Search Results (52)

The smut fungus Thecaphora frezzii causes severe yield losses in peanuts (Arachis hypogaea) in Argentina. Previous work established its fully intracellular biotrophic progression through subterranean organs and its exclusive sporulation within the seed coat, yet the ontogeny of teliospore formation in planta remained unresolved. Here, we applied a pragmatic correlative multiscale microscopy approach based on serial paraffin sections examined by stereomicroscopy, light microscopy, confocal laser scanning microscopy, and scanning electron microscopy, enabling spatial correlation of fungal structures within their tissue context. Using this integrative framework, we characterized the organization and progression of sporogenic structures associated with teliosporogenesis. Teliosporogenesis proved to be tightly synchronized with host tissue context and seed developmental stage, and was consistently preceded by a marked reorganization of sporogenous hyphae into three-dimensional coiled hyphal aggregates embedded in a mucilaginous matrix. These precursors undergo hyphal fragmentation followed by central–peripheral differentiation, whereby a small number of central units enlarge and individualize into teliospore initials while peripheral elements collapse, yielding stable teliospore balls as the final sporogenic product. This developmental sequence defines a distinct ontogenetic pattern not captured by current schemes of sporogenesis, here designated the Teliospore-ball type. Our results clarify the developmental pathways of T. frezzii sporulation in planta and demonstrate how accessible multiscale microscopy can be used to integrate structural information across spatial scales in complex plant–fungus interactions. Full article

Two fermentations in skim milk were used to evaluate the effectiveness of the bacteriocin Thermophilin-T produced “in situ” by Streptococcus thermophilus ACA-DC 0040 in inhibiting spore outgrowth of Clostridium sporogenes ACA-DC 3888 under conditions simulating Graviera cheese manufacture and ripening. In the experimental fermentation, S. thermophilus ACA-DC 0040 and Clostridium spores were used. In the control fermentation, a non-bacteriocin-producing S. thermophilus ACA-DC 004 strain and Clostridium spores were used. The temperature program used and the pH changes were similar to those observed in Graviera cheese production and ripening. Clostridium spore counts and organic acids were measured during both fermentations. In the experimental trial, bacteriocin production by S. thermophilus ACA-DC 0040 reduced Clostridium spores by 1.79 log units during the ripening phase. Conversely, the Clostridium spore count significantly increased in the control trial from the 15th day onward by about 2.9 log units and showed elevated levels of acetic, propionic, and butyric acids, along with decreased lactic acid, which is clearly linked to the “Late Blow Defect” profile. The results of our study on the inhibition of C. sporogenes spore outgrowth provide useful information for establishing an effective biological control system, in addition to other barriers used during Graviera cheese manufacturing. Full article

Globally, large quantities of animal waste and human sewage sludge are generated annually. Their application as soil amendments can enhance soil quality and support a circular economy. However, these wastes may harbour pathogenic bacteria, posing contamination risks to soil and water and potential transmission to animals and humans. This study investigated the survival of five bacterial pathogens during six months of storage in five types of organic waste and following their subsequent application to soil. During storage, T₉₀ values ranged as follows: Salmonella Typhimurium (2.3–17.7 days), Campylobacter jejuni (0 to 23.9 days), Escherichia coli O157:H7 (4.3 to 57.8 days), and Listeria monocytogenes (1.9 to 170.4 days). In soil, T₉₀ values were S. Typhimurium (4.2 to 17.4 days), C. jejuni (4.8 to 26.8 days), E. coli O157:H7 (4.3 to 52.9 days), and L. monocytogenes (2 to 83.7 days). Clostridium sporogenes remained stable throughout both experiments, preventing T₉₀ calculation. Contrary to our initial hypothesis that soil microbiota would accelerate pathogen decline, T₉₀ values were higher during storage in 11 cases and higher in soil in nine scenarios. These findings highlight the need for pre-treatment strategies for animal waste and biosolids before land spreading to consistently mitigate risks of pathogen transmission and environmental contamination. Full article

TCP transcription factors have long been known to play a crucial role in leaf development, but their significance in reproduction has recently been revealed. TCP5 is a member of class II of the TCP family, which predominantly regulates cell differentiation. This study used overexpression and SRDX fusion to evaluate the role of TCP5 in anther development. TCP5 overexpression resulted in lower fertility, primarily due to anther non-dehiscence. We also observed reduced lignin accumulation in the anther endothecium. In addition, TCP5 overexpression resulted in smaller anthers with fewer pollen sacs and pollen due to early-anther defects before meiosis. TCP5 showed expression in early anthers, including the epidermis, endothecium, middle layer, tapetum, sporogenous cells (pollen mother cells), and vascular bundles. Conversely, during meiosis, the TCP5 signal was only detected in the tapetum, PMCs, and vascular bundles. The TCP5 signal disappeared after meiosis, and no signal was observed in mature anthers. Interestingly, the TCP5-SRDX transgenic plants were also sterile, at least for the early-arising flowers, if not all of them. TCP5-SRDX expression also resulted in undersized anthers with fewer pollen sacs and pollen. However, the lignin accumulation in most of these anthers was comparable to that of the wild type, allowing these anthers to open. The qRT-PCR results revealed that several genes associated with secondary cell wall thickening had altered expression profiles in TCP5 overexpression transgenics, which supported the non-dehiscent anther phenotype. Furthermore, the expression levels of numerous critical anther genes were down-regulated in both TCP5 overexpression and TCP5-SRDX plants, indicating a comparable anther phenotype in these transgenic plants. These findings not only suggest that an appropriate TCP5 expression level is essential for anther development and plant fertility, but also improve our understanding of TCP transcription factor functioning in plant male reproduction and contribute information that may allow us to manipulate fertility and breeding in crops. Full article

Bacterial vectors for biomolecule delivery to targeted organelles, facilitating temporary or continuous protein production, have emerged as a promising approach for treating acquired and inherited diseases. This method offers a selective cancer eradication and targeting strategy with minimal side effects. Bacterial vectors provide an alternative to viral gene delivery, given their capacity to deliver large genetic materials while inducing minimal immunogenicity and cytotoxicity. Bacteria such as Bifidobacterium, Salmonella, Clostridium, and Streptococcus have demonstrated potential for tumor-targeted biomolecule delivery or serve as oncolytic bacteria. These vectors have also been used to transfer and amplify genes encoding biomolecules such as pro-drug-converting enzymes, toxins, angiogenesis inhibitors, and cytokines. The microenvironment of necrotic tumors offers a unique opportunity for targeted therapy with the non-pathogenic anaerobic bacterium. For example, Clostridium sporogenes can germinate selectively in the necrotic regions upon injection as endospores, which helps to enhance the specificity of Clostridium sporogenes, resulting in tumor-specific colonization. Also, E. coli and Salmonella sp. can be capacitated with a hypoxic sensing promotor gene for specificity delivery into the core region of solid tumors. The uniqueness of the tumor microenvironment, including hypoxia, immunosuppression, metabolite deficiency or enrichment, and necrosis, selectively enables bacteria in the tumor. Combining traditional cancer therapy with bacterial therapy will significantly complement and cover the limitations of other treatments. This review provides an overview of the use of the bacteria vector in cancer therapy, discussing strategies to maximize delivery efficiency and address potential challenges. In this review, we discuss the potential of bacteria vectors as anti-cancer therapeutics while focusing on therapeutic delivery strategies. We highlight the complementary use of bacteria therapy with other cancer therapies and the mechanism of bacteria cancer immunotherapy with limitations and perspectives for future use. Full article

Clostridial fermentation is the determining process causing the spoilage of direct-cut alfalfa silage, and the application of lactic acid bacteria (LAB) inoculant is considered as the most promising technology for inhibiting clostridial fermentation. In order to screen target-based LAB strains, identification and correlation analysis of key Clostridia and LAB species in alfalfa silage were conducted in this study. Three alfalfa cultivars (Sanditi, SD; Celsius, CE; SW5909, SW) were harvested at the early bloom stage and ensiled without (CK) or with LAB inoculant (LB) and sucrose (SC) for 60 d. Single-molecule real-time sequencing was used to identify dominant Clostridia and LAB species, and LAB with significant inhibitory effects on dominant Clostridia was screened via correlation network analysis. The results showed that silages CK and LB encountered severe clostridial fermentation as indicated by large amounts of butyric acid (BA) and ammoniacal nitrogen (NH₃-N) production. Compared to silages CK and LB, SC treatment decreased (p < 0.05) BA and NH₃-N concentrations, as well as decreasing (p < 0.05) the bacterial community indexes of Shannon and Chao1. Lactiplantibacillus pentosus was the first dominant LAB in silage CK of alfalfa SD and CE. The first dominant LAB in silage LB was also identified as L. pentosus, rather than Lentilactobacillus buchneri and Lactiplantibacillus plantarum in the used inoculant. L. buchneri became more abundant in silage SC of alfalfa SD and CE, accounting for the high fermentation quality of these silages. Clostridium tyrobutyricum, Clostridium luticellarii, Garciella sp._GK3, Clostridium sporogenes, Clostridium perfringens, and Clostridium sp._BTY5 were the most dominant Clostridia species in alfalfa silage. Furthermore, Enterococcus faecalis, L. buchneri, and L. pentosus exhibited significant (p < 0.05) inhibitory effects on C. tyrobutyricum, C. luticellarii, and Garciella sp._GK3, respectively, which were the top three Clostridia species associated with clostridial fermentation. In conclusion, E. faecalis, L. buchneri, and L. pentosus were screened and can be used as potential LAB inoculants for the targeted inhibition of clostridial fermentation. Full article

Probiotics are live microbiota that can confer their hereditary health benefits upon the host. They can positively alter the diversity of the host’s gut microbiota population. Bacillus clausii is a spore-producing potential probiotic. Its application in the food industry has been highly recommended. Clausii spores are greatly resistant to harsh food processing treatment (heat and industrial pressure) and they can maintain their physiological traits (acid, bile salt) inside the human gut. The utilization of spores can enhance the nutritional viability of livestock, as well as the functionality of on-shelf products. This contemporary review covers the great attributes of B. clausii, including physiological characteristics, modes of action, probiotics benefits, a safety assessment, and the bacteria food industry applications for the purposes of producing enhanced functional foods known as probiotic foods. To our knowledge, although B. clausii has been widely applied in food industry, the amount of the literature that is dedicated to its role in sustainable food production and addresses its potential sporogenous probiotic behavior is fewer as compared to non-sporogenous lactic acid bacteria (Lactobacillus spp. and Bifidobacterium spp.). In our review, we also discovered a lack of empirical evidence on the synbiotic and synergistic behavior of clausii in combination with other active nutrients. Therefore, our review suggests that more studies should be conducted on the potential probiotic contribution of B. clausii in sustainable food production and its synergistic and synbiotic behavior in conjunction with other active nutrients. Full article

The cluster of genes determining the production of botulinum toxins is an attribute of not only the Clostridium botulinum species. This cluster is also found in other members of the Clostridium genus, such as C. baratii, C. butyricum, and C. sporogenes. The occurrence of a botulinum-like cluster has also been recorded in strains of other genera, i.e., Enterococcus faecium, as well as in a Gram-negative species isolated from freshwater sediments; however, the biological activity of bont-related genes has not been noted. It can be said that the mentioned species have a dual nature. Another species with a dual nature is C. butyricum. This bacterium is a common human and animal gut commensal bacterium and is also frequently found in the environment. Although non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. Additionally, C. baratii strains are rare opportunistic pathogens associated with botulism intoxication. They have been isolated from food and soil and can be carried asymptomatically or cause botulism outbreaks in animals and humans. In addition to the mentioned clostridia, the other microorganisms considered as non-toxigenic have also been suspected of carrying botulinum cluster Gram-negative bacteria, such as Chryseobacterium piperi isolated from freshwater sediments; however, the biological activity of bont-related genes has not been noted. Additionally, Enterococcus faecium strains have been discovered carrying BoNT-related clusters (BoNT/En). Literature data regarding the heterogeneity of BoNT-producing strains indicate the requirement to reclassify C. botulinum species and other microorganisms able to produce BoNTs or possess botulinum-like gene clusters. This article aims to show the dual nature of Clostridium strains not belonging to the C. botulinum species that are sporadically able to carry bont clusters, which are usually considered saprophytic and even probiotic, and bont-like clusters in microorganisms from other genera. The aim was also to consider the genetic mechanisms of botulinum cluster expression in strains that are considered opportunistic and the microbiological safety aspects associated with their occurrence in the environment. Full article

Late blowing defects in semi-hard and hard cheeses caused by spore-forming clostridia (e.g., Clostridium tyrobutyricum, Clostridium butyricum, Clostridium sporogenes) pose a major issue for the dairy industry. With this study, we applied a multiplex PCR for the rapid and simultaneous detection of clostridia in raw milk for cheese production. Spore detection in milk usually relies on culture-dependent methods, among which the most probable number (MPN) technique is sensitive but time-consuming and nonspecific. We tested two PCR-based protocols: the one entailed direct milk analysis with results obtained within 24 h; the other included an enrichment step and gave results within 72 h. The second protocol was found to be more sensitive; it detected concentrations as low as 100 cells/L for C. sporogenes and C. butyricum and 800 cells/L for C. tyrobutyricum. Both protocols were applied to field samples (211 samples underwent protocol no. 1; 117 samples underwent protocol no. 2) collected from four dairy processing plants in Piedmont. The prevalence of C. butyricum (protocol no. 1: 9.5%; protocol no. 2: 23%) was higher than either C. sporogenes (0%; 9.4%) or C. tyrobutyricum (0%; 6.8%). Protocol no. 2 detected multiple targets in eight samples, indicating that more than one microorganism was present. Our findings underscore the importance of implementing preventive measures and early detection strategies to mitigate the risk of cheese spoilage due to clostridial contamination. Full article

This study utilized Aspergillus flavus to produce selenium nanoparticles (Se-NPs) in an environmentally friendly and ecologically sustainable manner, targeting several medicinal applications. These biosynthesized Se-NPs were meticulously characterized using X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, transmission electron microscope (TEM), and UV–visible spectroscopy (UV), revealing their spherical shape and size ranging between 28 and 78 nm. We conducted further testing of Se-NPs to evaluate their potential for biological applications, including antiviral, anticancer, antibacterial, antioxidant, and antibiofilm activities. The results indicate that biosynthesized Se-NPs could be effective against various pathogens, including Salmonella typhimurium (ATCC 14028), Bacillus pumilus (ATCC 14884), Staphylococcus aureus (ATCC 6538), Clostridium sporogenes (ATCC 19404), Escherichia coli (ATCC 8739), and Bacillus subtilis (ATCC 6633). Additionally, the biosynthesized Se-NPs exhibited anticancer activity against three cell lines: pancreatic carcinoma (PANC1), cervical cancer (Hela), and colorectal adenocarcinoma (Caco-2), with IC50 values of 177, 208, and 216 μg/mL, respectively. The nanoparticles demonstrated antiviral activity against HSV-1 and HAV, achieving inhibition rates of 66.4% and 15.1%, respectively, at the maximum non-toxic concentration, while also displaying antibiofilm and antioxidant properties. In conclusion, the biosynthesized Se-NPs by A. flavus present a promising avenue for various biomedical applications with safe usage. Full article

Botrytis cinerea is a necrotrophic fungus that causes considerable economic losses in commercial crops. Fungi of the genus Botrytis exhibit great morphological and genetic variability, ranging from non-sporogenic and non-infective isolates to highly virulent sporogenic ones. There is growing interest in the different isolates in terms of their methodological applications aimed at gaining a deeper understanding of the biology of these fungal species for more efficient control of the infections they cause. This article describes an improvement in the protoplast production protocol from non-sporogenic isolates, resulting in viable protoplasts with regenerating capacity. The method improvements consist of a two-day incubation period with mycelium plugs and orbital shaking. Special mention is made of our preference for the VinoTaste Pro enzyme in the KC buffer as a replacement for Glucanex, as it enhances the efficacy of protoplast isolation in B459 and B371 isolates. The methodology described here has proven to be very useful for biotechnological applications such as genetic transformations mediated by the CRISPR/Cas9 tool. Full article

Botrytis cinerea, a necrotrophic fungus responsible for grey rot disease, causes substantial economic losses. However, recent studies have discovered distinct non-sporogenic and non-infective isolates of this species, such as the B459 field strain. Examination of these isolates is particularly intriguing in the context of the development of methodological applications that could be useful in the biocontrol of this phytopathogenic species. This investigation revealed that B459 exhibited a reduced growth rate yet displayed enhanced resilience to stressors like oxidative stress agents, SDS, ethanol, and PhITC. Notably, its ability to generate reactive oxygen species (ROS) and acidic compounds, crucial in plant–pathogen interactions, was impacted. Bio-targeted toxin identification assays and the metabolomic study of extracts obtained from fermentations at seven and fourteen days revealed that this strain does not biosynthesize botrydial and derivatives neither botcinin family toxins. Furthermore, its inability to infect tomato fruits, grape, and gerbera petals coincides with its lack of toxin production under culture conditions typically adapted for reference strain B05.10. Full article

The search for natural food additives makes propolis an exciting alternative due to its known antimicrobial activity. This work aims to investigate propolis’ behavior as a nitrite substitute ingredient in cooked ham (a ready-to-eat product) when confronted with pathogenic microorganisms of food interest. The microbial evolution of Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Clostridium sporogenes inoculated at known doses was examined in different batches of cooked ham. The design of a challenge test according to their shelf life (45 days), pH values, and water activity allowed the determination of the mesophilic aerobic flora, psychotropic, and acid lactic bacteria viability. The test was completed with an organoleptic analysis of the samples, considering possible alterations in color and texture. The cooked ham formulation containing propolis instead of nitrites limited the potential growth (δ < 0.5 log₁₀) of all the inoculated microorganisms until day 45, except for L. monocytogenes, which in turn exhibited a bacteriostatic effect between day 7 and 30 of the storage time. The sensory analysis revealed the consumer’s acceptance of cooked ham batches including propolis as a natural additive. These findings suggest the functionality of propolis as a promising alternative to artificial preservatives for ensuring food safety and reducing the proliferation risk of foodborne pathogens in ready-to-eat products. Full article

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements. Full article

Contamination of milk and dairy products with pathogenic and spoilage bacteria may result in huge economic loss due to recalls of products. This study aimed to identify spore-forming bacteria from raw milk and characterise those for toxin production and their spoilage ability. Bovine raw milk collected from dairy farms in the Manawatu region of the North Island of New Zealand was tested for the presence of both aerobic and anaerobic spore-forming bacteria using standard culture-based techniques, as well as genomic analysis. The spore-forming bacteria were investigated for the presence of toxin genes and their spoilage potential. A low number of aerobic spore-forming bacteria were detected in raw-milk samples collected from the four farms in summer and winter. The 16S rRNA sequence types similar to important food spoilage bacteria like C. beijerinckii, C. sporogenes, B. licheniformis and members of the Paenibacillus genus, as well as potentially toxigenic bacteria such as B. cereus and C. perfringens were isolated. Genes responsible for important toxin production were present in some of the tested spore-forming bacteria. This pilot study highlights the presence of various spoilage and pathogenic spore-forming bacteria in raw milk from these farms. A low number of spore-forming bacteria indicates the implementation of good hygienic farm practices and management to reduce the contamination of raw milk with spore-forming bacteria. Full article