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Home , Aspergillus, Aspergillus ochraceus, Eurotiales, Laboulbeniomycetes, Mucor mucedo, Penicillium digitatum

Fungi morphology, cytology, vegetative and

Jarmila Pazlarová Micromycetes, , filamentous fungi

• Filamentous fungi - molds • In – molds – only the fungi of subphyllum Oomycota (ie. Phytophtora infestans – ), (ie. Synchytrium endobioticum) and (ie. mucedo ) • In some popular medical booklets is term mold used even for indication of yeasts. Alternative system of Simpson and Roger (2004) Today situation FUNGI

-Kingdom of Eukaryota -Eukaryotic organisms without plastids -Nutrition absorptive (osmotrophic) -Cell walls containing 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 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 Zygomycota

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 (), 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 formed from 6-deoxyhexoses (rhamnose -6-deoxy-L-mannose) - cellulose - compounds alike lignine (increase the wall firmness)

• • Fats • Waxes (responsible for minimal affinity to water)

Cytoplasmatic membrane • Responsible for transport of nutrients and osmoregulation • Site of synthesis - components of cell wall • 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

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 medical industry (Steroid transformation) Food production Rhizopus Cellulases (i.e.Tofu) R. oryzae, Rhizopus spp Fumaric acid Diverse Rhizopus sp. Lactic acid Diverse R. delemar Biotin Diverse romanniana, Mortierella vinacea and Linolenic acid Diverse 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 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: (syn. ARCHIASCOMYCOTINA) Class: Class: • Subdivision Class:

• Subdivision (syn. ASCOMYCOTINA)

Class: Laboulbeniomycetes Class: Class: Class: Class: Class: Class: 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 2. order Elaphomycetales 3. order Onygenales Order: Eurotiales

Order: Eurotiales • ochraceus • • fulva • variotii • Neosartorya fischeri • expansum • • Aspergillus

Foto: A. kubátová -frequently causes contamination of food and feed -manufacture of organic acids -mycotoxine () -osmophilic, osmotolerant - -opportunistic -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 1.Aflatoxiny B a G 2. 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 • 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: 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, , nerves, endocrine glands and cells of . They can cause acute toxic reaction, some have mutagenic, teratogenic, carcinogenic and estrogenic efect. MAIN GROUPS OF FUNGI AND THEIR MYCOTOXINES *

Aspergillus flavus SEM - Aspergillus flavus. microscopie. Aflatoxin B1, B2 and cyclopiazonic acid

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 formed by three types of secondary metabolites: A, B, a C. • All are produced by specia of Penicillium and Aspergillus. All three types are formed so, that Ochratoxin B (OTB) is nonchlorinated form of Ochratoxine A (OTA) and Ochratoxin C (OTC) is ethyl esther form of Ochatoxin A. • is contaminant of many comodits including beverages like beer and wine. • Aspergillus carbonarius is the main species found on grapes, where toxin is released during pressing. • OTA was qualified as carcinogen and nefrotoxin,probably is tumors promoting factor in human urinary tract. Citrinin • Citrinin – toxin originally isolated from , was identified in many other species of Penicillium and in some Aspergillus spp. • Some are used in food technology – cheeses (), sake, miso, soya sauces (). • Citrinin is responsible for disese of „yellow“ in Japan. It was prooved as nephrotoxin of all so far tested animals. • It occurs in many types of food (wheat, rice, , barley, oats, rye, and in food coloured by pigment) its full meaning for human health is not known. • Citrinin can synergisticaly cause together with Ochratoxinem A the suppresion of RNA synthesis in mice kidneys. Patulin • Patulin is toxin formed by P. expansum, Aspergillus, Penicillium, and Paecilomyces. • P. expansum is typical in rotten fruits and vegetable, namely rotten apples and figgs. • Its decomposition takes place during fermentation processes and is not find in beverages like cider. • Carcinogenity of patulin was not verified, but exist data about demage of immunity system in animals. • In 2004, E U set up limits for patulin concentration in food. • Maximal amount is 50 μg/kg for all types of fruit juices (concentrates), 25 μg/kg is limit for solid products from apples (ready to eat), 10 μg/kg for apple products for children nutrition, including apple juice. Mycotoxines of

• Fusaria belong to distinctive agents of human . Produce mycotoxins threatening human health. Namely trichotecens, zearalenon and fumonizin are common and dangerous.

• Zearalenon (C18H22O5) white, or pale yellow crystalic matter, without odour. • Temperature of thaw betwee 161 to 164 °C. Unsoluble in water, tetrachloromethan (CCl4) and alkoholes. Zearalenon is relatively lipophilic compound. Its content may be reduced in cereal technology processing. Zearalenon

• Isolated in 1966 • White or pale yellow crystalic matter • Temperature of thawing 161 – 164 °C • Found in flour and cereal products • estrogennic efect Trichothecens • 80 varieties (T-2 toxin, deoxynivalenol-DON…) • Chemicaly differnt • Tricyclic sesquiterpens • Symptoms of intoxication: inflammations of digestive tract, nausea, diarrhoea, inhibit proteosynthesis, destroy immunit systém.

deoxynivalenol T-2 toxin Trichothecens type A to D • Ergot alkaloids are produced as a mixture of toxic alkaloids in sclerocia of some Claviceps species, that are common grass . After digestion of ergot sclerocia from infected cereals, usually in bread made from contaminated flour, there is an onset of ergotisms – human disease, historicaly known as Fire of Saint Antonio. Two forms of ergotism: gangrenose affecting blood supply of limbs and convulsive, affecting central nervous system. Recent methods of corn cleaning reduced significantly the ergotisms as human disease. But it is still a veterinary problem. Ergot alkaloids has a wide pharmaceutical applications. Approaches to the risk analysis of mycotoxins in the food supply • While the complete elimination of mycotoxins from foods would be an impossible goal, it is important to ensure that their levels do not threaten health. In recent years, a variety of interrelated approaches have been developed to assess the hazards, monitor exposure and determine the associated risks. These processes need to be transparent so that the hazards and the need for risk management and intervention are clear. Risk assessments provide the scientific background and understanding for sound policy decisions that protect the public at an affordable cost and allow for public/international discussion, scrutiny and harmonization. Because there are differences in the significance of biological effects and in the available data, there is at present no single approach that can be used for all problems regarding mycotoxins, and a case-by-case approach is needed.