Agar Surface Research Articles

Removal of heavy metal ions (Pb2+, Cu2+, Cr3+, and Cd2+) from multimetal simulated wastewater using 3-aminopropyl triethoxysilane grafted agar porous cryogel

In this study, we have developed agar, a seaweed derived polysaccharide based green adsorbent for the removal of heavy metal ions (Pb2+, Cu2+, Cr3+ and Cd2+) from multimetal solution. Porous cryogels of agar grafted with 3-aminopropyl triethoxysilane (APTES) were prepared by freeze-drying. The adsorption capacity and selectivity of the optimized APTES-agar cryogel for heavy metal ions (Cu2+, Cr3+, Pb2+, Cd2+) were investigated in multimetal solutions. >95 % of all the cationic metal ions were removed from 400 mg/L multimetal metal solutions having equal concentrations of each metal at pH 5.5. The experimental adsorption capacities of Cr3+, Cu2+, Pb2+, and, Cd2+ were changed from 39.14, 39.0, 39.20, 37.93 mg/g, to 52.58, 52.70, 45.53, 31.10 mg/g, respectively, for the 400 mg/L and 800 mg/L multimetal solutions suggesting competitive adsorption of the metal ions for active sites. The competitive adsorption studies showed that Cd ions had lower affinity than other metal ions for active sites on APTES grafted agar surface, and adsorption followed in the order of Cu2+ ≈ Cr3+ > Pb2+ > Cd2+. The developed seaweed-derived agar-based porous adsorbent exhibits promise in the removal of several heavy metal ions from wastewater, and this approach would increase the use of natural polysaccharides that are sustainable.

Enhanced antimicrobial efficacy of hydroxyapatite-based composites for healthcare applications

Hydroxyapatite (HAp) and hydroxyapatite-based materials show promising potential in the healthcare sector due to their distinctive properties such as biocompatibility, antimicrobial efficacy, non-toxicity, and robust mechanical characteristics. This makes HAp materials play an important role in hindering infection spreading in healthcare provider institutions. This study assesses the antimicrobial efficacy of the developed hydroxyapatite-based composites incorporating copper, zinc, and silver nanoparticles. The synthesized HAp and its modified composite variants (Cu/HAp, Zn/HAp, and Ag/HAp) with varying ratios ranging from 0 to 15% (wt) were characterized using XRD, XPS, SEM, and TEM analyses. Furthermore, the antibacterial and antifungal properties of the synthesized HAp and HAp-based composites were evaluated. The antibacterial effectiveness of the HAp and its composites was evaluated using a modified disc diffusion test, where the resulting inhibition zones on the agar surface were observed. All the HAp and HAp-based composites (HAp, Cu/HAp, Zn/HAp, and Ag/HAp materials) elicited in the formation of inhibitory zones. The most substantial inhibition values were observed for the 5% Ag/HAp formulation, with values of 19.7 and 13.8, against E. coli and S. aureus, respectively. The 5% Ag/HAp concentration may strike an ideal balance, providing high antimicrobial activity without adverse effects on biocompatibility or material stability. These findings underscore the recommendation of the proposed HAp-based composites for infection control measures through their application on medical instruments, textiles, healthcare personnel attire, and patient garments.

Differential gliding motility responses of Chryseobacterium sp. strain PMSZPI isolated from uranium ore deposit on hard and soft substrates

The Bacteroidota bacterium, Chryseobacterium sp. strain PMSZPI isolated from sub-surface soil of uranium ore deposit was shown to move on solid surfaces via gliding motility resulting in the formation of thin spreading colonies. In this study, we attempted to understand the influence of the surfaces, soft or hard/rigid, on the motility behaviour of PMSZPI cells. The computational tool T9GPred in combination with LC-MS/MS analysis established the presence of orthologs of vital gliding motility proteins in PMSZPI. We analyzed the single cell or population motility phenotypes of PMSZPI under spreading and non-spreading conditions. A low percentage of agar or soft agar (0.35%) with low nutrient levels induced more active gliding motility in individual cells leading to increased colony spreading. Microscopic analyses indicated the self-assembly of the gliding cells into irregular edged or spherical microcolonies based on the agar concentration. Cells moved at a speed of 0.2 to 0.6 µm s-1 on low-percentage gliding permissive agar (0.35%) surface in contrast to significant inhibition of motility on rigid or hard agar (1.5%) surface. RNA sequencing and real-time quantitative PCR (qPCR) analysis revealed increased expression of gliding motility genes under low agar conditions consistent with increased spreading behaviour. These findings provide the first glimpse into the gliding motility behaviour of a Bacteroidota bacterium from metal enriched environment that apparently could have implications on bacterial adaptation to changing surface environments.

Innovative Pathogen Reduction in Exported Sea Bass Through Atmospheric Cold Plasma Technology.

The safety of sea bass is critical for the global food trade. This study evaluated the effectiveness of atmospheric cold plasma in reducing food safety risks posed by Salmonella Enteritidis and Listeria monocytogenes, which can contaminate sea bass post harvest. Cold plasma was applied to inoculated sea bass for 2 to 18 min, achieving a maximum reduction of 1.43 log CFU/g for S. Enteritidis and 0.80 log CFU/g for L. monocytogenes at 18 min. Longer treatments resulted in greater reductions; however, odor and taste quality declined to a below average quality in samples treated for 12 min or longer. Plasma treatment did not significantly alter the color, texture, or water activity (aw) of the fish. Higher levels of thiobarbituric acid reactive substances (TBARSs) were observed with increased exposure times. Cold plasma was also tested in vitro on S. Enteritidis and L. monocytogenes on agar surfaces. A 4 min treatment eliminated the initial loads of S. Enteritidis (2.71 log CFU) and L. monocytogenes (2.98 log CFU). The findings highlight the potential of cold plasma in enhancing the safety of naturally contaminated fish. Cold plasma represents a promising technology for improving food safety in the global fish trade and continues to be a significant area of research in food science.

Deciphering the influence of NaCl on social behaviour of Bacillus subtilis

Various environmental signals, such as temperature, pH, nutrient levels, salt content and the presence of other microorganisms, can influence biofilm’s development and dynamics. However, the innate mechanisms that govern at the molecular and cellular levels remain elusive. Here, we report the impact of physiologically relevant concentrations of NaCl on biofilm formation and the associated differences in an undomesticated natural isolate of Bacillus subtilis . NaCl exposure and its uptake by bacterial cells induced substantial changes in the architecture of pellicle biofilm and an upsurge in the expansion of biofilm colonies on agar surfaces. We have observed the upregulation of genes involved in motility and the downregulation of genes involved in the biosynthesis of extracellular matrix components through the transcription factor sigD, suggesting the possible underlying mechanisms. To further support these observations, we have used Δ sigD and Δ srfAC null mutants, which showed compromised NaCl-induced effects. Our results indicate that NaCl induces a lifestyle shift in B. subtilis from a sessile biofilm state to an independent unicellular motile state. Overall, we present evidence that NaCl can reprogramme gene expression and alter cellular morphology and the state of cells to adapt to motility, which facilitates the expansion of bacterial colonies.

First Report of Rhizoctonia solani AG1-IB Causing Tobacco Target Spot in China.

Tobacco target spot, caused by Rhizoctonia solani Kühn, induces shot-hole lesions on leaves that that significantly reduce yield and quality of tobacco. In July 2022, samples (n=5) with target spot were collected from three tobacco fields, one each in Puer (22.63°N, 100.72°E, cv. Yunyan87) and Mengzi (23.26°N, 103.36°E, cv. Yunyan87) of Yunnan province and one in Dandong (40.63°N, 124.18°E, cv. Liaoyan17) of Liaoning province, China; disease incidence in these fields was approximately 30%~40%. Initial symptoms (2- to 3-mm-diameter lesions) appeared on the middle to lower leaves, then expanded to 2 to 3 cm in diameter and developed the shot-hole appearance. Pieces of tissue (5×5 mm) were cut from the edge of lesions, surface sterilized, rinsed in sterile water, then placed on the surface of water agar (WA) and incubated at 25℃ for 2 days in the dark. Single hyphal tips were taken from fungal isolates identified as R. solani based on the morphological traits (Tsror 2010), then transferred onto potato dextrose agar (PDA) and cultured for 3 d as described above. A total of 15 pure cultures were obtained. With the exception of YN-3 (isolated from Puer), YN-62 (isolated from Mengzi) and LN-95(isolated from Dandong) strains, which exhibited hyphal fusion reaction with AG1-IB standard strain, all the other strains demonstrated hyphal fusion with AG-3 standard strain (Ogoshi 1987). Genomic DNA of these three strains were extracted by the CTAB method and ITS regions of rDNA were sequenced (White et al. 1990). The sequences were deposited in GenBank with accession No. OR770079, OR770080 and OR770082. All the three rDNA-ITS sequences exhibited 99.85% similar to AG1-IB found in GenBank, and a phylogenetic tree using a neighbor-joining method grouped the three strains within the R. solani AG-1 IB clade. Therefore, based on the hyphal fusion reaction and molecular methods, these isolates were identified as R. solani AG1-IB. To determine pathogenicity of the isolates, the healthy leaves of tobacco plants (cv. Yunyan 87) were used. Five-mm-diameter mycelial plugs of the strain on PDA were inoculated on leaves that had been previously wounded with a sterile needle, and cotton balls moistened with sterile water were used for moisturizing the inoculation sites. Ten leaves were inoculated for each strain and leaves inoculated with PDA plugs were as control. The experiment was conducted twice. All plants were incubated for 2 d at 15℃ to 25℃ and 90% relative humidity with a 12 h photoperiod/day. Irregularly shaped lesions appeared on the leaves around each of the inoculated sites, but not on control leaves. The pathogens were reisolated and confirmed be R. solani AG1-IB by hyphal fusion and molecular identification tests as previously described, thereby fulfilling Koch's postulates. It has been reported that AG-3, AG-2 (Mercado Cardenas et al. 2012), AG-5 (Wang et al. 2023) and AG-6 (Sun et al. 2022) of R. solani could cause tobacco target spot, but AG-3 is considered the main causal agent (Marleny Gonzalez et al. 2011). To our knowledge, this is the first report of AG1-IB causing tobacco target spot in China and worldwide. The AG1-IB strain has a wide host range including cabbage, mint, lettuce, beans, and rice (Gonzalez et al. 2006). The discovery poses a new challenge for the prevention and control of tobacco target spot, especially when contemplating disease management strategies such as crop rotation and fungicide treatments.

Transcriptome responses of Lactococcus paracarnosus to different gas compositions and co-culture with Brochothrix thermosphacta

Lactococcus (Lc.) paracarnosus and the phylogenetically closely related Lc. carnosus species are common members of the microbiota in meat stored under modified atmosphere and at low temperature. The effect of these strains on meat spoilage is controversially discussed. While some strains are known to cause spoilage, others are being studied for their potential to suppress the growth of spoilage and pathogenic bacteria. In this study, Lc. paracarnosus DSM 111017T was selected based on a previous study for its ability to suppress the growth of meat spoilers, including Brochothrix thermosphacta. The mechanism by which this bioprotective strain inhibits competing bacteria and how it contributes to spoilage are not yet known. To answer these two questions, we investigated the effect of four different headspace gas mixtures (simulated air (21 % O2/79 % N2); HiOx-MAP (70 % O2/30 % CO2); nonOx-MAP (70 % N2/ 30 % CO2); simulated vacuum (100 % N2) and the presence of Brochothrix (B.) thermosphacta TMW 2.2101 on the growth and transcriptional response of Lc. paracarnosus DSM 111017T when cultured on a meat simulation agar surface at 4 °C. Analysis of genes specifically upregulated by the gas mixtures used revealed metabolic pathways that may lead to different levels of spoilage metabolites production. We propose that under elevated oxygen levels, Lc. paracarnosus preferentially converts pyruvate from glucose and glycerol to uncharged acetoin/diacetyl instead of lactate to counteract acid stress. Due to the potential production of a buttery off-flavour, the strain may not be suitable as a protective culture in meat packaged under high‑oxygen conditions. 70 % N2/ 30 % CO2, simulated vacuum- and the presence of Lc. paracarnosus inhibited the growth of B. thermosphacta TMW 2.2101. However, B. thermosphacta did not affect gene regulation of metabolic pathways in Lc. paracarnosus, and genes previously predicted to be involved in B. thermosphacta growth suppression were not regulated at the transcriptional level. In conclusion, the study indicates that the gas mixture used in packaging significantly affects the metabolism and spoilage potential of Lc. paracarnosus and its ability to inhibit B. thermosphacta growth.

First report of Diaporthe eres causing leaf spot on Rosa roxburghii Tratt in Guizhou Province, China.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

Enhanced antimicrobial efficacy and energy efficiency of low irradiance 405-nm light for bacterial decontamination

Due to its increased safety over ultraviolet light, there is interest in the development of antimicrobial violet-blue light technologies for infection control applications. To ensure compatibility with exposed materials and tissue, the light irradiances and dose regimes used must be suitable for the target application. This study investigates the antimicrobial dose responses and germicidal efficiency of 405 nm violet-blue light when applied at a range of irradiance levels, for inactivation of surface-seeded and suspended bacteria. Bacteria were seeded onto agar surfaces (101–108 CFUplate−1) or suspended in PBS (103–109 CFUmL−1) and exposed to increasing doses of 405-nm light (≤ 288 Jcm−2) using various irradiances (0.5–150 mWcm−2), with susceptibility at equivalent light doses compared. Bacterial reductions ≥ 96% were demonstrated in all cases for lower irradiance (≤ 5 mWcm−2) exposures. Comparisons indicated, on a per unit dose basis, that significantly lower doses were required for significant reductions of all species when exposed at lower irradiances: 3–30 Jcm−2/0.5 mWcm−2 compared to 9–75 Jcm−2/50 mWcm−2 for low cell density (102 CFUplate−1) surface exposures and 22.5 Jcm−2/5 mWcm−2 compared to 67.5 Jcm−2/150 mWcm−2 for low density (103 CFUmL−1) liquid exposures (P ≤ 0.05). Similar patterns were observed at higher densities, excluding S. aureus exposed at 109 CFUmL−1, suggesting bacterial density at predictable levels has minimal influence on decontamination efficacy. This study provides fundamental evidence of the greater energy efficacy of 405-nm light for inactivation of clinically-significant pathogens when lower irradiances are employed, further supporting its relevance for practical decontamination applications.

An opportunity for post‐harvest seafood safety: atmospheric pressure air or helium cold plasma to control Salmonella Enteritidis in sea bass

SummarySalmonella contamination of fish after harvest is a significant food safety risk. To control this risk, it was aimed to reduce Salmonella Enteritidis in sea bass by helium and air plasma. Considering that preserving the fresh properties of sea bass is important for industrial applicability, determining the sensory effects of plasma treatments was also aimed. Since air plasma would provide a cheaper and easier alternative to helium plasma, their effects were compared. Additionally, the in vitro effects of these treatments on Salmonella Enteritidis were also investigated. Air and helium plasma completely inhibited the initial load of S. Enteritidis (2.79 Log CFU) inoculated on agar surface after 10 min treatment. Then, the inoculated sea bass samples were exposed to air or helium plasma for durations ranging from 1 to 15 min. Air and helium plasma treatments for 10 min provided 0.90 log cfu g−1 and 1.13 Log CFU/g reductions, respectively, but longer treatments did not lead to further significant reductions. Sensory scores were all above 7 (good) when samples were treated with air or helium plasma for 1–10 min, but longer treatments resulted in lower scores. The reduction of S. Enteritidis and changes in sensory characteristics were interpreted together and it was concluded that 10 min of air or helium plasma provided maximum reduction without causing any significant sensory changes. Overall, the current study showed that atmospheric pressure air and helium plasmas can be useful for ensuring post‐harvest safety in the fish trade.

Agar dehydration: a simple method for long-term storage of Acanthamoeba spp. collection at room temperature.

This study describes dehydration of agar containing cysts as a novel and inexpensive method for long-term storage of Acanthamoeba spp. collections at room temperature. Five hundred microliters of axenically cultured Acanthamoeba spp. trophozoites (106 cells/mL) in PYG media or 150µl of amoeba suspension (106 cells or cysts/mL) from monoxenic plate culture was spread onto the surface of non-nutritive agar (NNA, 2-3-mm thick) without or with a layer of heat-inactivated Escherichia coli, respectively. The plates were sealed and incubated at 30°C. After the encystment, the Parafilm® was removed, and the plates were kept at the same temperature until the NNA was completely dehydrated. The dehydrated cyst-containing NNA was cut in rectangles and stored in airtight tubes at room temperature for up to 3years. Cyst viability was assessed by inoculating them in fresh NNA with a layer of E. coli and in PYG followed by incubation at 30°C. One hundred percent of samples from all specimens (19) stored over the 3years allowed new cultures to be re-established; however, two strains showed reduced viability, at 66.7% and 62.5%, after 2years of room temperature storage. One hundred percent of the cyst samples produced axenically and maintained in dry NNA allowed the re-establishment of axenic cultures through direct incubation in PYG, with excystment occurring within 24 or 48h. For the first time, we report the dehydration of cyst-containing agar as an economical and effective method for the long-term storage of Acanthamoeba spp. collections at room temperature. It enables the creation of large collections using reduced space and economical transport of Acanthamoeba strains, in addition to allowing better organization of the collection.

Investigating the effect of acoustic waves on spoilage fungal growth and shelf life of strawberry fruit

The effects of acoustic waves on growth inhibition of food spoilage fungi (Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus and Botrytis cinerea) on the medium and strawberry surfaces were investigated. Firstly, single-frequency sound waves (250, 500, 1000, 2000, 4000, 8000, 12,000 and 16,000 Hz) were induced on inoculated medium with fungi spores for 24 h and growth diameter of each mold was evaluated during the incubation period. In the second stage, the sound waves with two frequencies of 250 Hz and 16,000 Hz were induced on inoculated strawberries with fungi spores at 5 °C for different times (2, 4, 6, 8 and 10 days). The results from the first stage indicated that the sound waves inhibited the growth of A. niger (20.02%) at 250 Hz and B. cinerea (4/64%) at 4000 Hz on potato dextrose agar (PDA) surface. Also, comparison of the growth diameter of some species of Aspergillus revealed various responses in presence of 250 Hz frequency. In the second stage, applying a frequency of 250 Hz over a period of 10 days proved to be more effective in inhibiting the growth of A. niger and B. cinerea on strawberries inoculated with fungal spores. Consequently, the shelf lives of the strawberries significantly increased to 26 days and 18 days, respectively, under this treatment. Based on the findings, it is concluded that sounding with acoustic waves can be used as a green and cheap technology along with other technologies to improve food safety.

First Report of Fusarium verticillioides causing Stem Blight of Schizonepeta tenuifolia in China.

Schizonepeta tenuifolia is an important medicinal plant in China. Over 10000 ha of S. tenuifolia is cultivated in the country annually. However, fungal diseases are a major limiting factor in S. tenuifolia production. In 2022, 50 ha in several S. tenuifolia fields in Hebei province were observed to be severely affected by a disease causing a yield loss of 30%. Results from field surveys suggested an epidemic during seedlings stages that affected S. tenuifolia stems, causing irregularly watery brown lesions. Lesions ranged from 1.5 to 2 × 2.5 to 3 cm. To isolate the causal agent, tissue was removed from the border of lesions and surface sterilized in 75% ethanol for 30 sec and 0.1% HgCl2 for 1 min, then rinsed three times with steriled distilled water(SDW), plated on potato dextrose agar(PDA) at 25℃, and incubated in the dark for 7 days. Five putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar(SNA) with a ~ 1 × 2-cm strip of sterile filter paper on the agar surface(Nirenberg 1976). Cultures were incubated for 7 to 10 days at 20℃ in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined at 400×. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Microconidia produced on polyphialides and aggregating in heads were unicellular, ovoidal or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 μm. Macroconidia were abundant, falcate to straight, three to five septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 μm. Based on morphological characteristics, isolates were tentatively identified as F. verticillioides(A1-Hatmi et al. 2016; Guarro 2013). Pathogenicity tests were performed by injection inoculation of 0.1 mL of conidial suspensions(1×106 conidia/mL) into three S. tenuifolia stems using a disposable needle and syringe. Distilled water was injected into three mock controls. Inoculated plants were placed in a greenhouse at 32 to 34℃ and 95% relative humidity. Typical lesions were observed 7 days after inoculation, except in the control samples. Each treatment was replicated three times. The suspected pathogen was consistently reisolated from diseased tissue according to Koch's postulates, and was found to be morphologically similar to F. verticillioides. Preliminary morphological identification of the pathogen was further confirmed by using genomic DNA extracted from the mycelia of a 7-day-old culture grown on PDA at 25℃. The translation elongation factor 1-α gene(TEF1) was amplified(O'Donnell et al. 1998) and the TEF region(Genbank Accession No. OR105502) was sequenced by Sangon Biotech Co., Ltd.(Shanghai, China) and displayed 100% nucleotide similarity with rDNA-TEF of F. verticillioides(JF740717) separately after a BLASTn search in Genbank. Based on the symptoms, fungal morphology, TEF sequence, and pathogenicity testing, this fungus was identified as F. verticillioides. to our knowledge, this is the first report of F. verticillioides infecting S. tenuifolia in China. This report will promote further research of F. verticillioides on this host and lead to better understanding of disease prevalence, extent of damage, and possible management options.

First report of Diaporthe eres causing leaf spot on Rhododendron latoucheae Franch. in China.

Rhododendron latoucheae Franch. is an evergreen shrub with charming fragrance and large and abundant flowers that make it highly attractive as an ornamental species. The species is distribution in southwest China covers several different habitats and environments (Zhang, et al. 2022). From May to July in 2023, symptoms of leaf spot were observed on R. latoucheae over a wide portion of the Baili Azalea Forest Area (27°10' to 27°20'N, 105°04' to 106°04'E), Guizhou Province, China. About 500 plants were surveyed, and the incidence of leaf spot on R. latoucheae leaves was 12%, significantly reducing their ornamental and economic value. The affected leaves had irregular, grey white lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar, and incubated at 25°C for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GULJ1-L7, GULJ1-L8, and GULJ1-L9) identical in morphology were obtained. The isolate GULJ 1-L7 was used for further study. Colonies on PDA irregular grew white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area on the upper surface, while the lower surface presents alternating white, tan and taupe. Colony with conidiomata irregularly distributed over agar surface. In the representative isolate, darkly pigmented pycnidia (flask-shaped) were produced over the colony surface on PDA after about 30 days, and oozed milky or yellowish mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 5.15-10.29 × 1.54-3.33 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.34-31.91 × 1.01-1.90 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes and the calmodulin (CAL) using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, and CAL-228F/CAL-737R, respectively (Tao et al. 2020). Sequences from PCR amplification were deposited in GenBank with accession numbers OR740563 (ITS), OR754301 (TUB), OR754298 (TEF), and OR754295 (CAL) respectively. BLAST searches of the sequences revealed 99.07% (533/538 nt), 100% (490/490 nt), 99.69% (317/318 nt) and 98.95% (376/380 nt) homology with those of D. eres AR5193T from GenBank (KJ210529.1, KJ420799.1, KJ210550.1 and KJ434999.1), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum-likelihood method placed the isolate GULJ1-L7 in a well-supported cluster with D. eres CBS 138694T. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). The pathogenicity of GULJ1-L7 was tested through a pot assay. Due to the difficulty of artificial planting wild R. latoucheae, we conducted a pot essay to detect the pathogenicity of GULJ1-L7 using a closely related Rhododendron delavayi Franch. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying α and β spore mixture suspension (106 spores ml-1) of GULJ1-L7 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times. After 14 days, the treated leaves developed brown lesions similar to those in the field, whereas the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. latoucheae caused by D. eres in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. latoucheae.

Argon cold atmospheric plasma eradicates pathogens in vitro that are commonly associated with canine bacterial keratitis.

To investigate the antimicrobial effect of cold atmospheric plasma (CAP) on pathogens associated with canine bacterial keratitis. Pseudomonas aeruginosa, Staphylococcus pseudintermedius, and Streptococcus canis strains, which were obtained from dogs with infectious keratitis, were subjected to testing. For each species, four isolates and a reference strain were cultivated on Columbia sheep blood agar and treated with the kiNPen Vet® plasma pen from Neoplas GmbH, Greifswald, Germany. Various continuous treatment durations (0.5, 2, and 5 min) were applied, along with a 0.5-min treatment repeated four times at short intervals. These treatments were conducted at distances of 3 and 18 mm between the agar surface and the pen. CAP treatment reduced bacterial growth in all three species. The most effective treatment duration was 5 min at 3 mm distance, resulting in inhibition zones ranging from 19 to 22 mm for P. aeruginosa, 26-45 mm for S. pseudintermedius and an overall reduction of bacterial growth for Str. canis. Inhibition zones were smaller with decreasing treatment duration and larger distance. Treatment times of 30 s repeated four times and 2 min showed comparable results. Treatment with argon alone did not lead to visible reduction of bacterial growth. Argon cold atmospheric plasma demonstrated a potent in vitro antimicrobial effect on P. aeruginosa, S. pseudintermedius and Str. canis strains with the latter showing the highest sensitivity.

Surface-active antibiotic production as a multifunctional adaptation for postfire microorganisms.

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments.

Emergent antibiotic persistence in a spatially structured synthetic microbial mutualism.

Antibiotic persistence (heterotolerance) allows a subpopulation of bacteria to survive antibiotic-induced killing and contributes to the evolution of antibiotic resistance. Although bacteria typically live in microbial communities with complex ecological interactions, little is known about how microbial ecology affects antibiotic persistence. Here, we demonstrated within a synthetic two-species microbial mutualism of Escherichia coli and Salmonella enterica that the combination of cross-feeding and community spatial structure can emergently cause high antibiotic persistence in bacteria by increasing the cell-to-cell heterogeneity. Tracking ampicillin-induced death for bacteria on agar surfaces, we found that E. coli forms up to 55 times more antibiotic persisters in the cross-feeding coculture than in monoculture. This high persistence could not be explained solely by the presence of S. enterica, the presence of cross-feeding, average nutrient starvation, or spontaneous resistant mutations. Time-series fluorescent microscopy revealed increased cell-to-cell variation in E. coli lag time in the mutualistic co-culture. Furthermore, we discovered that an E. coli cell can survive antibiotic killing if the nearby S. enterica cells on which it relies die first. In conclusion, we showed that the high antibiotic persistence phenotype can be an emergent phenomenon caused by a combination of cross-feeding and spatial structure. Our work highlights the importance of considering spatially structured interactions during antibiotic treatment and understanding microbial community resilience more broadly.

Fullerene C60-chitosan conjugate applied in the photoinactivation of Staphylococcus aureus

A novel fullerene/chitosan (C60-CS) conjugate was synthesized. The addition reaction of the amine groups of chitosan (CS) to fullerene (C60) was carried out in N,N-dimethylformamide (DMF), and triethylamine at room temperature. The conjugate C60-CS was purified by dialysis in water. C60-CS showed an absorption spectrum characteristic of modified fullerenes. The ability to produce O2(1Δg) was determined by the decomposition of 9,10-dimethylanthracene (DMA) in DMF, finding a quantum yield value of 0.07. Besides, to obtain information about the contributions of the photoreaction mechanisms, the L-tryptophan photodecomposition was studied in the presence of reactive oxygen species (ROS) scavengers, such as D-mannitol and sodium azide. The results indicated a significant contribution of the type II mechanism to the photooxidation of the amino acid. This effect was confirmed by studies of the kinetics of nitrotetrazolium blue photoreduction in the presence of nicotinamide adenine dinucleotide. This conjugate's photodynamic inactivation (PDI) capacity was investigated in Staphylococcus aureus. The photodynamic effect induced a 4 log decrease in cell viability when the cells were treated with 10 μM C60-CS and 30 min of irradiation with white light. The photoreaction mechanisms involved in the photosensitized inactivation of S. aureus by C60-CS were studied with the addition of azide ions, deuterated saline water, D-mannitol, and potassium iodide. It was confirmed that the photodynamic action of C60-CS was mainly mediated by type II photoprocess. Photooxidation of DMA induced by C60-CS in S. aureus cells indicated the production of O2(1Δg) in this biological medium. Finally, PDI studies were performed on agar surfaces, treating the cells with C60-CS before deposition on the surface. The subsequent inactivation of S. aureus sensitized by C60-CS after 30 min of irradiation with white light produced a marked elimination of bacteria as a function of increasing concentration. Therefore, this conjugate is a promissory photosensitizing agent to inactivate S. aureus.

Machine learning identification of Pseudomonas aeruginosa strains from colony image data.

When grown on agar surfaces, microbes can produce distinct multicellular spatial structures called colonies, which contain characteristic sizes, shapes, edges, textures, and degrees of opacity and color. For over one hundred years, researchers have used these morphology cues to classify bacteria and guide more targeted treatment of pathogens. Advances in genome sequencing technology have revolutionized our ability to classify bacterial isolates and while genomic methods are in the ascendancy, morphological characterization of bacterial species has made a resurgence due to increased computing capacities and widespread application of machine learning tools. In this paper, we revisit the topic of colony morphotype on the within-species scale and apply concepts from image processing, computer vision, and deep learning to a dataset of 69 environmental and clinical Pseudomonas aeruginosa strains. We find that colony morphology and complexity under common laboratory conditions is a robust, repeatable phenotype on the level of individual strains, and therefore forms a potential basis for strain classification. We then use a deep convolutional neural network approach with a combination of data augmentation and transfer learning to overcome the typical data starvation problem in biological applications of deep learning. Using a train/validation/test split, our results achieve an average validation accuracy of 92.9% and an average test accuracy of 90.7% for the classification of individual strains. These results indicate that bacterial strains have characteristic visual 'fingerprints' that can serve as the basis of classification on a sub-species level. Our work illustrates the potential of image-based classification of bacterial pathogens and highlights the potential to use similar approaches to predict medically relevant strain characteristics like antibiotic resistance and virulence from colony data.

Antibacterial Activities of Heterotrigona itama Honey Farmed in Ex-Mining Area

Honey is considered to be a valuable non-timber forest product due to its numerous preventative and therapeutic properties. The nutritional content in honey is the most complete so it can boost the immune system. This study aims to determine the potential inhibition of Heterotrigona itama stingless bee honey against the activity of Staphylococcus sp bacteria and Propionibacterium sp bacteria. This research adopted the agar diffusion method to conduct antibacterial testing. The nutrient agar media was aseptically put into a sterile petri dish and thereafter left to air-dry. Following this, the bacterial suspension was reconstituted and subsequently inoculated onto the solidified agar surface by creating perforations. The experiment involved conducting antibacterial tests using many concentrations of the samples. The experimental setup included a positive control using chloramphenicol and a negative control using distilled water. The test results showed that the activity of the inhibition of Heterotrigona itama kelulut honey against Staphylococcus sp bacteria and Propionibacterium sp bacteria belonged to the strong antibacterial category. The antibacterial activity of honey exhibits an enhanced potency against both types of bacteria in direct proportion to the content of the honey sample.