Protein synthesis inhibition by 8-oxo-12,13-epoxytrichothecenes

The Fusarium mycotoxin, 4-deoxynivalenol, is an abundant, natural contaminant of corn and wheat. 8-Oxo-12,13-epoxytrichothecenes related to 4-deoxynivalenol were synthesized; they either lacked the 7-hydroxyl but contained a hydroxyl at C-4 (7-deoxynivalenol) or lacked substituents at C-3 and C-7 (3,7-dideoxynivalenol). The ability of these synthetic analogs and their acetylated derivatives to inhibit protein synthesis by cultured mammalian cells was compared to that of 4-deoxynivalenol. Whereas the 50% inhibitory dose (ID50) for murine erythroleukemia cells was about 1 microgram/ml for 4-deoxynivalenol and 3,7-dideoxynivalenol, all of the other analogs were at least 10-fold less potent. When tested at their ID50 dose, all of the 8-oxotrichothecenes, except 4-deoxynivalenol and 3,7-dideoxynivalenol, caused polysome 'run-off', indicating that, at this dose, they are inhibitors of polypeptide chain initiation. With 4-deoxynivalenol and 3,7-dideoxynivalenol, polysomes remained at control levels indicating that these toxins prevent polypeptide chain elongation. From these results and comparisons to previous studies of 8-oxo-12,13-epoxytrichothecenes (trichothecolone, trichothecin, nivalenol and fusarenone X), trichothecenes with substituents at both C-3 and C-4 predominantly inhibit polypeptide chain initiation, whereas those lacking one substituent at either site are inhibitors of chain elongation.     

Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol,nivalenol, 4,7-dideoxynivalenol,and zearalenone in polish wheat

Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals.     

Toxinogenicity of<Emphasis Type="Italic">Microdochium nivale (Fusarium nivale)</Emphasis> isolates from cereals in Poland

Microdochium nivale (Fusarium nivale) was found to be frequently occuring in Poland pathogen of small grain cereals heads, causing symptoms similar to those observed after infection ofFusarium species. In consecutive years since 1985 till 1989 the following percentage of wheat and rye ears infected withM. Nivale and withFusarium head blight symptoms was found: 34%, 21%, 42%, 9%, 46% (wheat) and 57%, 43%, 65%, 4%, 47% (rye) heads.However, in naturally infected rye and wheat samples (kernels and chaff), we did not detect toxins usually present in samples infected with fungi of genusFusarium — such as deoxynivalenol and derivatives. TypicalFusarium trichothecene metabolites were also not present in cultures of 11M. nivale strains, growing 3–5 weeks on rice (45% water content) at 20°C. Cultures of two typical isolates on wheat grain (strain KF 1124) and on rice (KF 245) were found to be non toxic to broiler chickens when present in amount 20–40% in their diet. It can be concluded thatM. nivale (F. nivale) representatives in Poland did not produce toxic metabolites neither under laboratory condition nor after cereal ears infection under field conditions.     

Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment

Forty six bacterial isolates able to grow on crude oil were isolated from various hydrocarbon-contaminated sites in Kuwait. The extent of crude oil degradation varied over a wide range (1–87%) among the isolates. Isolates were predominantly Gram-positive bacteria (79% of total isolates) belonging to the genera Bacillus (93%) and Paenibacillus (7%). Among the few Gram-negative isolates were from the genera Acinetobacter, Alcaligenes, Klebsiella, Burkholderia, Pseudomonas, and Williamsia. Analyses of their cell-surface hydrophobicity (CSH) by various methods equally showed a wide variation among the isolates. About 74% of isolates that degraded significant amounts of crude oil (>40% degradation) possessed high level of CSH, while 58% of all the isolates exhibited high levels of CSH. Statistical analyses showed significantly high correlation between the ability to degrade crude oil and CSH. The ability of the isolates to bind to polystyrene and salt-aggregation test as measures of CSH were more strongly correlated with hydrocarbon-degrading ability than adherence to hydrocarbons.     

Isolation of Pseudomonas stutzeri in wastewater of catfish fish-ponds in the Mekong Delta and its application for wastewater treatment

The aim of this study was to explore the potential for reducing soluble N load in fishpond wastewater using naturally occurring denitrifying bacteria. Twenty-seven isolates were selected from in wastewater (liquid/solid) of catfish-ponds located along the Tien river, in the Mekong Delta, Vietnam in SW-LB medium (artificial seawater Luria-Britani medium) supplemented with 10 mM NH₄ and NO₃ and twenty-five isolates were identified as Pseudomonas stutzeri based on similarity of PCR-16S rRNA using universal primers and specific primers. Four isolates were effective in lowering soluble N (NH₄, NO₂ and NO₃) levels in fishpond water from 10 mg/L to negligible amounts after four days. Further experiments are underway to determine the fate of N lost from solution and the relative activity of ammonia oxidation, and nitrite and nitrate reduction by P. stutzeri isolates.     

Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol

The mycotoxin deoxynivalenol (DON) causes serious problems worldwide in the production of crops such as wheat and barley because of its toxicity toward humans and livestock. A bacterial culture capable of degrading DON was obtained from soil samples collected in wheat fields using an enrichment culture procedure. The isolated bacterium, designated strain WSN05-2, completely removed 1,000???g/mL of DON from the culture medium after incubation for 10?days. On the basis of phylogenetic studies, WSN05-2 was classified as a bacterium belonging to the genus Nocardioides. WSN05-2 showed significant growth in culture medium with DON as the sole carbon source. High-performance liquid chromatography analysis indicated the presence of a major initial metabolite of DON in the culture supernatant. The metabolite was identified as 3-epi-deoxynivalenol (3-epi-DON) by mass spectrometry and ¹H and ¹³C nuclear magnetic resonance analysis. The amount of DON on wheat grain was reduced by about 90% at 7?days after inoculation with WSN05-2. This is the first report of a Nocardioides sp. strain able to degrade DON and of the yet unknown 3-epi-DON as an intermediate in the degradation of DON by a microorganism.     

Screening of ecologically diverse fungi for their potential to pretreat lignocellulosic bioenergy feedstock

A widespread and hitherto by far underexploited potential among ecologically diverse fungi to pretreat wheat straw and digestate from maize silage in the future perspective of using such lignocellulosic feedstock for fermentative bioenergy production was inferred from a screening of nine freshwater ascomycetes, 76 isolates from constructed wetlands, nine peatland isolates and ten basidiomycetes. Wheat straw pretreatment was most efficient with three ascomycetes belonging to the genera Acephala (peatland isolate) and Stachybotrys (constructed wetland isolates) and two white-rot fungi (Hypholoma fasciculare and Stropharia rugosoannulata) as it increased the amounts of water-extractable total sugars by more than 50 % and sometimes up to 150 % above the untreated control. The ascomycetes delignified wheat straw at rates (lignin losses between about 31 and 40 % of the initial content) coming close to those observed with white-rot fungi (about 40 to 57 % lignin removal). Overall, fungal delignification was indicated as a major process facilitating the digestibility of wheat straw. Digestate was generally more resistant to fungal decomposition than wheat straw. Nevertheless, certain ascomycetes delignified this substrate to extents sometimes even exceeding delignification by basidiomycetes. Total sugar amounts of about 20 to 60 % above the control value were obtained with the most efficient fungi (one ascomycete of the genus Phoma, the unspecific wood-rot basidiomycete Agrocybe aegerita and one unidentified constructed wetland isolate). This was accompanied by lignin losses of about 47 to 56 % of the initial content. Overall, digestate delignification was implied to be less decisive for high yields of fermentable sugars than wheat straw delignification.     

Population divergence in the wheat leaf rust fungus Puccinia triticina is correlated with wheat evolution

Co-evolution of fungal pathogens with their host species during the domestication of modern crop varieties has likely affected the current genetic divergence of pathogen populations. The objective of this study was to determine if the evolutionary history of the obligate rust pathogen on wheat, Puccinia triticina, is correlated with adaptation to hosts with different ploidy levels. Sequence data from 15 loci with different levels of polymorphism were generated. Phylogenetic analyses (parsimony, Bayesian, maximum likelihood) showed the clear initial divergence of P. triticina isolates collected from Aegilops speltoides (the likely B genome donor of modern wheat) in Israel from the other isolates that were collected from tetraploid (AB genomes) durum wheat and hexaploid (ABD genomes) common wheat. Coalescence-based genealogy samplers also indicated that P. triticina on A. speltoides, diverged initially, followed by P. triticina isolates from durum wheat in Ethiopia and then by isolates from common wheat. Isolates of P. triticina found worldwide on cultivated durum wheat were the most recently coalesced and formed a clade nested within the isolates from common wheat. By a relative time scale, the divergence of P. triticinia as delimited by host specificity appears very recent. Significant reciprocal gene flow between isolates from common wheat and isolates from durum wheat that are found worldwide was detected, in addition to gene flow from isolates on common wheat to isolates on durum wheat in Ethiopia.     

Molecular Characterization of Fusarium globosum Strains from South African Maize and Japanese Wheat

The fungus Fusarium globosum was first isolated from maize in South Africa and subsequently from wheat in Japan. Here, multiple analyses revealed that, despite morphological similarities, South African maize and Japanese wheat isolates of the fungus exhibit multiple differences. An amplified fragment length polymorphism-based similarity index for the two groups of isolates was only 45%. Most maize isolates produced relatively high levels of fumonisins, whereas wheat isolates produced little or no fumonisins. The fumonisin biosynthetic gene FUM1 was detected in maize isolates by Southern blot analysis but not in the wheat isolates. In addition, most of the maize isolates produced sclerotia, and all of them produced large orange to dark purple sporodochia in carrot agar culture, whereas wheat isolates did not produce either structure. In contrast, individual isolates from both maize and wheat carried markers for both mating type idiomorphs, which indicates that the fungus may be homothallic. However, a sexual stage of F. globosum was not formed under standard self-fertilization conditions developed for other homothallic species of Fusarium. The inability to produce the sexual stage is consistent with the high similarity of 87–100% and G _ST index of 1.72 for the maize isolates, which suggests that these isolates are undergoing asexual but not sexual reproduction. Together, the results suggest that the South African maize and Japanese wheat isolates of F. globosum are distinct populations and could be different species.     

Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae

Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the “Black Box” wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2,22,25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4,7-dien-3-one (3-oxo-Δ^4,7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ^4,7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4,6,8(14)-trien-3-one, oxidized from 3-oxo-Δ^4,7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ⁴-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ⁴-steroids into ecdysteroids via 5β-reduction.     

Development of Genomic SSR Markers and Molecular Characterization of Magnaporthe oryzae Isolates from Wheat in Brazil

Magnaporthe oryzae, the causal agent of wheat blast, was characterized on a molecular level with 38 newly isolated genomic SSR loci. Among the 31 wheat isolates analyzed, 15 polymorphic loci were detected, with an average of 1.7 alleles per locus, 28.9% of them being highly or reasonably informative. The number of polymorphic loci was higher in isolates from Londrina in the Brazilian state of Paraná and Coromandel in Minas Gerais compared with Goiânia in Goiás and São Borja in Rio Grande do Sul. The rice isolate was clearly different from the wheat isolates, and the size difference in polymorphic SSR loci between one isolate from wheat and one isolate from rice was associated with the number of repeats. Some isolates collected from different states and in different years demonstrated similarities of 100%. The markers developed here are useful for the genetic analysis of M. oryzae isolated from wheat, and isolates representing the variability detected in the field can be used to search for better wheat blast resistance.     

Formation of coumarines during the degradation of alkyl substituted aromatic oil components by the yeast <Emphasis Type="Italic">Trichosporon asahii</Emphasis>

In this study, we investigated the ability of the yeast Trichosporon asahii SBUG-Y 833 to assimilate phenylalkanes with alkyl chain lengths from 7 to 12 carbon atoms, and we describe for the first time the formation of coumarines via a novel degradation pathway other than the normal terminal and ß-oxidation pathway of the alkyl residues. Besides benzoic acid and its further oxidation products, six new metabolites were identified. These were the three coumarines—4-hydroxycoumarin, 4,6-dihydroxycoumarin, 4,8-dihydroxycoumarin—and the three alkyl substituted aromatic acids—7-phenylheptanoic acid, 2-hydroxyphenylheptanoic acid, and 2-hydroxyphenylpropanoic acid. 4-Hydroxycoumarin was the main extracellular metabolite during the degradation of both odd- and even-chain phenylalkanes and was also produced during further biotransformation of 2-hydroxyphenylpropanoic acid and trans-2-hydroxycinnamic acid. Due to the ability of T. asahii to form hydroxylated coumarines, the transformation of 7-hydroxycoumarin and 2,4-dihydroxyphenylpropanoic acid was investigated. Yeast cells supplemented with 7-hydroxycoumarin formed 6,7-dihydroxycoumarin and 4,7-dihydroxycoumarin. The transformation of 2,4-dihydroxyphenylpropanoic acid yielded to 4,7-dihydroxycoumarin as the main product. All hydroxylated coumarines were continuously accumulated and are very resistant to further oxidation. The high potential of the yeast T. asahii SBUG-Y 833 to form different hydroxylated coumarines from alkylaromatics suggests possible applications in the biotechnological production of coumarine structures with medical potential as anticoagulative and antitumor pharmaceutical.     

Isolate Virulence and Cultivar Response in the Winter Wheat: Pyrenophora tritici-repentis (Tan Spot) Pathosystem in Oklahoma

Prevalence of tan spot of wheat caused by the fungus Pyrenophora tritici-repentis has become more prevalent in Oklahoma as no-till cultivation in wheat has increased. Hence, developing wheat varieties resistant to tan spot has been emphasized, and selecting pathogen isolates to screen for resistance to this disease is critical. Twelve isolates of P. tritici-repentis were used to inoculate 11 wheat cultivars in a greenhouse study in split-plot experiments. Virulence of isolates and cultivar resistance were measured in percent leaf area infection for all possible isolate x cultivar interactions. Isolates differed significantly (P < 0.01) in virulence on wheat cultivars, and cultivars differed significantly in disease reaction to isolates. Increased virulence of isolates detected increased variability in cultivar response (percent leaf area infection) (r = 0.56, P < 0.05) while increased susceptibility in cultivars detected increased variance in virulence of the isolates (r = 0.76, P < 0.01). A significant isolate × cultivar interaction indicated specificity between isolates and cultivars, however, cluster analysis indicated low to moderate physiological specialization. Similarity in wheat cultivars in response to pathogen isolates also was determined by cluster analysis. The use of diverse isolates of the fungus would facilitate evaluation of resistance in wheat cultivars to tan spot.     

Structural evolution of wheat chromosomes 4A, 5A,and 7B and its impact on recombination

The construction of comparative genetic maps of chromosomes 4A^m and 5A^m of Triticum monococcum and chromosomes of homoeologous groups 4, 5 and 7 of T. aestivum has provided insight into the evolution of these chromosomes. The structures of chromosomes 4A, 5A and 7B of modern-day hexaploid bread wheat can be explained by a 4AL/5AL translocation that occurred at the diploid level and is present both in T. monococcum and T. aestivum. Three further rearrangements, a 4AL/7BS translocation, a pericentric inversion and a paracentric inversion, have taken place in the tetraploid progenitor of hexaploid wheat. These structural rearrangements and the evolution of chromosomes 4A, 5A and 7B of bread wheat are discussed. The presence of the 4AL/5AL translocation in several Triticeae genomes raises two questions — which state is the more primitive, and is the translocation of mono- or poly-phylogenetic origin? The rearrangements that have occurred in chromosome 4A resulted in segments of both arms having different positions relative to the telomere, compared to 4A^m and to 4B and 4D. Comparisons of map length in these regions indicate that genetic length is a function of distance from the telomere, with the distal regions showing the highest recombination.     

Fusarium poae and Fusarium crookwellense, Fungi Responsible for the Natural Occurrence of Nivalenol in Hokkaido

To determine the reasons for the natural occurrence of nivalenol in the northernmost area of Japan, scabby wheat was harvested from 19 crop fields in Hokkaido. Mycological surveys and analysis for mycotoxin contamination were performed. Among 13 wheat grain samples harvested in seven locations, 9, 2, and 6 samples were positive for deoxynivalenol, nivalenol, and zearalenone, respectively, at levels ranging from 0.03 to 1.28 μg/g, 0.04 to 1.22 μg/g, and 2 to 25 ng/g, respectively. The predominant Fusarium species of the scabby wheat examined were F. sporotrichioides, F. avenaceum, F. poae, and F. crookwellense. Fifteen of 48 F. poae isolates and all four F. crookwellense isolates were screened for the production of seven derivatives of trichothecenes and zearalenone respectively, on rice culture. One isolate of F. poae produced diacetoxyscirpenol alone (4.3 μg/g); seven produced nivalenol (1.3 to 23.8 μg/g), 4-acetylnivalenol (0.1 to 4.6 μg/g), and diacetoxyscirpenol (0.9 to 99.5 μg/g); and five produced nivalenol alone (0.4 to 3.5 μg/g). The remaining two isolates produced no trichothecenes. Zearalenone production was not found in any isolate of F. poae tested. All isolates of F. crookwellense produced nivalenol (0.9 to 22.5 μg/g), 4-acetylnivalenol (0.5 to 25.0 μg/g), and zearalenone (1.4 to 162.5 μg/g). From these results, it is apparent that deoxynivalenol and zearalenone, and occasionally nivalenol, occur naturally throughout Hokkaido, and it is suggested that nivalenol-producing F. poae and F. crookwellense strains are responsible for the natural contamination with nivalenol found in the northernmost area of Japan. Furthermore, it was found for the first time that several isolates of F. poae distributed in Hokkaido possessed the ability to produce both type A and type B trichothecenes.     

Genetic Diversity Among Monoconidial and Polyconidial Isolates of Bipolaris sorokiniana

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat-growing regions of the world. This fungus shows a high genetic diversity and morphological and physiologic variability. In this study, 19 polysporic and 57 monosporic isolates of B. sorokiniana were characterized using universal rice primers—URP-PCR. The results obtained when the dendrogram was constructed with all the data produced with the amplification products showed very distinct clusters. However, the similarity among the isolates was low where 37 and 26.3 % of the monosporic and polysporic isolates, respectively, showed similarity above 70 %. All primers amplified multiple DNA fragments of polysporic as well as the monosporic isolates. Isolates fingerprints were constructed based on binary characters revealed by the three primers. An amplified fragment of approximately 750 bp was observed among 40 % of the isolates, when primer URP-1F was used. When primers URP-4R and URP-2R were used, a fragment of 450 and 400 bp was present in 31.5 and 29 % of the isolates, respectively. It was expected a higher similarity among the isolates since the monosporic cultures were originated from the polysporic. The dendrogram did not enable the separation of B. sorokiniana isolates by their geographic origin. This low correlation suggests that gene transfer may have occurred by parasexual combination in this fungus population. However, in spite of the research efforts for that end, it has not been possible to establish patterns that characterize the profile of B. sorokiniana.     

Characterization of Clinical and Environmental Mycobacterium avium Spp. Isolates and Their Interaction with Human Macrophages

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.