Black Fungus Research Articles

The β-glucan nanotube carrier achieves detoxification and efficacy enhancement of celastrol in intrahepatic cholangiocarcinoma therapy by increasing targeted controlled release and macrophage polarization

Celastrol (Cel) is a monomer from a famous traditional Chinese medicine named Tripterygium wilfordii Hook. f. Cel showed great potential for treating intrahepatic cholangiocarcinoma (ICC) but still faces problems, including poor water solubility, high toxicity, and lack of targeting ability. Thus, the present work constructed a drug-delivery system using polysaccharide self-assembled black fungus nanotubes (BFP). Cel-loaded nanotubes (BFP-Cel) were confirmed to have a high loading content of Cel (38 %), liver targeting, and enzyme-controlled release abilities. Moreover, BFP carriers can significantly increase the uptake efficiency of Cel by tumor cells. In vivo experiments showed that BFP-Cel could effectively inhibit tumor growth and reduce the physiological toxicity of Cel. Furthermore, BFP, as a carrier, could regulate the immune microenvironment in the liver through the activation of macrophages and play an immunomodulatory role. In summary, the BFP nanotube carrier achieves detoxification and efficacy enhancement in treating ICC by increasing the targetability, controlled release ability, uptake effect, and regulation of the immune microenvironment of Cel.

The Biphasic Activity of Auricularia Auricula-Judae Extract on Bone Homeostasis Through Inhibition of Osteoclastogenesis and Modulation of Osteogenic Activity.

Osteoporosis arises from the disturbance of bone homeostasis, a process regulated by osteoblasts and osteoclasts. The treatment and prevention of bone metabolic disorders resulting from an imbalance in bone homeostasis require the use of agents that effectively promote both bone formation and anti-resorptive effects. Therefore, an investigation was carried out to determine the potential of the edible mushroom Auricularia auricula-judae in modulating bone remodeling by inhibiting RANKL-induced osteoclastogenesis and enhancing BMP-2-stimulated osteoblast differentiation. Moreover, this study assessed the mode of action of the Auricularia auricula-judae extracts. The staining of tartrate-resistant acid phosphatase (TRAP), a marker for osteoclast activity, demonstrated that Auricularia auricula-judae water extract (AAJWE) inhibited the formation of multinucleated osteoclasts while exhibiting no cytotoxic effects. The study demonstrated that AAJWE reduced RANKL-induced osteoclast differentiation by inhibiting c-Fos/NFATc1 through the inhibition of ERK and JNK phosphorylation during the RANKL-induced osteoclast differentiation. Moreover, AAJWE exhibited a dose-dependent induction of ALP expression in the presence of BMP-2 during osteoblast differentiation. The AAJWE strengthened BMP-2-induced osteogenesis through the activation of Runx2 and Smad phosphorylation. Therefore, AAJWE emerges as a promising candidate for both prevention and therapy owing to its biphasic effect, which aids in the preservation of bone homeostasis.

Enhanced preservation of cut rose flowers through bacterial nanocellulose produced from legume wastewater and fortified with Auricularia auricula polysaccharide

Cut flowers of Rosa hybrida 'Lychee' are susceptible to rapid decay, resulting in a brief shelf life, a inherent feature to their floral physiology. This study evaluated the effects of bacterial nanocellulose (BNC), which was produced from the legume processing wastewater and loaded with Auricularia auricula polysaccharide and 8-hydroxyquinoline, in extending the shelf life of these cut rose flowers. Water-retaining cotton and floral foam blocks were employed as control materials. The bacterial strain Komagataeibacter rhaeticus CP050139.1 yielded BNC from legume waste at a rate comparable to that from a glucose-based medium: 0.107±0.004 g L−1 d−1 versus 0.157±0.004 g L−1 d−1, respectively. Characterization via FE-SEM, FT-IR, TGA, and XRD confirmed that the BNC from both sources exhibited similar microscopic morphology, chemical composition, thermal stability, and crystallinity. The use of BNC, particularly when enriched with Auricularia auricula polysaccharide (AAP), extended the shelf life of cut roses by 50 % and significantly minimized fresh weight loss in comparison to the control materials. BNC's superior water-holding capacity — 42.7 times greater than that of water-retaining cotton — proved advantages for maintaining hydration and preventing wilting during transportation. This study underscored the potential of BNC hydrogel, sourced from legume processing wastewater, as an innovative, effective, and environmentally sustainable solution for preserving cut rose flowers, promoting the recycling of agricultural by products and enhancing the preservation of horticultural products.

Investigation and determination of CoQ10(H2) and CoQ10(H4) species from black yeast-like fungi and filamentous fungi.

Coenzyme Q (CoQ) or ubiquinone functions as an electron transporter in the electron transport system in both prokaryotes and eukaryotes. The isoprenyl side chain of CoQ is modified in some organisms, especially in fungi, for optimal electron transport performance under various conditions. In this study, we investigated the side chain saturated dihydro CoQ (CoQ10(H2)) in Aureobasidium pullulans EXF-150, Sydowia polyspora NBRC 30562, and naturally isolated Plowrightia sp. A37, all of which are melanized Dothideomycetes species within the Ascomycota, and also in filamentous fungi Aspergillus oryzae and Aspergillus terreus. Plowrightia sp. A37 produced the rarely synthesized tetrahydro type CoQ10(H4), especially in glucose-rich medium, during extended cultivation in contrast to CoQ10(H2) in time-limited cultivation. Using liquid chromatography-mass spectrometry, we identified demethoxyubiquinone-H2 (DMQ(H2)) as an indicative intermediate that suggests that the side chain-saturation of CoQ occurs after the formation of DMQ and not always in the last step as previously considered.

Glucono-δ-lactone induced Auricularia auricula polysaccharide-casein composite gels for curcumin loading and delivery

Polysaccharides could be used to form the network structure of casein (CA) gel, and affect its gelling properties. Auricularia auricula polysaccharide (AAP) has good gel properties and activity, however, how the AAP affects gelling properties of CA gels remains unclear. In this study, AAP and CA were acid-induced by glucono-δ-lactone (GDL) to prepare composite gels for curcumin loading. The effects of different AAP additions on the gel structure were emphasized. Water holding capacity, rheology, texture profile analysis, sulfhydryl content, surface hydrophobicity and gel microstructure showed that the composite gels were most structurally stable upon addition of 0.4 % AAP. The composite gels exhibited a higher strength and a more regular network structure compared to the CA gels. The results of turbidity studies showed that CA and AAP formed gels through electrostatic interactions due to pH < pI. The results of FT-IR, X-ray diffraction, fluorescence spectroscopy, and UV–Vis spectroscopy indicated that curcumin interacted with the CA and was successfully encapsulated within the gel. In addition, in vitro simulated digestion experiments demonstrated that the composite gels exhibited better protection against curcumin than the single CA gel. The results suggested that composite gels can be used as a curcumin carrier, which may enhance its wider application in the food and health industries.

Clinical and pathological features of cerebrospinal meningitis caused by Pantoea agglomerans : a case report

BackgroundPantoea agglomerans (P. agglomerans) is a gram-negative bacterium that is commonly isolated from plant surfaces, seeds, and the environment. As an opportunistic pathogen, it can cause blood, urinary and soft tissue infections in immunocompromised patients. In central nervous system, P. agglomerans infection has been report in children and immune-compromised patients, however, infection by such bacterium in nontraumatized immune competent adults has not been reported. Here, we report a case of P. agglomerans cerebrospinal meningitis accompanied by positive anti-myeloperoxidase (MPO) antibody in a 49-year-old female who has a history of black fungus planting.Case presentationThe patient manifested with repeated fever, headache, generalized muscle pain, and neurological defects. Cerebrospinal fluid (CSF) tests revealed a moderately elevated number of polymorphonuclear leukocytes (50–193 × 106/L), low glucose levels (0.54–2.44 mmo1/L), and extremely high protein content (2.42–25.42 g/L). Blood tests showed positive anti-myeloperoxidase antibodies lasting for 1.5 year before turning negative. Spine MRI showed thickening and enhancement of the whole spinal meninges. CSF metagenomic next-generation sequencing (mNGS) revealed 75,189 specific DNA reads of P. agglomerans. The patient underwent spinal laminectomy due to meningeal adhesions. Pathological results revealed fibrous tissue proliferation, inflammatory infiltration with focal necrosis and calcification in the dura mater. The patient was successfully treated with sufficient antibiotics at 1-year follow-up.ConclusionsPeople should be alert to CNS infections caused by P. agglomerans which presented with relatively mild clinical symptoms at onset, especially for those who contucts relevant agricultural and forestry work. The CSF characterization of P. agglomerans meningitis is elevated multiple nuclei white blood cells, significantly reduced glucose content, and markedly increased protein level which may be related to the secondary spinal membrane adhesions.

Cultivation of a Wild Strain of Wood Ear Auricularia cornea from Brazil.

Auricularia cornea has become one of the most important cultivated mushrooms worldwide. Although not remarkably flavorful, Auricularia species are very versatile and rehydrate easily after drying, adding a unique and pleasing texture to the dishes. In this study, we collected, identified, and domesticated a wild strain of A. cornea from the Brazilian Atlantic Rainforest. The wild strain was evaluated for mycelial growth at different temperatures and substrates, biological efficiency, and nutritional composition. The temperature that best favored the A. cornea mycelium growth was 30°C, and the substrate was sterile Eucalyptus sawdust. The highest biological efficiency value obtained was 106.90 ± 13.28%. Nutritional analysis showed that the produced wood ears contained 71.02% carbohydrates, 19.63% crude fiber, 11.59% crude protein, 10.19% crude fat, and 4.24% ash on dry matter basis. For the mineral content profile, the elements K and P were the most abundant. This is the first report on cultivation of a wild strain of A. cornea from Brazil.

The effect of ionizing radiation on hydrated fungal cells: Implications for planetary protection and mars habitability

Liquid water is one of the essential conditions for life as we know it. Its presence has been currently reported beyond Earth. Geological and mineralogical evidence indicates that water once flowed on Mars. The recent discovery of present ice-water on the planet's surface is one of the driving factors for life-detection missions. The highly radiative Martian surface, combined with aqueous thin layers, is prohibitive for the presence of hypothetical forms of terrestrial-like life on the planet. In this context, we examined the survival of hydrated colonies of the Antarctic black fungus Cryomyces antarcticus, which thrives in the extreme environment of McMurdo Dry Valleys in Antarctica, after the exposure to increasing doses of space relevant γ-rays. Results suggest that water significantly reduces the number of survivors at the lowest doses, while at the higher dose (117 kGy) the cumulative damage caused by radiation could no longer be counteracted by dehydration.

Effect of microwave-puffed on Auricularia auricula polysaccharide and probiotic fermentation on its biotransformation and quality characteristics during storage period

In this study, probiotics with superior fermentation performance were screened, and the mixed-bacteria fermentation was carried out with Auricularia auricula treated with microwave-puffed process as fermentation substrate, and the changes in nutritional quality under different storage conditions were investigated. The results showed that the acid and bile salt resistance of Lactiplantibacillus plantarum 21,801 and 21,805 reached 95 % and 75 % respectively, and the intestinal adhesion was superior; microwave puffing treatment had the highest retention rate of A. auricula polysaccharides and the lowest loss of polyphenols, and no effect on soluble protein. Mixed bacterial fermentation significantly increased the total polyphenols and total flavonoids of A. auricula (p < 0.05), and the DPPH and ABTS radical scavenging reached 48.31 % and 73.21 % respectively. Furthermore, the viable counts, DPPH radical scavenging, color, and sensory quality of fermented A. auricula remained stable when stored at 4 °C. In contrast, when stored at 25 °C for 7 days, the taste was unfavorable, undesirable odor and spoilage occurred; by 21 days, DPPH clearance rate dropped below 40 % and color changed significantly (△E > 2). In conclusion, the probiotic mixed fermentation and storage conditions had a significant effect on the biometabolic transformation of macromolecules and other substances in A. auricula.

Auricularia auricula polysaccharide alleviates cyclophosphamide-induced liver injury in mice involving remodeling of the gut bacteriome, mycobiome, and metabolome

In this study, a novel polysaccharide (AHP) from Auricularia auricula was isolated and purified, showing protective effects against CTX-induced liver injury in mice. To study the action mechanism of AHP, a liver injury model was established by intraperitoneally injection 80 mg/kg of CTX for 3 consecutive days. The focus was on how AHP regulated the gut bacteriome and mycobiome to help alleviate metabolic disorders associated with liver injury. Results showed that AHP amended liver injury by improving liver function, stabilizing oxidative stress homeostasis, reducing inflammatory invasion and activating Akt/GSK3β/Nrf-2/HO-1 signaling pathway. The 16S ribosomal DNA (16S rDNA) and Internal Transcribed Spacer-1 (ITS1) sequencing results demonstrated that AHP supplementation significantly restored the gut bacteriome and mycobiome composition in CTX-induced liver injury mice, by enriching the abundance of beneficial bacteriome (unclassified_Muribaculaceae, Faecalibaculum and Alloprevotella) and mycobiome (Fusarium), reducing the abundance of harmful bacteriome (Akkermanisa) and mycobiome (Fusicolla and Cladosporium). Analysis of untargeted metabolomics indicated that AHP altered the levels of metabolites associated with both bile acid and arachidonic acid metabolism, showing a significant connection to the AHP-regulated bacteriome and mycobiome. To conclude, the findings suggested that AHP was a viable and secure candidate for use as a hepatoprotective medication.

A two-stage image segmentation method for harvest order decision of wood ear mushroom

A two-stage image segmentation method for harvest order decision of wood ear mushroom

Neglected Pulmonary Infection Caused by Exophiala dermatitidis Misidentified as Rhodotorula spp.

Exophiala dermatitidis is an emerging black fungus that causes pulmonary infections that may be underestimated by conventional culture methods. We encountered one case that initially appeared to be yeast and was misidentified as Rhodotorula spp. using a commercial identification kit. Thus, genetic identification and clinical background investigations were conducted on 46 strains of Rhodotorula spp. The sequences of the internal transcribed spacer and large-subunit RNA genes (D1/D2 regions) of 43 isolates, excluding two environmental isolates and one difficult-to-culture isolate, were determined and genetically identified. Notably, 22 isolates were identified as E. dermatitidis and misidentified as Rhodotorula spp. using the conventional method. Based on the exclusion criteria, the clinical information of 11 patients was retrospectively reviewed. Five cases (definite) had definite exacerbation of pulmonary infections due to E. dermatitidis, and six cases (possible) had undeniable infections. Of the 11 cases of pulmonary infection suggested to be caused by E. dermatitidis, comorbidities included two cases of chronic pulmonary aspergillosis (CPA), three cases of pulmonary non-tuberculous mycobacterial (NTM) infection and one case of pulmonary nocardiosis, suggesting a trend towards simultaneous detection of chronic pulmonary infections. Steroid and immunosuppressive drug use was observed in five cases, and β-D-glucan elevation was observed in three of five definite cases of pulmonary infections due to E. dermatitidis. The possibility of E. dermatitidis infection should be considered when Rhodotorula spp. are isolated from cultures of airway-derived specimens, and, in addition to CPA and NTM, identification of E. dermatitidis may be important in chronic pulmonary infections.

Dual sustainability of rGO/CuCoO2 nanocomposites with enhanced photocatalytic and antibacterial insights

New growth and photocatalytic activity of CuCoO2 and rGO/CuCoO2 nanocomposites were demonstrated for model organic pollutant (dye) and antibacterial studies. The rGO/CuCoO2 nanocomposites were designed by hydrothermal technique. CuCoO2 and rGO/CuCoO2 materials were investigated and characterized by several instrumental techniques like FT-IR spectroscopy (FT-IR), Raman spectroscopy, X-Ray diffractometer (XRD), DRS-UV Visible spectroscopy, HRTEM, FESEM analysis, Thermogravimetric analysis (TGA) and BET investigations. XRD confirmed the CuCoO2 cubic phase structure. It seems to have been evaluated for FT-IR and the Raman results were better than anything else that should be satisfied. Hence, Cu–O or Co-O (M−O−M) modes were confirmed peaks at 400 to 900 cm−1. We proposed CuCoO2 and rGO/CuCoO2 ((25/75), (50/50) & (75/25)) composite materials band gap energies at 2.8 eV to 2.0 eV. So, these results are also one of the benefits in the application area. A Sedum morganianum Burrito − Burro’s plant’s tail-like morphology was obtained in the pure CuCoO2 materials, and different weight percentages of rGO composites were also identified in FESEM-analysis as wood fungi, rods, and sheets-like structures. The rGO/CuCoO2 sheet-like morphology was confirmed by HRTEM measurements. The elemental composition of materials was investigated by EDX spectroscopy. We can prepare to suggest that the synthesis potential in the prepared matter is tbepensumed Bache formulation and much less explored, first-time declared phases and novelty of the synthetic designs, new morphologies, and bandgap energies. The synthesized rGO/CuCoO2 materials were achieved by 94 % Methylene blue (MB) dye degradation in photocatalytic applications, in which case composites were more efficient with environment-friendly when compared to CuCoO2 nanoparticles. The role of the rGO composite can be divided into three different principles such as absorbance, charge separator, and photosensitizer. This rGO material can reflect a big absorption capacity depending on the dye to rGO, with parallelly obeyed for π-π interactions. Hence, it’s favored for catalytic improvement. Secondly, rGO degradation was improved to the charge separation and may be foiling for the recombination of electron-hole pairs. Finally, transform their wide bandgap semiconductor into the catalyst to facilitate parameters. The rGO/CuCoO2 composite materials have performed well in reusability and radical abilities. The antimicrobial activity of the CuCoO2 nanoparticle was investigated on Gram-positive Staphylococcus aureus &Enterococcus faecalis and Gram-negative Escherichia coli &Pseudomonas aeruginosa bacterial strain. As promised rGO/CuCoO2 and CuCoO2 materials were examined for better degradation activities and antibacterial activities. Antibacterial efficacy studies play a major innovative role in the present work and have not been previously published. Hence, we have selected this kind of material.

Endophytic Fungi: A Treasure Trove of Antifungal Metabolites.

Emerging and reemerging fungal infections are very common in nosocomial and non-nosocomial settings in people having poor immunogenic profiles either due to hematopoietic stem cell transplants or are using immunomodulators to treat chronic inflammatory disease or autoimmune disorders, undergoing cancer therapy or suffering from an immune weakening disease like HIV. The refractory behavior of opportunistic fungi has necessitated the discovery of unconventional antifungals. The emergence of black fungus infection during COVID-19 also triggered the antifungal discovery program. Natural products are one of the alternative sources of antifungals. Endophytic fungi reside and co-evolve within their host plants and, therefore, offer a unique bioresource of novel chemical scaffolds with an array of bioactivities. Hence, immense possibilities exist that these unique chemical scaffolds expressed by the endophytic fungi may play a crucial role in overcoming the burgeoning antimicrobial resistance. These chemical scaffolds so expressed by these endophytic fungi comprise an array of chemical classes beginning from cyclic peptides, sesquiterpenoids, phenols, anthraquinones, coumarins, etc. In this study, endophytic fungi reported in the last six years (2018-2023) have been explored to document the antifungal entities they produce. Approximately 244 antifungal metabolites have been documented in this period by different groups of fungi existing as endophytes. Various aspects of these antifungal metabolites, such as antifungal potential and their chemical structures, have been presented. Yet another unique aspect of this review is the exploration of volatile antifungal compounds produced by these endophytic fungi. Further strategies like epigenetic modifications by chemical as well as biological methods and OSMAC to induce the silent gene clusters have also been presented to generate unprecedented bioactive compounds from these endophytic fungi.

Molecular Identification of Fungal Species Isolated from the External Surfaces of Ants Caught from Educational Hospitals in Babol City (2021-2022)

Background. Since the ants play a key role in transmitting microorganisms, it is important to identify the fungal species isolated from the external surfaces of ants caught from hospitals. Therefore, the present study aimed to identify the fungal species isolated from the external surfaces of ants caught from Babol hospitals. Methods. Ants were collected by trapping inside sterile tubes and were washed with normal saline for 20 seconds. Then, 100 µL of the resulting solution was cultured in Sabouraud Dextrose Agar (SDA) medium and incubated for one week at 35C°. Macroscopic, microscopic, and RFLP-PCR molecular techniques were employed to confirm the grown isolates. Results. A total of 79 fungal isolates including 47 isolates of hyaline fungi (59.49%) and 32 isolates of black fungi (40.51%) were obtained from 63 captured ants. Out of 47 cultured fungi, 15 fungi (31.91%) were Aspergillus niger and 15 (31.91%) were A. flavus, 12 (25.53%) were A. fumigatus and 5 isolates were Penicillium (10.63%). Conclusion. Taking into account the increasing number of immunocompromised patients, outbreak of COVID-19 in recent years, and numerous reports about the invasive fungal diseases among COVID-19 patients, it was found necessary to control the ants as carriers of these organisms. Practical Implications. It was necessary to control these insects effectively in order to prevent the increasing prevalence of the fungal diseases among the hospitalized people.

A single septin from a polyextremotolerant yeast recapitulates many canonical functions of septin hetero-oligomers.

Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septin homologues are conserved from yeasts to humans, the systems in which septins have been most studied. But there is also a fifth family of opisthokont septins that remain biochemically mysterious. Members of this family, Group 5 septins, appear in the genomes of filamentous fungi, but are understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can also serve as a model system for Group 5 septins. We have recombinantly expressed and biochemically characterized KpAspE, a Group 5 septin from K. petricola. This septin--by itself in vitro--recapitulates many functions of canonical septin hetero-octamers. KpAspE is an active GTPase that forms diverse homo-oligomers, binds shallow membrane curvatures, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher-order structures formed by canonical septins, which in K. petricola cells form extended filaments, and provide insight into how septin hetero-oligomers evolved from ancient homomers.

Disentangling how the opportunistic parasitic fungus Armillaria affects the flammability of coarse deadwood in exotic pine plantations

Parasitic wood fungi are important to forest carbon cycles globally. However, whether or how they affect the flammability of coarse deadwood is poorly understood. Given the predicted climate-driven increase in wildfires and associated carbon emissions into the atmosphere, potentially amplifying climate warming, filling this knowledge gap should have high priority. We thereto investigated coastal plantations of the exotic black pine, Pinus nigra J.F. Arnold, in the Netherlands, which are widely suffering from Armillaria infection. We hypothesized that branches from forest stands with a visible Armillaria infection will burn differently compared with branches from stands without a visible Armillaria infection, due to Armillaria infection having an additional effect on the branch traits. We tested this hypothesis by burning coarse Pinus nigra branches across a range of densities from infected and uninfected forest patches under standardized conditions in a fire lab and by measuring Armillaria biomass (via ddPCR), deadwood traits and key flammability parameters. Armillaria infection did enhance the flammability of Pinus nigra branches (e.g. more ignitable, longer flame duration and higher percentage mass loss). This higher flammability originated from both direct Armillaria influences, e.g. via changing wood structure (before and/or after wood death), and indirect influences, e.g. by facilitating nitrogen fixation in wood, thereby increasing wood decomposability and consequently reducing wood density. Our findings also have important implications for understanding the role of pathogens in fire regimes more broadly.

Screening for Bongkrekic Acid in Food Using a Monoclonal Antibody-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay (icELISA)

Pseudomonas cocovenenans subsp. farinofermentans is a pathogenic bacterium that causes the poisoning of fermented rice products. Its isomeric products, bongkrekic acid (BA) and isobongkrekic acid (IBA), have become popular research topics. In this study, we developed a heterologous-coating strategy for high-sensitivity indirect competitive enzyme-linked immunoassay (icELISA) for BA and IBA using monoclonal antibodies (mAbs). The optimized procedure exhibited an IC50 of 5.49 ng/mL, a limit of detection of 0.99 ng/mL, and a linear range from 2.12 to 14.21 ng/mL. Notably, this method exhibited negligible cross-reactivity to other small toxins, and the recoveries of the samples, including rice noodles, Auricularia auricula, and flat rice noodles, ranged from 92.0%–111.9%, 105.6%–115.0%, and 83.2%–109.0%, respectively, with coefficients of variation below 12%. A comparison of the recoveries of high-performance liquid chromatography-mass spectrometry (HPLC-MS) and icELISA demonstrated that the developed approach provides good accuracy and reliability. Overall, the simple preparation of IBA combined with the suitability of icELISA for high-throughput analysis is efficient and cost-effective, making it suitable for routine screening of food.

The Tet-on system for controllable gene expression in the rock-inhabiting black fungus Knufia petricola

Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K.petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis (melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K.petricola cell or for using K. petricola as host for the expression of heterologous genes.

Comparative growth kinetics of Listeria monocytogenes and Salmonella enterica on dehydrated enoki and wood ear mushrooms during rehydration and storage.

Specialty mushrooms have been implicated in foodborne illness outbreaks in the U.S. in recent years. These mushrooms are available to consumers in both their fresh and dried states. Dehydrating mushrooms is a convenient way to increase shelf life. The dehydration process results in a lowered water activity (aw) of the commodity, creating an environment where both spoilage and pathogenic bacteria cannot proliferate. Prior to food preparation and consumption, these mushrooms are typically rehydrated and possibly stored for later use which could lead to increased levels of pathogens. This study examined the survival and growth of Listeria monocytogenes and Salmonella enterica on dehydrated enoki and wood ear mushrooms during rehydration and subsequent storage. Mushrooms were heat dehydrated, inoculated at 3 log CFU/g, and rehydrated at either 5 or 25°C for 2 h. Rehydrated mushrooms were stored at 5, 10, or 25°C for up to 14 d. L. monocytogenes and S. enterica survived on enoki and wood ear mushroom types during rehydration at 5 and 25°C, with populations often <2.39 log CFU/g. During subsequent storage, no growth was observed on wood ear mushrooms, regardless of the rehydration or storage temperature, with populations remaining <2.39 log CFU/g for both pathogens. When stored at 5°C, no growth was observed for either pathogen on enoki mushrooms. During storage at 10 and 25°C, pathogen growth rates and populations after 14 d were generally significantly higher on the enoki mushrooms rehydrated at 25°C; the highest growth rate (3.56 ± 0.75 log CFU/g/d) and population (9.48 ± 0.62 log CFU/g) after 14 d for either pathogen was observed by S. enterica at 25°C storage temperature. Results indicate a marked difference in pathogen survival and proliferation on the two specialty mushrooms examined in this study and highlight the need for individual product assessments. Data can be used to assist in informing guidelines for time and temperature control for the safety of rehydrated mushrooms.