APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1994, p. 3543-3547 Vol. 60, No.

10
0099-2240/94/$04.00+0

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 of Agriculture,
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 subtrop- MATERUILS AND METHODS

ical 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. Tetranortriterpe-
noids 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 afla-
toxin 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 70179-0687. Phone: (504) 286-4379.
Fax: (504) 286-4419.
3543
Fungal growth conditions and culture. A wild-type aflatoxi-
genic 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-323255) 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 + 0.5°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 com-
pressed 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 contain-
ing 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-
3544 ZERINGUE AND BHATNAGAR
TABLE 1. Effect of neem leaf volatiles on A. parasiticus growtha and aflatoxin B, production in submerged culture
Biomass
Treatment Meanb (wg)
Wt of control Per g of mycelia
(g) ± SD
AC 0.54 ±0.01 41.6
B` 0.73 ± 0.03 55.8
Controld 1.31 + 0.01 100
"Determined after 3 days of incubation.
"Mean of at least three separate sets in each treatment.
cTreatments 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. parasiti-
cus 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 de-
scribed. 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
APPL. ENVIRON. MICROBIOL.
Aflatoxin B1
Total in cultures (,ug) % of control of
± SD total in cultures
145.6b 79.3 ± 2.8 8.6
157.5b 115.0 + 12.7 12.4
706.9" 923.6 ± 12.2 100
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 as-
say (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 ex-
panded 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 iden-
tified (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 concentra-
tion-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-Pente-
nal (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
VOL. 60, 1994 NEEM LEAF VOLATILE EFFECTS ON A. PARASITICUS CULTURES 3545

TABLE 2. Headspace volatiles from fresh neem leaves TABLE 2-Continued

Class and compound RRF %
Peak" Class and compound RRT pear
Alcohols Miscellaneous
2-Methyl-2-propen-1-ol 1.248 0.11 Styrene 1.000 0.45
2-Propen-1-ol 0.967 0.47 o-Cubebene 1.814 0.41
2,3-Butanediol 0.825 9.70 trans-Caryophyllene 1.860 0.74
1-Butanol 0.695 0.64 Sesquiterpene 1.947 0.48
2-Buten-1-ol 1.126 0.32 2-Methoxy-5-methyl-thiophene 2.005 1.04
2-Methyl-1-pentanol 1.516 0.28 2,5-Dihydro-furan 0.827 0.49
3-Methyl-2-hexanol 0.789 0.48 tert-Dodecanethiol 1.121 9.16
3-Hexen-1-ol 0.993 1.61 2-Hydroxy-propanoic acid 0.609 0.54
2,4-Hexadiene-1-ol 1.229 0.65 2-Hydroxy-ethyl ester propanic 0.887 0.41
3,5,5-Trimethyl-1-hexanol 1.243 1.17 acid
3-Heptanol 1.012 0.74 a RRT, retention time relative to that of styrene (retention time of compound/
1-Heptanol 1.022 5.43
2-Ethyl-1-hexanol 1.274 0.23 retention time of styrene in minutes from injection).
2-Ethyl-phenol 1.497 0.42 b Data are expressed as percentages of the total peak area.
Cyclopentanol 0.573 0.25
Hydrocarbons
Methoxyethane 0.880 0.81 identified and represented the predominant peak areas in this
2-Methyl-1-propene 0.679 0.35 class of compounds. In an earlier bioassay (20), it was observed
Butane 0.991 0.49 that all tested C6 to Cg monounsaturated aldehydes (60 to 86
2,3-Dimethyl-1,3-butadiene 0.527 0.12 ,uM) completely inhibited A. flavus radial growth and conse-
2,2,3,4-Tetramethyl-pentane 1.154 0.63 quently aflatoxin production. In the current study, a 10 ,uM
1,5-Hexadiene 1.196 0.58 concentration of 4-pentenal reduced radial growth by 26.4%
2,4-Dimethyl-hexane 1.422 0.38 and reduced aflatoxin production by 92.7% (Table 3). Also in
2-Methyl-hexane 1.501 0.16
3,4-Dimethyl-heptane 1.306 0.13 this study (Table 3), a 7.6 ,uM concentration of trans-2-
2,2-Dimethyl-3-heptene 1.480 0.36 heptenal reduced radial growth by 23.2% and reduced afla-
5-Ethyl-2-methyl-heptane 1.491 0.11 toxin production by 89.5%.
Octane 1.392 0.80 Ketones (43% of total peak area). A variety of C2 to C12, 2-
1-Octene 1.324 0.41 and 3-alkanones [saturated, unsaturated, and substituted; phe-
2,5,6-Trimethyl-octane 1.324 0.11 nyl-ethanone; and 5-hexyldihydro-2(3H)-furanone] were de-
2,5-Octadiene 1.108 0.68 tected. This class of compounds represented the largest class of
(E) 4-octene 1.153 1.61 the total peak areas identified. 3-Hydroxy-2-butanone (20% of
Nonane 1.473 0.32 the total peak area) and 2-propanone (13% of the total peak
1-Nonene 1.488 0.94
2,6,7-Trimethyl-decane 1.973 0.80 area) were the predominant ketones identified. 2-Heptanone
3,4-Dimethyl-1-decene 1.489 0.07 (71 ,uM)- and 3-heptanone (96 ,uM)-treated cultures had both
trans-Ociene 1.089 0.46 shown slightly reduced aflatoxin production (7.3 and 17%,
2-Methyl-undecane 1.495 0.28 respectively) in an earlier bioassay (20).
Dodecane 1.371 0.13 Hydrocarbons (11%) and miscellaneous compounds (14% of
4-Methyl-octadecane 1.367 0.23 total peak area). A variety of hydrocarbons were identified:
(E) 4-octene (1.6% of total peak area) and 1-nonene (0.9% of
Ketones total peak area) represented the predominant compounds in
1-Phenyl-ethanone 1.333 1.58 this class. tert-Dodecanethiol (9.2% of the total peak area) and
2-Propanone 0.511 12.84
2,3-Butanedione 0.389 1.44 5-methyl-2-methoxy-thiophene (1.0% of total peak area) con-
3-Hydroxy-2-butanone 0.639 19.67 stituted the predominant compounds in the miscellaneous
2-Pentanone 0.550 0.12 class of compounds identified. Sulfur-containing volatiles from
3-Pentanone 0.569 0.23 neem leaves have not been reported earlier. However, thio-
1-Penten-3-one 0.780 1.52 nimone (a sulfur-containing component) has been obtained
2-Hexanone 0.991 1.27 from neem seeds and was found to be highly inhibitory to the
3-Heptanone 0.987 0.34 growth of Fusanium oxysporum f. lycopersici Schlecht (11).
3-Methyl-2-heptanone 1.417 0.26 2,5-Dihydro-furan (0.5% of total peak area) probably repre-
4-Methyl-6-hepten-3-one 1.497 1.68 sented a fragment of the azadirachtin molecule (10). The
6-Dodecanone 1.973 0.94
5-Hexyldihydro-2(3H)-furanone 2.017 0.32 azadirachtins are the most interesting from both commercial
and biological points of view of the tetranortriterpenoids from
Aldehydes neem. The biological activities of isomeric forms of these
2-Methyl-2-propenal 1.114 0.27 compounds ranged from insect phagorepellent to insect
4-Pentenal 1.027 3.83 growth inhibitors (13).
2,2-Dimethyl-3,4-pentadienal 1.296 2.05
3,4-Pentadienal 0.767 0.29
trans-2-Heptenal 1.548 2.60 DISCUSSION
4-Heptenal 1.264 n cK
2-Isononenal 1.085 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).
Continued Both fungal growth and aflatoxin bioassay results with whole
3546 ZERINGUE AND BHATNAGAR APPL. ENVIRON. MICROBIOL.

TABLE 3. Radial growth and aflatoxin B1 production of A. an identical bioassay in the presence of an aflatoxigenic strain
parasiticus (SRRC 143)a as percentages of control results after 5 of A. parasiticus (SRRC 143).
days in atmospheres containing selected volatile compounds In summary, neem leaves contain specific volatile com-
pounds that have known fungicidal properties; a complex
Volatile Concn (1±M) Radial growthd Aflatoxin B1 mixture of volatiles was found to affect both fungal growth and
component testedb of tested
component
(%) productionda (%)
aflatoxin production in A. parasiticus. C2, C5 to C7, and Cg
2,3-Butanediol 10.0 92 ± 5 90.1 ± 1.5 alkenals representing 10% of the peak area of the total-ion
5.0 96 ± 3 97.3 ± 2.6 chromatogram from organic volatiles trapped on Tenax from
2.5 98 ± 2 102.8 ± 3.4 fresh neem leaves were observed in this study. In our earlier
1-Heptanol 7.0 136 ± 3 92.0 ± 3.2 studies (17, 20) as well as in those of others (8; for reviews, see
3.5 124 ± 5 98.3 ± 1.2 references 6 and 12), it has been observed that these small-
1.8 115±3 98.4±2.4 chain "gaseous" alkanals and alkenals exhibit strong fungicidal
4-Pentenal 10.0 73.6 ± 3 7.3 ± 2.8 properties. At specific concentrations, we observed some fun-
5.0 82.5 ± 2 47.3 ± 3.5 gicidal effects and a corresponding decrease in aflatoxin levels.
2.5 85.3 ± 2 54.8 ± 4.8 But, at specific concentrations we also observed a minor
trans-2-Heptenal 7.6 76.8 ± 5 10.5 ± 1.8 reduction in fungal growth with significant aflatoxin reduction,
3.5 87.5 ± 3 66.7 ± 3.5
1.8 88.9 ± 3 74.9 ± 4.1 an effect that may be due to the action of the aldehyde on a
Hexanal 8.3 61 ± 4 5.8 ± 3.2 surface-localized enzyme involved in aflatoxin biosynthesis
4.1 68 3 36.0 ± 1.5 (14). In all probability, the compounds responsible for the
2.0 76 ± 2 56.4 ± 2.8 bioactivity demonstrated in the current research effort resulted
Octanal 6.5 54 4 11.1 ± 4 from these small-chain gaseous aldehydes.
3.3 61 ± 5 22.1 ± 3.6
1.7 71 5 28.9 ± 2.1 ACKNOWLEDGMENTS
a
The initial inoculum was 50 ,tl of a spore suspension of A. parasiticus (SRRC We thank R. Knight, USDA-ARS Subtropical Horticulture Re-
143 containing 1.2 x 108 spores per ml) applied to a 10-mm potato dextrose agar search Station, for supplying us with the neem leaves and J. L. Bennett
extracted center well in a petri plate assay (20). for excellent technical assistance.
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). REFERENCES
c Initial concentration of component applied to petri plate bioassay (20).
d Results are the means ± standard deviations
1. Adye, J., and R. I. Mateles. 1964. Incorporation of labelled
expressed as percentages of compounds into aflatoxins. Biochim. Biophys. Acta 86:418-420.
control results for three replicates per tested level. Control values were 69.7 (± 2. Bhatnagar, D., and S. P. McCormick 1988. The inhibitory effect of
1.1) mm for growth and 2,072 (t 21) ng for aflatoxin B1 production. neem (Azadirachta indica) leaf extracts on aflatoxin synthesis in
eAflatoxin B1 was extracted from 20 g of potato dextrose agar medium per
petri plate, purified by thin-layer chromatography, and quantified by fluores- Aspergillus parasiticus. J. Am. Oil Chem. Soc. 65:1166-1168.
cence, as described in Materials and Methods. The limit of detection of aflatoxin 3. Bhatnagar, D., S. P. McCormick, L. S. Lee, and R. A. Hill. 1987.
B1 was 1 ng/,ul. Identification of O-methylsterigmatocystin as an aflatoxin B, and
G1 precursor in Aspergillus parasiticus. Appl. Environ. Microbiol.
53:1028-1033.
4. Bhatnagar, D., H. Zeringue, and S. P. McCormick. 1990. Neem
leaves were found to be different from our earlier results with leaf extracts inhibit aflatoxin biosynthesis in Aspergillus flavus and
blended and boiled neem leaf extracts (2, 4). Bhatnagar et al. A. parasiticus, p. 118-127. In Neem's potential in pest management
(2, 4) exposed volatiles from aqueous blended neem leaf programs. Proceedings of the USDA workshop, April 16 to 17,
extracts toA. parasiticus (SRRC 143) cultures growing on agar 1990, Beltsville, Md. USDA/ARS publication 86. U.S. Department
medium at a ratio of 1:5 (vol/vol) of the neem extract to the of Agriculture, Washington, D.C.
agar medium. They reported that volatiles from blended neem 5. Busby, W. F., and G. N. Wogan. 1979. Food-borne mycotoxins and
leaf extract did not affect either aflatoxin synthesis or fungal alimentary mycotoxicosis, p. 519-610. In H. P. Reimann and F. L.
Bryan (ed.), Food-borne infections and intoxications, 2nd ed.
growth after 4 days of incubation on the agar medium. When Academic Press, Inc., New York.
the blended neem extract was added directly to submerged 6. Fries, N. 1973. Effects of volatile organic compounds on the
cultures of A. parasiticus at concentrations greater than 10% growth and development of fungi. Trans. Br. Mycol. Soc. 60:1-21.
(vol/vol), no effect on fungal growth (i.e., mycelial dry weight) 7. Groopman, J. D., R. G. Croy, and G. N. Wogan. 1981. In vitro
was found but aflatoxin biosynthesis was essentially blocked reactions of aflatoxin Bl-adducted DNA. Proc. Natl. Acad. Sci.
(>98%). In this study, we have observed that fresh neem USA 78:5445-5499.
leaves contain volatile components (particularly highly antifun- 8. Gueldner, R. C., D. M. Wilson, and A. R. Heidt. 1985. Volatile
gal C3 to Cg monounsaturated aldehydes) that were not compounds inhibiting Aspergillus flavus. J. Agric. Food Chem.
present in the blended neem leaf extracts and were probably 33:411-413.
9. Hoagland, D. R., and D. I. Arnon. 1938. Calif Agric. Exp. Stn.
lost because of their high volatility and possible inactivation Circ. 347:32.
with released amino acid groups during maceration or boiling 10. Jones, P. S., S. V. Ley, E. D. Morgan, and D. Santafianos. 1989.
of neem leaves in preparation of the extracts in our earlier The chemistry of the neem tree, p. 19-45. In M. Jacobson (ed.),
study (2, 4). Also after boiling the leaves, we were not able to Focus on phytochemical pesticides. CRC Press, Inc., Boca Raton,
detect 4-pentenal and trans-2-heptenal which had been added Fla.
to a neem leaf extract prior to the boiling extraction procedure 11. Kahn, M. W., M. M. Alam, A. M. Khan, and S. K. Sarena. 1974.
(2, 4). The reported gas-liquid chromatograph of the volatile Effect of water-soluble fraction of oil-cakes and bitter principles of
profile of the blended neem leaf extract (2, 4) demonstrated neem on some fungi and nematodes. Acta Bot. Indica 2:120-126.
only one compound common to the total-ion chromatograph 12. Nandi, B., and N. Fries. 1976. Volatile aldehydes, ketones, esters
and terpenoids as preservatives against storage fungi in wheat. Z.
of the volatile profile of the fresh neem leaf, namely, 2,3- Pflanzenkr. Pflanzenschutz 83:284-294.
butanedione. No ketone bioactivity was found for an aflatoxi- 13. Rembold, H. 1989. Isomeric azadirachtins and their mode of
genic strain of A. flavus in the time- and concentration- action, p. 47-67. In M. Jacobson (ed.), Focus on phytochemical
dependent bioassay of the earlier study (20) or in this study, in pesticides. CRC Press, Inc., Boca Raton, Fla.
VOL. 60, 1994 NEEM LEAF VOLATILE EFFECTS ON A. PARASITICUS CULTURES
14. Rodriguez, S., and N. E. Mahoney. 1994. Inhibition of aflatoxin
production by surfactants. Appl. Environ. Microbiol. 60:106-110.
15. Singh, U. P., H. B. Singh, and R. B. Singh. 1980. The fungicidal
effect of neem (Azadirachta indica) extracts on some soil-borne
pathogens of gram (Cicea arietinumn). Mycologia 72:1077-1093.
16. Wiley & Sons, Inc. 1986. Wiley 13 K mass spectral data base. John
Wiley & Sons, Inc., New York. (Licensed to Hewlett-Packard
Company.)
17. Zeringue, H. J., Jr. 1991. Effect of C6 to C9 alkenals on aflatoxin
production in corn, cottonseed, and peanuts. AppI. Environ.
Microbiol. 57:2433-2434.
3547
18. Zeringue, H. J., Jr., and D. Bhatnagar. 1990. Inhibition of
aflatoxin production in Aspergillusflavus infected cotton bolls after
treatment with neem (Azadirachta indica) leaf extracts. J. Am. Oil
Chem. Soc. 67:215-216.
19. Zeringue, H. J., Jr., and S. P. McCormick. 1989. Relationships
between cotton leaf-derived volatiles and growth of Aspergillus
flavus. J. Am. Oil Chem. Soc. 66:581-585.
20. Zeringue, H. J., Jr., and S. P. McCormick. 1990. Aflatoxin
production in cultures of Aspergillus flavus incubated in atmo-
spheres containing selected cotton leaf-derived volatiles. Toxicon
28:445-448.