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Monacolin K is a valuable secondary metabolite produced after a period of fermentation by Monascus purpureus; however, our current understanding of the regulatory mechanisms of its synthesis remains incomplete. This study conducted functional analysis on the key transcription factor, comp54181_c0, that is involved in the synthesis of monacolin K in Monascus. Mutant strains with either knockout or overexpression of comp54181_c0 were constructed using CRISPR/Cas9. A comparison between the knockout and overexpression strains revealed changes in fungal morphology and growth, with a significant increase in the production of Monascus pigments and monacolin K when comp54181_c0 was absent. Real-time fluorescence quantitative PCR analysis revealed that comp54181_c0 significantly influenced the transcription of key genes related to monacolin K biosynthesis in Monascus. In conclusion, our study elucidates the crucial role of comp54181_c0 in Monascus, enriches our understanding of fungal secondary metabolite development and regulation, and provides a foundation for the development and regulation of Monascus and monacolin K production.

During the cultivation of button mushrooms, the green mold epidemic, which causes a decrease in productivity, is a very important problem. The environmental harm of chemicals used in the control of such epidemics and the demand of consumers for organic products without chemicals have brought environmentally friendly biological control to the fore. Biological control can be achieved by the use of antagonistic microorganisms and their metabolites. In this study, the effectiveness of Bacillus and Pseudomonas spp. for the biological control of the green mold disease agent Trichoderma aggressivum subsp. in Agaricus bisporus cultivation was examined. For this purpose, the antifungal effects of Bacillus and Pseudomonas spp. against T. aggressivum strains were examined by an in vitro dual culture test and the agar well diffusion method. Then, it was determined whether the bacterial strains showing antifungal activity showed antagonistic activity against A. bisporus. Although none of the Pseudomonas spp. showed antifungal activity against T. aggressivum strains, most of the Bacillus spp. were found to have high activity. It has been concluded that Bacillus sp. Ö-4-82, which shows high antifungal activity against T. aggressivum subsp. and low antagonism against A. bisporus, may be potential biological control agents for button mushroom cultivation.

The present study aimed to identify arsenic (As)-resistant bacterial strains that can be used to mitigate arsenic stress. A bacterium Bacillus mycoides NR5 having As tolerance limit of 1100 mg L -1 was isolated from Nag River, Maharashtra, India. It was also equipped with plant growth-promoting (PGP) attributes like P solubilization, siderophores, ammonia and nitrate reduction, with added antibiotic tolerance. Further, Scanning Electron Microscopy (SEM) and Transmission Electron Micrograph (TEM) suggested biosorption as possible mechanisms of arsenic tolerance. A strong peak in FTIR spectra at 3379.0 corresponding to amine in As-treated NR5 also indicated metal interaction with cell surface protein. Amplification of the arsenic reductase gene in NR5 further suggested intracellular transformation of As speciation. Moreover, the As tolerance capability of NR5 was shown in spinach plants in which the bacterium effectively mitigated 25ppm As by the production of defence-related proline molecules. Evidence from SEM, TEM and FTIR, concluded biosorption is possibly the primary mechanism of As tolerance in NR5 along with the transformation of arsenic. B. mycoides NR5 with PGP attributes, high As tolerance and antibiotic resistance mediated enhanced As tolerance in spinach plants advocated that the strain can be a better choice for As bioremediation in contaminated agricultural soil and water.

Duddingtonia flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and non-dormant chlamydospore of D. flagrans to investigate the differences in cAMP and protein content between the two types of spores. In this study, cAMP and soluble proteins were extracted from the non-dormant chlamydospore and dormant chlamydospore of D. flagrans isolates SDH035 and DH055, respectively. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in non-dormant spores (p<0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of non-dormant spores (p<0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of non-dormant spores, but the difference was not significant (p>0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus D. flagrans.

Phlebia genus is a relevant group of fungi with crucial role in numerous ecosystems. In tropical and subtropical areas this genus allows the efficient degradation of lignin and carbon recovery; however, the majority of these fungal species remains undiscovered. The main purpose of this work was to determine the enzymatic activity of extracellular proteins of a novel Phlebia floridensis strain isolated in Yucatan Peninsula, Mexico. The results that are reported here demonstrate that soluble protein extract of P. floridensis can degrade a broad spectrum of recalcitrant compounds. This induced protein extract is able to modify not only phenolic and non-phenolic compounds, but also anthroquinone dyes, even without addition of exogenous hydrogen peroxide. Using LC-MS/MS, we were able to identify a novel chloroperoxidase in enzymatic extract. As far as we know, this is the first report about the presence of this type of enzyme in Phlebia genus.

Salmonella Typhimurium (STM) is a facultative anaerobe of zoonotic importance and one of the causative agents of non-typhoidal salmonellosis (NTS). During infection, STM must adapt to the changes in oxygen concentration encountered in the crucial niches of host like gut lumen and intramacrophage environments. But being a chemo-organoheterotroph, STM is capable of obtaining its energy from organic sources via redox reactions. NarL, a transcription factor and the response regulator of the two-component regulatory system NarX/L, gets activated under nitrate rich anaerobic condition. Upon activation, it upregulates the nitrate reduction during anaerobic respiration. However, in this study, we observed a significant attenuation of virulence in the narL-knockout strain of STM, while the respective morphotypes got rescued upon genetic complementation. Along with motility and biofilm forming ability, the mutant strain displayed reduced intracellular replication in either intestinal epithelial cells or monocyte-derived macrophages of poultry origin. Further, in vivo competitive assay in the murine model showed that wild type STM significantly outcompeted its isogenic narL null mutant.

The aim of this study was to investigate the production, stability and applicability of colorants produced by filamentous fungi isolated from soil samples from the Amazon. Initially, the isolates were evaluated in a screening for the production of colorants. The influences of cultivation and nutritional conditions on the production of colorants by fungal isolates were investigated. The colorants produced by selected fungal isolates were chemically characterized using the LC-MS technique. The antimicrobial and cytotoxic activities, stability evaluation and applicability of the colorants were investigated. As results, we observed that the isolates Penicillium sclerotiorum P3SO224, Clonostachys rosea P2SO329 and Penicillium gravinicasei P3SO332 stood out since they produced the most intense colorants. Compounds produced by Penicillium sclerotiorum P3SO224 and Clonostachys rosea P2SO329 were identified as sclerotiorin and penicillic acid. The colorant fraction (EtOAc) produced by these species has antimicrobial activity, stability at temperature and at different pHs, stability when exposure to light and UV, and when exposed to different concentrations of salts, as well as being non-toxic and having the ability to dye fabrics and be used as a pigment in creams and soap. Considering the results found in this study, it was concluded that fungi from the soil in the Amazon have the potential to produce colorants with applications in the textile and pharmaceutical industries.

The morphological and structural differences of different types of chlamydospore of Duddingtonia flagrans, a nematophagous fungus, were studied under light microscope and electron microscope to provide reference for the biological control of parasitic nematodiasis. In this study, D. flagrans isolate F088 dormant chlamydospore and non-dormant chlamydospore were selected as the research objects. The structural differences of these spores were observed by optical microscopy through lactol cotton blue, Trypan blue and MTT staining. FUN-1, DAPI and CFW staining were used to observe the metabolic activity, cell wall and nucleus differences of the two types of spores under fluorescence microscope. Ultrastructure of the two kinds of spores was observed using scanning electron microscope (SEM) and transmission electron microscope (TEM). Since lacto phenol cotton blue, trypan blue staining cannot distinguish dormant spores from dead spores, MTT assay was performed. Fluorescence microscopy observation showed that the cytoplasmic metabolic activity of non-dormant spores was stronger than that of dormant spores. The nucleus of dormant spores was bright blue, and their fluorescence was stronger than that of non-dormant spores. The cell wall of non-dormant spores produced stronger yellow-green fluorescence than that of dormant spores. Ultrastructural observation showed that there were globular protuberances on the surface of the two types of spores, but with no significant difference between them. The inner wall of dormant spore possesses a thick zona pellucida with high electron density which was significantly thicker than that of non-dormant spores, and their cytoplasm is also changed. In this study, the microstructure characteristics of dormant and non-dormant chlamydospores of D. flagrans fungi were preliminarily clarified, suggesting that the state of cell wall and intracellular materials were changed after spores entered to dormancy.

Endophytic fungi are an important source of novel antitumour substances. Previously, we isolated an endophytic fungus, Alternaria alstroemeria, from the medicinal plant Artemisia artemisia, whose crude extracts strongly inhibited A549 tumour cells. We obtained a transformant, namely AaLaeAOE26, which completely loses its antitumour activity due to overexpression of the global regulator AaLaeA. Re-sequencing analysis of the genome revealed that the insertion site was in the non-coding region and did not destroy any other genes. Metabolomics analysis revealed that the level of secondary antitumour metabolic substances was significantly lower in AaLaeAOE26 compared to the wild strain, in particular flavonoids were more downregulated according to the metabolomics analysis. A further comparative transcriptome analysis revealed that a gene encoding FAD -binding domain protein (Fla1) was significantly downregulated. On the other hand, overexpression of AaFla1 led to significant enhancement of antitumor activity against A549 with a 7-fold higher inhibition ratio than the wild strain. At the same time, we also found a significant increase in the accumulation of antitumour metabolites including quercetin, gitogenin, rhodioloside, liensinin, ginsenoside Rg2 and cinobufagin. Our data suggest that the global regulator AaLaeA negatively affects the production of antitumour compounds via controlling the transcription of AaFla1 in endophytic Alternaria alstroemeria.

Ralstonia solanacearum is a rod-shaped phytopathogenic bacterium that causes lethal wilt disease in many plants. On solid agar growth medium, in the early hour of the growth of the bacterial colony, the type IV pili-mediated twitching motility, which is important for its virulence and biofilm formation, is prominently observed under the microscope. In this study, we have done a detailed observation of twitching motility in R. solanacearum colony. In the beginning, twitching motility in microcolonies was observed as a density-dependent phenomenon that influences the shape and sizes of the microcolonies. No such phenomenon was observed in Escherichia coli, where twitching motility is absent. In the early phase of colony growth, twitching motility exhibited by the cells at the peripheral region of the colony was more prominent than the cells towards the centre of the colony. Using a time scale photography and merging those into a video, twitching motility was observed as an intermittent phenomenon that progresses in layers in all directions as finger-like projections at the peripheral region of a bacterial colony. Each layer of bacteria twitches on top of the other and produces a multi-layered film-like appearance. We found that the duration between the emergence of each layer diminishes progressively as the colony becomes older. This study on twitching motility demonstrates distinctly heterogeneity among the cells within a colony regarding their dynamics and the influence of microcolonies on each other regarding colony shape and size.

Indiscriminate use of antibiotics has led to the emergence of antibiotic resistant microbes and the loss of natural flora in aquaculture systems ultimately necessitating the ban of many of the chemotherapeutants in aquaculture. Actinobacteria play a profound role in the biogeochemical cycling in the marine environment and they represent the principal source of secondary metabolites with antimicrobial property. In the present study, 98 marine derived actinomycete isolates were screened for antimicrobial activity against the common aquatic pathogens. A potent actinomycete isolate S26, identified as Streptomyces variabilis based on 16S rRNA gene sequencing was used for further study. Optimization of the fermentation medium for secondary metabolite production was carried out by response surface methodology (RSM) using DESIGN EXPERT. The ANOVA of the quadratic regression model demonstrated that the model was highly significant for the response concerned i.e., antimicrobial activity as evident from the Fisher’s F- test with a very low probability value [(P model>F) = 0.0001]. Of the 10 different solutions suggested by the software, the most suitable composition was found to be starch, 1.38 %; soy powder, 0.88 %; ammonium sulphate, 0.16 % and salinity, 27.76 ‰. S. variabilis S26 cultured in the optimized production medium was applied in the Penaeus monodon larval rearing system and the total Vibrio count and survival rate were estimated. S. variabilis S26 treatment showed a significant reduction in Vibrios and better survival in the Penaeus monodon culture system compared to the control.

The increasing growth of agro-industrial activity resulting in excessive amounts of agri-waste has led to the accumulation of a large quantity of lignocellulosic residues all over the world, in particular, deforestation initiatives for the removal of invasive trees in South Africa. These lignocellulosic residues are rich in energy resources consisting of a mixture of natural polymers based on lignin, cellulose and hemicellulose. The use of lignolytic fungi such as mushrooms in solid-state fermentation could sufficiently degrade the indigestible lignocellulosic components and add medicinal and nutritional value to otherwise unusable, high-energy waste material. The digestive type of animal for which the potential feed is developed must be identified and considered before deciding on the bioconversion method and process, since the outcomes for obtaining potentially high-quality feeds for non-ruminant and ruminant animals are different. The current study presents the data of the bioconversion of lignocellulosic substrate using solid-state fermentation with the edible and medicinal mushrooms, Ganoderma lucidum, Pleurotus ostreatus, and a possible new species, to increase digestibility and nutritional value to be applied as ruminant animal feed. The solid-state fermentation process was optimised and the resulting product analysed for the degradation of the lignocellulosic components. Results indicated that the solid-state fermentation duration and mushroom species were key components in achieving significant degradation. Data obtained after 18 weeks of degradation indicated a significant (p < 0.05) reduction in the acid detergent fibre, acid detergent lignin and neutral detergent fibre fractions of the biomass, with up to 20% reduction in indigestible components.

An underutilized experimental design was used to isolate adapted mutants of the model bacterium Pseudomonas putida KT2440. The experimental design consisted of subjecting a random pool of mini-Tn5 mutants of P. putida KT2440 to several rounds of selection in the rhizosphere of soybean irrigated with NaCl solution. Isolated adapted mutants (MutAd) showed a mutation in a gene encoding the membrane-binding protein LapA, which is involved in the early stages of biofilm formation on abiotic surfaces. Two MutAd bacteria (MutAd160 and MutAd185) and a lapA deletion mutant were tested to study the effect of this gene on salt tolerance, rhizosphere fitness, extracellular polymeric substances (EPS) production, and plant growth promotion. The inability of the mutants to form biofilm did not hinder attachment to soybean seeds and roots. MutAd bacteria showed an overproduction of EPS when grown under saline conditions, which would compensate for the lack of biofilm formation. MutAd185 bacteria showed increased root attachment and growth promotion of soybean in slightly saline soils. The proposed experimental design would be useful to accelerate bacterial adaptation to the rhizosphere of plants under a given environmental condition, identify genetic mutations that benefit bacterial fitness in that condition, and thus increase their ability to promote plant growth.

Aflatoxin and other mycotoxin contamination are major threats to global food security and present an urgent need to secure the global food crop against spoilage by mycotoxigenic fungi. Cocoa material is noted for naturally low aflatoxin contamination. This study was designed to assess the potential for harnessing cocoa-associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus. The candidate fungi were isolated from fermented cocoa beans collected from four cocoa-growing areas in Ghana. Molecular characterization included ITS-sequencing for identification and PCR to determine mating type. Effects of the candidate isolates on growth and aflatoxin-production by an aflatoxigenic A. flavus isolate (BANGA1) were assessed. Aflatoxin production was monitored by UV fluorescence and quantified by ELISA. Thirty-six filamentous fungi were cultured and identified as Aspergillus, Cladosporium, Lichtheimia or Trichoderma spp. isolates. The isolates generally interacted negatively with BANGA1 growth and aflatoxin production. The A. niger and A. aculeatus biocontrol candidates showed the strongest colony antagonism (54–94%) and reduction in aflatoxin production (12–50%) on agar. In broth, the A. niger isolates reduced aflatoxin production by up to 97%. Metabolites from the A. niger isolates showed the strongest inhibition of growth by BANGA1 and inhibited aflatoxin production. Four of the candidate isolates belonged to the MAT1-1 mating type and 12 identified as MAT1-2. This may be indicative of the potential for genetic recombination events between fungi in the field, and finding which is particularly relevant to the risk posed by A. flavus biocontrol measures that rely on atoxigenic A. flavus strains.

Background Plant mediated induced systemic resistance against the plant-parasitic nematode, M. incognita infecting tomato cv Pusa Ruby was evaluated on application of four nematicidal rhizobacterial isolates (Bacillus subtilis, B. pumilus, B. megaterium and B. cereus) as elicitors and compared with the application of a chemical nematicide, Velum Prime. Methods The bioefficacy trial was conducted in pots preinoculated with the above isolates followed by M. incognita inoculation tomato to observe the reduction in nematode infection at 60 days and effect on photosynthetic and transpiration rates. The mechanism of induced resistance was assessed using qRT-PCR for quantification of three key defense genes(PR-1b, JERF3 and CAT) at 0,2,4,8 and 16 days after inoculation (DAI). The defense enzymes viz., super oxide dismutase(SOD), polyphenol oxidase(PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) were quantified. Results Significant reduction in per cent root galling viz. 84.21 in B. pumilus, 83.70 in B. megaterium, 91.95 in B. subtilis, 81.8 in B. cereus, was observed compared to control. The reproduction factor was the lowest (15.83) in B. subtilis, followed by B. pumilus (21.00), compared to 48.16 in control, with enhanced photosynthetic and transpiration rate. The defense genes, PR-1b, JERF3 and CAT were expressed at 2.5 to 7.5 folds in rhizobacterial treated plants, but not in Velum Prime treatment. The increase in enzyme levels (μmol/min/mg) for SOD was from 1.5 to 17.5, PPO from 2.1 to 7.8, PO from 1.8 to 10.2, and PAL from 1.8 to 8.7 during 0 to 16 DAI.

The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. It is worth exploring the genetic function of C. neoformans, by which we may derive more antifungal strategies. Therefore, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. The RNAi system used pSilencer 4.1-CMV neo plasmid and short hairpin RNA to realize the effective transcriptional suppression. The CRISPR-Cas9 system used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, qRT-PCR, Transmission Electron Microscope (TEM) and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA were more powerful and lasted longer. The CRISPR-Cas9 system constructed an albino strain completely without the ability to produce melanin. Considering the weakening of transcriptional suppression, we recommend using a long loop for the RNAi system and 1st or 2nd passage of knockdown strains for the subsequent studies. Besides, the different capacities of melanin production might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, we recommend applying the PNK003 vectors to other serotypes of C. neoformans for quick screening of possible trait-regulating genes because of its easy construction and valid knockout effect.

Surfactin is a signal to trigger biofilm formation against harsh environments. Generally, harsh environments can result in change of the cellular redox state to induce biofilm formation, but we know little about whether the cellular redox state influences biofilm formation via surfactin. Here, the reductant glucose could reduce surfactin and enhance biofilm formation by a surfactin-independent way. The oxidant H2O2 led to a decrease of surfactin accompanying with weakened biofilm formation. Spx and PerR were both necessary for surfactin production and biofilm formation. H2O2 improved surfactin production but inhibited biofilm formation by a surfactin-independent manner in Δspx, while it reduced surfactin production without obvious influence on biofilm formation in ΔperR. The ability against H2O2 stress was enhanced in Δspx, but weakened in ΔperR. Thereby, PerR was favorable for resisting oxidative stress, while Spx played a negative role in this action. Knockout and compensation of rex also supported that the cells could form biofilm by a surfactin-independent way. Collectively, surfactin is not a unique signal to trigger biofilm formation, and the cellular redox state can influence biofilm formation by a surfactin- dependent or independent way in B. amyloliquefaciens.