Morphogenetic action of trichothecin on Trichothecium roseum]

Trichothecin and some conditions of cultivation, especially high concentrations of carbon favour differentiation of the submerged mycelium of Trichothecium roseum, i.e. formation of submerged conidia, bud forming cells, chlamidospores, chains of barrel-shaped cells capable of germination and development of new generations of the submerged mycelium. Biosynthesis of trichothecin is connected with growth of these generations of the mycelium which are characterized by a high dehydrogenase activity. Synthesis of fibrinolytic enzymes is also possible in the period of growth of a weakly differentiated mycelium.     

Synthesis of sesquiterpene metabolites in a Trichothecium roseum culture

During the cultivation Trichothecium roseum forms biologically active sesquiterpenes in particular trichothecin and trichothecolon. The quantitative ratio of these compounds in cultural liquid changes depending on the cultivation conditions. The compounds taking part in terpenoids shant specifically increase the synthesis of sesquiterpenes in fungus culture. A possibility of intertransformation of different trichothecenes provides the stability of the fungus-producent to its toxical metabolites. A fermental system carrying out the transformation of trichothecin into trichotecolon has been revealed.     

Biological effects of toxic products from Trichothecium roseum link

Extracts of rice cultures of 8 of 16 isolates ofTrichothecium roseum killed one or more ducklings or mice or both. One of the isolates (MC-156) obtained from shelled corn was the most toxigenic; extracts of this isolate killed all treated ducklings and mice.Doses of purified toxic fraction TR-1 of 166 mg/kg body weight given intraperitoneally killed all test mice but none of the mice given 100 mg/kg doses died. However, a partially purified fraction (Fraction VII), from which toxic fraction TR-1 was derived, killed two of three mice given 78 mg/kg doses.Crude ether extracts of rice cultures ofT. roseum (MC-156) produced death when injected intraperitoneally in rabbits and a 19-day-old pig and also produced dermal necrosis when applied to the skin of rabbits. The latter phenomenon was not observed when 5 mg of toxic fraction TR-1 was applied to the skin of rabbits.The greatest production of toxin occurred in rice cultures when incubated at 28° C and with 20 % (ml/g) water added to the rice. Incubation of rice cultures ofT. roseum (MC-156) under CO₂ tension suppressed toxin production.

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Zusammenfassung Auszüge von Reiskulturen von acht der 16 Stämme vonTrichothecium roseum haben mehrere Entchen und/oder Mäuse getötet. Einer der Auszüge (MC-156) von abgeschälten Mais war das toxischste; Auszüge dieses Isolates hat alle Entchen und Mäuse getötet. Dosen der gereinigten toxischen Fraktion TR-1, 166 mg/Kg Körpergewicht, intraperitonial verabreicht, haben alle Mäuse getötet, aber keine der Mäuse starb mit der Dose von 100 mg/Kg. Jedoch hat eine teilweise gereinigte Fraktion (Fraktion VII), von welcher die toxische Fraktion TR-1 gewonnen war, zwei von drei Mäusen mit 78 mg/Kg Dose getötet. Ungereinigte Ätherauszüge vonT. roseum (MC-156) waren tödlich, wenn sie in Kaninchen, in ein 19 Tage altes Schweinchen intraperitonel injiziert worden sind. Sie haben auch eine Hautnekrose hervorgerufen, wenn sie auf die Haut von Kaninchen gebracht worden sind. Das letztere Phänomen war nicht beobachtet, wenn 5 mg der toxischen Fraktion TR-1 an Kaninchenhaut gebracht worden ist. Die größte Produktion des Toxins fand in Reiskulturen statt, wenn sie bei 28° C und mit 20 Perzent (ml/g) Wasser bebrütet worden sind. Inkubation von Reiskulturen vonT. roseum (MC-156) unter CO₂-Druck hat die Toxinproduktion unterdrückt.

     

Hyperparasitic Attack of Trichothecium roseum on Conidia of Pestalotiopsis funerea

Trichothecium roseum (Tr) has been shown to be a highly effective hyperparasite on conidia of Pestalotiopsis funerea (Pf) in vivo and in vitro. The stages of this spore parasitism are: positive tropism of Tr towards Pf conidia, contact between Tr and Pf, formation of simple or lobed appressoria of Tr on the host conidial surface, penetration of the attacked host cells from the base of the appressoria, development of host-internal, mostly branching parasitic hyphae by Tr, desintegration, lysis and death of the parasitized host cells, exit of Tr from the destroyed host cells and its intensive sporulation over Pf remnants. Pf did not show any defence reactions against the attack by Tr. In addition to the antagonistic activities of Tr against Pf reported previously, which are due to extracellular toxins released by Tr, direct hyperparasitism is a second mechanism of antagonism, which contributes to the successful competitive ability of Tr in this fungal interaction.     

Release and regeneration of protoplasts from the fungus Trichothecium roseum

A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 x 10(4) protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose--yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.     

Qualitative and Quantitative Assay of Trichothecin: a Mycotoxin Produced by Trichothecium roseum

A method for quantitative determination of trichothecin in crude culture filtrates was presented. The method utilized an agar diffusion bioassay against Candida albicans, a colorimetric test involving a halochromatic reaction with sulfuric acid, and subsequent formation of blue color with methanol, and thin-layer chromatography of trichothecin and its dinitrophenylhydrazine derivative. A positive result in all three systems confirmed the presence of trichothecin. Quantitative results were generally in close agreement.     

Antagonism between Gliodadium roseum,Trichoderma harzianum,or Trichothecium roseum and Phytophthora megasperma f. sp. glydnea

The antagonism between Gliodadium roseum, Trichoderma harzianum, or Trichothecium roseum and Phytophthora megasperam f. sp. glycinea (Pmg), cause of Phytophthora rot of soybeans (Glycine max), was studied. G. roseum, T. harzianum, and 17 isolates of T. roseum were grown separately on modified Czapek-Dox medium (MCD) in the dark for 25 days at 25 °C. Culture filtrates of T. roseum at 0.5% and 20.0% concentrations inhibited mycelial growth of Pmg at 3.1% and 90.4%; and those of G. roseum at –0.7% and 44.0%; and of T. harzianum at 0.7% and 46.0%, respectively. Culture filtrates of T. roseum at 0.5% concentration inhibited zoosporangenesis of Pmg at 98.0% and at 1.0% concentration prevented it. Culture filtrates of G. roseum and T. harzianum at 5% concentration significantly (P=0.05) inhibited zoosporangenesis of Pmg, but differences were not significant from that on MCD. Culture filtrates of 17 isolates of T. roseum inhibited mycelial growth and zoosporangenesis of Pmg at different percentages with different concentrations with those of isolate 9 showing the greatest inhibition of both. Mycelial growth of most of 16 races of Pmg was prevented at 10% concentration of the culture filtrates of isolate SS-2 of T. roseum, and all 16 races, except race 6, was prevented at 20% concentration. Zoosporangenesis of all races of Pmg was prevented at 2% the culture filtrate of SS-2. Culture filtrates of SS-2 inhibited zoosporangenesis of Pmg in soil.     

Toxigenicity of Trichothecium roseum link: Isolation and partial characterization of a toxic metabolite

A toxic fraction (TR-1) was obtained from rice culture extracts of an isolate (MC-156) ofTrichothecium roseum from moldy corn. This toxic fraction was purified primarily by column and thin-layer chromatography and was chromatographically pure as evidenced by a single charred spot on an analytical thin-layer plate. The toxic fraction TR-1 possessed approximately 12.9 % nitrogen and an ethanol solution of this fraction possessed no ultraviolet absorbance in the range from 210 to 320 mµ. The infrared absorption spectrum exhibited strong absorption bands at 1180, 1380, 1445, 1630, 1670, 1730, 2930, 2960, and 3380 cm^–1; moderate absorption bands occurred at 775, 1230, 1410, 1515, 1540, and 2870 cm^–1. The dry residue from this fraction was readily soluble in ether, chloroform, ethanol and methanol.The toxic fraction Tr-1 appears to be a new and different toxic entity than has been described fromT. roseum.

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Zusammenfassung Eine toxische Fraktion (TR-1) ist von Reiskulturextrakten eines Stammes (MC-156) desTrichothecium roseum vom verschimmelten Mais erhalten worden. Diese toxische Fraktion war ursprünglich durch Säulen- und Dünnschicht-Chromatographie gereinigt worden und sie war chromatographically rein, wie ein einziger verkohlter Fleck an einer analytischen Dünnschichtplatte bewies. Die toxische Fraktion TR-1 hat ungefähr 12.9 % Stickstoff, und eine Ethanol-lösung dieser Fraktion besaß keine Absorption in der Spanne von 210 bis 320 mµ. Das infrarote Absorptionspektrum zeigte starke Absorptionstreifen bei 1180, 1380, 1445, 1630, 1670, 2930, 2960, und 3380 cm^–1; mäßige Absorptionstreifen erschienen bei 775, 1230, 1410, 151, 1540, und 2870 cm^–1. Der trockene Rückstand dieser Fraktion war leicht in Ether, Chloroform, Ethanol und Methanol auflösbar. Die toxische Fraktion TR-1 scheint eine neue und verschiedene Einheit zu sein, denn diejenigen, die vomTr. roseum beschrieben worden sind.

     

Trichothecene inactivation by proteolytic enzymes from Trichothecium roseum Lk ex Fr]

Trichothecium roseum Lk ex Fr produces simultaneously trichothecin and proteolytic enzymes possessing fibrinolytic, thrombolytic and esterase activity. In addition to the function of splitting and consuming the substrate, the proteolytic enzymes of T. roseum are probably able to hydrolize the other bond in the molecule of trichothecin, which results in partial inactivation of the antibiotic in both the fungus culture and the model experiment on trichothecin contact with the preparations of the proteolytic enzymes. Presence of proteases of T. roseum may be considered as a protective mechanism for detoxication of the metabolites toxic for the organism.     

Direct Evidence of Plant-pathogenic Activity of Fungal Metabolites of Trichothecium roseum on Apple

Apples were exposed to various concentrations of roseotoxins – metabolites of Trichothecium roseum and kinetic fluorescence imaging was used to detect the area influenced by the phytotoxin. Contrast was quantified within these images between the areas exposed to roseotoxins and the untreated areas. It was proved that roseotoxin B is able to penetrate apple peel and produce chlorotic lesions. Activity of roseotoxin B is similar as the activity of destruxins, host specific phytotoxins of Alternaria brassicae parasitic on canola.