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Toxic Fungi: Mycotoxins Mycology (Bio 594, Special Topics) M. Marshall 2013 Shippensburg University (See last slide for credits)

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1 Toxic Fungi: Mycotoxins Mycology (Bio 594, Special Topics) M. Marshall 2013 Shippensburg University (See last slide for credits)

2 A Mycotoxicosis is not a Mycosis Fungi can be a threat to plants, animals and humans either as parasites that incite disease, termed mycosies in animals & humans, or as toxin-producers that cause a mycotoxicosis. Although the later term is not commonly applied with plants, there are plant diseases for which the entire syndrome, the impairment of host biochemistry and the symptoms and death of host tissue that it causes, are explainable primarily on the basis of the effects produced by a toxic fungal metabolite. Most mycotoxins must be ingested to be toxic to humans and animals. In the case of plants, host cells are exposed to the secreted toxin as the parasite hyphae grow within the host.

3 Mycotoxin effects on Plants Mycotoxins produced by plant pathogenic fungi can be simple in structure and activity or complex. Many are secondary metabolites. They can serve to kill host tissue in a non-specific fashion, or be so specific that only certain host genotypes within a species are affected. The host-specific toxins produce all of the symptoms associated with their respective diseases and host disease resistance is based on resistance of host tissue to the biochemical activity of the specific fungal toxin.

4 Some representative fungal toxins Some fungal toxic products are relatively simple and non-specific, several necrotrophic fungi, Sclerotinia and Cryphonectria spp., for example, secrete large amounts of oxalic acid to kill host tissue which they then feed off of. Other fungal toxins can be quite complex. The more complex toxins are often produced as a family of similar structures with differing amounts of activity. oxalate Victorin, a non-ribosomal peptide specifically toxic to Victoria oats. T-toxins of C.heterostrophus

5 Some host-selective fungal toxins

6 Some Fungal toxin-induced diseases in the field Clockwise from upper left: Northern Corn leaf blight incited by Cochliobolus carbonum; S. corn leaf blight incited by C. heterostrophus; and C. victoriae blight on Victoria oats. S. corn leaf blight all but destroyed the American corn crop in the summer of 1970 due to the heavy use of cytoplasmically sterile male corn plants in corn breeding the previous several years

7 Some Selected Fungal toxins affecting Animals & Humans

8 Tall Fescue : Schedonorus phoenix (Scop.) Holub; formerly Lolium (Festuca) arundinacea ( Liliopsida>Cyperales>Poaceae ) Tall fescue was “discovered” on a Menifee County Kentucky farm in 1931 by a UK agronomist. It was notable for being vigorous and green in a time of drought. Material was harvested, evaluations were made, and “Kentucky 31” was eventually released to farmers who planted it widely. Similarly, the “Alta” variety was selected from a stand of tall fescue in Oregon in By the 1940s or so the grass became controversial; reports began to come in of animal toxicity. The grass proved to be very invasive as it tolerated insects and drought better than it’s competitors. Named varieties continued to be developed including turf selections for domestic and athletic field use in the 1970s. Eventually the grass proved to, in-fact, be a European to North African cool season perennial grass; an invader that was introduced to the U.S. by accident during the 1800’s. By the 1960’s it was obvious that many grazing animals did poorly on the grass; they ate less, salivated excessively, gained less weight, were heat intolerant, showed poorer reproduction, less milk production and in some cases came down with a cold weather hoof and tail necrosis that became known as “fescue foot”. The extent of these problems varied from one location to another.

9 Tall Fescue Facts “The moderately stout stem is unbranched with 1-3 swollen, light green nodes near the base. Leaves are mostly basal, flat, 4 to 18 in. ( cm) long with whitish to yellow-green, flared collars. The midvein is not noticeable. Flowers occur in loose panicles that are 4-12 in. ( cm) long. Tall fescue invades a variety of open habitats including fields, forest margins, roadsides, forest openings and savannas. It spreads mainly through rhizomes and can form extensive colonies that compete with and displace native vegetation.”(from: Schedonorus phoenix (Scop.) Holub Taxonomic history (synonyms)

10 Tall Fescue is the most widely grown pasture and turf grass in the humid central and eastern U.S.

11 Fescue-fed animals fail to thrive

12 Fescue ergot alkaloids mainly affect the peripheral circulation SyndromeSigns Fescue foot (ruminants) Time: 1-several weeks Cool weather temp and blood flow to distal limb Lameness, separation of hoof at coronary band, gangrene Bovine fat necrosis (ruminants) Time: several seasons Large masses of hardened fat in abdomen Summer slump (ruminants) Warm weather Animals spend less time grazing Lower weight gains, reduced milk production,heat intolerance* Reproductive and lactation problems (horses) Time: late gestation early postpartum [Prolactin]* Prolonged gestation, abortion, weak or stillborn young, thick placenta, agalactia

13 The Tall Fescue Endophyte In 1970, the cause for both the hardiness and toxicity of fescue was uncovered. It harbors an endophytic fungus, presently Neotyphodium coenophialum, or a related species, N. lolii (which also infects perennial ryegrass) that produces at least three toxic alkaloids. Over 90% of tall fescue pastures have infected plants. This fungus does not sporulate; the endophyte grows into the seeds and is propagated along with the grass.

14 Neotyphodium spp. Neotyphodium coenophialum, N. lolii and related species are sterile fungi that are propagated via mycelial fragments that grow into the seeds of their host plant. Neothyphodiums have been shown to be closely related to Epichloe spp. via DNA analysis. Epichloe spp. are alkaloid-producing endophytic parasites of grasses that do sporulate asexually and sexually. They are classified into the Clavaceptaceae. Epichloe typhina stroma and conidia on red fescue E. Typhina ascospore- bearing perithecia (flask- shaped chambers) in older stroma.

15 The endophyte mostly inhabits the main stem, seed head, and leaf sheaths. But the alkaloids produced can also be found in leaf tissue. Alkaloid contents are highest in June and under nitrogen fertilization. As little as a 20% endophyte infection rate can produce noticeable problems in grazing animals. The endophyte is thought to account for drought resistance, resistance to nematodes and insects, enhanced phosphorus uptake, seed production, and seed germination. Endophyte-free fescue can be produced, but it loses much of it’s desired vigor.

16 peramine Endophyte alkaloids Three types of alkaloids are produced, ergovaline and derivatives are the most likely candidate for the productuion of animal toxicity as neither loline, nor peramine are toxic to grazing animals; although loline is a natural insecticide, Alkaloid-less fescue is not nearly as competitive, so efforts are underway to alter the genes for ergot alkaloid production as a means to solve this problem

17 Fescue poisoning solutions General: Frequent mowing of fescue, heavy grazing pressure, chemical treatment to prevent seed head development Dilution of fescue pastures with >20% palatable legumes: dilutes ergot alkaloids Plant fields with old seeds: fungus dies >1 year under normal storage New cultivars of fescue have been developed that are relatively free of the endophyte Some states require labeling on % endophyte infection (<5%) Fescue foot : Remove from source at 1 st sign; reversal in ~1 week. Antibiotics, antiinflammatory With severe lameness, salvage Fat necrosis: Severe fat necrosis, salvage Reproductive problems in mares: Withdraw from fescue days before foaling Intensive therapy Domperidone (D2 dopamine receptor antagonism) for prolonged gestation (? Approved) in horses

18 Ergot of Rye and other grasses; Claviceps purpurea and others

19 C. purpurea on Rye ( Secale cereale ) life /disease cycle

20 Stroma cross section showing ascocarps and asci The sclerotia overwiner in the soil and produce stalks with stroma at the time the next crop is flowering. Stroma perithecia release ascospores to begin the next season’s infection. Flowers become infected and produce a spore-filled ooze or “honeydew;” insects spread these spores to yet more flowers. Infected flowers develop into sclerotia instead of seeds and the cycle starts anew when these are detached a fall to the soil during harvesting. Clavicepts spp. Are ascomycetes that produce asexual conidia as a summer cycling phase, then transition to replace the infected ovaries with hard sclerotia that detach and drop into the soil.

21 Ergot “summer stage” infected flowers producing conidia in “honeydew” C. africana on sorghum microconidia

22 Clavicepts purpurea sclerotia with stroma Stroma close-up dormant sclerotia Germinating sclerotia with stipes (necks) and stromas (heads)

23 Other Ergot Species Sorghum Ergot (C. africana) Millet Ergot Dallas Grass Ergot

24 St. Anthony’s Fire Ergotism results from consuming grain contaminated wih Clavicepts sclerotia; in humans it expresses itself as a combination of vasoconstrictive, convulsive and hallucinatory effects. Plagues of ergotism occurred commonly throughout Europe during ancient times up through the middle ages. The earliest clearly recognized account dates from 857 in the Rhine Valley. An1039 outbreak in Dauphine France, lead to the founding of the order of St. Anthony to care for those so afflicted. The cause of the crawlng feeling, gangrene of the extremities, convulsions and hallucinations was not definitely linked to ergoted rye until the work of the French physician Thuillier in 1670 and the botanist and physician Dodart in 1676.

25 Was Ergotism the “Devil loosed in Salem” in 1692? See: Ergotism: The Satan Loosed in Salem? Science 192 (1976) by Linda R. Caporael

26 Ergot alkaloids A host of alkaloids are produced by the sclerotia; the exact mixture varies with growing conditions, etc. Today ergotamine, is prescribed for various causes of headaches, including migraines. Ergonovine is used to control postpartum hemorrhage and to cause contraction of the uterus. Hydrolsis of these compounds yields lysergic acid, a starting point for the synthesis of LSD. Lysergic acid

27 Indoor “Mold” Problems Many fungal species have the ability to degrade cellulosic material when the conditions allow. Most often the sole missing ingredient is free water, so any situation in the home that creates wet or very damp conditions will be conducive to fungal growth. This growth is unsightly, creates musty odors and can lead to the marring or destruction of the substrate. More importantly, it can also create a health hazard. Many mold spores are highly allergenic and some ubiquitous indoor fungi have the potential to produce mycotoxins of various types. Overt infections of humans are rare, few fungal species are so adapted so most human mycosies result from opportunistic species colonizing (usually via the respiratory tract) immune-compromised individuals; uncontrolled diabetes is also a predisposing factor. Mycotoxicosies usually result from the ingestion of food stuffs contaminated by fungi. Although there have been several well-publicized episodes of presumed fungal poisoning via the inhalation of toxin-containing spores, the evidence for this link is weak at best (see CDC article: “Facts about Stachybotrys chartarum and other (indoor) molds.”

28 S. Chartarum “at home” Stachybotrys chartarum grows well on high cellulosic low nitrogen content materials where the environment is constantly or frequently wet. Wall board, paper and ceiling tiles are ideal substrates. In nature it will infest straw, hay, etc. where it has been found to be very toxic to animals. The organism is ubiquitous, as are its many potential substrates, so in the home, the only recourse is to prevent leaks or free water from contacting the substrate. Control the water and you control (prevent) the fungus. The alternative is to invite a clean-up nightmare.

29 Stachybotrys produces mycotoxins First described from Poland in 1837, S.chartarum is an imperfect (Deuteromycete) fungus in the Moniliales, and is common in nature. It’s unique conidiophores make it easy to identify. First seen as a problem with horses, other livestock, and people handling straw in the Ukraine in 1930s. Common symptoms in humans were rash, especially in areas subject to perspiration, dermatitis, pain and inflammation of the mucous membranes of the mouth and throat, conjunctivitis, a burning sensation of the eyes and nasal passages, tightness of the chest, cough, bloody rhinitis, fever, headache, and fatigue. Potentially linked to a cluster of cases of pulmonary hemorrhaging and hemosiderosis among infants in Cleveland in However, the CDC in 2000 published reports critical of these studies. At present the relationship of S chartarum to human health remains unclear, but it still provides ample fodder for sensational journalism, paranoids and hucksters (selling expensive clean-up schemes). Stay tuned.

30 S. chartarum tricothecene toxins These materials are known to promote inflamation and other immune perterbations in test animals the lab. Some are also neurotoxic to rodents. They are known to be present in the conidia of the fungus. These materials can be very toxic if ingested, but there is also some question as to the average amount of toxin exposure received by merely breathing in spores, due to the fact that the conidia are not especially mobile.

31 S. Chartarum in culture S.chartarum conidia are produced in a liquid droplet that tends to dry holding the spores in place.

32 S. Chartarum, another look showing conidiophore and conidia detail.

33 S. chartarum scanning E.M. micrographs

34 The great FSC Ceiling Tile Debacle During the summers of 1994 and 1995 we had episodes of significant mold growth on ceiling tiles in FSC due to constant condensation drips. A small research project was done to compare results obtained by culturing tile samples with contracted-for air sampling reports.

35 Stachybotrys chartarum on PDA agar (from FSC ceiling tile sample) We found S.chartarum a bit more frequently among the ceiling tile samples that we cultured from than one would have supposed judging from the frequency with which it’s spores were collected from FSC air samples. This was true for many other fungal species as well (see table next slide).

36 The evil mold right here in River City* * From Bushman and Marshall, 2004; numbers are % of samples isolated from. The column headings are the names of various firms contracted to do air sampling for fungal spores

37 Aflatoxins Aspergillus sp. Conidial head Penicillium sp. conidia on phialids

38 aflatoxins Aflatoxins B 1,B 2,G 1 and G 2 are fluorescent metabolites of Aspergillus flavus and other spp. Contamination usually results from field- infected grain or other products being stored at high moisture (>13%). Concern over these metabolites surfaced in the 1960s after several well publicized episodes of animal poisoning occurred after feeding on “moldy”grain, peanut by-products and cotton seed meal, etc. These materials are toxic at a few ppm, or even ppb with some animals. They (i.e. B 1 ) are some of the most potent carcinogens known, being modified in the liver to create carcinogenic compounds. They inhibit DNA and RNA synthesis. They may be responsible for high liver cancer rates in some parts of Africa where peanuts make up a significant part of the diet.

39 Aflatoxin control in ground nuts, Arachis hypogea (peanut)

40 Fusarium toxins The Fusaria are a diverse group of common imperfect (Deuteromycete) fungi that are grouped together solely on the basis of producing boat-shaped (fusiform) asexual conidia. Members of this genus are plant pathogens, opportunistic human pathogens, and saprophytes. Quite a few are commonly found associated with various grains in the field. Several can produce one or more mycotoxins.

41 Fusarium toxins Several Fusarium spp. can produce one or more mycotoxins. The Tricothecene Fusarium toxins are especially interesting; zearelanone (F. graminearum) has been associated with producing estrogen-like effects in swine, T2 toxin (F. sporotrichioides) produces a hemorrhagic syndrome if contaminated food is consumed, characterized by internal bleeding in many organs and oral necrotic lessions. T2 toxin was suspected as a “Yellow Rain” agent. In the mid to late 1970s reports from parts of Kampuchea and Laos alleged that planes were spraying a mysterious yellow substance that caused hemorrhaging on contact. (see Yellow Rain articles). The question of whether Russian-produced T2 toxin was being used as a chemical agent in Southeast Asia, or whether the material collected was little more than fungal-contaminated bee feces was debated throughout the early 1980s and was never fully resolved. In general, Aspergillus, Penicillium and Fusarium species have been found to produce a wide range of seconday metabolites toxic to animals. One theory is that these fungi evolved the abilty to produce these materials in order to compete with rodents for their cache of stored seeds (many of these substances can act as anti-feedants). The moral: if its moldy, throw it out.

42 Fusaric acid T2 Zearelenone Nivalenol Fusarium sp. mycotoxins Vomitoxin Monilliformin Fumonisins

43 Amanitas are poisonous Basidiomycetes Most Basidiomycetes form macroscopic fruiting structures (mushrooms, conks on trees, etc.) that contain club-like structures (basidia) with four sexual spores (basidiospores). Massive amounts of basidia are produced on the sides of mushroom gills or line the insides of pores. These fungi often grow for many years before they fruit. Mycelia of the opposite mating type fuse (plasmogamy) in the substrate to form an N+N dikaryotic mycelium that maintains itself in this form for years before nuclear fusion (karyogamy) and basidiocarp formation occurs>

44 A more detailed Basidiomycete life-cycle

45 Amanita spp. A. bispora A. pantherina A. phaloides A. muscaria

46 Amanita species Amanitas are fairly common mushroom-forming fungi of world-wide distribution. They are often mycorrhizal on various types of forest trees. They contain amatoxins which are deadly peptide hepatotoxins. Unfortunately several Amanitas closely resemble non-toxic edible mushroom species, and there are many other poisonous genera as well. Poisoning symptoms include nausea, diarrhea, cramps, vomiting and perhaps drowsiness, hallucinations and coma. The onset may be immediate, or may be delayed many days. Poisonous mushrooms have no distinctive taste or smell and the toxins are often not altered by cooking or drying. Various mushroom-hunting books contain all sorts of Do’s and Don‘ts in regard to collecting edible mushrooms. Given the high degree of phenotypic variability often seen in the field, and the finality of the consequences of making a mistake, these rules can (in my humble opinion) all be boiled down to one: DON”T do IT! Do your mushroom hunting in the supermarket! See the MMWR report of June 1997 for an impressive example.

47 Amatoxins are potent RNA synthesis inhibitors; ingestion results in hepatic and renal failure.

48 Muscimol: Muscimol's primary action is at GABA receptor sites as a potent GABA-A agonist. Muscimol is commonly used in lab research on GABA, which is a primary inhibitory neurotransmitter. Muscimol has been shown to be active in several parts of the brain including the cerebral cortex, hippocampus, and cerebellum. Ibotenic Acid: The current view is that some Ibotenic Acid does cross the blood brain barrier unchanged, but some is partially metabolised into muscimol and the rest excreted. Ibotenic acid, however, has been shown to be a potent neurotoxin when injected directly in the brains of mice and rats, and is used as a potent brain-lessoning agent. It is structurally similar to glutamate and activates NMDA receptors, but this is not likely to be involved significantly with the effects of A. muscaria. Muscarine: Muscarine is known to affect acetylcholine levels and acts at muscarinic receptors, named for this chemical. While the levels of muscarine in A. muscaria are quite low (.002% -.003% by dry weight), some of the effects of A. muscaria are characteristic of cholinergic involvement. Amanita hallucinogenic toxins

49 Hallucinogenic compounds in A. muscaria

50 Galleria autumnalis This is a common non-Amanita wood decaying species in this part of PA. The small brown basidiocarps occur on old logs in groups. Because of its small size it’s unlikely to be harvested to eat which is good as it also contains amatoxins.

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52 Credits APS Net Illustrated Glossary of Plant pathology Tom Volk’s Fungus Page

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55 Fungal symbiosies

56 Most micro-fungi are first encountered as the imperfect stage Although many fungi may in fact be the imperfect asexual (anamorphic) form of a fungus with an perfect (teleomorph) stage, usually the production of the latter stage requires two opposite mating types to unite on specific substrates and/or under limited conditions. Years may elapse between the discovery of an asexual isolate and its sexual form. So many fungi that are important in human affairs are known only by their asexual designation. Some later prove to have a sexual stage and some not. Some fungi produce important secondary metabolites that can function as useful antibiotics in humans: penicillins, cephalosporins, and so on. The inhibition shown in this photo seems to involve a fungus-like prokaryote,or streptomycete.


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