Alleviating effect of topical Paenibacillus polymyxa on Malassezia restricta-induced dermatitis by modulating immune response and skin microbiota.

Spore-forming bacteria are widely distributed in the environment and present a major challenge in healthcare settings due to their resistance to alcohol-based disinfectants. Understanding this resistance is essential for infection control education, especially in medical fields. Here, we propose a simple, low-cost laboratory exercise that combines hand hygiene training with a visual demonstration of microbial persistence using soil as a source of spore-forming bacteria. In this exercise, students imprint their hands on agar plates under three conditions: unwashed, soap-washed, and alcohol-disinfected. After 48 h of incubation, the plates reveal clear differences in microbial growth, highlighting the limited efficacy of alcohol-based sanitizers against spores. Gram staining of colonies provides presumptive evidence of the presence of spore-forming bacteria. This exercise addresses a common misconception among healthcare trainees-that alcohol sanitizers provide complete protection-and emphasizes the importance of proper handwashing with soap and water. Its simplicity, safety, and strong visual impact make it an effective and adaptable tool for microbiology education and healthcare professional training.

Introduction. The production of Taiwanese guava in Paquera is constrained by the fungus Pestalotiopsis sp., which causes fruit lesions that reduce quality. Although fungicides are applied for disease management, information on their efficacy remains limited. Objective. To evaluate the in-vitro efficacy of fungicides against Pestalotiopsis sp. associated with Taiwanese guava (Psidium guajava L.) cv. Tai-kuo-bar in the district of Paquera, Puntarenas. Materials and methods. Symptomatic fruits were collected, and fungal isolates were obtained on culture medium and identified morphologically at the genus level. ITS gene sequences were generated and compared against the NCBI (National Center for Biotechnology Information) database. In-vitro efficacy was assessed by inoculating the fungus onto potato dextrose agar (PDA) plates amended with fungicides at commercial dose, 50%, and 10% of the commercial dose. The percentage reduction in mycelial growth relative to the untreated control was calculated. Results and discussion. Pestalotiopsis sp. was confirmed to be associated with fruit lesions. ITS region sequences showed 100% identity with Neopestalotiopsis saprophytica, N. foedans, N. clavispora, and Pestalotiopsis versicolor from the NCBI database. Mycelial growth inhibition was greater than 90% when azoxystrobin, propineb, prochloraz, and difenoconazole were used at the commercial dose. In contrast, copper sulfate pentahydrate and carbendazim showed inhibition of 76.1% and 12.6%, respectively. Conclusions. Azoxystrobin, propineb, prochloraz, and difenoconazole demonstrated high potential for chemical control of Pestalotiopsis. The low efficacy of carbendazim suggests possible resistance in some isolates. Further studies are required to clarify the diversity of Pestalotiopsis species in the region.

Background: Dental caries remains one of the most prevalent oral diseases worldwide, largely driven by the virulence of Streptococcus mutans. Although plant phenolics from green tea and pomegranate are known for their antimicrobial properties, their molecular mechanisms of action against key S. mutans virulence targets remain insufficiently characterized. Aim: This study investigated the antibacterial and anti-virulence properties of Viroelixir, a phenolic-rich formulation derived from green tea (Camellia sinensis) and pomegranate (Punica granatum), against S. mutans, with particular emphasis on predictive molecular docking interactions with critical virulence-associated proteins. Methods: Viroelixir phytochemical composition was characterized by LC-MS using a C18 reverse-phase column and negative electrospray ionization mode. Antibacterial activity was evaluated using growth kinetics, agar plating, and crystal violet assays. Acidogenicity, hemolytic activity, and biofilm formation were assessed using pH modulation, hemolysis assays, SEM, and biofilm biomass quantification. Virulence gene expression was analyzed by RT-qPCR. In silico molecular docking was performed to explore potential interactions between major LC-MS-supported phenolic constituents and S. mutans virulence proteins, including glucosyltransferase B (GtfB), LuxS, and SpaP. Biocompatibility was evaluated in human gingival epithelial cells. Results: The LC-MS analysis revealed a complex mixture of phenolic compounds consistent with catechins and ellagitannins. Compound identification was considered tentative and based on mass spectral range and chromatographic behavior. Viroelixir significantly inhibited S. mutans growth, acid production, hemolytic activity, and biofilm formation in a concentration-dependent manner. Key virulence genes were markedly downregulated. Docking analyses suggested stable binding of selected phenolics-particularly punicalagin, catechin, and epigallocatechin-within the active sites of GtfB, LuxS, and SpaP. Importantly, Viroelixir showed no cytotoxic effects on gingival epithelial cells. Conclusions: Viroelixir exerts potent antibacterial and anti-virulence effects against S. mutans through a multi-target mechanism combining transcriptional suppression and predictive molecular inhibition of virulence proteins, supporting its potential as a safe, natural therapeutic for caries prevention.

Genomic characterization of a vancomycin-intermediate Staphylococcus aureus (VISA) small colony variant after long-term antibiotic therapy.

Atraphaxis bracteata is an important sand-fixing shrub in desert regions. In August 2024, an epidemic of leaf spot disease on A. bracteata was observed in Shapotou District, Zhongwei City, Ningxia, China (37°27′ N, 104°57′ E, altitude approx. 1250 m). Disease surveys, conducted on 100 randomly selected plants, revealed a high incidence of ~75%. Infected plants initially developed small yellow spots on leaves, which subsequently expanded into irregular necrotic lesions with yellow margins and dark brown centers. Severe infections led to premature leaf drop. Tissue sections from the lesion margins were surface-sterilized with 75% ethanol for 30 s, soaked in 1% NaClO solution for 1 min, rinsed three times with sterile distilled water, air-dried, and then placed on Potato Dextrose Agar (PDA) at 25°C for 3-7 days. A Botryosphaeria-like fungus was consistently isolated from all samples. Pure cultures were obtained via single-spore isolation (Cai et al., 2009), where individual spores from a diluted suspension were germinated on agar plates. Colony morphology was observed after 5 days of incubation, and spore morphology was examined after 14 days. Colonies transitioned from white to greyish-black, reaching 85-90 mm diameter. Hyphae were septate, branched, hyaline initially, turning dark brown with age. Pycnidia produced brown, elliptical, aseptate conidia [(9-18) × (2-8) μm; mean ± SD = 15.2 ± 1.8 × 6.3 ± 0.9 μm; n = 30]. The partial internal transcribed spacer region (ITS1), the partial β-tubulin gene (TUB2), and the partial translation elongation factor (TEF1-α) gene were amplified from pure cultures of isolates YFZ1 and YFZ2 (Espinoza-Lozano et al., 2023). The primer pairs used were ITS1 / ITS4 (White et al., 1990), Bt2a / Bt2b (Glass et al., 1995), and EF1-728F / EF1-986R (Carbone et al., 1999), respectively. Sequences were deposited in GenBank (YFZ1: ITS PV926689, TUB2 PX434322, TEF1-α PX434323; YFZ2: ITS PV926690, TUB2 PX434324, TEF1-α PX434325). BLASTn analysis revealed that the ITS, tub2, and tef1 sequences of both isolates shared over 99% homology with the corresponding sequences of the Botryosphaeria dothidea ex-type strain (e.g., CBS 110302 and CMW8000). A maximum likelihood phylogenetic tree was reconstructed based on the concatenated ITS-TEF1-TUB2 sequences using MEGA v.7 under the K2+G evolutionary model (selected by ModelTest) with 1000 bootstrap replicates. The analysis confirmed that both isolates YFZ1 and YFZ2 clustered within a well-supported clade comprising reference strains of Botryosphaeria dothidea, with Macrophomina phaseolina CBS 227.33 (ex-type strain) designated as the outgroup. Pathogenicity tests were conducted by spraying healthy A. bracteata leaves inoculated with 10 μL of spore suspension (106 /mL). Control plants were sprayed with sterile water. All potted plants were individually covered with transparent polyethylene bags to maintain high humidity and incubated in a greenhouse at 23-29 °C. Fourteen days post-inoculation, typical lesions consistent with field symptoms appeared on treated leaves, while control leaves remained asymptomatic. Fungal isolates re-isolated from the diseased leaves exhibited identical morphological characteristics and molecular identification results to the original inoculum strains, fulfilling Koch's postulates. This study provides the first confirmation of B. dothidea’s pathogenicity on A. bracteata in China, aiding diagnosis and management of this emerging disease.

Blueberry (Vaccinium corymbosum) is a temperate-climate fruit valued for its flavor and high content of bioactive compounds. Anthracnose, caused by Colletotrichum spp., is considered one of the most important diseases limiting blueberry yields (Liu et al. 2020). In Brazil, blueberry cultivation has expanded from the southern to the southeastern region. To date, two species, C. karsti and C. chrysophilum, have previously been associated with blueberry anthracnose in the country (Rios et al. 2015, Soares et al. 2022). In November 2024, an anthracnose epidemic was observed in an organic commercial blueberry orchard of the cultivar ‘Emerald’ (Piracicaba, SP, Brazil; 22°45′02.5″S, 47°31′59″W), where approximately 80% of the 2000 plants evaluated in a 0.6 ha production area were symptomatic, showing twig blight, anthracnose fruit rot, and predominantly black leaf spots. Acervuli without setae, bearing conidia masses, were collected and microscopically observed in leaf sections slides. These structures were carefully collected with a needle and transferred to Potato Dextrose Agar (PDA) plates. A single-spore Colletotrichum sp. isolate (LFN0500) was obtained, on PDA, and 15-day-old colonies colors were grayish white, with the reverse pale orange. Conidia (n = 100) were cylindrical with rounded ends, measuring 11.13 – 13.87 × 3.63 – 4.47 µm (mean 12.25 × 4.04 µm). DNA was extracted from aerial mycelium using the Wizard® Genomic DNA purification kit (Promega), following the manufacturer’s instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin 2 (TUB2), rDNA transcribed spacer region (ITS), and actin (ACT) regions were amplified using the primer pairs GDF/GDR (Guerber et al. 2003), Bt2a/Bt2b (Glass and Donaldson 1995), ITS-1F/ITS-4 (Gardes and Bruns 1993), ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. Amplicon sequences were deposited in GenBank (accession numbers PX461595 (ITS), PX531255 (ACT), PX531256 (TUB2), PX531254 (GAPDH)) and showed highest similarity (100%) with Colletotrichum siamense, a member of the Colletotrichum gloeosporioides species complex. Phylogenetic analysis placed isolate LFN0500 with C. siamense reference strain (ICMP 18578) (posterior probability = 0.96). To confirm pathogenicity, 6-months-old blueberry potted plants (cv. Emerald, n = 4) and detached fruits (average diameter, 15 mm, n = 6) were spray inoculated with a conidial suspension (1 × 10⁶ conidia/mL), incubated at 25 ± 5 °C in the dark for 48 hours, and then transferred to a growth chamber. The experiment was repeated twice. Blueberry potted plants exhibited typical black leaf spots 13 days after inoculation, whereas the negative controls (n = 4), sprayed with distilled water, remained asymptomatic. Anthracnose rot appeared on detached fruits 3 days after inoculation, with no symptoms observed on negative controls (n = 6). The pathogen was reisolated only from the edge of symptomatic tissues originated in both fruits and leaves, but not from asymptomatic negative controls, and its identity was confirmed through a new sequencing of the same gene regions previously analyzed. To our knowledge, C. siamense has previously been associated with blueberry in the United States and China (Liu et al. 2020; Talhinhas and Baroncelli 2023). The identification of C. siamense in this study expands the known diversity of Colletotrichum spp. affecting blueberry in Brazil and provides useful information for disease management in the country.

Ligustrum japonicum (Japanese privet) is an evergreen shrub native to East Asia. It is widely cultivated as an ornamental plant. Since September 2023, a necrotic leaf spot outbreak on L. japonicum was observed in Baihe Wetland Park (32°5'42"N, 112°28'13"E) in Nanyang, China. The disease caused sporadic and irregularly shaped brown lesions, with chlorotic halo and dark margin on leaves, and disease incidence was up to 29% among 120 plants surveyed. As the disease progressed, leaf lesions enlarged, became slightly sunken and dark brown, and sometimes coalesced. In severe cases, necrosis extended from leaves into adjacent shoots. To isolate the pathogen, small tissue pieces excised from the margin of necrotic lesions on symptomatic leaves were surface sterilized by immersing in 75% ethanol for 30 s followed by 1% NaClO for 1 min, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 3 days. A total of 22 fungal isolates were obtained. Three representative isolates (Bd1, Bd6, and Bd17) sampled at separate sites were selected for further investigation. On PDA, the colonies were white at first and gradually turned dark gray with irregular margins within 7 days at 25°C. Conidia were hyaline, aseptate, fusiform, measuring 19.6-24.4 × 4.4-8.6 μm (n = 100). These morphological characteristics were connected to Botryosphaeriaceae fungi (Slippers et al. 2014). For molecular identification, the rDNA internal transcribed spacer (ITS), 28S large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha gene (tef1-α), and β-tubulin genes (tub) were amplified using primers ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), EF1-688F/EF1-1251R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were deposited in GenBank under accession numbers OR922737, OR976075, OR976076 (ITS), OR977584, OR977713, OR978107 (LSU), OR993993 to OR993995 (tef1-α), OR947471, OR993991, OR993992 (tub). A phylogenetic tree was constructed using neighbor-joining method based on concatenated ITS, LSU, tef1-α, and tub sequences, together with sequences from type or reference strains. The three isolates clustered closely with the reference strains CBS 115476 and CBS 110302 of Botryosphaeria dothidea in phylogenetic analysis and were clearly separated from other Botryosphaeria species. The molecular analyses confirmed that the isolates are B. dothidea (Slippers et al. 2014). Pathogenicity experiments were performed on healthy L. japonicum seedlings (three-month-old, 60 to 80 cm tall) using conidial suspensions (106 conidia ml-1) as inoculum. For each isolate (Bd1, Bd6, and Bd17), the conidial suspension was sprayed onto the aerial parts (leaves and young shoots) of 10 seedlings. Control plants were sprayed with sterile distilled water. All plants were maintained in controlled-environment chambers at 25°C and 90% relative humidity under a 12 h light/12 h dark photoperiod. Initial symptoms on leaves were first observed at 5 days post inoculation. At 10 dpi, inoculated seedlings developed necrotic brown lesions mainly at the petiole bases and leaf margins. By 21 dpi most affected leaves had abscised, and some lesions occurred on young shoots leading to partial shoot dieback. All inoculated seedlings developed symptoms (30/30 total), whereas control plants remained symptomless. The three isolates caused similar symptoms with comparable disease incidence. B. dothidea was consistently reisolated from symptomatic leaves and shoots, thereby fulfilling Koch’s postulates. B. dothidea is a well-known pathogen that causes diseases in various plants, including Camellia oleifera (Hao et al. 2023), Quercus dentata (Liu et al. 2023), and Cornus officinalis (Zhang et al. 2023). This is the first report of B. dothidea as the foliar disease causal agent on L. japonicum in China. Identifying this pathogen contributes to a better understanding of the disease affecting L. japonicum in this region and provides a basis for future disease management strategies.

This study investigated the antifungal performance of copper-based antimicrobial coatings developed by Gencoa Ltd., previously validated against bacterial ESKAPE pathogens, alongside newly formulated titanium oxide coatings, against key agricultural fungal pathogens: Alternaria alternata, Botrytis cinerea, Cladosporium cucumerinum, and Fusarium oxysporum. Testing was conducted both in vitro and in field trials within an actively used polytunnel. In vitro assays included a modified ISO 846 agar plate protocol and a six-well plate fungal colonization assay simulating high humidity conditions. Field trials assessed coating performance under real-world exposure. Copper-containing coatings: pure copper, copper oxynitride, and copper-doped titanium oxide, consistently demonstrated significant antifungal activity, effectively reducing spore germination and colonization. Titanium oxide coatings without copper showed minimal effect, performing similarly to uncoated polyethylene. While copper-based coatings were highly effective, some susceptibility to surface degradation under prolonged moisture was observed. However, antifungal activity often persisted in degraded areas of samples with high copper content. Copper-based antimicrobial coatings offer strong potential for preventing fungal colonization on agricultural surfaces, outperforming titanium oxide formulations under both laboratory and field conditions. Optimization to enhance durability will further improve their suitability for long-term use in protected cultivation systems.

Virtual reality (VR) devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections. The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing. This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility. The external components and internal facial interface were swabbed and separately cultured on four AGAR plates (Horse Blood, Nutrient, bile Esculin, and Mannitol Salt). Colonies were counted, sampled, pooled and subsequently processed for shotgun metagenomic sequencing. A higher number of colonies were present on surfaces closest to the eyes and facial interface compared to the external components. Metagenomic analysis identified 27 pathogenic bacteria including 4 "ESKAPE" pathogens (Enterobacter sp., Staphylococcus aureus, Klebsiella spp. and, Escherichia coli) and numerous organisms associated with ocular infections. A broad range of antimicrobial resistance genes were identified conveying resistance to Methicillin, Aminoglycosides, Macrolides, Tetracyclines, and Polymixins. Further research is required to ensure that current sanitization practices of VR head mounted displays are appropriate within high-risk hospital settings.

Introduction: Indirect Pulp Capping (IPC) is defined as a procedure wherein a small amount of carious dentin is retained in deep areas of the cavity to avoid exposure of pulp, followed by placement of a suitable medicament and restorative material that seals off the carious dentin and encourages pulp recovery. Calcium hydroxide has been the gold standard for pulp capping material due to its exceptional biocompatibility, antibacterial properties, and ability to encourage the formation of reparative dentin. Fish Bone (FB) powder is a naturally occurring substance that has shown promise as a pulp capping material. It contains a lot of calcium, phosphates, and other bioactive substances that could help with dentinogenesis and repair. To ascertain whether it can be a suitable substitute for or addition to calcium hydroxide, its antimicrobial activity and sealing capacity must be thoroughly assessed. Need of the study: The current study can demonstrate the antimicrobial effectiveness and sealing ability of FB paste as a pulp capping material. Aim: To evaluate and compare the anti-microbial efficacy and sealing ability of FB paste with calcium hydroxide paste as pulp capping materials through an in-vitro study. Materials and Methods: An in-vitro study will be conducted in Department of Paediatric and Preventive Dentistry at Sharad Pawar Dental College, Wardha, Maharashtra, India, from May 2025 to December 2025, in which FB paste and calcium hydroxide paste will be prepared to evaluate and compare the anti-microbial efficacy and sealing ability for an in-vitro investigation in the Department of Paediatric and Preventive Dentistry at Sharad Pawar Dental College, Wardha, Maharashtra, India. The agar diffusion method will be used to evaluate the antibacterial efficacy of calcium hydroxide paste and FB paste against Enterococcus faecalis. After being cultivated in brain-heart infusion broth for the entire night, the E. faecalis stock culture will be injected onto Muller-Hinton agar plates. A copper puncher will be used to create wells on the plates that are 4 mm in diameter and 4 mm deep. The wells will then be promptly filled with freshly created, specially designed test materials. After being incubated at 37°C for 24 and 72 hours, the agar plates will be evaluated. The diameter of the zone of inhibition will be measured in millimetres after incubation. The dye penetration method will be used to evaluate the sealing ability of calcium hydroxide paste and FB paste. After preparing a Class I cavity, pulp capping material consisting of calcium hydroxide and powdered FBs will be inserted into the cavity. All samples will undergo 500 thermocycling cycles with 60-second dwell intervals to replicate dental conditions, and each tooth will receive two coats of nail polish with a 1 mm margin around the restoration border. Following a 24-hour incubation period at room temperature in 5% methylene blue dye, the samples will be rinsed under running water and then sectioned mesiodistally using a diamond disk with water cooling. To assess the material's capacity to seal, the degree of dye penetration will be evaluated. The Chi-square test, student paired and unpaired t-test, One-way Analysis of Variance (ANOVA), and Tukey test will all be used in the statistical analysis.

Novel diethylthiosulfinate suppresses postharvest peach brown rot in vitro and vivo by disrupting carbon metabolism and virulence pathways in Monilinia fructicola.

Strongyloides stercoralis in type 2 diabetes mellitus: discordant stool and serologic findings in an endemic Brazilian population.

The overuse of antibiotics in healthcare has accelerated the emergence of antimicrobial resistance (AMR) and the development of multi drug-resistant “superbugs” worldwide. The study aimed to induce resistance in standard ATCC Escherichia coli strain by fixed concentration of different antimicrobial groups through repeated exposure in our institutional laboratory. E. coli, ATCC 25922 strain which was initially susceptible to antibiotics was serially exposed to five groups of antibiotics (ampicillin, co-trimoxazole, tetracycline, gentamicin, and amoxicillin–clavulanate) over 60 daily passages. Antibiotic susceptibility testing (AST) was performed by Kirby–Bauer disk diffusion method on Müller-Hinton agar with a standardised inoculum (~10^6 CFU). On day 0, a Muller Hinton Agar (MHA) plate was inoculated with the broth and mentioned antibiotic disks were placed for zone diameter estimation. Thereafter, for each next passage, colonies from the edge of the inhibition zone for each drug were picked and inoculated on fresh MHA plates and were further rechallenged with the same antibiotic discs. Inhibition zone diameters were measured after overnight incubation at 37°C. On day 0, E. coli was fully susceptible to all five antibiotics. After 60 serial passages, the E. coli population exposed to ampicillin developed resistance, with the inhibition zone decreasing from 19 mm initially to resistant zone of 6 mm. This work provides insights into the mechanisms by which E. coli adapts to sustained antibiotic pressure and offers a basis for developing strategies to prevent the rise of multi drug-resistant bacteria.

The use of natural compounds that are extracted from plants is currently gaining attention as an alternative means of treating bacterial infections with antimicrobial agents that are synthetically produced. One plant that has recently garnered scientific attention is the chayote plant (Sechium edule) and the substances that naturally exist within its fruits. The antibacterial potential of the crude 95% ethanolic extract that is prepared from the fruits of the chayote plant has been tested against two bacterial species: Staphylococcus aureus and Escherichia coli, using the Kirby-Bauer disc diffusion method. The aim of this study was to determine if varying concentrations of the crude 95% ethanolic extract from the fruits of Sechium edule would have inhibitory effects upon these bacterial species. The fruits of the chayote plant were firstly washed, dried, and ground into powder. This powdered form of the plants’ fruits was soaked in 95% ethanol for three days in order to extract the active compounds from the plant – the maceration method. The resulting solution was prepared into four different concentrations of the crude ethanolic extract: 25%, 50%, 75%, and 100%. These concentrations of the crude ethanolic extract were placed onto nutrient agar plates that were seeded with either S. aureus or E. coli bacterial suspensions. Each of these seeded agar plates was incubated for 24 hours at 37 °C. The diameter of the zones of inhibition for each of these bacterial species was determined after incubation. Erythromycin was used as the positive control for cultures of S. aureus at a concentration of 15 µg discs, whereas ciprofloxacin was used as the positive control for cultures of E. coli at a concentration of 5 µg discs. A negative control was prepared with 95% ethanol. Antimicrobial activity was only visible with the highest concentration of the 95% ethanolic extract from the fruits of the chayote plant. At the highest concentration of 100% crude ethanolic extract, inhibition zones of 8.33 mm were achieved against both S. aureus and E. coli. For the S. aureus bacteria, inhibition zones at the 75%, 50%, and 25% concentrations of the ethanolic extract were 8, 8, 9 mm for E. coli; 9, 8, and 8 mm for S. aureus, respectively. No antibacterial activity was visible at the 25%, 50%, and 75% concentrations of the ethanolic extract against either bacterial species, and no antibacterial activity was visible in the negative control preparation with the crude ethanolic extract preparation of S. edule fruits’ fruits. These results indicate that the crude ethanolic extract preparation from the fruits of the chayote plant does contain antibacterial activity against both Gram-positive and Gram-negative bacteria. However, the zone of antibacterial activity associated with the extract is weaker than that of the positive control antimicrobial agents. Therefore, additional studies can be performed in relation to this preparation of antibacterial compound from S. edule fruits, such as determining its minimum inhibitory concentration (MIC), isolating the active compounds from the fruits that contain those antibacterial properties, and testing the antibacterial properties of this natural compound.

Leymus chinensis is a dominant plant in the Eurasian steppe and plays a crucial role in ecological protection and restoration due to its functions in sand fixation, soil conservation, and ecosystem stabilization (Liu et al. 2022). In August 2024, leaf spot disease was first observed on L. chinensis in a grassland of Abaga Banner, Xilinhot City, Inner Mongolia, China (43°54′47″N, 115°34′1″E). Investigations covering 0.25 hectares revealed that the disease incidence stood at 15%. Initial symptoms appeared as elliptical spots with brown edges and grayish-white centers. These spots gradually expanded into elongated lesions at later stages, eventually causing leaf wilting and, in severe cases, plant death. Thirty leaves displaying typical leaf spot symptoms were collected for pathogen isolation. Tissue pieces (5 mm×5 mm) from lesion margins were surface-sterilized (70% ethanol, 20 s; 1% NaClO, 2 min), rinsed three times with sterile distilled water, and placed on PDA. The plates were incubated at 25 ℃ in the dark for 5 days. Hyphal tips from actively growing colonies were picked and transferred onto fresh potato dextrose agar (PDA) plates for purification, yielding 10 isolates with consistent morphological characteristics. All isolates formed gray, circular colonies with gray edges, and the reverse sides of the culture plates showed gray pigmentation.Two representative isolates, YH1 and YH2, were selected for detailed characterization. Conidia were clavate to drum-shaped, measuring 10.85-34.96 × 5.86-13.76 μm (n=30), with 3-5 transverse septa, 0-2 longitudinal septa. These morphological characteristics were consistent with descriptions of Alternaria species (Simmons 2007). For molecular identification, genomic DNA of isolates YH1 and YH2 was extracted using a commercial kit (Aidlab Novel Plant Genomic DNA Extraction Kit). The internal transcribed spacer (ITS) region, translation elongation factor (TEF1-α) gene, and Alternaria major allergen (Alt a 1) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), Ef728M/Tef1R (Stępień et al. 2012), and Alt-for/Alt-rev (Woudenberg et al. 2015), respectively. The obtained sequences were deposited in GenBank under accession numbers: ITS (PX644766, PX644767), TEF1-α (PX653172, PX653174), and Alt a 1 (PX653171, PX653173). BLASTn analysis revealed 100% (ITS), 99.73% (TEF1-α), and 100% (Alt a 1) sequence similarity with Alternaria alternata. A phylogenetic analysis based on the combined dataset of the three genes placed isolates YH1 and YH2 within the A. alternata clade. Pathogenicity tests were conducted by spraying leaves of fifteen healthy L. chinensis plants (three plants per pot, five pots total) with a conidial suspension (1×10⁶ conidia/mL, containing 0.2% Tween 20) until runoff. Fifteen control plants were sprayed with sterile water containing 0.2% Tween 20. All plants were maintained in a greenhouse at 25 ℃, 80% relative humidity, under a 12 h light/dark cycle. The experiment was repeated twice. Ten days post-inoculation, symptoms identical to those observed in the field developed on inoculated leaves, while control plants remained symptomless. The fungus re-isolated from the lesions was confirmed as A. alternata based on morphology and molecular analysis, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of A. alternata infecting L. chinensis in China. This study provides essential information for the diagnosis, understanding of disease epidemiology, and development of control strategies for this newly identified disease.

Air-borne mycoflora significantly influences the environment and human health due to its ability to spread vigorously through spores. These spores can act as allergens and may produce mycotoxins which can be injurious to human health. This study evaluates the diversity of fungal species beyond different polluted areas of Karachi, Pakistan. Research conducted from September to December 2025 which explored air borne variations detected at ten different localities by using the agar plate method. Statistical analysis using ANOVA was also applied to check the significant difference in fungal variation and abundance among selected localities. The result showcased the clear dominance of Aspergillus niger throughout study period, with its consistent and widespread instance across various sites.This indicates its ecological adaptability and dominance. On the other hand Alternaria solani prevails during the winter season (November to December) showing seasonal variation in distribution. Other pinpointed fungal genera include Aspergilus fumigatus, Aspergillus flavus, Aspergillus terreus, Penicillium spp., Curvularia verruculosa, Curvularia affins, Drechselera musaesapientum, Drechselera (state of Cochliobolus sativus), Alternaria raphani, Alternaria alternata. This study provides important insights into the fungal ecology of polluted urban environments of Karachi which can enhance the knowledge of airborne fungal communities and their impact on human health and environment.

Triadica sebifera (L.) Small (Euphorbiaceae), commonly known as the Chinese tallow tree, is an endemic deciduous species of significant economic importance in China (Gao et al. 2016). In October 2023, leaf spot disease was observed on T. sebifera in the Longquan Mountain planting area (30.56°N, 104.27°E), Sichuan Province. Field surveys were performed across three representative planting plots, where a total of 150 trees were randomly examined (50 trees per plot). The mean disease incidence was approximately 83%. Initial symptoms included brown to dark brown lesions on leaves, progressing from small, oval to irregular spots (1–3 mm in diameter) to coalesced large necrotic patches with distinct margins. To isolate the causal agent of the disease, infected tissues were collected from 10 diseased leaves, with each leaf sampled from an individual tree. These infected tissues were cut into 5 × 5 mm pieces, and 3 such tissue pieces were plated per 90 mm-diameter potato dextrose agar (PDA) plate. The infected tissues were surface-disinfected with 75% ethanol and 3% sodium hypochlorite for 60 s and 30 s, respectively (Chen et al. 2025), and rinsed three times in sterile water. Subsequently, the tissues were blot-dried with autoclaved filter paper and cultured on PDA amended with streptomycin sulfate (50 μg/mL), and incubated at 25 ± 2 ℃ in the dark for 8 days. A hyphal tip was taken from the edge of the emerging fungal colony and transferred onto fresh PDA plates for purification. A total of nine fungal isolates were recovered from these ten diseased leaves, seven of which (WJ-1 to WJ-7) exhibited consistent morphological characteristics and were selected as representative strains. For morphological observation and conidiation induction, the purified strains were cultured on PDA at 25 ± 2 ℃ under a 12 h light/12 h dark photoperiod for 8 days. Their colony diameters ranged from 68.9 to 76.9 mm (n=7). The colonies on PDA initially exhibited white, cottony mycelium with undulate margins and concentric rings. Black conidial masses formed on the mycelium. Conidia were fusoid, slightly curved, five-celled with four septa and three dark brown median cells; a total of 50 mature conidia were measured, with an average size of 25.6 × 8.8 μm and a range of 22.3–28.9 × 7.5–9.7 μm. The apical cells were hyaline and conical, bearing 2-3 tubular appendages, while the basal cells were hyaline, conical, and tuberculate with a single central appendage. These morphological traits aligned with Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). For molecular identification, the genomic DNA of a representative isolate (WJ-1) was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The ITS, TEF1-α, and TUB2 regions were amplified and sequenced. The primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995) were used to amplify ITS, TEF1-α, and TUB2, respectively. The sequences were concatenated and analyzed together with morphological characteristics to determine the species identity of the isolate (Shu et al. 2020). The sequences were deposited in GenBank (accession nos. PQ222635 [ITS], PV686787 [TUB2], PV730094 [TEF1-α]). BLASTn analysis revealed 98 - 100% identity with N. ellipsospora reference strains (PP838333.1, MT044311.1, OK594056.1). In this study, we used Pestalotiopsis arengae as an outgroup to construct a multigene phylogenetic tree based on the maximum likelihood method and OFPT (Zeng et al. 2023) software, and the isolate clustered in the N. ellipsospora clade with a bootstrap support greater than 90%. The fungi isolated from the diseased T. sebifera leaves were confirmed as N. ellipsospora using morphological and three-gene sequence analysis. Subsequently, pathogenicity testing was conducted by inoculating leaves of ten T. sebifera trees under field conditions. Ten healthy leaves per tree were surface-sterilized with 75% ethanol. No wounding was performed before inoculation. A conidial suspension of the WJ-1 isolate (1 × 10⁷ conidia/mL) was sprayed onto the leaves, with sterile water serving as the control. Transparent plastic bags were used to maintain relative humidity. After 15 days (25 ± 2 ℃, 14 h light/10 h dark), disease severity was assessed qualitatively by observing lesion appearance and expansion. Inoculated leaves developed symptoms similar to those observed on naturally diseased plants, whereas the control leaves remained asymptomatic. N. ellipsospora was re-isolated from the artificially infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity tests were repeated three times with similar results, confirming Koch’s postulates. To our knowledge, this is the first report of N. ellipsospora infecting T. sebifera globally. This study provides a reference for future research on T. sebifera leaf spot disease management, based on the identification of its causal agent.

Erythromycin-resistant Bordetella pertussis (ERBP) has increased sharply in China, posing significant challenges to clinical treatment. The lack of a standardized antimicrobial susceptibility testing (AST) system for B. pertussis makes it difficult to screen out effective antibiotics, which further aggravates the generation of drug-resistant strains and causes a vicious cycle. In this study, we modified a superior charcoal-free medium for antibiotic susceptibility testing, enabling more reliable evaluation of antibiotic efficacy. The minimum inhibitory concentrations (MICs) of erythromycin (E), trimethoprim-sulfamethoxazole (TMP-SMX), levofloxacin (LVF), and piperacillin (PIP) were determined using E-test strips on the agar plates with and without charcoal. The performance of these two media for AST was compared to establish a more accurate laboratory detection method. Subsequently, the superior charcoal-free medium was used to assess the antibiotic susceptibility of 98 B. pertussis strains isolated from clinical specimens after the COVID-19 pandemic, analyzing the sensitivity distribution of these first-line and alternative antibiotics. The MIC values of TMP-SMX and LVF were significantly higher on charcoal-containing agar plates than on charcoal-free medium. E-test results revealed that the MICs of B. pertussis strains to TMP-SMX and LVF had increased by approximately five gradient levels on the charcoal-containing agar compared to charcoal-free agar. Quality control (QC) testing demonstrated that the MICs of TMP/SMX and LVX fell outside the acceptable QC range due to adsorption by charcoal particles. Antimicrobial susceptibility of 98 B. pertussis clinical isolates showed that all strains were resistant to E with the MIC90 exceeded the maximum value (> 256 μg/mL). The overall MIC90 values for TMP-SMX, LVF and PIP were 1/0.75/<0.016 μg/mL on charcoal-containing agar and 0.023/0.008/<0.016 μg/mL on charcoal-free medium, respectively. Statistical analysis of violin plots confirmed that the MIC values of the second-line antibiotic TMP-SMX and LVF were significantly elevated on charcoal-containing agar. An accurate AST method can provide crucial guidance for optimal antibiotic selection in patients with B. pertussis infections. Our findings demonstrate that charcoal-free agar serves as an effective alternative medium for reliable AST of B. pertussis clinical isolates.

Cannabis-related chemistry is gradually becoming incorporated into the undergraduate curriculum, and laboratory experiments focusing on chemical analysis of cannabis products have appeared in this Journal; however, experiments focusing on the reactions and chemical synthesis of cannabinoids have yet to appear. We report a project-like, undergraduate organic chemistry teaching lab experiment in which students convert cannabidiol (CBD) to cannabinol (CBN) on a small scale using an experimentally straightforward, one-pot reaction. Students then establish the extent of conversion by TLC co-spotting their crude reaction mixture versus authentic samples of CBD and CBN. CBN is then isolated from the crude product mixture via a small-scale modification of dry column vacuum chromatography (DCVC), a convenient and rapid reduced-pressure alternative to flash column chromatography that employs common lab glassware. Finally, students qualitatively assess the antibacterial activities of CBD and CBN by evaluating inhibition of bacterial growth on nutrient agar plates, using cannabinoid samples they recrystallized from commercially available crude distillates.