally Novel Species of Colletotrichum in the Magnum Complex in South Korea.

Euonymus japonicus Thunb., also known as the evergreen spindle tree, is an evergreen tree, which is widely planted as a hedge plant along streets in South Korea. In April 2022, severe anthracnose symptoms were observed on the leaves of this tree in Jangsu in the Jeonbuk Province of the country (35°43'49.44″N, 127°34'53.7″E). About 80% of the leaves of each affected tree within a 0.03-ha area showed incidence of the disease on approximately 30 trees were planted along the roadside (~30 m). These symptoms typically included circular or irregularly shaped whitish-gray lesions with a diameter of 2.0 to 3.0 cm. In cases where some leaves were severely affected, larger blotches formed. To isolate the pathogen, about ten leaves showing anthracnose symptoms on each tree were randomly selected and brought to the laboratory. Fungal isolations were made from acervuli filled with conidial masses on infected evergreen tissues, followed by plating onto 2% potato dextrose agar (PDA) as well as incubated at 25℃. On the PDA, colonies were circular, raised, green-grey or dark grey, and had a distinct white margin. The conidia were single-celled, transparent, cylindrical with rounded ends, had smooth walls, with a length ranging from 12 µm to 16.7 µm and a width raging from 4 µm to 6.5 µm (av. = 14.1 X 5.0 µm, n=40). Of those that were successfully recovered with approximately 90% frequency, two monoconidial isolates were deposited to the culture collection at Chungnam National University in South Korea (Accession number: CDH059-060). To ensure the identity of the fungus, genomic DNAs were extracted from the selected isolates, CDH059-060, and were sequenced. This was achieved based on partial sequences of the internal transcribed spacer (ITS), actin and beta-tubulin (TUB2) gene regions which were amplified using ITS1F / ITS4 (Gardes and Bruns 1993; White et al. 1990), ACT-512F / ACT-783R (Carbone and Kohn 1999), and T1 / Bt2b (O'Donnell and Cigelnik 1997; Glass and Donaldson 1995) primer pairs, respectively. The resulting sequences were deposited to GenBank (OR984424-425) for ITS, (OR996289-290) for actin, and (OR996291-292) for TUB2. For a phylogenetic analysis, sequences from different gene regions (ITS, actin and TUB2) retrieved from GenBank were aligned, concatenated, and analyzed as a single dataset based on a maximum likelihood analysis. The phylogenetic result revealed that the fungus isolated in this study was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Colletotrichum, which is most closely related to Colletotrichum liaoningense (Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, CGMCC3.17616 (KP890104 for ITS, KP890097 for actin, and KP890111 for TUB, Diao et al. 2017). Sequence comparisons revealed that this pathogen differed from C. liaoningense at 20 of 494 characters (∼4.0%) in the ITS and 2 of 251 (∼1.0%) in the actin sequences. For pathogenicity tests, three seedlings of E. japonicus were used. The leaves for each tree were treated with 10 ml of a conidial suspension by spraying (1x106 conidia ml-1 of the isolate, CDH059), while the three seedlings were treated with distilled water as control. After sprayed, the treated areas were sealed with plastic bags for a day to maintain humidity. Anthracnose symptoms identical to those observed in the field appeared seven days after inoculations, while no symptoms were observed in the control. Re-isolations were successfully achieved from the treatments, fulfilling Koch's postulates. Anthracnose associated with the provisionally novel species of Colletotrichum sp. on E. japonicus has not been recorded elsewhere, and in this regard, this is the first report of anthracnose caused by Colletotrichum sp. on E. japonicus in Korea. To effectively control the disease, more attention should be paid to the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

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.

Identification and Characterization of Xanthomonas arboricola pv. juglandis Causing Bacterial Blight of Walnuts in Korea.

The present study describes the bacterial blight of walnut, caused by Xanthomonas arboricola pv. juglandis (Xaj) in the northern Gyeongbuk province, Korea. Disease symptoms that appear very similar to anthracnose symptoms were observed in walnut trees in June 2016. Pathogens were isolated from disease infected leaves, fruits, shoots, bud, flower bud of walnut, and cultured onto yeast dextrose carbonate agar plates. Isolated bacteria with bacterial blight symptoms were characterized for their nutrient utilization profiles using Biolog GN2 and Vitek 2. In addition, isolates were subjected to physiological, biochemical, and morphological characterizations. Furthermore, isolates were identified using 16S rDNA sequence analysis, and multi-locus sequence analysis using atpD, dnaK, efp, and rpoD. To confirm pathogenicity, leaves, fruits, and stems of 3-year-old walnut plants were inoculated with bacterial pathogen suspensions as a foliar spray. One week after inoculation, the gray spots on leaves and yellow halos around the spots were developed. Fruits and stems showed browning symptoms. The pathogen Xaj was re-isolated from all symptomatic tissues to fulfill Koch's postulates, while symptoms were not appeared on control plants. On the other hand, the symptoms were very similar to the symptoms of anthracnose caused by Colletotrichum gloeosporioides. When walnut plants were inoculated with combined pathogens of Xaj and C. gloeosporioides, disease symptoms were greater in comparison with when inoculated alone. Xaj population size was more in the month of April than March due to their dormancy in March, and sensitive to antibiotics such as oxytetracycline and streptomycin, while resistant to copper sulfate.

Endophytic Trichoderma citrinoviride isolated from mountain-cultivated ginseng (Panax ginseng) has great potential as a biocontrol agent against ginseng pathogens.

BACKGROUND: Ginseng (Panax ginseng Meyer) is an invaluable medicinal plant containing various bioactive metabolites (e.g., ginsenosides). Owing to its long cultivation period, ginseng is vulnerable to various biotic constraints. Biological control using endophytes is an important alternative to chemical control. METHODS: In this study, endophytic Trichoderma citrinoviride PG87, isolated from mountain-cultivated ginseng, was evaluated for biocontrol activity against six major ginseng pathogens. T. citrinoviride exhibited antagonistic activity with mycoparasitism against all ginseng pathogens, with high endo-1,4-ß-D-glucanase activity. RESULTS: T. citrinoviride inoculation significantly reduced the disease symptoms caused by Botrytis cinerea and Cylindrocarpon destructans and induced ginsenoside biosynthesis in ginseng plants. T. citrinoviride was formulated as dustable powder and granules. The formulated agents also exhibited significant biocontrol activity and induced ginsenosides production in the controlled environment and mountain area. CONCLUSION: Our results revealed that T. citrinoviride has great potential as a biological control agent and elicitor of ginsenoside production.

Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus is a migratory endoparasitic nematode known to cause severe environmental damage and economic losses in pine forest ecosystems. This present study investigated the nematicidal metabolites of actinomycetes in vitro and evaluated the disease control efficacy of the active compound and metabolites under greenhouse and field conditions. RESULTS: Five thousand types of actinobacteria from Korean forest soil samples were screened to identify novel nematicidal agents against the pine wood nematode. Streptomyces sp. AN091965 showed the strongest nematicidal activity. One active compound, spectinabilin, was obtained by nematicidal asssy-directed fractionation, and it showed significant nematicidal activity against B. xylophilus, with an LC50 value of 0.84 µg mL-1 . Spectinabilin effectively suppressed the development of pine wilt disease in 5-year-old Pinus densiflora trees, even at 0.9 mg per tree under greenhouse conditions. Moreover, the acetone extract of the active strain's mycelia efficiently suppressed the development of pine wilt disease under field conditions. CONCLUSION: To the best of our knowledge, this the first report to describe the nematicidal activity of spectinabilin against B. xylophilus. The cell extracts described herein merit further field studies as potential nematicides against the pine wood nematode. © 2018 Society of Chemical Industry.

Genomic Insights into Nematicidal Activity of a Bacterial Endophyte, Raoultella ornithinolytica MG against Pine Wilt Nematode.

Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, is one of the most devastating conifer diseases decimating several species of pine trees on a global scale. Here, we report the draft genome of Raoultella ornithinolytica MG, which is isolated from mountain-cultivated ginseng plant as an bacterial endophyte and shows nematicidal activity against B. xylophilus. Our analysis of R. ornithinolytica MG genome showed that it possesses many genes encoding potential nematicidal factors in addition to some secondary metabolite biosynthetic gene clusters that may contribute to the observed nematicidal activity of the strain. Furthermore, the genome was lacking key components of avermectin gene cluster, suggesting that nematicidal activity of the bacterium is not likely due to the famous anthelmintic agent of wide-spread use, avermectin. This genomic information of R. ornithinolytica will provide basis for identification and engineering of genes and their products toward control of pine wilt disease.

Lactones from the pericarps of Litsea japonica and their anti-inflammatory activities.

Five new lactones, litsenolide F1 (1), lisealactone H1 (10), lisealactone H2 (11), akolactone D (13), and akolactone E (14), along with thirteen known compounds were isolated from the pericarps of Litsea japonica (Thunb.) Jussieu. Their chemical structures were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR, HRMS, and chemical methods. The isolated compounds were evaluated for their inhibitory effects on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among them, 2-alkylidene-3-hydroxy-4-methylbutanolide derivatives (compounds 1-9) exhibited the most potent activity, with IC50 values in the range of 2.9-12.8 µM. In additon, compounds 1, 3, 4, and 6 showed inhibition of iNOS and COX-2 expression in concentration-dependent manner. Compound 3 suppresses mRNA expression of iNOS, COX-2, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells. Based on these evidence, the isolated lactones from L. japonica could be promissing candidates for the development of new anti-inflammatory agents.

Enzymatic synthesis of avermectin B1a glycosides for the effective prevention of the pine wood nematode Bursaphelenchus xylophilus.

Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP)-glycosyltransferase from Bacillus licheniformis with various UDP sugars. As a result, the following four avermectin B1a glycosides were produced: avermectin B1a 4″-ß-D-glucoside, avermectin B1a 4″-ß-D-galactoside, avermectin B1a 4″-ß-L-fucoside, and avermectin B1a 4″-ß-2-deoxy-D-glucoside. The avermectin B1a glycosides were structurally analyzed based on HR-ESI MS and 1D and 2D nuclear magnetic resonance spectra, and the anti-nematodal effect of avermectin B1a 4″-ß-D-glucoside was found to exhibit the highest activity (IC50 = 0.23 µM), which was approximately 32 times greater than that of avermectin B1a (IC50 = 7.30 µM), followed by avermectin B1a 4″-ß-2-deoxy-D-glucoside (IC50 = 0.69 µM), avermectin B1a 4″-ß-L-fucoside (IC50 = 0.89 µM), and avermectin B1a 4″-ß-D-galactoside (IC50 = 1.07 µM). These results show that glycosylation of avermectin B1a effectively enhances its in vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.

The Effect of Raffaelea quercus-mongolicae Inoculations on the Formation of Non-conductive Sapwood of Quercus mongolica.

In Korea, mass mortality of Quercus mongolica trees has become obvious since 2004. Raffaelea quercus-mongolicae is believed to be a causal fungus contributing the mortality. To evaluate the pathogenicity of the fungus to the trees, the fungus was multiple- and single-inoculated to the seedlings and twigs of the mature trees, respectively. In both the inoculations, the fungus was reisolated from more than 50% of inoculated twigs and seedlings. In the single inoculations, proportions of the transverse area of non-conductive sapwood at inoculation points and vertical lengths of discoloration expanded from the points were significantly different between the inoculation treatment and the control. In the multiple inoculations, no mortality was confirmed among the seedlings examined. These results showed that R. quercus-mongolicae can colonize sapwood, contribute to sapwood discoloration and disrupt sap flows around inoculation sites of Q. mongolica, although the pathogenicity of the fungus was not proven.

Effects of Water Stress on the Endophytic Fungal Communities of Pinus koraiensis Needles Infected by Cenangium ferruginosum.

To examine the effects of water stress and Cenangium ferruginosum (CF) on the fungal endophytic community of needles of Pinus koraiensis (PK), fungal endophytes isolated from the needles of 5-year-old PK seedlings were compared before and after exposure to water stress conditions and artificial inoculation with CF ascospores. Artificial CF inoculation was successfully confirmed using PCR with CF-specific primers (CfF and CfR). For comparison of the degree of water deficit in water-stressed and control groups of PK seedlings infected with CF, the water saturation deficit and water potential were measured. Lower water potential estimates were found in the water-stressed seedlings than in the control group. The fungal endophytes isolated from the second-year needles of non-water-stressed seedlings before and after CF inoculation revealed that primary saprobes were approximately 30% and 71.7%, respectively, and the remaining endophytes were rot fungi or pathogens. Sixty days after CF inoculation, diverse fungal endophytes in the first-year needles were isolated from the water-stressed seedlings. However, some fungal endophytes isolated from the non-water-stressed seedlings were also identified. Fungal endophytes in the second-year needles of the water-stressed and non-water-stressed seedlings were approximately 8% and 71.7% of saprobes, respectively, and the remaining endophytes were rot fungi or pathogens. On the basis of the results, we conclude that water deficit and CF can have an effect on fungal endophytic communities in the needles of PK seedlings.

A New Record of Candida kashinagacola (Synonym Ambrosiozyma kashinagacola) from Galleries of Platypus koryoensis, the Oak Wilt Disease Vector, in Korea.

The ambrosia beetle, Platypus koryoensis, is an economically important pest affecting oak trees in Korea. Candida kashinagacola was isolated from galleries of the beetle in oak wood and identified by analyses of morphology, physiological properties, and nucleotide sequence of the large subunit ribosomal DNA. This is the first report on Candida species associated with oak wilt disease vectored by the ambrosia beetle, Platypus koryoensis, in Korea.

Filamentous Fungi Isolated from Platypus koryoensis, the Insect Vector of Oak Wilt Disease in Korea.

The ambrosia beetle, Platypus koryoensis, is a serious pest of oak trees in Korea. In this study we investigated filamentous fungi present in the body of the beetle. Fourteen genera of filamentous fungi belonging to Ascomycota and Basidiomycota were isolated. Among the isolated fungi, some were able to produce wood degrading enzymes. This is first report of fungi associated with P. koryoensis.

PCR-based specific detection of Ralstonia solanacearum by amplification of cytochrome c1 signal peptide sequences.

A polymerase chain reaction (PCR)-based method was developed to detect the DNA of Ralstonia solanacearum, the causal agent of bacterial wilt in various crop plants. One pair of primers (RALSF and RALSR), designed using cytochrome c1 signal peptide sequences specific to R. solanacearum, produced a PCR product of 932 bp from 13 isolates of R. solanacearum from several countries. The primer specificity was then tested using DNA from 21 isolates of Ralstonia, Pseudomonas, Burkholderia, Xanthomonas, and Fusarium oxysporum f. sp. dianthi. The specificity of the cytochrome c1 signal peptide sequences in R. solanacearum was further confirmed by a DNA-dot blot analysis. Moreover, the primer pair was able to detect the pathogen in artificially inoculated soil and tomato plants. Therefore, the present results indicate that the primer pair can be effectively used for the detection of R. solanacearum in soil and host plants.

Genotypic characterization of Burkholderia cenocepacia strains by rep-PCR and PCR-RFLP of the fliC gene.

Thirty-five strains of Burkholderia cenocepacia from clinical and environmental sources were characterized genotypically by repetitive sequence PCR (ERIC- and BOX-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC). In cluster analysis based on the repetitive PCR profiles the strains were composed of five clusters, of which clusters 1, 2 and 3 were more closely related to each other than to clusters 4 and 5. It has been reported that the majority of Burkholderia cepacia complex strains can be separated into two types on the basis of fliC size (types I and II correspond to 1.4 and 1.0 kb, respectively). When the strains were analysed by PCR of fliC, all strains yielded amplified products of 1.0 kb except for three strains. The latter strains gave PCR products of 0.7 kb (atypical type), which belonged to repetitive PCR cluster 5. These results indicated that the majority of B. cenocepacia strains belonged to flagellin type II. In the RFLP analysis of the large fliC amplicons with HaeIII, 10 patterns were observed indicating remarkable variation. Strains grouping in repetitive PCR cluster 4 had a unique fliC RFLP pattern. The results of repetitive PCR typing and PCR-RFLP analysis of fliC showed a strong correlation. Strains belonging to the repetitive PCR clusters 4 or 5 were distinctly different from other B. cenocepacia strains as shown by PCR-RFLP analysis of the fliC gene and phenotypic assays.