Increased susceptibility and reduced phytoalexin accumulation in drought-stressed peanut kernels challenged with Aspergillus flavus

Three genotypes of peanut (Arachis hypogaea L.), with ICG numbers 221, 1104, and 1326, were grown in three replicate plots and drought stressed during the last 58 days before harvest by withholding irrigation water. Within each plot there were eight levels of stress ranging from 1.1 to 25.9 cm of water. Kernels harvested from the plots were hydrated to 20% moisture and challenged with Aspergillus flavus. Fungal colonization, aflatoxin content, and phytoalexin accumulation were measured. Fungal colonization of non-drought-stressed kernels virtually ceased by 3 days after inoculation, when the phytoalexin concentration exceeded 50 micrograms/g (fresh weight) of kernels, but the aflatoxin concentration continued to rise exponentially for an additional day. When fungal colonization, aflatoxin production, and phytoalexin accumulation were measured 3 days after drought-stressed material was challenged, the following relationships were apparent. Fungal colonization was inversely related to water supply (r varied from -0.848 to -0.904, according to genotype), as was aflatoxin production (r varied from -0.876 to -0.912, according to genotype); the phytoalexin concentration was correlated with water supply when this exceeded 11 cm (r varied from 0.696 to 0.917, according to genotype). The results are discussed in terms of the critical role played by drought stress in predisposing peanuts to infection by A. flavus and the role of the impaired phytoalexin response in mediating this increased susceptibility.     

Increased susceptibility and reduced phytoalexin accumulation in drought-stressed peanut kernels challenged with Aspergillus flavus.

Three genotypes of peanut (Arachis hypogaea L.), with ICG numbers 221, 1104, and 1326, were grown in three replicate plots and drought stressed during the last 58 days before harvest by withholding irrigation water. Within each plot there were eight levels of stress ranging from 1.1 to 25.9 cm of water. Kernels harvested from the plots were hydrated to 20% moisture and challenged with Aspergillus flavus. Fungal colonization, aflatoxin content, and phytoalexin accumulation were measured. Fungal colonization of non-drought-stressed kernels virtually ceased by 3 days after inoculation, when the phytoalexin concentration exceeded 50 micrograms/g (fresh weight) of kernels, but the aflatoxin concentration continued to rise exponentially for an additional day. When fungal colonization, aflatoxin production, and phytoalexin accumulation were measured 3 days after drought-stressed material was challenged, the following relationships were apparent. Fungal colonization was inversely related to water supply (r varied from -0.848 to -0.904, according to genotype), as was aflatoxin production (r varied from -0.876 to -0.912, according to genotype); the phytoalexin concentration was correlated with water supply when this exceeded 11 cm (r varied from 0.696 to 0.917, according to genotype). The results are discussed in terms of the critical role played by drought stress in predisposing peanuts to infection by A. flavus and the role of the impaired phytoalexin response in mediating this increased susceptibility.     

Estimation of Fungal Infection of Peanut Kernels by Determination of Free Glutamic Acid Content

Peanut kernels (Tainan 9, a Spanish cultivar) inoculated with Aspergillus parasiticus, A. flavus, A. niger, or A. ochraceus as well as noninoculated kernels were incubated in a humidified environment (relative humidity, 100%) at 25(deg)C for 7 weeks. Internal fungal populations and changes in moisture and sucrose content and free amino acid composition of the kernels were determined periodically. Fungal populations determined by using A. flavus and A. parasiticus agar and rose bengal chlortetracycline agar as enumerating media were closely correlated. Moisture content in the kernels increased from 5.8 to 20.4% (dry basis), and changes in individual free amino acid contents varied, depending upon the incubation time and type of fungus used as an inoculum. In the early infection period (up to 5 weeks), sucrose contents and logarithms of threonine and tyrosine contents increased while logarithms of free glutamic acid content decreased linearly with incubation time. A negative linear relationship was further obtained between logarithms of fungal populations and the logarithm of free glutamic acid content (R(sup2) > 0.80) of the infected peanut kernels.     

Aflatoxin variability in pistachios.

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.     

Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds

Soil is a reservoir for Aspergillus flavus and A. parasiticus, fungi that commonly colonize peanut seeds and produce carcinogenic aflatoxins. Densities of these fungi in soil vary greatly among fields and may influence the severity of peanut infection. This study examined the relationship between soil density of Aspergillus species and the incidence of peanut seed colonization under laboratory conditions. Viable peanut seeds were wounded and inoculated with 20 soils differing in composition and density of Aspergillus species and were then incubated for 14 days at 37 degrees C (seed water activity = 0.92). The effect of soil density of individual section Flavi species (A. flavus strains L and S, A. parasiticus, A. caelatus, and A. tamarii), section Nigri, and A. terreus on the incidence of seed colonization was best expressed as a function of exponential rise to maximum. Exponential curves often rose to maximum percentages of seed colonization by section Flavi species that were well below 100% despite high species densities in some soils. Competition primarily among section Flavi species may explain the reduced incidences of seed colonization. An average of two or fewer propagules of each Aspergillus species in the soil at the wound site was required for colonization of 20% of peanut seeds. Other fungal species were capable of invading peanut seeds only when soil densities of sections Flavi and Nigri species were low.     

Colonization of rye green manure and peanut fruit debris by Aspergillus falvus and Aspergillus niger group in field soils.

Aspergillus flavus and Aspergillus niger group colonization of deep-plowed, decomposing rye green manure cover crops in peanut field soils was studied in four fields during 1972 and 1973; colonization of decomposing peanut fruits was studied in 1972 in two fields. A. flavus colonization of rye and peanut fruits was greater in soils of heavy texture, and an A. flavus population as high as 165 propagules per g of soil was observed in soil adjacent to rye, whereas A. flavus populations in soils not associated with rye were 18 propagules per g of soil or lower. Highest A. flavus populations in soil adjacent to decomposing peanut fruits were usually comparable to populations associated with rye. Little decomposing rye or peanut fruit colonization was generally observed by the A. flavus competitor, A. niger group. A. flavus may maintain or increase its inoculum potential by colonization of these and other moribund plant tissues.     

Lack of host specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

Effects of natamycin and potassium sorbate on growth and aflatoxin production in olives

Aspergillus flavus NRRL 6555 was inoculated onto whole olives and olive paste samples containing variable amounts of either natamycin or potassium sorbate and incubated at 15 degrees, 25 degrees, and 35 degrees C for 7, 14 and 21 days for whole olives and at 15 degrees and 25 degrees C for 8 and 16 days for olive pastes. The initiation time of growth was parallel to the concentrations of either preservatives applied. However, at 15 degrees C, natamycin at 160 and 320 micrograms/g (ppm) completely inhibited the growth of mold on whole olives for 21 days and olive paste for 7 and 15 days, respectively. All levels of potassium sorbate inhibited mold growth at 15 degrees C, but at 25 degrees C, 6000 micrograms/g (ppm) only, delayed growth for 15 days. The extent of growth at the end of the incubation periods was parallel to the temperatures of incubation. The analyses for aflatoxin B1 production in all samples at all levels of preservatives and control were negative.     

Colonization of wounded peanut seeds by soil fungi: selectivity for species from Aspergillus section Flavi

Soil is a source of primary inoculum for Aspergillus flavus and A. parasiticus, fungi that produce highly carcinogenic aflatoxins in peanuts. Aflatoxigenic fungi commonly invade peanut seeds during maturation, and the highest concentrations of aflatoxins are found in damaged seeds. A laboratory procedure was developed in which viable peanut seeds were wounded and inoculated with field soil containing natural populations of fungi, then incubated under different conditions of seed water activity and temperature. Densities of Aspergillus section Flavi in soil used for inoculating seeds were low relative to the total numbers of filamentous fungi (<1%). Aspergillus species from section Flavi present in soil included A. flavus morphotypes L and S strains, A. parasiticus, A. caelatus, A. tamarii and A. alliaceus. Wounding was required for high incidences of fungal colonization; viability of wounded seeds had little effect on colonization by Aspergillus species. Peanut seeds were colonized by section Flavi species as well as A. niger over broad ranges of water activity (0.82-0.98) and temperature (15-37 C), and the highest incidences of seed colonization occurred at water activities of 0.92-0.96 at 22-37 C. A. parasiticus colonized peanut seeds at lower temperatures than A. flavus, and cool soil temperatures relative to temperatures of aerial crop fruits might explain why A. parasiticus is found mostly in peanuts. Other fungi, dominated by the genera Penicillium, Fusarium and Clonostachys, colonized seeds primarily at water activities and temperatures suboptimal for section Flavi species and A. niger. Eupenicillium ochrosalmoneum frequently sporulated on the conidial heads of section Flavi species and showed specificity for these fungi. The inoculation of wounded viable peanut seeds with soil containing natural populations of fungi provides a model system for studying the infection process, the interactions among fungi and those factors important in aflatoxin formation.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii.

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to aw 0.93 were more rapid than in unsupplemented media (aw 0.99). Although growth in unsupplemented medium was lower at 35 degrees C, incubation at 21 degrees C or 35 degrees C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 micrograms potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35 degrees C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35 degrees C than in cultures incubated at 21 degrees C in media both with and without 300 micrograms potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at -18 degrees C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 micrograms potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21 degrees C. Sensitivity to freezing increased when cultures were incubated at 21 degrees C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Dusky Sap Beetle Mediated Dispersal of Bacillus subtilis to Inhibit Aspergillus flavus and Aflatoxin Production in Maize Zea mays L.

Laboratory assays were performed with detached milk stage maize ( Zea mays L.) ears and dusky sap beetles ( Carpophilus lugubris Murray) carrying the Kodiak Concentrate formulation of the bacterium, Bacillus subtilis (Ehrenberg) Cohn. After 1 day of exposure to the B. subtilis- contaminated C. lugubris , the colonization of mechanically damaged kernels by Aspergillus flavus Link ex. Fries was reduced from 82% (if the A. flavus was inoculated first) to 41% (if B. subtilis was added by C. lugubris before the A. flavus ). Field cage studies were performed with an autoinoculative device containing B. subtilis into which C. lugubris beetles were introduced. C. lugubris -dispersed B. subtilis reduced visible A. flavus colonization by 97% when the A. flavus was added to purposely damaged maize ears 4 days after C. lugubris were released from the autoinoculator. In 1993 field studies, none of the purposely damaged ears that allowed access to C. lugubris beetles emerging from autoinoculators containing B. subtilis had visible sporulating A. flavus compared with 92% of ears that did not allow access of C. lugubris but that subsequently had the A. flavus inoculum added. In 1994 field studies, 70% of the ears that excluded C. lugubris had aflatoxin levels greater than 200 ppb in purposely damaged kernels, as opposed to less than 10% of kernels that permitted access by natural populations of C. lugubris that probably acquired B. subtilis from a single autoinoculator. Aflatoxin levels in these ears were negatively correlated with the presence of both B. subtilis and C. lugubris . The B. subtilis was widely dispersed over a 16-ha area as indicated by maize ear and C. lugubris trap sampling. These studies indicate that autoinoculative dispersal of B. subtilis by natural populations of C. lugubris is a potentially useful means for reducing A. flavus and aflatoxin in maize.     

Increase of enzyme activities in Neurospora crassa during incubation at low temperatures.

The effect of lowering the incubation temperature of sucrose-grown cultures of Neurospora crassa on the level of various enzyme activities was investigated. Of twelve inducible/derepressible activities studied, three, in addition to glycerol kinase, were found to increase during 48 h of incubation at 4-6 degrees C: trehalase (increase in specific activity of 3-10-fold), beta-glucosidase (6-12-fold) and beta-N-acetylglucosaminidase (4 to 6-fold). The maximum increases occurred at 6 degrees C and no increases took place in mycelia incubated at 0 degrees C. The kinetics of the changes in activity were markedly different from those observed previously with glycerol kinase. The increases were inhibited by cycloheximide. Trehalase, beta-glucosidase and beta-N-acetylglucosaminidase activities were not rapidly lost when cultures incubated at 6 degrees C were returned to 26 degrees C.     

Aspergillus flavus and other mycoflora of groundnut kernels in Israel and the absence of aflatoxin

More than 300 groundnut (peanut) samples collected from different regions of Israel were examined by ELISA for aflatoxin contamination. Samples were designated for export, local consumption or for sowing. None of the samples were contaminated with the toxin. However, when kernels were kept at high humidity (RH?99%), aflatoxin could be frequently detected seven days after incubation and the toxin was not uniformly distributed among kernels.Aspergillus niger, A flavus, Penicillium citrinum andP pinophilum were the dominant fungi and no differences were observed among cultivars. Almost half of the commercial samples examined were devoid ofA flavus. Other fungi identified wereA tamaril, A amstelodami, P rubrum, Rhizoctonia solani, Macrophomina phaseolina, Rhizopus spp., Sclerotium rolfsll, Fusarium andAlternaria spp; the two last ones comprising a group of low incidence. Although groundnut samples that containA flavus—infected kernels are moderately common, the local climate and agrotechniques In use in Israel are not conducive to aflatoxin accumulation. Nevertheless infected kernels may become a threat to health if stored under inadequate conditions.     

Effect of ADP on ATPASE from a strain of Bacillus stearothermophilus.

Bacillus stearothermophilus ATCC 12016 was unable to grow at temperatures below 40 degrees C. On incubating the bacteria at the temperatures, ATP in cells disappeared, ADP was accumulated and ATPase (EC 3.6.1.3) was inactivated. When the purified ATPase was incubated at the temperatures for 1 h with 0.17 mM ADP in the presence of MgCl2, the enzyme was completely inactivated. The inactivated enzyme was reactivated on dilution or dialysis or on warming at 65 degrees C. During the incubation of the enzyme sample, the absorbance spectrum of the enzyme changed. On further incubating the sample over 1.5 h, the second step of spectral change occurred together with the change of the circular dichrosim and the dissociation into a lower molecular weight species of the protein. When the enzyme was treated with ADP-MgCl2 at 65 degrees C, the inactivation and conformational change of the enzyme was not observed.     

An improved medium for the detection of Aspergillus flavus and A. parasiticus

An effective selective medium for the enumeration of Aspergillus flavus and Aspergillus parasiticus has been developed by modification of Bothast and Fennell's Aspergillus Differential Medium. Results can be obtained with the new medium, Aspergillus flavus and parasiticus Agar (AFPA), after 42 h incubation at 30 degrees C. The medium is thus suitable for use in quality control as a guide to the presence of A. flavus and, potentially, of aflatoxins. AFPA has been extensively tested on peanuts and soils. Results were reproducible and comparable with those on standard fungal enumeration media incubated for much longer periods. A very low percentage of false positive or negatives was found.     

Concanavalin A-induced redistribution of surface receptors in Acanthamoeba castellanii at different growth phases

Concanavalin A (ConA)-induced redistribution of surface receptors has been studied in Acanthamoeba castellanii at different growth phases utilizing double fluorescent techniques and transmission electron microscopy. When the amoebae were incubated with 2 micrograms and 10 micrograms tetramethylrhodamine isothiocyanate (TRITC)-ConA/ml for 4 min and 15 min at 28 degrees C the staining pattern was characterized by various numbers of scattered aggregates of fluorescent ConA. Double labeling of the amoebae showed that the fluorescent aggregates represented internalized label, and the internalization was not preceded by any aggregation of ConA receptors on the cell surface as visualized by incubating with anti-ConA serum followed by fluorescein isothiocyanate-conjugated anti-IgG. Following exposure of the amoebae to 10 micrograms TRITC-ConA/ml for 4 min and 15 min at 28 degrees C intracellular accumulation of some of the fluorescent aggregates in cap-like structures occurred at the logarithmic and postlogarithmic growth phases but not at the early stationary growth phase. Electron microscopic observation of amoebae labeled with ferritin-conjugated ConA at 28 degrees C revealed a uniform surface labeling and an intracellular accumulation of the label in vesicular and tubular structures, and occasionally in cap-like structures. Surface capping of ConA receptors in Acanthamoeba was induced by treating the amoebae with ConA and anti-ConA serum at 0 degrees C followed by incubation at 28 degrees C. The formation of surface caps in Acanthamoeba showed growth-phase dependency, too. The visualization of the surface caps at the electron microscopic level was performed by indirect staining utilizing protein A-colloidal gold.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH, pCO2 and NADP changes during aerobic and anaerobic incubation of the brain cortex of young and adult rats

Wistar rats of both sexes, aged 5 and 90-110 days, bred in the author's department, were used for experiments in which pH, pCO2 and NADP changes induced by incubating brain cortex homogenate (30 min at 37 degrees C) under aerobic and anaerobic conditions were studied. The medium was S?rensen's buffer solution, with succinate as substrate. NADP was assayed by determining glucose-6-phosphate dehydrogenase activity, which is NADP-dependent. The pH and pCO2 were determined with an ABL-2 radiometer (Denmark). The NADP extinction curves showed that the adult rat cortex, with or without substrate, always contained significantly less NADP under anaerobic conditions (the amount of its reduced form increased). In 5-day-old rats, the course of changes in the extinction curves was completely analogous to that for adult rats if the tissue was incubated without substrate; in the presence of substrate, the amount of the oxidized form, i.e. of NADP, decreased and there was no difference between the course of extinction changes under anaerobic and aerobic conditions. Similarly, the pH fell significantly less in 5-day-old rats during anaerobic incubation than in adult rats and was practically no different from the pH changes found during aerobic incubation. In addition, we found that the increase in pCO2 during anaerobic incubation of the brain cortex of 5-day-old rats was very small (also compared with aerobic conditions) whereas the increase in pCO2 under aerobic and anaerobic conditions in adult rats was the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature- and dose-dependent internalization of concanavalin A in monolayer culture

When rat hepatoma cells (R117-21B) were incubated for 20 h at 37 degrees C with 125I-labeled concanavalin A at low concentrations (0.5-10 micrograms/ml), only 20-30% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside, but at high concentrations (50-500 micrograms/ml), 60-80% of the cell-associated radioactivity was released. At 4 degrees C, the cell-associated radioactivity decreased with the increase in concentration of concanavalin A, and more than 80% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside. These results suggest that the amount of cell-associated concanavalin A is related to the physicochemical state of the plasma membrane, which can be altered by the incubation temperature or by the concentration of concanavalin A, the transitional concentration being 5-10 micrograms/ml.     

Influences of incubation temperature and various saccharides on the production of organic acids and gases by gut microbes of rainbow trout Oncorhynchus mykiss in a micro-scale batch culture

We studied the influence of incubation temperature and additional saccharides on the metabolism of hindgut microbes of the rainbow trout Oncorhynchus mykiss in a 50 microl-scale batch culture system. Intestinal contents of rainbow trout reared at 15 degrees C were incubated with glucose, lactosucrose, sodium alginate or colloidal chitin (each 10 g/l) at 15 degrees C or 25 degrees C for 12 h. Levels of organic acids at 0 h and 12 h of incubation were quantified with HPLC. We also monitored gas release from these cultures during incubation. The main product was iso-butyric acid, except for the cultures with colloidal chitin where no net production of organic acids was observed. We detected higher levels of iso-butyric acid in cultures with lactosucrose than in the other cultures. Net production of this acid was less in cultures with colloidal chitin than in blank cultures. The volume of released gas was larger when incubated at 25 degrees C than at 15 degrees C. Cultures with colloidal chitin released more gas than blank cultures when they were incubated at 15 degrees C. Cultures with sodium alginate released less gas than blank cultures irrespective of incubation temperature. These results indicate that the hindgut microbes of this carnivorous fish mainly produce branched-chain fatty acids, very likely by microbial digestion of nitrogenous materials rather than saccharides. However, additional saccharides affected production of branched-chain fatty acids. The influence of incubation temperature in the present study also suggested that the environmental temperature of host fish should affect microbial digestion in the fish gut.