Degeneration of zearalenone production inFusarium graminearum

EXPERIMENTAL

9, 133-140 (1985)

MYCOLOGY

Degeneration

of Zearalenone JANICE

Weizmann Microbial

Production

S. DUNCAN~

in Fusariutn

graminearu

AND JOHN D. BU'LOCK

Chemistry Laboratory, Department of Chemistry, Manchester Manchester Ml3 9PL. England Accepted for publication

University,

February 5, 1985

DUNCAN, I. S., AND BU’LOCK, J. D. 1985. Degeneration of zearalenone production in Fusarium graminearum. Experimental Mycology 9, 133-140. Macroconidia of a high-zearalenone-yielding Fusarium graminearum, NRRL 3198, were exposed to uv radiation and 0.02% nitrosoguanidine. The organism was also serially transferred in malt extract broth for up to 10 transfers. High proportions of morphologically distinctive low-toxin-producing variants (RUP variants) were isolated via unstable intermediates after mutagen treatments. The unstable intermediates readily reverted to high zearalenone producers with wild-type morphology. Stable RUP variants and more degenerate isolates (PR variants) were isolated in large numbers from the serial transfer experiments. Stable RUP variants did not revert to wild type even after uv exposure. Wild-type and RUP auxotrophs showed no reassortment in a heterokaryon test, implying that the RUP variants were not cytoplasmic mutants of NRRL 3 198. Toxin production appeared to be augmented in WTI RUP heterokaryons despite the overall RUP morphologies of these cultures. Experiments with a further toxigenic and a nontoxigenic strain demonstrated that the lability found in NRRL 3198 was not common to all strains of Fusarium graminearum 0 1985 Academic Press, Inc. INDEX DESCRIPTORS: degeneration; Fusarium graminearcun; morphological variants; mycotoxin; zearalenone.

The mycotoxin zearalenone (F-2) is produced by species of Fusarium including some strains of F. graminearum. This species displays considerable variation in culture morphology, particularly pigmentation (Booth, 1971). In addition, the mycotoxin yields from Fusaria can vary substantially between strains and in individual strains under different culture conditions (Eugenio et al., 1970). The variability of mycotoxin production in fungi has significant implications for industries interested in these, or closely related, organisms. Knowledge of the mechanisms by which the toxigenic capabilities of an isolate are lost or can be induced might help to explain the diversity of mycotoxin production that is found among isolates of a species and to define the differences between toxigenic and nontoxigenic strains. The relatively low toxicity of zear-

alenone provides a system whereby these mechanisms can be studied without undue concern about the hazards normally associated with mycotoxins.

’ Present address: Department of Pharmacy, Wisconsin University, Madison, Wisconsin 53706.

* Abbreviations used: MM, minimal medium; RUP, red underpigment; WT, wild type; PP., pale RUP.

MATERIALS

AND METHODS

Organisms. A high-zearalenone-producing strain of F. graminearum, 3198, a less toxigenic strain, NRR and a nontoxigenic strain, @MI 14.5425, were used. These were estimated to produce 700, 140, and less than 1 pg zearalenone per plate culture, respectively. Media and culture techniques. Malt extract agar (Oxoid) was used for routine plating and colony selection. Filtration enrichment for auxotroph isolation employed the minimal medium (MM)’ of Westergaard and Mitchell (1947), with agar (15 g/liter) to provide a solid minimal medium. Serial transfers for the induction of

133 0147-597.5185 $3.00 Copyright All rights

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

134

DUNCAN

AND

generation in toxigenic strains were conducted in malt extract broth. This medium was also employed for the measurement of specific growth rates. All the above cultures were incubated at 22°C. Macroconidial suspensions (3 to 8 nucleate) were obtained from 7-day shake flask cultures of a carboxymethylcellulose medium (Cappellini and Peterson, 1965) at 26°C under a fluorescent strip light. Zearalenone was assayed on plates containing 20 ml of GRCS agar medium, comprising ground rice (50 g/liter), cornsteep liquor (5 g/liter), and agar (15 g/liter), incubated 2 weeks at 22°C and 1 week at 15°C. Mutagenesis by uv light. Macroconidia were aseptically filtered through glass wool to remove mycelial fragments. For the production of auxotrophs, macroconidia were filter-concentrated on three layers of filter paper (Whatman No. 1) and resuspended in 15 ml sterile Ringer’s solution (giving a final conidial concentration of lo7 to 108/ml). Stirred 15-ml aliquots of conidial suspensions were placed 8 cm below a shortwave (254 nm) uv light source (9-W Hanovia germicidal lamp) for predetermined time intervals that resulted in >90% kill. Tenfold dilutions were made in sterile Ringer’s solution prior to plating out. Nitrosoguanidine treatment. Approximately lo8 macroconidia were washed and suspended in 5 ml sterile pH 6.8 sodium phosphate buffer, N-Methyl-N’-nitro-N-nitrosoguanidine (2 mg) was dissolved in a further 5 ml sterile buffer and both this solution and the conidial suspension were placed in a 28°C water bath for 30 min, before the conidia were aseptically transferred to the vessel containing nitrosoguanidine. After 60 min, 0.2-ml aliquots were withdrawn from the suspension and added to 9.8 ml sterile 0.1% (w/v) aqueous cysteine hydrochloride solution, to inactivate the mutagen (Detroy et al., 1973). Further lo-fold dilutions were made in cysteine solution prior to plating (kill rate about 96%).

BU’LOCK

Multiple passage. Triplicate flasks containing 30 or 100 ml of malt extract broth were inoculated with 10 ml conidial suspensions and shaken (150 t-pm, 22°C) for 2 or 4 days. The contents of each flask were macerated for 45 s in a Waring blender (100 ml at a time) and combined. The combined mycelial macerates were then used to inoculate a fresh set of flasks (1 ml to each flask). Ten such transfers were made in each experiment and lo-fold dilutions of the mycelial macerates were plated out onto malt extract agar during each transfer and after incubation and maceration of the 10th transfer. Formation of heterokaryons. Auxotrophs were produced by the filter-enrichment method of Catcheside (1954) and defined according to the method of Holliday (1956). Heterokaryons were forced between complementing auxotrophs by plating washed germinating conidia on minimal medium and transferring presumptive heterokaryotic colonies to fresh MM plates. Component homokaryons were retrieved by macerating mycelia from heterokaryotic colonies suspended in 50 ml sterile Ringer’s solution, inoculating carboxymethylcellulose shake flasks with 10 ml of these macerates, and, after 7 days growth, filtering off the conidia and plating on complete medium. Nutritional requirements of individual colonies were tested using appropriately supplemented minimal medium. Zearalenone assay. Agar and mycelium from each plate were extracted with 40 ml chloroform; the extracts were dried with anhydrous sodium sulfate and filtered. The residues were washed with a further 20 ml chloroform which was filtered and combined with the first extract. The extracts were concentrated to 1 ml and l-3 ~1 of these extracts (diluted where appropriate) were spotted onto 50 x 100 x 0.25-mm precoated silica gel 60 F,,, TLC plates (Merck). A lOO-ng zearalenone reference spot was similarly applied to each TLC

DEGENERATION

OF

ZEARALENONE

plate. Plates were developed 4 cm in diethyl ether, dried, and sprayed with Fast Violet B Salt reagent (Scott et al., 1978). The comparative densities of sample and standard zearalenone spots were measured, after spraying, with an Helena Laboratories Quick Scan densitometer and used to calculate zearalenone quantities. RESULTS

Exposure of NRRL 3 198 macroconidia to uv light or to nitrosoguanidine resulted in the frequent isolation of highly pigmented variants with severely reduced zearalenone production (Figs. 1 and 2). These were designated “RUP” strains (red underpigment). On further incubation, all these RUP colonies were observed to throw out sectors with morphology similar to that of the wildtype (WT) parent isolate. Analysis revealed that these sectors produced high levels of zearalenone. Repeated subculture of hyphal tips from these unstable RUP colonies

135

PRODUCTION

eventually produced stable RUP and stable WT isolates with low and high zearalenone yields, respectively. Unstable RUP cultures passed through a conidial stage (and therefore a uninucleate stage, since in Fusartum the conidia originate from a single nucleus; Booth, 1971) and plated in such a way that colonies were produced from single macroconidia, resulting in the recovery of RUP colonies which also threw out WT sectors. The unstable characteristic therefore appears to have been retained through t uninucleate stage and could not result from RUP/WT heterokaryon formation ~o~low~~g mutagen treatment of the rnult~~uc~cat~ macroconidia. RUP variants were also produced from NRRL 3198 after several successive transfers in malt extract broth. A further set of morphological variants, designated ’ “P strains (pale RUP), were isolated during multiple passage. These isolates were less pigmented than the RUP variants and

A

RANGE

OF F-2

0

w-r

m

RUP

u5IlIl

WT

m

unsegregated

Ez2

others

PRODUCTION

FIG. 1. Zearalenone production of morphological conidia.

lpg

from

unstable

RUP

WT/RUP

plateS’l

variants produced by uv exposure of NRRL 3 1978

136

DUNCAN

RANGE

2. Zearalenone production macroconidia to nitrosoguanidine. FIG.

OF

AND BU’LOCK

F-2

LO

w-f

m

RUP

DlITIa

WT

m

unsegregated

m

others

PRODUCTION

of morphological

not produce detectable levels of zearalenone. The production of RUP and PR strains during these serial transfers suggested a sequential degeneration of WT through RUP to PR (Fig. 3) and multiple

0

from

unstable

RUP

WT/RUP

IPg plate?

variants produced by exposure of NRRL 3198

passage of a RUP variant resulted in the isolation of PR strains after only three transfers. The production, or sequence of isolation, of RUP and PR variants was not affected by changing the volume of medium in each flask or the incubation period between transfers, and after 10 such transfers these variants comprised 86 to 100% of the population. Comparison of the specific growth rates of WT, RUP, and PR cultures, measured under these conditions (Table l), indicated that the sequential degeneration with respect to toxin production was ac-

TABLE 1 Specific Growth Rates of Variants from Multiple Passage of NRRL 3 198, Measured during the Phase of Exponential Growth in Shake Flask Cultures

2ol

r, 0

m 1

P

R

2

3

4

5

6 TRANSFER

7

8

9

10

.

NO

FIG. 3. Variants arising during multiple passage of NRRL 3198 mycelium. q , WT; 0, RUP; 0, PR.

Isolate type

Growth rate (h-t)

WT RUP PR

0.346 0.289 0.277

DEGENERATION

Proportions Total colonies

OF ZEARALENONE

137

PRODUCTION

TABLE 2 of Phenotypes Recovered from Segregated WT il vallRUP inos heterokaryons WT il val

WT inos

RUP il val

RUP inos

195 180 19.5

116 128 127

0 0 0

0 0 0

79 52 68

Average (%)

65

0

0

35

companied by a parallel reduction of growth rate. RUP and PR variants from the multiple passage did not produce WT sectors and retained their low or nontoxigenic properties even after uv irradiation. All RUP variants from uv- or nitrosoguanidine-exposed NRRL 3198 macroconidia, when subcultured to produce nonsectoring RUP cultures, were also stable to uv irradiation. No reassortment was observed in heterokaryon tests between WT il val and RUP inos or RUP hypox auxotrophs (Tables 2 and 3). The high frequency of generation of RUP cultures from mutagen-treated NRRL 3 198 macroconidia and their inherent instability could not therefore be explained by a cytoplasmic mutation. Since no protrophic colonies were isolated during segregation of the heterokaryons, there was no evidence of the formation of heterozygous diploids or parasexual recombinants. The heterokaryons grew more slowly than either WT or RUP prototrophs on minimal medium, and they rapidly broke down to give the component homokaryons when transferred on to nonselective media. Despite the apparent higher proportion of WT colonies recovered from the WTiRUP het-

Proportions

erokaryons, it was observed that the heterokaryotic cultures displayed little of the abundant aerial mycelium associated with WT strains, and they accumulated red pigments which were similar to RUP variants. It was not possible to screen zearalenone production by the forced heterokaryons on GRCS agar (owing to their instability on this medium), but the zearalenone yields of the WT il val/RUP inos heterokaryon an of NRRL 3 198 could be compared on MM ~ while those from the components of tke heterokaryon and NRRL 3198 could be compared on GRCS agar (Table 4). On GRCS agar the RUP inos component produced no zearalenone even when inositol was supplied, whereas the WT ileu val cornponent was zearalenone-positive as expected. On MM agar, somewhat surprisingly, the heterokaryon produced significantly more zearalenone than did the parental NRRL 3198 culture. The low level of conidiation from the PR strains prevented the completion of heterokaryon tests between PR and RUP auxotrophs. A few heterokaryotic colonies were isolated following protoplast fusion of adenine-requiring auxotrophs of the nontoxigenie strain, CM1 145425 (iVZad), and the

TABLE 3 of Phenotypes Recovered from Segregated WT il vaNRiP

hypox Heterokaryons

Total colonies

WT il val

WT hypox

RUP il val

RUP hypox

99 88

78 68

0 0

0 0

21 22

Average (%)

78

0

0

22

138

DUNCAN

Zearalenone Production by WT il val/RUP

AND &LOCK TABLE 4 inos Heterokaryon

Culture NRRL 3198

and by Auxotrophic

Components

F-2 produced on GRCS ((*g/plate)

F-2 produced on MM (pg/plate)

800

50

50 400

-

0 0

-

WT il val

Without lieu + val With lieu + val R UP inos

Without inos With inos WT il vallRUP

inos

heterokaryon

RUP inos variant. No zearalenone was detected in the cultures of this heterokaryon, which was also very unstable and grew extremely slowly. The extreme lability of the NRRL 3198 strain, under defined conditions, was not found in the two other strains tested. Although some variation in toxin yields was obtained after uv exposure and multiple passage of the zearalenone-producing strain, NRRL 5883, the number of nontoxigenie strains isolated and the range of zearalenone production in these isolates were greatly reduced compared to the NRRL 3198 results. Out of 74 isolates screened after uv irradiation of NRRL 5883 macroconidia, only 3 variants with significantly reduced zearalenone production were isolated. Only 1 nontoxigenic variant was isolated from the multiple passage of this strain, out of 28 tested. Furthermore, uvirradiated macroconidia of the nontoxigenic strain, CM1 145425, retained the negative zearalenone-producing characteristics of the organisms, although several morphological variants were produced among the 110 isolates tested. DISCUSSION

The high frequency (about 10%) with which the RUP strains were isolated after mutagen treatments of NRRL 3198 macroconidia seems to preclude the involvement of a normal chromosomal mutation.

-

90

For comparison, experiments to select for pimaricin- and cycloheximide-resistant mutants using either uv light or nitrosoguanidine yielded less than 1:106 resistant:nonresistant phenotypes. The RUP variants were always isolated in the unstable form after either treatment of NRRL 3198 conidia. This unstable RUP strain, however, was not found among the isolates from the multiple passage experiments, but since in these experiments only microcolonies of the variants were counted after successive transfers, it is possible that any instability not immediately apparent was overlooked. Moreover, in these experiments, it would have been difficult to distinguish such colonies from heterokaryons. It is thus probable that the stable RUP variants induced from the unstable isolates by subculture on agar medium arose in a way similar to the stable RUP strains obtained from the serial transfer experiments. The lack of reassortment in the heterokaryon tests clearly demonstrated that the RUP condition was associated with changes in the nucleus rather than the cytoplasm. However, as shown in Tables 2 and 3, the ratios of WT:RUP colonies that were recovered following sporulation of the heterokaryotic cultures were not compatible with the overall RUP appearance of the original heterokaryon. Certainly, the procedures employed to segregate heterokaryons would not be expected to reflect

DEGENERATION

OF

ZEARALENONE

the nuclear composition of each heterokaryotie culture accurately, but since sporulation by the RUP component was also consistently found to be more prolific than by the WT strains, it is intriguing that the numbers of WT colonies recovered should be consistently greater than RUP colonies. Significantly, a nuclear-based control of toxin production was demonstrated by Bennett et al. (1980) in Aspergillus parasiticus and was also suggested for F. tricinctum and F. solani by Cullen et al. (1983). Zearalenone production by the WT il vail RUP inos heterokaryon on MM was observed to be greater than from the NRRL 3198 culture (Table 4). This is significant since both the WT il vu1 and the RI/P inos auxotrophs produced markedly less zearalenone than the parent on GRCS agar, implying that the RUP trait does not in itself suppress zearalenone biosynthesis and in fact toxin production was augmented in the heterokaryon. Such observations would appear to contradict the association of RUP morphology with reduced zearalenone production. If there were disproportionately higher numbers of WT over RUP nuclei in the heterokaryon (as indeed was suggested by the greater numbers of WT colonies recovered following segregation of all WT/ RUP heterokaryons), then this could perhaps partly account for the high toxin yield of the WT il vallRUP inos heterokaryon, but on the other hand the RUP morphology, slow growth and yet increased zearalenone production compared to the WT prototrophic culture, suggests that it is likely that some regulatory or cosynthesis mechanism was also involved. The absence of toxin production by the RUP inoslNZ ad heterokaryon clearly demonstrates an absence of complement&ion between these two strains. In contrast to NRRL 3198, the comparative stability of NRRL 5883 and the absence of recognizable, sectoring, morpho-

PRODUCTION

13

logical variants indicated that the previously observed instability was not common to all toxigenic variants of F. graminearum. Admittedly, the zearalenone yields from this strain were found to be markedly lower than from NRRL 3198; however, variants with significantly increased toxin production were also absent after uv exposure or multiple passage of NRRL 5883. The zearalenone-producing characteristics of this strain were therefore notably more stable than those of NRRL 3 198. Similarly the nontoxigenic variant, CMI 145425, could not be induced to produce even low levels of zearalenone. It would appear that toxin production is apparently more easily lost from, than induced in, a species like F. graminearum. While no definite mechanism can be given for the instability of NRRL 3198, there are several plausible explanations. Many of the characteristics of RUP are similar to those described by Booth (1971) an attributed to “‘senescence” and “degeneration,” but little is known about the mechanisms that govern such changes an ability in this species. In Podospora anserina, senescence has been associate with a plasmid-like cccDNA originati the mitochondria (Esser et al., 198(b). nett et al. (1980) suggested the involvement of plasmids, episomes, trans~oso~s) or other such dispensable genetical factors in the regulation of mycotoxin production in A. flaws, and such elements may well be involved here. An intriguing phenomenon was the consistent isolation of the intermediate unstable RUP variants. This implies the involvement of a labile transient state duri degeneration. Interestingly, transfer of hyphae to, or plating WT spores on, m containing 0.007% of the haploi~~zati~~ agent p-fluorophenylal ine also resulted in the isolation of unst le RUP variants, whereas both RUP and PR were u~af~e~~~ under these conditions. Although this is in-

140

DUNCAN

AND

sufficient evidence to conclude anything concerning the ploidy of NRRL 3198, a mechanism involving haploidization of WT cultures via unstable aneuploids to haploid RUP strains can be envisaged. This theory does not, however, accommodate the further degeneration to the PR strains in the multiple passage, and clearly additional work is required to understand the mechanisms involved. Burnett (1984) discusses various aspects of Fusarium genetics, including sequential morphological variation in F. oxysporum and F. solani arising during prolonged culture and mass mycelial transfers. The various forms described are not unlike the degenerate variants from NRRL 3198 with respect to morphology and stability. However, since there are practically no data on the genetic basis of either morphological variation or the vast array of secondary products synthesized by the genus Fusarium, the results presented here take on some significance. ACKNOWLEDGMENTS

This work was supported by a grant from the Science Engineering Research Council under the Total Technology Programme. Further assistance from the Lord Rank Centre is also gratefully acknowledged. REFERENCES BENNETT, J. W., WHEELER, D. G., AND DUNN, J. J. 1980. Genetic analysis of aflatoxin production by Aspergillus

parasiticus.

In Advances

in Biotech-

BU’LOCK nology, Vol. 3, Fermentation Products (M. MooYoung, C. Vezina, and K. Singh, Eds.), pp. 41?422. Pergamon Press, New York. BOOTH, C. 1971. The Genus Fusarium. Commonwealth Agricultural Bureau. BURNE’IT, J. H. 1984. Aspects of Fusarium genetics. In The Applied Mycology of Fusarium (M. 0. Moss and J. E. Smith, Eds.), pp. 39-69. Cambridge Univ. Press, Cambridge. CAPPELLINI, R. A., AND PETERSON,J. L. 196.5.Macroconidium formation in submerged cultures by a nonsporulating strain of Gibberella zeae. Mycologia 57: 962-968. CATCHESIDE, D. G. 1954. Isolation of nutritional mutants of Neurospora crassa by filtration enrichment. J. Gen. Microbial 11: 34-36. CULLEN, D., SMALLEY, E. B., AND DIMOND, R. L. 1983. Heterokaryosis in Fusarium tricinctum and F. sporotrichoides. J. Gen. Microbial. 129: 3035-3041. DETROY, R. W., FREER, S., AND CIEGLER, A. 1973. Aflatoxin and anthraquinone biosynthesis by nitroguanidine-derived mutants of Aspergillus parasiticus.

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ESSER, K., TUDZYNSKI, P., STAHL, II., AND KUCK, U. 1980. A model to explain senescence in the filamentous fungus Podospora anserina by the action of plasmid-like DNA. Mol. Gen. Genet. 178: 213-216. EUGENIO, C. P., CHRISTENSEN, C. M., AND MIROCHA, C. J. 1970. Factors affecting production of the mycotoxin F-2 by Fusarium roseum. Phytopathology 60: 1055-1057. HOLLIDAY. R. 1956. A new method for the identification of biochemical mutants of microorganisms. Nature (London)

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SCOTT, P. M., PANALAKS, T., KANHERE, S., AND MILES, W. F. 1978. Determination of zearalenone in cornflakes and other corn-based foods by thin layer chromatography, HPLC and GLC/high resolution mass spec. J. Assoc. Off. Anal. Chem. 61: 593-600. WESTERGAARD, M., AND MITCHELL, H. K. 1947. A synthetic medium favouring sexual reproduction. Amer.

J. Bot.

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