Survival dynamics of stick insect and the impact of environmental factors on natural fungal infection during the rainy season.

Phasmatodea, commonly known as stick insects, are recognized as noteworthy pests globally, impacting agriculture and forest ecosystems. Among them, the outbreak of Ramulus mikado has emerged as a notable concern in East Asian forests. Recently, Metarhizium phasmatodeae has been identified as utilizing stick insects as hosts. We have observed evidence of this entomopathogenic fungus infecting stick insects. Given the increase in these occurrences during the rainy period, this study investigated the relationship between the survival of R. mikado and the M. phasmatodeae infection during the rainy seasons of 2022 and 2023. We collected stick insects in two representative forests of the Republic of Korea and examined insect survival, fungal infection, and various environmental factors. No infections were detected in specimens collected in June before the rainy season, but from July onwards, both the mortality of R. mikado and the fungal infection substantially increased. By the last sampling date of each year, 75% (2022), 71.4% (2023) of the specimens were infected, and over 90% of the total individuals succumbed as a result. Fungi isolated from deceased R. mikado were successfully identified as M. phasmatodeae using morphological and taxonomic approaches. Various statistical analyses, including principal component analysis and modeling, revealed a robust association between fungal infection and the survival of stick insects. The results highlight the correlation between mass deaths of stick insects and fungal infection, particularly during the summer rainy season. These findings offer valuable insights for forecasting R. mikado population in the upcoming year and developing effective pest control strategies.

Con7 is a key transcription regulator for conidiogenesis in the plant pathogenic fungus Fusarium graminearum.

The mycelium of the plant pathogenic fungus Fusarium graminearum exhibits distinct structures for vegetative growth, asexual sporulation, sexual development, virulence, and chlamydospore formation. These structures are vital for the survival and pathogenicity of the fungus, necessitating precise regulation based on environmental cues. Initially identified in Magnaporthe oryzae, the transcription factor Con7p regulates conidiation and infection-related morphogenesis, but not vegetative growth. We characterized the Con7p ortholog FgCon7, and deletion of FgCON7 resulted in severe defects in conidium production, virulence, sexual development, and vegetative growth. The mycelia of the deletion mutant transformed into chlamydospore-like structures with high chitin level accumulation. Notably, boosting FgABAA expression partially alleviated developmental issues in the FgCON7 deletion mutant. Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, the chitin synthase gene Fg6550 (FGSG_06550) showed significant upregulation in the FgCON7 deletion mutant, and altering FgCON7 expression affected cell wall integrity. Further research will focus on understanding the behavior of the chitin synthase gene and its regulation by FgCon7 in F. graminearum. This study contributes significantly to our understanding of the genetic pathways that regulate hyphal differentiation and conidiation in this plant pathogenic fungus. IMPORTANCE: The ascomycete fungus Fusarium graminearum is the primary cause of head blight disease in wheat and barley, as well as ear and stalk rot in maize. Given the importance of conidia and ascospores in the disease cycle of F. graminearum, precise spatiotemporal regulation of these biological processes is crucial. In this study, we characterized the Magnaporthe oryzae Con7p ortholog and discovered that FgCon7 significantly influences various crucial aspects of fungal development and pathogenicity. Notably, overexpression of FgABAA partially restored developmental defects in the FgCON7 deletion mutant. ChIP-qPCR analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, our research revealed a clear correlation between FgCon7 and chitin accumulation and the expression of chitin synthase genes. These findings offer valuable insights into the genetic mechanisms regulating conidiation and the significance of mycelial differentiation in this plant pathogenic fungus.

A mitochondrial NAD/NADH kinase governs fungal virulence through an oxidative stress response and arginine biosynthesis in Fusarium graminearum.

NADP/NADPH plays an indispensable role in cellular metabolism, serving as a pivotal cofactor in numerous enzymatic processes involved in anabolic pathways, antioxidant defense, and the biosynthesis of essential cellular components. NAD/NADH kinases (NADKs) phosphorylate NAD/NADH, constituting the sole de novo synthetic pathway for NADP/NADPH generation. Despite the pivotal role of NADP/NADPH in cellular functions, the physiological role of NADK remains largely unexplored in filamentous fungi. In this study, we identified three putative NADKs in Fusarium graminearum-FgNadk1, FgNadk2, and FgNadk3-responsible for NAD/NADH phosphorylation. NADK-mediated formation of intracellular NADPH proved crucial for vegetative growth, sexual reproduction, and virulence. Specifically, FgNadk2, the mitochondrial NADK, played a role in oxidative stress resistance and the maintenance of mitochondrial reactive oxygen species levels. Moreover, the deletion of FgNADK2 resulted in arginine auxotrophy, contributing to the reduced fungal virulence. These findings underscore the necessity of mitochondrial NADK in fungal virulence in F. graminearum, revealing its involvement in mitochondrial redox homeostasis and the arginine biosynthetic pathway. This study provides critical insights into the interconnectedness of metabolic pathways essential for fungal growth, stress response, and pathogenicity.

Proteomic Analysis of Cell Wall Proteins with Various Linkages in Fusarium graminearum.

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.

Infectivity and stress tolerance traits affect community assembly of plant pathogenic fungi.

Understanding how ecological communities assemble is an urgent research priority. In this study, we used a community ecology approach to examine how ecological and evolutionary processes shape biodiversity patterns of plant pathogenic fungi, Fusarium graminearum and F. asiaticum. High-throughput screening revealed that the isolates had a wide range of phenotypic variation in stress tolerance traits. Net Relatedness Index (NRI) and Nearest Taxon Index (NTI) values were computed based on stress-tolerant distance matrices. Certain local regions exhibited positive values of NRI and NTI, indicating phenotypic clustering within the fungal communities. Competition assays of the pooled strains were conducted to investigate the cause of clustering. During stress conditions and wheat colonization, only a few strains dominated the fungal communities, resulting in reduced diversity. Overall, our findings support the modern coexistence theory that abiotic stress and competition lead to phenotypic similarities among coexisting organisms by excluding large, low-competitive clades. We suggest that agricultural environments and competition for host infection lead to locally clustered communities of plant pathogenic fungi in the field.

Past and Future Epidemiological Perspectives and Integrated Management of Rice Bakanae in Korea.

In the past, rice bakanae was considered an endemic disease that did not cause significant losses in Korea; however, the disease has recently become a serious threat due to climate change, changes in farming practices, and the emergence of fungicide-resistant strains. Since the bakanae outbreak in 2006, its incidence has gradually decreased due to the application of effective control measures such as hot water immersion methods and seed disinfectants. However, in 2013, a marked increase in bakanae incidence was observed, causing problems for rice farmers. Therefore, in this review, we present the potential risks from climate change based on an epidemiological understanding of the pathogen, host plant, and environment, which are the key elements influencing the incidence of bakanae. In addition, disease management options to reduce the disease pressure of bakanae below the economic threshold level are investigated, with a specific focus on resistant varieties, as well as chemical, biological, cultural, and physical control methods. Lastly, as more effective countermeasures to bakanae, we propose an integrated disease management option that combines different control methods, including advanced imaging technologies such as remote sensing. In this review, we revisit and examine bakanae, a traditional seed-borne fungal disease that has not gained considerable attention in the agricultural history of Korea. Based on the understanding of the present significance and anticipated risks of the disease, the findings of this study are expected to provide useful information for the establishment of an effective response strategy to bakanae in the era of climate change.

Two Previously Unrecorded Fungal Species Isolated from Muui Island in Korea.

Fungi are cosmopolitan and they occupy diverse niches as consumers, producers, and decomposers. They play critical roles in the environment by enabling nutrient cycling and generating a plethora of secondary metabolites. This study aimed to identify and characterize fungal strains isolated from diverse sources on Muui Island, Republic of Korea. In 2023, a total of 86 fungal strains were collected and examined. Investigation of the morphological features and phylogenetic analyses of multiple barcode loci identified one putative novel species and two species previously unrecorded in the Republic of Korea: Colletotrichum sp., Colletotrichum guizhouense, and Fusarium brachygibbosum. This study provides a comprehensive description of their molecular phylogenies and morphological characteristics. These findings will contribute to the existing knowledge about fungal species in the Republic of Korea and future research on the fungal diversity on Muui Island.

Identification and Characterization of Fungal Pathogens Associated with Boxwood Diseases in the Republic of Korea.

Boxwood is a representative ornamental shrub that is widely used in landscaping horticulture. After pruning, damaged leaves or stems of boxwoods are unavoidably vulnerable to infection by various plant pathogens. Several boxwood diseases caused by fungi, such as Volutella blight and Macrophoma leaf spot, have been reported worldwide including Republic of Korea. In this study, we isolated and identified fungal pathogens of boxwood diseases that occurred in Korea and characterized their morphological and taxonomic characteristics. Boxwood samples showing blight symptoms were collected in Seoul, Republic of Korea, and the putative fungal pathogens Pseudonectria buxi, P. foliicola, and Neofusicoccum buxi were successfully identified. Investigation of the morphological features of the field isolates, including mycelial growth and conidial morphology, and phylogenetic analysis of multiple DNA barcode loci revealed that there were some morphological and genetic variations among isolates, but all of the analyzed isolates were closely related to the corresponding reference strains. We also found that P. foliicola strains were more virulent than P. buxi, and the N. buxi strains isolated in this study were weak pathogens or saprophytes. The results of our study will contribute to the development of control strategies for boxwood diseases caused by fungi and accelerate research on the complex ecology of boxwood diseases.

First Report of Pseudonectria buxi causing Volutella blight on Boxwood (Buxus microphylla) in the Republic of Korea.

Boxwood (Buxus spp.) are evergreen landscaping plants commonly used as hedges and fresh greenery. In July and August 2020, boxwood (Buxus microphylla Sieb. et Zucc) samples with blight symptoms were collected from the parterres of Seoul National University (Seoul, Republic of Korea). Diseased leaves and stem tissues were soaked in 1% sodium hypochlorite for 1 min, rinsed with sterile water, cultured on potato dextrose agar (PDA; Difco, Sparks, MD, USA) and incubated at 25 ℃ for 5 days. Four isolates (B2S72-1, B2S7-3, B3B2, and B4L3-3) were pure-cultured using the single-spore isolation method. Light pink-colored sporodochia containing one-celled, fusoid conidia were observed on PDA. Mean conidial size was 9.11 × 3.79 µm and ranged from 7.68 to 10.71 × 3.18 to 4.92 µm. Morphological features suggested that these isolates possessed the same characteristics as previously described for P. buxi (Bezerra, 1963, Yang et al., 2021). Genomic DNA was extracted from each isolate and the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), ß-tubulin coding sequence, and D1/D2 region of the large subunit of the rDNA gene cluster (LSU) were amplified using the primer pairs ITS4/ITS5 (White et al., 1990), Btub4Rd/Btub2Fd (Woudenberg et al., 2009), and LR0R/LR5 (Rehner & Samuels, 1994, Vilgalys & Hester, 1990), respectively. The sequences were deposited in GenBank (GenBank accessions No. OM169273-OM169276, OM176571-OM176574, OM176566-OM176569). BLAST search showed high similarity to GenBank sequences of the ex-type strain of P. buxi for ITS (≥99.50%, MH892589.1 and MH892583.1). In addition, neighbor-joining (NJ) phylogeneticnalysis was performed using MEGA-X based on the combined sequences. The four isolates were clustered on the same clade with Pseudonectria buxi reference strains with a high bootstrap value (100 %). For the pathogenicity test, leaves of 1-year-old boxwood were given a single cut with a razor blade and a 5-mm PDA mycelial plug was placed on the wound site. Plants were kept in a transparent box at 25°C with a 12-h photoperiod. After 21 days, inoculated leaves had turned yellow and orange to pink sporodochia were observed. P. buxi was successfully reisolated from the symptomatic tissues but not from the control leaves, therefore Koch's postulates were completed. To our knowledge, this is the first report of Volutella blight caused by P. buxi in the Republic of Korea.

Histomorphometric analysis of microcrack healing after the installation of mini-implants.

PURPOSE: The goal of this study was to investigate the histomorphometric characteristics of the healing process of microcracks in the cortical bone after the installation of mini-implants (MIs). METHODS: Self-drilling MIs were inserted into the tibial diaphysis of twelve adult male New Zealand rabbits. Four MIs per rabbit were placed randomly. The animals were divided into four groups according to the length of the healing period: group A was sacrificed immediately, group B was sacrificed after one week, group C was sacrificed after two weeks, and group D was sacrificed after four weeks. Cortical bone thickness was measured using micro-computed tomography, and histomorphometric analyses of the cumulative length of the microcracks (CLCr) and the total number of microcracks (NCr) were performed using hematoxylin and eosin staining. RESULTS: The microcracks were radially and concentrically aligned in the peri-MI bone. The CLCr decreased significantly one week after the surgery, mainly due to healing of the concentrically aligned microcracks. The CLCr showed another significant decrease from two weeks after the surgery to four weeks after the surgery, mainly reflecting healing of the radially aligned microcracks. A statistically significant decrease in the NCr occurred as the microcracks healed from zero weeks to two weeks. However, no significant difference in the NCr was found between groups C and D. CONCLUSIONS: In order to improve the primary stability of MIs, delayed loading and a healing period of a certain length are recommended to ensure the optimal healing of microcracks and bone remodeling.