Fusarium Graminearum Schwabe [Telemorph Gibberella

Mvcotoxins No. 41, 1995 41

Stimulation of deoxynivalenol and nivalenol production by dimethyl sulfoxide

Nantarit CHOKETHAWORN, Yasuko WATANABE, Naoko MORI and Takumi YOSHIZAWA

ナンタリチョタヲン,渡邊靖子,森直子,芳澤宅實:ジメチルスルフナキシド

によるdeoxynivalenolとnivalenolの産生促進

Summary

Stimulatory effect of dimethyl sulfoxide (DMSO) on trichothecene biosynthesis by

Fusarium graminearum KU 1352 [deoxynivalenol (DON) producer] and F. graminearum KU 1383 [nivalenol (NIV) producer)] were investigated in yeast extract-polypepton-sucrose

medium. DMSO at concentrations subinhibitory to fungal growth significantly enhanced

production of trichothecenes in both strains. Maximum yields of trichothecenes were 162% (660 ƒÊg/ml) of control at 5% DMSO in the DON producer and 141% (778 ƒÊg/ml) at

2% DMSO in the NIV producer. This is the first report on the enhancement of tricho-

thecene production by DMSO without inhibition of biomass formation.

Key words:Dimethyl sulfoxide,ジメチルスルフォキシド;Deoxynivalenol,デオキシニバレ

ノール;Nivalenol,ニバレノール;Trichothecene mycotoxin,トリコテセンマイコトキシン;Fusarium graminearum,フザリウムグラミネアラム

Fusarium graminearum Schwabe [telemorph Gibberella zeae (Schw.) Petch] is one of the major causative fungi of scb in wheat and barley and rot in corn, and is well-known as a representative species producing nivalenol (NIV) and deoxynivalenol (DON), trichothecene mycotoxins of sesquiterpenoids. The mycotoxins occur worldwide in these plant products intended for human and animal consumption. Hence, the management of these mycotoxins is an important problem in many countries. Regarding the chemical control of trichothecenes, several papers described the inhibition of trichothecene biosynthesis by various chemicals such as flavonoids and furanocoumarines, cytochrome P450 inhibitors ancymidol and menadione, sodium bicarbonate, derivatized biosynthetic precursors of trichothecenes, and fungicide. Some of these inhibitors block the biosynthetic pathway after formation of trichodiene, the hydrocarbon precursor of trichothecenes, to accumulate variable amounts of this precursor. Contrary to these reports, little is known about enhancement of biosynthesis of trichothecenes. Accumulation of informations of this point may provide a basic under- standing of factors influencing formation of mycotoxins. This paper reports the stimulation of biosynthesis of nivalenol (NIV), deoxynivalenol (DON) and their derivatives by dimethyl sulfoxide (DMSO).

* Department of Bioresource Science , Faculty of Agriculture, Kagawa University, Miki, Kagawa 761- 07. 香川大学農学部生物資源科学科(〒761-07香川県木田郡三木町) 42 Mycotoxins

Materials and Methods

Organisms and culture medium Two strains of F. gyaminearum, F. gyamineayum KU 1352 producing DON and its 3-acetate (3-ADON) and F. gyamineayum KU1383 producing NIV and its 4-acetate (4-ANIV), were used in all tests. Stock cultures were maintained on potato dextrose agar slants and stored at 4•Ž. To measure mycotoxin production and growth, the fungi were grown in YEPS medium (pH 7.0) containing 0.25% yeast extract S

(Wako Pure Chem. Ind.), 0.25% polypepton (Wako Pure Chem. Ind.) and 2.0% sucrose. Culture condition One hundred milliliters of YEPS broth was dispensed into a 500 ml shake flask and autoclaved. Each flask was inoculated with mycelia and incubated for 7 days at 27•}1•Ž on a reciprocating shaker (Thomas Kagaku Co.) operating at 110 rpm. To examine the enahcement of toxin production, individual organic chemical was added to the broth at concentrations of 0.5% to 5% (v/v) before inoculation. Chemicals Trichothecene mycotoxins including DON, NIV, 3-ADON and 4-ANIV were prepared in our laboratory (purity, over 97%). Organic chemicals tested were acetone, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, N,N-dimethylacetamide, acetamide and 2-butanone and were of reagent grade. For the derivatization of trichothecenes to tri- methylsilyl (TMS) ethers, a formulation of N-TMS-imidazole, N,O-bis-TMS-acetamide and TMS-chloride (3: 3: 2) was used.

Measurement of fungal growth At the end of incubation period, the mycelia were filtered off and dried at 80•Ž for 22 hr to determine the yield of biomass. In addition, total ergosterol level as an indicator of fungal growth was determined according to the method previously reported. Briefly, a portion of dried mycelia was sponified with 10% KOH in

60% ethanol at 90•Ž for 2 hr followed by extraction with n-hexane. An aliquot of this extract was subjected to high-performance liquid chromatography with an UV detector (282 nm). A reversed phase column (TSKgel ODS-80, 100 mm by 4.6 mm inside diameter,

TOSOH) was used, and a mobile phase was MeOH-H20 (96: 4) at a flow rate of 1 ml/min.

Culture filtrates were also analyzed for ergosterol in a similar manner as above. Total ergosterol was the summation of its levels in mycelia and the filtrate.

Analysis of trichothecenes An aliquot (2 ml) of the culture filtrate was mixed with acetonitrile (6 ml) followed by addition of sodium chloride (ca. 1.5 g). None of detectable amount of trichothecenes was found in the mycelia of two strains. A portion (300 ,al) of the acetonitrile layer salted out was evaporated to dryness in a small test tube under air stream and reacted with 25 ƒÊl of the TMS reagent for 10 min at 60•Ž. The reaction solution was diluted with 500 ƒÊl of n-hexane followed by treatment with 500 ƒÊl of water to decompose unreacted reagent. An aliquot of resulting n-hexane solution was analyzed by gas chromatography with a flame ionizing detector under the following conditions: fused silica capillary column (Hicap CBP1-M25-025, 0.33 mm by 25 m, chemical bonded type); carrier, nitrogen gas at a flow rate of 2.1 ml/min; column temperature, 2 min at 70•Ž, increased to 160•Ž at

15•Ž/min, held for 5 min, and increased to 270•Ž at 4•Ž/min. Quantification of trichothecenes was based on externally similarly derivatized authentic standards. A Shimadzu No. 41, 1995 43

GCMS-QP 2000 gas chromatograph-mass spectrometer was used for the confirmation of trichothecenes under the conditions previously described"'

Results and Discussion

Although it is known that nutrient factors such as carbon and nitrogen sources were re- quired for optimum yield of trichothecene mycotoxins, very limited informations are so far available for chemical stimulators of the toxin production. In the course of our studies on chemical control of trichothecene biosynthesis, we found that the toxin production by F. graminearum was stimulated by DMSO used as a solvent. DMSO is amphiphilic and fre- quently used as a carrier solvent. Its dipole moment, D=3.9, is quite high among organic solvents." We selected several other compounds with relatively high dipole moments including N,N-dimethylformamide, N,N-dimethylacetamide, acetamide, acetone and 2-butanone, and their stimulatory effects on the production of DON and its acetate by F. graminearum KU 1352 were examined. As shown in Table 1, DMSO and N,N-dimethylformamide at concentrations of 0.5% and 1.0% significantly enhanced the production of DON and 3-ADON, of which the latter level was more than three times higher than the former in cultures after 7-days incubation. As for other compounds tested, N,N-dimethylacetamide

Table 1. Effects of several chemicals on growth and production of DON and its derivative by Fusarium graminearum KU1352

1 The fungus was incubated in YEPS medium containing 0% (control), 0.5% and 1.0% of individual chemical. All data are means of three replications. *2 The total amount of DON and 3-ADON produced after 7-days incubation. *3 Numbers followed by the same letter are not significantly different within treatments at P=0.05.

* 44 Mycotoxins

(1%) and 2-butanone (1%) inhibited the formation of both toxins and biomass. Acetamide did not show any significant effect on the formation of toxins and biomass, but DON level was 3.5 times higher than 3-ADON, suggesting the stimulation of hydrolytic bioconversion of 3-ADON to DON by added acetamide. Subinhibitory concentrations of acetone showed a tendency of increase in trichothecenes formation, though statistically insignificant. Similarly, stimulatory effect of low concentrations of acetone was also reported on the biosynthesis of aflatoxins and versicolorin by Aspergillus flavus. Thus, among six chemicals examined, the production of DON and its acetate by F. graminearum KU1352 was enhanced by DMSO and N,N-dimethylformamide up to 132% and 124%, respectively, in comparison to control. It is particularly noteworthy that the stimulatory effect of DMSO was shown at concentrations subinhibitory to fungal growth. Further experiments were carried out to study the stimulatory effect of DMSO using two strains (KU 1352 and KU1383, DON and NIV producers, respectively) of F. graminearum in YEPS media, in which various concentrations of DMSO were added before inoculation. Influences of DMSO on mycelial growth of the two strains, as determined by measurement of ergosterol level, were not the same with different concentrations of DMSO: addition of 10% DMSO resulted in the complete inhibition of mycelial growth of both strains, and 5% DMSO was also inhibitory to KU1383 strain but no effect to the other (data not shown). As shown in Table 2, there was insignificant difference in the ergosterol level at below 5% and below 2% in KU1352 and KU1383 strains, respectively. At these subinhibitory concentrations, stimulation was observed at DMSO levels ranging from 0.5 to 5% in the DON producer and

Table 2. Enhancement of DON and NIV production by DMSO in two strains of Fusarium graminearum

*1 Fungi were incubated in PEPS media for 7 days . DMSO was added to the media before inoculation. All data are means of three replications. *2 The total amount of trichothecenes produced: DON plus 3-ADON for the DON producer , and NIV plus 4-ANIV for the NIV producer. DON and NIV levels were less than 25% and 5% of the total trichothecenes, respectively, in individual cases. *3 The concentration of ergosterol after 7-days incubation was used as an index of fungal

growth. *4 Numbers followed by the same letter are not significantly different within treatments at

P=0.05. No. 41. 1995 45

from 0.5 to 2% in the NIV producer, and these effects were dependent to DMSO levels in

both producers as shown in Table 2. Maximum yields of trichothecenes were 162% (660

ƒÊ g/ml) of control at 5% DMSO in KU1352 strain and 141% (778 ƒÊg/ml) at 2% DMSO in KU1383 strain. Enahcement of trichothecene production by DMSO when administered to

vegetative cultures was also exhibited: 138% and 124% increases in KU1352 and KU1383

strains, respectively, by addition of 5% DMSO at 50 hr (29% of the final toxin level was

produced in KU1352) or 70 hr (20% of the final toxin level in KU1383) after inoculation

(data not shown). Based on these data, it is suggested that DMSO may not affect a biosynthetic pathway from farnesyl pyrophosphate, a common and key precursor of sesquiter-

penoids and steroids, to ergosterol but stimulate a pathway from the precursor to trichothecenes. This is the first report on the enhancement of production of DON, NIV and their derivatives by two chemotypes (DON-and NIV-producers) of F, gyamineayum at subinhibitory concentrations of DMSO. Desjardins et al. reported insignificant effect of 2% DMSO on production of T-2 toxin and diacetoxyscripenol by Gibberella pulicaris (=F.sambucinum) in a define liquid medium. These data suggest an interspecies difference in stimulatory effect of DMSO, which need further investigation. It is reported that DMSO can stimulate lysine production by Brevibacterium flavum, probably due to an enhancement of cell permeability because of its amphiphilic propertity. Although it is also suggested that DMSO may alter the permeability of the cell wall and (or) membrane of F. gyamineayum to enhance the secretion of trichothecenes, secondary metabolites, this point should be clarified by further study. Effect of DMSO on production of secondary metabolites other than trichothecenes by F. gyamineayum will be reported in a subsequent paper.

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