Monitoring of Benzimidazole Resistance in Botrytis cinerea Isolates from Strawberry in Korea and Development of Detection Method for Benzimidazole Resistance.

Botrytis cinerea is a major fungal plant pathogen that causes gray mold disease in strawberries, leading to a decrease in strawberry yield. While benzimidazole is widely used as a fungicide for controlling this disease, the increasing prevalence of resistant populations to this fungicide undermines its effectiveness. To investigate benzimidazole resistant B. cinerea in South Korea, 78 strains were isolated from strawberries grown in 78 different farms in 2022, and their EC50 values for benzimidazole were examined. As a result, 64 strains exhibited resistance to benzimidazole, and experimental tests using detached strawberry leaves and the plants in a greenhouse confirmed the reduced efficacy of benzimidazole to control these strains. The benzimidazole resistant strains identified in this study possessed two types of mutations, E198A or E198V, in the TUB2 gene. To detect these mutations, TaqMan probes were designed, enabling rapid identification of benzimidazole resistant B. cinerea in strawberry and tomato farms. This study utilizes TaqMan real-time polymerase chain reaction analysis to swiftly identify benzimidazole resistant B. cinerea, thereby offering the possibility of effective disease management by identifying optimum locations and time of application.

First report of Diaporthe eres causing leaf spot disease on Machilus thunbergii in Korea.

Machilus thunbergii (Japanese bay tree) is native to warm temperate and subtropical regions in East Asia such as China, Japan, Korea, Taiwan, and Vietnam (Wu et al., 2006). This tree is used for landscape trees, windbreaks, and furniture because the wood is hard and dense (Hong et al., 2016). In May 2020, a leaf spot disease was observed on M. thunbergii in an arboretum on Wando Island, Korea. Among 25 trees surveyed in the arboretum, 7 trees showed 5 to 30% leaf spot disease. Symptoms consisted of gray and dry leaf spots up to approximately one to two centimeters in diameter, surrounded by a deep black margin. Leaf samples containing lesions were collected from the seven diseased trees. Pieces of leaf tissue (5mm × 5mm) were cut from the lesion margins and surface disinfected with 1% sodium hypochlorite (NaOCl) for 1 min and rinsed with sterile distilled water three times, patted dry on sterile paper towel and placed on Potato Dextrose Agar (PDA) in Petri dishes. From the cultures, ten fungal isolates were obtained and two representative isolates (CMML20-5 and CMML20-6) were stored at the Molecular Microbiology Laboratory, Chonnam National University, Gwangju, Korea. Colony morphology of the two isolates on PDA was observed after 7 days at 25°C in the dark. Conidiomata were induced after 7days in a 14h-10h light-dark condition using sufficiently grown mycelium in PDA, and both alpha and beta conidia were observed. Alpha conidia were 7.6 ± 0.9 × 2.8 ± 0.4 µm (n = 30), fusiform, aseptate, and hyaline. Beta conidia were 28.1 ± 3.6 × 2.7 ± 0.4 µm (n = 30), aseptate, hyaline, linear to hooked. Genomic DNA of the two isolates was extracted using the CTAB DNA extraction method (Cubero et al., 1999), followed by PCR using primer sets of the internal transcribed spacer (ITS1/ITS4) (White et al., 1990), elongation factor 1-α (EF1-728F/EF1-986R), calmodulin (CAL228F/CAL737R) (Carbone and Kohn, 1999), and TUB2 (Bt2a/Bt2b) (Glass and Donaldson 1995). PCR products were sequenced and analyzed to confirm species identity. The obtained sequences were deposited in GenBank (accession numbers OM049469, OM049470 for ITS, OM069429, OM069430 for EF1-α, OP130141, OP130142 for CAL, and OP130139, OP130140 for TUB2). BLASTn search analyses for ITS, EF1-α, CAL, and TUB2 sequences of two isolates selected resulted in near identical match (>97% for ITS, 100% for EF1-α, >99% for CAL, and >96% for TUB2) to sequences of Diaporthe eres strain AR4346 (=Phomopsis fukushii) (JQ807429 for ITS, JQ807355 for EF1-α, KJ435003 for CAL, and KJ420823 for TUB2). Phylogenetic analysis using maximum likelihood indicated that the two isolates grouped with reference strains (AR4346, AR4349, and AR4363) of D. eres with 76% bootstrap support. Based on morphological and phylogenetic analyses, the two isolates characterized in this study are members of the Diaporthe eres species complex as described by Udayanga et at. 2014. Pathogenicity tests were conducted using both detached leaf and whole plant assays. Mycelial PDA plugs (5-mm in diameter) or 10µl of 106 conidia suspensions were inoculated on detached leaves of M. thunbergii from 2-year-old trees and placed in 90 mm Petri-dishes containing wet filter papers or water agar medium. Mock inoculated controls used water in place of conidial suspensions. The plates were sealed with Parafilm and incubated at 25°C in the dark. Two year old M. thunbergii trees were inoculated with wet mycelia (1.5g) that was ground with a homogenizer and mixed with 50ml of sterile water and sprayed onto wounded leaves and stems with a needle. Mock inoculated controls were sprayed with water only. The inoculated seedlings were placed in plastic containers at 25 to 30°C to maintain high humidity. The pathogenicity tests were repeated three times with three replications. In detached leaves, symptoms of black spots were observed 6 days after mycelial plug inoculation and 20 days after conidia inoculation. In whole plants, typical symptoms were observed 9 days after inoculation. Symptoms were not observed on the control leaves and plants. Diaporthe eres was re-isolated from the inoculated leaf and whole plants and morphologically identified, fulfilling Koch's postulates. Diaporthe eres has been reported to cause a leaf spot on Photinia × fraseri 'Red Robin' in China (Song et al. 2019). To our knowledge, this is the first report of leaf spot disease caused by Diaporthe eres on Japanese bay tree (Machilus thunbergii) in Korea. It is expected that use of this tree will expand given its utility, however infection with D. eres can cause serious diseases to the leaves and stems. Therefore, further studies on disease management are needed.

Prediction models for early diagnosis of actinomycotic osteomyelitis of the jaw using machine learning techniques: a preliminary study.

BACKGROUND: This study aimed to develop and validate five machine learning models designed to predict actinomycotic osteomyelitis of the jaw. Furthermore, this study determined the relative importance of the predictive variables for actinomycotic osteomyelitis of the jaw, which are crucial for clinical decision-making. METHODS: A total of 222 patients with osteomyelitis of the jaw were analyzed, and Actinomyces were identified in 70 cases (31.5%). Logistic regression, random forest, support vector machine, artificial neural network, and extreme gradient boosting machine learning methods were used to train the models. The models were subsequently validated using testing datasets. These models were compared with each other and also with single predictors, such as age, using area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: The AUC of the machine learning models ranged from 0.81 to 0.88. The performance of the machine learning models, such as random forest, support vector machine and extreme gradient boosting was significantly superior to that of single predictors. Presumed causes, antiresorptive agents, age, malignancy, hypertension, and rheumatoid arthritis were the six features that were identified as relevant predictors. CONCLUSIONS: This prediction model would improve the overall patient care by enhancing prognosis counseling and informing treatment decisions for high-risk groups of actinomycotic osteomyelitis of the jaw.

Genome-wide transcriptional response of the causal soybean sudden death syndrome pathogen Fusarium virguliforme to a succinate dehydrogenase inhibitor fluopyram.

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs) have been widely used to manage plant diseases caused by phytopathogenic fungi. Although attention to and use of SDHI fungicides has recently increased, molecular responses of fungal pathogens to SDHIs have often not been investigated. A SDHI fungicide, fluopyram, has been used as a soybean seed treatment and has displayed effective control of Fusarium virguliforme, one of the causal agents of soybean sudden death syndrome. To examine genome-wide gene expression of F. virguliforme to fluopyram, RNA-seq analysis was conducted on two field strains of F. virguliforme with differing SDHI fungicide sensitivity in the absence and presence of fluopyram. RESULTS: The analysis indicated that several xenobiotic detoxification-related genes, such as those of deoxygenase, transferases and transporters, were highly induced by fluopyram. Among the genes, four ATP-binding cassette (ABC) transporters were characterized by the yeast expression system. The results revealed that expression of three ABCG transporters was associated with reduced sensitivity to multiple fungicides including fluopyram. In addition, heterologous expression of a major facilitator superfamily (MFS) transporter that was highly expressed in the fluopyram-insensitive F. virguliforme strain in the yeast system conferred decreased sensitivity to fluopyram. CONCLUSION: This study demonstrated that xenobiotic detoxification-related genes were highly upregulated in response to fluopyram, and expression of ABC or MFS transporter genes was associated with reduced sensitivity to the SDHI fungicide. This is the first transcriptomic analysis of the fungal species response to fluopyram and the finding will help elucidate the molecular mechanisms of SDHI resistance. © 2021 Society of Chemical Industry.

Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.

Treatment of intramuscular lipoma of tongue with enveloped mucosal flap design: a case report and review of the literature.

BACKGROUND: Lipomas are benign soft tissue neoplasms of mature adipose tissue commonly occurring in the trunk or extremities. But, intraoral lipomas are rare entities which may be only noticed during routine dental examinations. Especially intramuscular lipomas on the tongue have been reported very rarely. In this study, we report a case of intramuscular lipoma on tongue, with a review of the literature from 1978 to 2019, providing data on age, gender, location, presenting symptoms, size, surgical methods, and recurrence. CASE PRESENTATION: A case of intramuscular lipoma occurring in tongue region in a 65-year-old male is reported. Surgical excision is the mainstay of treatment for the lesion. In order to decrease the deformity and discomfort after the excision, we tried to modify surgical technique using enveloped mucosal flap. This technique provided more comfortable healing procedure on the operative site without recurrence. CONCLUSION: This is a rare case of large intramuscular lipoma on tongue. Surgical excision with enveloped mucosal flap design was performed to diminish postoperative raw surface and discomfort and a 24-month follow-up showed excellent healing without any recurrence. A case of intramuscular lipoma on tongue and relevant literature reviews are presented in this study.

Dispersion-Solvent Control of Ionomer Aggregation in a Polymer Electrolyte Membrane Fuel Cell.

In this study, we examined the influence of the dispersion solvent in three dipropylene-glycol/water (DPG/water) mixtures, with DPG contents of 0, 50, and 100 wt%, on ionomer morphology and distribution, using dynamic light scattering (DLS) and molecular-dynamics (MD) simulation techniques. The DLS results reveal that Nafion-ionomer aggregation increases with decreasing DPG content of the solvent. Increasing the proportion of water in the solvent also led to a gradual decrease in the radius of gyration (Rg) of the Nafion ionomer due to its strong backbone hydrophobicity. Correspondingly, MD simulations predict Nafion-ionomer solvation energies of -147 ± 9 kcal/mol in water, -216 ± 21 kcal/mol in the DPG/water mixture, and -444 ± 9 kcal/mol in DPG. These results suggest that higher water contents in mixed DPG/water solvents result in increased Nafion-ionomer aggregation and the subsequent deterioration of its uniform dispersion in the solvent. Moreover, radial distribution functions (RDFs) reveal that the (-CF2CF2-) backbones of the Nafion ionomer are primarily enclosed by DPG molecules, whereas the sulfonate groups (SO3-) of its side chains mostly interact with water molecules.

Tuning the Ionomer Distribution in the Fuel Cell Catalyst Layer with Scaling the Ionomer Aggregate Size in Dispersion.

With the demands for better performance of polymer electrolyte membrane fuel cells, studies on controlling the distribution of ionomers have recently gained interest. Here, we present a tunable ionomer distribution in the catalyst layer (CL) with dipropylene glycol (DPG) and water mixtures as the ionomer dispersion medium. Dynamic light scattering and molecular dynamics simulation demonstrate that, by increasing the DPG content in the dispersion, the size of the ionomer aggregates in the dispersion is exponentially reduced because of the higher affinity of DPG for Nafion ionomers. The ionomer distribution of the resulting CLs dictates the dimensional feature of the ionomer dispersion. Although the ionomer distribution becomes more uniform with increasing the DPG content, an optimal power performance is obtained at a DPG content of 50 wt % regardless of feed humidity because of balanced proton and mass transports. As a guide for tuning the ionomer distribution, we suggest that the ionomer aggregates in the dispersion with a size close to that of the Pt/C aggregates form a highly connected ionomer network and maintain a porosity in the catalyst/ionomer aggregate, resulting in high power performance.

In-Plane Channel-Structured Catalyst Layer for Polymer Electrolyte Membrane Fuel Cells.

In this study, we present a novel catalyst layer (CL) with in-plane flow channels to enhance the mass transports in polymer electrolyte membrane fuel cells. The CL with in-plane channels on its surface is fabricated by coating a CL slurry onto a surface-treated substrate with the inverse line pattern and transferring the dried CL from the substrate to a membrane. The membrane electrode assembly with the in-plane channel-patterned CL has superior power performances in high current densities compared with an unpatterned, flat CL, demonstrating a significant enhancement of the mass-transport property by the in-plane channels carved in the CL. The performance gain is more pronounced when the channel direction is perpendicular to the flow field direction, indicating that the in-plane channels increase the utilization of the CL under the rib area. An oxygen-transport resistance analysis shows that both molecular and Knudsen diffusion can be facilitated with the introduction of the in-plane channels. The direct CL patterning technique provides a platform for the fabrication of advanced CL structures with a high structural fidelity and design flexibility and a rational guideline for designing high-performance CLs.

Three-Dimensional Interlocking Interface: Mechanical Nanofastener for High Interfacial Robustness of Polymer Electrolyte Membrane Fuel Cells.

A scalable nanofastener featuring a 3D interlocked interfacial structure between the hydrocarbon membrane and perfluorinated sulfonic acid based catalyst layer is presented to overcome the interfacial issue of hydrocarbon membrane based polymer electrolyte membrane fuel cells. The nanofastener-introduced membrane electrode assembly (MEA) withstands more than 3000 humidity cycles, which is 20 times higher durability than that of MEA without nanofastener.