Ochratoxin A accumulation in cultures of Penicillium verrucosum with the antagonistic yeast Pichia anomala and Saccharomyces cerevisiae

Mycol. Res. 102 (8) : 1003–1008 (1998)

1003

Printed in the United Kingdom

Ochratoxin A accumulation in cultures of Penicillium verrucosum with the antagonistic yeast Pichia anomala and Saccharomyces cerevisiae

S T I N A P E T E R S S O N1, M A R I A N N E W I T T R U P H A N S EN2, K A R I N A X B E R G2, K A R L H U L T2 A N D J O H A N S C H N U> R E R1* " Swedish University of Agricultural Sciences, SLU, Department of Microbiology, Box 7025, S-750 07 Uppsala, Sweden # Royal Institute of Technology, KTH, Department of Biochemistry and Biotechnology, Teknikringen 34, S-100 44 Stockholm, Sweden

Growth and ochratoxin A accumulation by two isolates of Penicillium verrucosum, IBT5010 and IBT12803, were examined in co-cultures with the antagonistic yeast Pichia anomala or baker’s yeast Saccharomyces cerevisiae. Each yeast was co-cultured with Penicillium verrucosum in malt extract agar supplemented with 1 % yeast extract and incubated at 25 °C for 14 d. Ochratoxin A was found to be stable at 25°, whether or not yeast was present. Pichia anomala and Saccharomyces cerevisiae reduced mould growth to about the same extent in vitro. Both yeasts significantly reduced the growth of Penicillium verrucosum isolates at 3¬10$ yeast c.f.u.}plate. In all co-cultures in vitro accumulated ochratoxin A was reduced to below the detection limit (100 ng}plate) by yeast amounts as low as 3¬10# c.f.u.}plate. In wheat, the growth of Penicillium verrucosum IBT5010, measured as c.f.u., was not reduced in the presence of Pichia anomala, whereas the growth of Penicillium verrucosum IBT12803 was clearly inhibited by this yeast. A similar pattern was observed for ochratoxin A accumulation in wheat when the two mould strains were co-cultured with Pichia anomala. Mycotoxin production was more sensitive to the presence of yeasts than was mould growth. Co-cultivation with yeasts gave no detectable stimulation of ochratoxin A accumulation in agar or wheat.

Antagonistic yeast can considerably reduce the growth of filamentous spoilage moulds both in vitro and under conditions simulating full-scale storage (McGuire, 1994 ; Petersson & Schnu$ rer, 1995). In vitro, the fungal biomasses of Penicillium roquefortii and Aspergillus candidus, quantified both as hyphal length and number of colony forming units (c.f.u.), are reduced by the antagonistic yeast Pichia anomala (Bjo$ rnberg & Schnu$ rer, 1993). Although the growth of spoilage fungi is inhibited by biocontrol micro-organisms, the metabolic activity of the existing hyphae is not necessarily reduced. One characteristic feature of secondary metabolism is its frequent association with a state of stress or a morphogenic change in the mould (Moss, 1991). Competition could be expected to have a profound effect on secondary metabolism since micro-organisms in co-culture compete for all essential environmental factors, including space and nutrients. The nutrient supply strongly affects secondary metabolism, including mycotoxin production (Luchese & Harrigan, 1993). Co-culturing micro-organisms can result in either stimulation or inhibition of mycotoxin production. Presence of the yeast Hyphopichia burtonii significantly increased aflatoxin production by Aspergillus flavus on maize compared with pure cultures (Cuero, Smith & Lacey, 1987). Candida guilliermondii (anamorph Pichia guilliermondii) was the only one of 14 fungi

* Corresponding author.

tested that did not inhibit aflatoxin production in maize by A. flavus (Wicklow et al., 1980). Both ochratoxin A and aflatoxin are produced in higher amounts when toxigenic fungi are inoculated in sterile wheat than when inoculated in unsterilized wheat (Vandegraft et al., 1973 a, b ; Chelack et al., 1991). Mycotoxin production can thus decrease in response to competition between the natural microbiota and inoculated mycotoxin producers. Biocontrol micro-organisms should not enhance mycotoxin production by the inhibited fungi. Sub-inhibitory levels of antagonistic micro-organisms might, however, stimulate secondary metabolism, and thus also mycotoxin production. Consequently, in the evaluation of potential biocontrol agents, the effects of sub-inhibitory concentrations on mycotoxin production should also be considered. Due to the temperate climate of Sweden the ochratoxins, particularly ochratoxin A, are mycotoxins of great concern (Holmberg et al., 1990 ; Breitholtz et al., 1991 ; Olsen, Mo$ ller & AI kerstrand, 1993). Ochratoxin A has nephrotoxic, carcinogenic, teratogenic and immunotoxic properties (KupierGoodman & Scott, 1989). The climate of Sweden favours the growth of P. verrucosum, resulting in a high daily intake of ochratoxin A by the population, in some parts of of the country exceeding the tolerable daily intake (Breitholtz et al., 1991 ; Olsen et al., 1993). The antagonistic activity of the yeast P. anomala may have practical value in the post-harvest control of storage fungi in airtight storage of high-moisture

Ochratoxin A accumulation during yeast-mould interaction feed grains. It is thus important to determine whether P. anomala can reduce ochratoxin A accumulation in airtight storage of high-moisture grains intended as feed. We investigated ochratoxin A accumulation by two isolates of P. verrucosum when grown in co-culture with the yeasts P. anomala and S. cerevisiae in vitro. In wheat, we studied the effect of P. anomala on ochratoxin A accumulation by the two isolates of P. verrucosum. P. verrucosum was chosen because it is a common spoilage mould in high-moisture grains in Sweden and also a producer of the potent mycotoxin ochratoxin A (Holmberg et al., 1991). P. anomala was chosen in this investigation owing to its ability to act as a biocontrol agent, both in vitro and in air tight stored grains (Bjo$ rnberg & Schnu$ rer, 1993 ; Petersson & Schnu$ rer, 1995). S. cerevisiae is commonly used for baking and brewing and is regarded as a safe micro-organism. The effects of yeasts on growth and accumulation of ochratoxin A by P. verrucosum have not been evaluated earlier. MATERIALS AND METHODS Micro-organisms studied Pichia anomala (E. C. Hansen) Kurtzman (J121) was originally isolated from airtight-stored grain (Bjo$ rnberg & Schnu$ rer, 1993). Saccharomyces cerevisiae Meyen ex E. C. Hansen (baker’s yeast strain P1 : 6, Svenska Ja$ stfabriks AB, Sollentuna, Sweden) was a gift from Dr Bjo$ rn Lindman, Penicillium verrucosum Dierckx IBT12803 and IBT5010 were gifts from Dr Ole Filtenborg, Department of Biotechnology, Technical University of Denmark, Lyngby, Denmark. Inocula Yeast suspensions were prepared by inoculating 100 ml of a yeast extract malt extract sucrose broth (0±2 g yeast extract [BBL, Meyland, France], 1±5 g malt extract [Oxoid, Basingstoke, England], 1±0 g sucrose [BDH, Poole, England] l−" distilled water) with a loopfull of cells from a culture stored on malt extract media (MEA) (Oxoid) at 4 °C. After incubation on a rotary shaker (100 rpm) at 25° for 24 h, the number of yeast cells was enumerated using a haemocytometer. Spore suspensions were prepared by collecting spores from 7-d-old colonies (grown on MEA at 25°) in peptone water (2 g peptone l−" distilled water) with 0±015 % Tween 80 added to assist in the dispersal of spores. The spore concentration was determined using a haemocytometer. Stability of ochratoxin A in malt extract agar A 0±23 mg amount of pure ochratoxin A was dissolved in 2±3 ml of 0±2 % Na CO , and 10 µl (corresponding to 1000 ng # $ ochratoxin A) was mixed with 6 ml autoclaved malt extract agar (Oxoid) supplied with 1 % yeast extract (Sigma), cooled to 45°. Yeast extract was included to stimulate ochratoxin A production by P. verrucosum (Filtenborg, Frisvad & Svendsen, 1983). This ochratoxin A-containing agar portion was poured onto a bottom agar of the same constitution, resulting in 1000 ng ochratoxin A per plate. Plates were either inoculated with 5¬10' or 10( c.fu of surface-spread P. anomala or left

1004 uninoculated. Plates were incubated at 25° for 10 or 15 d and stored at ®20° before ochratoxin A analysis. Influence of different initial yeast levels on ochratoxin A production by Penicillium verrucosum in agar Yeast and spore suspensions were prepared as described above. A top agar was prepared by mixing 8 ml of the malt yeast extract agar and 1 ml of yeast suspension with either 0, 10", 10#, 10$, 10%, 10& or 10' cells ml−". The agar–yeast suspension was poured into Petri dishes containing 15–20 ml solidified malt yeast extract agar. Once the top agar had set, 10 µl portions of a P. verrucosum IBT5010 suspension with 10& spores ml−" were inoculated at three spots on each plate, with three replicates for each mould isolate. Five plates of each treatment were prepared, two intended for fungal growth measurement and three for ochratoxin A analysis. Plates were incubated at 25° for 14 d. A preliminary study of the accumulation of ochratoxin A during growth of P. verrucosum IBT5010 in agar, with and without inoculation of 5¬10" c.f.u. of P. anomala per plate, showed the highest amount of accumulated ochratoxin A between days 12 and 16 (data not shown). The degree of growth inhibition was determined after mixing the agar from each Petri dish with peptone water (0±005 % Tween 80, 1 g peptone l−" distilled water) to obtain a 10-fold dilution. The mixture was then homogenised for 40 s with a mixer (Philips HR1390}92, Austria). Samples (0±1 ml) of 10-fold dilutions of the homogenates were surface spread on MEAC plates (MEA, supplemented with 100 ppm of chloramphenicol (Sigma Chemical Co.)) and MEACC (MEAC, supplemented with 10 ppm cycloheximide, Sigma Chemical Co., No. C-6255, St Louis, Mo., U.S.A.). Chloramphenicol is a broad-spectrum bacteriostatic antibiotic, while cycloheximide inhibits growth of P. anomala (Bjo$ rnberg & Schnu$ rer, 1993). Yeast c.f.u. were counted on MEAC after 3 d, and mould c.f.u. were counted on MEACC after 7 d at 25°. The limit of detection for the fungal quantification method was 2¬10$ c.f.u. per agar plate. The coefficients of variation for log-transformed c.f.u. values in Figs 1 and 2 were less than 6 %. Plates intended for ochratoxin A analysis were stored at ®20° until analysis. Ochratoxin A was analysed as described below. Influence of different initial yeast levels on growth and ochratoxin A production by Penicillium verrucosum in wheat Non-sterile winter wheat (cv. Kosack) stored at a water content of 8 % at 20° was moistened with tap water to obtain a water activity (aw) of 0±96 (25 % water). To equilibrate the moisture content the grains were stored for " 48 h at 2° and frequently mixed. Water activity was measured at 25° with a Novasina TH-2RTD-33}BS equipped with a temperature controller (Defensor AG, formerly Novasina AG, Pfa$ ffikon, Switzerland). Wheat grains with an aw of 0±96 were inoculated with a spore suspension of either P. verrucosum IBT5010 or IBT12803 in order to obtain about 10% spores g−". Spores were applied in the form of drops of suspension added to the grain. The grain was then mixed to obtain a uniform spore distribution.

Stina Petersson and others

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Table 1. Stability of ochratoxin A in malt yeast extract agar plates in the absence or presence of Pichia anomala. Plates were supplemented with 1000 ng ochratoxin A per plate. All plates were incubated at 25° for 10 or 15 d. Values represent means of two replicates (³..) P. anomala (c.f.u. per plate)

Incubation (d)

Ochratoxin A (ng per plate)

Recovery (%)

0 5¬10' 10( 5¬10' 10(

10 10 10 15 15

810³24 820³150 760³60 1040³30 750³140

81 82 76 104 75

inoculated grain and sealed with a paper stopper. The tubes were incubated at 25°. On day 21, two tubes from each treatment were stored at ®20° until ochratoxin A analysis (see below), and two tubes from each treatment were analysed by c.f.u. plating. The contents of the tubes were diluted 10-fold with peptone water, soaked for 0±5 h and homogenized for 2 min at normal speed in a Stomacher 400 (Colworth, U.K.). Aliquots of the dilutions were plated as described above. The limit of detection for the fungal quantification method was 1¬10# colony forming units (c.f.u.) g−" wheat. The coefficients of variation for log-transformed c.f.u. values in Fig. 3 were less than 6 %, in Fig. 4 standard deviations are shown.

11

1 Penicillium verrucosum IBT5010 (log c.f.u./plate)

10

9

8

7

6

5 0

1

2

3

4

5

6

7

11

2 Penicillium verrucosum IBT12803 (log c.f.u./plate)

10

9

8

7

6

5 0

1

2

3

4

5

6

7

8

Ochratoxin A analysis Extraction from agar. Ochratoxin A was extracted from the agar of each Petri dish with 100 ml methanol supplemented with 0±2 ml formic acid. The mixture was incubated on a rotary shaker (120 rpm) at 30° for 1 h and passed through a filter paper (Whatman filter). In a test tube 1 ml of the filtrate was mixed with 2 ml chloroform and 2 ml of 1 % sodium bicarbonate solution. Ochratoxin A was extracted into the water phase by turning the test tube end over end (20 rpm) for 15 min. The organic phase was then removed and the water phase acidified with HCl to reach pH ! 3, whereupon it was mixed with 2 ml chloroform. Again, the solution was mixed as described above. This time the water phase was removed, and the organic phase was washed with distilled water. The sample was dried with sodium sulphate and filtered through glass wool, whereafter the chloroform was evaporated under a stream of nitrogen gas at ! 30°. The residue was dissolved in 1 ml of mobile phase as described under HPLC analysis. Each plate was analysed three times. The average recovery of ochratoxin A from the agar was 84 %. Extraction from cereal. Two 5 g subsamples were taken from each tube with wheat. The kernels were crushed before mixing with 1 ml of 2 M HCl and 25 ml toluene for 1 min in a Sorvall Omnimixer, Model 17106 (Du Pont Instruments, Newton, Conn. USA). Of this extract, 5 ml was passed through 200 mg silica gel (Si-60, 40–63 µm) packed in a Pasteur pipette. The silica gel was then washed with 3 ml chloroform. Ochratoxin A was eluted with 3±5 ml chloroform : formic acid (95 : 5, vol : vol). The eluate was washed with 1 ml water and dried with anhydrous sodium sulphate. The chloroform was evaporated under a stream of nitrogen. The recovery of ochratoxin A was 90–95 %.

yeast at day 0 (log c.f.u. per plate)

Figs 1, 2. Effects of increasing initial levels of Pichia anomala (D) or S. cerevisiae (E) on the growth of Fig. 1. Penicillium verrucosum IBT5010 ; and Fig. 2. P. verrucosum IBT12803, measured as colony forming units (c.f.u.), in co-cultures on malt yeast extract media at 25° for 14 d (n ¯ 2).

The inoculation procedure did not significantly change the water activity of the grain. P. anomala was inoculated to reach 0, 10", 10#, 10$, 10%, 10& and 10' yeast cells g−" grain. Thick-walled glass tubes (27 ml) were filled with approx. 17 g portions of

Hplc analysis. Liquid chromatography analysis was performed using ion-pair technique described by Breitholtz et al., 1991. The wavelengths for excitation and emission were 380 nm and 450 nm respectively. The separations were run on a 4±6¬150 mm Sperisorb S30DS2 (C-18) column (Hichrom Ltd, Reading, Berkshire, UK) with 3 µm particles. The column temperature was 30° and total flow rate 0±8 ml}min. Samples were dissolved in the mobile phase, and 100 µl of the sample was injected into the column. The mobile phase was 38±25 m phosphate buffer pH 7±5 and methanol 48 : 52 (v}v) supplemented with 10 m tetrabutylammonium bromide. The

Ochratoxin A accumulation during yeast-mould interaction retention time of ochratoxin A was 4–7 min. The detection limit of ochratoxin A was 100 ng per agar plate and 1 ng g−" wheat.

1006 Table 2. Amounts of ochratoxin A accumulated in malt yeast extract agar plates following inoculation with Penicillium verrucosum IBT12803 or IBT5010 and either Pichia anomala or S. cerevisiae. Plates were incubated at 25° for 14 d. Values represent means of two replicates (³..)

RESULTS

Ochratoxin A accumulation (µg}plate)

Stability of ochratoxin A in the presence of Pichia anomala

P. verrucosum IBT5010

Influence of yeast on the growth of Penicillium verrucosum in agar Penicillium verrucosum IBT5010 reached higher c.f.u. values than P. verrucosum IBT12803, presumably owing to a higher degree of sporulation. Both P. anomala and S. cerevisiae reduced growth of the two isolates of P. verrucosum in a dosedependent manner in vitro (Figs 1 and 2). P. verrucosum IBT5010 growth was reduced to a higher degree by P. anomala than by S. cerevisiae at initial yeast levels below 1¬10$ c.f.u. g−". No clear difference in growth-suppressing ability between the two yeast species was detected in vitro at higher inoculation levels (Fig. 1). The growth of P. verrucosum IBT12803 was reduced less by P. anomala than by S. cerevisiae (Fig. 2). At an initial level of 1¬10' c.f.u. per plate P. anomala and S. cerevisiae reduced P. verrucosum IBT12803 to 3¬10' c.f.u. and 3¬10& per plate, respectively. Influence of different initial yeast levels on ochratoxin A accumulation in agar with Penicillium verrucosum Both P. anomala and S. cerevisiae strongly affected mycotoxin accumulation by the two tested strains of P. verrucosum, i.e. IBT5010 and IBT12803 (Table 2). Ochratoxin A accumulation in plates with P. verrucosum IBT5010 was reduced at 3¬10" c.f.u. P. anomala or 5¬10" c.f.u. S. cerevisiae, and mycotoxin concentrations were reduced to non-detectable levels by an initial yeast level of 3¬10# c.f.u. per agar plate. Accumulated concentrations of ochratoxin A produced by P. verrucosum IBT12803 were below the detection limit in plates inoculated with the lowest amount of yeast, i.e. 1¬10# c.f.u. P. anomala and S. cerevisiae per agar plate. The yeast reached approximately 10"! c.f.u. per plate regardless of the dose of inoculant at levels above 1¬10$ c.f.u. per plate (data not shown). At lower inoculation levels yeast colonies reached a maximum diameter of 1 cm, and at inoculant doses of 1¬10% c.f.u. per plate a dense yeast layer was formed. At higher initial yeast levels, accumulated ochratoxin A concentrations remained below the detection limit. No enhancement of ochratoxin A accumulation was detected at any yeast level. Influence of different initial doses of yeast inoculant on the growth and ochratoxin A production of Penicillium verrucosum in wheat To obtain a measurable amount of ochratoxin A in the wheat,

Yeast day 0 (c.f.u.}plate)

P. anomala

S. cerevisiae

P. anomala

S. cerevisiae

0 1¬10" 3¬10" 5¬10" 1¬10# 3¬10# 1¬10$ 3¬10$ 1¬10% 3¬10% 1¬10& 3¬10&

2±5³2±1 —* 1±8³0±3 0±3³0±2 — ! 0±1* — ! 0±1 — ! 0±1 — ! 0±1

5±7³1±6 2±4³1±5 — 1±8³0±9 — ! 0±1 — ! 0±1 — ! 0±1 — ! 0±1

1±8³0±7 — — — ! 0±1 — ! 0±1 ! 0±1 ! 0±1 — ! 0±1 —

4±0³1±2 — — — ! 0±1 — ! 0±1 ! 0±1 ! 0±1 ! 0±1 — —

* — ¯ not measured ; ! 0±1 ¯ below detection limit.

12

11 Penicillium verrucosum (log c.f.u. g–1)

Ochratoxin A was stable in malt extract agar (Table 1). No degradation or adsorption of ochratoxin A by the yeast was observed. The average recovery was 84 % ranging from 75 to 104 %.

P. verrucosum IBT12803

10

9

8

7

6 0

1

2

3

4

5

6

7

Pichia anomala at day 0 (log c.f.u. g–1)

Fig. 3. Effects of increasing numbers of Pichia anomala on the growth and sporulation of P. verrucosum IBT5010 (D) and IBT12803 (E), measured as c.f.u., in co-cultures in glass tubes with wheat (aw 0±96) at 25° for 21 d (n ¯ 2).

tubes were incubated for 3 wk with the only restriction of the air supply being that a tube was used as inoculation container. During week 1 P. anomala, as judged by the naked eye, totally inhibited the growth of P. verrucosum. During week 2, weak growth was observed, and during week 3 mould growth was clearly visible on the grain. After 3 weeks, isolate IBT5010 showed no inhibition in either growth or ochratoxin A accumulation, even at the highest initial concentration of P. anomala (3¬10' c.f.u. g−") (Figs 3 and 4). Numbers of P. verrucosum IBT12803, however, were reduced from 2±5¬10) to 2±0¬10' c.f.u. g−" wheat by 1¬10' c.f.u. of P. anomala g−" (Fig. 3), and ochratoxin A accumulation was reduced in a dosedependent manner (Fig. 4).

Stina Petersson and others

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100 000

ochratoxin A (ng g–1 wheat)

10 000

1 000

100

10

0 0

1

2

3

4

5

6

7

Pichia anomala at day 0 (log c.f.u. g–1)

Fig. 4. Effects of increasing numbers of Pichia anomala on the accumulation of ochratoxin A, ng g−" wheat logarithmic scale, produced by P. verrucosum IBT5010 (D) or IBT12803 (E) in cocultures on wheat in glass tubes (aw 0±96) at 25° for 21 d. Values represent means of two replicates (³..).

DISCUSSION P. anomala and S. cerevisiae both reduced the growth of P. verrucosum IBT5010 and IBT12803 and their accumulation of ochratoxin A in vitro. In wheat P. verrucosum IBT12803, but not IBT5010, was reduced in growth and ochratoxin A accumulation. The level of yeast needed to reduce mycotoxin accumulation seemed to be lower than the level needed to reduce growth both in vitro and in grains. No stimulation of mycotoxin accumulation in the tested co-cultures was observed in contrast to earlier findings with other yeastmould co-cultures (Wicklow et al., 1980 ; Cuero et al., 1987). In vitro P. anomala and S. cerevisiae reduced the growth of P. verrucosum to similar extents. Only P. anomala, however, suppresses mould growth on non-sterile, high moisture wheat (Petersson & Schnu$ rer, 1995). Isolate IBT5010 produced more ochratoxin A in wheat, and showed more pronounced growth and sporulation, than IBT12803. In co-cultures consisting of a toxigenic mould and other microorganisms, degradation, adsorption, or uptake of mycotoxin by the other microorganisms could affect the amount of mycotoxin detected. In studies on ochratoxin A in beer fermentation about 10 % of the added ochratoxin A is found to be taken up by S. cerevisiae (Nip et al., 1975 ; Scott et al., 1995). The agar substrate can also influence the detection of mycotoxin. The recovery of ochratoxin A from malt extract agar in our experiments was similar to the previously reported recovery from Czapek-yeast agar (Abramson & Clear, 1996). Abiotic factors have a great influence on mycotoxin accumulation in both mono-cultures and co-cultures. Generally, the accumulation of particular metabolites occurs in a more narrow interval of each abiotic factor compared with growth of the toxigenic fungi (Samson et al., 1995). Mechanisms underlying the factors affecting mycotoxin accumulation when micro-organisms are co-cultured have not

been thoroughly studied, but they probably vary depending on the species. S. cerevisiae reduces aflatoxin production by Aspergillus parasiticus in broth by depleting the amounts of fermentable carbohydrates available as substrate for mycotoxin production (Weckbach & Marth, 1977). In our tests nutrient depletion was probably only an important factor at yeast levels higher than 1¬10$ per agar plate since P. anomala and S. cerevisiae were co-cultured with P. verrucosum on a very rich medium. At the lower inoculation levels, the yeast formed isolated colonies, whereas at higher inoculation levels a confluent layer of yeast formed on the agar. Since competition for nutrients might only have been important at yeast levels higher than those that reduced mycotoxin accumulation, other mechanisms might also have affected the accumulation of ochratoxin A. Fungal competition can both stimulate and inhibit mycotoxin production depending on species and environmental conditions. Comparisons between sterilized and nonsterile wheat and barley have shown that aflatoxin and ochratoxin accumulation associated with mould growth tend to be higher on sterilized substrate than on nonsterile substrate (Vandegraft et al., 1973 a, b ; Chelack et al., 1991). Due to competition between the natural microbiota and inoculated mycotoxin producers, both mould growth and mycotoxin production are reduced. Growth reduction caused by the presence of specific bacterial or yeast species can also influence mycotoxin production. In addition, certain substances produced by competing micro-organisms can reduce mycotoxin production by toxigenic fungi. Thus, for example, Streptococcus lactis produces a heat-stable, low-molecular-weight compound that inhibits synthesis of aflatoxin by A. flavus in broth culture (Coallier-Ascah & Idziak, 1995). Co-cultivation of a mycotoxigenic isolate with an atoxigenic isolate of same species can affect mycotoxin production by the former. For instance, atoxigenic isolates of both A. flavus and Pithomyces chartarum have been shown to be able to reduce mycotoxin production by the mycotoxigenic isolates both in vitro and in natural systems (Brown, Cotty & Cleveland, 1990 ; Chourasia & Sinha, 1994 ; Cotty & Bhatnagar, 1994 ; Collin & Towers, 1995). Biological methods of controlling moulds cannot be considered safe unless microbial interactions at sub-inhibitory levels of the antagonistic micro-organisms are understood. Once the biocontrol agent has stopped growing, owing to age or nutrient depletion, spores of spoilage moulds can germinate and grow on nutrients leaking out from the dead cells. Since not all cells of the antagonist die simultaneously, its levels may be high enough to stress the target microorganisms without suppressing their growth. It is well known that sub-inhibitory concentrations of antimycotic chemicals, such as sorbate, propionic acid, Benomyl (Dupont), and Bavistin (BASF), can stimulate mycotoxin production (Mu$ ller & Ho$ rber, 1982 ; Marshall & Bullerman, 1986 ; Sharma & Padwal-Desai, 1989). Patulin production by Penicillium patulum is totally reduced in the presence of potassium sorbate at concentration 0±10 %, but at 0±15 % variable amounts were produced late in the incubation period. Potassium sorbate suppresses the growth of P. roquefortii more strongly than it suppresses patulin production (Bullerman, 1984). If the

Ochratoxin A accumulation during yeast-mould interaction effectiveness of the preservative decreases with time, mould growth can resume, and mycotoxin formation may be enhanced in the somewhat stressful environment. For example, over time, as ammonia slowly loses its preserving ability in high-moisture corn, moulds can grow and mycotoxin can be produced (Vandegraft, Hesseltine & Shotwell, 1975). We have found that both P. anomala and S. cerevisiae strongly reduced ochratoxin A accumulation in vitro by two isolates of P. verrucosum. Since the substrate has an important influence on mycotoxin accumulation, results from experiments in vitro are only indications of what might happen in vivo. In wheat neither ochratoxin A accumulation nor growth of P. verrucosum IBT5010 was clearly reduced. Both growth and ochratoxin A production of P. verrucosum IBT12803 were, however, reduced. P. anomala did not enhance ochratoxin A accumulation in any of the tests but, before it can be affirmed that P. anomala is safe to use in biological control, further mycotoxin studies have to be conducted with other toxigenic grain spoilage moulds, both in vivo and in vitro. We wish to thank Drs Ole Filtenborg and Bjo$ rn Lindman for providing isolates. Prof. Martin Lindberg offered helpful comments on the manuscript. This investigation was financed by the Swedish Council for Forestry and Agricultural Research (SJFR), the Swedish Farmers Foundation for Agricultural Research (SLF), and the Foundation for Strategic Environmental Research (MISTRA).

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