Occurrence and distribution of Fusarium graminearum and deoxynivalenol in sweet corn ears.

Unusual wet and cool weather conditions during the 1994 growing season in Maryland and Delaware resulted in a severe outbreak of Fusarium graminearum on sweet corn ears ('Moore' variety) prior to harvesting and canning. The number of ears visibly infected with Fusarium spp. ranged from less than 5% to 25% in some fields. Infection typically occurred at the tassel end of the ears. Fusarium graminearum was isolated from surface disinfected kernels, both those which were visibly infected and those kernels which appeared disease-free in an area up to 5 cm from the edge of the visibly moulded areas. Infected ears were cut into four sections and the kernels only were analysed for deoxynivalenol (DON) using liquid chromatography (LC) and mass spectrometry/gas chromatography (GC/MS). Kernels from the visibly mouldy area of the ears contained DON at levels of approximately 446 mg/g DON on average; whereas in the non-visibly infected portion of the ears adjacent to the mouldy tips, DON levels averaged approximately 10 mg/g. Sections of ears closest to the base contained no detectable DON or less than 1 microgram/g. This is the first reported natural occurrence of the mycotoxin DON in sweet corn prior to harvest and canning.

Effects of Fusarium moniliforme culture extracts and fumonisin B1 on DNA, RNA and protein synthesis by baby hamster kidney cells.

Baby hamster kidney cells (BHK-21) were exposed to culture filtrates of 4 Fusarium moniliforme isolates containing varying levels of fumonisin B1 (FMB1) and the effects upon RNA, DNA and protein synthesis were monitored. Cells were also grown on medium amended with FMB1 only for comparison. After 24 h incubation FMB1 (100 micrograms/100 ml medium) reduced protein synthesis by 4% and by 18% after 48 h. Culture filtrates containing the highest levels of FMB1 also caused the greatest inhibition in protein synthesis after 24 h but after 48 h protein synthesis levels were the same as controls even though the FMB1 level was 360 micrograms/100 ml. Only FMB1 reduced DNA synthesis, by 8% after 24 h but after 48 h DNA levels had increased by 40% over controls. The culture filtrates containing the highest levels of FMB1 (360 micrograms/100 ml) reduced DNA synthesis more than 50% after 24 h and 48 h. Culture filtrates containing lesser amounts of FMB1 in some instances stimulated DNA synthesis and inhibited it in others. There was also no correlation in the level of FMB1 with the inhibition of RNA synthesis by BHK cells. It appears that metabolites other than fumonisin produced by F. moniliforme in culture can affect and both stimulate and inhibit RNA, DNA and protein synthesis by BHK cells.

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells.

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.

A biological assay for the detection of Myrothecium spp. produced macrocyclic trichothecenes.

A rapid, inexpensive bioassay to detect Myrothecium spp.-produced macrocyclic trichothecenes was developed. Media containing Myrothecium isolates were inoculated with Chlorella vulgaris, Ustilago maydis and Trichoderma viride. Based on width of the inhibition zone, isolates could be classified as highly toxigenic, non-toxigenic and intermediate. Whereas, C. vulgaris and U. maydis showed significant differences in their response to toxigenic and non-toxigenic isolates, T. viride did not. Production of roridins and verrucarins by the toxigenic isolates (by bioassay) was confirmed by thin layer chromatography and high performance liquid chromatography. This bioassay system, combined with confirmation chemical analyses, increases our ability to detect toxigenic fungal isolates.

Occurrence of aflatoxins in parboiled rice in Sri Lanka.

In Sri Lanka, rice is the main staple which is mostly processed into parboiled rice. The levels of aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) in parboiled and raw milled rice collected from major rice producing areas and rice consuming townships were estimated. In almost all the samples of parboiled rice examined, the AFB1 and AFG1 contents were significantly higher than in raw milled rice. The highest AFB1 content was 185 micrograms/kg and AFG1 content 963 micrograms/kg. These samples were collected from a major rice producing/milling district where the mean relative humidity is 78% and mean annual temperature 27 degrees C which is the highest amongst the rice growing areas in Sri Lanka. Raw rice was either free of aflatoxins or when toxins were detected, they occurred in less than 10% of the samples. The frequency of occurrence of surface fungal flora (Aspergillus/Penicillium) and aflatoxin content in market samples was closely related. Brownish or greenish moldly rice samples with fermented odour contained over 1000 micrograms/kg of AFB1.

Effect of parboiling and bran removal on aflatoxin levels in Sri Lankan rice.

Commercial parboiling of rice in Sri Lanka and many south Asian countries provides ideal conditions for the occurrence of aflatoxins because the rice is steeped (allowing fermentation) thus providing ideal conditions for growth of toxigenic Aspergillus species. However the traditional 'cottage' method of parboiling rice, which does not involve steeping, appears to reduce Aspergillus growth even after long storage periods. Preferential infection of parboiled rice by Aspergillus flavus was observed. Aflatoxin contents in inoculated rice produced by commercial parboiling (AFB1 60-92 mg/kg) were significantly higher than that in inoculated 'cottage' processed rice (AFB1 12-29 micrograms/kg). The steeping (precooking/soaking) process in commercial parboiling appears to increase the susceptibility of rice grains to fungal infection. Aflatoxin content in grains increased considerably with the increase in duration of soaking. However, the addition of 10 ppm calcium hypochlorite (bleach) to soaking water appreciably reduced A. flavus contamination and subsequent aflatoxin content in parboiled rice. No significant reduction in aflatoxin levels were observed after bran removal of contaminated rice.

Toxigenic Aspergillus flavus and aflatoxins in Sri Lankan medicinal plant material.

The fungal flora of 6 Asian medicinal plants, Aerva lanata (Linn.) Juss. Alyssicarpus vaginalis D.C., Tribulus terrestris Linn. Adhatoda vasica Nees., Centella asciatica (L.) Urb., Cardiospermum halicacabum Linn. was determined. After surface disinfection Aspergillus spp. were most frequently observed. Aspergillus flavus, isolated from Alyssicarpus vaginalis and Aerva lanata produced aflatoxins in culture. Aflatoxin B1 was also detected in a sample of Aerra lanata at a level of 0.5 micrograms/g. Plant material destined for medicinal use should be stored carefully prior to its use to prevent growth of naturally occurring toxigenic mold fungi.

Macrocyclic trichothecenes produced by Stachybotrys isolated from Egypt and eastern Europe.

Twenty seven isolates of Stachybotrys chartarum, S. albipes, S. kampalensis and S. microspora from Egypt and Eastern Europe were tested for production of macrocyclic trichothecenes. Twenty of the 27 isolates, grown on rice seeds, were toxic to brine shrimp larvae. Based on TLC and HPLC analyses, 5 macrocyclic trichothecenes (verrucarin J, roridin E, satratoxins F, G & H) as well as trichoverrols were identified. When grown in liquid culture on rice extract medium, only 3 isolates were toxic and produced verrucarin J, roridin E and satratoxins G & H. Extracts from mycelial mats were more toxic than culture filterates of two isolates grown on rice extract and both contained the same macrocyclic trichothecenes (285.5 mg/4 L), in addition to trichoverrols A & B (31 mg/4 L) found in mycelial mats only. When grown on 3% sucrose Czapek's medium supplemented with peptone and yeast extract (still cultures), all isolates were non-toxic to brine shrimp and no trichothecenes could be detected in the extracts.

Allelochemical regulation of reproduction and seed germination of two BrazilianBaccharis species by phytotoxic trichothecenes.

The potent phytotoxic trichothecene roridins and baccharinoids occur naturally in the Brazilian plants,Baccharis coridifolia andB. megapotamica. Biosynthesis of roridins inB. coridifolia appears to be linked to pollination, and the phytotoxins then accumulate in the seed. The roles of the phytotoxins in pollination, seed maturation, and germination of theBaccharis species were investigated. The high production of roridins occurred only in seeds resulting from intraspecific pollination, and the concentration of the toxins in the seeds generally increased with seed maturity. Removal of seed coats from trichothecene-producing BrazilianBaccharis species (B. coridifolia andB. megapotamica) and non-trichothecene-producing AmericanBaccharis species (B. halimifolia andB. glutinosa) resulted in improved seed germination ofB. halimifolia andB. glutinosa but complete inhibition of seed germination ofB. coridifolia andB. megapotamica. Addition of seed coat extracts of the BrazilianBaccharis species of dilute solutions (10(-6)µg/ml) of roridins or baccharinoids to the decoated seeds ofB. coridifolia andB. megapotamica resulted in germination, while seeds ofB. halimifolia andB. glutinosa were killed by the phytotoxins. Roridins interacted with gibberellic acid, a germination promoter, but not with abscisic acid, a germination inhibitor. The results from this study suggest that macrocyclic trichothecenes have a regulatory role(s) on reproduction and germination of BrazilianBaccharis species in their natural habitat.

The mystery of trichothecene antibiotics in Baccharis species.

The Brazilian higher plant Baccharis coridifolia has been shown to synthesize de novo a series of highly toxic macrocyclic trichothecene antibiotics heretofore found to be produced only by fungi. These compounds are produced only by female plants that have undergone pollination. Neither the male nor female plant is sensitive to the toxic effects of trichothecenes, whereas North American Baccharis species are. The macrocyclic trichothecenes found in B. coridifolia are the same as those produced by Myrothecium fungi, and it is suggested that the plant has acquired the toxin-producing genes from this fungus.

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus.

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.

Interaction Between the Antibiotic Trichothecenes and the Higher Plant Baccharis megapotamica.

The Brazilian shrub Baccharis megapotamica contains significant amounts of antibiotic trichothecenes. When these plants are grown in the United States, they are devoid of the mycotoxins. Feeding experiments with fungus-produced trichothecenes show that Baccharis megapotamica absorbs, translocates, and chemically alters these compounds to ones with structures analogous to those found in the plant in its native habitat. The mycotoxins, which have no apparent ill effect in Baccharis megapotamica, kill tomatoes, peppers, and artichokes.

Minicolumn detection methods for aflatoxin in raw peanuts: collaborative study.

The Holaday-Velasco method and a modified Holaday method have been compared. The former method combines the speed and simplicity of the Holaday extraction and cleanup with the sensitivity of the minicolumn originally described by Velasco. The combination method has been approved by the AOAC and the AACC for determining aflatoxin in corn. The Holaday method was modified by substituting toluene for benzene in the solvent partition, and methylene chloride for chloroform in the minicolumn development to eliminate use of hazardous solvents. The neutral alumina in the Holaday minicolumn was changed from activity V to activity III to provide a more stable column. At aflatoxin levels in raw peanuts of 13-20 ng/g, the presence of aflatoxin was missed by the modified Holaday method in 4 analyses (3 laboratories) of 42 reported. There were no misses in this contamination range by the Holaday-Velasco method. There were no misses by either method with samples containing greater than 20 ng total aflatoxins/g. Analysis of uncontaminated raw peanuts by the modified Holaday method resulted in 2 false positives of 14 reports; the Holaday-Velasco method produced no false positive reports from 15 analyses of uncontaminated peanuts. The Holaday-Velasco method was adopted official first action for peanuts.

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum.

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Delta(5) to Delta(7) or to Delta(5,7). Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24alpha-ethyl = trans-Delta(22)-24alpha-ethyl > trans-Delta(22)-24beta-ethyl = 24alpha-E-ethylidene = 24alpha-methyl > 24beta-methyl = trans-Delta(22)-24beta-methyl = 26-methyl = saturated C(7) side chain and C-20 R (17-alphaH, 20-alphaH, right-handed conformer) = cis-Delta(22)-C(7) side chain and C-20 R > saturated C(7) side chain and C-20 S (17-alphaH, 20-betaH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20alpha-hydroxycholesterol = 24xi-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20alpha-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.