Mycol. Res. 96 (6): 518-523 (1992) Prinled in Greal Brita;n

Cultural and toxigenic variability in Fusarium acuminafum

A. LOGRIECO, C. AL TOMARE, A. MORETTI AND A. BOTTALICO"

Istituta Tossine e Micotassine da Parassiti Vegetali del Consiglio Nazionale delle Ricerche, Bari, Italy and 'Dipartimento di Patologia vegetaledel/'Universita degli Studi, Bari, Italy

518

Twenty-five isolates of F. acuminatum from different sources and geographic origins were used for studying the cultural andtoxigenic characteristics of the species. The isolates showed a large cultural variability, and the features ranged from slow-growingisolates with carmine-red pigmentation and relatively short 5-septate conidia to fast-growing yellow-brown isolates and long5-septate conidia. The cultural differences corresponded to differences in production of secondary metabolites. On the basis of theirmetabolite production, the isolates were grouped into three categories: (a) enniatin B and/or moniliformin producers (up to 1000 and2340 mg kg-t, respectively); (b) T-2 toxin, HT-2 toxin and/or neosolaniol producers (up to 450, 40 and 20 mg kg-I, respectively); (c)

no-toxin producers. The toxicity of the culture extract towards Artemia salina could usually be related to the occurrence of the abovetoxins.

Fusarium acuminatum Ell. & Ev. sensu Gordon, a member ofthe Gibbosum Woll. section, is a fungus with a widespreadgeographic distribution occurring on a wide range of hosts(e.g. plants, soil and insects), mostly as a saprobe and as asecondary invader (Booth, 1971).

Although taxonomically F. acuminatum is considered a'well-documented' species (Nelson, Toussoun & Marasas,1983), conflicting data still exist in the literature on somemorphophysiological characteristics (e.g. growth rate, pigmen­tation, toxin production). On the basis of their studies onFusarium, Gerlach & Nirenberg (1982) and Nelson et al. (1983)considered F. acuminatum a species with rapid growth onpotato dextrose agar (PDA). On the other hand, Booth (1971)and Burgess & Liddell (1983), who worked on potato sucroseagar (PSA) and PDA respectively, considered F. acuminatum tohave a relatively slow growth. In addition, literature reportssuggest a large variability in pigmentation (Gerlach &

Nirenberg, 1982; Nelson et aI., 1983) with the presence ofsome variants (Kommedahl, Windels & Stucker, 1979; Rabieet al., 1986).

In recent years, F. acuminatum has acquired increasedimportance from the mycotoxicological point of view(Marasas, Nelson & Toussoun, 1984). Conflicting data exist onthe ability of F. acuminatum to produce some specific toxins.Several reports support the ability of F. acuminatumto produce type A trichothecenes, including T-2 toxin,(Burmeister et al., 1981; Echinoe, Kurata & Ueno, 1982;Marasas et al., 1984; Rabie ef al., 1986), but recently Abbas,Mirocha & Gunther (1989) tested 25 isolates, and none wasable to synthesize trichothecenes. This species has also beenreported to synthesize a cyclic peptide 'swelling factor',butenolide (Marasas et al., 1984), moniliformin (MF) (Rabie

ef al., 1982), enniatins (Deol, Ridley & Singh, 1978) andzearalenone (ZEA) (Richardson et al., 1985). However, theproduction of ZEA has not been confirmed (Thrane, 1989).

The object of this study was to examine several isolates ofF. acuminatum from different geographical origins and sourcesin order to evaluate both the morphophysiological andtoxigenic variability of the species.

MATERIALS AND METHODS

Cultures

The geographic origin, habitat, source and strain number ofthe twenty-five isolates of F. acuminafum investigated in thisstudy are given in Table 1. The isolates were kindly suppliedby W. F. O. Marasas (National Research Institute forNutritional Diseases, South Africa), P. E. Nelson (FusariumResearch Center, Pennsylvania State University), D. T. Wick­low (National Center for Agricultural Utilization Research,Illinois), J. Chelkowski (Agricultural University of Warsaw,Poland), H. Nirenberg (Biologische Bundesanstalt fur Land­und Forstwirtschaft, Berlin). All the strains were re-isolatedfrom single conidia at the beginning of this study and thendepOSited in the Culture Collection of the Istituto Tossine eMicotossine da Parassiti Vegetali (ITEM), Bari, Italy.

Cultural characteristics

Twenty-five isolates were tested for some culture character­istics (pigmentation and growth) on freshly prepared PDA(800 ml filtrate from 200 g peeled, sliced and autoclavedpotatoes, 20 g dextrose, 15 g Bacto agar (Difco), up to1000 ml with distilled water). Small squares (2 mm) of water

A. Logrieco and others

Table 1. Strains of F. acuminatum examined

519

ITEM

728791792793794795796797800804987988990991992993994995996997998999104210441045

Origin

Maize kernels, PeruMedicago seeds, South AfricaMedicago seeds, South AfricaMedicago seeds, South AfricaMedicago seeds, South AfricaMedicago seeds, South AfricaBarley, South AfricaOats, South AfricaSouth AfricaWheat roots, ItalyCrownvetch, Pennsylvania, U.s.A.Carnation, AustraliaSoil, AustraliaSoil, AustraliaFescue hay, u.s.A.Aspergillus sclerotia, u.s.A.Aspergillus sclerotia, U.s.A.PolandPolandPolandPolandPolandSoil, DenmarkCucurbita, GermanyArtemisia vulgaris, Germany

Other reference nos.

MRC-3308MRC-3309MRC-331OMRC-33I1MRC-3312MRC-3397MRC-3826MRC-3936

R-2I09R-2136R-6365R-6638NRRL-6227NRRL-13908NRRL-13909KF-332KF-1I50KF-403KF-359KF-958646416214965106

Source

A. LogriecoW. F. O. MarasasW. F. O. MarasasW. F. O. MarasasW. F. O. MarasasW. F. O. MarasasW. F. O. MarasasW. F. O. MarasasW. F. 0 MarasasA. LogriecoP. NelsonP. NelsonP. NelsonP. Nelson

A. Logrieco (D. T. Wicklow)A. Logrieco (D. T. Wicklow)j. Chelkowskij. Chelkowskij. Chelkowskij. Chelkowskij. CheikowskiH. NirenbergH. NirenbergH. Nirenberg

Table 2, Physiological and morphological characteristics of F. acuminatum

strains

Grown Colour of Production of 5-septate conidia(em)' colony' EB T-2 length (..m)'

ITEM-728 4'1 Carmine red + 39'1 (1'1)ITEM-795 4'2 Carmine red + 38'2 (0'4)ITEM-804 4'3 Carmine red + 46'0 (0'7)ITEM-9S7 4'4 Carmine red + 50'2 (1'4)ITEM-794 4'5 Carmine red + 37'1 (0'4)ITEM-991 4'6 Reddish brown + 40'2 (0'7)ITEM-I044 4'8 Carmine red + 39'0 (0'6)ITEM-I045 5'2 Carmine red + 49'6 (1'3)

ITEM-793 5'2 Carmine red 36'9 (0'6)ITEM-792 5'3 Carmine red 43'4 (1'0)ITEM-996 5'3 Carmine red 49'2 (1'4)ITEM-I042 5'6 Carmine red + 51'4 (1'6)lTEM-990 5'8 Carmine red + 40'5 (1'3)ITEM-791 6'0 Reddish brown 50'6 (1'0)lTEM-995 6'1 Reddish brown + 51'2 (1'6)lTEM-994 6'2 Reddish brown + 50'0 (1'4)ITEM-997 6'4 Reddish brown + 43'0 (0'9)ITEM-999 6'4 Reddish brown 50'1 (1'8)ITEM-993 6'5 Carmine red + 59'3 (0'9)ITEM-800 9'0 Yellow ochre + 52'5 (1'8)ITEM-796 9'0 Yellow ochre + 48'1 (1'0)ITEM-9S8 9'0 Yellow ochre + 62'4 (1'3)ITEM-992 9'0 Yellow ochre + 52'2 (1'0)ITEM-797 9'0 Yellow brown + 57'6 (1'1)ITEM-998 9'0 Yellow brown + 63'2 (1'4)

I Colony diameters on PDA. after 7 d in the dark at 25 0.

, The colony colour is based on the underside of the PDA plates.3 Length are means. the standard errors are in parentheses.

agar containing 12-h-old single germinating conidia (ten foreach strain) were cut out with a dissecting needle andinoculated into the centre of 90 mm Petri dishes containing20 ml of PDA. The growth rate of each strain was calculatedas the mean of the colony diameters after 7 d in the dark at25 dc. Microscopic features were observed from subculturesgrown on water agar with sterile carnation leaf (CLA) (Nelsonef ai" 1983), The length of fifty 5-septate macroconidia perstrain was measured, We chose these conidia because theyappeared to be the most variable. The CLA cultures wereincubated at 250 under fluorescent and black (uv) lamps(2700 lux) with a 12 h photoperiod,

Toxin production

The toxigenic potential of 25 isolates was determined on cornkernels, although the strains were isolated from many matrices,Corn proved to be a good medium for Fusarium mycotoxinsynthesis (Bottalico, Lerario & Visconti, 1983), Fifty g ofyellow corn kernels cv, 'Plata', brought overnight to about45 % moisture, was autoclaved in a 250 ml Erlenmeyer flaskfor 30 min at 1200 and then inoculated with five small pieces(3 x 3 mm) of fresh F. acuminafum culture on PDA. Thecultures were incubated for 21 d at 250 under a 12 hphotoperiod, oven-dried at 500 for 2 d and finely ground,Uninoculated media treated in the same way were used ascontrols, The isolation, identification and quantitative analysesby thin-layer chromatography (TLC) and high-performancethin-layer chromatography (HPTLC) of T-2, diacetoxy­sCirpenol (DAS), HT-2 toxin (HT-2), neosolaniol (NEOS),fusarenone (FUS), deoxynivalenol (DON), 3-acetyldeoxy­nivalenol (3-AcDON), ZEA, enniatin B (EB) and MF wereperformed according to the methods previously described

Cultural and toxigenic variability in Fusarium acuminafum 520

Fig. 1. Long (a, c) and short (b, d) representative conidia of F. acuminatum. a, b, bar = 20 IJrn; c, d, bar = 12'5 IJrn.

(Bottalico ef ai., 1983; Bottalico, Logrieco & Visconti, 1989).Detection limits for EB, trichothecenes and ZEA were 5 IJgg-t, 1 IJg g-l and 1 IJg g-l com culture, respectively.

Bioassay

The toxicity of each culture extract was tested on brine shrimps(Arfemiasalina L.) according to Harwig & Scott (1971). Briefly,the bioassays were performed in cell culture plates (Coming,New York) with 24 wells containing about 30-40 larvae in

500 IJI sea water with 1 % methanolic extract of fungal cultureper well (4 replicates per extract). The number of dead shrimpswas recorded after incubation at 27° for 36 h. The totalnumber of shrimps per well was measured after killing theremaining shrimps by freezing at - 20° for 12 h.

RESULTS

The morphophysiological characteristics of 25 F. acuminafum

isolates are reported in Table 2.

A. Logrieco and others 521

EB MF T-2 HT-2 NEOS

ITEM-728 400ITEM-795 250 2.340ITEM-804 1000ITEM-987 ISOITEM~794 300 670ITEM-991 200ITEM-1044 60ITEM-1045 100ITEM-793 670ITEM-792ITEM-996ITEM-1042 60 335ITEM-990 100ITEM-791ITEM-995 225 335ITEM-994 150ITEM-997 125ITEM-999ITEM-993 20ITEM-800 80 5ITEM-796 450 40 10ITEM-988 80ITEM-992 300 20 20ITEM-797 450 40 10ITEM-998 300 20 20

1 Strains grown on autoclaved corn kernels at 25° for 21 d.2 EB. enniatin B; MF, moniliformin; T-2. T-2 toxin; HT-2, HT-2 toxin;

NEOS, neosolaniol. Determined by TLC and HPTLC. (-) = not detected.

Table 4. Toxicity of F. acuminalum strains on Arlemia salina'

No. of strains PercentageNo. of strains causing 100% of toxic

Producing/strain tested mortality strains

Trichothecenes 7 7 100EB and/or MF 14 2 14No tested toxins 4 0 0

, Four replicates per strain.

Length of 5-septate macroconidia

The 25 isolates presented a great variability in the length of5-septate conidia (39'1-63'2 >.lm). Overall, the 5-septateconidia were produced more abundantly by the fast~growing

than by the slow-growing isolates. The latter producedheterogeneous macroconidia (mostly 3- to 4-septate). Fur­thermore, the longer macroconidia, mostly produced by thefast-growing isolates, were thinner, and characterized by along tapering apical cell (Fig. I).

Colony pigmentation Table 3. Secondary metabolite production by F. acuminalum 1

The pigmentation of the F. acuminatum isolates on PDA Toxin production (mg kg~l)2

proved to be diverse, summarizable in two distinct limitgroups and one intermediate group. Thirteen isolates produceda deep carmine red, six produced an additional brown colourand six a diffuse yellow ochre-brown colour.

Colony growth

The growth rate (diameter of the colony after 7 days) of 25F. acuminatum isolates showed a great range of variability. Thesmallest colony diameter was 4'1 cm (ITEM-728), while thelargest proved to be 9'0 cm (ITEM-800, -796, ~988, -992,-797, -998). Furthermore, when listed in order (Table 2), theincrease in the diameter of isolates was continuous, with amaximum difference between sequential isolates measuring2'5 cm (between ITEM-993 and ITEM-800). Overall, theslow-growing isolates were characterized by a carmine redpigmentation and formed a dense greyish~pink floccosemycelium, while the fast-growing isolates were characterizedby a yellow-brown pigmentation and produced a brownish­orange floccose mycelium. The reddish brown cultures seemintermediate among the above-cited characteristics. Some~

times, on PDA, the Single-conidia carmine red culturemutated or degenerated into a brown mycelium with a fastergrowth rate.

Mycotoxin production

The results of the mycotoxin analysis of autoclaved cominoculated with 25 F. acuminatum isolates are summarized inTable 3. None of the tested isolates produced type Btrichothecenes or zearalenones. EB was detected in 13 cultures(60-1000 mg kg-I), five of which were associated with MF(335-2340 mg kg-I). All of these EB or MF producing isolatesshowed relatively slow growth, and none was able tosynthesize the tested type A trichothecenes. On the contrary,the fast-growing isolates, including ITEM-993, producedtype A trichothecenes. In particular, 2 isolates (ITEM-993,-988) produced only T-2 (20 and 80 mg kg-I respectively),I isolate produced T-2 and HT-2 (80 and 5 mg kg-I

respectively) and 4 produced T-2, HT-2 and NEOS (up to 450,40 and 20 mg kg-I respectively). Finally, 4 isolates were notable to produce any toxin. However, due to the detectionlimits used in this study, our results do not exclude thepossibility that the isolates could synthesize small amounts oftoxins.

Biological assays

The toxicity of the culture methanolic extracts of the testedisolates, compared with the mycotoxins detected, issummarized in Table 4. Briefly, the trichothecene-producingstrains showed a 100% mortality of the larvae, and only twoout of fourteen isolates (14 %) of the EB and/or MF producingstrains were toxic to A. salina. Finally, none of the four isolateswhich did not produce the tested toxins was toxic to A. salina.

DISCUSSION

Several isolates of F. acuminatum from different sources andgeographical origin showed a large variability in bothmorphological and toxicological aspects. Our findings aresupported by a certain variability already reported in thisspecies. Kommedahl et al. (1979), who studied the occurrenceof Fusarium species in com fields in Minnesota, pointed outtwo variants of F. acuminatum, a typical' red' one and anotherone that was light pink to peach, rather fast growing and with

Cultural and toxigenic variability in Fusarium acuminatum

macroconidia larger in size than the 'red' type. Similarly,Rabie et aI. (1986) indicated some trichothecene producerstrains from oats and barley in South Africa, which proved tobe different from the typical representatives of F. acuminatum.Finally, in Australia, Burgess & Liddell (1983) proposed thespecies name of 'armeniacum' for a variant population ofF. acuminatum with larger macroconidia and a faster growthrate. On the other hand, no variants were pointed out byNelson et aI. (1983) and Gerlach & Nirenberg (1982), becausethey considered only the rapid-growing strains belonging tothis species. Booth (1971), who considered the species to havea relatively slow growth, indicated mutant strains showingvarious degrees of loss of red pigment with some strainsbecoming buff-brown, bay or peach, usually accompanied bya reduction in aerial mycelium.

Although groups of isolates separated on the basis ofdifferent morphological and toxicological features overlap,our findings indicated some discontinuity in the hetero­geneity of the isolates studied, suggesting the possibilitythat the species could be subdivided into varieties.

The other goal of the present study involved a preliminaryinvestigation of the ability of F. acuminatum to producemycotoxins. Our results suggest that F. acuminatum includesisolates with different pattern of toxin biosynthesis.

This is the first time that the different toxigenic F.acuminatum strains have been culturally characterized. As faras we are aware, F. acuminatum T-2 producers have beenisolated in the u.s.A. from fescue hay (Burmeister, Ellis &

Vesonder, 1981); in Germany from oats (Gedek & Bauer,1983); in Japan from wheat and barley (Echinoe et aI., 1982)

and in South Africa from oats and barley (Rabie et aI., 1986).

On the other hand, F. acuminatum MF and/or EB producershave been reported in South Africa from dried bean leaves,millet and sorghum (Rabie et aI., 1982); in Norway from anunspecified matrix (Abbas et aI., 1989); and in Australia (Deolet aI., 1978). Consequently, this is the first report of T-2 toxinproduction by Australian isolates of F. acuminatum as well asthe production of EB and/or MF by North and SouthAmerican isolates.

The highest level of EB (1000 mg kg-I) was produced byITEM-804 (Table 3). It is interesting that ITEM-804, whichwas isolated from wheat roots, together with other isolatesfrom the soil or the rhizosphere (ITEM-990, -991, -987,-1042), were found to produce mostly EB, which is consideredan antagonistic antibiotic (Vesonder & Golinski, 1989).Wicklow (1985) observed that the physiological attributesand secondary metabolites are ecologically relevant charactersthat define the fungal niche.

Our results are in accordance with previous reports on theproduction of type A trichothecenes by the same F.acuminatum strains. In particular, the ability of MRC 3826(ITEM-797) and MRC 3397 (ITEM-796) to produce T-2 hasalready been established in a previous paper (Rabie et aI.,1986), although the T-2 yields were almost ten times lower.Our results can partly explain the contrasting data in literatureon the production of toxins (including those on trichotheceneproduction) by this species.

The high toxicity shown by trichothecene producers can beexplained by the amounts of toxin found. Literature reports

522

50% lethal concentrations (LC50) of T-2, HT-2 and NEOS,after an incubation period of 24 h: 0'65, 1'00, 3'20 ~g ml-I,respectively (Schmidt, 1989). The concentration of tricho­thecenes in the assays ranged from 2 to 45 ~g/ml for T-2, 0'5to 4 ~g ml-I for HT-2 and 1'0 to 2'0 ~g ml- I for NEOS. Thefact that only two out of fourteen isolates producing EBand/or MF (ITEM-804 and -728) were toxic to A. salina maybe due to the large amount of EB detected in their methanolicextracts. The toxicity of EB towards A. salina is relatively low(LC50 = 8'6 ~g ml-I) (Altomare et aI., unpublished data).

The significant cultural and toxigenic variability in F.acuminatum demonstrated by the present paper shows thenecessity of specifying the culture features of the isolatesunder study to give a better understanding of the biology ofthe fungus (e.g. epidemiology, ecology, toxigenicity).

The authors are grateful to J. Chelkowski, W. F. O. Marasas,P. Nelson, H. Nirenberg and D. T. Wicklow for supplying F.acuminatum isolates; and to V. Ricci for his competenttechnical assistance.

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