Fumonisins are polyketide mycotoxins that are produced in maize kernels by the fungus Gibberella fujikuroi mating population A (MP-A) (anamorph, Fusarium moniliforme, syn. F. verticillioides). These toxins are carcinogens in rats and mice and there is an epidemiological correlation between the consumption of fumonisin contaminated maize and human esophageal cancer in some areas of the world. Researchers are employing biochemical, genetic, and molecular genetic approaches to study fumonisin biosynthesis. We used PCR with a cDNA template and degenerate primers to isolate a gene (FUM5) that has sequence homology to bacterial and fungal Type I PKS genes. Inactivation of FUM5 via gene disruption reduced fumonisin production by over 99% in G. fujikuroi MP-A. These data are consistent with FUM5 encoding a PKS that catalyzes the synthesis of the linear carbon backbone of fumonisins. Because classical genetic studies indicate that fumonisin biosynthetic genes may be clustered, we have begun sequencing the DNA flanking FUM5 in search of other genes. To date, we have identified four open reading frames (ORFs). Comparisons of the predicted translation products of these ORFs with protein sequence databases suggest that the ORFs may function in fumonisin biosynthesis. We are beginning to understand the process of fumonisin biosynthesis as a result of the studies described above and others. We anticipate that a clear understanding of this process will reveal weak links that can be exploited to control fumonisin contamination in maize.

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Aflatoxins are produced from acetyl CoA through more than 18 enzyme steps. Although most of all enzymatic reactions have already been reproduced by in vivo and in vitro experiments, a few enzyme activities such as one catalyzing reaction from O-methylsterigmatocystin to aflatoxin G₁ remained to be clarified. We recently detected biosynthetic activities for aflatoxins G₁ and G₂ in cell-free extracts of A, parasiticus NIAH-26. In the presence of NADPH, aflatoxins G₁ and G₂ were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. Also, we for the first time determined that G- and B-aflatoxins are independently produced from the same substrate. At least three reactions, catalyzed respectively by the ordA gene product, an unstable microsome enzyme, and an 220-kDa cytosol protein, are involved in the enzymatic formation of G-aflatoxins from either O-methylsterigmatocysin or dihydro-O-methylsterigmatocystin. On the other hand, many of the genes involved in aflatoxin biosynthesis have been isolated from A. parasiticus and A. flavus, and most of them have been found to be clustered. We recently purified O-methyltransferase catalyzing the conversion of demethylsterig-matocystin to sterigmatocystin in aflatoxin biosynthesis. Based on the N-terminal amino acid sequence of the enzyme, genomic and cDNA genes (dmtA) of the enzyme were then cloned and sequenced from A. parasiticus NIAH-26 through the use of polymerase chain reaction (PCR) strategies. The dmtA gene was found to be placed between the omtA and ord-2 genes in the aflatoxin biosynthesis cluster region with the same orientation. Clarification of the aflatoxin biosynthetic mechanism using the biochemical and molecular biological approaches will provide promising clues to eliminate aflatoxin contamination from crops.

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Our group found that some strains, but not all, of A, oryzae and A. sojae have homologs of ver-1, a gene essential for aflatoxin biosynthesis. We compared the nucleotide sequences of ver-1 homologs in A, oryzae, A. sojae, A. flavus and A. parasiticus, and that the homologs in A. oryzae and A. sojae exhibited an extremely high degree of sequence identity with that of A. flavus and A. parasiticus. Transcripts of the homologs in A. oryzae and A. sojae are not detected. Moreover, we examined the expression of the aflR-homolog genes in A, oryzae strains having this gene, which encodes a regulatory protein for other aflatoxin synthesis genes, and demonstrated that no transcripts of the aflR-homolog genes in the examined strains of A. oryzae were detected under the aflatoxin producing condition. Considering the above observations together with the information from other research groups, non-aflatoxigenic A. oryzae and A. sojae basically have some homologs of aflatoxin synthesis genes. Thus, they are possibly domesticated, differentiated from aflatoxin producer evolutionally. Nevertheless, their transcripts are not found in any of the strains and some homologs are not found in the examined strains. Therefore these species do not produce aflatoxin because the synthesis genes are neither transcribed nor existent, probably due to mutations during repetitive use as domesticated strains in manufacturing fermented foods. Further characterization of the regulatory mechanism for aflatoxin production might resolve why koji molds having the homologs do not produce aflatoxin.

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The genera Aspergillus, Penicillium and Fusarium contain many mycotoxin producers and the identification and classification of these genera are confused because they lack distinct morphological character for each species. New taxonomic criteria for these genera are needed. The sequences (402 bp) of mitochondrial (mt) cytochrome b gene from these genera were determined and the phylogenetic relationship was analyzed. The phylogenetic tree based on cytochrome b (amino acid sequences) showed that Zygomycota, Ascomycota, Basidiomycota, Ascomycetous yeasts, Basidiomycetous yeasts and Schizosaccharomyces branched at early period in the evolution, respectively. The relationship in the genus Aspergillus based on cytochrome b showed that A. clavatus belonged to the subgenus Clavati was placed in cluster of subgenus Fumigati. A. terreus and A. flavipes separated from other species of subgenus Nidulantes, and they were placed in the cluster of subgenus Circumdati. Aspergillus section Flavi, including aflatoxin producers, were divided into 6 DNA types. A. sojae has individual sequence, different from that of A. parasiticus, in spite of the same conidial wall texture. A. oryzae and A. flavus bad the same sequences of the cytochrome b gene. Aflatoxin productivity did not strictly correlate with the classification. In the genus Penicillium, the relationship based on cytochrome b gene almost agreed with the classification by Pitt (1980). P. citreonigrium, which had been classified as subgenus Aspergilloides by Pitt, was placed in subgenus Furcatum. On the other hand, P, janczewskii belonged to subgenus Furcatum was placed in the cluster of subgenus Penicillium. The relationship of the genus Fusarium based on cytochrome b gene almost correlated with classification of Nelson et al (1983), whereas there were some differences. F. dimerum belonged to the section Eupionnotes was placed in the same cluster with F. solani (section Martiella and Ventricosum). F. sambucinum belonged to the section Discolor was placed in the same cluster with section Sporotrichiella. F. oxysporum was separated to 6 DNA types. Our results showed that the sequences of cytochrome b gene (402 bp) are useful for identification, classification and determination of phylogenetic relationship of these fungi.

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During the search for the hemorrhagic factors from the Fusarium species, we could isolate several toxins other than trichothecenes. Fusarium sambucinum PZF-4 produced an unknown toxin in wheat culture. This toxin, given the trivial name as sambutoxin, was isolated by silica gel chromatography and preparative HPLC. Sambutoxin was elucidated as 3-{5-methyl-6-[E]-1, 3, 5-trimethylhept-1-enyl}tetrahydropyranyl}-5-(p-hydroxyphenyl)-1-methyl-4-hydroxy-2(1H)-pyridone mainly by NMR analyses. Sambutoxin caused toxic effects in rats, including body weight loss, feed refusal, hemorrhage in stomach and intestines, and finally death when rats were fed diets supplemented with 0.05 and 0.1% sambutoxin. It also showed a potent in vitro cytotoxicity against human and mouse tumor cell lines, and the 50% inhibitory dose values ranged from 46 to 174 ng/ml. Fusarium sp. KTTC 16677 from soybean seeds produced apicidin and its derivative, apicidin B. The chemical structure of apicidin B was elucidated as cyclo-[pipecolyl-isoleucyl-(N-methoxy-tryptophan)-(2-amino-8-oxo-3-hydroxy- decanoic acid)] by NMR analyses. Apicidin caused the hemorrhage in stomach and intestines when rats were fed diets supplemented with 0.1 apicidin. Apicidin exhibited acute toxicity with LD₅₀ values of 300 mg/kg by oral and 327 mg/kg by intraperitoneal administration, respectively. Apicidin caused significant cellular electrolyte leakage and chlorophyll reduction in a duckweed bioassay at concentration of 10-100μM. Apicidin also showed in vivo antitumor activity against P388 murine leukemia cell implanted mice with the effective dosages of 30-100 mg/kg by intraperitoneal administration that it produced significant prolongation of survival time of mice with T/C values of 120-148%.

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We studied the relationship between the effect of trichothecenes on susceptibility of mice to infectious diseases and that on intestinal local defense systems. It was found that a relative low dose (0.2-2 ppm) of deoxynivalenol (DON) and diacetoxyscirpenol (DAS) reduced the susceptibility to Salmonella infection among five trichothecenes (T-2 toxin, DON, DAS, fusarenon X (FX) and nivalenol (NIV)). T-2 toxin, DON, DAS, FX and NIV modulated cell-mediated and also humoral immune systems. DON and DAS inhibited IL-8 secretion from the intestinal cells at a concentration lower than that causing protein synthesis inhibition. IL-8 is considered to inhibit the overgrowth and translocation of bacteria through induction of neutrophil migration to apical surface of intestinal cells. Therefore, our results suggest that the impairment of local defense system caused by DON and DAS exposure via the oral route is primarily involved in induction of susceptibility increases to food borne Salmonella infection.

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Fusaria are the most common fungi that infect corn (maize) crops world-wide. Ingestion of feed contaminated with Fusarium verticillioides (=F. moniliforme) causes leukoence-phalomalacia in horses, pulmonary edema in pigs, cardiotoxicity in baboons, and hepatic and renal injury in several species. Some isolates of F. verticillioides are rodent carcinogens. Elevated levels of F, verticillioides have been found in corn in areas of the world that have high incidences of esophageal cancer, and led to the discovery of fumonisin B₁ in 1987 as a Fusaria metabolite. Fumonisin B₁ was isolated from F, proliferatum cultures, and was purified as the ammonium salt with a final purity of >96%. The fumonisin B₁ was mixed with rodent feed (autoclaved NIH-31; <60μg/kg fumonisin B₁) and fed to rodents (48/dose/sex) at the following levels : female F344/N Nctr rats -0, 5, 15, 50, 100 mg/kg diet ; male F344/N Nctr rats -0, 5, 15, 50, 150 mg/kg diet; female B6C3F₁/Nctr mice -0, 5, 15, 50, 80 mg/kg diet; male B6C3F₁/Nctr mice -0, 5, 15, 80, 150 mg/kg diet. There were no dose-related changes in the weights or survival in the rats in the 2-year study. Increased incidence of renal tubule adenomas and carcinomas were observed in male F344 rats, but did not occur in the female rats. Renal tubule adenomas and carcinomas did not occur in male rats consuming diets with 0, 5, or 15 mg/kg fumonisin B₁, and occurred in 2/48 and 7/48 of rats consuming 50 mg/ kg fumonisin B₁, and in 5/48 and 10/48 rats consuming 150 mg/kg fumonisin B₁, respectively. Body weights were not affected by dose in the mice ; however, female B6C3F₁/Nctr consuming 80 mg/kg diet fumonisin B₁ had reduced survival. An increase in hepatocellular adenomas and carcinomas was detected in female B6C3F₁ mice, but not in male mice. The frequency of occurrence of adenomas and carcinomas were 16/47 and 10/47 at 50 mg/kg fumonisin B₁ and 31/45 and 9/45 at 80 mg/kg fumonisin B₁, respectively. The mechanism of action of fumonisin B₁ is interruption of de novo sphingolipid synthesis through inhibition of ceramide synthase. Since this enzymatic inhibition results in apoptosis in vivo and in vitro, it suggests that tumor formation arises as a result of compensatory regeneration in the tissues in vivo following fumonisin B₁-induced apoptosis.

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We attempted to estimate whether mycotoxins might account for the increased risk of human esophageal cancer (HEC) in Henan province, China. Corn and wheat as staple foods were collected in 1989, 1995 and 1997 from Linxian and Shangqiu counties, the high- and low-risk areas of HEC in Henan, respectively, and analyzed for the occurrence of trichothecenes, fumonisins, zearalenone and aflatoxin B₁. Among these mycotoxins examined, fumonisin B₁ (FB₁) and deoxynivalenol (DON) were major toxins exposing populations in both areas through corn consumption. According to the risk assessment of FB₁ and DON in both areas based on their levels in the staples, the estimated probable daily intake (PDI) values of FB₁ and DON for the high-risk area were 1.6 to 1.9 and 4.9 to 9.2 times higher, respectively, than those for the low-risk area. As compared with the tolerable daily intake (TDI) values of FB₁, which is estimated 800 ng/kg bw per day based on the published toxicological data, PDI values of FB₁ especially in 1995 and 1997 for both areas were 3.0 to 6.8 times of the TDI value. The PDI value of DON for the high-risk area was above its TDI (1, 000 ng/kg bw per day) in 1989 and 1995, whereas the PDI for the low-risk area was below the TDI in three years. The results obtained in the present study hardly support the hypothesis that FB₁ contamination increases the risk of HEC in Henan province, China. Considering HEC as a multifactorial disease, however, FB₁ as well as DON may be considered to be one of factors involved in HEC.

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The Central Science Laboratory (CSL) has been heavily involved over the last decade in carrying out surveillance exercises to estimate the exposure of the population to mycotoxins in the UK food supply. Data produced on behalf of the Ministry of Agriculture, Fisheries and Food for range of mycotoxins important to the UK food supply are given together with the actions taken to reduce exposure to minimal levels. Particular emphasis has been placed on UK studies on patulin in apple juice which is of particular concern to the UK and ochratoxin A in food which is of general concern in the European Union. Regular surveys for patulin in apple juice have been carried out in the UK since 1992. The results and regulatory strategies to reduce exposure are presented. Similarly a number of surveys have been carried out to assess the amount of ochratoxin A in the UK diet. These results and assessments of human exposure through a duplicate diet study are presented, in addition Hazard Analysis Critical Control Point strategies used to reduce contamination of the food supply are discussed.

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Aflatoxin contamination of peanuts, which results from infection and growth by Aspergillus flavus and A. parasiticus, is a serious economic problem in developed countries and a potentially serious human and animal health problem in developing countries. Cyclopiazonic acid is another mycotoxin produced by A, flavus that has been found as a contaminant of peanuts. A biological control strategy has been developed for reducing contamination of peanuts by these toxins. In involves inoculation of fields with competitive, non-toxigenic strains of the same fungi. The methodology has been tested for several years and has produced reductions in aflatoxin ranging from 30 to 90%. Field studies conducted in 1997 yielded an aflatoxin reduction of 91.6% and a reduction in cyclopiazonic acid contamination of 85.7%.

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Transmittance near infra red (NIR) spectra (500-1500 nm) of individual peanut was measured to detect the internal moldy nut. The moldy nut whose appearance had little difference from the sound nut with visual observations could be distinguished from each other by comparing the transmittance ratio of 700 nm to 1100 nm on NIR spectrometry. The fungal hydrolysis of the triglycerides which were contained in the nut seemed to account for these differences on the NIR spectra. Because of the higher incidence of aflatoxin (AF) contamination on these moldy nuts, taking out the internal moldy nut detected by NIR, could drastically reduce AF content of over all lot of peanuts.

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Pistachio nuts with shell defects detract from consumer acceptance and, in some cases, may be more prone to insect damage, mold decay, and/or aflatoxin contamination. Automated color sorters in use at California pistachio processing plants are not able to accurately distinguish normal nuts from nuts with oily stains, dark stains, and adhering hull. Improved sorting of these defects not only can increase consumer acceptance but can also reduce losses of high grade product during processing. Sorting based on images, rather than color, can facilitate more accurate sorting of small defects. The primary objective of this study was to develop imaging algorithms to improve sorting of nuts with oily stains, dark stains and adhering hull. A second objective of this study was to assess the ability of the image sorter to remove nuts with navel orangeworm (NOW) damage, fungal decay, and Aspergillus molds, all of which indicate risk of aflatoxin contamination. An imaging algorithm was developed to distinguish normal nuts from nuts with oily stains, dark stains, and adhering hull. Testing of the image algorithm on a validation data set showed that nuts having oily stain, dark stain, or adhering hull can be distinguished from normal nuts with an accuracy of 98%. Sorting for oily stain, dark stain, and adhering hull will remove 98.7% of NOW positive nuts, 89.7% of nuts with kernel decay, and 93.8% of nuts with Aspergillus molds present.

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In order to learn whether there might be a risk to human health from the intake of mycotoxins contaminating agricultural products, the stabilities of mycotoxins under various cooking conditions employed in daily life and the possibility of removal of mycotoxins during manufacturing processes were investigated.1) Stability of mycotoxins against heat. By heating mycotoxins within the range of 100-210°C, temperatures which are seen during a normal cooking process, we investigated the stability of mycotoxins against heat. When boiled (100-120°C), mycotoxins remained almost 100% after 45 minute heating. As the temperature rose, such as when frying (150-180°C), mycotoxins started decomposition. In the case of grilling (about 210°C), the nivalenol type of toxin rapidly decreased to 10% after 15 minute heating. In conclusion, we hypothesize that mycotoxins decompose as the temperature rise, but that part of the mycotoxins remains through ordinary cooking methods in terms of heating temperature as well as heating time.2) Influence on mycotoxins by a cooking process. Mycotoxins were added artificially to raw materials, which were then processed. In the case of noodles, only 10 to 12% of the added aflatoxins were transferred into water. Other foods were made from spaghetti, barley, coix seed, popcorn which were naturally contaminated with deoxy nivalenol, nivarenol, zearalenone. The contents of these mycotoxins were essentially unaffected by cooking. These experiments indicate that there is a high probability of intake of mycotoxins from such cooked foods.3) Influence of manufacturing process on mycotoxins. The possibility that mycotoxins may be removed in manufacturing processes was investigated. Edible oil (aflatoxins, Fusarium toxins) and cornstarch (fumonisins) at various stage of the manufacturing processes was examined to determine the fate of mycotoxins. Even when food materials are contaminated by mycotoxins, they can be completely removed during manufacture.

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The elimination of DON and NIV from barley during pearling and from wheat during milling was studied. In barley samples, DON and NIV contents of the original barley and the pearling-treated barley at the level of 55% were analyzed. The original hull-less barley and the pearling-treated hull-less barley at the level of 60% were analyzed. The wheat was milled with test mill and each of the fractions was analyzed. Whereas the original barley contained 0.51 ppm of DON and 0.65 ppm of NIV, inedible parts such as hull and bran contained 95% of DON and 97% of NIV by pearling at the level of 55%. Whereas the original hull-less barley contained 0.31 ppm of DON and 0.48 ppm of NIV, inedible part such as bran contained 93% of DON and 95% of NIV by pearling at the level of 60%. Whereas the original wheat contained 0.37 ppm of DON and 0.55 ppm of NIV, inedible parts such as bran contained 83% of DON and 86% of NIV by milling at the level of 60%. Therefore, the values of DON and NIV contamination were apparently reduced in edible parts of wheat after milling. We investigated the removal of fumonisins in corn by dry milling. Dry milling was done as follows. The raw corn's samples were cleaned and tempered. The endosperm was separated from hull and germ by degerming. After the separated endosperm was ground, separated and dried, the corn grit, the corn flour, the germ and the feed were obtained by treating the endosperm. The former three portions are edible part and the feed is inedible part. By dry milling of corn contaminated with fumonisins, inedible part of corn contained 87% of fumonisin B₁ and 88% of fumonisin B₂. Therefore, the values of fumonisins contamination were apparently reduced in edible part of corn after dry milling.

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In meat mycotoxins can occur primarily as a result of indirect transmission from animals exposed to natural contaminated feed. In case of meat products the possible occurrence of mycotoxins is strongly influenced by the particular recipe, i.e. the origin of meat and edible tissues used and the quality of ingredients such as spices, which are often highly contaminated with different mycotoxins. Other sources of contamination are the use of fungal derivatives in food processing, the use of toxigenic species or strains of fungal starter cultures for fermented products or when undesired moulding takes place. Among the farmed animals relevant mycotoxin residues could be found in edible tissues and blood sera from pigs and poultry, while organs or muscle meat from ruminants are of no importance because of the decomposition activity of the rumen flora. Highest toxin concentrations could mainly be expected in liver and kidney, being often the target organs for mycotoxins. Transmission of mycotoxins and/or their metabolites to animal tissues has been proven for several mycotoxins among which aflatoxins and ochratoxins are of most importance because of their carcinogenic and genotoxic properties. In Europe, special interest is focused on ochratoxin A, for which the setting of maximum tolerable levels in various food stuffs and beverages is discussed. Ochratoxin A has been shown to occur in edible tissues of swine, a variety of sausages and spices. To ensure the safety of meat, prevention strategies have to include all stages of food production with the aim to avoid fungal as well as mycotoxin contamination. The most important step lies at the stage of animal feeding and the reduction of the carry - over risk of mycotoxins from feed to animals. For safe meat products concepts for risk management and prevention measures are needed, which allow the detection and exclusion of the use of contaminated raw materials and spices. Fungal derivatives such as pigments used as colouring agents have to be evaluated toxicologically. Strategies for minimising the mycotoxin risk in mould-ripened meat products require fungi which are generally regarded as safe (GRAS status). Selection criteria therefore must be focused particularly on the absence of mycotoxin production on the fermented food substrate and should include the use of bioassays.

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Neotyphodium endophytes occur as intercellular mycelium within their grass hosts and are transmitted as mycelium in seed. They have a mutualistic relationship with the grass and assist in the survival of their host plants. Plant growth and competitive ability can be enhanced through better utilization of nutrients and through production of phytohormones by the endophyte. Endophytes produce compounds which deter predation from some insects and nematodes and make the plant resistant to some diseases. A major benefit of endophyte-infection is the protection they give their hosts from drought stress. Although endophytes are beneficial to the persistence of grasses some of the alkaloids they produce affect the health of animals which feed on the grass. Lolitrem B is the cause of Ryegrass Staggers and ergovaline is believed to be responsible for Fescue Toxicosis in animals which eat endophyte-infected perennial ryegrass and tall fescue. Thus endophytes are beneficial to plant persistence but not to livestock production. Strains of the two Neotyphodium endophytes that infect these grasses have been found which do not produce these toxins and they have been infected into seedlings of ryegrass and tall fescue. Agronomic and animal grazing trials with cultivars infected with these strains have shown the endophytes still protect the plants from environmental stresses but they do not appear to affect the health of animals. Cultivars infected with these novel strains will be available to farmers in the next 1-2 years.

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A characteristic of seed-borne clavicipitaceous symbionts (endophytes) that mediates their mutualisms with host grasses is production of anti-herbivore metabolites. Ergot alkaloids and indolediterpenes are potent neurotoxins in vertebrates ; saturated 1-aminopyrrolizidines (lolines) and the pyrrolopyrazine alkaloid, peramine, are active against insects. All except lolines are reported to be produced in cultures of fungal endophytes free of plant material. We identified lolines in defined-medium cultures of Neotyphodium uncinatum. We have also developed Epichloë festucae and Epichloë typhina as Mendelian genetic models to test the effects on aphids of lolines and peramine, respectively. In each case, the phenotypic difference of expression or non-expression was apparently governed by a single locus. Genotypes of E. typhina expressing peramine caused killing of greenbug aphid (Schizaphis graminum) on the host plants. Lolines were associated with killing of both greenbug and bird-cherry oat aphid (Rhopalosiphum padi). Statistically, the anti-aphid activities of the endophytes were entirely attributable to their alkaloids. Recent progress on genetic control of ergot alkaloid and indolediterpene expression holds promise for analogous tests for roles of these alkaloids in host benefits. Mendelian segregation and molecular knockouts can be used eventually to test the ecological importance of all known endophyte alkaloids in the many established endophyte effects, including increased drought tolerance, competitiveness, resistance to nematodes, and resistance to vertebrate and insect herbivores. Genetic knockouts in endophytes of genes for anti-vertebrate alkaloids will likely become an integral part of forage cultivar development.

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Grass infected with endophytes is known to be protected against several enemies by means of mycotoxins. Many fungitoxic compounds were isolated and the chemical structures were determined in order to understand the resistance mechanisms against pathogens.

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Among the perennial ryegrass and tall fescue in Japan, the turf-type grasses, ecotype collections and imported grasses were found to be infected with Neotyphodium endophytes, but few commercial pasture-type grasses were infected. The occurrence of ryegrass stag-gers was limited to the beef cattles and cows which had been fed with the imported ryegrass straw. Therefore, it was strongly suggested that the ryegrass staggers occurring in Japan was caused by the imported endophyte-infected grasses. On the other hand, the endophyte-infected grasses showed feeding deterrence to insects such as bluegrass webworm and Tribolium confusum, and disease resistance to Bipolaris sorokiniana, Rhizoctonia solani, Drechslera dictyoides f. sp. perenne and so on. In Japan, Neotyphodium endophytes have been isolated from 27 species of cool-season grasses in 13 genera during the last three years, and most of them belonged to the subfamily Pooideae. Among the warm season grasses, Ephelis endophytes were detected in 15 species such as bermudagrass, Pangola-grass and paragrass. Paspalum thunbergii infected with Ephelis sp. showed feeding deterrence to a rice grass hopper, and Digitariaa decumbens (Pangola-grass) also had acquires tolerance to Japanese army worm (Pseudaletia separata) and grasshopper (Aiolopus thalassimus). However, the toxicity of the endophyte-infected grasses to animals is not known. It is important to know whether non-host plants acquired the resistance to insects and plant pathogens by artificial inoculation with fungal endophytes or not, which depends on the specificity between plant and endophyte associations. Here the compatibility and incompat-ibility between them are discussed.

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From 1997 to 1999, we experienced 29 cases of disorders in cattle and horses that had been fed ryegrass straw imported from the USA. These animals showed symptoms resem-bling ryegrass staggers and the clinical signs disappeared after removal of the straw. To clarify the cause of these cases, we examined the suspected straw for the presence of endophytic fungi and toxins. Endophytic hyphae were detected in the seeds of all straw samples that were responsible for the clinical cases. Lolitrem B concentrations in the straw ranged between 972 and 3740 ppb. Ergovaline concentrations were between 355 and 1300 ppb. It is known that N. lolii-infected perennial ryegrass contains both lolitrem B and ergovaline. Our result was comparable with the observations of previous reports. Turner et al. (1995) proposed the threshold levels of lolitrem B for toxicity as 1800-2000 ppb in the total diet. Even though the concentrations of lolitrem B were lower than this proposed threshold in almost all clinical cases, we diagnosed the episodes as ryegrass staggers from the clinical signs and other epidemiological findings. Namely, neurologic disorders were diminished by deprivation of the suspected straw. Vitamin B1, calcium or magnesium deficiency, white muscle disease and nitrate poisoning were ruled out by the clinical exami-nations. Furthermore, experimental feeding of the straw produced typical neurologic signs.

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A rapid method for the fluorometric analysis of aflatoxins in corn, corn meal, popcorn, rice, wheat, cottonseed and peanuts was developed. This method proved to be quantitative, inexpensive and very efficient. Ground samples of the commodity being tested are extracted with methanol-water (80+20) and cleaned up by passage through a solid-phase separatory column. Five hundred μl of purified extract is derivatized with a bromine reagent and the fluorescence of the solution is quantified with a calibrated fluorometer. This method will quantify aflatoxin from 5 to 5000 ppb without dilution and was linear when applied to samples of noncontaminated corn spiked at 0 to 5000 ng aflatoxin B₁/g. Correlation coefficients of the method with HPLC for multiple analyses for corn (n=34), cottonseed (n=32) and peanuts (n=11) were 0.999, 0.995 and 0.980, respectively. Individual analyses can be conducted in less than 5 min and grouping of samples is unnecessary. The sensitivity of the method for corn is 5 ppb and the fluorometer, operating under the prescribed conditions, has a limit of detection of 0.6 ng of aflatoxin B₁.

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Endophytic fungi are associated with many common pasture grasses, in an association beneficial to both organisms, with endophyte-infected grasses demonstrating greater persistence under environmental stress, than endophyte-free grasses. However, this agronomic advantage is obtained at the expense of animal health. Perennial ryegrass staggers (RGS), common in New Zealand, and fescue toxicoses (FT), prevalent in the eastern USA, are the most widely recognised examples of such toxicoses. The compounds responsible for the toxicity of these grasses have been widely studied. A number of these toxins have been conjugated to protein, and used to raise antibodies capable of binding the toxin, and closely related structures, with high specificity for use in immunoassays such as ELISA for detection and quantitation of toxin. The ELISA, performed in 96-well PVC microtitre plates, typically requires only 50 μL sample per well, and has a detection limit in the pg/ml to ng/ml range. The ELISA is generally able to cope with crude extracts without extensive (and expensive) cleanup. The low cost and high throughput that this permits has allowed researchers to analyse samples from large-scale field trials. Immunological techniques have been used in the study of toxin accumulation in localised plant tissue, and for screening of endophytes for toxin production in vitro. They have also been used to probe plant material for compounds structurally related to the toxins to give an insight into the biosynthetic pathways of toxin synthesis. This paper details the production of antisera, and development and application of ELISA for endophyte toxins.

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We have developed clean withont and rapid analytical methods for aflatoxins, ochratox-in A, fumonisins and patulin wlthout using chloroform and an immunoaffinity column. With regard to the fumonisin analysis, we developed a postcolumn derivatization HPLC method. Since the mobile phase does not contain a non-volatile buffer, we can utilize it for LC/MS analysis to confirm the fumonisins.

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Abbreviation BSA, bovine serum albumin ; OVA, ovalbumin ; KLH, keyhole limpet hemocyanin ; i.p., intraperitoneal ; ELISA, enzyme-linked immunosorbent assay ; CI-ELISA, competitive indirect enzyme-linked immunosorbent assay ; CD-ELISA, competitive direct enzyme-linked immunosorbent assay ; FB₁(or FB₂), fumonisin B₁ (or B₂) ; PBS, phosphate-buffered saline.

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To minimize the interference of co-extracted compounds from foods and feeds in which mycotoxins occur, the conventional methods for chemical analysis of mycotoxins consume large amount of time and solvent, need several cleanup steps, and sometimes require practi-cal experiences. From the environmental point of view, toxic solvents such as chloroform and benzene used for mycotoxin analysis should be saved from now on. The immunoaffinity column (IAC) using anti-mycotoxins antibodies are highly specific, simple and rapid, and saving toxic solvents. Moreover, recently, with the availability of commercial IACs for aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), fumonisins and deoxynivalenol (DON), these IACs have become the powerful tools in cleanup stage of mycotoxin analysis. This paper describes the performances of IACs in mycotoxin analysis, reuse of IAC, and multimycotoxin analysis using linked different IACs.

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A rapid and sensitive tandem immunoaffinity column (IAC) cleanup and high performance liquid chromatography (IAC-HPLC) for the determination of deoxynivalenol (DON) in wheat and corn is described. A monoclonal antibody specific to DON conjugated to the CNBr- activated Sepharose 4B gel was used in construction of the IAC with a capacity of 2.5 μg DON per column. In the analysis, ground sample was extracted with acetonitrile-water (84+16) to which 4 ml (or less) were withdrawn and evaporated. The residue was dissolved in 0.01 M phosphate buffered saline, and subjected to the IAC. After washing with 10% methanol, DON was eluted from the IAC with 100% methanol, which was evaporated, redissolved in acetonitrile-water (2+8) and then subjected to HPLC using a reversed-phase C₁₈ column and diode array detection (DAD). The analysis was completed in 6 min after injection of each sample with a detection limit of 0.03 μg DON/g. The chromatogram and the DAD spectrum for DON peak showed no interference substances in the sample treated with IAC. The average recoveries of DON added to wheat and corn extracts at levels from 0.5 to 3 μg/g were found to be 94.4 and 87.1%, respectively. Analysis of 6 naturally contaminated wheat samples (0.7 to 12 μg DON/g) showed good agreement between the IAC-HPLC data and those from direct competitive enzyme linked immunosorbent assay (dc-ELISA) (r=0.9970; slope=0.8533).

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A modified method for extracting tenuazonic acid (TA) from cereals and rice culture was developed. The method gave a distinct increase in TA concentration in comparison with the published one, at an average of 6.1 and 1.8 times higher for weathered wheat and rice culture materials of Alternaria alternata, respectively. It will be applicable to extract low levels of TA from cereals.

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Corn and commercial corn products obtained from different regions of the Philippines were analyzed for total aflatoxins using the validated “lahar” minicolumn method, originally designed for copra meal, and for aflatoxin B₁(AFB₁) by high performance liquid chromatography (HPLC). The whole corn samples showed an incidence of 48% aflatoxin contamination, 30% of which were above than 20 μg/kg permissible level for aflatoxins; the whole kernel feed corn showed higher levels of aflatoxins than the corn grits and cracked corn; while for the commercial corn products, a consistent aflatoxin contamination was observed with fried corn, the other processed corn products showed less than 20μg/kg aflatoxin contamination. Results showed that there is a good agreement between the minicolumn responses and the HPLC data. Therefore, the minicolumn method is an efficient screening tool for aflatoxin detection in the samples.

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In this study we studied the contamination of AFB₁ in corn seeds (N=28) and ground peanut (N=28) in northern Thailand. The samples were extracted with chloroform for TLC and with 80% methanol for competitive enzyme-linked immunosorbent assay (cELISA). The levels of AFB₁ in corn seeds determined by TLC and ELISA method were ranged from 0 to 540 ppb and 0 to 576 ppb; and in ground peanut from 0 to 695 ppb and 4 to 450 ppb, respectively. The minimum detectable limit of the ELISA with the most sensitive monoclonal antibody (MAb) AF5 was 1 ppb while that of TLC was 10 ppb. The correlation coefficients between our own ELISA with commercial ELISA kits, and TLC method were 0.92 and 0.87 for corn seeds (p<0.05), and 0.94 and 0.84 for ground peanut (p<0.05), respectively.

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A successful ELISA using monoclonal antibody (MAb) and one-step extraction method was established for aflatoxin (AF) B₁ determination. Eight clones of stable hybridoma cells secreting IgG anti-AFB₁ MAbs were prepared from spleen of BALB/c mice. The hybridoma cells were proliferated in culture and infused into pristane-induced BALB/c mice. Two weeks later, their ascites fluid were withdrawn and precipitated by 45% saturated ammonium sulfate. The IgG precipitate was dissolved in PBS and kept at -80°C for further test. The direct competitive ELISA (direct cELISA) system was developed for AFB₁ determination in microplates by AFB₁-horseradish peroxidase conjugated by carbodiimide method. The quality control was done. The minimum detectable limits of the direct cELISA with those monoclonal antibodies : AF 1, 2, 3, 4, 5, 6, 7 and 8 were 10, 20, 2, 2.5, 2, 5, 2 and 2.5 pg of standard AFB₁ per assay, respectively. The specificity or cross reactivity to other aflatoxin derivatives was that MAbs were reactive with AFG₁ as well as AFB₁, weakly with aflatoxicol (AFL) I>AFL II>AFB₂>AFG₂>AFM₁, respectively, except AF5 weakly reactive with AFG₁. The result showed that MAb (AF5) was the best MAb for further use in the ELISA kit.

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Thailand is in a tropical zone with much rains and high humidity which are suitable for rapid growth of fungi including Aspergillus. Aflatoxins (AF) mainly AFB₁ are frequently observed in agricultural commodity products which are good substrates, especially peanuts, corn, copra, rice flour, noodle, dried plants, and shrimp. Problems are the high contamination of AF in Thai food and agricultural products. Approaches to the AF decontamination at pre- and post-harvesting will be explained and discussed, and the contaminant detoxification of AF and its mutagenicity which were mostly destroyed by ammonium salts to reduce human exposure and health risk will be presented. It is our goal and plan that the AF contamination problem in dietary products and animal feeds and health risk will be reduced and solved in the near future. Hopefully, if no other factors much involved, the incidence of liver cancer and diseases among Thai population would be subsequently somewhat decreased. All these control measures for AF risk have been done nationwide by research, education, presonnel training and restrict regulation.

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Rice noodle has been commonly and popularly consumed by Asians and Thais. In the noodle factories, noodles are daily and freshly made from rice flour, by pasting, steaming and drying. Before marketing out to customers in the same day, the noodle preparations are traditionally coated with raw peanut oil for the reduction of the stickiness and flavoring of the noodle. Uncooked noodle samples (N=10) from Chiang Mai market were randomly bought and analysed for aflatoxin content by TLC and fluorometry. It was found that aflatoxin B₁ (AFB₁) content in the samples before and after cooking in boiling water was 20.24±41.16 and 15.75±37.88 ppb, respectively. The effect of heating of oil-coated noodle at 100°C could decrease the AFB₁ contamination only about 5%. AFB₁ in separated peanut oil, which was contained about 1.8% by wt. in noodle samples, was significantly high averaged 343.35±298.17 ppb. The peanut oil was mainly contaminated with aflatoxin B₁. Such aflatoxin contamination in peanut oil and noodle samples was harmful, over the safe level limit (=20 ppb). The noodle-coating peanut oil should be previously detoxified by alkalization. The high-grade peanut oils should be selected for using in order to maximize the health safety of consumers.

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Aflatoxin contamination of agricultural commodities is of great concern for both public health and the economy in southeast Asian countries. Near the end of 1998, we collected twenty-six agricultural samples from three different markets in central and east Java and Bali in Indonesia and analysed for aflatoxins. Also, 22 Aspergillus flavus group isolates from these commodities and soil samples were tested for their mycotoxin productivity. Aflatoxin B₁ was detected in five of eight peanut and four of five maize samples. No aflatoxins were detected from six rice, three soybean, or four other bean samples. The highest amount of aflatoxin B₁ detected was close to 6 ppm in one peanut sample followed by 300 ppb in one maize sample. Aflatoxin G₁ and G₂ were not detected in any of the samples. Seven out of 22 Aspergillus f lavus group isolates produced detectable amounts of aflatoxin B₁ and B₂ in liquid culture medium (0.18 to 21 ppm of aflatoxin B₁ and 0.002 to 0.8 ppm of aflatoxin B₂), but none produced detectable amounts of aflatoxin G₁ or G₂. A11 22 isolates produced kojic acid (0.07 to 3.18%) and 21 produced cyclopiazonic acid (trace to 54 ppm)

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Commercial samples of food and feed collected at Bangkok markets were assayed for the presence of aflatoxins (B₁, B₂, G₁, G₂ and M₁), ochratoxins (A and B), citrinin, patulin and fumonisins (B₁, and B₂). Aflatoxins were detected in 11 samples from the total of 36 samples of peanut, corn, rice, coix seed and dairy products, aflatoxin B₁ was found in the range of 0.01-626 ppb. Eight corn and cereals products were assayed for ochratoxins, citrinin and fumonisins. No ochratoxins were detected. Citrinin were detected in 4 samples in the range of 0.4-4 ppb, while fumonisins were detected in 2 samples in the range of fumonisins B₁ 130-630 ppb, fumonisins B₂ 20-160 ppb. Five kinds of juice products were assayed for patulin. No patulin was detected.

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From a total of 131 samples of raw and processed peanut commercialized in several supermarkets of Florianopolis city, the capital of Santa Catarina State, collected from December, 1998 to July, 1999 and surveyed for aflatoxin contamination, only 12% of them were contaminated (being the most contaminated the pacoquinha followed by fried peanut with testae). The levels of aflatoxin B₁ and G₁ in the pacoquinha samples reached up to 127.3 and 57.1 μgkg^-1 respectively which were higher than the MRL allowed by the Brazilian legislation (30 μgkg^-1). On the other hand, no aflatoxin contamination was detected in the following peanut products : fried and roasted peanut without testae, roasted peanut covered with honey, peanut with chocolate, Japanese peanut, and peanut butter of all brands surveyed, as well as on all raw peanut samples, corresponding to 88% of the total samples.

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A survey on esophageal cancer exposure from 1995 to 1997 was carried at the radiotherapy service of the Charity Hospital in Florianopolis city, capital of Santa Catarina State, southern region of Brazil. From a total of 2, 495 medical registers, 134 cases were of esophageal cancer affecting 80.5% of males and only 19.5% of females. Most of the patients were from the South and West regions of Santa Catarina (28 and 27%, respectively), regions of high corn flour consumption and maize production. It was observed that farmers presented the highest incidence of esophageal cancer, followed by bricklayers, miners, drivers, and carpenters. Most of the patients had the habit of smoking and drinking alcohol regularly, and 27% of patients used to drink chimarrao. Some patients had those habits associated i.e., they used to smoke, drink alcohol, and also have chimarräo.

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The hepatic diseases (HD) and hepatocellular carcinoma (HCC) that affected children and adults patients from Santa Catarina State, Southern region of Brazil were studied. For children, the incidence of cirrhosis and cepatitis was 46 and 40% of the cases, respectively, and that of HCC was 14%. Sixty four % of the cases of hepatitis were caused by virus infection and 22% were autoimmune. The virus A and B hepatitis was 7.1 and 21.4% of the total of virus hepatitis cases, respectively. The percentage of deaths by cirrhosis was 71.4 and that by HCC was 28.6%. Although the HCC incidence in Santa Catarina has been low in relation to other HD studied between 1980 and 1997, it is important to emphasize that the virus hepatitis had a high participation especially by B virus infection (HBV). In adults, the incidence of cirrhosis and hepatitis was higher (70.5 and 14.3% of the cases, respectively) than HCC (11.7%). The percentage of deaths by cirrhosis and HCC were 77.7 and 17.3%, respectively. Apart from other factors that could be leading to the HD reported in Santa, Catarina State, one should take into account other factors such as the diet and mycotoxin contamination. Aflatoxin B1 is a potent carcinogen and the diet in the south of the country includes food that has been reported being highly contaminated by that toxin. The food that are preferred and mostly eaten, either by children or adults, as snacks are: peanuts (pacoquinha = ground peanut paste with sugar) and corn (high variety of corn snacks and polenta = cornflour porridge).

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The study was conducted to relate quantitatively the aflatoxin residue found in table eggs via feed intake, of poultry layers fed with aflatoxin contaminated feeds with and without mycotoxin binder. The experimental animals were given feeds naturally contaminated with aflatoxin B1 at a level of 20.27 to 167.96 ppb for seven weeks on a commercial farm in the Philippines which started on the post peak period. Egg collection started at week 36 with egg production at 82.69% for the control group and 70.13% for the treated group ; and ended at week 43 with egg production performance at 71.53% and 62.28% respectively. Treated group given with the mycotoxin binder produced eggs with aflatoxin B1 contamination at a level of 0.016 to 0.045 ppb and 0.03 to 0.069 ppb for the control group. Aflatoxin M1 contamination for the treated group ranged from 0.04 to 1.08 ppb while the control group registered a 0.49 to 2.54 ppb level. Comparison of the overall production performance between groups proved favorable with the use of the mycotoxin binder in layer feeds. Results showed that egg production and egg quality, in terms of aflatoxin M₁ contamination, was improved.

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We investigated whether endogenous glucocorticoid and/or tumor necrosis factor-α (TNF-α) are involved in T-2 toxin (T-2)-induced apoptosis by using adrenalectomized mice and anti-TNF-α antibody-injected mice. The magnitude of DNA fragmentation found in T-2-treated-adrenalectomized mice was similar to sham-operated T-2-injected group. Agarose gel electrophoresis showed that thymocytes from mice treated with T-2 alone and those from mice treated with T-2 together with anti-TNF-α antibody exhibited a similar pattern of DNA fragmentation. LPS-induced DNA fragmentation was markedly inhibited by the anti-TNF-α antibody. Taken together, T-2-induced thymic apoptosis appears to be independent of endogenous TNF-α and glucocorticoid in vivo in mice.

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The objective of this study was to relate the diversity of a naturally occurring population of Aspergillus flavus to their ability to contaminate the grain with aflatoxin and produce bright greenish- yellow fluorescent (BGYF) kernels. Nineteen strains of A. Flavus isolated from a corn field near Kilbourne, Illinois were used as inoculum, including 16 genotypes (DNA fingerprinting), and representing both aflatoxin producers and non-producers. A commercial corn hybrid (Pioneer 3394) was grown in this field in 1996 and 1998 and twenty ears in the late milk to early dough stage of maturity were inoculated with each A. Flavus strain using a toothpick wound procedure. At harvest, 20-24 kernels nearest each wounded site were separated into three categories : wound-inoculated kernels, intact BGYF kernels, and all other intact kernels. Sample weights of intact BGYF kernels in 1996 and 1998 grain samples averaged 5.0% and 9.5% of the total sample weight, respectively. Aflatoxin producing strains were associated with a higher frequency (P<0.05) of BGYF kernels for grain samples harvested in 1998. Removal of the individual wound-inoculated kernels and the intact BGYF kernels from corn ears inoculated with 13 aflatoxin-producing strains of A. Flavus, lowered mean aflatoxin values from 115 ng/g (range=<1 to 387 ng/g) to 2ng/g for 1996 grain samples and from 744 ng/g (range=20 to 1416 ng/g) to 33 ng/g for 1998 grain samples. Results indicated substantial variation among A. flavus genotypes in their ability to produce aflatoxin in the germ and endosperm of infected BGYF kernels. The naturally occurring A. Flavus population may include a majority of strains that produce no aflatoxin but exhibit BGYF and are thus aflatoxin “false positives” when corn grain is examined with an ultra violet light at 365 nm. Intraspecfic competition between aflatoxin producing and non-producing strains would be expected to naturally suppress the severity of aflatoxin outbreaks within the Midwestern corn belt.

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Penicillium expansum, rotting blue mold, is frequently isolated from spoiled apple fruits during storage and transportation. Besides spoiling the fruit, the fungus produces a mycotoxin, patulin. Therefore there have been many investigations to prevent the infection and spread of P.expansum. But relationship between the growth characteristics of the fungus and the preferential spread of it into the fruit has not been revealed. This study deals with the growth specificity on fruit constituents of P. expansum, the productivity of enzymes by the fungus and the action site of the enzymes on fruit tissue.

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The blooming of Microcystis aeruginosa Kütz. in the reservoir of Mae Kuang Udomtara dam, Chiang Mai, Thailand was investigated for 18 months during August 1996-January 1998. The water quality in the reservoir classified by trophic level was found to be mesotrophic to eutrophic. The main problem of water quality in the reservoir was the proliferation of phytoplankton, Microcystis aeruginosa Kütz. which secreted microcystin (hepatotoxin). It was found throughout the investigation in large amount during July 1996-January 1997. The factors effecting the proliferation were the amount of soluble reactive phosphorus and the total phosphorus which showed negative correlation with the volume of water in the reservoir. The vertical changes of the biovolume of M. aeruginosa was more obvious at the water surface and decreased at the lower levels. Six types of microcystin were isolated from the samples. Microcystin RR was present in the highest concentration in this investigation followed by microcystin LR, Z-RR, YR, Z-LR and ThtyrR. Toxicity tests with animals showed an LD50 for microcystin RR of 700-800 mg.kg^-1 body weight, whilst the value for microcystin LR was 70 mg.kg^-1 body weight.

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Aflatoxin-producing fungi were isolated from harvested sugarcane, peanuts and green coffee in Hochiminh City, Vietnam. A, parasiticus was found on cut surface of the sugarcane stems as well as A. flavus. A. flavus was also isolated from peanuts and green coffee, but A. parasiticus was not found in both of the samples examined.

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The genus Chaetomium was targeted as a potentially rich source of antiinsectan natural products because different Chaetomium species have been shown to be avoided by or harmful to fungus-feeding insects. Bioassay guided studies using both antiinsectan assays (Helicoverpa zea) and antifungal assays (Aspergillus flavus) led us to a variety of known Chaetomium metabolites (i.e. antibiotic 1233A; chaetochromins ; chaetoglobosins A, B, D, and F ; 19-O-acetylchaetoglobosins A and D; chaetoviridins A, B, and X; chetocin; chetomin ; dethiotetra (methylthio) chetomin; cochliodinol, cyclosporins A and C; eugenitin; sterigmatocystin; O-methylsterigmatocystin). Two novel compounds, closely related to cytochalasins, but with previously undescribed ring systems, were discovered to have antiin-sectan activity. Among the compounds isolated in this work, only cyclosporin A and sterigmatocystin had been previously reported to have toxicity to insects. Most of these compounds were originally isolated as mycotoxins. The study confirmed our hypothesis that Chaetomium spp. produce potent insecticides as chemical defenses.

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Aflatoxin contamination in food, especially ground peanut and ground chili, is one of the serious problem for health of Thai people. Both ground peanut and ground chili are always added in many varieties of dishes. Consequently, the accumulation of aflatoxin occurred in human consumption with unavoidable. To reduce the risk of human consumption of aflatoxin contaminated food, we try to use the convenient and simple method which everybody can do by themselves. Hence, we try to utilize the microwave oven as the easy tool for decontamination of aflatoxin in food. The experiments were conducted using naturally contaminated ground peanut and ground chili from domestic market. The sample was mixed properly and divided for microwave oven treated and control. A set of samples was directly exposed to microwave oven at medium power level for 1, 3, 5 and 7 minutes. Whereas the other set all the sample were covered with glass lid while exposed in microwave oven. Aflatoxin was detected from both treated and untreated samples by direct competitive ELISA. Percentage of aflatoxin reduction in ground peanut when treated with microwave oven at medium power for 3 and 5 minutes were 25% and 49.25%, respectively. Aflatoxin was reduced 39% at 3 minutes heating time in ground chill. Whereas, percentage of aflatoxin reduction was 3.94, 23.8, 44.97 and 71.42 in ground peanut covered sample when treated with microwave oven for 1, 3, 5 and 7 minutes respectively. The results showed that microwave oven at medium power can reduce aflatoxin contamination in ground peanut and ground chili. Though it can not remove all but at least it can reduce risk of human consumption to take high amount of aflatoxin

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