Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities

Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation.     

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.     

Temperature and the Life Cycle of Heterodera zeae

Development of the corn cyst nematode, Heterodera zeae, was studied in growth chambers at 20, 25, 29, 33, and 36 ± 1 C on Zea mays cv. Pioneer 3184. The optimum temperature for reproduction appeared to be 33 C, at which the life cycle, from second-stage juvenile (J2) to J2, was completed in 15-18 days; at 36 C, 19-20 days were required. Juveniles emerged from eggs within 28 days at 29 C and after 42 days at 25 C. Although J2 were present within eggs after 63 days at 20 C, emergence was not observed up to 99 days after inoculation. Female nematodes produced fewer eggs at 20 C than at higher temperatures.     

Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains

The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed. The xylanase produced by T. lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C. In comparison seven ATCC strains and the DSM 5826 strain of T. lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C. Culture filtrates of T. lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C. The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C. The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP. The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C. At 50 degrees C, the enzyme of T. lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0. Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h. The xylanase of T. lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.     

Cytoplasm-specific Effects of Helminthosporium maydis Race T Toxin on Survival of Corn Mesophyll Protoplasts

High yields of mesophyll protoplasts were obtained from leaves of corn (Zea mays L., inbred W64A). Many protoplasts survived a week in the dark in a simple osmoticum. Culture filtrate from Helminthosporium maydis race T at dilutions of 1:10,000 to 1:20,000 destroyed protoplasts with Texas male-sterile (T) cytoplasm. Substantial damage to protoplasts with nonmale-sterile (N) cytoplasm occurred only at a 1:20 dilution. High concentrations of partially purified H. maydis race T (HMT) toxin (32.5-130 μg dry weight/ml) did not reduce survival of protoplasts with N cytoplasm or C or S male-sterile cytoplasms after 6 days of exposure. Protoplasts with T or TRf (fertility restored) cytoplasm collapsed within 1 to 3 days after treatment with 0.13 μg of HMT toxin/ml, which was one-fifth the level causing 50% inhibition of T cytoplasm seedling root growth. Protoplasts with T cytoplasm which were washed after 30 minutes or more of exposure to HMT toxin also collapsed within a few days. Cultured W64A T protoplasts and freshly isolated protoplasts from inbreds C103 and Mo17 with T cytoplasm were less sensitive to HMT toxin than freshly isolated W64A T protoplasts. Toxin-treated protoplasts survived longer in the light than in the dark. The sensitivity and specificity of the system described will facilitate physiological, ultrastructural, and genetic studies of toxin action.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Growth of and toxin production by nonproteolytic Clostridium botulinum in cooked puréed vegetables at refrigeration temperatures.

Seven strains of nonproteolytic Clostridium botulinum (types B, E, and F) were each inoculated into a range of anaerobic cooked puréed vegetables. After incubation at 10 degrees C for 15 to 60 days, all seven strains formed toxin in mushrooms, five did so in broccoli, four did so in cauliflower, three did so in asparagus, and one did so in kale. Growth kinetics of nonproteolytic C. botulinum type B in cooked mushrooms, cauliflower, and potatoes were determined at 16, 10, 8, and 5 degrees C. Growth and toxin production occurred in cooked cauliflower and mushrooms at all temperatures and in potatoes at 16 and 8 degrees C. The C. botulinum neurotoxin was detected within 3 to 5 days at 16 degrees C, 11 to 13 days at 10 degrees C, 10 to 34 days at 8 degrees C, and 17 to 20 days at 5 degrees C.     

Viriditoxin Production by Aspergillus viridi-nutans and Related Species

Bioproduction of viriditoxin on various substrates by strains of the Aspergillus fumigatus group was determined under several incubation conditions. Aspergillus viridi-nutans strains NRRL 4365 and 576 produced the largest quantities of toxin, A. brevipes gave reduced yields, and there was no detectable synthesis by isolates of four related species. After 30 days in static culture at 20 C on various autoclaved agricultural commodities, optimal yields of 440 and 380 mg of toxin were observed per kilogram of sorghum and rice. Toxin levels were reduced on corn, rye, and wheat (40-200 mg/kg); yields were low on cottonseed, barley, and oats. Incubation at 10 C restricted biosynthesis of viriditoxin, and no toxin accumulated on substrates maintained at 5 C for 120 days. In a liquid, yeast extract-sucrose medium, maximal mycotoxin production developed in shake flasks; after 156 h, 10 mg of toxin accumulated per gram of mycelium. Viriditoxin produced in submerged culture was associated with the mycelium; less than 1% was detected in the filtered broth after 156 h of incubation.     

Control of Penicillium martensii Development and Penicillic Acid Production by Atmospheric Gases and Temperatures

The effects of various gaseous environments and temperatures on development of Penicillium martensii NRRL 3612 and production of penicillic acid (PA) were determined. Accumulation of PA in mold-inoculated corn was measured following incubation under air; 20% CO(2), 20% O(2), 60% N(2); 40% CO(2), 20% O(2), 40% N(2); and 60% CO(2), 20% O(2), 20% N(2). Although reduced temperature initially inhibited PA production, at the end of the trial the largest quantity of PA (120 mug/g of corn) was found in air-incubated corn at the lowest test temperature (5 C). Atmospheres enriched with 60% CO(2) reduced PA accumulation below a detectable level at 5 and 10 C after a 4-week incubation period. Spore germination tests were carried out in a liquid growth medium incubated for 16 hr under several test conditions. Germ tube outgrowth at 30 C ranged from 36% in air to 2% in 60% CO(2), whereas no germination was observed in CO(2)-enriched gases at 10 C. When spore respiration rates were measured in air and O(2) in a liquid growth medium, complete removal of CO(2) from the reaction atmosphere did not reduce O(2) uptake.     

A Comparison of Purified Host Specific Toxin from Helminthosporium maydis, Race T, and Its Acetate Derivative on Oxidation by Mitochondria from Susceptible and Resistant Plants

NADH or succinate oxidation and malate oxidation were differentially affected in mitochondria from both susceptible and resistant corn by a purified and chemically characterized preparation of host-specific toxin from Bipolaris (Helminthosporium) maydis, race T. NADH and succinate oxidation by susceptible T corn mitochondria were stimulated 50 to 200% with apparent uncoupling from the cytochrome chain at approximately 10(-9)m toxin (5 to 20 ng/ml). Significant inhibition of malate oxidation was observed at slightly higher toxin concentrations, but oxidation was still coupled to ADP utilization. Inhibition of malate oxidation also was observed in N corn (resistant) and soybean mitochondria at approximately 1,000-fold greater concentrations, but stimulation of NADH and succinate oxidation was not found at any toxin concentration tested.A fully acetylated toxin derivative at approximately 1 microgram per milliliter also caused stimulation of NADH or succinate oxidation in T corn mitochondria, but not those of N corn or soybean mitochondria at 100 micrograms per milliliter. Malate oxidation was inhibited to the same extent by toxin acetate with mitochondria from T corn, N corn, and soybean. The blocking of hydroxyl groups in race T toxin by acetyl functions eliminated selectivity toward malate oxidation only. The data suggest that inhibition of malate oxidation is either a separate or secondary effect of selective action of toxin on T corn mitochondria, perhaps by interference with transport in or out of the matrix. Sensitivity of T, but not N, corn mitochondria to purified toxin decays within minutes after pellets are suspended in aqueous osmotica, with no obvious change in mitochondrial integrity. The action of race T toxin seems to involve a labile process, such as ion gradient(s), or an unstable structural conformation of T corn mitochondria.     

Degradation of the Cry1Ab protein within transgenic Bacillus thuringiensis corn tissue in the field

Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem. Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues. Therefore, it is important to know how long the toxin persists in plant residues. In two field studies in the temperate corn-growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme-linked immunosorbent assay (ELISA). In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month. During the second month, Cry1Ab toxin concentrations decreased to approximately 20% of their initial values. During winter, there was no further degradation. When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June. In the second field trial, representing a no-tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil-incorporated Bt plants, to 38% of the initial concentration during the first 40 days. They then continued to decrease until the end of the trial after 200 days in June, when 0.3% of the initial amount of Cry1Ab toxin was detected. Our results suggest that extended pre- and post-commercial monitoring are necessary to assess the long-term impact of Bt toxin in transgenic plant residues on soil organisms.     

Swine refusal factors elaborated by Fusarium strains and identified as trichothecenes.

Fusarium poae (Peck) Wollenw. NRRL 3287, F. nivale (Fr.) Ces. NRRL 3289, and F. moniliforme Sheldon NRRL 3197, each grown on cracked corn (13 days at 28 degrees C), produced refusal factors in pig bioassays. Substantial quantities of trichothecenes were detected in the refused corn: T-2 toxin (30 micrograms/g) was detected in corn fermented with the F. poae strain; the level of vomitoxin (1 microgram/g) in corn cultured with F. nivale did not account for the 48% refusal response in the pigs tested. The F. moniliforme concomitantly produced T-2 toxin (33 micrograms/g) and vomitoxin (1.5 micrograms/g). This strain's taxonomic position was reexamined, and it is shown to be a cultural variant of the species F. tricinctum (Cda.) Sacc.     

T-2 Toxin Production by Fusarium tricinctum on Solid Substrate

A method has been developed to produce and purify gram quantities of T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3alpha-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C.     

Bioproduction of [14C]ochratoxin A in submerged culture.

A number of Aspergillus and Penicillium species were tested for production of ochratoxin A (OA) in several media. After 8 days of static incubations of submerged cultures at 28 degrees C, toxin yields of 25 and 30 micrograms/ml were obtained with Aspergillus alliaceus NRRL 4181 in Ferreirás and 2% yeast extract-4% sucrose media, respectively. However, the largest production observed in the preliminary screening was 54 micrograms/ml; this highest level was produced by A. sulphureus NRRL 4077 in a modified Czapek solution. The medium contained the basal salts and sucrose of Czapek plus urea (3%) and corn steep liquor (0.5% solids). A time study of toxin production demonstrated maximum yield of 350 micrograms/ml by the A. sulphureus isolate in the modified Czapek medium after 11 days of static incubation at 28 degrees C. The optimal production conditions were employed in additional tests designed to measure the efficiency of 14C incorporation from sodium [1-14C]-acetate into OA. Samples (20 microCi) of sodium acetate were added to separate culture flasks at 24-h intervals during the initial 9 days of the fermentation. Addition of [14C]acetate on day 4 of incubation provided the maximum yield of labeled OA. The highest specific activity of labeled toxin obtained was 0.07 microCi/mg of OA and the maximum incorporation rate of labeled acetate was 5.3%.     

Potentiation effect of corn extract on the production of eremofortin C, EC oxidase, and PR toxin by Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Their structures are similar and differ only by an alcohol and an aldehyde group at the C-12 position. EC has been demonstrated to be the precursor of PR toxin, and EC is transformed to PR toxin by EC oxidase. These two compounds and EC oxidase are secreted by P. roqueforti in the culture medium, which is usually composed of 15% sucrose and 2% yeast extract. Recently, we discovered that the addition of corn extract to this medium increased the production of EC and PR toxin and the activity of EC oxidase in a coordinative manner. In a time-course study, we found that the peak yield of EC and PR toxin and the maximum activity of EC oxidase in the culture medium containing 7.5% sucrose, 1% yeast extract, and 20% corn extract were increased 6.2, 4.6, and 4.7-fold, respectively, as compared with those obtained in the medium without corn extract. Moreover, corn extract increased the production of EC and PR toxin and the activity of EC oxidase by P. roqueforti in a dose-dependent manner. On the other hand, when the concentrations of sucrose and yeast extract were increased while fixing the ratio of corn extract, we found that the levels of EC and PR toxin and the enzyme activity were decreased concomitantly. We thus conclude that corn extract can enhance the production of EC, PR toxin and EC oxidase by P. roqueforti when grown in a minimal medium and that the potentiation effect of corn extract is suppressed when the fungi are grown in a rich medium.     

Aflatoxins: Production of the Toxins In Vitro in Relation to Temperature

The production or accumulation of aflatoxins in vitro by four isolates on three substrates (acid-delinted cottonseed, shelled Spanish peanut, and rough rice) was studied in relation to temperature in the range of 10 to 40 C. Within the first 10 days after inoculation, the optimal temperature range for aflatoxin production was between 20 and 35 C. Only small amounts of the toxins were produced at 10 and 40 C. Within the optimal temperature range, the time required for toxin production and for significant accumulation decreased as the temperature increased. More aflatoxin G was produced or accumulated in relation to aflatoxin B at low temperatures (within the optimal range), and the G aflatoxins were metabolized more rapidly at the higher temperatures.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.     

The effect of elevated temperature on the toxicity of the laboratory cultured dinoflagellate Ostreopsis lenticularis (Dinophyceae)

Ostreopsis lenticularis Fukuyo 1981, is the major benthic dinoflagellate vector implicated in ciguatera fish poisoning in finfish on the southwest coast of Puerto Rico. Clonal laboratory cultures of O. lenticularis (clone 301) exposed to elevated temperatures (30-31 degrees C) for 33 and 54 days showed significant increases in the quantity of extractable toxin they produced as compared to their toxicities versus cells grown at temperatures of 25-26 degrees C. O lenticularis samples collected directly from the field following exposure to elevated temperatures for comparable periods of time also showed significant increases in extractable toxin. The increased toxicity of both field sampled and laboratory grown O. lenticularis exposed to elevated temperatures may result from the effects of elevated temperatures on their metabolism and/or the bacterial symbionts found associated with these microalgae. The number of bacteria associated with cultured O. lenticularis exposed to elevated temperatures was significantly reduced. Increased toxin recovery from O. lenticularis exposed to elevated temperatures may have resulted from the direct effect of temperature on toxin production and/or the reduction of Ostreopsis associated bacterial flora that consume toxin in the process of their growth. This reduction in the quantity of associated bacterial flora in temperature treated cultures may result in increased toxin recovery from O. lenticularis due to a reduction in the consumption of toxin by these symbiont bacteria.