Pathogenic Fungal Species Research Articles

Cryptococcus extracellular vesicles properties and their use as vaccine platforms.

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting‐edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein‐based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane‐bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

Advancement in specific strand scission of DNA and evaluation of in-vitro biological assessment by pharmacologically significant tetraaza macrocyclic metal complexes constrained by triazole

A novel ligand, tetra-azamacrocyclic, L(C24H16N12O2S4) and its complexes with the structures, [MLCl2] and [CuL]Cl2 (where M = Ni(II), Fe(II); L = S,S′-[benzene-1,3-diylbis(4H-1,2,4-triazole-5,3-diyl)]bis{[(5-benzene-1,3-diyl-4H-1,2,4-triazol-3yl)sulfanyl] ethanethioate} were described and depicted by the analytical and spectral techniques. These studies revealed an octahedral geometry for Ni(II) and Fe(II) structures, while the Cu(II) complex exhibited a square planar structure. All the integrated metal structures were screened against targeted species of pathogenic fungi and bacterial strains to evaluate in vitro antimicrobial activities. The binding capability of the complexes with CT-DNA was considered by supporting detailed examination followed by viscosity measurements and thermal denaturation studies. The mode of DNA cleavage using gel electrophoreses revealed that the complexes possess photonuclease characteristics against pUC19 DNA under UV–visible irradiation. The newly synthesized novel Cu(II)-L complex has been proven to be a potential DNA cleavage agent and a potent inhibitor against both the bacterial and fungi strains. Introduction

In Vitro Antimicrobial Activity of 80% Methanol Extract and Solvent Fractions of Urtica simensis Hochst. ex. A. Rich. (Urticaceae) Leaves against Pathogenic Bacteria and Fungi

Medicinal plants have been used for the treatment of infectious diseases since a long time. Urtica simensisHochst. ex. A. Rich. is one of the endemic medicinal plants used for the treatment of bacterial and fungalinfections in Ethiopian folklore medicine. The aim of this study was to investigate the antimicrobial activityof 80% methanol extract and solvent fractions of the leaves of U. simensis. The 80% methanol extract wasprepared by maceration and the solvent fractions were obtained by successive fractionation of the 80%methanol extract with chloroform, ethyl acetate, n-butanol and distilled water. The antimicrobial activity ofthe extracts was evaluated on eight bacterial and two fungal species using agar well diffusion method atconcentrations of 200, 400 and 800 mg/ml. Minimum inhibitory concentrations (MICs) were determined bybroth microdilution method. From the gram positive bacteria tested, the most susceptible species wasStreptococcus pneumoniae (MIC of 1.36 mg/ml), while Klebsiella pneumoniae and Aspergillus niger weremost susceptible gram negative bacterial and fungal species with MIC values of 2.54 and 3.13 mg/ml,respectively. The least values of MIC, MBC and MFC were 1.36, 5.21 and 6.25 mg/ml, respectively. Resultsof the present study revealed that the 80% methanol extract and solvent fractions, of the leaves of U.simensis, with the exception of the aqueous fraction, possess activity against the growth of selected pathogenic bacterial and fungal species. Among the solvent fractions, the n-butanol fraction was the most active fraction against the gram positive bacteria tested, while the ethyl acetate fraction was the most active fraction against the other tested organisms. Further studies are needed to isolate the active principles, elucidate the mechanism of antimicrobial action, determine in vivo antimicrobial activity and effect of the extracts on other pathogens including clinical isolates.

Biscogniauxia rosacearum the charcoal canker agent as a pathogen associated with grapevine trunk diseases in Zagros region of Iran

Grapevine trunk diseases (GTDs) are well-known and significant fungal diseases of Vitis vinifera with a worldwide distribution. During August to November 2016 in a survey to characterize fungi associated with grapevine trunk diseases in Kermanshah Province (west of Iran) vineyards, 286 fungal isolates were obtained. Based on morphology and DNA sequences data eight species were identified, of which Biscogniauxia rosacearum, Neoscytalidium hyalinum and Phaeoacremonium minimum were the most aggressive fungal pathogenic species characterized in this research. N. hyalinum was the most prevalent species. N. hyalinum and Ph. minimum have previously been reported from Vitis vinifera. Thus far, there are two records of Biscogniauxia mediterranea and Biscogniauxia capnodes on grapevine in the world with no data on pathology aspects. To our knowledge, it is the first time B. rosacearum is reported from grapevine across the globe. Pathogenicity test with three strains of B. rosacearum on 2-year-old potted grapevines confirmed the pathogenicity of B. rosacearum on grapevine. The proximity of vineyards to the oak trees in Zagros forests as one of the plant hosts of Biscogniauxia spp. further highlights the need for extensive studies on B. rosacearum as a new fungal pathogen.

Genetic sources of resistance to root rots chicory

Relevance. One of the significant problems in the technology of obtaining root chicory is the protection of root vegetables from the defeat of root rots. The most common lesions of root vegetables chicory various pathogenic species of fungi causing phomosis (Phoma rostrupii Sacc.), gray rot (Botrytis cinerea (R.) Fr.J), wet bacterial rot (Erwinia carotovora (Jones) Holt.). An important place in solving this problem is the breeding of new varieties that are resistant to major diseases and adapted to the conditions of cultivation in the soil-climatic conditions of the chicory-seeding zone. This requires, first of all, to determine the range of donor varieties of the sign of resistance to root rots, both during the growing of culture, and during long-term storage.Material and methods. In 2018-2020, studies were carried out to assess gene sources of different ecological and geographical origin (including against a provocative background) in the Non-Black Earth Zone of the Russian Federation and to isolate donors of resistance to root rot from them. Research on the Rostov Vegetable experimental station on chicory.Results. Studies have shown that the defeat of root vegetables chicory root rots genetic sources were very different from each other, so Harpachi, Luxor did not affect the. root rots neither on the usual nor on provocative backgrounds.

CANDIDA DIAGNOSTIC PLATFORMS: ESSENTIAL IN EARLY MANAGEMENT OF CANDIDA INFECTIONS

Invasive candidiasis (IC) is associated with high morbidity and mortality. Candida species have significant roles in invasive candidiasis andthe emergence of clinical strains that are difficult to treat due tovirulent and resistant properties. Expansion of fungal pathogen diversity necessitates the development and adoption of new methods for prompt diagnosis and management. The review aimed to highlight the relevance of different Candida diagnostic methods for prompt management of Candida infections. The conventional gold standard diagnostic phenotypic-based (culture)method is time-consuming, associated withlow specificity and subjective interpretation.The specificity and sensitivity performances for candidaemia or deep-seated candidiasis of biochemical-based methods, including VITEK, API 20C AUX, and latex agglutination, have higher resolution than the culture. Nucleic acid-basedpolymerase chain reaction diagnostic techniques have been rapidly evolving. PCR will improve the diagnostic performance and patient outcome. The PCR technique uses different ribosomal DNA gene complexes, including D1/D2, ITS1/ITS4, or IGS1/IGS2, ashelpful markersto delineate the main pathogenic fungal species belonging to different genera. Sensitive and specific diagnostic methods for Candida speciesare significant for clinical decision and effective clinical outcome.

Inter- and Intra-Continental Genetic Variation in the Generalist Conifer Wood Saprobic Fungus Phlebiopsis gigantea

Phlebiopsis gigantea (Fr.) Jülich is a well-known generalist conifer wood saprobe and a biocontrol fungus used in several world countries to prevent stump infection by tree pathogenic Heterobasidion fungal species. Previous studies have reported the presence of regional and continental genetic differentiation in host-specific fungi, but the presence of such differentiation for generalist wood saprobes such as P. gigantea has not been often studied or demonstrated. Additionally, little information exists on the distribution of this fungus in western North America. The main purposes of this study were: (I) to assess the presence of P. gigantea in California, (II) to explore the genetic variability of P. gigantea at the intra and inter-continental levels and (III) to analyze the phylogeographic relationships between American and European populations. Seven loci (nrITS, ML5–ML6, ATP6, RPB1, RPB2, GPD and TEF1-α) from 26 isolates of P. gigantea from coniferous forests in diverse geographic distribution and from different hosts were analyzed in this study together with 45 GenBank sequences. One hundred seventy-four new sequences were generated using either universal or specific primers designed in this study. The mitochondrial ML5–ML6 DNA and ATP6 regions were highly conserved and did not show differences between any of the isolates. Conversely, DNA sequences from the ITS, RPB1, RPB2, GPD and TEF1-α loci were variable among samples. Maximum likelihood analysis of GPD and TEF1-α strongly supported the presences of two different subgroups within the species but without congruence or geographic partition, suggesting the presence of retained ancestral polymorphisms. RPB1 and RPB2 sequences separated European isolates from American ones, while the GPD locus separated western North American samples from eastern North American ones. This study reports the presence of P. gigantea in California for the first time using DNA-based confirmation and identifies two older genetically distinct subspecific groups, as well as three genetically differentiated lineages within the species: one from Europe, one from eastern North America and one from California, with the latter presumably including individuals from the rest of western North America. The genetic differentiation identified here among P. gigantea individuals from coniferous forests from different world regions indicates that European isolates of this fungus should not be used in North America (or vice versa), and, likewise, commercially available eastern North American P. gigantea isolates should not be used in western North America forests. The reported lack of host specificity of P. gigantea was documented by the field survey and further reinforces the need to only use local isolates of this biocontrol fungus, given that genetically distinct exotic genotypes of a broad generalist microbe may easily spread and permanently alter the microbial biodiversity of native forest ecosystems.

Post-translational regulation of autophagy is involved in intra-microbiome suppression of fungal pathogens

BackgroundMicrobiome interactions are important determinants for ecosystem functioning, stability, and health. In previous studies, it was often observed that bacteria suppress potentially pathogenic fungal species that are part of the same plant microbiota; however, the underlying microbe-microbe interplay remains mostly elusive. Here, we explored antagonistic interactions of the fungus Fusarium graminearum and bacterium Streptomyces hygroscopicus at the molecular level. Both are ubiquitous members of the healthy wheat microbiota; under dysbiosis, the fungus causes devastating diseases.ResultsIn co-cultures, we found that Streptomyces alters the fungal acetylome leading to substantial induction of fungal autophagy. The bacterium secrets rapamycin to inactivate the target of rapamycin (TOR), which subsequently promotes the degradation of the fungal histone acetyltransferase Gcn5 through the 26S proteasome. Gcn5 negatively regulates fungal autophagy by acetylating the autophagy-related protein Atg8 at the lysine site K13 and blocking cellular relocalization of Atg8. Thus, degradation of Gcn5 triggered by rapamycin was found to reduce Atg8 acetylation, resulting in autophagy induction in F. graminearum.ConclusionsAutophagy homeostasis plays an essential role in fungal growth and competition, as well as for virulence. Our work reveals a novel post-translational regulation of autophagy initiated by a bacterial antibiotic. Rapamycin was shown to be a powerful modulator of bacteria–fungi interactions with potential importance in explaining microbial homeostasis in healthy plant microbiomes. The autophagic process provides novel possibilities and targets to biologically control pathogens.3Zu6EQMv78RbwrwJHF2G1TVideo abstract

Plant quarantine measures for the safe global distribution of germplasm of ICRISAT mandate crops

ICRISAT safely exported 182,605 seed samples to 127 countries during 1998–2019. The maximum number of exported samples were of sorghum (51,309) followed by chickpea (48,513) and pearl millet (39,771). A total of 2749 seed samples (chickpea 1301, groundnut 300, minor millets 50, pearl millet 338, pigeonpea 395 and sorghum 365) were rejected due to presence of seed-borne pathogens of quarantine significance. Thirty-one pathogenic fungal species were detected in the blotter test and seven pathogens detected in seed washing and sedimentation test. Maximum fungal/oomycetes species were detected in sorghum seed samples (18) followed by chickpea (16), pigeonpea (15), groundnut (10), pearl millet (9) and minor millets (6). Acremonium sp., Botrytis sp., Cercospora sp., Colletotrichum sp., Exserohilum turcicum, Fusarium sp., Phoma medicaginis, Phomopsis sp., Pyricularia grisea, Sclerotium rolfsii, Peronosclerospora sp., Sporisorium sorghi, Puccinia purpurea, Moesziomyces penicillariae, Claviceps fusiformis and Sphacelotheca sp. were detected in the seed samples.

Rational Design of an Antifungal Polyacrylamide Library with Reduced Host-Cell Toxicity.

Life-threatening invasive fungal infections represent an urgent threat to human health worldwide. The limited set of antifungal drugs has critical constraints such as resistance development and/or adverse side effects. One approach to overcome these limitations is to mimic naturally occurring antifungal peptides called defensins. Inspired by their advantageous amphiphilic properties, a library of 35 synthetic, linear, ternary polyacrylamides was prepared by controlled/living radical polymerization. The effect of the degree of polymerization (20, 40, and 100) and varying hydrophobic functionalities (branched, linear, cyclic, or aromatic differing in their number of carbons) on their antifungal activity was investigated. Short copolymers with a calculated log P of ∼1.5 revealed optimal activity against the major human fungal pathogen Candida albicans and other pathogenic fungal species with limited toxicity to mammalian host cells (red blood cells and fibroblasts). Remarkably, selected copolymers outperformed the commercial antifungal drug amphotericin B, with respect to the therapeutic index, highlighting their potential as novel antifungal compounds.

Anti-Fungal Activity of Cressa cretica, Leptadenia pyrotechnica and Pulicaria crispa, Indigenous Plants of Cholistan Desert, Pakistan

Background: Fungal infections have always remained a problem and they are getting worse with the passage of each year due to their resistance against available antibiotics. Natural ways of treatment with homeopathic medicines made of mother tinctures of plants have no reported side effects and have been proved effective against many bacterial and fungal infections. Materials & Methods: Preparation of mother tinctures of plants Cressa cretica, Leptadenia pyrotechnica, and Pulicaria crispa was done and used to evaluate the anti-fungal potential of these plants against potentially pathogenic fungal species like Aspergillus niger, Aspergillus flavus, Aspergillus ustus, and Candida albicans by agar disc diffusion method. Each tincture was evaluated at 0.25 ml, 0.5 ml, and 1 ml volume per disc and zone of inhibition was measured in millimeters and compared with commercial drug Fluconazole (2 mg/ml), which was used as standard. Results: The results showed the satisfactory anti-fungal activity of these plants in comparison to the standard drug Fluconazole. The ratio of anti-fungal activity of Cressa cretica measured as the zone of growth inhibition of these cultures against Aspergillus niger, Aspergillus flavus, Aspergillus ustus, and Candida albicans was 24 mm / 27 mm, 30mm / 28 mm, 23 mm/ 30 mm, and 32 mm/ 30 mm, respectively. In the case of Leptadenia pyrotechnica, the mother tincture’s antifungal activity was 32 mm / 27 mm, 30 mm / 28 mm, 17 mm/ 30 mm, and 24 mm/ 30 mm, respectively. In the case of our third plant Pulicaria crispa, anti-fungal activity was found to be 23 mm/ 27 mm, 26 mm/ 28 mm, 26 mm / 30 mm and 24 mm/ 30 mm, respectively. Conclusion: Our study proved that the mother tincture of these plants could be a potential new therapy to treat fungal infections and has the potential to rule out fungal problems. Further research using the mother tincture of these plants against other fungal species has the potential to prove them a safer and widespread anti-fungal homeopathic medicine.

English

Early blight incurs huge losses to solanaceous crops at both pre- and post-harvest stages by reducing yields up to 35–78%. Large-spored Alternaria spp. were isolated and characterized from the symptomatic tomato and potato plants. Morphological characterization revealed heterogeneity in the different traits of the isolates, which were subsequently confirmed by molecular characterization and two different Alternaria spp. viz. A. solani and A. linariae were characterized. A multi-locus phylogenetic analysis was conducted to infer the taxonomic position of the isolates within Alternaria spp. The five conserved genome regions used to infer phylogenetic lineage and evolutionary relationship were the internal transcribed spacer (ITS), the translation elongation factor (TEF), the glyceraldehyde-3-phosphate dehydrogenase (GADPH), the major allergen Alt a1 (Alt a1) and the RNA-polymerase 2 (RPB2) genes. All A. linariae isolates were closely related and formed a clade to A. linariae isolated from the United States. Similarly, A. solani isolates were closely related to each other and formed an independent clade indicating their unique nature. Koch’s postulates were fulfilled for all the isolates. Among six different tested fungicides, aveet (azoxystrobin 5% + mancozeb 70%) inhibited the mycelial growth the most and saver (cymoxanil 5% + chlorothalonil 37%) the least. All the isolates infected tomato and potato leaves with varying severity except A. linariae isolate Egy-T2 was non-pathogenic on potato leaves, while the rest of A. linariae isolates were more aggressive. Here, we demonstrated that early blight in Egypt is caused by two different species of fungal pathogens, A. linariae and A. solani, and reported isolation of A. linariae from Egypt. © 2021 Friends Science Publishers

Antimycotic potential of Kawayang tinik against pathogenic fungal species

The importance of discovering and obtaining new, natural and sustainable sources of potential drugs have been the focus of scientific communities due to the emergence of increasing cases of microbial resistance, one of the biggest health threats in our society today. This study aimed to determine the antimycotic potential of Bambusa blumeana (kawayang tinik) specifically its leaf, rhizome, root, inner culm and outer culm extracts using the agar well diffusion assay. Results of the study revealed that all kawayang tinik extracts produced statistically equal size zone of inhibition (ZOI) against Aspergillus niger at 1 mg/ ml concentration while the ethanolic root and leaf extracts showed larger ZOI against Penicillium chrysogenum compared to other kawayang tinik extracts. Furthermore, the results of the antifungal assay showed comparable activity of kawayang tinik extracts to Fluconazole, a pharmaceutically approved antifungal drug, at 1 mg/ml concentration. Phytochemical studies further revealed the presence of alkaloids, tannins, phenols, sterols, triterpenes and flavonoids in its different parts which may support its potential antimycotic properties.

Kirinyuh (Chromolaena odorata): species distribution modeling and the potential use of fungal pathogens for its eradication

Chromolaena odorata is one of the weeds that is difficult to control and causes loss both economically and ecologically. This study aims to predict the distribution of C. odorata which affected by climate change and the potential of using fungal pathogens for its eradication efforts in Indonesia. The Biodiversity and Climate Change Virtual Laboratory application was used to create Habitat Suitability Index of C. odorata. Species data were obtained from the Global Biodiversity Information Facility and the variable environment parameters were obtained from the Worldclim current condition (1950-2000). Results show that Generalized Linear Model has values of 0.98. C. odorata most likely spreads to eastern parts of Indonesia. The model predicted it to be suitable to inhabit Bali-Lombok Islands (HSI ±0.8) as well as Lesser Sunda Islands, Flores (HSI ±0.76) and Sumba (HSI ±0.82), and also Papua which share similar HSI with Sumba. Four species of putative fungal pathogens were isolated from the diseased leaf tissue of C. odorata. One species of fungus that causes severe infection in the leaves of C. odorata was morphologically identified as Septoria sp. It considered as a biological control agent. Further studies are needed to confirm this association and their potential for biological control.

The growth inhibitory potential of eudicot‐based preparations against plant pathogenic fungi

The infection of certain plants with pathogenic fungi species results in a significant loss in the production yield of these plants, some of which are with high economical importance. In the current study, we studied the growth inhibitory potential of several eudicot plant preparations (Lavandula angustifolia, Matricaria chamomilla L. and Saponaria officinalis L.) against certain plant pathogenic fungi species, namely, Phytophthora infestans, Rhizoctania solani, Fusarium oxysporum. f. sp. lycopersici and Macrophamina phaseolina. We found that solutions prepared with these plants mostly show concentration-dependent inhibitory effect on the growth of these fungi species. These plant-based anti-fungal preparations might potentially be used in the control of fungi species studied in the present study, in order to increase the production yield of their host plants. Further research is required to identify the mechanisms of action of these plant-based solutions on pathogenic fungi growth.

Accessory Chromosome-Acquired Secondary Metabolism in Plant Pathogenic Fungi: The Evolution of Biotrophs Into Host-Specific Pathogens.

Accessory chromosomes are strain- or pathotype-specific chromosomes that exist in addition to the core chromosomes of a species and are generally not considered essential to the survival of the organism. Among pathogenic fungal species, accessory chromosomes harbor pathogenicity or virulence factor genes, several of which are known to encode for secondary metabolites that are involved in plant tissue invasion. Accessory chromosomes are of particular interest due to their capacity for horizontal transfer between strains and their dynamic “crosstalk” with core chromosomes. This review focuses exclusively on secondary metabolism (including mycotoxin biosynthesis) associated with accessory chromosomes in filamentous fungi and the role accessory chromosomes play in the evolution of secondary metabolite gene clusters. Untargeted metabolomics profiling in conjunction with genome sequencing provides an effective means of linking secondary metabolite products with their respective biosynthetic gene clusters that reside on accessory chromosomes. While the majority of literature describing accessory chromosome-associated toxin biosynthesis comes from studies of Alternaria pathotypes, the recent discovery of accessory chromosome-associated biosynthetic genes in Fusarium species offer fresh insights into the evolution of biosynthetic enzymes such as non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and regulatory mechanisms governing their expression.

Targeting the ATP Synthase in Staphylococcus aureus Small Colony Variants, Streptococcus pyogenes and Pathogenic Fungi.

The ATP synthase has been validated as a druggable target with the approval of the ATP synthase inhibitor, bedaquiline, for treatment of drug-resistant Mycobacterium tuberculosis, a bacterial species in which the ATP synthase is essential for viability. Gene inactivation studies have also shown that the ATP synthase is essential among Streptococci, and some studies even suggest that inhibition of the ATP synthase is a strategy for the elimination of Staphylococcus aureus small colony variants with deficiencies in the electron transport chain, as well as pathogenic fungi, such as Candida albicans. Here we investigated five structurally diverse ATP synthase inhibitors, namely N,N′-dicyclohexylcarbodiimide (DCCD), oligomycin A, tomatidine, resveratrol and piceatannol, for their growth inhibitory activity against the bacterial strains Streptococcus pyogenes, S. aureus and two isogenic small colony variants, as well as the pathogenic fungal species, C. albicans and Aspergillus niger. DCCD showed broad-spectrum inhibitory activity against all the strains (minimum inhibitory concentration (MIC) 2–16 µg/mL), except for S. aureus, where the ATP synthase is dispensable for growth. Contrarily, oligomycin A selectively inhibited the fungal strains (MIC 1–8 µg/mL), while tomatidine showed very potent, but selective, activity against small colony variants of S. aureus with compromised electron transport chain activity (MIC 0.0625 µg/mL). Small colony variants of S. aureus were also more sensitive to resveratrol and piceatannol than the wild-type strain, and piceatannol inhibited S. pyogenes at 16–32 µg/mL. We previously showed that transposon inactivation of the ATP synthase sensitizes S. aureus towards polymyxin B and colistin, and here we demonstrate that treatment with structurally diverse ATP synthase inhibitors sensitized S. aureus towards polymyxin B. Collectively, our data show that ATP synthase inhibitors can have selective inhibitory activity against pathogenic microorganisms in which the ATP synthase is essential. The data also show that the inhibition of the ATP synthase in Streptococcus pyogenes may be a new strategy for development of a narrow-spectrum antibiotic class. In other major bacterial pathogens, such as S. aureus and potentially Escherichia coli, where the ATP synthase is dispensable, the ATP synthase inhibitors may be applied in combination with antimicrobial peptides to provide new therapeutic options.

Chemical composition and antioxidant and antimicrobial activities of Lactarius sanguifluus, a wild edible mushroom from northern Morocco

Lactarius sanguifluus is part of the diverse mycological heritage in the Koudiat Taifour forest (a site of biological and ecological interest in the northern region of Morocco), although this fungus is in danger of extinction in this forest. The main objective of the research reported here was to perform an advanced chemical analysis of a methanolic extract from L. sanguifluus and to investigate its biological activities. Spectrophotometric determination of the chemical compounds present in the extract showed that this fungus contains significant amounts of phenolics and flavonoids [18.71–18.81 mg GAE per g of dry methanolic extract (dme)], moderate quantities of ascorbic acid and tannins [3.50–3.86 mg/g of dry weight (dw)], and low levels of β-carotene and lycopene (0.11–0.10 mg/g dme). The phenolic compounds were identified and quantified via LC–MS, and the results showed that p-hydroxybenzoic acid (134.10 µg/g dw) and paraben acid (85.35 µg/g dw) were the main phenolic compounds in the sample. GC–MS analysis of the methanol extract led to the identification of more than 77 chemical compounds in the sample, which were categorized into five main groups: sugars (48.15%), fatty acids (20.66%), amino acids (11.92%), organic acids (9.30%), and alcohols (8.42%). The antioxidant activity of the fungal methanol extract was demonstrated using three methods: the DPPH assay (IC50: 2.045 mg/ml), the β-carotene bleaching test (IC50: 0.56 mg/ml), and the ferric reducing antioxidant power assay (FRAP; RC50: 0.85 mg/ml). The antimicrobial activity was evaluated using microdilution methods. Antifungal susceptibility tests indicated that the extract was able to inhibit the growth of five fungal pathogen species. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) ranged from 1 to 16 mg/ml for Epidermophyton floccosum, Trichophyton rubrum, and Microsporum canis, and from 21 to 64 mg/ml for Candida albicans and Aspergillus fumigatus. Likewise, antibacterial tests showed that the extract inhibited the growth of Escherichia coli and Staphylococcus aureus at concentrations ranging from 21 to 64 mg/ml for the MIC and at 64 mg/ml for the MFC. Considering their nutritional and nutraceutical value, fruiting bodies of L. sanguifluus and extracts from them could be used as nutritional supplements and as natural antioxidant and antimicrobial agents in food preservation and human health.

Evaluation of Chitin as a Biomarker of Pathogenic Fungal Isolates

Chitin is a polysaccharide component of the inner cell wall of fungi that has been used to estimate fungal invasion in plant products. However, its detection in major pathogenic fungal species has not been investigated. The present study aimed to determine the chitin contents of pathogenic fungal species in order to evaluate its diagnostic potential as a biomarker for fungal infections. High performance liquid chromatography (HPLC) was used to measure chitin content. Pure chitin was acid hydrolyzed and the fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine produced were measured. The chitin contents of ten pathogenic fungal isolates were determined per mycelial dry weight. They varied from 18.61 (± 0.09) to 47.12 (± 0.50) μg/mg dry mycelial weight. Candida albicans and Cryptococcus neoformans exhibited the highest and lowest levels of chitin, respectively. Based upon relative amounts of chitin produced, three groups namely: high (Candida albicans, Cryptococcus gattii, Aspergillus niger and Penicilliumat 47.12, 46.98, 46.05, and 44.15 μg/mg respectively), medium (Rhizopus, Aspergillus fumigatus, Fusarium solani, andMucor at 36.61, 36.30, 35.03, and 34.84 μg/mg, respectively), and low (Candida tropicalis and Cryptococcus neoformans at 20.78 and 18.61 μg/mg, respectively), were identified. Chitin was not detectable in bacterial isolates used as controls. The chitin detection method offers a sensitive and specific tool for the quantification of chitin in pathogenic fungal isolates. The detection of chitin may be a useful assay for the diagnosis of fungal infections in clinical samples.

The Spectrum of Weed Species and Fungal Pathogens in Stands of Alternative Fodder Crop Lolium Multiflorum Var. Westerwoldicum Growing up in a Drought-Prone Area in the Czech Republic

Annual ryegrass (Lolium multiflorum var. westerwoldicum) is cultivated in various areas of the world as a feed for live stock. Efforts to improve the production of high-quality feed in the face of deepening climate change have increased the demand for growing non-traditional types of crops, including ryegrass. The aims of the research were to determine the spectrum of weed and fungal pathogen species and to assess dangerous species of weeds and pathogens which negatively affect forage crops of annual ryegrass grown in dry conditions. In 2018-2019 the occurrence of weeds and fungal pathogens of ryegrass was evaluated in field conditions. The assessed lands were located in dry climatic conditions of South Moravia (Czech Republic). The vegetation recording was done on phytosociological plots of 12 m2. A total of 57 weed species were identified in the plots, where 12 species were toxic and 16 species were undesirable. The most problematic weeds and poisonous species were: Aethusa cynapium, Datura stramonium, Equisetum palustre, Euphorbia helioscopia, Fumaria officinalis and Solanum nigrum, which can cause health problems if present in cattle feed. A pathogenic infection caused by Blumeria graminis, Puccinia graminis var. graminicola and P. coronata var. coronata, Gaeumannomyces graminis and Drechslera sp. was identified on the ryegrass plants in the experimental plots during the vegetation. Blumeria graminis was the most abundant, but it did not endanger the final feed. The data was processed by CCA analysis.