Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature

The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus’ survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores’ size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

How do visible and UV light affect the structure and function of leaf-associated aquatic fungal communities polluted by TiO2 nanoparticles?

We assessed the phototoxicity of TiO2 nanoparticles on fungal structure and function. The fungal diversity was reduced by long-term exposure. However, the leaf decomposition rate was not inhibited rather than promoted under natural photoperiod.

Mycorrhizal Patterns in the Roots of Dominant Festuca rubra in a High-Natural-Value Grassland

Grassland ecosystems occupy significant areas worldwide and represent a reservoir for biodiversity. These areas are characterized by oligotrophic conditions that stimulate mycorrhizal symbiotic partnerships to meet nutritional requirements. In this study, we selected Festuca rubra for its dominance in the studied mountain grassland, based on the fact that grasses more easily accept a symbiotic partner. Quantification of the entire symbiosis process, both the degree of colonization and the presence of a fungal structure, was performed using the root mycorrhizal pattern method. Analysis of data normality indicated colonization frequency as the best parameter for assessing the entire mycorrhizal mechanism, with five equal levels, each of 20%. Most of the root samples showed an intensity of colonization between 0 and 20% and a maximum of arbuscules of about 5%. The colonization degree had an average value of 35%, which indicated a medium permissiveness of roots for mycorrhizal partners. Based on frequency regression models, the intensity of colonization presented high fluctuations at 50% frequency, while the arbuscule development potential was set to a maximum of 5% in mycorrhized areas. Arbuscules were limited due to the unbalanced and unequal root development and their colonizing hyphal networks. The general regression model indicated that only 20% of intra-radicular hyphae have the potential to form arbuscules. The colonization patterns of dominant species in mountain grasslands represent a necessary step for improved understanding of the symbiont strategies that sustain the stability and persistence of these species.

Molecular Characterization of Distinct Fungal Communities in the Soil of a Rare Rarth Mining Area

The exploitation of ion absorbed rare earth elements (REEs) has caused serious ecological destruction and environmental pollution. Effect on soil fungal structure and diversity caused by mining activities are usually ignored, although fungi are one of the most important components in soil ecosystems. In the present research, quantitative polymerase chain reaction (qPCR) and high-throughput Illumina MiSeq sequencing were conducted to characterize fungal community composition and structure in soil of a rare earth mining area after in-situ leaching. Statistical analyses, Network and FUNGuild were used to conduct in-depth analysis. Ascomycota, Basidiomycota, Glomeromycota and unclassified fungi were the most abundant phyla in the mining soils. Organic matter, TC and TN contents, but not pH or REEs contents, were the vital factors to determine soil fungal abundances and diversities. Fungal community structures were stable after leaching practice, but nutrition contents significantly and positively contributed to fungal abundances and diversities. Fungi could mediate the interaction between species to enhance their ability to resist the harsh environment of REEs toxicity or ammonium caused by in-situ leaching practice. Saprotroph in phyla Ascomycota and Basidiomycota were the dominant fungal trophic mode in the mining soils, and they played a critical role in nutrient cycling, transformation processes and reducing metal toxicity. Symbiotrophs of phyla Glomeromycota contributed to soil aggregation and slowing down nutrient losses after in-situ leaching practice.

Tolerance to Abiotic Factors of Microsclerotia and Mycelial Pellets From Metarhizium robertsii, and Molecular and Ultrastructural Changes During Microsclerotial Differentiation

Metarhizium species fungi are able to produce resistant structures termed microsclerotia, formed by compact and melanized threads of hyphae. These propagules are tolerant to desiccation and produce infective conidia; thus, they are promising candidates to use in biological control programs. In this study, we investigated the tolerance to both ultraviolet B (UV-B) radiation and heat of microsclerotia of Metarhizium robertsii strain ARSEF 2575. We also adapted the liquid medium and culture conditions to obtain mycelial pellets from the same isolate in order to compare these characteristics between both types of propagules. We followed the peroxisome biogenesis and studied the oxidative stress during differentiation from conidia to microsclerotia by transmission electron microscopy after staining with a peroxidase activity marker and by the expression pattern of genes potentially involved in these processes. We found that despite their twice smaller size, microsclerotia exhibited higher dry biomass, yield, and conidial productivity than mycelial pellets, both with and without UV-B and heat stresses. From the 16 genes measured, we found an induction after 96-h differentiation in the oxidative stress marker genes MrcatA, MrcatP, and Mrgpx; the peroxisome biogenesis factors Mrpex5 and Mrpex14/17; and the photoprotection genes Mrlac1 and Mrlac2; and Mrlac3. We concluded that an oxidative stress scenario is induced during microsclerotia differentiation in M. robertsii and confirmed that because of its tolerance to desiccation, heat, and UV-B, this fungal structure could be an excellent candidate for use in biological control of pests under tropical and subtropical climates where heat and UV radiation are detrimental to entomopathogenic fungi survival and persistence.

Peritoneal Cryptococcosis in a Dog

Background: Cryptococcosis is an opportunistic, systemic mycotic disease caused by a yeast of Cryptococcus neoformans. These pathogens cause serious public health problems, as they can be transmitted to humans, domestic and wild animals. In cats and dogs, the main site of infection is the upper lower respiratory tract, however, the infection can extend and affect other sites in the organism, however, the peritoneal manifestation of the disease is rare. Thus, the present work aims to report a case of cryptococcosis in a canine, mixed breed, female, adult, from the Metropolitan Region of Belém.Case: A mixed breed dog, female, adult was admitted in a veterinary hospital at Belém, presenting abdominal discomfort and, after the clinical examination, emergency exploratory laparotomy was indicated, and it were observed disseminated lesions in the abdominal cavity, with multiple nodules attached to the peritoneum and intestinal serosa and bladder, uterus, among other organs. Fragments from the surgical biopsy were fixed, and processed routinely according to the techniques for histological tissue processing. Histological examination revealed a predominance of granulomatous lesions in peritoneum and mesentery organs. It was observed a discrete inflammatory reaction of macrophages, epithelioid cells and giant cells, many in phagocytosis of spherical or ovoid organisms, with a thick capsule and a clear perinuclear halo. In the PAS staining, the Cryptococcus conidia presented eosinophilic characteristics and some of them had the capsule well demarcated, which was shown to be radiated. Also it was possible to see the budding yeast and in the Grocott stain, the Cryptococcus stained in black.Discussion: Cryptococcosis with peritoneal involvement in animals is rarely reported in the literature. Cryptococcus is an agent that presents tropism by the central nervous system and nasal cavity, however atypical presentations have been reported, especially at the level of the abdominal cavity (intestines and mesentery). It is believed that the gastrointestinal tract is the gateway for ascending contamination, or that the contamination can occur by fungal ingestion. The lesions presented a multiple distribution, with isolated and coalescent nodules adhered to the peritoneum and adjacent structures. The literature reports two cases of dogs with intra-abdominal cryptococcosis, in which, macroscopically, granulomatous formations were observed in the jejunum, with lymph nodes and mesentery. Microscopically, a discrete inflammatory infiltrate of macrophages and lymphocytes were observed. Some areas with granulomatous reaction, lymphocytes, epithelioid macrophages and giant cells and, in other areas, yeasts in the cytoplasm were observed. The yeasts were spherical or ovoid, surrounded by a thick capsule of polysaccharide. The periodic acid Schiff (PAS) and silver impregnation (Grocott) staining were used to highlight structures of the Cryptococcus wall which were presented by a strong stained polysaccharide capsule. It must be emphasized the zoonotic potential of cryptococcosis, a disease that can affect both animals and humans. Cryptococcosis in humans normally occurs when the fungus accesses the airways through inhalation and ranges from asymptomatic pulmonary colonization to compromised meninges, causing the human patient's life-threatening condition. In the present report, the diagnosis of cryptococcosis was established through histopathological and histochemical examination of the fungal structure, which were determinant in the etiological diagnosis of cryptococcosis.

Early life dietary intervention in dairy calves results in a long-term reduction in methane emissions

Recent evidence suggests that changes in microbial colonization of the rumen prior to weaning may imprint the rumen microbiome and impact phenotypes later in life. We investigated how dietary manipulation from birth influences growth, methane production, and gastrointestinal microbial ecology. At birth, 18 female Holstein and Montbéliarde calves were randomly assigned to either treatment or control (CONT). Treatment was 3-nitrooxypropanol (3-NOP), an investigational anti-methanogenic compound that was administered daily from birth until three weeks post-weaning (week 14). Samples of rumen fluid and faecal content were collected at weeks 1, 4, 11, 14, 23, and 60 of life. Calves were tested for methane emissions using the GreenFeed system during the post-weaning period (week 11–23 and week 56–60 of life). Calf physiological parameters (BW, ADG and individual VFA) were similar across groups throughout the trial. Treated calves showed a persistent reduction in methane emissions (g CH4/d) throughout the post-weaning period up to at least 1 year of life, despite treatment ceasing three weeks post-weaning. Similarly, despite variability in the abundance of individual taxa across weeks, the rumen bacterial, archaeal and fungal structure differed between CONT and 3-NOP calves across all weeks, as visualised using sparse-PLS-DA. Similar separation was also observed in the faecal bacterial community. Interestingly, despite modest modifications to the abundance of rumen microbes, the reductive effect of 3-NOP on methane production persisted following cessation of the treatment period, perhaps indicating a differentiation of the ruminal microbial ecosystem or a host response triggered by the treatment in the early development phase.

A novel putative microtubule-associated protein is involved in arbuscule development during arbuscular mycorrhiza formation

The formation of arbuscular mycorrhizal (AM) symbiosis requires plant root host cells to undergo major structural and functional reprogramming in order to house the highly branched AM fungal structure for the reciprocal exchange of nutrients. These morphological modifications are associated with cytoskeleton remodelling. However, molecular bases and the role of microtubules (MTs) and actin filament dynamics during AM formation are largely unknown. In this study, the tomato tsb gene, belonging to a Solanaceae group of genes encoding MT-associated proteins for pollen development, was found to be highly expressed in root cells containing arbuscules. At earlier stages of mycorrhizal development, tsb overexpression enhanced the formation of highly developed and transcriptionally active arbuscules, while tsb silencing hampers the formation of mature arbuscules and represses arbuscule functionality. However, at later stages of mycorrhizal colonization, tsb OE roots accumulate fully developed transcriptionally inactive arbuscules, suggesting that the collapse and turnover of arbuscules might be impaired by TSB accumulation. Imaging analysis of the MT cytoskeleton in cortex root cells overexpressing tsb revealed that TSB is involved in MT-bundling. Taken together, our results provide unprecedented insights into the role of novel MT-associated protein in MT rearrangements throughout the different stages of the arbuscule life cycle.

Fungal mycoflora dysbiosis in gastric cancer

Background: Bacterial infection is associated with gastric carcinogenesis. However, the relationship between nonbacterial components and gastric cancer (GC) has not been fully explored. We aimed to characterize the fungal mycobiome in GC.Results: We observed significant gastric fungal dysbiosis in GC. Principal component analysis revealed separate clusters for the GC and control groups, and Venn diagram analysis indicated that the GC group showed a lower OTU abundance than the control. At the genus level, the abundances of 15 fungal biomarkers distinguished the GC group from the control, of which Candida (p = 0.000246) and Alternaria (p = 0.00341) were enriched in GC, while Saitozyma (p = 0.002324) and Thermomyces (p = 0.009158) were decreased. Combining the results of Welch’s t test and Wilcoxon rank sum test, C. albicans was significantly elevated in GC. The species richness Krona pie chart further revealed that C. albicans occupied 22% and classified GC from the control with an area under the receiver operating curve (AUC) of 0.743. Random forest analysis also confirmed that C. albicans could serve as a biomarker with a certain degree of accuracy. Moreover, compared with that of the control, the alpha diversity index was significantly reduced in the GC group. The Jaccard distance index and the Bray abundance index of the PCoA clarified separate clusters between the GC and control groups at the species level (p = 0.00051). Adonis (PERMANOVA) analysis and ANOVA showed that there were significant differences in fungal structure among groups (p = 0.001). Finally, FUNGuild functional classification predicted that saprotrophs were the most abundant taxa in the GC group.Conclusions: This study revealed GC-associated mycobiome dysbiosis characterized by an altered fungal composition and ecology and demonstrated that C. albicans can be a fungal biomarker for GC. In addition, C. albicans may mediate GC by reducing the diversity and richness of fungi in the stomach, contributing to the pathogenesis of GC.

Early endocytosis as a key to understanding mechanisms of plasmalemma tension regulation in filamentous fungi

ABSTRACTTwo main systems regulate the plasmalemma tension and provide a close connection of the protoplast with the cell wall in fungi: turgor pressure and actin cytoskeleton. These systems work together with the plasmalemma focal adhesion to the cell wall and their contribution to fungal cell organization has been partially studied, but remains controversial in model filamentous ascomycetes and oomycetes, and even less investigated in filamentous basidiomycetes. Early endocytosis, in which F-actin is actively involved, can be used to research of mechanisms regulating the plasmalemma tension, since the latter influences on the primary endocytic vesicles formation. This study examined the effects of actin polymerization inhibitors and hyperosmotic shock on early endocytosis and cell morphology in two filamentous basidiomycetes. The main obtained results: (i) depolymerization of F-actin leads to the fast formation of primary endocytic vesicles but to inhibition of their scission; (ii) moderate hyperosmotic shock does not affect the dynamics of early endocytosis. These and a number of other results allowed offering a curtain model of regulation the plasmalemma tension in basidiomycetes. According to this model, the plasmalemma tension in many nonapical cells of hyphae is more often regulated not by turgor pressure, but by a system of actin driver cables that are associated with the proteins of focal adhesion sites. The change in the plasmalemma tension occurs similar to the movement of the curtain along the curtain rod using the curtain drivers. This model addresses the fundamental properties of the fungal structure and physiology and requires confirmation, including through the yet technically unavailable high quality labeling of the actin cytoskeleton of basidiomycetes.