Fungal Microbiome of Alive and Dead Korean Fir in its Native Habitats.

A rapid decline of Abies koreana has been reported in most of the natural alpine habitats in Korea. It is generally accepted that this phenomenon is due to climate change even though no clear conclusions have been drawn. Most research has focused on abiotic environmental factors, but studies on the relationships between A. koreana and soil fungal microbiomes are scarce. In this study, the rhizoplane and rhizosphere fungal communities in the alive and dead Korean fir trees from its three major natural habitats including Mt. Deogyu, Mt. Halla, and Mt. Jiri in Korea were investigated to identify specific soil fungal groups closely associated with A. koreana. Soil fungal diversity in each study site was significantly different from another based on the beta diversity calculations. Heat tree analysis at the genus level showed that Clavulina, Beauveria, and Tomentella were most abundant in the healthy trees probably by forming ectomycorrhizae with Korean fir growth and controlling pests and diseases. However, Calocera, Dacrymyces, Gyoerffyella, Hydnotrya, Microdochium, Hyaloscypha, Mycosymbioces, and Podospora were abundant in the dead trees. Our findings suggested that Clavulina, Beauveria, and Tomentella are the major players that could be considered in future reforestation programs to establish ectomycorrhizal networks and promote growth. These genera may have played a significant role in the survival and growth of A. koreana in its natural habitats. In particular, the genus Gyoerffyella may account for the death of the seedlings. Our work presented exploratory research on the specific fungal taxa associated with the status of A. koreana.

Fungal Load of Groundwater Systems in Geographically Segregated Islands: A Step Forward in Fungal Control.

The fungal distribution, diversity, and load were analyzed in the geographically segregated island groundwater systems in Korea. A total of 79 fungal isolates were secured from seven islands and identified based on the internal transcribed spacer (ITS) sequences. They belonged to three phyla (Ascomycota, Basidiomycota, and Chlorophyta), five classes, sixteen orders, twenty-two families, and thirty-one genera. The dominant phylum was Ascomycota (91.1%), with most fungi belonging to the Cladosporium (21.5%), Aspergillus (15.2%), and Stachybotrys (8.9%) genera. Cladosporium showed higher dominance and diversity, being widely distributed throughout the geographically segregated groundwater systems. Based on the diversity indices, the genera richness (4.821) and diversity (2.550) were the highest in the groundwater system of the largest scale. As turbidity (0.064-0.462) increased, the overall fungal count increased and the residual chlorine (0.089-0.308) had low relevance compared with the total count and fungal diversity. Cladosporium showed normal mycelial growth in de-chlorinated sterilized samples. Overall, if turbidity increases under higher fungal diversity, bio-deterioration in groundwater-supplying facilities and public health problems could be intensified, regardless of chlorine treatment. In addition to fungal indicators and analyzing methods, physical hydrostatic treatment is necessary for monitoring and controlling fungal contamination.

Worker Protection Scenarios for General Analytical Testing Facility under Several Infection Propagation Risks: Scoping Review, Epidemiological Model and ISO 31000.

Infectious disease is a risk threating industrial operations and worker health. In gastrointestinal disease cases, outbreak is sporadic, and propagation is often terminated within certain populations, although cases in industrial sites are continuously reported. The ISO 31000 international standard for risk management, an epidemiological triad model, and a scoping review were the methods used to establish response procedures (scenarios) to protect workers from the risk of the propagation of a gastrointestinal disease. First, human reservoirs and transmission routes were identified as controllable risk sources based on a scoping review and the use of a triad model. Second, the possibility of fomite- or surface-mediated transmission appeared to be higher based on environmental characterization. Thus, the propagation could be suppressed using epidemiological measures categorized by reservoirs (workers) or transmission routes during a primary case occurrence. Next, using results of a matrix, a strengths-weaknesses-opportunities-threats analysis and a scoping review, the risk treatment option was determined as risk taking and sharing. According to epidemiology of gastrointestinal infections, systematic scenarios may ensure the efficacy of propagation control. Standardized procedures with practicality and applicability were established for categorized scenarios. This study converged ISO 31000 standards, an epidemiological model, and scoping review methods to construct a risk management scenario (non-pharmaceutical intervention) optimized for the unique characteristics of a specific occupational cluster.

Rhizoplane and Rhizosphere Fungal Communities of Geographically Isolated Korean Bellflower (Campanula takesimana Nakai).

Fungal communities in the rhizoplane (RP) and rhizosphere (RS) of geographically isolated C. takesimana habitats in different environments such as oceanic (Seodo, the Dokdo Islands), coastline (Sadong, Ulleungdo Island), and inland (Taeha, Ulleungdo Island) regions were analyzed by MiSeq sequencing. In total, 1279 operational taxonomic units (OTUs) were obtained and they were further classified into 185 genera belonging to five phyla. The total number of fungal taxa in the RP samples was lower than those in the RS samples in all the sampled locations, providing an indication of the existence of a certain level of the selective pressures from the host plant. The richness of the RP in the Dokdo Islands was higher than that of Ulleungdo Island, but the richness of the RS in the Dokdo Islands was lower than that of Ulleungdo Island. These results suggest evidence for strong effects of a harsh geo-climate on the RP and RS fungal diversities in the Dokdo Islands. Additionally, a total of 82 fungal genera were identified in all three RP samples and 63 genera (77%) were uniquely found in each of the geographical regions and 43 genera (52.4%) showed high dependency on the C. takesimana vegetation. It was found that the genus Mortierella was the most dominant taxon in all the samples. The geo-ecological isolation of the Korean bellflower may have caused unique formation of the RP and RS fungal communities in the natural habitats.

Adhaeribacter radiodurans sp. nov., isolated from the rhizospheric soil of Elymus tsukushiensis, a plant native to the Dokdo Islands, Republic of Korea.

KUDC8001T was isolated from the rhizosperic soil of Elymus tsukushiensis in the Dokdo Islands, Republic of Korea. Strain KUDC8001T was Gram-stain-negative, non-motile and rod-shaped. KUDC8001T was catalase- and oxidase-positive. This strain is capable of growing at 4-37 °C and pH 7.0-8.0 and exhibited optimal growth at 25 °C and pH 7.0. It could be grown in R2A, nutrient agar and ISP2 agar plates. The cell width ranged from 0.7 to 1.0 µm, and length ranged from 2.5 to 5.5 µm. The genomic G+C content was 40.8 mol%. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that the strain KUDC8001T belongs to the genus Adhaeribacter, which is most closely related to the strain A. pallidiroseus HMF7616T (97.5%). The DNA relatedness of KUDC8001T with the type strains of A. pallidiroseus HMF7616T, A. swui 17 mud1-7T and A. arboris HMF7605T was ≤80.3 % based on average nucleotide identity calculations and ≤86.9 % based on average amino acid identity calculations. In silico DNA-DNA hybridization values of the strain KUDC8001T with the most closely related strains were 22.1, 24.0 and 24.4 %. Based on its phenotypic, phylogenetic, genetic and chemotaxonomic features, the strain KUDC8001T should be considered as a novel species in the genus Adhaeribacter, for which we have proposed the name Adhaeribacter radiodurans sp. nov. The type strain is KUDC8001T (=KCTC 82078T=CGMCC 1.18475T).

Geographical Isolation and Root-Associated Fungi in the Marine Terrains: A Step Toward Establishing a Strategy for Acquiring Unique Microbial Resources.

This study aimed to understand whether the geo-ecological segregation of native plant species affects the root-associated fungal community. Rhizoplane (RP) and rhizosphere (RS) fungal microbiota of Sedum takesimense native to three geographically segregated coastal regions (volcanic ocean islands) were analyzed using culture-independent methods: 568,507 quality sequences, 1399 operational taxonomic units, five phyla, and 181 genera were obtained. Across all regions, significant differences in the phyla distribution and ratio were confirmed. The Chao's richness value was greater for RS than for RP, and this variance coincided with the number of genera. In contrast, the dominance of specific genera in the RS (Simpson value) was lower than the RP at all sites. The taxonomic identity of most fungal species (95%) closely interacting with the common host plant was different. Meanwhile, a considerable number of RP only residing fungal genera were thought to have close interdependency on their host halophyte. Among these, Metarhizium was the sole genus common to all sites. These suggest that the relationship between potential symbiotic fungi and their host halophyte species evolved with a regional dependency, in the same halophyte species, and of the same natural habitat (volcanic islands); further, the fungal community differenced in distinct geographical regions. Importantly, geographical segregation should be accounted for in national culture collections, based on taxonomical uniqueness.

Monitoring Culicine Mosquitoes (Diptera: Culicidae) as a Vector of Flavivirus in Incheon Metropolitan City and Hwaseong-Si, Gyeonggi-Do, Korea, during 2019.

The flaviviruses are small single-stranded RNA viruses that are typically transmitted by mosquitoes or tick vectors and are etiological agents of acute zoonotic infections. The viruses are found around the world and account for significant cases of human diseases. We investigated population of culicine mosquitoes in central region of Korean Peninsula, Incheon Metropolitan City and Hwaseong-si. Aedes vexans nipponii was the most frequently collected mosquitoes (56.5%), followed by Ochlerotatus dorsalis (23.6%), Anopheles spp. (10.9%), and Culex pipiens complex (5.9%). In rural regions of Hwaseong, Aedes vexans nipponii was the highest population (62.9%), followed by Ochlerotatus dorsalis (23.9%) and Anopheles spp. (12.0%). In another rural region of Incheon (habitat of migratory birds), Culex pipiens complex was the highest population (31.4%), followed by Ochlerotatus dorsalis (30.5%), and Aedes vexans vexans (27.5%). Culex pipiens complex was the predominant species in the urban region (84.7%). Culicine mosquitoes were identified at the species level, pooled up to 30 mosquitoes each, and tested for flaviviral RNA using the SYBR Green-based RT-PCR and confirmed by cDNA sequencing. Three of the assayed 2,683 pools (989 pools without Anopheles spp.) were positive for Culex flaviviruses, an insect-specific virus, from Culex pipiens pallens collected at the habitats for migratory birds in Incheon. The maximum likelihood estimation (the estimated number) for Culex pipiens pallens positive for Culex flavivirus was 25. Although viruses responsible for mosquito-borne diseases were not identified, we encourage intensified monitoring and long-term surveillance of both vector and viruses in the interest of global public health.

Fungal Clusters and Their Uniqueness in Geographically Segregated Wetlands: A Step Forward to Marsh Conservation for a Wealth of Future Fungal Resources.

Here, we investigated fungal microbiota in the understory root layer of representative well-conserved geographically segregated natural wetlands in the Korean Peninsula. We obtained 574,143 quality fungal sequences in total from soil samples in three wetlands, which were classified into 563 operational taxonomic units (OTU), 5 phyla, 84 genera. Soil texture, total nitrogen, organic carbon, pH, and electrical conductivity of soil were variable between geographical sites. We found significant differences in fungal phyla distribution and ratio, as well as genera variation and richness between the wetlands. Diversity was greater in the Jangdo islands wetland than in the other sites (Chao richness/Shannon/Simpson's for wetland of the Jangdo islands: 283/6.45/0.97 > wetland of the Mt. Gariwang primeval forest: 169/1.17/0.22 > wetland of the Hanbando geology: 145/4.85/0.91), and this variance corresponded to the confirmed number of fungal genera or OTUs (wetlands of Jangdo islands: 42/283 > of Mt. Gariwang primeval forest: 32/169 > of the Hanbando geology: 25/145). To assess the uniqueness of the understory root layer fungus taxa, we analyzed fungal genera distribution. We found that the percentage of fungal genera common to two or three wetland sites was relatively low at 32.3%, while fungal genera unique to each wetland site was 67.7% of the total number of identified fungal species. The Jangdo island wetland had higher fungal diversity than did the other sites and showed the highest level of uniqueness among fungal genera (Is. Jangdo wetland: 34.5% > wetland of Mt. Gariwang primeval forest: 28.6% > wetland of the Hanbando geology: 16.7%).

Talaromyces halophytorum sp. nov. Isolated from Roots of Limonium tetragonum in Korea.

Talaromyces halophytorum sp. nov. was isolated from the roots of halophyte Limonium tetragonum collected from Seocheon-gun, Korea in November 2015. It showed a slow growth on yeast extract sucrose agar at 25 °C, no growth at 4 °C or 37 °C and produced smooth-walled and globose to sub-globose conidia. T. halophytorum is phylogenetically distinct from the other reported Talaromyces species of section Trachyspermi based on multi-locus sequence typing results using partial fragments of ß-tubulin, calmodulin, ITS, and RNA polymerase II genes.

Microbial control measures for soft ice cream in franchise brands: A comparative analysis of microbial analysis and manufacturing practices.

The aim of this study was to establish a complementary direction of the franchise food hygiene guideline to control microbial risks. We evaluated current measures of preventing microbial spoilage and ensuring microbiological safety of food in the food franchise industry. Manufacturing practices were assessed using microbiological analyses, third-party food safety audits, and existing hygiene guidelines. Microbial load indicators of food, manufacturing processes, work environment, and workers were also analyzed to track microbial proliferation. We audited manufacturing practices and processes and analyzed the cleaning and sanitation clauses. We found high contamination of heterotrophic bacteria and detected coliforms in some products. There was no direct contamination by food handlers, and the sterility of raw materials was satisfactory. The main issues were structural complexities of equipment, which hindered cleaning and disinfection, and disinfection practices of franchise brands. Store-level microbial control may be compromised due to the industrial nature of small stores operating collectively. We thus issued complementary guidelines. Improved collective microbiological safety may be ensured through implementation of the revised cleaning and sterilization regulations.

Distribution, Characterization, and Diversity of the Endophytic Fungal Communities on Korean Seacoasts Showing Contrasting Geographic Conditions.

This study analyzed the distribution of endophytic fungi in 3 coastal environments with different climatic, geographical, and geological characteristics: the volcanic islands of Dokdo, the East Sea, and the West Sea of Korea. The isolated fungal endophytes were characterized and analyzed with respect to the characteristics of their host environments. For this purpose, we selected common native coastal halophyte communities from three regions. Molecular identification of the fungal endophytes showed clear differences among the sampling sites and halophyte host species. Isolates were also characterized by growth at specific salinities or pH gradients, with reference to previous geographical, geological, and climate studies. Unlike the East Sea or West Sea isolates, some Dokdo Islands isolates showed endurable traits with growth in high salinity, and many showed growth under extremely alkaline conditions. A smaller proportion of West Sea coast isolates tolerate compared to the East Sea or Dokdo Islands isolates. These results suggest that these unique fungal biota developed through a close interaction between the host halophyte and their environment, even within the same halophyte species. Therefore, this study proposes the application of specific fungal resources for restoring sand dunes and salt-damaged agricultural lands and industrialization of halophytic plants.

Cutaneous Microflora from Geographically Isolated Groups of Bradysia agrestis, an Insect Vector of Diverse Plant Pathogens.

Larvae of Bradysia agrestis, an insect vector that transports plant pathogens, were sampled from geographically isolated regions in Korea to identify their cutaneous fungal and bacterial flora. Sampled areas were chosen within the distribution range of B. agrestis; each site was more than 91 km apart to ensure geographical segregation. We isolated 76 microbial (fungi and bacteria) strains (site 1, 29; site 2, 29; site 3, 18 strains) that were identified on the basis of morphological differences. Species identification was molecularly confirmed by determination of universal fungal internal transcribed spacer and bacterial 16S rRNA gene sequences in comparison to sequences in the EzTaxon database and the NCBI GenBank database, and their phylogenetic relationships were determined. The fungal isolates belonged to 2 phyla, 5 classes, and 7 genera; bacterial species belonged to 23 genera and 32 species. Microbial diversity differed significantly among the geographical groups with respect to Margalef's richness (3.9, 3.6, and 4.5), Menhinick's index (2.65, 2.46, and 3.30), Simpson's index (0.06, 0.12, and 0.01), and Shannon's index (2.50, 2.17, and 2.58). Although the microbial genera distribution or diversity values clearly varied among geographical groups, common genera were identified in all groups, including the fungal genus Cladosporium, and the bacterial genera Bacillus and Rhodococcus. According to classic principles of co-evolutionary relationship, these genera might have a closer association with their host insect vector B. agrestis than other genera identified. Some cutaneous bacterial genera (e.g., Pseudomonas) displaying weak interdependency with insect vectors may be hazardous to agricultural environments via mechanical transmission via B. agrestis. This study provides comprehensive information regarding the cutaneous microflora of B. agrestis, which can help in the control of such pests for crop management.

Foodborne Infectious Diseases Mediated by Inappropriate Infection Control in Food Service Businesses and Relevant Countermeasures in Korea.

OBJECTIVES: The objective of this review is to propose an appropriate course of action for improving the guidelines followed by food handlers for control of infection. For this purpose, previous epidemiological reports related to acute gastroenteritis in food service businesses mediated by food handlers were intensively analyzed. METHODS: Relevant studies were identified in international databases. We selected eligible papers reporting foodborne infectious disease outbreaks. Among primary literature collection, the abstract of each article was investigated to find cases that absolutely identified a causative factor to be food handlers' inappropriate infection control and the taxon of causative microbial agents by epidemiological methodologies. Information about the sites (type of food business) where the outbreaks occurred was investigated. RESULTS: A wide variety of causative microbial agents has been investigated, using several epidemiological methods. These agents have shown diverse propagation pathways based on their own molecular pathogenesis, physiology, taxonomy, and etiology. CONCLUSION: Depending on etiology, transmission, propagation, and microbiological traits, we can predict the transmission characteristics of pathogens in food preparation areas. The infected food workers have a somewhat different ecological place in infection epidemiology as compared to the general population. However, the current Korean Food Safety Act cannot propose detailed guidelines. Therefore, different methodologies have to be made available to prevent further infections.

Specific rhizobacterial resources: characterization and comparative analysis from contrasting coastal environments of Korea.

This study analyzed the rhizobacterial distribution from two coasts, which show contrasting climates and geographical and geological characteristics, to secure specific microbial resources. Furthermore, rhizobacteria were characterized and the results were comparatively analyzed with reference to the characteristics of two coastal environments. For this purpose, three representative halophyte species communities native to the Dokdo Islands and the East Sea coast of Korea were selected. Partial identification of rhizobacteria showed a clear difference between each sampling site and halophyte. Furthermore, isolates were characterized by their growth properties under NaCl or pH gradients related with previous geographical, geological, and climatic studies of the Dokdo Islands and the East Sea coast. A high proportion of the East Sea isolates showed halotolerance, but a high proportion of Dokdo isolates shared halophilic traits. Meanwhile, a higher proportion of East Sea isolates grew at a wider range of pH values than those of the Dokdo Islands. The results of our study suggest that unique rhizobacterial resources developed under specific rhizospheric conditions derived from halophytes interacting with their specific environment, even within the same coastal halophytic species. Therefore, this study proposes the necessity of securing characterized and unique microbial resources to apply to specific environments for the purpose of recovering and restoring sand dunes or salt-damaged agricultural lands.

Diversity of Endophytic Fungi Associated with the Roots of Four Aquatic Plants Inhabiting Two Wetlands in Korea.

A total of 4 aquatic plants, Eleocharis kuroguwai Ohwi, Hydrocharis dubia Backer, Salvinia natans All., and Zizania latifolia Turcz., were sampled from representative two wetlands of South Korea. A total of 38 endophytic fungal strains were isolated from aquatic plants native to the Daepyeong wetland, and 27 strains were isolated from the Jilnal wetland. The internal transcribed spacer regions of fungal isolates were sequenced and a phylogenetic analysis was performed. In addition, endophytic fungal diversity from each wetland and host plant species was deduced. A total of 25 fungal genera were purely isolated, and 16 fungal genera were isolated from each of the two wetlands. Commonly isolated genera from both wetlands were Aspergillus, Cladosporium, Clonostachys, Fusarium, Leptosphaeria, Penicillium, and Talaromyces. This study revealed that fungal diversity varied with environmental conditions and by host plant in representative two wetlands.

Characterization of three antifungal calcite-forming bacteria, Arthrobacter nicotianae KNUC2100, Bacillus thuringiensis KNUC2103, and Stenotrophomonas maltophilia KNUC2106, derived from the Korean islands, Dokdo and their application on mortar.

Crack remediation on the surface of cement mortar using microbiological calcium carbonate (CaCO3) precipitation (MICP) has been investigated as a microbial sealing agent on construction materials. However, MICP research has never acknowledged the antifungal properties of calcite-forming bacteria (CFB). Since fungal colonization on concrete surfaces can trigger biodeterioration processes, fungi on concrete buildings have to be prevented. Therefore, to develop a microbial sealing agent that has antifungal properties to remediate cement cracks without deteriorative fungal colonization, we introduced an antifungal CFB isolated from oceanic islands (Dokdo islands, territory of South Korea, located at the edge of the East Sea in Korea.). The isolation of CFB was done using B4 or urea-CaCl2 media. Furthermore, antifungal assays were done using the pairing culture and disk diffusion methods. Five isolated CFB showed CaCO3 precipitation and antifungal activities against deteriorative fungal strains. Subsequently, five candidate bacteria were identified using 16S rDNA sequence analysis. Crack remediation, fungi growth inhibition, and water permeability reduction of antifungal CFB-treated cement surfaces were tested. All antifungal CFB showed crack remediation abilities, but only three strains (KNUC2100, 2103, and 2106) reduced the water permeability. Furthermore, these three strains showed fungi growth inhibition. This paper is the first application research of CFB that have antifungal activity, for an eco-friendly improvement of construction materials.

Application of Bacillus subtilis 168 as a multifunctional agent for improvement of the durability of cement mortar.

Microbiological calcium carbonate precipitation (MCCP) has been investigated for its ability to improve the durability of cement mortar. However, very few strains have been applied to crack remediation and strengthening of cementitious materials. In this study, we report the biodeposition of Bacillus subtilis 168 and its ability to enhance the durability of cement material. B. subtilis 168 was applied to the surface of cement specimens. The results showed a new layer of deposited organic-inorganic composites on the surface of the cement paste. In addition, the water permeability of the cement paste treated with B. subtilis 168 was lower than that of non-treated specimens. Furthermore, artificial cracks in the cement paste were completely remediated by the biodeposition of B. subtilis 168. The compressive strength of cement mortar treated with B. subtilis 168 increased by about 19.5% when compared with samples completed with only B4 medium. Taken together, these findings suggest that the biodeposition of B. subtilis 168 could be used as a sealing and coating agent to improve the strength and water resistance of concrete. This is the first paper to report the application of Bacillus subtilis 168 for its ability to improve the durability of cement mortar through calcium carbonate precipitation.

Application of antifungal CFB to increase the durability of cement mortar.

Antifungal cement mortar or microbiological calcium carbonate precipitation on cement surface has been investigated as functional concrete research. However, these research concepts have never been fused with each other. In this study, we introduced the antifungal calciteforming bacteria (CFB) Bacillus aryabhattai KNUC205, isolated from an urban tunnel (Daegu, South Korea). The major fungal deteriogens in urban tunnel, Cladosporium sphaerospermum KNUC253, was used as a sensitive fungal strain. B. aryabhattai KNUC205 showed CaCO3 precipitation on B4 medium. Cracked cement mortar pastes were made and neutralized by modified methods. Subsequently, the mixture of B. aryabhattai KNUC205, conidiospore of C. sphaerospermum KNUC253, and B4 agar was applied to cement cracks and incubated at 18 degrees C for 16 days. B. aryabhattai KNUC205 showed fungal growth inhibition against C. sphaerospermum. Furthermore, B. aryabhattai KNUC205 showed crack remediation ability and water permeability reduction of cement mortar pastes. Taken together, these results suggest that the CaCO3 precipitation and antifungal properties of B. aryabhattai KNUC205 could be used as an effective sealing or coating material that can also prevent deteriorative fungal growth. This study is the first application and evaluation research that incorporates calcite formation with antifungal capabilities of microorganisms for an environment-friendly and more effective protection of cement materials. In this research, the conception of microbial construction materials was expanded.