Control of mycotoxin production by chemically inhibiting fungal growth

An effective method to control aflatoxin produced by toxic strains of Aspergillus parasiticus fungi is disclosed. An effective amount of Beta-ionone is applied to said fungi to inhibit the growth and sporulation of the fungi and thereby control production of aflatoxin from the fungi without killing the fungi.

BACKGROUND OF THE INVENTION 
(1) Field of the Invention 
This invention relates to the control of mycotoxins by means of chemical 
treatment. 
(2) Description of the Prior Art 
Heretofore, it has been known that "turkey X" disease has been caused by a 
toxin produced by some strains of the fungi Aspergillus parasiticus. This 
aflatoxin which is produced by strains of A. parasiticus is acutely toxic 
as well as carcinogenic. However, much of the research on aflatoxin dealt 
solely with the detection of aflatoxin and relatively little research has 
been done on the prevention of formation of aflatoxins. 
A review of the control or suppression of fungi producing aflatoxin reveals 
efforts of fumigating with high level dosages of methyl bromide, ethylene 
dibromide, propane/propene ethylene oxide, sulfur dioxide, and phosphine 
and did show some effects of fungicidal activity. Ammonia proved to be 
fungicidal but demonstrated a lack of any residual effect. Propionic, 
acetic, and isobutyric acids also have antifungal activity. However, all 
the above chemicals have the definite disadvantage of toxicity to humans 
and animals, corrosiveness, and lowered nutritional quality. Therefore, 
they are not acceptable to either humans or animals. 
The problem is magnified because fungal invasion of a crop begins in the 
field and either remains or increases during storage. Thus, levels of 
aflatoxin usually increase during storage. 
SUMMARY OF THE INVENTION 
Some strains of Aspergillus parasiticus fungi produce serious amounts of 
aflatoxin in agricultural crops during both preharvest and postharvest 
periods. This invention consists of a method for inhibiting and 
controlling said aflatoxin which is produced by aflatoxin producing 
strains of aspergillus parasiticus fungi and comprises: treating the crop 
and associated fungi with an effective amount of Beta-ionone to inhibit 
and control the growth and sporulation of said fungi without filling the 
fungi, and thus eliminate the aflatoxin which would be produced by the 
fungi is left untreated. Effective amounts of beta-ionone have been 
determined to be about 1 .mu.L to 100 .mu.l. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Aspergillus parasiticus Speare is on deposit with the Agricultural Research 
Culture Collection (NRRL) in Peoria, Illinois, and has been assigned the 
following accession number: NRRL 2999. The address of the Agricultural 
Research Culture Collection (NRRL) is: 
A. J. Lyons, Curator, ARS Patent Collection Culture Collection Research 
NRRC, 1815 N. University Street, Peoria, Ill. 61604.

Beta-ionone has a definite effect on the growth and development of A. 
parasiticus fungi as demonstrated in the following examples: 
EXAMPLE 1 
Asperguillus parasiticus Speare (NRRL 2999)was maintained on potato 
dextrose agar (pda) slants and used in these experiments. The A. 
parasiticus isolate (NRRL 2999)produces aflatoxins. 
Direct contact tests with .beta.-ionone were done using petri dishes 
(15.times.100 mm) containing 20 ml pda and maintained at 26.degree. C. 
Varying amounts (1-20 .mu.l) of .beta.-ionone were pipetted directly onto 
the pda surface just before inoculation. Some of the cultures were placed 
in plastic bags during incubation. Gross observations, diameter 
measurements and microscopic observations were periodically taken. 
Bioassays of volatile effects of .beta.-ionone were also done using pda in 
divided plates. These bioassays were conducted using the following 
procedure: (a) all 4 quadrants were inoculated with stabs of a spore 
suspension; (b) different concentrations of .beta.-ionone (1.50 .mu.l) 
were placed only in 1 quadrant; (c) plates were stored in separate plastic 
bags and incubated at 26.degree. C.; (d) gross observations and diameter 
measurements were recorded after 3, 4, or 5 days of incubation; (e) slides 
were made and microscopic observations recorded. 
Shake cultures of A. parasiticus (NRRL 2999) were used to determine the 
effects of .beta.-ionone on growth and aflatoxin production. The medium 
was prepared by dissolving 50 g Bacto mycological broth w/low pH (Difco), 
15 g sucrose and 2 g yeast extract in 1,000 ml H.sub.2 O. One hundred ml 
of the medium were placed in 125 ml Erlenmeyer flasks and autoclaved. When 
the medium was cool, varying amounts of .beta.-ionone (0.100 .mu.l/flask) 
were pipetted into the flasks. All experiments were replicated at least 4 
times. The cultures were inoculated with a spore suspension of A. 
parasiticus. The flasks were sealed with aluminum foil and rubber bands. 
The cultures were grown on a rotary shaker and dry weights taken at 
1,2,3,4,5,6,7, and 10 days. 
Aflatoxin was determined from 7-day cultures after extraction of the liquid 
medium with CHCl.sub.3. Twenty-five ml, of medium was removed from each 
flask and mixed with 25 ml of saturated sodium chloride solution. This 
mixture was extracted twice with 25 ml CHCl.sub.3. The CHCl.sub.3 layers 
were collected and taken to dryness using a rotary evaporator. The residue 
was suspended in 1 ml CHCl.sub.3 then diluted with 1 ml hexane. The 
suspension was placed on a silica gel Sep-Pak (Water Assoc.) and cluted 
with 5 ml hexane, 5 ml anhydrous ethyl ether, and 3 ml CHCl.sub.3 
/CH.sub.3 OH (90:10). The CHCl.sub.3 /CH.sub.3 OH fraction was collected, 
taken to dryness under N.sub.2 and reconstituted in HPLC mobile phase. The 
aflatoxins were determinied by HPLC using the method of Thean et al. 
RESULTS AND DISCUSSIONS 
Direct contact of A. parasiticus with 1,2.5,5,10,20, and 100 .mu.l of 
.beta.-ionone placed on the surface of pda resulted in severely restricted 
growth and arrested sporulation. The colonies remained light brown-white 
and the growth habit was compact. No sporulation occurred at levels of 5 
.mu.l or above even after 4 weeks' incubation. Sporulation occurred after 
1-2 weeks with 1 and 2.5 pl .mu.l of applied .beta.-ionone. These colonies 
were restricted and many conidial heads were atypical. Frequently, 
vesicles of reduced size were formed, sometimes with irregular sterigmata. 
When mycelial fragments from any treatments (1-100 .mu.l) were transferred 
to fresh pda, normal growth and sporulation ocurred, therefore no fungus 
was killed. 
The volatile effects of .beta.-ionone on opposite or adjacent quadrants 
were somewhat different than the effects of direct contact. One .mu.l of 
.beta.-ionone produced effects only in the quadrant containing 
.beta.-ionone; the growth and sporulation of A. parasiticus was not 
affected in the other quadrants. In plates with 5,10, 15,20,25,30,35,40,45 
and 50 .mu.l .beta.-ionone in 1 quadrant, an increasing effect on growth 
and sporulation in other quadrants was noted in plates containing 5-20 
.mu.l of .beta.-ionone. The effects of increasing from 5 to 20 .mu.l 
included increasing restriction of colony diameters and decreasing levels 
of sporulation after 7 days. Radial growth in plates receiving 20-50 .mu.l 
.beta.-ionone was about half that of the controls. Little sporulation 
occurred after 7 days in any quadrant of these plates as long as they 
remained in unopened bags. 
Direct contact with .beta.-ionone at levels of 1-20 .mu.l, resulted in very 
restricted growth, little or no sporulation, and arrested asexual 
reproductive development. Few, if any, mature conidia were produced. The 
primary thallus consisted of vegetative hyphae and conidiophore initials 
that were atypical or of reduced size. The volatile effects of 
.beta.-ionone were evidenced by morphological changes, growth inhibition, 
and sporulation reduction in adjaent and opposite quadrants. Microscopic 
observations included: 
Reduced size of vesicle and conidiophore diameter, arrested asexual 
reproduction with many immature conidiophores; increased vegetative growth 
when compared to direct contact; atypical distribution of sterigmata, 
similar to direct contact; elongated, irregular sterigmata; atypical 
branching of conidiophores and abnormal conidiophore appearance. These 
effects are concentration-dependent at levels of 1-5 .mu.l/plate for 
direct contact and at levels of 5-20 .mu.l/plate for volatile effects in 
divided plates. 
The effects of .beta.-ionone on growth (dry wt) and aflatoxin synthesis of 
A. parasiticus are given in Table 1. 
TABLE I 
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Effects of .beta.-ionone on Growth and Aflatoxin B.sub.1 
Accumulation In Shake Liquid Cultures of 
Aspergillus parasiticus. 
.beta.-Ionone added 
Dry wt Aflatoxin B.sub.1 
(.mu.l/l) (g).sup.b 
(ng/ml).sup.b 
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0 1.92 9528 
10 1.93 10200 
50 1.50 11240 
100 1.23 2496 
200 1.29 1568 
250 1.02 1368 
300 0.79 176 
400 0.84 280 
500 0.71 16 
1000 0.74 2 
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.sup.b Numbers are averages from four flasks per treatment 
The effects on growth were noticeable beginnin at 50 .mu.l/l of medium. 
Concentrations above 250 .mu.l/l had little further effect on growth. The 
primary effect of .beta.-ionone on growth in shake culture seemed to be on 
the rate of growth; however, sporulation of A. parasiticus in shake or 
submerged culture is inhibited and was not measured. Concentrations of 100 
.mu.l and above of .beta.-ionone/l inhibited aflatoxin accumulations 
whereas 10 and 50 .mu.l/l slightly stimulated aflatoxin production. This 
shows that the ability of the toxigenic strain of A. parasiticus to 
produce aflatoxin is not necessarily linked to growth; but aflatoxin 
synthesis may be positively correlated with the asexual reproductive 
process.