Analysis of 98 moldy corn samples collected in Wisconsin between November 1992 and January 1993 for Fusarium toxins by various immunochemical assays revealed overall average mycotoxin concentrations of 305.6, 237.7, and 904.3 ng/g for type A trichothecenes (TCTCs), deoxynivalenol (DON)-related type B TCTCs (total DON), and zearalenone (ZE), respectively. A small portion (5.1%) of the samples was found to be contaminated with high levels ( > 1 microgram/g) of type A TCTCs and total DON during the whole survey. Over 40% of the samples had 100 to 1,000 ng of total DON per g, while 17% of the samples had the same levels of type A TCTCs. The analytical data were consistent with those from mycological examinations for the samples in which various toxic Fusarium spp., including F. sporotrichioides, F. poae, and F. graminearum, were found. The samples received in November 1992 had relatively low concentrations of toxin; the average levels of type A TCTCs and total DON were 9.9 and 79 ng/g, respectively. The toxin concentrations became progressively higher in the samples received in December. The average levels for the type A TCTCs and total DON increased to 920 and 335 ng/g, respectively. However, the levels of ZE were higher in the samples collected earlier. The average levels for samples collected in November and late December were 1,195 and 242 ng/g, respectively. Analysis of selected samples by high-performance liquid chromatography monitoring with an enzyme-linked immunosorbent assay revealed that T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and T-2 tetraol (T-2-4ol) were common in these samples. Statistical analysis revealed a weak correlation between the levels of total type A TCTCs and total DON in the samples (r = 0.18, P = 0.09), but a strong correlation between the levels of ZE and total type B TCTCs (r = 0.75, P < 0.0001) was found. The mycotoxin levels of total type A TCTCs, total DON-related type B TCTCs, and ZE in the cobs (5.2, 3.9, and 21 micrograms/g, respectively) were considerably higher than those in the kernels (1.0, 0.5, and 0.5 microgram/g, respectively). The type A toxin levels increased from a range of 14 to 35 ng/g to a range of 110 to 538 ng/g after the moldy corn samples were held at 5 degrees C for 8 days in the laboratory.

Insects play an important role as facilitators of the aflatoxin-producing fungus, Aspergillus flavus Link, in both preharvest and postharvest corn. The current study investigated the role of maize weevils, Sitophilus zeamais Motschulsky, in enhancing aflatoxin B1 content in stored corn. In laboratory experiments, aflatoxin B1 was quantified with an indirect enzyme-linked immunosorbent assay (ELISA) on corn following artificial infestation with adult weevils that had each been topically treated with 100 spores of A. flavus. Corn kernels infested with A. flavus-contaminated weevils had significantly higher levels of aflatoxin B1 than A. flavus-inoculated corn without weevils. The presence of maize weevils resulted in increased kernel moisture content during incubation, and grain moisture was positively correlated with aflatoxin content across treatments receiving spores. Aflatoxin B1 levels were higher in corn treated with fungus-contaminated weevils compared with corn that was mechanically damaged and inoculated with spores, which in turn had more aflatoxin than undamaged corn treated with spores. Aflatoxin B1 content in corn increased with time of weevil exposure from 7 to 21 d, but decreased after 28 d of exposure. Aflatoxin levels in infested corn increased significantly with increased numbers of A. flavus-contaminated weevils. Maize weevils carried spores both internally and externally; however, substantial numbers of spores were intimately associated with the exoskeleton of adult weevils. These findings indicate that maize weevils facilitate the growth of A. flavus and aflatoxin production in corn by increasing surface area susceptible to fungal infection and increasing moisture content as a result of weevil metabolic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of the elm phytoalexin, mansonone E, on linear growth of 17 fungal species was examined to determine whether the Dutch elm disease fungus Ophiostoma novo-ulmi is more tolerant of mansonone E than other fungi. Linear growth of O. novo-ulmi was less inhibited by mansonone E than that of most, but not all, other fungi examined, suggesting that O. novo-ulmi is relatively tolerant of mansonone E. To determine whether this tolerance is required for pathogenicity, we generated mutants of O. novo-ulmi with reduced tolerance to mansonone E by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis followed by a screen for reduced tolerance to 1,2-naphthoquinone, a compound similar in structure to mansonones. Reduced tolerance to 1,2-naphthoquinone and mansonone E was accompanied by reduced tolerance to another elm phytoalexin, mansonone F. Two mutants exhibited wild-type levels of virulence on three elms, while three other mutants exhibited markedly reduced virulence compared with the wild-type field isolate from which they were derived. When the weakly virulent mutants were crossed with wild-type isolates, progeny that had wild-type levels of virulence but low tolerance to mansonone E were recovered. These results suggest that although O. novo-ulmi is relatively tolerant of mansonone E, this tolerance may not be required for high levels of virulence on elm. Key words: Ophiostoma novo-ulmi, mansonone, phytoalexin, Dutch elm disease.

Heterokaryons were formed in intra- and interspecific crosses between Fusarium sporotrichioides and F. tricinctum auxotrophs. Segregant homokaryons were evaluated for trichothecene toxin production in culture. Results were consistent with nuclear control of toxin synthesis. The sexual compatibility of auxotrophs and 30 additional F. tricinctum sensu Snyder & Hansen strains was tested. Perithecial production was restricted to crosses between Florida isolates pathogenic to English ivy (Hedera helix). The linkage of several auxotrophic markers was determined by analysis of progeny of certain crosses. No T-2 toxin was produced by sexually compatible F. tricinctum isolates.

Strains of Fusarium produced high levels of T-2 toxin when cultured on certain media absorbed into vermiculite. Modified Gregory medium was nutritionally complex (2% soya meal, 0.5% corn steep liquor, 10% glucose) and, when inoculated with the appropriate fungal strain, yielded maximum T-2 toxin within 24 days of incubation at 19 degrees C. On Vogel synthetic medium N (H. J. Vogel, Microb. Genet, Bull. 13:42-43, 1956) supplemented with 5% glucose, optimal toxin levels were synthesized after incubation for 12 to 14 days at 15 degrees C. Fusarium tricinctum T-340 produced 714 and 353 mg/liter on modified Gregory medium and Vogel synthetic medium N plus 5% glucose, respectively. Improved analytical procedures were developed and involved aqueous methanol extraction, purification by liquid-liquid partitions, and gas-chromatographic quantitation.

Fusarium tricinctum, a cause of stalk or ear rot of corn, and its T-2 toxin produced necrotic lesions and hemorrhages in the intestines, liver, and kidneys of rats. When rats were given the fungus or toxin orally, the chief site of absorption was the intestines. Sufficient T-2 toxin could be absorbed by the skin to cause local inflammation or even death. The median lethal dose (LD50) of the fungus when given orally was between 160 and 730 mg./kg., and that of T-2 toxin was 3.8 mg./kg. Clotting and prothrombin times and vascular permeability were increased. Although O2 intake was not materially inhibited, there were evidences that O2 utilization was not normal. The exact sites of interference need further study. Hepatic microsomal oxidative enzyme activity was inhibited and glucuronide excretion was increased. Activation of the microsomal enzymes with phenobarbital reduced prothrombin times, lessened toxicosis, and increased glucoronide excretion. This avenue of detoxification is interpreted to be of low magnitude. Neither tolerance nor carcinogenesis was evident.

Proline and γ-amino-n-butyric acid were present in considerable amounts in the sap of resistant species of the Ulmaceae such as Ulmus pumila, Hemiptelea davidii, and Celtis occidentalis, but occurred only in trace amounts in the sap of susceptible U. americana, U. japonica, and U. carpinifolia. The xylem sap of U. japonica, C. occidentalis, and H. davidii contained large amounts of alanine. Total concentrations of amino acids and ammonia were higher in resistant species than in susceptible species. Fructose, glucose, and sucrose were present in the sap of all the six species. Sucrose was the main sugar in the sap of U. japonica, U. pumila, and C. occidentalis; in U. americana and U. carpinifolia sucrose and fructose were present in about equal amounts; in H. davidii fructose was the major sugar.

Amino acid concentrations in the xylem sap of Ulmus americana L. decreased gradually from early spring to summer. Twenty amino acids, ammonia, γ-amino-n-butyric acid, ethanol-amine, and β-alanine were always present. Asparagine + glutamine constituted the most abundant organic nitrogenous components in the xylem sap during June when elms in Wisconsin are most susceptible to Dutch elm disease. Proline was present only in the sap samples collected during May, and disappeared in early June with the onset of susceptibility to Dutch elm disease. Ammonia was present in high concentration; and ornithine, lysine, histidine, ethanolamine, arginine, glycine, methionine, isoleucine, leucine, tyrosine, and phenylalanine were present in measurable amounts during May and early June, but all declined to small amounts in later months.

Thirty-two ninhydrin-positive nitrogenous compounds were present in the xylem sap of 6- to 9-year- old American elms. Twenty-six of these compounds were identified. Total concentration of nitrogenous materials was several times higher in diseased than in healthy sap after inoculation during the susceptible period in the spring, but was lower after inoculations during the late resistant period. Percentage compositions of γ-amino-n-butyric acid, proline, and alanine in diseased sap increased after both spring and mid-summer inoculations. Proline, which was present in trace amounts in healthy sap, constituted 14 to 38% of the total in diseased sap. Percentage concentrations of amide nitrogen in diseased sap were reduced 50% or more in all inoculated trees. Percentages of ammonia, aspartic acid, glutamic acid, and several other amino acids did not change.