Ecotoxicity effect of aspirin on the larvae of Musca domestica through retinol metabolism.

Aspirin is a widely used multi-efficiency pharmaceutical, and its environmental residues are frequently detected. However, limited information is available on its effects on the development of the public health pest and saprophytic insect Musca domestica. In this study, it was demonstrated that aspirin inhibits the larval growth of house flies in a concentration-dependent manner. Microbiome analysis indicated that the composition of larval intestinal bacteria was influenced by aspirin but not greatly. The dominant bacterial genus in the aspirin group was still Klebsiella, as in the control group. Transcriptome sequencing and gene set enrichment analysis showed that retinol metabolism was activated after aspirin treatment. High performance liquid chromatography indicated that the content of retinol in larvae was decreased and that of retinoic acid was increased. The addition of ß-carotene, a precursor substance of retinol, in feeding promotes larval development and alleviates the inhibitory effect caused by aspirin. In contrast, retinoic acid delayed the larval development of house flies as well as aspirin. Gene expression analysis after aspirin exposure demonstrated that genes involved in the transformation from retinol to retinoic acid were upregulated. Overall, aspirin exposure impairs larval development by activating retinol metabolism in house flies and can be utilized as an effective pesticide. This work uncovers the mechanism underlying the larval development inhibition induced by aspirin in terms of metabolism and genetics, and provides novel functional exploration of a traditional drug for pest management.

Pathogenic and Saprophytic Growth Rates of Fusarium oxysporum f. sp. vasinfectum Interact to Affect Variation in Inoculum Density and Interannual Infection Risk.

Fusarium wilt of cotton caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a contemporary epidemic affecting cotton production in Far West Texas. The spatial distribution of soilborne FOV4 can be heterogeneous at small scales, and the factors that lead to this heterogeneity require investigation. Hypothetical causes include dissemination of spores through soils and variable saprophytic growth of the fungus. In the field, FOV4 DNA was quantified from soil during and after the cotton-growing season, and though the average amounts of DNA were not different between these time points, the variances of DNA across space were significantly different. Variability was higher when pathogenic growth of the fungus was expected owing to the presence of live cotton plants and lower when saprophytic growth was expected after cropping. In sterile-environment growth chamber experiments, the abundance of organic matter influenced the fungal vegetative growth rate and maximum amount as measured through quantitative PCR and the timing of the fungus' increasing its rate of spore production as measured through dilution plating. To investigate movement of spores in soils, spore mobility in experimental columns was quantified. Soil composition and organic matter abundance affected spore mobility, indicating that the timing of spore production relative to the availability of growth resources will affect the spatial spread of FOV4 and suggesting that soil properties affect the retention of conidia. The spatial spread of FOV4 through soil varies temporally and is affected by the shift between pathogenic and saprophytic growth modes of the fungus.

New Spirostane from a Fungus Neohelicomyces hyalosporus and Its Bioactivity.

A new spirostane, namely neohelicomyine B (1), together with six known steroids (2-7) were isolated from the fermentation of fungus Neohelicomyces hyalosporus. The structures of these compounds were elucidated by extensive analyses of spectroscopic methods including 1D and 2D NMR and HR-ESI-MS. The absolute configuration of 1 was confirmed by single-crystal X-ray diffraction. The bioactivities of compounds 1-7 were evaluated using cellular assays. Compound 1 displayed moderate cytotoxicity against HepG2 cells (hepatoma cells) with IC50 value of 8.4±2.1 µM. Compound 7 also exhibited cytotoxic activity against HepG2 cells with the IC50 value of 3.0±0.2 µM.

Antibacterial activity and mechanism of slightly acidic electrolyzed water against Shewanella putrefaciens and Staphylococcus saprophytic.

The purpose of this study was to investigate the antimicrobial effect and mechanism of slightly acidic electrolyzed water (SAEW) against Shewanella putrefaciens (S. putrefaciens) and Staphylococcus saprophytic (S. saprophyticus). The results showed that SAEW exhibited strong antimicrobial activity against tested bacteria, which was positively correlated to the available chlorine concentration (ACC) of SAEW. The mortality rate of S. putrefaciens and S. saprophyticus was up to 96% and 85%, respectively, when the ACC of SAEW was 60.0 mg/L. The results of scanning electron microscopy (SEM) showed that the cell morphology and structure were destroyed by SAEW. Besides, the results of confocal laser scanning microscopy (CLSM), leakage of DNA and protein provided evidence that SAEW induced membrane permeabilization in cells. Compared with the control, the intracellular reactive oxygen species (ROS) generated by SAEW was strengthened significantly with the increase of ACC, and the cells were injured to death accordingly.

Mycodegradation of diazinon pesticide utilizing fungal strains isolated from polluted soil.

The current study aimed to isolate biodegradable soil fungi capable of metabolizing diazinon. The collected soil samples were investigated for diazinon pollution to detect the pesticide level in the polluted soil samples. Food poisoning techniques were utilized to preliminary investigate the biodegradation efficiency of the isolated fungal strains to diazinon pesticide using solid and liquid medium and also to detect their tolerance to different concentrations. GC-MS analysis of control and treated flasks were achieved to determine the diazinon residues for confirmation of the biodegradation efficiency. The total diazinon residues in the collected soil samples was found to be 0.106 mg/kg. Out of thirteen fungal strains isolated form diazinon polluted soils, six strains were potentially active in diazinon biodegradation. Food poisoning technique showed that A. niger, B. antennata, F. graminearum, P. digitatum, R. stolonifer and T. viride strains recorded fungal growth diameters of 65.2 ± 0.18, 57.5 ± 0.41, 47.2 ± 0.36, 56.5 ± 0.27, 85.0 ± 0.01, 85.0 ± 0.06 mm respectively in the treated group which were non significantly different compared to that of control (P > 0.05), indicating the high efficiency of these strains in diazinon degradation compared to the other isolated strains. GC-MS analysis revealed that B. antennata was the most efficient strain in diazinon degradation recording 32.24 ± 0.15 ppm concentration after 10 days incubation. Linear regression analysis confirmed that B. antennata was the most effective biodegradable strain recording the highest diazinon dissipation (83.88%) with the lowest T1/2 value of 5.96 days while T. viride, A. niger, R. stolonifer and F. graminearum exhibited a high biodegradable activities reducing diazinon to 80.26%, 78.22%, 77.36% and 75.43% respectively after 10 days incubation. In conclusion, these tolerant fungi could be considered as promising, eco-friendly and biodegradable fungi for the efficient and potential removal of hazardous diazinon from polluted soil.

Fusarium Head Blight of Small Grains in Pennsylvania: Unravelling Species Diversity, Toxin Types, Growth, and Triazole Sensitivity.

Fusarium graminearum is the main causal species of Fusarium head blight (FHB) globally. Recent changes in the trichothecene (toxin) types in the North American FHB pathogens support the need for continued surveillance. In this study, 461 isolates were obtained from symptomatic spikes of wheat, spelt, barley, and rye crops during 2018 and 2019. These were all identified to species and toxin types using molecular-based approaches. An additional set of 77 F. graminearum isolates obtained from overwintering crop residues during winter 2012 were molecularly identified to toxin types. A subset of 31 F. graminearum isolates (15 15-acetyl-deoxynivalenol [15ADON] and 16 3-acetyl-deoxynivalenol [3ADON]) were assessed for mycelial growth, macroconidia, perithecia, and ascospore production, and sensitivity to two triazoles. Ninety percent of isolates obtained from the symptomatic spikes (n = 418) belonged to F. graminearum, with four other species found at a lower frequency (n = 39). The F. graminearum isolates from symptomatic spikes were mainly of the 15ADON (95%), followed by 3ADON (4%), nivalenol (0.7%), and NX-2 (0.3%) toxin types. All F. graminearum isolates obtained from overwintering residue were of the 15ADON type. The toxin types could not be differentiated based on the multivariate analysis of growth and reproduction traits. All isolates were sensitive to tebuconazole and metconazole fungicides in vitro. This study confirms the dominance of F. graminearum and suggests ecological and environmental factors, to be further identified, that lead to similar composition of toxin types in the northern United States. Our results may be useful to assess the sustainability of FHB management practices and provide a baseline for future FHB surveys.

Saprophytic Bacillus Accelerates the Release of Effective Components in Agarwood by Degrading Cellulose.

The value of Agarwood increases with time due to the gradual release of its major components, but the mechanism behind this remains unclear. Herein we reveal that the potential driving force of this process is the degradation of cellulose in Agarwood by its saprophytic Bacillus subtilis. We selected 10-year-old Agarwood from different places and then isolated the saprophytic bacteria. We confirmed these bacteria from different sources are all Bacillus and confirmed they can degrade cellulose, and the highest cellulase activity reached 0.22 U/mL. By co-cultivation of the bacterium and Agarwood powder, we found that three of the strains could release the effective components of Agarwood, while they had little effect in increasing the same components in living Aquilaria sinensis. Finally, we demonstrated that these saprophytic Bacillus subtilis have similar effects on Zanthoxylum bungeanum Maxim and Dalbergiaod orifera T. Chen, but not on Illicium verum Hook. f, Cinnamomum cassia Presl and Phellodendron chinense Schneid. In conclusion, our experiment revealed that the saprophytic Bacillus release the effective components of Agarwood by degrading cellulose, and we provide a promising way to accelerate this process by using this bacterial agent.

Characterization and Genome Study of Novel Lytic Bacteriophages against Prevailing Saprophytic Bacterial Microflora of Minimally Processed Plant-Based Food Products.

The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from -20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.

Aerosol Transmission of Aspergillus fumigatus in Cystic Fibrosis Patients in the Netherlands.

We collected sputum samples and cough plates from 15 cystic fibrosis patients in the Netherlands who were colonized with Aspergillus fumigatus; we recovered A. fumigatus of the same genotype in cough aerosols and sputum samples from 2 patients. The belief that transmission of A. fumigatus from cystic fibrosis patients does not occur should be reconsidered.

Contribution of chitooligosaccharides to biofilm formation, antibiotics resistance and disinfectants tolerance of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen present in various environmental reservoirs. It exhibits resistance and tolerance to antibiotics and sanitizing agents used in several food processing industries. It has been reported that L. monocytogenes chitinase can catalyze hydrolysis of chitin polymeric carbohydrate present in the environment and act as a virulence factor that support its survival in mammalian host cells. By taking advantage of chitinase, L. monocytogenes has both saprophytic and pathogenic lifestyles in the soil and the living host, respectively. The objective of the present study was to determine the involvement of chitin degradation products such as chitooligosaccharides (COS) in biofilm formation of L. monocytogenes. Results showed that different concentrations of COS with various molecular weight enhanced biofilm formation of L. monocytogenes. Such enhancement in biofilm formation contributed to the development of antibiotics resistance and disinfectants tolerance of cells present in the biofilm. The present article also described diverse roles of chitin, chitinase, and degradation of chitin and chitin-like substrates in saprophytic and pathogenic lifestyles of L. monocytogenes. This study offers a new direction for further exploration of the mechanisms of pathogenesis caused by L. monocytogenes.

Production of melanin pigments in saprophytic fungi in vitro and during infection.

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin-specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin-specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.

Characterization of an aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea with broad substrate specificity against aromatic alcohols.

OBJECTIVES: The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3_2). RESULTS: The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol. CONCLUSIONS: Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

The saprophytic fungus Fusarium solani increases the insecticidal efficacy of the entomopathogenic nematode Steinernema diaprepesi.

In two field surveys, high proportions of Galleria mellonella L. (Lepidoptera: Pyralidae) sentinel larval cadavers were infected by Fusarium solani without evidence of concomitant entomopathogenic nematode (EPN) or entomopathogenic fungus (EPF) reproduction. Because F. solani is not considered entomopathogenic, the survey suggested the possibility that F. solani competes with EPNs. We tested the hypotheses that F. solani attracts the EPN, Steinernema diaprepesi, to facilitate infection of Diaprepes root weevils (Diaprepes abbreviatus L.) and thereafter competes with the nematode in the insect cadaver. In two-choice olfactometer assays where one side was treated with F. solani mycelia and conidia, juvenile S. diaprepesi were attracted to the fungus, in either raw soil, or in autoclaved soil in the presence or absence of insects. However, this attraction was attenuated as the habitat became more complex, by using raw soil in combination with insect larvae. Fusarium oxysporum did not recruit the nematode. When soil microcosms were tested with F. solani conidia and S. diaprepesi, the concomitant infection increased the mortality of the insect (P = 0.02) to 83%, compared to 58% and 0% mortality when nematodes or fungi were individually applied, respectively. Concomitant inoculation also increased the number of cadavers that supported nematode reproduction and increased the population density of fungus in soil. The number of IJs entering the host insect was not affected by F. solani. These results support the possibility that F. solani can facilitate the insecticidal efficiency of S. diaprepesi in order to exploit the resources in the cadaver.

Identification of saprophytic and allergenic fungi in indoor and outdoor environments.

The airborne spores of some saprophytic and allergenic fungi such as Aspergillus, Alternaria, and Cladosporium are found throughout the world and exposure to these agents may result in various types of allergic diseases. The aim of this study, therefore, was to investigate the frequency of different saprophytic, allergenic, and pathogenic fungi in indoor and outdoor environments. During a 6-month period, 780 samples were obtained from a number of houses, mosques, parks, public restrooms, grocery stores, laboratories, and hospitals. An open-plate method was applied for air sampling by exposing 90-mm plates containing chloramphenicol/potato dextrose agar and malt extract agar were exposed to air for 30 min. Alternatively, the sampling from surfaces was performed using sterile wet swab and tape-stripe method. All samples were then inoculated in media and incubated at 28 °C for 2-3 weeks. The isolated fungi were purified in order to detect the genus, and if possible, species level of the targeted fungi based on morphological and microscopic features using standard methods. The findings revealed that the dominant indoor and outdoor fungal species were Aspergillus, Penicillium, and Cladosporium whose frequency values were 16.42%, 16.17%, and 14.92% respectively. The lowest frequency was related to Acrophialophora and Madurella (0.25%). More notably, the results for air and surface were similar. It was also found out that the three dominant genera were Aspergillus (16.53%), Penicillium (15.50%), and Cladosporium (11.93%), with Basidiobolus and Acrophialophora having the lowest frequency. It was observed that different environmental spaces have a great bearing on the spreading of such allergic agents, especially in subtropical humid climates.

[Laboratory confirmation of staging and volume of preventive measures at work under the conditions of microbial pollution of work environment air.]

Conceptual and methodological basics of the medical surveillance system with confirmed staging and regulated volume of laboratory studies on workers of diverse industrial sectors exposed to increased microbial load with opportunistic infections have been developed.

Fungal partner shifts during the evolution of mycoheterotrophy in Neottia.

PREMISE OF THE STUDY: Few previous studies have examined how mycobionts change during the evolution from autotrophy to mycoheterotrophy based on phylogenetic hypotheses. Neottia (Orchidaceae) comprises leafy species that are autotrophic and related leafless mycoheterotrophic species, and the phylogenetic relationships among them have been clarified. Accordingly, Neottia is a suitable taxon for investigating the question above. Here we clarified the diversity of mycobionts in Neottia plants and elucidated changes in the character of symbiotic associations during the evolution of mycoheterotrophy. METHODS: We sequenced the internal transcribed spacer (ITS) regions of nuclear ribosomal (nr) DNA for mycobionts of Neottia plants. Furthermore, we selected one representative DNA sample from each fungal operational taxonomic unit (OTU) and used it to amplify the large subunit (LSU) nrDNA sequences. Phylogenetic analyses of Sebacinales (basidiomycetes), the dominant mycobiont of Neottia, were conducted and sample-based rarefaction curves generated for the observed mycobiont richness on each OTU. KEY RESULTS: Leafy and leafless species in Neottia were associated with Sebacinales Group B and Sebacinales Group A, respectively. The composition and specificity level of fungal partners varied among Neottia species. CONCLUSIONS: Fungal partner composition and specificity level changed with speciation in both leafy and leafless Neottia species. In particular, mycorrhizal associations likely shifted from Sebacinales Group B to Group A during the evolution from autotrophy to mycoheterotrophy. Partner shifts to Sebacinales Group A have also been reported in the evolution of mycoheterotrophy of other plant groups, suggesting that convergence to this fungal group occurs in association with the evolution of mycoheterotrophy.

Massive turnover rates of fine root detrital carbon in tropical Australian mangroves.

Dead fine roots are the major component of organic carbon (C) stored in mangrove forests. We measured the mass and decomposition of fine root detritus in three mangrove forests along an intertidal gradient in tropical Australia to provide the first integrated estimates of the rate of turnover of fine root detritus. The grand mean dry masses of dead fine roots in the forests decreased in the order mid-intertidal Rhizophora (mean 28.4 kg m(-2)), low-intertidal Rhizophora (16.3 kg m(-2)) and high-intertidal Ceriops (mean 8.9 kg m(-2)), and were some of the highest on record. The first-order decay coefficients (day(-1)) for dead fine roots in the low Rhizophora, mid Rhizophora and high Ceriops forest sites were 0.0014, 0.0017 and 0.0007, respectively, and were the lowest on record. The estimated mean fluxes of C via decomposition of dead fine roots were very high in all forests, decreasing in the order mid Rhizophora (18.8 g C m(-2) day(-1)), low Rhizophora (8.4 g C m(-2) day(-1)) and high Ceriops (2.5 g C m(-2) day(-1)). There were relatively low levels of uncertainty in these estimates when all sources of error were considered. The fluxes of C for the two Rhizophora sites integrate all losses from saprophytic decay and leaching of dissolved C and were 50-200 % higher than the estimated total annual loss of C derived by summing rates of bacterial metabolism and export via groundwater and surface waters in these forests. The significant difference reflects both the very high dead root masses and the incorporation of the impact of fungi in our estimates.

The old 3-oxoadipate pathway revisited: new insights in the catabolism of aromatics in the saprophytic fungus Aspergillus nidulans.

Aspergilli play major roles in the natural turnover of elements, especially through the decomposition of plant litter, but the end catabolism of lignin aromatic hydrocarbons remains largely unresolved. The 3-oxoadipate pathway of their degradation combines the catechol and the protocatechuate branches, each using a set of specific genes. However, annotation for most of these genes is lacking or attributed to poorly- or un-characterised families. Aspergillus nidulans can utilise as sole carbon/energy source either benzoate or salicylate (upstream aromatic metabolites of the protocatechuate and the catechol branches, respectively). Using this cultivation strategy and combined analyses of comparative proteomics, gene mining, gene expression and characterisation of particular gene-replacement mutants, we precisely assigned most of the steps of the 3-oxoadipate pathway to specific genes in this fungus. Our findings disclose the genetically encoded potential of saprophytic Ascomycota fungi to utilise this pathway and provide means to untie associated regulatory networks, which are vital to heightening their ecological significance.