Cultural degeneration in two Fusarium species and its effects on toxigenicity and cultural morphology

Mycol. Res. 99 (5): 615-620 (1995)

615

Printed in Great Britain

Cultural degeneration in two Fusarium species and its effects on toxigenicity and cultural morphology

N. WING!, L. W. BURGESS! AND W. L. BRYDEN 2 1 2

Fusarium Research Laboratory, Department of Crop Sciences, University of Sydney, NSW 2006, Australia Mycotoxin Research Group, University of Sydney, Camden, NSW 2570, Australia

Ten isolates each of Fusarium compactum and Fusarium acuminatum subsp. armeniacum were sub-cultured using single hyphal tips (HT) and single germinated macroconidia (SM) for ten generations. Cultural degeneration to intermediate, mycelial or pionnotal cultures was observed in a large proportion of cultures of both fungi, but pionnotal cultures were only produced in sub-cultures using SM transfers. Some reversion of intermediate and mycelial cultures to the wild type was observed. However, pionnotal cultures once formed were stable and did not revert to other cultural types in later generations. The toxicity and trichothecene production of degenerate cultures were comparable to those of wild type cultures. Colony diameters of pionnotal cultures were reduced in comparison with wild type cultures. The pigmentation of all degenerate cultures remained consistent with that observed in wild type cultures. Chlamydospore production was independent of cultural degeneration, as all cultural types produced chlamydospores equally well as the parental wild types.

Wild type cultures of Fusarium species may degenerate with repeated sub-culturing to give rise to pionnotal or mycelial cultures (Hansen, 1938; Snyder & Hansen, 1954; Nelson, Toussoun & Marasas, 1983; Burgess, Liddell & Summerell, 1988). Pionnotal cultures are characterized by an abundance of sporodochia and a lack of mycelium, while mycelial cultures produce abundant mycelium and few if any sporodochia. Cultures that partially degenerate are termed intermediate (Hansen, 1938; Oswald, 1949). The frequency of degeneration of Fusarium species varies both between and within species (Oswald, 1949; Snyder & Hansen, 1954; Snyder & Toussoun, 1965; Toussoun & Nelson, 1975; Puhalla, 1981). Cultural degeneration has been associated with the nature of the culture medium (Brown, 1926; Booth, 1971), the age of the culture (Booth, 1975) and the methods by which the culture is propagated. The transfer of cultures by single germinated macroconidia, for example, has been associated with the occurrence of degenerate cultures (Hansen & Smith, 1932; Leonian, 1932; Oswald, 1949; Snyder & Toussoun, 1965; Booth, 1971). Some workers have suggested that subculturing by means of a single hyphal tip or single microconidium may reduce the occurrence of cultural degeneration (Puhalla, 1981; Burgess et al., 1988). However, other workers have argued that a single macroconidium and a single hyphal tip have an equal probability of producing degenerate cultures (Brown, 1926; Ullstrup, 1935). Cultural degeneration has also been associated with changes in the pathogenicity of some Fusarium species (Tu, 1929; Ullstrup, 1935; Wellman & Blaisdell, 1941; Oswald, 1949;

Cormack 1951; Bolton & Donaldson, 1972; Awuah & Lorbeer, 1988). Degenerate cultures may be more or less pathogenic than the wild type parental cultures. Some workers have found that mycelial cultures tend to be more pathogenic than pionnotal cultures (Ullstrup, 1935; Awuah & Lorbeer, 1988).

There have been relatively few studies on the effect of repeated transfer of cultures of Fusarium species on the toxigenicity of those species. The use of chemical and uv radiation techniques to induce permanent mutations in cultures of some species has resulted in either a loss of ability to produce a specific toxin (Duncan & Bu'Lock, 1985; Giesen, Glenn & Leistner, 1990) or a change in the type of toxins produced (Plattner, Tjarks & Beremand, 1989). However, the effect of degeneration induced by repeated transfer on toxin production may be quite different from that of mutation induced by chemical or other means. Some authors have suggested that repeated sub-culturing of some Fusarium species is associated with a loss in toxigenicity Goffe, 1962; Kirksey & Cole, 1974; Duncan & Bu'Lock 1985) The studies reported in this paper were designed to assess both the morphological and physiological changes that result from repeated transfer using single germinated macroconidia and single hyphal tips in Fusarium compactum (Wollenw.) W. L. Gordon and Fusarium acuminafumEllis & Everh. subsp. armeniacumG. A. Forbes, Windels & L. W. Burgess, which are known to degenerate readily in culture (Burgess ef aI., 1988) and have been shown to be highly toxigenic (Nelson ef aI., 1990; Wing ef aI., 1993 a).

Cultural degeneration in Fusarium species

616

MATERIALS AND METHODS Cultures. Cultures of F. compacfumwere isolated from a sample of cultivated soil from Alice Springs in the Northern Territory, Australia using the soil dilution plate technique and the selective medium of Nash & Snyder (1962). The fungi were sub-cultured onto carnation leaf piece agar (CLA) for identification (Burgess et al., 1988) and from these cultures, ten representative wild type isolates of F. compactumwere selected and defined as the original cultures. Ten representative wild type isolates of F. a. armeniacum were established from lyophilized cultures stored at 5 °C in the Fusarium Research Laboratory. Three isolates of F. a. armeniacum(FI454, F6885, F7972) originated from soil samples and two from wheat stem (F6637, F6963) from New South Wales, Australia; two from cornstalk (F5188, F5190) from Minnesota, U.S.A.; two from oat seed (F9470, F9472) from the Transvaal and one from soil (F9691) from Natal, South Africa. The term 'isolate' is used to distinguish original cultures from those produced by repeated transfer in subsequent generations. All cultures were grown in a controlled environment room with an alternating temperature of 25° day, 20° night and a photoperiod of 12 h. Each original culture on CLA was then used as the source of inoculum from which repeated transfers were made by two methods, a single germinated macroconidium (SM) and a single hyphal tip (HT). For the transfer of SM, a water agar (WA) plate (8 em diam.) was seeded with 1 ml of a suspension of spores in sterile water (Hansen & Smith, 1932). Excess water was shaken off and the plate incubated on an angle for 18 h at 25°. Single germinated conidia were transferred to CLA and potato dextrose agar (PDA) and grown for 10-14 d under the growth conditions described above. The procedure for transfer by HT was adapted from that described by Burgess et al. (1988). WA was poured into a Petri dish (10 em diam.) which was then placed at an angle of 30-40° until the agar had set. A 0'5 em mycelial plug from a CLA culture was then placed on the deeper sector of the water agar and incubated for 4 d in the dark at 25°. Hyphae developing across the shallow sector were sparse,

First generation

HT Second generation

SM Ten SM cultures

HT

SM

Tenth generation

Fig. 1. General outline of transfer procedures by a Single germinated macroconidium (8M) and a single hyphal tip (HT) for ten isolates of Fusarium compaetum and Fusarium acuminafum subsp. armeniacum over ten generations.

G3

Assessment of morphology and inoculum for toxicity testing

Fig. 2. Outline of the transfer procedure of a single germinated macroconidium (8M) and a single hypha! tip (HT) from the reference culture in each generation (G). The media referred to are carnation leaf agar (CLA), water agar (WA) and potato dextrose agar (PDA).

enabling transfer of individual hyphal tips onto new media to establish the next generation. The transfer procedures are summarized diagrammatically in Fig. 1. The ten isolates of each of F. compacfum and F. a. armeniacumwere transferred repeatedly by both SM and HT for ten generations. At each generation, a total of five single hyphal tips and five single macroconidia were individually transferred from WA plates to new agar plates. Two of the five were transferred to two CLA plates (5 em diam.), one per plate, and the remaining three were individually transferred to three separate PDA plates (10 em diam.) (Fig. 2). One of the CLA cultures, called the 'reference culture', was used for all morphological assessments, including culture type, spore measurements and chlamydospore production. This culture was also used as inoculum for toxicity testing and for initiating the next generation. Reference cultures derived from SM were grown for 10-12 d before each transfer. Reference cultures derived from HT were grown for 8-10 d before being inoculated onto WA plates, in preparation for the next transfer. The other CLA culture at each generation was lyophilized. Colony pigmentation and general morphology were assessed on one PDA culture. The nature of the culture on this plate was also noted if morphology differed from that of the reference culture. The other two PDA cultures were used for determining colony diameters at 25 and 30°.

Assessment of cultural morphology. The morphology of the Reference Culture initiated from each transfer procedure in each generation was examined and classified in terms similar to those described by Hansen (1938), Oswald (1949) and Awuah & Lorbeer (1988). Four cultural types were recognized; wild type, where cultural morphology was consistent with that observed in wild type cultures; intermediate, where partial degeneration was evident; mycelial, where cultures

N. Wing, L. W. Burgess and W. L. Bryden produced abundant aerial mycelium and few or no sporodochia; and pionnotaL where cultures were characterized by an abundance of sporodochia and a lack of aerial mycelium.

Toxicity testing. Original cultures and the reference cultures of F. compactum and F. a. armeniacum in selected generations were used as inoculum to establish Weet Bix® cultures (WB) for toxicity testing (Fig. 2), as described by Wing et al. (1993 b). Briefly, cultures were grown on a WB medium at 25° for 14 d in the dark. extracted and tested for toxicity using a chick bioassay as described by Kirksey & Cole (1974). Trichothecene analysis. Selected culture extracts of F. a. armeniacum were also screened for trichothecene derivatives to determine whether the level of trichothecenes produced by the fungus declined after repeated transfer. Trichothecene analysis was done using a method adapted from Lauren & Agnew (1991), and described by Wing et al. (1993 b). Briefly, the solid medium was extracted with solvent and after preliminary clean-up, the extract was hydrolysed to convert all trichothecenes to the parent alcohols, then passed through a carbon column for final clean-up. The samples were analysed for the four main trichothecene families, nivalenol (NIV), deoxynivalenol (DON), scirpentriol (Sctol) and T-2 tetraol (T2tol) by gas chromatography (GO/electron capture detection (ECD). Colony diameters. The colony diameters of one PDA culture incubated at 25° and one at 30° in each generation were measured after 72 h incubation in the dark. Pigmentation. Pigmentation in the agar of the PDA cultures grown under alternating light conditions was assessed according to Kornerup & Wanscher (1978). Macroconidia. The shape and length of macroconidia were assessed at each generation using the reference culture on CLA (Fig. 2). Spore suspensions of macroconidia were prepared from each of three sporodochia (two on carnation leaf pieces and one on the water agar) and the lengths of macroconidia were measured. Measurements were made on six to eight macroconidia that appeared the shortest, those that were intermediate and those that appeared the longest in each spore suspension. Chlamydospore production. The production of chlamydospores in the reference culture was assessed semiquantitatively. The mean of the chlamydospores observed in three fields of view under the 10 x objective on a compound microscope was noted and assessed according to the following index: 0, No chlamydospores; 1, less than five chlamydospores; 2, 5-15 chlamydospores and 3, more than 15 chlamydospores per field of view.

RESUL IS Cultural morphology. All isolates of F. compactum and all but one of F. a. armeniacum degenerated following repeated transfer for ten generations. The cultural types produced by

617 each transfer method were generally similar after ten generations, although cultures transferred by HT usually degenerated at least one generation later than those transferred by SM (Fig. 3). A culture of an isolate on CLA was generally classified as the same cultural type as that observed on PDA, but degeneration was usually more obvious on PDA. No pionnotal cultures were produced following HT transfer of isolates of both fungi. There was some reversion of both intermediate and mycelial cultures to wild type cultures. However, pionnotal cultures remained stable with no reversion back to parental wild types. The appearance of germinated macroconidia on the water agar plate prior to transfer provided some indication of the nature of cultures produced in the next generation. The germtubes of macroconidia that gave rise to pionnotal or partly pionnotal cultures were knotted and distorted in appearance on the water agar and germinated slowly compared with macroconidia that produced wild type cultures in the next generation. The appearance of hyphae and hyphal tips on the water agar plates gave no indication of the nature of the cultures produced in the next generation.

Toxicity. All isolates of F. compactum and all but one of F. a. armeniacum remained highly toxic in the bioassay after ten generations of transfer. Wild type and degenerate cultures of most isolates produced 100% mortality in the bioassay. Pionnotal cultures of both fungi only partially colonized the WB medium yet proved to be highly toxic in the bioassay. Trichothecene analysis. T-2tol was the only trichothecene family detected in hydrolysed culture extracts of F. a. armeniacum. The levels of T-2tol detected in culture extracts of an isolate varied (Table 1), with some cultures from the tenth generation prodUcing higher levels of T-2tol and some producing lower levels. Mean colony diameters. Intermediate and mycelial cultures of F. compactum grew more slowly at 25° than wild type cultures, but had similar growth rates at 30° (Table 2). In contrast, mycelial cultures of F. a. armeniacum on average grew more rapidly at both 25 and 30° compared with wild type cultures. Pionnotal cultures of both fungi grew more slowly than wild type cultures at both 25 and 30°. Pigmentation. The pigmentation of degenerate cultures of both fungi on PDA did not vary greatly from that produced by wild type cultures. Macroconidia. Suspensions of macroconidia taken from sporodochia formed on carnation leaf pieces and the agar revealed that most cultural types of both fungi produced a wide range of macroconidia. Macroconidia taken from sporodochia on the agar varied more in length than those taken from sporodochia on the carnation leaf pieces. In all cultural types of F. compactum most macroconidia were 25-37'5 IJm in length, although the size range of macroconidia varied from 12 to 75 IJm. Cultures of F. a. armeniacum mostly produced macroconidia of 37'5-75 IJm in length, although the size range of macroconidia produced varied from 20 to

Cultural degeneration in Fusarium species

618 ISOLATE

(a)

F8391

F8393

F8397

F8400

6

o

6

o

Q

6

Q

6

G3

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G4

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o

Q

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G8

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G9

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G2

G6 G7

6

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G

A T

F8396

o

Gl

G5

E N E R

F8395

GI0

(b)

I

• F1454

F5190

o

F8405

o

F8407

F8408

Q

6

o

o

D

F5188

F8402

•

o F6637

F6885

F6963

F7972

o F9470

F9472

F9691

o N (G)

6

o

Gl

o

6

Q

6

6

Q

G2

[j

6

Q

6

6

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G3

[j

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6

6

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o

Q

G4

[j

Q

o

6

[j

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G5

[j

Q

6

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G6

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6

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Q

G7

[j

[j

6

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G8

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6

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o

G9

[j

6

o

o

[j

GIO

[j

6

o

o

o

o

o

6

o

• • • • •

Fig. 3. Degenerate cultural types produced in ten isolates of (a) Fusarium compactum and (b) Fusarium acuminaiumsubsp. armeniacum repeatedly transferred by a single germinated macroconidium and a single hyphal tip for ten generations. - , HT; ... " SM; 0, wild type; D, intermediate; O. mycelial; e. pionnotal.

150 j.lm. The greatest variation in the length of macroconidia was usually observed in degenerate cultures. These cultures often produced very long macroconidia which were usually thin and spindle-like. Short, broad conidia were also observed more frequently in degenerate cultures. These shod conidia were usually 2-3 septate, rounded at both ends, and lacked a foot-shaped basal cell.

Chlamydospore production. The production of chlamydospores on CLA remained relatively consistent over the ten

generations regardless of cultural type (Table 3). Most cultural types of F. compaetum produced a moderate amount (at least five per field of view) of chlamydospores, while most cultural types of F. a. armeniacumproduced low to moderate numbers of chlamydospores in each generation (mostly less than five per field of view).

DISCUSSION Cultural degeneration occurred commonly in isolates of

N. Wing, L. W. Burgess and W. L. Bryden

619

Table 1. T-2tol derivatives produced by cultures of Fusarium acuminarnm subsp. armeniacum in the original (GO) and tenth generation (G 10) after transfer by a single germinated macroconidium (SM) and a single hyphal tip (HT). Values in parentheses indicate the number of deaths that occurred in four chicks dosed with each culture extract T-2tol production (~g

Isolate

g-l)

Go

F1454

23 (4)

F5I88

42 (4)

F7972

255 (4)

F9470

30 (4)

GlO

58" 188" 34" 113 b 118" 158" 10" 3'8"

(4) (4) (4) (4) (4) (4) (3) (1)

" Culture derived from SM transfer. " Culture derived from HT transfer.

Table 2. Mean colony diameters of wild type (WT), intermediate (I), mycelial (M) and pionnotal (P) cultures of Fusarium compaclum and Fusarium acuminalum subsp. armeniacum after 3 d growth on potato dextrose agar at 25 and 30°

°c

M

WT

F. compaclum

25 5'0 ± 0'06' 30 5'2±0'04 F. a. armeniacum 25 5'6 ± 0'06 5'6±0'06 30 " Standard error of the mean.

4'5±0'05 5'I±0'05 5'7±0'06 5'5±0'06

P

4'6±0'06 3'I±0'07 5'0 ± 0'05 3'3±0'06 6'2±0'08 3'3±0'24 6'1±0'08 2'8±0'40

Table 3. Chlamydospore production by wild type (WT), intermediate (I), mycelial (M) and pionnotal (P) cultures of Fusarium compaclum and Fusarium acuminalum subsp. armeniacum Number of cultures Chlamydospores/ field of view"

F. compaclum

Culture type

Total number of cultures

WT I M

48' 70 73

0 0 0

P

F. a. armeniacum

o 7 5 9

2

3

17 30 17

24 35 47

8

0

0

1

7

WT I M

73 84 33

2 12

5

43 38 28

24 28 0

4 6 0

P

10

0

8

2

0

" Chlamydospore production as classified by the following index: 0, no chlamydospores; 1, less than five chlamydospores; 2, 5-15 chlamydospores and 3, more than 15 chlamydospores per field of view. , One culture was not assessed.

F. compactum and F. a. armeniacum following transfer by SM and HT. While some reversion of intermediate and mycelial cultures to wild type cultures was observed in both fungi, no reversion of pionnotal cultures occurred, a conclusion which supports earlier findings (Eide, 1935; Ullstrup, 1935; Oswald, 1949).

Transfer of wild type cultures of both fungi by HT was

effective in reducing degeneration in cultures in the first few generations, as suggested previously by some workers. The lack of pionnotal cultures produced by HT transfer of both fungi also suggests that this may be the preferred method of transfer when handling cultures of a species that is known to degenerate. The similar toxigenic ability of wild type and degenerate cultures of F. compactum and F. a. armeniacum indicates that degeneration is not necessarily associated with a loss of toxicity of a culture, despite dramatic morphological changes occurring within the culture. Interestingly, these findings are in direct contrast to reports on the relationship between degenerate cultures of some Fusarium species and pathogenicity. A number of workers have found that degenerate cultures, in particular pionnotal cultures, of some Fusarium species are less pathogenic than parental wild types (Wellman & Blaisdell, 1941; Awuah & Lorbeer, 1988). The variations in T-ltol detected in hydrolysed culture extracts of F. a. armeniacum are indicative of the inherent variation in toxin production that can occur within cultures of a species and especially of Fusarium species (Nelson ef al., 1991). Such variation makes it difficult to determine trends in toxin production. However, the ability of some degenerate cultures of F. a. armeniacum to produce higher levels of trichothecenes than wild type cultures in this study indicates that degeneration is not necessarily associated with a decline in toxin production. This finding is contrary to earlier reports suggesting that toxin production by F. graminearum (Duncan & Bu'Lock, 1985) and A. flavus (Torres ef aI., 1980) declines with repeated transfer. However, it should be noted that crude methods of detecting and quantifying toxin production were employed in these earlier studies. The colony diameters of pionnotal cultures were significantly lower than those recorded for wild type cultures, which supports findings by Ullstrup (1935) that cultures which have less abundant aerial mycelium grow more slowly. However, these findings are contrary to those reported by Awuah & Lorbeer (1988), who found no difference in the radial growth of mycelial and pionnotal cultures of Fusarium oxysporum. It was also observed in this study that the germtubes of germinating macroconidia that gave rise to pionnotal cultures in the next generation often appeared knotted on water agar plates, a consequence of repeated branching in different directions, and germinated slowly, an observation similar to that reported by Cormack (1951). There was large variation in the length of macroconidia observed in wild type cultures of F. compactum and F. a. armeniacum, but the shape of the macroconidia was characteristic of these species. In contrast, macroconidia in degenerate cultural types were highly variable with respect to shape and length as has been reported by many workers (Snyder & Toussoun, 1965; Toussoun & Nelson, 1975). The presence of short, broad conidia was most obvious in degenerate cultures although these conidia were observed infrequently in some wild type cultures. These conidia appear to be intermediate to macroconidia and microconidia and in previous reports may have been referred to as microconidia (Rabie ef al., 1986). However, they may also be immature macroconidia. The function of these short, broad conidia is not

Cultural degeneration in Fusarium species known and although they appear more prevalent in degenerate cultures, no definite relationship could be established between their presence and the morphology of a degenerate culture. There was little variation in the pigmentation of wild type and degenerate cultures for both fungi. Similarly, chlamydospore production, while reported elsewhere to be reduced in degenerate cultures (Awuah & Lorbeer, 1988), was found to be independent of cultural degeneration in our study. The study reported in this paper emphasizes the frequency with which cultural degeneration occurs in some Fusarium species when transferred using standard laboratory procedures. Such findings suggest that many taxonomic problems associated with Fusarium species have probably been compounded by the ability of some Fusarium species to degenerate in culture. However, in this study we found that the toxigenicity of a species is not lost as a result of complete cultural degeneration. The ability of degenerate cultures to sustain toxin production suggests that either the mycotoxins or the secondary metabolism involved in their production may contribute to the survival and proliferation of these fungi in the soil ecosystem. The authors would like to thank S. Suksupath for technical assistance and Dr H. Rose and J. Zhang for their assistance with trichothecene analysis by GCjECD. This work was supported by the Australian Pig Research and Development Corporation. REFERENCES Awuah, R. T. & Lorbeer, J. W. (1988). Nature of cultural variability in Fusarium oX!fsporum f.sp. apii race 2. Phytopathology 78. 385-389. Bolton. A. T. & Donaldson, A. G. (1972). Variability in Fusarium solani f. pisi and F. oxysporum f. pisi. Canadian Journal of Plant Science 52, 189-196. Booth, C. (1971). The genus Fusarium. The Commonwealth Mycological Institute; Kew, Surrey. Booth. C. (1975). The present status of Fusarium taxonomy. Annual Review of Phytopathology 13, 83-93. Brown, W. (1926). Studies in the genus Fusarium. IV. On occurrence of saltations. Annals of Botany 40, 223-243. Burgess, L. W., Liddell, C. M. & Summerell, B. A. (1988). Laboratory Manual for Fusarium Research. The University of Sydney. Cormack, M. W. (1951). Variation in the cultural characteristics and pathogenicity of Fusarium avenaceum and F. arthrosporioides. Canadian Journal of Botany 29, 32-45. Duncan, J. S. & Bu'Lock. J. D. (1985). Degeneration of zearalenone production in Fusarium graminearum. &perimental Mycology 9. 133-140. Eide, C. J. (1935). The pathogenicity and genetics of Gibberella saubinetii (Mont.) Sacco University of Minnesota Agricultural Experiment Station Technical Bulletin 106. 3-55. Geisen. R.. Glenn, E. & Leistner. L. (1990). Two Penicillium camembertii mutants affected in the production of cyclopiazonic acid. Applied and Environmental Microbiology 56. 3587-3590. Hansen, H. N. (1938). The dual phenomenon in imperfect fungi. Mycologia 30, 442-455. Hansen, H. N. & Smith, R. E. (1932). The mechanism of variation in imperfect fungi; Botrytis cinerea. Phytopathology 22, 953-964.

(Accepted 20 September 1994)

620 Joffe. A. Z. (1962). Biological properties of some toxic fungi isolated from overwintered cereals. Mycopathologia et Mycologia Applicata 16. 202221. Kirksey. J. W. & Cole. R. J. (1974). Screening for tOXin-producing fungi. Mycopathologia et Mycologia Applicata 54. 291-296. Komerup, A. & Wanscher, J. H. (1978). Methuen Handbook of Colour. Methuen & Company; London. Lauren, D. R. & Agnew, M. P. (1991). Multitoxin screening method for Fusarium mycotoxins in grains. Journal of Agricultural and Food Chemistry 39, 502-507. Leonian. L. H. (1932). The pathogenicity and the variability of Fusarium moniliforme from com. West Virginia Agricultural &perimental Station Bulletin 248, 2-16. Nash. S. M. & Snyder, W. C. (1962). Quantitative estimations by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopathology 52. 567-572. Nelson. P. E.• Cole. R.J., Toussoun. T. A.. Domer, J. W. & Windingstad. R. M. (1990). Fusarium species recovered from waste peanuts associated with sandhill crane mortality. Mycologia 82, 562-565. Nelson. P. E., Plattner, R. D.. Shackelford, D. D. & Desjardins. A. E. (1991). Production of fumonisins by Fusarium moniliforme strains from various substrates and geographic areas. Applied and Environmental Microbiology 57. 2410-2412. Nelson, P. E., Toussoun, T. A. & Marasas. W. F. O. (1983). Fusarium Species. An Illustrated Manual for Identification. The Pennsylvania State University Press: University Park and London. Oswald. J. W. (1949). Cultural variation. taxonomy and pathogenicity of Fusarium species associated with cereal root rots. Phylopathology 39, 359-376. Plattner, R. D., Tjarks, L. W. & Beremand. M. N. (1989). Trichothecenes accumulated in liqUid culture of a mutant of Fusarium sporolrichioides NRRL 3299. Applied and Environmental Microbiology 55. 2190-2194. Puhalla, J. E. (1981). Variation in Fusarium cultures. In Fusarium; Diseases. Biology and Taxonomy (ed. P. E. Nelson, T. A. Toussoun & R. J. Cook), pp. 293-305. The Pennsylvania State University Press: University Park. Rabie, C. J., Sydenham, E. W., ThieL P. G., Lubben. A. & Marasas. W. F. O. (1986). T-2 toxin production by Fusarium acuminalum isolated from oats and barley. Applied and Environmental Microbiology 52, 594-596. Snyder, W. C. & Hansen. H. N. (1954). Variation and speciation in the genus Fusarium. Annals of the New York Academy of Sciences 60, 16-23. Snyder, W. C. & Toussoun. T. A. (1965). Current status of taxonomy in Fusarium species and their perfect states. Phytopalhology 55, 833-837. Torres, J., Guarro, J., Suarez, G., Sufie. N., Calvo, M. A. & Ramirez. C. (1980). Morphological changes in strains of Aspergillus flavus Link ex Fries and Aspergillus parasiticus Speare related with aflatoxin production. Mycopalhologia 72. 171-174. Toussoun, T. A. & Nelson, P. E. (1975). Variation and speciation in the fusaria. Annual Review of Phytopathology 13. 71-82. Tu. C. (1929). Physiologic specialization in Fusarium spp. causing headblight of small grains. Phylopathology 19. 143-154. Ullstrup, A. J. (1935). Studies on the variability of pathogenicity and cultural characters of Gibberella saubinetii. Journal of Agricultural Research 51, 145-162. Wellman, F. L. & Blaisdell. D. J. (1941). Pathogenic and cultural variation among single-spore isolates from strains of the tomato-wilt Fusarium. Phytopathology 31. 103-120. Wing, N., Bryden. W. L., Lauren. D. R. & Burgess. L. W. (1993a). Toxigenicity of Fusarium species and subspecies in section Gibbosum from different regions of Australia. Mycological Research 97. 1441-1446. Wing. N., Lauren, D. R., Bryden. W. L. & Burgess, L. W. (1993 b). Toxicity and trichothecene production by Fusarium acuminatum subsp. acuminalum and Fusarium acuminatum subsp. armeniacum. Natural Toxins 1, 229--234.