Pathological studies on Bovine papilloma virus-fern interaction in hamsters.

Early pathological changes of Bovine papilloma virus (BPV-2)-fern (Pteridium aquilinum and Onychium contiguum fern) interaction in hamsters were studied. In bracken-exposed cattle, BPV induces malignancy in gastrointestinal and urinary bladder mucosa. Cutaneous warts were transmitted successfully in hamsters approximately after 3 months post inoculation while urinary bladder tumour of enzootic bovine haematuria cases were not transmitted. Histologically, tumour was diagnosed as fibroma. Onychium produced more pronounced effects than bracken fern which was characterized by significant reduction in body weight and testicular atrophy. BPV-fern interaction was not appreciable during early period of tumour induction and requires long-term studies for 12 to 18 months.

Efficient method for determining the glycyrrhizin content of fresh and dried roots, and root extracts, of Glycyrrhiza species.

A reliable and efficient method suitable for the selective analysis of the glycyrrhizin content of fresh and dried roots of Glycyrrhiza species and of root extracts is described. The method was optimised for recovery and analysis of glycyrrhizin after conversion to its aglycone, 18 beta-glycyrrhetinic acid. The reversed-phase HPLC system was developed using a sterically protected C8 column that allowed UV detection of the aglycone at 254 mm without interference from co-occurring components. The average recovery through the method was determined at 83%, with a range of 65-99%. Analysis of 150 dried root samples gave levels of glycyrrhizin consistent with earlier reported values. A comparison of post-harvest treatment conditions. showed that the drying of root samples at up to 65 degrees C did not result in any measurable reduction in glycyrrhizin.

Stability of the fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in ground maize under typical cooking environments.

The effects of moisture, pH and heat on the stability of nivalenol (NIV), deoxtnivalenol (DON) and zearalenone (ZEN) present as natural contaminants of ground maize were measured for different periods. Standard solution tests were also performed to measure pH, salt and temperature effects on NIV and DON. The solution tests showed NIV and DON to be relatively stable in buffer solutions over the pH range 1-10. Quite harsh conditions (pH 12, high salt concentration, 80 degrees C, prolonged exposure) were needed to give substantial breakdown. In the ground maize substrate, these toxins were further stabilized relative to the solution tests. NIV and DON were both reduced (range 60-100%) by treatment with aqueous bicarbonate solution at 10, 20 or 50% of the ground maize dry weight, and subsequent heating at 80 or 110 degrees C for 2 and 12 days. There was no measurable reduction at lower test temperatures (20, 40 degrees C). NIV (but not DON) also showed some reduction following addition of water and heating at 80 or 110 degrees C for 12 days. ZEN content was not reduced even by 12 days of heating at 110 degrees C after treatment with a sodium bicarbonate solution.

Impact of competitive fungi on Trichothecene production by Fusarium graminearum.

Bioassays were used to determine the production of the trichothecene mycotoxin, deoxynivalenol (DON), by two isolates of Fusarium graminearum when grown in association with potentially competitive fungi and an antifungal chemical, 6-pentyl-alpha-pyrone (6PAP). The presence of 6PAP in the culture medium reduced DON production by as much as 80%, but this effect was reduced for the F. graminearum isolate that most efficiently metabolized the added 6PAP. A 6PAP-producing Trichoderma isolate grown in a competition assay system with the F. graminearum isolates was also able to substantially reduce DON production. When Fusarium isolates (F. crookwellense, F. culmorum, F. subglutinans, F. poae, F. equiseti, F. avenaceum, and F. sambucinum), which co-occur with F. graminearum in New Zealand maize plants (Zea mays), were grown in competition assays, the effect on DON production was variable. However, all isolates of F. subglutinans tested were shown to cause reductions in DON production (by 13-76%, mean = 62%). F. subglutinans frequently co-occurs with F. graminearum, but its presence can vary with location and time of the season. When the competitive fungus tested was also a trichothecene producer (e.g., of nivalenol), both toxins were produced in the assay medium. The results indicate that mycotoxin production by F. graminearum can be affected by the presence of particular competitive fungi. These results have implications for an ecological understanding of pathogenicity and of mycotoxin accumulation in plants. Early establishment of F. subglutinans, for example, may act as a biological control mechanism providing a temporary protection against invasion by more commonly toxigenic fusaria such as F. graminearum.

Biotransformation of the Trichoderma metabolite 6-n-pentyl-2H-pyran-2-one by cell suspension cultures of Pinus radiata.

Cell suspension cultures of Pinus radiata metabolize the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) (1) via hydroxylation of the pentyl side chain. Examination of the culture medium following dosing studies with 1 revealed that 79-85% of this bioactive compound had been metabolised after 144 h. At that time, 34-40% of the metabolized dose was recovered as a series of monohydroxylated isomers of 1, the principal metabolite being 5-(2-pyron-6-yl)pentan-5-ol (7).

Effect of bracken (Pteridium a quilinum) and dryopteris (Dryopteris juxtaposita) fern toxicity in laboratory rabbits.

Experimental studies with Bracken and Dryopteris ferns @ 25% concentrate ration mixture were conducted in rabbits. Fern fed rabbits showed progressive anaemia, leukopaenia, lymphopaenia and relative heterophilia. Significant elevations in serum enzymes like serum glutamate oxaloactate transminase (SGOT), serum glutamate pyruvate transminase (SGPT), alkaline phosphatase (ALP), urea and creatinine levels were seen. Histopathologically, rabbits showed mild to moderate vascular changes in most of visceral organs, vacuolar degenerative changes in hepatocytes, hypersecretory activity in intestine, presence of casts in renal tubules and degenerative changes in renal tubular lining epithelial cells. Dryopteris fed rabbits showed somewhat more severe degenerative and vascular changes in different intervals. A low level of toxic principle ptaquiloside was detected in Bracken and Dryopteris ferns by thin layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) methods.

Trichoderma/pathogen interactions: measurement of antagonistic chemicals produced at the antagonist/pathogen interface using a tubular bioassay.

A tubular bioassay was used to measure analytically the local production and concentration of the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) at the Trichoderma antagonist/pathogen interface. 6PAP levels significantly increased in the presence of the pathogen Botrytis cinerea, typically 300-700%, and were highest near the pathogen source. The level of response for a particular Trichoderma isolate was found to vary with the test organism used. Two products produced by biotransformation of 6PAP by B. cinerea in response to the interaction were also detected.

Determination of the fate of three Fusarium mycotoxins through wet-milling of maize using an improved HPLC analytical technique.

The fate of three Fusarium mycotoxins, nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN), all common contaminants in New Zealand-grown maize, has been measured in fractions of maize after passage through a commercial wet-milling plant. Distribution of the three toxins follows a pattern reasonably expected from their physical solubility characteristics. The highly water-soluble mycotoxins, NIV and DON, were found at high concentrations (up to 8.8 mg/kg) in concentrated steep liquor (CSL) fractions, but at low levels (less than 0.3 mg/kg) in the solid (germ, fibre and gluten) fractions. The converse was true for ZEN, which is relatively insoluble in water. For ZEN, the maximum concentration found in CSL was 0.6 mg/kg compared with 2.2-4.8 mg/kg in germ, fibre and gluten fractions. Accordingly, an animal food byproduct composed mainly of pressed fibre and concentrated steep liquor was usually found to contain concentrations of all three mycotoxins above those existing in the input maize. A single sample of corn oil recovered during the study also had a high concentration (4.6 mg/kg) of ZEN. The analytical clean-up method used converts all trichothecenes present to parent alcohols, therefore results are indicative of total trichothecene content. HPLC analytical conditions suitable for the analysis of NIV and DON in complex process grain products are also described.

Fusaria and Fusarium toxins in New Zealand maize plants.

A time course study was made of the development of Fusarium infection and the appearance of the three Fusarium toxins, nivalenol (NV), deoxynivalenol (DON) and zearalenone (ZEN), in various fractions of maize plants from two sites in New Zealand, one in the Manawatu region and one in the Waikato. Fusarium infection was seen in leaf axil fractions in January, at the time of tassel emergence, and was detectable in stalks, leaf blades, rachis and peduncles during February and in kernels in April. NV, DON and ZEN were only detectable some time after infection was demonstrable. NV, in high concentrations relative to DON (up to 287 mg/kg for NV and up to 8 mg/kg for DON), was found in fractions from the Manawatu site where F. crookwellense and F. culmorum were the predominant toxigenic species. NV and DON at similar levels (up to 25 mg/kg) were found in fractions from the Waikato site at which F. graminearum and F. subglutinans predominated. Highest levels of NV and DON were in rachis and peduncle. ZEN was found most consistently in leaf axils and blades at both sites (up to 8 mg/kg at the Manawatu site and up to 75 mg/kg at the Waikato site) but at times there were high levels in rachis fractions (up to 417 mg/kg at the Manawatu site).

Ruakuric acid: a natural product from Aspergillus fumigatus.

A new chroman derivative, named ruakuric acid, was isolated from a strain of Aspergillus fumigatus growing in conjunction with a coral lichen. The structure was determined as 6-acetyl-5-hydroxy-4-methoxy-chroman-2-carboxylic acid (mixture of 2,4-cis,trans isomers).

Toxicity and trichothecene production by Fusarium acuminatum subsp. acuminatum and Fusarium acuminatum subsp. armeniacum.

The toxicity of cultures of Fusarium acuminatum subsp. acuminatum and Fusarium acuminatum subsp. armeniacum grown on Weet-Bix medium was assessed using a chick bioassay. Thirty-nine of 45 cultures of F. a. armeniacum tested produced at least 50% mortality in the chick bioassay. In contrast, of the 26 cultures of F. a. acuminatum tested, only nine produced at least 50% mortality. Selected extracts of both subspecies were analyzed by gas chromatography after clean-up and hydrolysis for the four main trichothecene families, namely; nivalenol (NIV), deoxynivalenol (DON), scirpentriol (Sctol), and T-2 tetraol (T-2tol). Levels of up to 500 micrograms/g and 7 micrograms/g of T-2tol were detected in F. a. armeniacum and F. a. acuminatum extracts respectively. Four cultures each of F. a. armeniacum and F. a. acuminatum were also grown on two solid media (Weet-Bix and Vermiculite) and two liquid media (MYRO and GYEP). Culture extracts were again tested for toxicity and analyzed for trichothecene production. Cultures of F. a. armeniacum grown on the solid media and on MYRO produced the highest toxicity. Levels of up to 168, 129, 150, and 8 micrograms/g of T-2tol were detected in cultures of F. a. armeniacum on Weet-Bix, Vermiculite, MYRO, and GYEP respectively. In contrast, only trace amounts of T-2tol were detected in extracts of F. a. acuminatum on all media. Sctol levels of less than 0.5 microgram/g were also detected in some cultures of both subspecies on solid media, but only F. a. armeniacum produced trace levels of Sctol on liquid media.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense.

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060-5010 mg/kg dry wt of culture) at ambient temperature (16-29 degrees C) in daylight than at ambient temperature (18-23 degrees C) in darkness or at controlled temperatures of 11 degrees C, 20 degrees C or 25 degrees C in darkness. Yields at 25 degrees C were low. Incubation at 11 degrees C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 degrees C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 degrees C (2460-21 360 mg/kg), 1 strain at 11 degrees C (6570 mg/kg). Yields at a temperature oscillating daily from 10-20 degrees C were less than at 15 degrees C. One of the 5 strains produced appreciable amounts of a-zearlaenol (1645 mg/kg at 20 degrees C) and 2 of nivalenol (340 and 499 mg/kg at 20 degrees C).

A survey of the natural occurrence of Fusarium mycotoxins in cereals grown in New Zealand in 1986-1989.

Fusarium mycotoxins, principally trichothecenes, occurred commonly in grain from crops in the North Island of New Zealand, but were much less common and also at the much lower levels in grains from South Island regions. The principal contaminants were trichothecenes of the nivalenol (NIV) and deoxynivalenol (DON) types. Trichothecenes derived from scirpentriol and T-2 tetraol were not common. Moniliformin occurred very rarely, while zearalenone contamination was not uncommon, but the levels were generally low. Maize kernels were commonly contaminated by moderate levels of both NIV- and DON-type trichothecenes, with levels up to 3.6 mg/kg and 11.95 mg/kg respectively recorded. The occurrence of NIV-type trichothecenes as a general contaminant in the range of 0.3-0.8 mg/kg, and frequently as the main contaminant, is unusual.

Carcinogenicity of bracken fern (Pteridium esculentum) in New Zealand.

Bracken fern (Pteridium esculentum) was harvested from two sites LB and TB one of which (TB) was on a central North Island New Zealand farm where bovine enzootic haematuria (BEH) was known to occur. The fern was dried, ground and incorporated (25% w/w) into a pelleted diet and fed to female rats for a total of 162 days. Fifteen weeks later when the rats were autopsied it was found that numerous tumours, mainly of the ileum and urinary bladder were present in the animals fed the bracken fern from the TB site. Neoplasms were found in 85% of rats from the TB group compared with 11% in the LB group while only a single tumour (a haemangioma of the uterus) was observed in the controls. In all, there were neoplasms in 42, 5 and 1 organ/s from the TB, LB and control rat groups respectively (p<0.001). Analysis of the fern and pellets for ptaquiloside, the carcinogen in bracken fern, showed much higher levels present in the material from the TB site. There was 26 and 2270 microg ptaquiloside/g of dried fern and, for pellets from the same fern, 6.5 and 355 microg ptaquiloside/g of pellets, for one collection from the LB and TB sources respectively.