Production of 4,15-diacetylnivalenol by <Emphasis Type="Italic">Fusarium Sambucinum</Emphasis> Fuckel var. <Emphasis Type="Italic">minus</Emphasis>

Fusarium sambucinum Fuckel var. minus isolate produced unusual for F. sambucinum Fuckel trichothecene metabolite 4,15-diacetylnivalenol (9 mg/l) in conditions of deep cultivation on Myro medium. This compound was identified by TLC, GLC, HPLC, and ¹H NMR spectroscopy. Other trichothecenes, 4-acetylnivalenol (3 mg/l) and nivalenol (1 mg/l), were also found in the culture. The observed feature of the studied isolate is assumed to be due to the presence of an additional gene, which encodes cytochrome P450 oxygenase responsible for the introduction of keto group at C-8 and hydroxyl group at C-7 of the trichothecene structure.     

Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum Fuckel.

Fusarium sambucinum Fuckel 8099-1 was grown on Czapek-Dox peptone-supplemented medium at 15 degrees C for 14 days, and the cultures were investigated for diacetoxyscirpenol (DAS) production by liquid-liquid extraction and gas chromatography. The addition of 150 mg of sorbic acid, a tricarboxylic acid cycle inhibitor, per liter stimulated both fungal growth and DAS production. Among the beta-hydroxy-beta-methylglutaryl coenzyme A precursors tested, isovaleric acid completely inhibited fungal growth and DAS production, ethyl isovalerate did not support a significant increase in DAS production, and L-leucine partially inhibited DAS production, showing that L-leucine and isovaleric acid catabolisms do not induce trichothecene biosynthesis. Solid particles (cork powder) were necessary for DAS production in stationary cultures but did not influence DAS production in shaken cultures. Shaking strongly stimulated DAS production and fungal growth.     

Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum Fuckel

Fusarium sambucinum Fuckel 8099-1 was grown on Czapek-Dox peptone-supplemented medium at 15 degrees C for 14 days, and the cultures were investigated for diacetoxyscirpenol (DAS) production by liquid-liquid extraction and gas chromatography. The addition of 150 mg of sorbic acid, a tricarboxylic acid cycle inhibitor, per liter stimulated both fungal growth and DAS production. Among the beta-hydroxy-beta-methylglutaryl coenzyme A precursors tested, isovaleric acid completely inhibited fungal growth and DAS production, ethyl isovalerate did not support a significant increase in DAS production, and L-leucine partially inhibited DAS production, showing that L-leucine and isovaleric acid catabolisms do not induce trichothecene biosynthesis. Solid particles (cork powder) were necessary for DAS production in stationary cultures but did not influence DAS production in shaken cultures. Shaking strongly stimulated DAS production and fungal growth.     

Trichothecene biosynthesis inGibberella pulicaris: Inheritance of C−8 hydroxylation

Summary Naturally occurring strains ofGibberella pulicaris (Fusarium sambucinum) produce different kinds and levels of trichothecene toxins. Progeny from crosses between strains which produce trichothecenes with an oxygen-containing group at C–8 (C8+) and those that do not (C8–) can segregate in a 11 ratio for this trait. These results define a genetic locus, which we have designatedTox1. The segregation patterns observed for progeny obtained from crosses between high-toxin producers and low-toxin producers indicate that the level of toxin production is determined by several loci. One gene which controls quantitative aspects of toxin production segregates independently of both theTox1 locus and another locus which controls toxin levels. These results suggest that multiple, unlinked nuclear loci are involved in the control of trichothecene biosynthesis.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Sambutoxin-Producing Isolates of Fusarium Species and Occurrence of Sambutoxin in Rotten Potato Tubers

A total of 50 Fusarium isolates representing 13 species from various sources were surveyed to determine their potential to produce sambutoxin. Sambutoxin production was restricted to Fusarium sambucinum and F. oxysporum, with the exception of one isolate of F. semitectum. Sambutoxin was produced by high percentages of F. sambucinum (80.0%) and F. oxysporum (84.6%) isolates at levels of 1.1 to 101.0 (mu)g/g. In addition, 9 (42.9%) of 21 rotten potato samples were contaminated with sambutoxin at levels of 15.8 to 78.1 ng/g.     

A fungal symbiont of plant-roots modulates mycotoxin gene expression in the pathogen Fusarium sambucinum

Fusarium trichothecenes are fungal toxins that cause disease on infected plants and, more importantly, health problems for humans and animals that consume infected fruits or vegetables. Unfortunately, there are few methods for controlling mycotoxin production by fungal pathogens. In this study, we isolated and characterized sixteen Fusarium strains from naturally infected potato plants in the field. Pathogenicity tests were carried out in the greenhouse to evaluate the virulence of the strains on potato plants as well as their trichothecene production capacity, and the most aggressive strain was selected for further studies. This strain, identified as F. sambucinum, was used to determine if trichothecene gene expression was affected by the symbiotic Arbuscular mycorrhizal fungus (AMF) Glomus irregulare. AMF form symbioses with plant roots, in particular by improving their mineral nutrient uptake and protecting plants against soil-borne pathogens. We found that that G. irregulare significantly inhibits F. sambucinum growth. We also found, using RT-PCR assays to assess the relative expression of trichothecene genes, that in the presence of the AMF G. irregulare, F. sambucinum genes TRI5 and TRI6 were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We conclude that AMF can modulate mycotoxin gene expression by a plant fungal pathogen. This previously undescribed effect may be an important mechanism for biological control and has fascinating implications for advancing our knowledge of plant-microbe interactions and controlling plant pathogens.     

Sambutoxin, a new mycotoxin produced by toxic Fusarium isolates obtained from rotted potato tubers.

Ninety-nine isolates of Fusarium species were obtained from rotted potato tubers from various parts of Korea. Of these isolates, 80 were identified as Fusarium oxysporum, F. solani, or F. sambucinum. The isolates of these species were grown on autoclaved wheat grains and examined for toxicity in a rat-feeding test. A total of 8 of 57 F. oxysporum isolates, 3 of 14 F. solani isolates, and 5 of 9 F. sambucinum isolates caused the death of the rats. Of the 16 toxic isolates, 1 isolate of F. oxysporum produced a substantial amount of moniliformin, which could account for its toxicity. None of the other 15 isolates produced trichothecenes, moniliformin, fusarochromanone, fumonisin B1, or wortmannin. F. sambucinum PZF-4 produced an unknown toxin in wheat culture. This new toxin, given the trivial name sambutoxin, caused toxic effects in rats, including body weight loss, feed refusal, hemorrhage in the stomach and intestines, and, finally, death when rats were fed diets supplemented with 0.05 and 0.1% sambutoxin. The toxin was also toxic to chicken embryos, and the 50% lethal concentration was 29.6 micrograms per egg. Sambutoxin formed as white crystals that turned purple when combined with reagents such as sulfuric acid and p-anisaldehyde. It exhibited a green color immediately after treatment with potassium ferricyanide-ferric chloride. Its UV spectrum had absorption maxima at 213, 233, and 254 nm, and its infrared spectrum showed an amide group at 1,650 and 1,560 cm-1 and a hydroxy group at 3,185 cm-1. Mass spectrometry showed that the molecular weight of the toxin was 453 and the molecular formula was C28H39NO4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrocarbon Degradation and Biosurfactant Production by an Acenaphthene-degrading Pseudomonas Species

An acenaphthene-degrading bacterium putatively identified as Pseudomonas sp. strain KR3 and isolated from diesel-contaminated soil in Lagos, Nigeria was investigated for its degradative and biosurfactant production potentials on crude oil. Physicochemical analysis of the sampling site indicates gross pollution of the soil with high hydrocarbon content (2100 mg/kg) and detection of various heavy metals. The isolate grew luxuriantly on crude oil, engine oil and acenaphthene. It was resistant to septrin, amoxicillin and augmentin but was susceptible to pefloxacin, streptomycin and gentamycin. It was also resistant to elevated concentration of heavy metals such as 1–15 mM lead, nickel and molybdenum. On acenaphthene, the isolate exhibited specific growth rate and doubling time of 0.098 day^?1 and 3.06 days, respectively. It degraded 62.44% (31.2 mg/l) and 91.78% (45.89 mg/l) of 50 mg/l acenaphthene within 12 and 21 days. On crude oil, the specific growth rate and doubling time were 0.375 day^?1 and 1.85 days with corresponding percentage degradation of 33.01% (903.99 mg/l) and 87.79% (2403.71 mg/l) of crude oil (2738.16 mg/l) within 9 and 18 days. Gas chromatographic analysis of residual crude oil at the end of 18 days incubation showed significant reductions in the aliphatic, alicyclic and aromatic fractions with complete disappearance of benzene, propylbenzene, pristane, phytane, and nC₁₈-octadecane fractions of the crude oil. The isolate produced growth-associated biosurfactant on crude oil with the highest emulsification index (E₂₄) value of 72% ± 0.23 on Day 10 of incubation. The partially purified biosurfactant showed zero tolerance for salinity and had its optimal activity at 27°C and pH 2.0.     

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production inGibberella pulicaris (Fusarium sambucinum)

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production inG. pulicaris are described in the present study. A highly fertile, isogenic line was constructed forG. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Engineered bakers yeast as a sensitive bioassay indicator organism for the trichothecene toxin deoxynivalenol

The aim of this study was to increase the sensitivity of Saccharomyces cerevisiae towards trichothecene toxins, in particular to deoxynivalenol (DON), in order to improve the utility of this yeast as a bioassay indicator organism. We report the construction of a strain with inactivated genes (PDR5, PDR10, PDR15) encoding ABC transporter proteins with specificity for the trichothecene deoxynivalenol, with inactivated AYT1 (encoding a trichothecene-3-O-acetyltransferase), and inactivated UBI4 and UBP6 genes. Inactivation of the stress inducible polyubiquitin gene UBI4 or the ubiquitin protease UBP6 increased DON sensitivity, the inactivation of both genes had a synergistic effect. The resulting pdr5 pdr10 pdr15 ayt1 ubp6 ubi4 mutant strain showed 50% growth inhibition at a DON concentration of 5 mg/l under optimal conditions. The development of a simple two step assay for microbial DON degradation in 96 well microtiter format and its testing with the DON detoxifying bacterium BBSH 797 is reported.     

Susceptibility of a Brazilian wild rodent isolate of Schistosoma mansoni to praziquantel in mice

The therapeutic effects of praziquantel (PZQ) against a Schistosoma mansoni isolate derived from Nectomys squamipes (isolate R) and a susceptible isolate (BH) were analyzed in Swiss mice by fecal egg counting, adult worm reduction and oogram pattern. Infected mice were orally administrated with 62.5mg/kg (group 1), 125mg/kg (group 2), 250mg/kg (group 3) and 500mg/kg (group 4), each dose divided over 3 days (49, 50 and 51 days after infection). The data were analyzed using one-way analysis of variance (ANOVA). In regard to isolate R, no fecal eggs were observed with 250 mg/Kg and 500 mg/kg (p<0.05), whereas BH excretion reached zero with all doses. Mean worm burden reduction was significantly (p<0.05) higher at the two highest concentrations, regardless of isolate. At 62.5mg/kg, the percentage of immature eggs varied from 17% (isolate R) to 38% (isolate BH). At 125 mg/kg, the percentage of immature eggs varied from 20% (isolate R) to 16% (isolate BH). At 250 mg/kg, immature eggs dropped significantly to 1% (isolate R) and 4% (isolate BH). At 500 mg/kg, no immature eggs were found in isolate R, whereas in BH was 8%. No dosage significantly (p>0.05) affected the percentage of mature eggs, regardless of isolate. There was a large increase (p<0.001) in the percentages of dead eggs in all treated groups of 62% and 64% in groups 3 and 4, respectively (isolate R). The percentage of dead eggs rose from 34% (group 1) to 58% (group 3) in isolate BH. Although group 4 showed lowest increase in the percentage of dead eggs (46%), it was higher (p<0.001) compared to the 8% in the control. Our findings indicate that the wild isolate from N. squamipes is susceptible to PZQ.     

Bioremediation of pulp and paper mill effluent by a novel fungal consortium isolated from polluted soil

Two basidiomycetous fungi (Merulius aureus syn. Phlebia sp. and an unidentified genus) and a deuteromycetous fungus (Fusarium sambucinum Fuckel MTCC 3788) were isolated from soils affected with effluents of a pulp and paper mill over several years. These isolates were immobilized on nylon mesh and the consortium was used for bioremediation of pulp and paper mill effluent in a continuously aerated bench-top bioreactor. The treatment resulted in the reduction of color, lignin and COD of the effluent in the order of 78.6%, 79.0% and 89.4% in 4 days. A major part of reductions in these parameters occurred within first 24h of the treatment, which was also characterized by a steep decline in the pH of the effluent. During this period, total dissolved solids, electrical conductivity and salinity of the effluent also registered marked decline. It is pertinent to note that this is the first report of bioremediation of pulp and paper mill effluent by an immobilized fungal consortium.     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Diacetoxyscirpenol production by Fusarium sambucinum strain KF 735 on solid and liquid media

The yield of diacetoxyscirpenol (DAS) production by F. sambucinum strain No KF 735, isolated from potato tuber with dry rot symptoms, cultured on solid media and on liquid medium, has been examined.The amount of DAS produced within 28 days at 25°C in the cultures grown on solid media (wheat, rye, rice, oats, corn, barley, triticale and malt) reached 238mg/kg±9 to 789±16mg/kg (mean ± standard error; n=3), on potato cubes ?55±3mg/kg and on the potato extract ?147±5mg/dcm³.The best substracts for crystalline compound production were malt and barley grain.     

Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene in Fusarium sporotrichioides and Gibberella pulicaris.

Ancymidol, a plant growth regulator, inhibited biosynthesis of diacetoxyscirpenol by Gibberella pulicaris (Fusarium sambucinum) in a defined liquid medium. Ancymidol also inhibited biosynthesis of T-2 toxin by a wild-type strain of Fusarium sporotrichioides and biosynthesis of diacetoxyscirpenol, deacetylated calonectrin, and dideacetylated calonectrin by mutant strains of this species. Ancymidol-treated cultures accumulated the hydrocarbon trichodiene, a biosynthetic precursor of the trichothecenes. Ancymidol did not block trichodiene accumulation by a trichodiene-producing mutant strain of F. sporotrichioides. Ancymidol appears to block the trichothecene biosynthetic pathway after formation of trichodiene and before formation of trichothecenes containing four or more oxygen atoms.     

Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene in Fusarium sporotrichioides and Gibberella pulicaris

Ancymidol, a plant growth regulator, inhibited biosynthesis of diacetoxyscirpenol by Gibberella pulicaris (Fusarium sambucinum) in a defined liquid medium. Ancymidol also inhibited biosynthesis of T-2 toxin by a wild-type strain of Fusarium sporotrichioides and biosynthesis of diacetoxyscirpenol, deacetylated calonectrin, and dideacetylated calonectrin by mutant strains of this species. Ancymidol-treated cultures accumulated the hydrocarbon trichodiene, a biosynthetic precursor of the trichothecenes. Ancymidol did not block trichodiene accumulation by a trichodiene-producing mutant strain of F. sporotrichioides. Ancymidol appears to block the trichothecene biosynthetic pathway after formation of trichodiene and before formation of trichothecenes containing four or more oxygen atoms.     

An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand

Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100?% similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02?g/l) was obtained when the cells were grown on minimal salt medium containing 15?g/l molasses and 1?g/l commercial monosodium glutamate supplemented with 1?g/l NaCl, 0.1?g/l leucine, 5?% (v/v) inoculum size at 30?°C and 150?rpm after 54?h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5?mN/m), a small CMC value (8?mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.     

Competitive interactions between Fusarium sambucinum Fuckel and Phoma glomerata (Corda) Wollenweber & Hochapfel under in vitro conditions

The effects of temperature (15 and 25 degrees C), water activity (0.85, 0.90, 0.95, 0.98 y 0.995) and the interaction between Fusarium sambucinum and Phoma glomerata in rice extract agar on fungus growth were investigated. Fungi interactions were given a numerical score to obtain an Index of Dominance (ID) and to observe possible variations under different conditions of temperature and water activity (aw) changed. F. sambucinum and P. glomerata grew most rapidly -both individually and paired- at 0.995 aw and 25 degrees C. On the other hand, F. sambucinum presented higher growth rates than P. glomerata and it was dominant over P. glomerata under all the tested conditions. Water activity and temperature showed a significant effect on fungus growth.     

Canker and wilt of black locust (Robinia pseudoacacia L.) caused by Fusarium species

From the pathological material of black locust trees showing symptoms of wilting of the foliage or canker of the bark the following Fusarium species were isolated: Fusarium avenaceum (Fr.) Sacc., Fusarium lateritium Nees., Fusarium semitectum Berk. & Rav., Fusarium solani (Mart.) Sacc., Fusarium sulphureum Schlecht. (syn.: Fusarium sambucinum Fuckel f. 6 Wollenw.) The results of the provocation infections of one-year-old black locust seedlings showed that all of the species--except Fusarium solani--are able to cause considerable necrosis in living bark and phloem. Fusarium sulphureum had by far the highest pathogenecity among the tested species. Fusarium semitectum isolated from withered black locust tree also caused necrosis on significant bark area. In the course of the penetration assay Fusarium sulphureum and Fusarium avenaceum were the most successful, and these species can cause cankers on the stem and twigs of black locust without frost effect.