Clostridium perfringens epsilon toxin (ETX) is a potent pore-forming exotoxin responsible for severe enterotoxemia and necrotizing enterocolitis in ruminants. To elucidate the molecular mechanisms underlying ETX pathogenicity and attenuation, several site-directed mutants—ETX-R25A, ETX-F92A, ETX-Y133A, ETX-F206A, ETX-D210A, and ETX-G221A—were constructed based on structural analysis. Cytotoxicity assays revealed reduced virulence in ETX-Y133A, ETX-F92A, and ETX-F206A, with Y133A exhibiting the most significant attenuation. To further investigate the role of residue Y133, additional mutants (Y133E, Y133F, Y133S, Y133W, and Y133G) were generated. Selected mutants were evaluated for cytotoxicity, pathogenicity in BALB/c mice, and in vivo safety through histopathological analysis. Furthermore, their pore-forming ability, binding affinity to MDCK cells, and oligomerization properties were assessed. Results demonstrated that residue Y133 is critical for ETX activity, likely due to the necessity of its aromatic side chain for pore formation. In contrast, F92 and F206 appear to be involved in host-cell interactions via distinct mechanisms. These findings provide insights into ETX structure–function relationships and offer potential strategies for rational attenuation in vaccine development.

Rhizosphere engineering offers a promising strategy to improve crop productivity and soil health by optimizing plant-microbe interactions through targeted modulation of rhizosphere functioning. A key step in this approach is the effective recruitment and functional activation of inoculated plant growth-promoting rhizobacteria (PGPR), mainly driven by root exudate-mediated signaling. This study investigates the response of five phylogenetically diversified PGPR strains viz., Azotobacter chroococcum (Ac1), Azospirillum lipoferum (Az204), Pseudomonas chlororaphis (ZSB15), Bacillus altitudinis (FD48), and Pristia endophytica (NE14) to root exudates derived from three different rice cultivars (BPT5204, Co51, and Co55) at two developmental stages (45 and 75 days after sowing). Functional traits including growth, chemotaxis, biofilm formation, and cell wall degrading enzyme activity of rhizobacteria were assessed. The results revealed strain- and cultivar-specific modulation of these traits, with NE14 and FD48 showing significant upregulation of all the assessed traits in response to exudates from BPT5204 and Co51. Gas chromatography-mass spectrometry profiling of root exudates confirmed compositional differences between cultivars and developmental stages, highlighting key metabolite signals likely involved in regulating these bacterial functional traits. Principal component and correlation analyses identified cell wall-degrading enzymes and chemotaxis as major contributors to strain variability, underscoring their primary role in establishing rhizosphere competence. These findings strengthen the importance of functional trait-based screening for identifying PGPR strains with high adaptability to the rhizosphere environment. By demonstrating that root exudate-mediated modulation of PGPR traits can enhance bacterial colonization and functionality, this study offers a conceptual foundation and experimental framework for PGPR-mediated rhizosphere engineering.

Mycorrhizae contribute significantly for plant growth and development, providing nutrients such as phosphate and nitrogen, while also enhancing stress tolerance and resistance against phytopathogens. Plant symbiotic mycorrhizal networks harbour bacterial communities that aid mycorrhizal functions and augment plant nutrition and development. Various bacterial associations of mycorrhiza bring forth advantageous traits to mycorrhizal symbiosis and host colonization, which involves a wide range of signalling and regulatory molecules. These regulatory molecules play an important role in adapting and responding to new microenvironments with different hosts through the production of metabolites and expression of favourable genes. Small molecular components such as non-coding RNA (miRNA and sRNA) are also involved in the regulation and adaptation to these microenvironments. Regulatory pathways involving protein kinases play an important role in the tripartite association of bacteria and mycorrhiza with plants, and the subsequent promotion of symbiotic interactions. This mini-review highlights potential bacterial regulatory candidates which can influence mycorrhiza-plant interactions to significantly benefit plant growth and development. An understanding of these bacterial regulatory mechanisms may suggest new strategies for knowledge-based application in crop productivity improvement programs.

Withania somnifera (Ashwagandha), a vital medicinal plant, faces significant losses due to fungal diseases such as root rot, wilt, and leaf spot caused by Fusarium annulatum and Alternaria alstroemeriae. To manage these pathogens, metabolites of Cordyceps militaris were extracted following methods from Vinale et al. (2006) and others, with modifications. These metabolites were tested for antifungal efficacy using the poison food technique. Results showed the minimum inhibitory concentrations (MIC) against F. annulatum and A. alstroemeriae were 15 mg/mL and 20 mg/mL, respectively, with cidal effects observed at 20 mg/mL and 30 mg/mL. In-silico investigations revealed that Cordycepin, a metabolite, exhibited strong binding affinity to the fungal chitin synthetase protein. These findings suggest that C. militaris metabolites could serve as a potential alternative to synthetic fungicides, pending further research.

Monascus is a widely used natural microorganism in our country, which can produce useful secondary metabolites. Studies have shown that medium composition plays an important role in the primary and secondary metabolism of Monascus, especially the nitrogen source directly affects the growth, reproduction and secondary metabolites of Monascus. As a global transcriptional regulator of nitrogen metabolism, AreA gene is involved in the regulation of secondary metabolism. In this study, a MareA gene with high sequence homology to AreA gene was found by alignment analysis of the C100 genome. C100 knockout and overexpression mutants were obtained by Agrobacterium tumefaciens transformation. In this study, four nitrogen sources (Glutamine, Urea, NaNO 3, (NH 4) 2SO 4) were used to grow C100 and mutant strains. The yield of monascus rice under different nitrogen sources was compared by solid-state fermentation, and the types and yields of Mps produced were analyzed. HPLC and RT-qPCR were used to analyze the secondary metabolites of the three strains. Notably, growth differences between these monochorus strains were most pronounced when NaNO 3 medium was added. The fermentation product yield and gene expression level of the knockout strain were significantly lower than those of the C100 strain, while the opposite was observed for the overexpression strain. In conclusion, MareA gene had different regulatory effects on monochorus growth and metabolism under different nitrogen sources.

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.