Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Effect of environmental factors on tenuazonic acid production by Alternaria alternata on soybean-based media

Aims: To determine the effects of water activity (a_W; 0·995–0·90), temperature (5, 18, 25 and 30°C), time of incubation (7–35 days) and their interactions on tenuazonic acid (TA) production on 2% soybean‐based agar by two Alternaria alternata strains isolated from soybean in Argentina. Methods and Results: TA production by two isolates of A. alternata was examined under interacting conditions of a_W, temperature and time of incubation on 2% soybean‐based agar. Maximum TA production was obtained for both strains at 0·98 a_W, but at 30 and 25°C for the strains for RC 21and RC 39, respectively. The toxin concentration varied considerably depending on a_W, temperature, incubation time and strain interactions. TA was produced over the temperature range from 5 to 30°C and a_W range from 0·92 to 0·995, however at 5 and 18°C little TA was produced at a_W below 0·94. Contour maps were developed from these data to identify areas where conditions indicate a significant risk for TA accumulation. Conclusions: The optimum and marginal conditions for TA production by A. alternata on soybean‐based agar were identified. The results indicated that TA production by A. alternata is favoured by different temperatures in different strains. Significance and Impact of the Study: Data obtained provide very useful information for predicting the possible risk factors for TA contamination of soybean as the a_W and temperature range used in this study simulate those occurring during grain ripening. The knowledge of TA production under marginal or sub‐optimal temperature and a_W conditions for growth are relevant as improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality.     

Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity

The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile lyases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohols or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes.

The temperature effects on enantioselectivity were studied in detail. From measurements of the enantiomeric ratio (E) at different temperatures the activation parameters ΔΔH^# and ΔΔS^# were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, ΔΔH^# and ΔΔS^# had opposite signs and therefore the effects cooperated to give high E values (−RTlnE = ΔΔG^# = ΔΔH^# − TΔΔS^#). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations was discussed.

In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions, while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict.     

Effect of water activity and temperature on the growth of Eurotium species isolated from animal feeds

Background

Xerophilic fungi represent a serious problem due to their ability to grow at low water activities causing the spoiling of low and intermediate moisture foods, stored goods and animal feeds, with the consequent economic losses.

Temperature effect on a high stearic acid sunflower mutant.

Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M(2) progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 degrees C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 degrees C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.     

Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

The effect of temperature on viability of imbibed weed seeds

Imbibed seed of 10 common arable weeds were placed in trays in initially moist soil and, after imbibing for 2h, heated in ovens/incubators set to 31^oC, 42^oC, 56^oC, 75^oC or 100^oC for 0.5, 1, 2, 4, 8 or 16 days or at 102^oC, 155^oC, 204^oC or 262^oC for 0.5, 1, 2, 5, 7.5 or 10 min. After heating, seeds were incubated for 28 days at 10/20^oC or 20/30^oC on a 12 h dark/light regime, depending on species, and germination recorded. At the lower temperatures, germination of all species was prevented by temperatures of 75^oC or higher for periods of 0.5 days or more. Germination was lower after treatment at 56^oC than at 31^oC or 42^oC for all species except Rumex obtusifolius. The maximum temperature required to prevent germination varied among species and was of greater importance than the duration of heating. Germination was variable with duration of heating. At the higher temperatures, there was very little germination of any species after heating at 204^oC for 7.5 min or 262^oC for 5 min or more. Seeds were greatly buffered from the air temperature by 3 mm of soil, throughout the shorter duration of heating. The average temperature of the soil, over the 10 min heating required to prevent over 90% germination, varied among species and ranged from 48^oC for Avena fatua to 65^oC for R. obtusifolius. This work implies that composting systems maintained at 65^oC are unlikely to provide an efficient method of weed control. Recommendations for improvement of the laboratory technique are suggested.     

Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain.

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

Microbial interaction ofAspergillus parasiticus andBacillus subtilis withalternaria alternata. production of alternariol, alternariol monomethylether and tenuazonic acid on sunflower seeds

Production of alternariol, alternariol mono-methylether and tenuazonic acid byAlternaría alternata was studied in competition withAspergillus parasiticus andBacillus subtilis on irradiated sunflower seeds at 0.90 a_w. In cultures co-inoculated withAlternaría alternata andAspergillus parasiticus alternariol production decreased by 64%. Similar results were observed in cultures co-inoculated withAlternaría alternata andBacillus subtilis.     

Induction of secondary dormancy in sunflower seeds by high temperature. Possible involvement of ethylene biosynthesis

High temperature (45°C) inhibits seed germinition and seedling sunflower ( Helianthus annuus L. cv. Mirasol). Treatment of imbibed seeds at 45°C for more than 48 h induces a secondary dormancy, which is associated with progressive decrease of germination ability at optimal temperature (25°C) as well as with abnormal seedling growth. Ethylene (55μl l⁻¹) and 2-chloroethylphosphonic acid (ethephon) (2.5 m M ) improve germination of thermodormant seeds at 25°C. but the abnormal growth of the seedlings remains. O₂-enriched atmosphere and dry storage improve germination and normal seedling growth. The induction of thermodormancy in sunflower seeds seems associated with loss of their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. Possible effects of high temperature on membranes and ethylene forming enzyme (EFE) are discussed.     

Changes in mass and dimensions of sunflower (Helianthus annuus L.) Achenes and seeds due to carbonization

When analyzing sunflower (Helianthus annuus L.) remains, which are often carbonized, archaeobotanists commonly differentiate between wild and domesticated achenes and seeds based on the measured length (L) and width (W) or the calculated index L*W. Carbonization reduces the dimensions. To compensate for these reductions, archaeobotanists use a single correction factor proposed by Richard Yarnell (1978) for all cases. The use of a single correction factor can bias the reconstructed dimensions as carbonization is a highly variable process. The current study determines the relationship between carbonization and the dimensions of length and width. Measurements established that a decrease of 2.5-22.5% in achene length and 10-29% in achene width can occur, depending on temperature, heating rate, and variety. For seeds, temperature is of most importance, and shrinkage ranges from 0-27% for the length and from 0-20% for the width. These ranges make the use of a single correction factor problematic. A method is developed in which reflectance (an optical property applied in coal technology to determine coal rank) is used to measure the carbonization temperature, and in turn the shrinkage can be calculated. Subsequently, correction factors are calculated to reconstruct the original length and width. When applied to an assemblage of carbonized sunflower achenes, the newly developed method shows that the Yarnell single correction factor may bias the dimensions towards classifications of “wild” or “ruderal” forms of sunflower     

Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

温度对压力-容积分析和对植物组织吸水影响的研究

以25年树龄的挪威云杉树冠中部生长1年的小枝为测试材料,应用电子控温压力室,分别在15、20、25、30和35℃的恒温条件下进行PV分析所得到的水分参数表明;温度升高将引起π₀和π_p值下降,ROWC_πp和RWC_πp却几乎不受温度的影响;在不控温条件下应用Hammel逐渐升压法进行PV分析时,压力室温度比室温高1-2℃,所得结果与同样温度控温所测结果相似。在5-30℃不同温度条件下的植物组织吸水试验表明,在一定限度内升高温度可以增强植物组织的吸水量。     

IAA production during germination of Orobanche spp. seeds

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.     

Purification and characterization of the sunflower seed (Helianthus annuus L.) major aminopeptidase

The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at pH 7.5–8.0 and 45–50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either α- or β-position and l-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P₁-position and its catalytic efficacy is affected by the structure of both P₁ and P₁′ parts of the substrate.     

Organization of lipid reserves in cotyledons of primed and aged sunflower seeds

Imbibing sunflower (Helianthus annuus L., cv. Briosol) seeds at water potentials between –2 MPa and –5 MPa leads to faster (priming) or slower (accelerated ageing) germination depending on the temperature and duration of treatment. Mobilization of food reserves may be associated with the changes in seed vigor. To study this, morphological, biochemical and phase properties of lipid, the major food reserve in sunflower, were compared in freshly harvested (i.e., control), primed and aged sunflower cotyledons using electron microscopy, biochemical analyses and differential scanning calorimetry, respectively. Lipid bodies became smaller and more dispersed throughout the cytoplasm during priming and ageing. Despite ultrastructural changes, there were few measured changes in biochemistry of the neutral lipid component; lipid content, proportion of saturated and unsaturated fatty acids and level of free fatty acids were unchanged in primed and slightly aged seeds, with only severely aged seeds showing a net decrease in polyunsaturated fatty acids and an increase in free fatty acids. Subtle changes in the calorimetric behavior of lipids within sunflower cotyledons were observed. Sunflower lipids exhibited polymorphic crystalline and amorphous solid phases when cooled to <–100°C, but priming decreased the rate of crystallization in vivo and ageing increased the rate of crystallization, but decreased percentage crystallinity. The observed changes in thermal behavior in vivo are consistent with losses and gains, respectively, of interacting non-lipid moieties in the triacylglycerol matrix.     

Endophytic bacteria in sunflower (<Emphasis Type="Italic">Helianthus annuus </Emphasis>L.): isolation,characterization, and production of jasmonates and abscisic acid in culture medium

This study was designed to isolate and characterize endophytic bacteria from sunflower (Helianthus annuus) grown under irrigation and water stress (drought) conditions, to analyze growth of isolated bacteria under drought condition, and to evaluate the ability of bacteria isolated from plants cultivated under drought to produce jasmonates (JAs) and abscisic acid (ABA). Bacteria were isolated from soil samples collected when sunflower plants were at the end of the vegetative stage. A total of 29 endophytic strains were isolated from plants grown under irrigation or drought condition. Eight strains (termed SF1 through SF8) were selected based on nitrogen-fixing ability. All eight strains showed positive catalase and oxidase activities; five strains (SF2, SF3, SF4, SF5, SF7) solubilized phosphates; none of the strains produced siderophores. Strains SF2, SF3, SF4, and SF5, the ones with the highest phosphate solubilization ability, strongly inhibited growth of the pathogenic fungi Verticillum orense and Sclerotinia sclerotiorum but had less inhibitory effect on Alternaria sp. Among the eight strains, SF2 showed 99.9% sequence homology with Achromobacter xiloxidans or Alcaligenes sp., while the other seven showed 99.9% homology with Bacillus pumilus. Strains SF2, SF3, and SF4 grown in control medium produced jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), and ABA. These three strains did not differ in amount of JA or OPDA produced. ABA content was higher than that of JA, and production of both ABA and JA increased under drought condition. The characteristics of these isolated bacterial strains have technological implications for inoculant formulation and improved growth of sunflower crops.     

ACC conversion to ethylene by sunflower seeds in relation to maturation,germination and thermodormancy

Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene was studied in sunflower (Helianthus annuus L., cv. Mirasol) seeds in relation to germinability. Ethylene production from ACC decreased during seed maturation, and non-dormant mature seeds were practically unable to synthesize ethylene until germination and growth occurred, indicating that ethylene forming enzyme (EFE) activity developed during tissue imbibition and growth. ACC conversion to ethylene was reduced by the presence of pericarp, and in young seedlings it was less in cotyledons than in growing axes.ACC conversion to ethylene by cotyledons from young seedlings was optimal at c. 30°C, and was strongly inhibited at 45°C. Pretreatment of imbibed seeds at high temperature (45°C) induced a thermodormancy and a progressive decrease in EFE activity.Abscisic acid and methyl-jasmonate, two growth regulators which inhibit seed germination and seedling growth, and cycloheximide were also shown to inhibit ACC conversion to ethylene by cotyledons of 3-day-old seedlings and by inbibed seeds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - CH cycloheximide - EFE ethylene forming enzyme - IAA indole-3-acetic acid - Me-Ja methyl-jasmonate     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.