Elevated plasma glycosaminoglycans in chickens with tibial dyschondroplasia induced by a Fusarium oxysporum isolate.

Chicken plasma glycosaminoglycans (GAGs) were isolated and digested. Their building block molecules, namely, glucosamine and galactosamine, were quantified by gas chromatography. The levels of these two amino sugars were elevated in broiler chickens with tibial dyschondroplasia (TD) induced by culture material of Fusarium oxysporum (FO), a mold isolated from corn originated from the endemic region of Kaschin-Beck disease (KBD) in China. As the TD severity score changed from 1 (healthy) to 2, 3, and 4, glucosamine increased by 10%, 33%, and 57% and galactosamine by 9%, 13% and 48%, respectively. The elevated plasma GAGs correlated to TD severity but not to the amount of FO material in the diets. This correlation of plasma GAGs to TD in chickens parallels the reported correlation of urinary GAGs to KBD in humans. The possibility of TD as an animal model for KBD is discussed.

Natural occurrence of Fusarium mycotoxins in field samples from the 1992 Wisconsin corn crop.

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.

Combined chemical defenses against an insect-fungal complex.

This study considered how host plant allelochemicals may contribute to defense against insects and fungi that jointly colonize the subcortical tissues of trees, the relative roles of constitutive and inducible chemistry in these defenses, and how the actions of two different feeding guilds might be interrelated. Our model consisted of the coniferous treePinus resinosa, the root- and lower stem-colonizing beetlesHylastes porculus andDendroctonus valens, and their associated fungiLeptographium procerum andL. terebrantis, and the stem-colonizing bark beetleIps pini and its associated fungusOphiostoma ips. In a novel bioassay, extracts from reaction tissue elicted by wound inoculation withL. terebrantis were more repellent to beetles than were similar extracts from constitutive or mechanically wounded tissue. The effect on beetle behavior was more pronounced in nonpolar extracts, which contain mostly monoterpenes, than in polar extracts, which contain mostly phenolics. Synthetic monoterpenes at concentrations present in the various tissues exerted similar effects and were likewise repellent in dose-response experiments. Growth ofL. procerum andL. terebrantis was inhibited by polar extracts from constitutive and reaction tissue. Inhibition was higher in wounded than control tissue, but the inhibition response did not vary with the type of wounding. Synthetic monoterpenes strongly inhibited spore germination and mycelial growth of both fungi. Colonization of red pine roots byLeptographium spp. altered the subsequent effects of extracts of stem phloem tissue onI. pini. These effects varied with host condition. Beetles preferred extracts from constitutive stem phloem tissue of healthy trees to that of root-diseased trees. However, extracts from reaction tissues of healthy trees were more repellent toI. pini than were the reaction tissues of root-diseased trees. The implications of these results to plant defense against insect-fungal complexes and interactions among different feeding guilds are discussed.

Effects of maize weevils (Coleoptera: Curculionidae) on production of aflatoxin B1 by Aspergillus flavus in stored corn.

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)

Induction of tibial dyschondroplasia and suppression of cell-mediated immunity in chickens by Fusarium oxysporum grown on sterile corn.

An isolate of Fusarium oxysporum from corn associated with Kaschin-Beck disease in humans was tested for its ability to induce tibial dyschondroplasia (TD) and toxicity in chicks. Both leghorn and broiler chicks were fed diets in which corn was replaced with varied amounts (0% to 50%) of the F. oxysporum culture grown on sterile corn, or with known TD-inducing agents. F. oxysporum did not affect body weight in either type of chicks. In leghorn chicks, neither F. oxysporum nor the known TD-inducing agents (F. equiseti, 4%; tetramethylthiuram disulfide [Thiram], 35 ppm) caused TD. However, F. oxysporum at high levels (50%) and the two known TD-inducing agents reduced interdigital cutaneous response to phytohemagglutinin-P challenge. In addition, Thiram also reduced body-weight gain by more than 17%. In female broiler chicks (Cornish Rock), F. oxysporum not only decreased cell-mediated cutaneous response to phytohemagglutinin-P but also increased TD incidence; these same effects were observed with F. equiseti and Thiram. Histological examinations revealed similar pathological changes among dyschondroplastic lesions induced by F. oxysporum, F. equiseti, and Thiram. Results of this experiment indicate that the isolate of F. oxysporum from the region in which Kaschin-Beck disease is endemic can induce TD in broiler chicks and that it is immunosuppressive.

Identification of RAPD markers linked to a black leaf spot resistance gene in Chinese elm.

Black leaf spot (Stegophora ulmea) is a common foliage disease on Chinese (Ulmus parvifolia) and Siberian elms (U. pumila), two species which have been widely used as sources of Dutch-elm disease-resistance genes for interspecific elm hybrids. A dominant gene controlling resistance to black leaf spot was identified in a population derived from self-pollination of a single U. parvifolia tree. Using RAPD markers, in combination with bulked segregant analysis, we have identified three markers linked to this resistance gene. A survey of Chinese-elm hybrids revealed that the same gene is likely to confer a high level of resistance to black leaf spot in interspecific elm hybrids, although other genetic factors may also be involved in the determination of a disease phenotype.

Effects of biotic and abiotic stress on induced accumulation of terpenes and phenolics in red pines inoculated with bark beetle-vectored fungus.

This study characterized the chemical response of healthy red pine to artificial inoculation with the bark beetle-vectored fungusLeptographium terebrantis. In addition, we sought to determine whether stress altered this induced response and to understand the implications of these interactions to the study of decline diseases. Twenty-five-year-old trees responded to mechanical wounding or inoculation withL. terebrantis by producing resinous reaction lesions in the phloem. Aseptically wounded and wound-inoculated phloem contained higher concentrations of phenolics than did constitutive tissue. Trees inoculated withL. terebrantis also contained higher concentrations of six monoterpenes,α-pinene,ß-pinene, 3-carene, limonene, camphene, and myrcene, and higher total monoterpenes than did trees that were mechanically wounded or left unwounded. Concentrations of these monoterpenes increased with time after inoculation. Total phenolic concentrations in unwounded stem tissue did not differ between healthy and root-diseased trees. Likewise, constitutive monoterpene concentrations in stem phloem were similar between healthy and root-diseased trees. However, when stem phloem tissue was challenged with fungal inoculations, reaction tissue from root-diseased trees contained lower concentrations ofα-pinene, the predominant monoterpene in red pine, than did reaction tissue from healthy trees. Seedlings stressed by exposure to low light levels exhibited less extensive induced chemical changes when challenge inoculated withL. terebrantis than did seedlings growing under higher light. Stem phloem tissue in these seedlings contained lower concentrations ofα-pinene than did nonstressed seedlings also challenge inoculated withL. terebrantis. It is hypothesized that monoterpenes and phenolics play a role in the defensive response of red pine against insect-fungal attack, that stress may predispose red pine to attack by insect-fungal complexes, and that such interactions are involved in red pine decline disease. Implications to plant defense theory and interactions among multiple stress agents in forest decline are discussed.

Decreased cell-mediated immunity and lack of skeletal problems in broiler chickens consuming diets amended with fusaric acid.

Young female broiler chickens fed diets amended with 0, 35, 75, and 150 mg fusaric acid (FA)/kg diet for 3 weeks showed no aberrations in behavior, feed intake, weight gain, or appearance of the visceral organs. Furthermore, there was no correlation between the dietary concentration of FA and incidence of tibial dyschondroplasia and leg-shape deformities. Ash content of dry fat-free tibiae was not influenced by FA; thus, no rickets was present in these chickens. FA enhanced the humoral response to sheep erythrocytes but significantly reduced cell-mediated cutaneous response to phytohemagglutinin-P.

Tibial dyschondroplasia of chickens induced by Fusarochromanone, a mycotoxin.

Chicks were fed for 3 weeks a practical broiler starter diet amended with fusarochromanone (FC), a mycotoxin. In Hubbard broiler chicks, the highest test dose of FC (75 ppm) suppressed body weight by 33% and humoral response to sheep erythrocytes by 34% to 50%, and increased the incidence of tibial dyschondroplasia (TD) to 100%. The intermediate dose (35 ppm) significantly increased the incidence of TD in both types of broiler chicks (Hubbard and Indian River), but its effect on humoral response to sheep erythrocytes was not consistent across several experiments. A minimum dietary concentration greater than 20 ppm was required to significantly increase the incidence of TD. Excessive dietary copper or zinc (200 ppm) alleviated the effect of FC on TD. However, activities of copper-dependent ceruloplasmin and superoxide dismutase was not affected by FC. Cartilage mineral profiles in FC- or Thiram-induced TD differed from that of 1-day-old hypertrophic embryonic cartilage core (less K and Mn). FC did not induce TD in leghorn chicks.

Decreased immune response and increased incidence of tibial dyschondroplasia caused by fusaria grown on sterile corn.

Corn, feed, and litter samples reported to be associated with feed refusal, diarrhea, leg weakness, and mortality were evaluated for the presence of toxic substances. Intubated residues of ethyl acetate extracts of these samples did not cause gross lesions, diarrhea, or mortality in young New Hampshire x Single Comb White Leghorn crossbred chicks. Fusarium moniliforme was the predominant fungal species found in unpelleted feed and corn samples. Young broiler chicks, fed diets supplemented with 2 or 8% corn cultures of selected F. moniliforme isolates from a suspected toxic corn sample, failed to develop clinical signs of mycotoxicosis. However, some isolates resulted in decreased antibody responses to SRBC. Corn cultures of some Fusarium equiseti and Fusarium semitectum strains also decreased the immune response. Cultures of three F. equiseti strains from barley and potato induced tibial dyschondroplastic lesions in young broiler chicks. Other F. equiseti strains and strains of other Fusarium species did not cause this skeletal abnormality.

Fusarochromanone production by Fusarium isolates.

Sixty two Fusarium isolates representing nine species from many parts of the world were screened for fusarochromanone production. A simplified method for the detection of fusarochromanone in culture filtrates or grain cultures was used. Under UV irradiation (364 nm) the chloroform phase from fusarochromanone-positive culture extracts fluoresced a characteristic bright blue color. Results were confirmed by thin-layer-chromatography comparison with pure fusarochromanone standards. Detection was possible in cultures as young as 1 week old. Biosynthesis of fusarochromanone was rare in Fusarium spp. and was only detected in three isolates of Fusarium equiseti, namely R-4482 (barley [Federal Republic of Germany]), R-6137 (barley [Alaska]), and R-8508 (potato [Denmark]), among all the isolates tested from various geographic sources.

Immune and bone properties of chicks consuming corn contaminated with a Fusarium that induces dyschondroplasia.

A study was conducted to contrast the dyschondroplasia-inducing capability of several species of Fusarium with that of the natural fungi found in poultry diets and litter. Day-old broiler chicks were fed pure corn cultures of specific fungal isolates for 3 weeks. Humoral immunity to sheep red blood cells (SRBC), body weight, valgus and varus leg deformities, incidence of dyschondroplasia, and mechanical properties of the tibiotarsi were examined. F. equiseti #15 was the only fungal isolate to induce dyschondroplasia to any significant degree, which confirmed previous work in Minnesota. This isolate also suppressed growth rate and humoral immunity, although dyschondroplasia sometimes did occur without associated immunosuppression and growth depression. Bone strength of the tibiotarsal diaphysis was normal, and gross lesions of valgus and varus leg deformities did not appear to be related to the incidence of dyschondroplasia in the 3-week-old chicks.

Heterokaryosis in Fusarium tricinctum and F. sporotrichioides.

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.

New process for T-2 toxin production.

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.

Feed refusal factors in pure cultures of Fusarium roseum 'graminearum'.

Isolations from 1972 Wisconsin feed refusal corn yielded predominantly cultures of Fusarium roseum 'graminearum.' With one possible exception, none of the selected isolates of this fungus induced emesis in pigeons, whereas six of nine isolates produced feed refusal responses in all test animals. A single isolate of F. roseum 'equiseti' also induced a severe refusal response and possibly slight emesis. None of the other fungi isolated from this corn (F. moniliforme, Acremoniella atra) or controls caused either emesis or feed refusal. Zearalenone was detected in all isolates and was shown to be partially responsible for refusal activity. The remaining activity was ascribed to one or more nonvolatile, neutral, relatively polar molecules. T-2 toxin, although not detected in these isolates, was shown to have dramatic refusal activity in rats.

Isolation of acetyl T-2 toxin from Fusarium poae.

Acetyl T-2 toxin (3,4,15-triacetoxy-8-isovaleroxy-12,13-epoxy-delta9-trichothecene) was isolated and characterized as a naturally occurring emetic trichothecene from liquid cultures of Fusarium poae (NRRL 3287). Acetyl T-2 toxin was shown to be much less toxic than T-2 toxin in pigeon assays.

Isolation and partial characterization of a mycotoxin from Penicillium roqueforti.

Extracts of pure cultures of Penicillium roqueforti isolated from toxic feed samples and of P. roqueforti NRRL 849 were lethal to rats by either intraperitoneal or oral administration. Purification studies guided by this test led to the isolation of a major toxin which showed intraperitoneal and oral median lethal dose values in weanling rats of 11 and 115 mg/kg, respectively. Partial characterization of the crystalline compound, C(17)H(20)O(6), by infrared, ultra violet, PMR, and mass spectroscopy, and by several chemical transformations indicated the presence of three C-methyl substituents plus one acetoxy, one aldehyde, and one alpha,beta-unsaturated ketone group. Two oxygen atoms are present either in epoxide or ether form.

Identification of T-2 toxin in moldy corn associated with a lethal toxicosis in dairy cattle.

Over a 5-month period during the winter of 1970-71, 20% of the lactating Holstein cows in a Wisconsin dairy herd died after prolonged ingestion of a diet containing 60% moldy corn infested with Fusarium tricinctum (2 x 10(5) propagules per g of moldy corn). Ethyl acetate extracts of the ground dried corn induced severe dermal reactions when applied to the skin of shaved 60-g albino rats and killed four of five 100-g rats that were force fed 1 ml in 2 ml of pure corn oil. T-2 toxin (3-hydroxy-4, 15-diacetoxy-8-[3-methylbutyryloxy]-12, 13-epoxy-Delta(9)-trichothecene) at concentrations of 2 mg per kg of dry corn was identified in purified extracts of the moldy corn by means of gas-liquid chromatography and mass spectrometry. This concentration of T-2 toxin in the moldy feed and the nature of the toxic effects observed strongly suggest a major causal relationship.

Improved skin test for detection of T-2 toxin.

A modified rat skin test based on dermatitic properties of trichothecenes is described which is quick, convenient, and sensitive to 0.05 mug of T-2 toxin.