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.

Isolation and Identification Antagonistic Bacterium Paenibacillus tianmuensis YM002 against Acidovorax citrulli.

In this study, we aimed to screen antagonistic microorganisms against Acidovorax citrulli, the causal agent of bacterial fruit blotch, which is known to induce sever diseases in cucurbit crops. From 240 bacterial strains isolated, only one unknown bacterial isolate, named YM002, showed significant antagonistic activity against A. citrulli KACC17909. Further experiments revealed that YM002 shows antagonistic activity against all tested A. citrulli strains, including KACC17000, KACC17001 and KACC17005, to different degrees. The phylogenetic analysis of 16S rRNA sequences identified YM002 as Paenibacillus tianmuensis. Importantly, pretreatment of cucumber (Cucumis sativus) leaves with YM002 enhanced disease resistance as observed by significantly reduced necrotic symptom development and bacterial growth. YM002-induced resistance accompanied by enhanced expression of defense-related genes, such as PAL1, PR1-1a and CTR1. Importantly, culture filtrate of YM002 significantly suppressed biofilm formation and swimming motility of A. citrulli, which is indispensable for its full virulence. In addition to its antagonistic activity, YM002 showed a various plant growth promotion (PGP)-related traits, such as production of ammonia production, amylase production, ACC deaminase production, inodole-3-acetic acid production, extracellular protease production, siderophore production, and zinc solubilization activities. Indeed, treatment of cucumber roots with YM002 significantly enhanced plant growth parameters, such as fresh and dry weight of leaves or roots. This study suggests the potential of YM002 as an effective PGPR with biological control activity against Acidovorax citrulli in cucumber plants.

Biological Control Activity of Plant Growth Promoting Rhizobacteria Burkholderia contaminans AY001 against Tomato Fusarium Wilt and Bacterial Speck Diseases.

Plant growth promoting rhizobacteria (PGPR) is not only enhancing plant growth, but also inducing resistance against a broad range of pathogens, thus providing effective strategies to substitute chemical products. In this study, Burkholderia contaminans AY001 (AY001) is isolated based on its broad-spectrum antifungal activity. AY001 not only inhibited fungal pathogen growth in dual culture and culture filtrate assays, but also showed various PGPR traits, such as nitrogen fixation, phosphate solubilization, extracellular protease production, zinc solubilization and indole-3-acetic acid (IAA) biosynthesis activities. Indeed, AY001 treatment significantly enhanced growth of tomato plants and enhanced resistance against two distinct pathogens, F. oxysporum f.sp. lycopersici and Pseudomonas syringae pv. tomato. Real-time qPCR analyses revealed that AY001 treatment induced jasmonic acid/ethylene-dependent defense-related gene expression, suggesting its Induced Systemic Resistance (ISR)-eliciting activity. Gas chromatography-mass spectrometry (GC-MS) analysis of culture filtrate of AY001 revealed production of antimicrobial compounds, including di(2-ethylhexyl) phthalate and pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl). Taken together, our newly isolated AY001 showed promising PGPR and ISR activities in tomato plants, suggesting its potential use as a biofertilizer and biocontrol agent.

Chemical Fungicides and Bacillus siamensis H30-3 against Fungal and Oomycete Pathogens Causing Soil-Borne Strawberry Diseases.

Chemical and biological agents were evaluated to inhibit Colletotrichum fructicola, Phytophthora cactorum, and Lasiodiplodia theobromae causing strawberry diseases. Mycelial growths of C. fructicola were gradually arrested by increasing concentrations of fungicides pyraclostrobin and iminoctadine tris (albesilate). P. cactorum and L. theobromae were more sensitive to pyraclostrobin compared to C. fructicola, but iminoctadine tris (albesilate) was not or less effective to limit P. cactorum or L. theobromae, respectively. Bacillus siamensis H30-3 was antagonistic against the three pathogens by diffusible as well as volatile molecules, and evidently reduced aerial mycelial formation of P. cactorum. B. siamensis H30-3 growth was declined by at least 0.025 mg/ml of pyraclostrobin. The two fungicides additively inhibited mycelial growths of C. fructicola, but not of P. cactorum and L. theobromae. B. siamensis H30-3 volatiles led to less growth of C. fructicola than one reduced by the fungicides. Taken together, in vitro antimicrobial activities of the two fungicides together with or without B. siamensis H30-3 volatiles may be cautiously incorporated into integrated management of strawberry diseases dependent on causal pathogens.

Salicylic Acid as a Safe Plant Protector and Growth Regulator.

Since salicylic acid (SA) was discovered as an elicitor of tobacco plants inducing the resistance against Tobacco mosaic virus (TMV) in 1979, increasing reports suggest that SA indeed is a key plant hormone regulating plant immunity. In addition, recent studies indicate that SA can regulate many different responses, such as tolerance to abiotic stress, plant growth and development, and soil microbiome. In this review, we focused on the recent findings on SA's effects on resistance to biotic stresses in different plant-pathogen systems, tolerance to different abiotic stresses in different plants, plant growth and development, and soil microbiome. This allows us to discuss about the safe and practical use of SA as a plant defense activator and growth regulator. Crosstalk of SA with different plant hormones, such as abscisic acid, ethylene, jasmonic acid, and auxin in different stress and developmental conditions were also discussed.