Fungi morphology, cytology, vegetative and sexual reproduction Jarmila Pazlarová Micromycetes, molds, filamentous fungi • Filamentous fungi - molds • In mycology – molds – only the fungi of subphyllum Oomycota (ie. Phytophtora infestans – potato mold), Chytridiomycota (ie. Synchytrium endobioticum) and Zygomycota (ie. Mucor mucedo ) • In some popular medical booklets is term mold used even for indication of yeasts. Alternative system of Kingdom Simpson and Roger (2004) Today situation FUNGI -Kingdom of Eukaryota -Eukaryotic organisms without plastids -Nutrition absorptive (osmotrophic) -Cell walls containing chitin and β-glucans -mitochondria with flattened cristae -Unicelullar or filamentous -Mostly non flagellate -Reproducing sexually or asexually -The diploid phase generally short-lived -Saprobic, mutualialistic or parasitic Size of micromycetes • 1,5 milions species, only 5% of them was formaly classified • Great diversity of life cycles and morphology • Recent taxonomy is based on DNA sequences Fungi and pseudofungi Kingdom: PROTOZOA Division Acrasiomycota Myxomycota Plasmodiophoromycota Kingdom: CHROMISTA Division Labyrinthulomycota Peronosporomycota Hyphochytriomycota Kingdom: FUNGI Division Chytridiomycota Microsporidiomycota Glomeromycota Zygomycota Ascomycota Basidiomycota kingdom: Fungi Division: Eumycota – true fungi Subdivision: Zygomycotina Ascomycotina Basidiomycotina Supporting subdivision: Deuteromycotina Kingdom: Fungi (Ophisthokonta) • Division: • Chytridiomycota • Microsporidiomycota • Zygomycota • Glomeromycota • Ascomycota • Basidiomycota Characteristics of forms • Majority of filamentous fungi grow as fibres (hypha), that are cylindrical thread-like structures 2–10 µm in diameter, long up to several cm. • Hyphae grow at their ends (apices); new hyphae are formed by origination of new ends on existing hypha, process is designated branching. • Sometimes growing end of hypha splits in two, and then grow two parallel hyphae. Structure of hyphae cell Growing tip of hypha Mikromycetes – cells Cell wall – chemical composition • Polysacharides - chitin(N-acetylglukosamin) - chitosan (deacetylated chitin) - β-glucans, mannans - other polysaccharides formed from 6-deoxyhexoses (rhamnose -6-deoxy-L-mannose) - cellulose - compounds alike lignine (increase the wall firmness) • Proteins • Fats • Waxes (responsible for minimal affinity to water) Cytoplasmatic membrane • Responsible for transport of nutrients and osmoregulation • Site of synthesis - components of cell wall • Enzymes of oxidative phosphorylation are not placed there • Invaginations are not so frequent as in yeasts Basic cytoplasma • Composition and function does not differ from yeasts cytoplasma • Main storage compound are lipids • Lipids are placed in vacuoles • In older cells lipids are released from the cells, maz be considered for spores Principal organelles • Nucleus • Mitochodria (mtDNA) • Endoplasmatic reticulum • Golgi apparatus • Vacuoles Mycelium - substrate and aerial Aerial mycelium Substrate mycelium agar Kingdom: Fungi (Ophisthokonta) • Division: • Chytridiomycota • Microsporidiomycota • Glomeromycota • Zygomycota • Ascomycota • Basidiomycota • Zygomycetes are a diverse fungal class of the phylum Zygomycota that can be found worldwide. • There are well over 800 species of Zygomycetes fungi within the 124 genera, 32 families and 10 orders that make up Zygomycota. Industrial purposes of Zygomycetes Species Product Uses Leather, detergent and Several Mucor and Lipases and proteases medical industry (Steroid Rhizopus transformation) Food production Rhizopus Cellulases (i.e.Tofu) R. oryzae, Rhizopus spp Fumaric acid Diverse Rhizopus sp. Lactic acid Diverse R. delemar Biotin Diverse Mortierella romanniana, Mortierella vinacea and Linolenic acid Diverse Mucor indicus Mortierella alpina Arachidonic acid Diverse Blakeslea trispora β-carotene Diverse Kingdom: Fungi (Ophisthokonta) • Division: • Chytridiomycota • Microsporidiomycota • Zygomycota • Glomeromycota • Ascomycota • Basidiomycota Characteristics of Ascomycota • Most species grow as filamentous, microscopic structures – hyphae. • Many interconnected hyphae form a mycelium, which—when visible to the naked eye (macroscopic)—is commonly called mold (or, in botanical terminology, thallus). • During sexual reproduction, many Ascomycota typically produce large numbers of asci. The asci is often contained in a multicellular, occasionally readily visible fruiting structure, the ascocarp (also called an ascoma). • Ascocarps come in a very large variety of shapes - solitary or clustered. • Some ascomyceous fungi, such as Saccharomyces cerevisiae, grow as single-celled yeasts, which—during sexual reproduction— develop into an ascus, and do not form fruiting bodies. Kingdom: Fungi (Ophistokonta) Ascomycota -The biggest group of Fungi -Shared attribute - ascus with ascospore (endospore) Division ASCOMYCOTA Schizosaccharomyces pombe • Subdivision: Taphrinomycotina (syn. ARCHIASCOMYCOTINA) Class: Schizosaccharomycetes Class: Taphrinomycetes • Subdivision Saccharomycotina Class: Saccharomycetes • Subdivision Pezizomycotina (syn. ASCOMYCOTINA) Class: Laboulbeniomycetes Class: Eurotiomycetes Class: Pezizomycetes Class: Leotiomycetes Class: Lecanoromycetes Class: Sordariomycetes Class: Dothideomycetes Division ASCOMYCOTA • Subdivision: Taphrinomycotina (syn. ARCHIASCOMYCOTINA) Class: Schizosaccharomycetes Class: Taphrinomycetes • Subdivision Saccharomycotina Class: Saccharomycetes • Subdivision Pezizomycotina (syn. ASCOMYCOTINA) Class: Laboulbeniomycetes Class: Eurotiomycetes Class: Pezizomycetes Class: Leotiomycetes Class: Lecanoromycetes Class: Sordariomycetes Class: Dothideomycetes Subdivision Pezizomycotina Class Eurotiomycetes 1. order Eurotiales 2. order Elaphomycetales 3. order Onygenales Order: Eurotiales Order: Eurotiales • Aspergillus ochraceus • Aspergillus niger • Byssochlamys fulva • Paecilomyces variotii • Neosartorya fischeri • Penicillium expansum • Penicillium chrysogenum • Penicillium digitatum Aspergillus Foto: A. kubátová -frequently causes contamination of food and feed -manufacture of organic acids -mycotoxine (Aflatoxins) -osmophilic, osmotolerant -allergens -opportunistic pathogen -warmer parts of the world Aspergillus ochraceus Important (toxic) metabolites: penicillic acid, ochratoxin A, xanthomegnin, viomellein Conidial heads yellow, globose, when young The most important mycotoxins 1.Aflatoxiny B a G 2.Aflatoxin M1 3.Patulin 4.Ochratoxin A 5.Deoxynivalenol (DON) 6.Zearalenon 7.Fumonisiny 8.T-2 toxin Vesicles globose, hyaline. Phialides borne on metulae. Conidia globose to subglobose. Penicillium • Over 300 species • One of the most widespread fungus • Frequently causes contamination of food and feed • mycotoxine • allergens • Pathogen – only one species P. marnefei • Food processing (P. camemberti, P. roqueforti, P. nalgiovense) • Antibiotic (Penicilin) conidia phialides metulae Branch (ramus) stipe stipe Penicillium chrysogenum Conidiophore and conidia Exudate typically produced Odour aromatic, fruity Important (toxic) metabolited: roquefortine C (P. chrysogenum); tryptoquivalins Penicillium digitatum (P.digitatum) Conidiophore irregularly branched. Colonies velvety Phialides often solitary. Kingdom: Fungi (Ophisthokonta) • Division: • Chytridiomycota • Microsporidiomycota • Zygomycota • Ascomycota • Basidiomycota Rhodotorula sp. Basidium Basidiomycetes basidiospore sterigma karyogamy basidia basidiospore basidiospore Plasmogamy Dikaryotic Primare mycelium basidium mycelium sterigma Taxonomy of Basidiomycetes Hibbett & al. (2007) Mycol. Res. Subdivision: Pucciniomycotina Ustilaginomycotina Agaricomycotina Basidiomycete yeasts Mycotoxines • Mycotoxines are toxic secondary metabolites of many species of micromycetes (molds), that can contaminate wide spectrum of food and feed. These dangerous natural contaminants cause various toxic syndroms, called mycotoxikoses. • Impact of mycotoxines depends on the type of toxine, duration of its effect, dose and and the age of man, his nutrition and actual heath status. • Target organs of mycotoxine effects are cells of liver, kidney, lungs, nerves, endocrine glands and cells of immune system. They can cause acute toxic reaction, some have mutagenic, teratogenic, carcinogenic and estrogenic efect. MAIN GROUPS OF FUNGI AND THEIR MYCOTOXINES * Aspergillus flavus Aspergillus flavus SEM - Aspergillus flavus. microscopie. Aflatoxin B1, B2 and cyclopiazonic acid Penicillium expansum patulin Fusarium graminearum deoxynivalenol, zeralenone Most frequent mycotoxines -AFLATOXINES -OCHRATOXINES -TRICHOTECENS -ZEARALENONS STRUCTURE OF AFLATOXINS Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 Monitoring of mycotoxines • Many international authorities are trying to accomplish the global standardization of regulatory limits for mycotoxines. Today, more then 100 countries set up regulatory limits for mycotoxines in feed industry. • In total 13 mycotoxines (or groups of mycotoxines) has been followed. Evaluation process of mycotoxine regulation includes a large battery of laboratory tests using extractions, purifications and techniques of separation. • Majority of official control methods is based on High Pressure Liquid Chromatography (HPLC). • Many standards for mycotoxines analytics has been guaranteed by European Committee for Standardization (CEN). Ochratoxin • Ochratoxin is mycotoxin formed by three types of secondary metabolites: A, B, a C. • All are produced by specia of genus Penicillium and Aspergillus. All three types are formed so, that