Reprinted irom MYCOLOGIA, Vol. LXXIII, No.2, pp. 216-228, Mar.-Apr. 1981Printed in U. S. A.

AFLATOXIN AND MOLD FLORA IN NORTHCAROLINA IN 1977 CORN CROP

c. W. HESSELTINE, R. F. ROGERS, AND ODETTE L. SHOTWELL

AFLATOXIN AND MOLD FLORA IN NORTHCAROLINA IN 1977 CORN CROP

c. W. HESSELTINE, R. F. ROGERS, AND ODETTE L. SHOTWELL

Northern Regional Research Center, Agricultural Research,Science and Education Administration,

U.S. Department of Agriculture, I

Peoria, Illinois 61604

SUMMARY

A 1977 survey of 238 corn samples from the Piedmont and Coastal Plainarea of North Carolina revealed that 187 samples contained aflatoxin; ofthese, 139 were above the 20 ppb guideline. Aflatoxin G 1 was found in 32samples from the Piedmont/mountain area. The highest level of aflatoxinfound was 3,622 ppb. In general. the higher the percentage of insect-damagedcorn the higher the aflatoxin levels found. The sample with the most insect­damaged corn (11%) showed a level of 2,208 ppb, and all samples with 4.5% ormore insect damage contained aflatoxin. In general, the greater the infectionof corn by Aspergillus flavus the higher the level of aflatoxin, with 7 sampleshaving 40-44 undamaged kernels out of 50 infected. Aspergillus niger wasfound in 10.9% of the samples, but there was no positive correlation with theamounts of aflatoxin found. Strains of Penicillium funiClilosum series iso­lated from South Carolina and reported by us earlier were found again in25.3% of the total kernels examined. Few other Penicillium or Aspergillusspecies were seen. Corn germination was high in most samples. Assumingthat most of the aflatoxin G 1 was produced by A. parasitiClls, we found a def­inite ecological correlation of G 1 with an ecological area, namely the Pied­mont/mountain area.

That aflatoxin forms in corn in the field is now an established fact(1, 3, 5, 10), The occurrence of aflatoxin in corn in the Southern andSoutheastern parts of the United States is a continuing problem andprobably exists wherever corn is grown in the warmer and humidparts of the world, Various surveys made in the Midwest indicate thatthe problem is not a serious one in the Corn Belt (8), except as it ap­plies to the development of aflatoxin from the growth of Aspergillusflavus Link ex Fries in storage (2, 9). The source of the A. flavus co­nidia and the manner of incubation of the corn kernels in the fieldstill have eluded discovery; but, by elimination of infective vehicles, it

I The mention of firm names or trade products does not imply that they are en­dorsed or recommended by the U.S. Department of Agriculture over other firms or sim­ilar products not mentioned.

216

HESSELTINE ET AL.: AFLATOXIN AND MOLD FLORA 217

can be concluded that most of the infection is brought about by inver­tebrates. The species of invertebrates, however, are still to be deter­mined. Other factors involved in the field problem are the amount ofdamage to the kernels, the amount of A. flavus infection, and the in­teraction of A. flavus infection in areas of high aflatoxin contamina­tion with other fungi in the corn kernels, especially fungi that occurin large numbers. We were fortunate to obtain 238 samples of 1977corn from sites scattered over the Coastal Plain and the Piedmont areaof North Carolina for study of the microflora from Dr. W. ]. Dickens(Weaver Laboratory, North Carolina State University, Raleigh, NorthCarolina). This study was made to determine: (a) if aflatoxin was aproblem in North Carolina in the field as we had found it in SouthCarolina in 1973; (b) if the amount of aflatoxin correlated positivelywith the A. flavus infection; and (c) the relation of A. flavus to otherAspergillus and Penicillium species in the corn kernels; and (d) theamount of insect damage.

1\1ATERIALS AND METHODS

Sample collection.-Ten samples (10 Ib each) were collected on twodifferent dates at least I wk apart in September 1977 from 21 countiesin the Coastal Plain and 9 counties in the Piedmont area and moun­tains of North Carolina. All samples were shelled and dried within 24h of collection so that no further A. flavus growth or aflatoxin forma­tion would occur.

About 200 g of kernels was taken from shelled corn from each 10­Ib sample and dried 6 h at 60 C so the fungi would not be destroyed.The remainder of the sample was dried at 90 C, ground, and mixed toobtain 50-g subsamples for aflatoxin analysis.

Analyses of aflatoxin in corn samples.-Subsamples (50 g) of grainwere extracted with 250 ml chloroform, 25 ml water, and 25 g diato­maceous earth for analysis by the CB method (6). Extracts were puri­fied by column chromatography for thin-layer chromatography.Amounts of aflatoxin on thin-layer plates were measured densito­metrically.

Mold studies.-The unground portion of each sample (200 g) wasseparated into broken, insect-infected kernels and undamaged, intactkernels. Fifty undamaged kernels were surface disinfected in a I% so­dium hypochlorite solution for I min, washed twice in sterile water,and placed on malt agar (30 g malt extract, 20 g agar in I liter of wa­ter). Five kernels were placed on each plate and incubated for 5 to 6 da

218 MYCOLOGL\, VOL. 73, 1981

at 28 C. The percentage of insect-damaged kernels was determined byweighing the damaged kernels and expressing this as percent of totalsample. A record was made of the number of whole kernels that ger­minated on the agar plates. The only molds recorded that grew fromkernels were Aspergillus flavus/ parasiticus (no attempts were made toidentify the species except to recorq them as members of the A. flavusseries), Aspergillus niger series, and the predominant Penicillia; alsorecorded was whether A. flavus occurred with these fungi. Observa­tions were made using a dissecting microscope.

RESULTS AND DISCUSSION

Of the 238 corn samples analyzed, 187 were aflatoxin positive; ofthese, 142 samples (60%) were above the 20 ppb guidelines (TABLE I).Aflatoxin B1 was the predominant aflatoxin, but 32 samples also con­tained G l . Because Aspergillus parasiticus Speare is known to pro­duce large quantities of G l but A. flavus does not (4), one can assumethat A. parasiticus was present, and it was identified in isolates fromsome corn kernels. In 22 samples in which B1 was found, no B2 wasdetected; but B2 never occurred unless B l was present. However, inmost instances when B2 was not found, the B1 levels were low; thehighest was 109 ppb. If G 1 was not present, no G2 was found, but ifG2 was found, Gl was always present.

The highest aflatoxin levels were 1,180, 1,341, 2,208, and 3,622.The sample with 3,622 ppb total aflatoxin had 1,173 ppb of aflatoxin

TABLE I

TOTAL AFLATOXIN FROM 238 CORN SAMPLES FROM NORTH CAROLINA(1977 CROP)

Number of samples analyzed foraflatoxin B, and GI

Aflatoxin Totalrange (ppb) B, 07' G, cr samples '"0 10 '0

ND' 51 21 206 87 51 211-19 48 20 14 6 45 19

20-40 44 18 4 2 40 1750-99 36 15 5 2 38 16

100-249 37 16 5 2 36 15250-499 10 5 2 I 15 6500-1;000 9 4 I 0.5 9 4>1,000 3 1 1 0.5 4 2

Aflatoxin positive 187 32 187

'ND = not detected.

HESSELTINE ET AL.: AFLATOXIN AND MOLD FLORA 219

G 1• These samples came from the Piedmont/mountain area of NorthCarolina.

The distribution of aflatoxin G 1 found in corn samples is shownin FIG.I. This figure shows assay values in ppb for the 32 positivesamples found in 14 counties. Examination of this figure shows thatthe samples from the southeastern portion of the state were G 1

negative.We compared the percentage of damaged kernels of corn with the

total amount of aflatoxin found (TABLE II). In general, as the percen­tage of damaged corn kernels increased, so did the average level ofaflatoxin. In one sample with 9.33% damage, all the corn kernels onthe plates were overrun with Trichoderma, which probably inhibitedaflatoxin formation. In several instances, a sample with a high afla­toxin content made the average aflatoxin level seem quite high. Forexample, among the samples with 6.0 to 6.4% damage, 1 sample con­tained 3,622 ppb. Most of the damage to the corn kernels appeared tobe caused by insect activity.

TABLE III summarizes an examination of corn kernels infectedwith Aspergillus flavus and shows the number of kernels infected andthe number of samples with this level of infection. This is comparedto the average total aflatoxin and B1 present. The assay figures foraflatoxin G 1 also are given. In 9 samples, no infection by A. flavus oc­curred; of these, 3 samples were positive for aflatoxin and 1 samplehad 149 ppb. In 15 samples with only 1 kernel in 50 infected, only 4contained aflatoxin; the highest was 36 ppb. In 8 samples containing2 infected kernels, 5 were aflatoxin positive, and the highest was 42ppb.

When the total aflatoxin content was high, then G 1 was present,which confirmed the fact that A. parasiticus usually produces moreaflatoxin than A. flavus (4). For example, in the 5 samples containing30 kernels infected with A. flavus, the average total aflatoxin was 618ppb (range 204-1,341), and four of the samples contained aflatoxinG 1• A total of 3,780 kernels were infected with A. flavus, either by it­self or with one or more other fungi, as listed in TABLE IV.

Predominant Aspergillus and Penicillium isolates were recordedfrom surface-sterilized kernels placed on malt agar plates, as well asthe combination of A. flavus growing with A. niger van Tiegh. andwith Penicillium. Only the more abundant fungi in the genera As­pergillus and Penicillium were recorded, although species of Fusar­ium were very common. Aspergillus flavus was observed growingfrom 3,780 kernels. Besides A. flavus, A. niger was the most abundantAspergillus species, with a total of 1,301 kernels infected. Penicillium

Mountain Plateau Coo, tal Plain

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Flc. I. Distrihulion of aflatoxin G, in North Carolina in H connlies. Numhers are assay values in pph.

TABl.E II

REl.ATION OF nA~IAGEn KERNEl.S OF CORN TO TilE l.EVEl.S OF AFl.ATOXIN

% Damaged ppb Aflaloxin% Damaged Number of

kernels samples Range Average Range Average

O.O-IU :> 0.21-0.,17 0.28 0-,17 :>0':>-0.9 19 (U9-0.92 0.:,9 058 61.0-l/l 22 0.%-1/1'1 1.2:, 0-'189 581.!">-1.9 26 1.'17-I.~H 1.69 0-211 'Iii2.0-2.'1 ·IIl 1.97-2.'15 2.IH 0-+11 7025-2.9 22 2.'18-2.92 2.65 0-'106 ·19'LO-'l.'! ,12 2.96-:U:> :l.I 8 0-1,1% 171:1.:>-,1.9 17 ,1.:>0-,1.92 :1.7:1 0-25Il 87'1.0-,1.'1 1:1 ,1.96-'1.'11 ·1.17 0-919 1:,2'1.:>-'1.9 10 '1.51-'!.89 4.7:l 'I-I,:HI 179:>.0-:>.'1 :1 :'.:1'1-:>.% 5.:15 :111-266 117:>5-5.9 ,I 5.'19-5.77 :>.Ii'! 1:1-490 'II!">6.0-6.'1 8 5.~H-6.'I'1 6.25 :17-:1,622 722'1li.5-6.9 I 6.78 6.78 182 1827.0-7.'1 6 7.07-7.:17 7.2'1 9-851 2lil75-7.9 !l 7/16-7.67 7.58 8-1-920 '1808.0-8/1 2 8.2:'-8.112 8.29 '12-97-1 :>088.:'-8.9 I 8.77 8.77 liO 609.0-9.'1 I 9.:1:1 9.,1:1 'I lt b

11.0-1 !.'l I 11.2,1 11,24 2,208 2,208

"This number is high because of one sample conlaining a high level of aflalOxin.h All kernels were infeCled wilh Trichoderma,

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222 MYCOLOGIA, VOL. 73, 1981

TABLE III

REL\TIO~ OF KER~ELS I~FECTED WITH A. flavtls TO TOTAL,BI, AND G 1 AFLATOXt~ PRESE~T'

Aflatoxin present (ppb)Number of

kernels Number of Total B1 G1

infected samples (average) (average) (actual)

0 9 18 IjI 15 3 32 8 15 12 163 10 4 44 3 8 j

5 8 29 ')0 19-;)

6 14 18 Ijj 14 26 ')._J8 2 ')" 21-;)

9 4 39 29 2810 6 18 ]jII I 20 13 412 9 154 136 5513 5 206 ]j2 14,9414 9 94 86 9, 1015 j 68 6316 5 50 45Ij 8 41 3718 6 50 45 1319 5 112 89 5820 5 j3 6521 j 68 55 1622 5 165 143 3423 3 66 6524 6 135 114 jl'). 4 32 26_J

26 II j2 j5 3, 182j j 132 119 1028 j 31j 236 34629 5 341 260 31930 5 618 512 14, 2j, 114, 19631 5 184 145 11532 4 311 2j2 4633-39 9 610 460 j, 46, 92, 130, 135, 50540-44 j 800 55j 14, 4j, 1,lj3

'Fifty kernels were examined for each sample.

was represented by three distinct taxa. Although typical P. funiculo­sum Thorn was found in 185 kernels, another member of the P. funi­culosum series-somewhat variable but distinct in gross morphologyfrom P. funiculosum-was observed in 3,020 kernel infections, and itappeared to be antagonistic to other molds. The data on this portionof the study is shown in TABLE IV. The only other prevalent speciesof Penicillium was P. oxalicum Currie & Thorn. Out of the thousandsof corn kernels examined, less than I% were sterile, and the majorityof the kernels were infected with Fusarium moniliforme Sheldon. The

HESSELTINE ET AL.: AFUTOXIN AND MOLD FLORA 223

TABLE IV

INCIDENCE OF FUNGI FOUND IN THE 11,900 KERNELS OF CORN'

Fungi

Total kernels withA. flavus

Kernels with A. flavusonlv

Total 'kernels withA. niger

Kernels with A. flavusand A. niger

Total kernels withPenicillium spp.

Kernels with A. flal}uSand Penicillium spp.

Kernels with Penicilliumftmiculoswn series

Kernels with typicalP. fWliculoswn

Kernels with Penicilliumoxalicwn

Kernels with otherPenicillium spp.

Kernels with otherAspergillus

Sterile kernels

Kernels infectedTotal Percent

3,780 31.8

2,268 19.1

1,301 10.9

781 6.6

3,401 28.6

731 6.1

3,020 25.4

185 1.6

142 1.2

54 0.5

II 0.121 0.2

'Fifty kernels per sample.

corn coming directly from the field in the Southeast is distinct fromstored corn in the Midwest where a large number of different speciesof Aspergillus and Penicillium are always encountered. Also the ac­tual numbers of fungi undoubtedly are low, because in some instancesspecies of Trichoderma, Fusarium, and Rhizopus overgrew the ker­nels to such an extent that Aspergillus and Penicillium were obscuredor inhibited from growing. Besides A. flavus and A. niger, a few ker­nels developed growth of A. clavatus Desm. and A. terreus Thorn.Trichoderma sp. was observed growing from at least one kernel in 58samples. Rhizopus sp. also was seen growing from one or more ker­nels in 37 samples.

FIGURE 2 shows the average aflatoxin level (ppb) per county andthe average number of kernels infected with A. flavus based upon ex­amination of 50 kernels of corn. Aspergillus flavus was found in 10 ormore kernels in 22 of the 30 counties.

FIGURE 3 shows the average number of kernels infected by Asper­gillus niger and the number infected with isolates of the Penicilliumfuniculoswn series, characteristic of the type of infection in much ofthis corn. This is the same Penicillium reported in our earlier study of

Mountain Plateau Coastal Plain

I>:)I>:)

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1'1(;, 2. Average aflaloxin levels in corn samples for each Nonh Carolina cOllllly

(lOp nlllnber) and average nnmber of kernels infected wilh A. Jim/lis (lower number).

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<000

FIG. !l. Average infeclion of corn kernels by Penicillium !lIniculosum for each NOr/h Carolina counly (lOp Ilumber)and average infection by As/Jagillus niga (lower number).

Mountain Plateau Coostal Plain::r:tTlCJlCJltTl

9:zMtTl.>-1;...t-

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226 MYCOLOGIA, VOL. 73, 1981

corn from the field in South Carolina (3) as PeniCl"llum No. I, andtypical isolates in the Agricultural Research Culture Collection areNRRL 6420, NRRL 6417, and NRRL 6455. There appears to be noparticular region where either of these two fungi predominate, exceptthat A. niger seemed to occur in greater incidence in the southeasternregion of the state whereas P. funiculosum was less prevalent in thisregion.

Our ability to distinguish initially between two forms of P. funi­culosum is based on their colony appearance (texture and color) fol­lowing invasion of the malt extract agar upon which individual cornkernels were plated. One kind, regarded as typical and represented byNRRL 6420 (11) is strongly funiculose and produces an abundance ofsterile, coarse, yellow-encrusted hyphae on either Czapek or malt ex­tract agars. The yellow-encrusted aerial hyphae give these heavilysporing colonies a yellow-green appearance near Andover Green orSlate Olive (Ridgway PI. XLVIe).

The other kind, recognized as P. funiculosum I (11) and repre­sented by strains NRRL 64 I7, NRRL 6454, and NRRL 6455, is lessstrongly funiculose, appearing lanose in marginal zones on Czapekagar and velvety to tufted in marginal areas, giving a granular ap­pearance on malt agar. What particularly distinguishes these isolatesfrom the more typical kind is the absence of sterile, coarse, yellow-en­crusted, vegetative hyphae, giving the heavily sporing areas a gray­green appearance near Tea Green to Pea Green (Ridgway PL· XLVII)in some strains. In other strains shades were near Andover Green toSlate Olive, but without a bright yellowish tint that we consider to bethe more typical form. Strains NRRL 64 I7 and NRRL 6455 also pro­duce numerous small, dark brown sclerotia embedded in the surfaceof malt agar. These cultures are very similar in appearance to NRRL1132, which was described (7) as a strain of P. funiculosum producingsclerotium-like bodies.

TABLE V shows the amount of infection by A. niger and P. flmi­culosum series in the North Carolina samples, and each species iscompared to the average total aflatoxin found. Aspergillus niger wasfound in 63 samples, whereas P. funiculosum occurred in one or morekernels in all but 5 samples. However, there appears to be no correla­tion with either mold and the average total aflatoxin. Thus, in thefive samples not infected with P. funiculosunl the average aflatoxinwas 436 ppb.

'Ridgway, R. 1912. Color standards and color nomenclature. Washington, D.C.43 p.

HESSELTINE ET AL.: AFLATOXIN AND MOLD FLORA 227

TABLE V

KERNELS INFECfED WITH Aspergillus niger AND Penicillium funiculosumSERIES COMPARED TO AVERAGE AFUTOXIN LEVELS

Aspergillus niger Penicillium funiculosum

Number Average Number Averageof Number total of Number total

kernels of aflatoxin kernels of alfatoxininfected samples (ppb) infected samples (ppb)

0 63 34 0 5 436I 34 31 I 4 982 23 102 2 10 1613 20 84 3 7 6924 10 91 4 13 1375 II 92 5 10 3236 14 87 6 6 1787 8 75 7 II 1028 8 262 8 17 589 7 116 9 8 126

10 5 584 10 19 12011-15 9 11-15 4416-20 10 16-20 2621-30 10 21-30 3631-40 6 31-40 16

41-47 6

When the plated kernels were examined for mold growth, thenumber of germinated kernels were recorded (TABLE VI). Germina­tion was relatively good in most samples. The samples with 80% ger­mination showed only 9 ppb of total aflatoxin. In the samples with78, 76, and 28% germination, no aflatoxin could be detected.

TABLE VI

GERMINATION OF WHOLE KERNELS OF CORN PLACED ON ~IALT AGAR AND EXAMINEDAT 6 DA

% Germination

10098969492908886848280787628

Number of samples

4159532819II65445III

228 MYCOLOGIA, VOL. 73, 1981

ACKNOWLEDGMENT

We acknowledge with great thanks the study on the colony ap­pearance of isolates of Penicillium funiculosum by Dr. D. T. Wick­low of our laboratory.

LITERATURE CITED

I. Anderson, H. W., E. W. Nehring, and W. R. Wichser. 1975. Aflatoxin contamina­tion of corn in the field. ]. Agric. Food Chern. 23: 775-782.

2. Bothast, R. J., Marion L. Goulden, Odette L. Shotwell, and C. W. Hesseltine. 1976.Aspergillus flavus and aflatoxin production in acid-treated maize. J. StoredProd. Res. 12: 177-183.

3. Hesseltine, C. W., O. L. Shotwell, W. F. Kwolek, E. B. Lillehoj, W. K. Jackson, andR. J. Bothast. 1976. Aflatoxin occurrence in 1973 corn at harvest. II. Mycologicalstudies. Mycologia 68: 341-353.

4. --, --, M. Smith, J. J. Ellis, E. Vandegraft, and G. Shannon. 1970. Productionof various aflatoxins by strains of the Aspergillus flavus series. Pp. 202-210. In:Proc. First U.S. Japan Conference on toxic microorganisms. Washington, D.C.

5. Lillehoj, E. B., W. F. Kwolek, G. M. Shannon, O. L. Shotwell, and C. W. Hessel­tine. 1975. Aflatoxin occurrence in 1973 corn at harvest. I. A limited survey in thesoutheastern U.S. Cereal Chern. 52: 603-611.

6. Official Methods of Analysis of the Association of Official Analytical Chemists.12th Ed. 1975. Washington, D.C. Secs. 26.014-26.019, 26.037. 26.055.

7. Raper, K. B., and C. Thorn. 1949. A maTwal of the Penicillia. Williams and Wil­kins Co., Baltimore. 875 p.

8. Shotwell, O. L. 1977. Aflatoxin in corn. ]. Amer. Oil Chern. Soc. 54: 216A-224A.9. --, M. L. Goulden, R. J. Bothast, and C. W. Hesseltine. 1975. Mycotoxins in hot

spots in grains. I. Aflatoxin and zearalenone occurrence in stored corn. CerealChern. 52: 687-697.

10. --, --, E. B. Lillehoj, W. F. Kwolek, and C. W. Hesseltine. 1977. Aflatoxin oc­currence in 1973 corn at harvest. III. Aflatoxin distribution in contaminated, in­sect-damaged corn. Cereal Chern. 54: 620-626.

II. Wicklow, D. T., and C. W. Hesseltine. 1979. Fluorescence produced by Aspergillusflavus in association with other fungi in autoclaved corn kernels. Phytopathol­ogy 69: 589-591.

Accepted for publication July 2. 1980