Aflatoxigenic Aspergillus parasiticus

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1994, p /94/$ Vol. 60, No. 10 Effects of Neem Leaf Volatiles on Submerged Cultures of Aflatoxigenic Aspergillus parasiticus H. J. ZERINGUE, JR.,* AND D. BHATNAGAR Southem Regional Research Center, Agricultural Research Service, U.S. Department ofagriculture, New Orleans, Louisiana Received 23 March 1994/Accepted 28 July 1994 Microbe-free compressed air was passed continuously for a 3-day test period through an enclosed system containing fresh neem leaves; the resultant emitted volatiles were passed over the surface of submerged liquid cultures of a wild-type aflatoxigenic isolate of AspergiUlus parasiticus. Aflatoxin determinations for the fungal culture that received neem-derived volatiles, after a 3-day incubation period, resulted in a 90% overall reduction in aflatoxin production and a 51% reduction in fungal biomass when compared with cultures that did not receive neem volatiles. In a separate experiment but in a similarly enclosed system, volatiles from fresh neem leaves were collected on a small Tenax column and were thermally desorbed and cryogenically focused on a capillary gas chromatography column. The neem volatiles were subsequently separated and identified by gas chromatography-mass spectrometry. Sixty-eight compounds were identified by comparison of retention times and mass spectra with either authentic compounds or spectra from a computer-assisted library database of mass spectra. It was found that 10%o of the total headspace volatiles were composed of C3 to C9 alkenals, which are toxic to aflatoxigenic Aspergillus spp., which could explain the bioactivity that resulted in reduced biomass in the neem-treated cultures. Neem, Azadirachta indica A. Juss (Meliaceae) is a subtropical tree native to the drier regions of Asia and Africa. Components taken from the neem tree (bark, leaves, and seeds) have demonstrated an unusual effectiveness against a wide spectrum of pests (insects, fungi, and viruses) (13, 15). Chemical components, mostly tetranortriterpenoids, from neem tree parts have been identified as active principles involved with this effectiveness toward pests. Tetranortriterpenoids found in neem tree seeds have been reported to be active as insect feeding deterrents, toxicants, and/or disruptants of growth and development against a variety of insects and nematodes (13). AflatoxigenicAspergillus spp. produce secondary metabolites called aflatoxins, which are carcinogenic to both humans and animals (5, 7). Aflatoxigenic Aspergillus spp. infection is found in many commercially important food and/or feed crops (corn, cotton, peanuts, and tree nuts). We demonstrated earlier that nonvolatile neem leaf extracts in in vitro studies inhibit aflatoxin biosynthesis in the early stages of the biosynthetic pathway (2, 4). Injection of first the neem leaf extract and then an aflatoxigenic Aspergillus flavus spore suspension onto the carpel surface of developing cotton bolls did not affect fungal growth in the bolls, but the cotton seeds from the locules exhibited almost total inhibition in aflatoxin production (18). As a continuation of our studies on the bioactivity of neem tree components, the purpose of the current study was to determine and identify those volatile components of neem leaves that can affect the production of aflatoxin in aflatoxigenic strains of Aspergillus parasiticus. * Corresponding author. Mailing address: USDA, ARS, SRRC, P.O. Box 19687, New Orleans, LA Phone: (504) Fax: (504) MATERUILS AND METHODS Fungal growth conditions and culture. A wild-type aflatoxigenic isolate of A. parasiticus designated SRRC 143 was cultured on potato dextrose agar in petri plates. Spores were harvested from cultures after 6 days of incubation at 28 C, and a spore suspension was prepared with 2.6 x 109 spores per ml in sterile deionized water with 1% Triton X-100. To prepare cultures for neem leaf volatile evaluations, 1 ml of the A. parasiticus suspension (2.6 x 109 spores) was added to 200 ml of Adye and Mateles growth medium (1) contained in 1.2-liter glass storage bottles (Kontes K ) fitted with Teflon inlet and outlet valves; the inlet tube extended to 2 cm above the level of the culture medium. The cultures were incubated in a static condition at 28 C C. Neem leaf treatment and volatile transfer apparatus. Fresh neem leaves were provided by R. J. Knight, Jr., USDA-ARS Subtropical Horticulture Research Station (Miami, Fla.). The surfaces of detached leaves with their attached petioles were sterilized by immersion in 10% sodium hypochlorite for 30 s followed by thorough rinsing in sterile deionized water. Twenty grams of surface-sterilized neem leaves were positioned in each of three separate sterilized 150-mm-internal-diameter Wheaton dry-seal desiccators equipped with desiccator covers containing sleeve valves as described earlier for use with cotton leaves (19). Sterile techniques were used in positioning the leaves within the desiccators. The petiole of each leaf was placed through a hole of the porcelain desiccator plate, and the basal cut areas of the petioles were immersed in sterile Hoagland's plant nutrient solution (9). Microbe-filtered compressed air passed through desiccators containing the neem leaves, and by means of glass tubing interconnected with short pieces of Tygon tubing, volatiles were transferred over the surfaces of static cultures of A. parasiticus. Each of the desiccators containing neem leaves or the desiccator containing only plant nutrient solution (the control) was connected by a three-way adapter to three separate A. parasiticus cultures contained in 1.2-liter solvent storage bottles. The culture- 3543

2 3544 ZERINGUE AND BHATNAGAR APPL. ENVIRON. MICROBIOL. TABLE 1. Effect of neem leaf volatiles on A. parasiticus growtha and aflatoxin B, production in submerged culture Biomass Aflatoxin B1 Treatment Meanb(wg) Wt of control Per g of mycelia Total in cultures (,ug) % of control of (g) ± SD ± SD total in cultures AC 0.54 ± b 79.3 ± B` 0.73 ± b Controld " ± "Determined after 3 days of incubation. "Mean of at least three separate sets in each treatment. c Treatments A and B were identical, each consisting of volatiles from 20 g of neem leaves directed to three separate cultures of A. parasiticus. d Same as treatments A and B, except there were no neem leaves in the dry-seal desiccator. containing bottles were fitted with Teflon vacuum valves and inlet and outlet tubes. Neem leaf volatiles and volatiles from control desiccators were purged to the surfaces of A. parasiticus cultures continuously for 3 days at a flow rate of 2 cm3/min. Measurement of fungal biomass and extraction and assay of aflatoxins. After a 3-day incubation, mycelia and medium were extracted with aqueous acetone and then with methylene chloride (3). Aflatoxins were separated on silica gel thin-layer chromatography plates in ether-methanol-water (96:3:1, vol/ vol/vol). The toxins were quantitated by fluorometric scans (360 nm) of thin-layer chromatography plates containing the extracted samples and comparison with aflatoxin standards run on the same plates (20). Collection, separation, and identification of major volatiles emitted from neem leaves. Twenty grams of fresh neem leaves was placed in Wheaton dry-seal desiccators, as already described. Volatiles were purged by microbe-filtered compressed air from the neem leaf-containing desiccators onto small Tenax glass traps (0.1 g; Tenax GC; 60/80 mesh) packed between plugs of glass wool in tubes (84 by 9 mm; 7-mm internal diameter). Air was purged through the system at a flow rate of 20 cm3/min, and the volatiles were collected for 1-h periods. The Tenax tubes were loaded into an external inlet device (Scientific Instrument Service, River Ridge, La.) which was interfaced to an HP-5890A-5971A gas chromatography-mass spectrometry system. Neem volatiles were thermally desorbed at 1 10 C for 3 min at a helium flow rate of 20 cm3/min from the Tenax tubes and were cryogenically focused (-30 C) on a 50-m, Hewlett-Packard HP-5 cross-linked 5% PhMe Silicone capillary column. The injector valve on the external inlet device was then switched to the vent position, helium linear velocity was adjusted to 30 cm3, the oven temperature was raised at a rate of 15 C/min to 250 C, and that temperature was maintained for 5 min. The separated components were scanned from 10 to 650 atomic mass units at a threshold of 500 on the HP 5971A mass selector detector. Data acquisition was accomplished with an HP G1034A MS Chem Station program, and separated unknown peak identifications were based on retention times and mass spectra of authentic compounds or comparisons with the HP G1035A AA9 Wiley 130 K Mass Spectra database (16). Quantitative analyses of the peak areas of 2,3-butanediol, 1-heptanol, 4-pentenal, and trans-2-heptenal were obtained from calibration curves computer generated by the HP G1034A MS Chem Station program, using authentic samples. RESULTS The mean results of two identical treatments (A and B; Table 1) exposing A. parasiticus cultures to volatiles derived from fresh neem leaves yielded a 51 % reduction in fungal biomass and a 79% reduction in aflatoxin production per mg of mycelia, which yielded a combined reduction in aflatoxin of 90%. To study which volatiles might be responsible for the bioactivity demonstrated by fresh neem leaves, headspace volatiles of these leaves were trapped on small glass columns of Tenax. Gas chromatography-mass spectrometry separations and identifications of the organic volatiles trapped on Tenax from fresh neem leaves resulted in the identification of 68 major compounds (Table 2). The compounds were principally alcohols, aldehydes, hydrocarbons, ketones, and miscellaneous compounds, including terpenes, styrene, sulfur-containing compounds, and 2,5-dihydro-furan. A previously reported aflatoxigenic A. flavus time- and concentration-dependent assay (20) was utilized to compare the bioactivity of neem leaf components identified by gas chromatography-mass spectrometry with individually selected, commercially obtained volatile compounds. Recently, this assay (20) was repeated with the same commercially obtained volatile compounds with aflatoxigenic A. parasiticus (SRRC 143). The A. parasiticus-containing assay demonstrated results similar to those reported earlier for the aflatoxigenic A. flavus bioassay. The previously reported aflatoxigenic Aspergillus time- and concentration-dependent assay (20) was also expanded with A. parasiticus (SRRC 143) to explain lesser dose amounts of reference volatile compounds on the growth and aflatoxin production of the fungus and thereby correlate chromatographic results of emitted volatiles with bioassay results (Table 3). The following volatile components were separated and identified, and concentrations were determined by procedures outlined in Materials and Methods. Reported concentrations represent the amounts collected over the 1-h sampling periods. Alcohols (23% of total peak area). A variety of C2 to C7 alcohols (primary, secondary, saturated, unsaturated, and branched chain), 2-ethyl-phenol, and cyclopentanol were identified (Table 2). 2,3-Butanediol (21.8 iim), representing 9.7% of the total peak area, was followed by 1-heptanol (18.4 F.M) at 5.4% of the total peak area. In a related time- and concentration-dependent bioassay (20), we reported that 1-heptanol reduced radial fungal growth by 38% in potato dextrose agar petri solid-culture plates and aflatoxin production by 56% when an aflatoxigenic A. jiavus strain was exposed to an atmosphere containing 70,uM 1-heptanol. In the same study, 70 p.m 3-hepten-1-ol reduced A. flavus radial growth by 38% and aflatoxin production by 45%. However, as demonstrated by data reported in Table 3, 2.5 to 10.0 ipm 2,3-butanediol or 1.8 to 7.0 F.M 1-heptanol had little effect on the growth or aflatoxin production of A. parasiticus. Aldehydes (10% of total peak area). C2, C5, C6, C7, and C9 (all unsaturated) aldehydes were detected (Table 2). 4-Pentenal (15.1,uM; 3.8% of total peak area) and trans-2-heptenal (12.5 p.m; 2.6% of total peak area) were separated and

3 VOL. 60, 1994 NEEM LEAF VOLATILE EFFECTS ON A. PARASITICUS CULTURES 3545 TABLE 2. Headspace volatiles from fresh neem leaves Class and compound RRF % Peak" Alcohols 2-Methyl-2-propen-1-ol 2-Propen-1-ol 2,3-Butanediol 1-Butanol 2-Buten-1-ol 2-Methyl-1-pentanol 3-Methyl-2-hexanol 3-Hexen-1-ol 2,4-Hexadiene-1-ol 3,5,5-Trimethyl-1-hexanol 3-Heptanol 1-Heptanol 2-Ethyl-1-hexanol 2-Ethyl-phenol Cyclopentanol Hydrocarbons Methoxyethane 2-Methyl-1-propene Butane 2,3-Dimethyl-1,3-butadiene 2,2,3,4-Tetramethyl-pentane 1,5-Hexadiene 2,4-Dimethyl-hexane 2-Methyl-hexane 3,4-Dimethyl-heptane 2,2-Dimethyl-3-heptene 5-Ethyl-2-methyl-heptane Octane 1-Octene 2,5,6-Trimethyl-octane 2,5-Octadiene (E) 4-octene Nonane 1-Nonene 2,6,7-Trimethyl-decane 3,4-Dimethyl-1-decene trans-ociene 2-Methyl-undecane Dodecane 4-Methyl-octadecane Ketones 1-Phenyl-ethanone 2-Propanone 2,3-Butanedione 3-Hydroxy-2-butanone 2-Pentanone 3-Pentanone 1-Penten-3-one 2-Hexanone 3-Heptanone 3-Methyl-2-heptanone 4-Methyl-6-hepten-3-one 6-Dodecanone 5-Hexyldihydro-2(3H)-furanone Aldehydes 2-Methyl-2-propenal 4-Pentenal 2,2-Dimethyl-3,4-pentadienal 3,4-Pentadienal trans-2-heptenal 4-Heptenal 2-Isononenal n ck Continued TABLE 2-Continued Class and compound RRT pear Miscellaneous Styrene o-cubebene trans-caryophyllene Sesquiterpene Methoxy-5-methyl-thiophene ,5-Dihydro-furan tert-dodecanethiol Hydroxy-propanoic acid Hydroxy-ethyl ester propanic acid a RRT, retention time relative to that of styrene (retention time of compound/ retention time of styrene in minutes from injection). b Data are expressed as percentages of the total peak area. identified and represented the predominant peak areas in this class of compounds. In an earlier bioassay (20), it was observed that all tested C6 to Cg monounsaturated aldehydes (60 to 86,uM) completely inhibited A. flavus radial growth and consequently aflatoxin production. In the current study, a 10,uM concentration of 4-pentenal reduced radial growth by 26.4% and reduced aflatoxin production by 92.7% (Table 3). Also in this study (Table 3), a 7.6,uM concentration of trans-2- heptenal reduced radial growth by 23.2% and reduced aflatoxin production by 89.5%. Ketones (43% of total peak area). A variety of C2 to C12, 2- and 3-alkanones [saturated, unsaturated, and substituted; phenyl-ethanone; and 5-hexyldihydro-2(3H)-furanone] were detected. This class of compounds represented the largest class of the total peak areas identified. 3-Hydroxy-2-butanone (20% of the total peak area) and 2-propanone (13% of the total peak area) were the predominant ketones identified. 2-Heptanone (71,uM)- and 3-heptanone (96,uM)-treated cultures had both shown slightly reduced aflatoxin production (7.3 and 17%, respectively) in an earlier bioassay (20). Hydrocarbons (11%) and miscellaneous compounds (14% of total peak area). A variety of hydrocarbons were identified: (E) 4-octene (1.6% of total peak area) and 1-nonene (0.9% of total peak area) represented the predominant compounds in this class. tert-dodecanethiol (9.2% of the total peak area) and 5-methyl-2-methoxy-thiophene (1.0% of total peak area) constituted the predominant compounds in the miscellaneous class of compounds identified. Sulfur-containing volatiles from neem leaves have not been reported earlier. However, thionimone (a sulfur-containing component) has been obtained from neem seeds and was found to be highly inhibitory to the growth of Fusanium oxysporum f. lycopersici Schlecht (11). 2,5-Dihydro-furan (0.5% of total peak area) probably represented a fragment of the azadirachtin molecule (10). The azadirachtins are the most interesting from both commercial and biological points of view of the tetranortriterpenoids from neem. The biological activities of isomeric forms of these compounds ranged from insect phagorepellent to insect growth inhibitors (13). DISCUSSION U.28 Specific volatiles from fresh neem leaves were found to have significant effects on fungal growth and aflatoxin production in submerged cultures of aflatoxigenica. parasiticus (SRRC 143). Both fungal growth and aflatoxin bioassay results with whole

4 3546 ZERINGUE AND BHATNAGAR TABLE 3. Radial growth and aflatoxin B1 production of A. parasiticus (SRRC 143)a as percentages of control results after 5 days in atmospheres containing selected volatile compounds Volatile Concn (1±M) Radial growthd Aflatoxin B1 component testedb of tested (%) productionda (%) component 2,3-Butanediol ± ± ± ± ± ± Heptanol ± ± ± ± ±3 98.4±2.4 4-Pentenal ± ± ± ± ± ± 4.8 trans-2-heptenal ± ± ± ± ± ± 4.1 Hexanal ± ± ± ± ± 2.8 Octanal ± ± ± ± 2.1 a The initial inoculum was 50,tl of a spore suspension ofa. parasiticus (SRRC 143 containing 1.2 x 108 spores per ml) applied to a 10-mm potato dextrose agar extracted center well in a petri plate assay (20). b The tested volatile component was added to a 1-ml glass beaker positioned along the margin edge of the bottom lid of the petri plate in the petri plate bioassay (20). c Initial concentration of component applied to petri plate bioassay (20). d Results are the means ± standard deviations expressed as percentages of control results for three replicates per tested level. Control values were 69.7 (± 1.1) mm for growth and 2,072 (t 21) ng for aflatoxin B1 production. eaflatoxin B1 was extracted from 20 g of potato dextrose agar medium per petri plate, purified by thin-layer chromatography, and quantified by fluorescence, as described in Materials and Methods. The limit of detection of aflatoxin B1 was 1 ng/,ul. leaves were found to be different from our earlier results with blended and boiled neem leaf extracts (2, 4). Bhatnagar et al. (2, 4) exposed volatiles from aqueous blended neem leaf extracts toa. parasiticus (SRRC 143) cultures growing on agar medium at a ratio of 1:5 (vol/vol) of the neem extract to the agar medium. They reported that volatiles from blended neem leaf extract did not affect either aflatoxin synthesis or fungal growth after 4 days of incubation on the agar medium. When the blended neem extract was added directly to submerged cultures of A. parasiticus at concentrations greater than 10% (vol/vol), no effect on fungal growth (i.e., mycelial dry weight) was found but aflatoxin biosynthesis was essentially blocked (>98%). In this study, we have observed that fresh neem leaves contain volatile components (particularly highly antifungal C3 to Cg monounsaturated aldehydes) that were not present in the blended neem leaf extracts and were probably lost because of their high volatility and possible inactivation with released amino acid groups during maceration or boiling of neem leaves in preparation of the extracts in our earlier study (2, 4). Also after boiling the leaves, we were not able to detect 4-pentenal and trans-2-heptenal which had been added to a neem leaf extract prior to the boiling extraction procedure (2, 4). The reported gas-liquid chromatograph of the volatile profile of the blended neem leaf extract (2, 4) demonstrated only one compound common to the total-ion chromatograph of the volatile profile of the fresh neem leaf, namely, 2,3- butanedione. No ketone bioactivity was found for an aflatoxigenic strain of A. flavus in the time- and concentrationdependent bioassay of the earlier study (20) or in this study, in APPL. ENVIRON. MICROBIOL. an identical bioassay in the presence of an aflatoxigenic strain of A. parasiticus (SRRC 143). In summary, neem leaves contain specific volatile compounds that have known fungicidal properties; a complex mixture of volatiles was found to affect both fungal growth and aflatoxin production in A. parasiticus. C2, C5 to C7, and Cg alkenals representing 10% of the peak area of the total-ion chromatogram from organic volatiles trapped on Tenax from fresh neem leaves were observed in this study. In our earlier studies (17, 20) as well as in those of others (8; for reviews, see references 6 and 12), it has been observed that these smallchain "gaseous" alkanals and alkenals exhibit strong fungicidal properties. At specific concentrations, we observed some fungicidal effects and a corresponding decrease in aflatoxin levels. But, at specific concentrations we also observed a minor reduction in fungal growth with significant aflatoxin reduction, an effect that may be due to the action of the aldehyde on a surface-localized enzyme involved in aflatoxin biosynthesis (14). In all probability, the compounds responsible for the bioactivity demonstrated in the current research effort resulted from these small-chain gaseous aldehydes. ACKNOWLEDGMENTS We thank R. Knight, USDA-ARS Subtropical Horticulture Research Station, for supplying us with the neem leaves and J. L. Bennett for excellent technical assistance. REFERENCES 1. Adye, J., and R. I. Mateles Incorporation of labelled compounds into aflatoxins. Biochim. Biophys. Acta 86: Bhatnagar, D., and S. P. McCormick The inhibitory effect of neem (Azadirachta indica) leaf extracts on aflatoxin synthesis in Aspergillus parasiticus. J. Am. Oil Chem. Soc. 65: Bhatnagar, D., S. P. McCormick, L. S. Lee, and R. A. Hill Identification of O-methylsterigmatocystin as an aflatoxin B, and G1 precursor in Aspergillus parasiticus. Appl. Environ. Microbiol. 53: Bhatnagar, D., H. Zeringue, and S. P. McCormick Neem leaf extracts inhibit aflatoxin biosynthesis in Aspergillus flavus and A. parasiticus, p In Neem's potential in pest management programs. Proceedings of the USDA workshop, April 16 to 17, 1990, Beltsville, Md. USDA/ARS publication 86. U.S. Department of Agriculture, Washington, D.C. 5. Busby, W. F., and G. N. Wogan Food-borne mycotoxins and alimentary mycotoxicosis, p In H. P. Reimann and F. L. Bryan (ed.), Food-borne infections and intoxications, 2nd ed. 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5 VOL. 60, 1994 NEEM LEAF VOLATILE EFFECTS ON A. PARASITICUS CULTURES Rodriguez, S., and N. E. Mahoney Inhibition of aflatoxin production by surfactants. Appl. Environ. Microbiol. 60: Singh, U. P., H. B. Singh, and R. B. Singh The fungicidal effect of neem (Azadirachta indica) extracts on some soil-borne pathogens of gram (Cicea arietinumn). Mycologia 72: Wiley & Sons, Inc Wiley 13 K mass spectral data base. John Wiley & Sons, Inc., New York. (Licensed to Hewlett-Packard Company.) 17. Zeringue, H. J., Jr Effect of C6 to C9 alkenals on aflatoxin production in corn, cottonseed, and peanuts. AppI. Environ. Microbiol. 57: Zeringue, H. J., Jr., and D. Bhatnagar Inhibition of aflatoxin production in Aspergillusflavus infected cotton bolls after treatment with neem (Azadirachta indica) leaf extracts. J. Am. Oil Chem. Soc. 67: Zeringue, H. J., Jr., and S. P. McCormick Relationships between cotton leaf-derived volatiles and growth of Aspergillus flavus. J. Am. Oil Chem. Soc. 66: Zeringue, H. J., Jr., and S. P. McCormick Aflatoxin production in cultures of Aspergillus flavus incubated in atmospheres containing selected cotton leaf-derived volatiles. Toxicon 28: