Occurrence of Aflatoxins and Aflatoxin-Producing Strains of Aspergillus spp. in Soybeans

Above average rainfall in Maryland during August, September, and October 1971 resulted in heavy mold growth in soybeans while still in the field. Of 28 samples of soybean seed, aflatoxins were found in 14, 2 of which had been used in poultry feed. Aflatoxins were identified by thin-layer chromatography, spectrophotometry, and chicken embryo bioassay. Aspergillus spp. were isolated from 11 samples, and 5 of these isolates produced aflatoxins when grown in liquid culture.     

Occurrence and distribution of lettuce mosaic disease in Tehran province from Iran

Lettuce mosaic virus (LMV) Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in fields of Tehran province. In this study 452 leaf samples were collected from the fields throughout the Tehran province during 2002 and 2003. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV)and Arabis mosaic virus (ArMV) was determined with DAS-ELISA. Percentage of single Infection to LMV. CMV or TSWV was 20.58, 15.93 and 9.96% respectively. Also 15.28% of samples were co- infected with LMV+CMV, 8.19% with LMV+TSMV and 7.74% with CMV+TSWV. 4.65% of samples were Infected to all of these three viruses. LMV was found in 48.69%, CMV in 43.59% and TSWV in 30.54% of samples totally. Therefore LMV is major dominant agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and first report of CMV and LMV in Tehran province.     

Occurrence of the upper triassic bivalveMonotis in Iran

Monotis (Monotis) haueri Kittl is described from the Nayband Formation of the western Lut area (Central Iran) indicating uppermost Norian. This is also the first established record for the species between its disjunct occurrences in Europe and North America.     

Occurrence of Aggregate Sheath Spot of Rice in Iran

Aggregate sheath spot of rice caused by Rhizoctonia oryzae-sativae (Swa.) Mordue was found in several locations in Mazandaran Province. The polyacrylamide gel electrophoretic pattern of cell proteins of isolates from different regions were identical. The protein profiles of Iranian isolates were also identical with that of an anastomosis group (AG)-Bb tester isolate of binucleate Rhizoctonia from Japan, but distinctly different from an AG-Ba tester strain.     

The Natural Occurrence of Ethionine in Bacteria

Two unknown radioactive areas appeared after radioautography and two dimensional paper chromatography of culture medium in which Escherichia coli was grown. These materials were studied by paper chromatography and paper electrophoresis of several derivatives and identified as ethionine and ethionine sulfone, the latter an artifact. Chromatographic coincidence of the unknowns and their derivatives with authentic materials establishes the identification. Ethionine was found in cellular extracts and in the growth media of Escherichia coli, Bacillus megaterium, Pseudomonas aeruginosa, and Aerobacter aerogenes but not in Scenedesmus, Saccharomyces cerevisiae, or bovine lymphosarcoma cells. Ethionine was synthesized by resting E. coli cultures from radioactive sulfate and from radioactive methionine. Growing cells labeled ethionine within 1 minute after addition of radioactive sulfate to cultures. Levels of radioactivity in ethionine increased with time. No incorporation of this amino acid could be detected in the cellular proteins formed under the conditions of this study.     

Widespread Natural Occurrence of Hydroxyurea in Animals

Here we report the widespread natural occurrence of a known antibiotic and antineoplastic compound, hydroxyurea in animals from many taxonomic groups.Hydroxyurea occurs in all the organisms we have examined including invertebrates (molluscs and crustaceans), fishes from several major groups, amphibians and mammals. The species with highest concentrations was an elasmobranch (sharks, skates and rays), the little skate Leucoraja erinacea with levels up to 250 μM, high enough to have antiviral, antimicrobial and antineoplastic effects based on in vitro studies. Embryos of L. erinacea showed increasing levels of hydroxyurea with development, indicating the capacity for hydroxyurea synthesis. Certain tissues of other organisms (e.g. skin of the frog (64 μM), intestine of lobster (138 μM) gills of the surf clam (100 μM)) had levels high enough to have antiviral effects based on in vitro studies. Hydroxyurea is widely used clinically in the treatment of certain human cancers, sickle cell anemia, psoriasis, myeloproliferative diseases, and has been investigated as a potential treatment of HIV infection and its presence at high levels in tissues of elasmobranchs and other organisms suggests a novel mechanism for fighting disease that may explain the disease resistance of some groups. In light of the known production of nitric oxide from exogenously applied hydroxyurea, endogenous hydoxyurea may play a hitherto unknown role in nitric oxide dynamics.     

Natural Occurrence of Helenium Virus S in Impatiens holstii

Samples of commercially grown Impatiens holstii from several sources in Minnesota contained a carlavirus which serologically was closely related, if not identical with Helenium virus S described earlier in Germany. The two viruses have similar experimental host ranges and caused similar cytopathogenic effects. After mechanical transmission the Impatiens isolate produced less severe symptoms in Impatiens than the Helenium isolate. Only the latter isolate was transmitted by Myzus persicae in the non-persistent manner.     

Natural Occurrence of Seiridium cardinale on Thuja in Germany

Seiridium cardinale (Wagener) Sutton & Gibson (syn. Coryneum cardinale Wagener) is the most dangerous parasitic fungus of cypress and other Gupressaceae, already appearing in epidemic proportions, especially in the mediterranean area. Recently (in 1989, 1990 and 1991) this pathogen has been detected in the northern part of Germany (Hamburg and Schleswig-Holstein) naturally infecting Thuja. The geographical distribution and the progressive spread of S. cardinale is reviewed. Diagnostic features of the fungus, modes of host penetration, principal symptoms of the disease, ways of dispersal and control methods are described. Possible causal factors for the recently observed occurrence of 5. cardinale in the north of Germany are discussed.     

Natural Occurrence of Entomogenous Nematodes in Tennessee Nursery Soils

To isolate potential insect biocontrol agents, entomogenous nematodes were surveyed in Tennessee plant nurseries in 1991. Soil samples from 113 nursery sites were baited with greater wax moth (Galleria mellonella) larvae, house cricket (Acheta domesticus) adults, lesser mealworm (Alphitobius diaperings) adults, and house fly (Musca domestica) larvae. Heterorhabditis bacteriophora and Steinernema carpocapsae were each recovered from 17 soil samples. Heterorhabditis bacteriophora was more common in habitats with crape myrtle (Lagerstroemia indica) and Chinese juniper (Juniperus chinensis) than other nursery plants, and S. carpocapsae was more frequently recovered from habitats with juniper and Southern magnolia (Magnolia grandiflora). Bulk density, electrical conductivity, organic matter, pH, temperature, and moisture content of the entomogenous-nematode positive soil samples were compared. Other nematode genera recovered with insect baits included Rhabditis sp., Pelodera sp., Cryptaphelenchoides sp., and Mesodiplogaster sp., which was recovered from a greater percentage of soil samples than the other five genera.     

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants

Abstract The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed. Received: 16 March 1999; Accepted: 27 August 1999; Online Publication: 23 February 2000     

Natural Occurrence of 16 Fusarium Toxins in Grains and Feedstuffs of Plant Origin from Germany

A total of 220 samples comprising cereals, cereal byproducts, corn plants and corn silage as well as non-grain based feedstuffs was randomly collected during 2000 and 2001 from sources located in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol (SCIRP), 15-monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol (NEO), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivealenol (15-ADON), nivalenol (NIV) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry. Zearalenone (ZEA) and α- and β-zearalenol (α- and β-ZOL) were analysed by high performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 μg/kg. Out of 125 samples of a group consisting of wheat, oats, corn, corn byproducts, corn plants and corn silage only two wheat samples did not contain any of the toxins analysed. Based on 125 samples the incidences were at 2–11% for DAS, NEO, T-2 Triol, FUS-X, α- and β-ZOL, at 20–22% for SCIRP, MAS, T-2 tetraol and 3-ADON, at 44–74% for HT-2, T-2, 15-ADON, NIV and ZEA, and at 94% for DON. Mean levels of positive samples were between 6 and 758 μg/kg. Out of 95 samples of a group consisting of hay, lupines, peas, soya meal, rapeseed meal and other oilseed meals, 64 samples were toxin negative. DAS, T-2 triol, NEO and FUS-X were not detected in any sample. The incidences of DON and ZEA were at 14 and 23% respectively, those of the other toxins between 1–4%, mean levels of positive samples were between 5 and 95 μg/kg.     

Occurrence and physico-serological properties of potato virus S (PVS) in Iran

1818 collected samples of potato plant showing virus infection symptoms from 85 fields were tested for PVS infection using DAS-ELISA. Average of infection to this virus varied from 0 to 100%. Least infection was belonging to fields with new introduced varieties. On the other hand native and old introduced cultivars showed heavy infection. In field condition, PVS infected plants didn't show very obvious symptoms, so some infected plants may be missed in field sample collecting. The physical properties of 3 isolates, Avaj, Stanboly and Agria No 15 were determined. TIP 55-60 degrees C, DEP 10(-3) and Liv measured 3-4 days. Ouchterlony agar double diffusion test using SDS was useful for virus detection and precipitation lines didn't show any spur between isolates, although isolates differs slightly in symptomatology. SDS-Page and Western blotting methods used successfully for virus detection and determining and measuring viral protein components.     

Liquid Chromatography of Aflatoxins

There are numerous reports on studies of aflatoxins, but there are only a few reports on the isolation and mutual separation of aflatoxins by liquid chromatography. Following the previous reports on the liquid chromatography of four kinds (B₁, B₂, G₁ and G₂) of aflatoxins, the authors carried out the chromatography of six kinds of aflatoxins including aflatoxins B_2a and G_2a. Various kinds of adsorbents and eluting solvents were examined, and then the good mutual separation of aflatoxins was obtained by using the Sephadex G-10 (CM), a newly prepared adsorbent containing carboxymethyl group, and pure water for eluting solvent. Aflatoxins G_2a, B_2a, G₂, B₂, G₁ and B₁ were eluted in this order.     

Production of Aflatoxins in Submerged Culture

Aflatoxins can be produced on a synthetic medium in submerged culture. Glucose, sucrose, or fructose are the preferred carbon sources, and Casamino Acids are the preferred nitrogen source. Ammonia is almost as good a nitrogen source. Zinc is required at levels of at least 0.4 mg per liter. Concentrations of aflatoxin of 60 to 80 mg per liter (as determined by optical-density measurements of a chloroform extract of the unfiltered broth) can readily be obtained in indented shake flasks; somewhat lower yields were obtained in 5-liter fermentors.     

The Natural Occurrence of Turnip Mosaic Potyvirus in Allium ampeloprasum

An isolate of turnip mosaic potyvirus (TuMV) was obtained from Allium ampeloprasum grown in commercial greenhouses in Israel. Symptoms on infected plants include systemic chlorosis and yellow stripes, accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically, and in a non-persistent manner by aphids, among Allium, Chenopodium. Gomphrena and some Nicotiana spp. Purified preparations contained numerous filamentous particles similar to those observed in crude extracts of infected leaves. Particles from crude plant extracts had a normal length of 806 nm. Cells of infected plants contained cylindrical cytoplasmic inclusions with pinwheel, scrolls and laminated aggregates which indicated the presence of a potyvirus of Edwardson's subgroup III. and which resemble those of turnip mosaic virus (TuMV), The virus reacted strongly with antiserum to typical isolates of TuMV in immunoelectron microscopy and western blotting but not with antisera to several other potyviruses. Based on serological reactivity, electron microscopy, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV.     

Antimicrobial Activity of Aflatoxins

Antimicrobial activity of a crude aflatoxin preparation and of aflatoxin B(1) was studied. They were found inactive against common gram-positive and gram-negative bacteria at a concentration of 100 mug/ml. Both samples of aflatoxin did, however, exhibit antimicrobial activity, though narrow and limited, against various strains of Streptomyces and Nocardia. The antibiotic action of aflatoxin B(1) was confirmed by bioautogram after thin-layer chromatography. Among seven strains of microorganisms, including aflatoxin-sensitive and -resistant strains, N. asteroides IFM 8 was found to reduce aflatoxin B(1), in addition to other minor fluorescent components in the crude preparation.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

We detected biosynthetic activity for aflatoxins G₁ and G₂ in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G₁ and G₂ were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B₁, aflatoxin B₂, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G₂ from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G₁ was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G₁ and G₂. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.     

The Occurrence and Development of Amylase Enzymes in Incubated, De-embryonated Maize Kernels

The development of amylase activity in extracts from de-embryonated and GA₃-treated de-embryonated maize kernels (Zea mays L.) was determined during a 10-day incubation period. The increase in activity was compared with activity extracted from endosperms dissected from germinating whole kernels. Chromatographic analysis of reaction products as well as physicochemical characterization demonstrated that the activities from GA₃-treated and nontreated tissue were comparable and that part of the activity was attributable to α-amylase.