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Trans-2-hexenal, a volatile aldehyde, is produced by soybean (Glycine max [L.] Merr) and other plants via the lipoxygenase pathway. In vitro tests showed it significantly (P < 0.001) reduced Aspergillus flavus germinating conidial viability at 10 μM, with approximately 95% viability reduction observed at 20 μM. The viability of nongerminated conidia was not reduced. To test the effectiveness of this volatile to prevent fungal growth in stored corn, trans-2-hexenal was pumped intermittently into glass jars containing corn. Experiments were performed to determine the ability of 2 different pump cycle time-courses to prevent A. flavus growth on sterile corn (23% moisture). Intermittently (30-min pumping period) over 7 d, this volatile was pumped through 350 g of corn kernels inoculated with 1 mL of 3 × 104 conidia of A. flavus. Controls consisted of (1) sterile corn, (2) corn inoculated with A. flavus with no pumped air, and (3) corn inoculated with A. flavus with intermittently pumped air. Aflatoxin B1 (AFB1), viability counts, and aldehyde concentration in the headspace were performed in each experiment. To determine whether an increased time period between volatile pumping would prevent A. flavus growth, a 2nd series of experiments were performed that were similar to the 1st series except that trans-2-hexenal (only) was pumped for a 30-min period every 12 h. Experiments were performed 3 times for each time course. Both experiments showed that intermittent pumping of volatile trans-2-hexenal significantly (P < 0.001) prevented A. flavus growth and aflatoxin B1 production over a 7-d period. Practical Application: Results from this study indicate that intermittent pumping of volatile trans-2-hexenal could be used to protect stored corn from A. flavus growth and aflatoxin contamination.

Control of preharvest aflatoxin contamination of susceptible crops such as corn, cotton, peanut, and tree nuts is possible through time-consuming and expensive agronomic practices. Breeding for disease-resistant crops is also very time consuming and does not lend itself readily to combat the evolution of new virulent fungal races. Moreover, availability of known genotypes with natural resistance to mycotoxin-producing fungi is a prerequisite for the successful breeding program. While it is possible to identify a few genotypes of corn or peanuts that are naturally resistant to Aspergillus, we do not know whether these antifungal factors are specific to Aspergillus flavus. In crops like cotton, there are no known varieties naturally resistant to Aspergillus spp. So far, the best options available to us are through biocontrol and/or genetic engineering. Availability of transgenic varieties with antifungal traits is extremely valuable as a breeding tool. Use of fungicides or chemicals can add to the cost of production. Moreover, the growing concerns regarding environmental safety and groundwater quality demand less dependence on agrochemicals. Disease-resistant transgenic crops would not only control mycotoxin-producing organisms such as A. flavus, A. parasiticus and Fusarium spp. but also other microbial (fungal, bacterial, and viral) diseases that cause significant economic losses in crop production. Above all, transgenic crops resistant to aflatoxin-producing fungi offer the promise of negating the adverse effects caused by the toxin on immunocompromised humans and animals. This review explores recent advances regarding genetic engineering approaches towards the control of aflatoxin contamination using native and heterologous genes.

CAY-1, a novel saponin from Capsicum frutescens (commercially known as cayenne pepper) was investigated to determine its in vitro antifungal activity, mechanism of action and mammalian cell cytotoxicity. CAY-1 was active against 16 different fungal strains, including Candida spp. and Aspergillus fumigatus [minimum inhibitory concentrations (MIC) ranging from 4 to 16 microg ml(-1)], and was especially active against Cryptococcus neoformans (90% inhibition at 1 microg ml(-1)). Synergistic activity was also observed between CAY-1 and amphotericin B against Candida albicans and A. fumigatus. No significant cytotoxicity was demonstrated when CAY-1 was tested against 55 mammalian cell lines at up to 100 microg ml(-1). Importantly, CAY-1 appears to act by disrupting the membrane integrity of fungal cells.

Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal saponin, CAY-1, with a molecular weight of 1243.35 Da, was isolated and purified to homogeneity from commercially available dry, ground fruit of Capsicum frutescens. CAY-1 was shown to be a potent fungicide for the germinating conidia of Aspergillus flavus, A. fumigatus, A. parasiticus and A. niger with species-dependent LD90 values between 3 and 20 microM. Activity against some Aspergillus species was affected by the test medium used. In vitro assays, CAY-1 was effective against Pneumocystis carinii (IC50): 9.5 microM) and Candida albicans (IC90: 6.2 microM). CAY-1 had no effect on the viability of the nongerminating conidia of the two filamentous fungi, P. carinii and C. albicans, nor on the conidial type of Fusarium oxysporum. It was ineffective against the bacteria Enterobacter agglomerans, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. CAY-1 was not cytotoxic to A 549 lung carcinoma cells or HeLa cells at effective fungicidal concentrations. The results indicate that CAY-1 is an effective fungicide for Aspergillus species, C. albicans and P. carinii at concentrations below the threshold for mammalian cell toxicity.

L-Cecropin B (LCB) is a potent fungicidal peptide that is subject to proteolytic degradation by extracellular enzymes produced by Aspergillus flavus. We hypothesized that D-cecropin B (DCB), containing all D-amino acids, should resist proteolysis while retaining its fungicidal and target specificities. DCB was synthesized by solid phase methods using Fmoc chemistry. In vitro, at pH 6 x 0, DCB was lethal against the germinating conidia of A. flavus (LD90, 25 microM) and A. fumigatus (LD98, 25 microM) and for nongerminating and germinating conidia of Fusarium moniliforme (LD98, 1 x 25 microM) and F. oxysporum (LD95, 2 x 5 microM) at concentrations similar to those previously reported for LCB. It was lethal for Candida albicans with an LD98 at 12 x 5, microM. DCB was not active for the nongerminating conidia of A. fumigatus or A. flavus. Papain, trypsin, pepsin A and Staphylococcus aureus V8 protease degraded LCB but not DCB. Binding assays and circular dichroism showed DCB and LCB bound to cholesterol, ergosterol, beta-1,3-glucan, mannan and chitin. Data show that DCB retains the potent fungicidal properties of the L-form while being resistant to proteolytic enzymes that degrade the latter peptide. This study demonstrates that D-enantiomerization of cecropin B yields a novel fungicidal peptide, which resists proteolytic degradation and is lethal for pathogenic fungi.

The fungicidal properties of the synthetic peptide D4E1 were studied with nongerminated and germinating conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium moniliforme, and Fusarium oxysporum. The minimal lethal concentrations (MLC) needed to kill 100% of germinating conidia ofA. fumigatus, A. flavus, and A. niger were 12.5, 12.5, and 25 µM, respectively. The MLC value for nongerminated and germinating conidia of both Fusarium spp. was 3.0 µM. Except for A. fumigatus, D4E1 was inactive against the nongerminated conidia of the Aspergillus spp. Physicochemical studies showed D4E1 complexed with ergosterol, a sterol present in conidial walls. Cholesterol, present in nongerminated conidia of F. moniliforme, had a greater affinity for D4E1 than did ergosterol. D4E1 was more resistant to fungal and plant protease degradation than the natural peptide, cecropin A. These in vitro results suggest D4E1 is a candidate for transgenic expression in plants to enhance host resistance to fungal infection.Key words: peptides, fungicidal, Aspergillus spp., Fusarium spp., sterols.

Background: CAY-1 is a fungicidal saponin from cayenne pepper whose mode of action differs from amphotericin B (AB) and itraconazole (IT). This work determined CAY-1 synergism with AB or IT. Methods: CAY-1 was purified and used in checkerboard microdilution studies where CAY-1 and AB or IT were mixed with nongerminated (NG) and germinating (G) conidia of three Aspergillus species and Candida albicans. Inhibition was visually determined at 24 and 48 h. Results: CAY-1 had predominantly additive-synergistic interaction with AB or IT against the Aspergillus NG and G conidia. Excellent synergy between CAY-1 and AB occurred at 24 and 48 h against C. albicans. Results suggest CAY-1 enhances AB and IT efficacy.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

UNLABELLED Blue light (470 nm) LED antimicrobial properties were studied alone against bacteria and with or without the food grade photosensitizer, erythrosine (ERY) against filamentous fungi. Leuconostoc mesenteroides (LM), Bacillus atrophaeus (BA) or Pseudomonas aeruginosa (PA) aliquots were exposed on nutrient agar plates to Array 1 (AR1, 0·2 mW cm(-2)) or Array 2 (AR2, 80 mW cm(-2)), which emitted impure or pure blue light (0-300 J cm(-2)), respectively. Inoculated control (room light only) plates were incubated (48 h) and colonies enumerated. The antifungal properties of blue light combined with ERY (11·4 and 22·8 μmol l(-1)) on Penicillium digitatum (PD) and Fusarium graminearum (FG) conidia were determined. Conidial controls consisted of: no light, room light-treated conidia and ERY plus room light. Light-treated (ERY + blue light) conidial samples were exposed only to AR2 (0-100 J cm(-2)), aliquots spread on potato dextrose agar plates, incubated (48 h, 30°C) and colonies counted. Blue light alone significantly reduced bacterial and FG viability. Combined with ERY, it significantly reduced PD viability. Blue light is lethal to bacteria and filamentous fungi although effectiveness is dependent on light purity, energy levels and microbial genus. SIGNIFICANCE AND IMPACT OF THE STUDY Light from two arrays of different blue LEDs significantly reduced bacterial (Leuconostoc mesenteroides, Bacillus atrophaeus and Pseudomonas aeruginosa) viabilities. Significant in vitro viability loss was observed for the filamentous fungi, Penicillium digitatum and Fusarium graminearum when exposed to pure blue light only plus a photosensitizer. F. graminearum viability was significantly reduced by blue light alone. Results suggest that (i) the amount of significant loss in bacterial viability observed for blue light that is pure or with traces of other wavelengths is genus dependent and (ii) depending on fungal genera, pure blue light is fungicidal with or without a photosensitizer.

Soybean homogenates produced volatile compounds upon exposure to lipase. These induced volatiles were identified by SPME. Seventeen volatile compounds identified by SPME were chosen for determination of their ability to inhibit Aspergillus flavus growth and aflatoxin B(1) (AFB1) production in a solid media assay. These volatiles included aldehydes, alcohols, ketones, and furans. Of the tested compounds, the aldehydes showed the greatest inhibition of fungal growth and AFB1 production. These compounds inhibited up to 100% of the observed growth and AFB1 production as compared to the controls. The greatest activity by the aldehydes to disrupt growth was ranked as follows: 2,4 hexadienal > benzaldehyde > 2-octenal > (E)-2-heptenal > octanal > (E)-2-hexenal > nonanal > hexanal. The greatest activity by the aldehydes to reduce AFB1 was ranked as follows: (E)-2-hexenal > 2,4 hexadienal > (E)-2-heptenal > hexanal > nonanal. (E)-2-hexenal and (E)-2-heptenal were tested further in an A. flavus-inoculated corn kernel assay. Both compounds prevented colonization by A. flavus and eliminated AFB1 production when exposed to compound volumes < 10 muL as also shown in the solid media assay. The results suggest that soybeans react to lipase by production of potent antifungal volatiles.

Syringomycin-E (SE) was significantly lethal to Aspergillus and Fusarium species at between 1.9 and 7.8 μg/ml. SE complexed with the following fungal wall constituents (in order of binding): β-1,3-glucan > chitin > mannan > ergosterol = cholesterol. Cytotoxicity in HeLa cells was proportional to the SE concentration, while the amount required for cytotoxicity was 3 to 20 times that needed to kill 95% of the fungi tested.

Two steroidal saponins have been purified from cayenne pepper (Capsicum frutescens). Both have the same steroidal moiety but differ in the number of glucose moieties: the first saponin has four glucose moieties (molecular mass 1081 Da) and the second contains three glucose moieties (molecular mass 919 Da). Solubility in aqueous solution is less for the saponin containing three glucose moieties than for the one containing four glucose moieties. The larger saponin was slightly fungicidal against the nongerminated and germinating conidia of Aspergillus flavus, A. niger, A. parasiticus, A. fumigatus, Fusarium oxysporum, F. moniliforme, and F. graminearum, whereas, the second saponin (molecular mass 919 Da) was inactive against these fungi. Results indicate that the absence of one glucose molecule affects the fungicidal and aqueous solubility properties of these similar molecules.Key words: fungicidal, saponin, structure–function.

Plants produce potent constitutive and induced antifungal compounds to complement the structural barriers to microbial infection. Approximately 250 000 – 500 000 plant species exist, but only a few of these have been investigated for antimicrobial activity. Nevertheless, a wide spectrum of compound classes have been purified and found to have antifungal properties. The commercial potential of effective plant-produced antifungal compounds remains largely unexplored. This review article presents examples of these compounds and discusses their properties.Key words: antifungal, peptides, phytopathogenic, plants, proteins.

Nonheme chloroperoxidase (CPO-P) of Pseudomonas pyrrocinia catalyzes the oxidation of alkyl acids to peracids by hydrogen peroxide. Alkyl peracids possess potent antifungal activity as found with peracetate: 50% killing (LD(50)) of Aspergillus flavus occurred at 25 microM compared to 3.0 mM for the hydrogen peroxide substrate. To evaluate whether CPO-P could protect plants from fungal infection, tobacco was transformed with a gene for CPO-P from P. pyrrocinia and assayed for antifungal activity. Leaf extracts from transformed plants inhibited growth of A. flavus by up to 100%, and levels of inhibition were quantitatively correlated to the amounts of CPO-P activity expressed in leaves. To clarify if the peroxidative activity of CPO-P could be the basis for the increased resistance, the antifungal activity of the purified enzyme was investigated. The LD(50) of hydrogen peroxide combined with CPO-P occurred at 2.0 mM against A. flavus. Because this value was too small to account for the enhanced antifungal activity of transgenic plants, the kinetics of the enzyme reaction was examined and it was found that the concentration of hydrogen peroxide needed for enzyme saturation (K(m) = 5.9 mM) was already lethal. Thus, the peroxidative activity of CPO-P is not the basis for antifungal activity or enhanced resistance in transgenic plants expressing the gene.

Respirable cotton dust, implicated in the pathogenesis of byssinosis, contains a number of bioactive compounds. These include lipopolysaccharide (LPS), tannins, bacterial peptides, byssinosin, iacinilene C, and 1,3-beta-D-glucan. The exact aetiological agent of byssinosis in such dust has not been definitively identified nor has its mechanism of action on lower lung surfaces been determined. In the present study 1,3-beta-D-glucan, Enterobacter agglomerans LPS, and ovine pulmonary surfactant were mixed in varying combinations. After incubation, their characteristics were determined by sucrose density centrifugation, TLC, and carbohydrate analysis. Precipitates were found in mixtures containing surfactant-glucan and surfactant-glucan-LPS, but not in surfactant-LPS. Precipitates were not seen in the surfactant, LPS, and glucan controls. The formation of a precipitate did not increase the density of the surfactant glucan mixture when compared by density gradient centrifugation with the surfactant control. The interaction between surfactant and glucan was analysed by molecular modelling. The energy of a surfactant-glucan complex (60.07 kcal/mol) was calculated to be much lower than the sum of glucan (47.09 kcal/mol) and surfactant (30.98 kcal/mol) when added separately. The results indicate that 1,3-beta-D-glucan does interact with surfactant and this complex may play a part in the pathogenesis of byssinosis by altering lung physiology maintained by pulmonary surfactant.

Twelve fatty acid derivatives of glycolic acid containing erucic acid, or other selected vegetable oil fatty acids, were synthesized. These were screened for antimicrobial activity against a gram-positive bacterium,Staphylococcus aureus; a gram-negative bacterium,Escherichia coli; a mold,Penicillium notatum, and a yeast,Candida utilis. All of the compounds inhibited at least one of these organisms. These compounds were derivatives of glycolic acid prepared by esterification of the carboxyl function of glycolic acid with a long-chain fatty alcohol and reaction of the hydroxyl function of glycolic acid with a long-chain fatty acyl group.