Investigations of the capability of Fusarium isolates from corn for biosynthesis of fusariotoxins

Abstract

The aim of this paper was to investigate the potential of zearalenone (ZEN) and type A trichothecenes (T-2 toxin and diacetoxyscirpenole - DAS) for biosynthesis by Fusarium spp. isolated from corn kernels contaminated by mycotoxins. The samples of corn kernels (2004 and 2005 harvest) originating in different regions of Bačka (Vojvodina, Serbia) were tested. Mycotoxicological investigations showed in most cases a significant contamination with deoxynivalenol (DON 200 - 2,460 μg/kg) and ZEA (520 1,680 μg/kg). Isolations and identifications of fusaria established only the presence of species F. verticillioides, after one month storage in freezer conditions, in fusariotoxin positive samples. The control cultures known as ZEA producers - F. graminearum GZ-LES, i.e. T-2 toxin and DAS producer - F. sporotrichioides KF-38/1/R were also tested. In vitro toxicological investigations of isolated fusaria were performed in liquid semisynthetic media (GPK or SPK), and on wet sterilized corn kernels, respectively. Under testing conditions, analyzed F. verticillioide and F. sporotrishioides isolates were not ZEA producers. Contrary to them, F. graminearum GZ-LES pure culture was very good producer of fusariotoxins; it biosynthesized max. 465,900 μg/kg DON, and 4,416 μg/kg ZEA, respectively. Cultivation conditions influenced a great deal of T-2 toxin production under laboratory conditions. In most cases, higher yields were obtained during the cultivation of F. verticillioides in liquid glucose medium (80-240 μg/L). Contrary to the control strain F. sporotrichioides KF-38/1/R that under the same conditions synthesized, besides T-2 toxin (4.000 μg/L) and DAS (240 μg/L), isolates of F. verticillioides from corn grain did not show that ability.

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Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad,
¥ 113, 125—133, 2007
UDC 633.15:615.98
I g o r M. J a j i ã1
A l e k s a n d r a S. B o å a r o v - S t a n å i ã2
M i l o r a d B. B i j e l i ã2
1Faculty of Agriculture, Department of Animal Science,
Trg D. Obradoviãa 8, 21000 Novi Sad, Serbia, jajic@polj.ns.ac.yu
2Holding Company “Center for Bio-Ecology", Petra Drapšina 15,
23000 Zrenjanin, Serbija
INVESTIGATIONS OF THE CAPABILITY OF FUSARIUM
ISOLATES FROM CORN FOR BIOSYNTHESIS
OF FUSARIOTOXINS
ABSTRACT: The aim of this paper was to investigate the potential of zearalenone
(ZEN), and type A trichothecenes (T-2 toxin and diacetoxyscirpenole — DAS) for bi-
osynthesis by Fusarium spp. isolated from corn kernels contaminated by mycotoxins.
The samples of corn kernels (2004 and 2005 harvest) originating in different regions
of Baåka (Vojvodina, Serbia) were tested. Mycotoxicological investigations showed in most
cases a significant contamination with deoxynivalenol (DON 200 — 2,460 mg/kg) and ZEA
(520 — 1,680 mg /kg).
Isolations and identifications of fusaria established only the presence of species F.
verticillioides, after one month storage in freezer conditions, in fusariotoxin positive sam-
ples. The control cultures known as ZEA producers — F. graminearum GZ-LES, i.e. T-2
toxin and DAS producer — F. sporotrichioides KF-38/1/R were also tested. In vitro
toxicological investigations of isolated fusaria were performed in liquid semisynthetic media
(GPK or SPK), and on wet sterilized corn kernels, respectively.
Under testing conditions, analyzed F. verticillioide and F. sporotrishioides isolates
were not ZEA producers. Contrary to them, F. graminearum GZ-LES pure culture was very
good producer of fusariotoxins; it biosynthesized max. 465,900 mg/kg DON, and 4,416
mg/kg ZEA, respectively.
Cultivation conditions influenced a great deal of T-2 toxin production under labora-
tory conditions. In most cases, higher yields were obtained during the cultivation of F. ver-
ticillioides in liquid glucose medium (80—240 mg/L). Contrary to the control strain F. spo-
rotrichioides KF-38/1/R that under the same conditions synthesized, besides T-2 toxin
(4.000 mg/L) and DAS (240 mg/L), isolates of F. verticillioides from corn grain did not
show that ability.
KEY WORDS: biosynthesis, corn grains, fusariotoxins, Fusarium spp.
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INTRODUCTION
Corn is predominantly attacked by Fusarium graminearum, F. verticillioi-
des, F. proliferatum and F. subglutinans (D o o h a n et al., 2003). These re-
presentatives of Fusarium genus differ in their climatic distribution, optimal
conditions for growth and toxin production, as well as in pathogenicity to dif-
ferent cerealia. Several Fusarium species known as causal agents of corn ear
rot are capable to biosynthesize toxic metabolites in infected kernels, some of
which have notable impact on human and animal health. Zearalenone (ZEA)
and deoxynivalenol (DON) are commonly found in red ear rot of corn, a dise-
ase essentially caused by species of Discolor section, usually F. graminearum
(L o g r i e c o et al., 2002). Diacetoxyscirpenol (DAS) and T-2 toxin are ma-
inly associated with the presence of representatives of Sporotrichiella section
F. poae and F. sporotrichioides. According to the data presented by M a r a -
s a s et al. (1984) F. verticillioides can also biosynthesize ZEA, DAS and T-2
toksin, although these authors regarded them as unlikely.
MATERIAL AND METHODS
Fungal cultures. Four F. verticillioides isolates from corn grains of 2004
and 2005 harvest were investigated (Table 1). Isolations were performed from
coarse corn grain after one month of storage in freezer conditions. Coarsed
material was placed on the surface of Petri dish (10 cm diameter), on potato
dextrose agar supplemented with straeptomycin sulphate (500 mg/L) (PDAS),
and incubated at room temperature (23—26°C) for 7 days. Grown fungal colo-
nies, were purified by the method of obtaining monosporous colonies which
were further on used for the identification of Fusarium spp. Determination of
the obtained species was done according to N e l s o n et al. (1983), and con-
firmed by dr J. L e v i ã (Maize Research Institute “Zemun Polje", Belgrade—
Zemun). The obtained isolates were afterwards kept on PDA slants at 4°C, un-
til in vitro investigation of their toxigenic potential was not performed in
liquid culture.
Control isolates. Fusarium cultures, known as fusariotoxin producers,
were also under present investigation: 1) Fusarium graminearum GZ-LES pro-
ducer of ZEA, according to B o å a r o v - S t a n å i ã et al. (1986), and 2) F.
sporotrichioides KF-38/1/R reisolate of original strain known as T-2 toxin and
DAS producer (M a š i ã et al., 1997). Stock cultures of the fungi were mainta-
ined on potato dextrose agar at 4°C.
Conditions for toxin production. 1) Control isolates Fusarium graminea-
rum GZ-LES and F. sporotrichioides KF-38/1/R were cultivated on naturaly
dried corn grain (hybrid NS-444, locality Stajiãevo — Vojvodina) with 53.12%
of initial moisture. Substrate inoculation (250 g) in flasks (1750 ml) was per-
formed with fungal fragments cultivated for 7 days at 26 ± 1°C in PDA Petri
dishes (1/2 of the fungal growth, 10 cm in diameter). Cultivation was perfor-
med in darkness at 30 ± 1°C for 35 days. 2) All F. verticilioides isolates, as
well as both control cultures, were parallely grown in GPY liquid medium
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(5% of glucose + 0.1% of peptone + 0.1% of yeast extract) pH 5.8; and SPY
medium (5% of saccharose + 0.1% of peptone + 0.1% of yeast extract) pH 6.5
respectively. Media were poured in Erlenmeyer flasks (500 ml) in volume of
100 ml each (A), or 250 ml each (B), and sowed with 5 fragments (5 x 5 mm)
of fungus cultivated on PDA in Petri dishe at 26 ± 1°C for 7 days. After ino-
culation, Erlenmeyer flasks were cultivated on rotary shaker under submerged
conditions (180 revolutions per min-rpm) at room temperature (23—26°C).
Fusarium graminearum GZ-LES was also cultivated under stacionary conditi-
ons in 100/500 ml GPY for 13 days at room temperature (23—26°C) (C).
Determination of fusariotoxins. 1) After cultivation on corn grains, the
samples were dried for 24 h or more at 60°C, until constant weight was not
achieved. After pulverization of dried samples, DON determination was done
according to A b r a m o v i ã et al. (2005), and ZEA by the use of multitoxin
method applied by B a l z e r et al. (1978). Type A trichothecenes were isola-
ted and purified according to the method of R o m e r et al. (1978). Thin layer
chromatography of ZEA, T-2 toxin and DAS was was done according to P e -
p e l j n j a k and B a b i ã (1991).
2) After cultivation on rotary shaker, liquid cultures were filtered. Quali-
tative and quantitative ZEA determination in filtrates of Fusarium cultures was
carried out by applying multitoxin thin layer chromatographic method of
C v e t n i ã et al. (2005). Crude extracts of type A trichothecenes (DAS i T-2
toxin) were obtained by the use of ethyl acetate. Further purification was done
by the method of R o m e r et al. (1978). Thin layer chromatography was per-
formed according to P e p e l j n j a k and B a b i ã (1991) with toluole/ethyl
acetate/formic acid developing solvent (5:4:1, v/v/v).
RESULTS AND DISCUSSION
Results of the present investigations are shown in Tables 1—4.
Tab. 1 — Presence of deoxynivalenole (DON) and zearalenone (ZEA) in samples of corn kernels
(harvest 2004 and 2005).
No.
Sample
design.
Type of sample
Quantity of fusariotoxins
(?g/kg)
DON
1,130
200
—
2,460
ZEA
1,680
520
1.
2.
3.
4.
N001/06
N002/06
N003/06
N004/06
Coarse corn grain KPUT4, Putinci 2005
Coarse corn grain KŠID, Šid 2005
Coarse corn grain 57002
Corn grain 12168, Kupusina 2004
n.d. (< 0.037)
800
Seventy five percentages of tested samples of coarsed/whole corn grain
originating from 2004 and 2005 were contaminated with DON. Detected
quantities of this mycotoxin ranged from 200 to 2,460 mg/kg (Table 1). Higher
concentrations of fusariotoxin ZEA than DON were found in 2 samples (2005
harvest) out of three positive samples for this type B trichothecene. The obtai-
ned results can be explained by the assumption that in 2005 the harvest was
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carried out in later vegetative phase of corn than in 2004. According to the in-
vestigations of L e w (1995), DON can be found very early and in high
quantities in cereal grains attacked by Fusarium spp., contrary to ZEA, fusa-
riotoxin which appears only in the later phase of fungal growth.
Tab. 2 — Yield of DON, ZEA and type A trichothecenes (DAS i T-2) in control isolates F. gra-
minearum and F. sporotrichioides cultivated on sterilised corn grains under laboratory conditions
No.
Cultivation
period
Control
isolate
Moisture
(%)
Yield of fusariotoxins (?g/kg)
ZEADON DAS T-2
1.
Starting
sample
— 53.1 n.d.*n.d.** n.d.***n.d.***
2.17 days
GZ-LES
KF-38/1/R
GZ-LES
KF-38/1/R
62.5
58.4
80.4
—
—
—
220
n.d.**
4,416
n.d.**
n.d.***
n.d.***
n.d.***
1,600
n.d.***
n.d.***
n.d.***
2,400
3.35 days
465,900
n.d.*
Legende: n.d* — not detected (< 45 mg/kg),
n.d.** — not detected (< 37 mg/kg),
n.d.*** — not detected (< 60 mg/kg).
The results obtained during cultivation of the control strains F. graminea-
rum GZ-LES and F. spototrichioides KF-38/1/R on natural solid substrate at
30°C are shown in are Table 2. Corn kernels used as the substrate for in vitro
investigations of toxin production were not contaminated with fusariotoxins.
Isolate F. graminearum produced only type B trichothecene (DON) and ZEA
in that conditions. During prolongated cultivation, the yield of ZEA increased
from 220 mg/kg after 17 days to 4,416 mg/kg after 35 days. The obtained re-
sults are in accordance with the previously quoted opinion of L e w (1995).
F. sporotrichioides KF-38/1/R biosynthesized only type A trichothecenes after
35 days on wet sterilized corn grain substrate (1,600 mg/kg DAS, and 2,400
mg/kg T-2 toxin, respectively). These results are in accordance with the data
presented by M a r a s a s et al. (1985) concerning toxigenic potential of Fusa-
rium spp., as well as with data concerning optimal temperature conditions for
the production of fusariotoxins presented by other authors. B o å a r o v - S t a n -
å i ã et al. (1986) point out that most (50%) of F. graminearum isolates bio-
synthesized ZEA during cultivation at rather high constant temperature (28°C).
Similar temperature conditions favoured DON production by the same fusaria
species: 28°C (L l o r e n s et al., 2004) and 25°C (H o p e et al., 2005, R a -
m i r e z et al., 2006) respectively.
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Tab. 3 — Yield (mg/L) of zearalenone and tipe A trihotecenes (DAS, T-2 toxin) in GPY liquid
culture of four F. verticillioides originated from corn grains and control isolates F. graminearum
(No. 5) and F. sporotrichioides (No. 6)
No.
Isolate
designition
Aeration pH
Yield of fusariotoxins (?g/L)
ZEADAS
n.d.*n.d.**
n.d.*n.d.**
n.d.* n.d.**
n.d.* n.d.**
n.d.*n.d.**
n.d.*n.d.**
n.d.* n.d.**
n.d.*n.d.**
37 n.d. **
75—
n.d. *240
T-2
80
240
n.d.**
240
160
n.d.**
n.d.**
80
n.d. **
—
4. 000
1. N001/06
A
B
A
B
A
B
A
B
A
C
A
4.71
4.29
4.77
4.72
4.96
4.33
4.76
4.55
4.40
5,70
4.20
2. N002/06
3. N003/06
4.N004/06
5. GZ-LES
6.KF-38/1/R
Legende: n.d.* — not detected (< 37 mg/L),
n.d.** — not detected (< 80 mg/L)
Tab. 4 — Yield (mg/L) of zearalenone and tipe A trihotecenes (DAS, T-2 toxin) in SPY liquid
culture of four F. verticillioides originated from corn grains and control isolates F. graminearum
(No. 5) and F. sporotrichioides (No. 6)
No.
Isolate
designition
Aeration pH
Yield of fusariotoxins (?g/L)
ZEADAS
n.d.*n.d.**
n.d.*n.d.**
n.d.* n.d.**
n.d.* n.d.**
n.d.*n.d.**
n.d.*n.d.**
n.d.* n.d.**
n.d.*n.d.**
n.d.*n.d.**
n.d.*n.d.**
T-2
n.d.**
120
n.d.**
80
80
n.d.**
n.d.**
80
n.d.**
160
1.N001/06
A
B
A
B
A
B
A
B
A
A
4.95
4.22
5.14
4.80
5.17
4.07
5.04
4.45
4.73
4.69
2.N002/06
3.N003/06
4. N004/06
5.
6.
GZ-LES
KF-38/1/R
Legende: n.d.* — not detected (< 37 mg/L),
n.d.** — not detected (< 80 mg/L)
On the basis of the results presented in Table 1, high concentrations of
DON and ZEA, it should be expected that one can isolate from these corn ker-
nels mainly species F. culmorum or F. graminearum which are, according to
the literature data, the best producers of the same mycotoxins (M a r a s a s et
al., 1984). However, having in mind storage conditions of the samples and low
viability of F. graminearum conidia in the first place, as well as higher viabi-
lity of conidia of other fusaria pathogenic to corn (D o o h a n et al., 2003), it
is not so surprising that F. verticillioides was obtained solely.
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pH value. During submerged cultivation (Tables 3 and 4), with the
exception of the control isolate F. graminearum GZ-LES, the pH value decre-
ase was observed in all other liquid cultures after incubation. In both cases of
liquid media (GPY/SPY) more outstanding changes of the same paramether
were noted in higher volume of fermentation media (250/500 ml).
ZEA biosynthesis. Although C v e t n i ã et al. (2005) found that some F.
verticillioides isolates from agricultural regions of Croatia can produce ZEA,
during our investigation the production of this fusariotoxin was noted only in
liquid GPY culture of F. graminearum (Table 3), but in much lower quantities
than during incubation on the natural solid substrate (37 and 75 mg/L, and 220
and 4,659 mg/kg, respectively) (Tables 2 and 3). Higher yield of ZEA was ob-
tained during stationary cultivation in the liquid medium of the same composi-
tion. Last result is in accordance with our previous investigations of different
liquid nutrient media (B o å a r o v - S t a n å i ã and Š k r i n j a r, 1995).
DAS biosynthesis. It was not recorded in any of the tested F. verticillioi-
des isolates, but was found in liquid GPY culture of the control isolate F. spo-
rotrichioides KF-38/1/R — 240 mg/L (Table 3). Although again, much lower
yields of DAS were achieved in liquid medium than on sterilized corn grains
(Tables 2 and 3), the period of incubation was much shorter (3 days in compa-
rison with 35 days). That fact can become a great advantage in the case of
screening toxicity of a large number of Fusarium spp. under laboratory condi-
tions.
T-2 toxin biosynthesis. It was observed (Tables 3 and 4), besides control
strain F. sporotrichioides KF-38/1/R, in all tested F. verticillioides isolates
from corn (80 do 240 mg/l in GPY, and 80—120 mg/l in SPY, respectively). In
the last case, found concentrations were rather low, i.e. near the detection
limit. During submerged cultivation in both liquid media in 75% of the te-
sted cases, higher yields of this type A trichothecene were achieved under the
conditions of smaller aeration (250/500 mL). An exception was the isolate
N003/06 that produced more T-2 toxin during cultivation in higher aeration
conditions (Tables 3 and 4). The obtained results are in accordance with the
investigation of M a š i ã et al. (1997). These authors recorded, with one F.
verticillioides isolate from corn grains, T-2 toxin biosynthesis (500 mg/L) un-
der similar laboratory conditions.
Influence of the sugar type. Eighty percentages of the tested isolates, in-
cluding the control one KF-38/1/R, regardless of volume of fermentation me-
dium, achieved higher yields of T-2 toxin in glucose medium GPY (80 —
4,000 mg/L) than in sucrose medium SPY (80 — 160 mg/L). In contrast to our
findings, U e n o et al. (1975) didn't observe the same influence of sugar type
during cultivation of other fusaria in the liquid media with the same composi-
tion.
On the basis of the presented results it can be concluded that isolates of
F. verticillioides originating from Vojvodina possess weak potential for T-2
toxin production.
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CONCLUSIONS
Only F. verticillioides cultures were isolated from corn kernels harvested
in 2004/2005, contaminated with rather high quantities of DON (200 — 2,460
mg/kg) and ZEA-a (520 — 1,680 mg/kg).
During submerged cultivation of these isolates in liquid media (GPY and
SPY) all 4 tested F. verticillioides cultures biosynthesized T-2 toxin (80 —
240 mg/L), as well as the control strain F. sporotrichioides KF-38/1/R.
Production of DAS and ZEA was not observed with F. verticillioides iso-
lates, but it was with control cultures F. graminearum (ZEA) and F. sporotri-
chioides (DAS) during cultivation in the same liquid media (GPY and SPY).
The influence of glucose, as sugar source, was more favourable than sac-
charose on T-2 toxin biosynthesis in most (80%) of the tested samples.
After 35 days of cultivation on solid natural substrate, the control strain
F. graminearum GZ-LES produced high DON (469,900 mg/kg) and ZEA
(4,416 mg/kg) quantities. Under the same cultivation conditions another control
isolate F. sporotrichioides KF-38/1/R, biosynthesized only type A trichothece-
nes (1,600 mg/kg DAS, and 2,400 mg/kg T-2 toxin, respectively).
ACKNOWLEDGEMENTS
The paper is part of the investigations realized in the scope of the project
No. TR-6826B financially supported by the Ministry for Science and Environ-
ment Protection of SR Serbia.
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ISPITIVAWE SPOSOBNOSTI ZA BIOSINTEZU
FUZARIOTOKSINA KOD FUSARIUM IZOLATA SA KUKURUZA
Igor Jajiã1, Aleksandra S. Boåarov-Stanåiã2, Milorad B. Bijeliã2
1Poqoprivredni fakultet, Departman za stoåarstvo,
Trg D. Obradoviãa 3, 21000 N. Sad, Srbija
2A.D. „Bio-ekološki centar", Petra Drapšina 15,
23000 Zrewanin, Srbija
Rezime
Iako pripada grupi najmawe toksiånih trihotecena (tip B), fuzarijumski
mikotoksin dezoksinivalenol (DON) je od velikog znaåaja za kvalitet hrane i
stoåne hrane, s obzirom da moÿe biti indikator prisustva drugih, toksiånijih
jediwewa kao što je npr. zearalenon (ZEA). S druge strane znaåajno je i pra-
ãewe prisustva trihotecena tipa A (T-2 toksin i DAS) s obzirom na wihovu
izrazitu toksiånost i u mawim koncentracijama.
Ciq ovog istraÿivawa je bio da se ispita potencijal za biosintezu zeara-
lenona (ZEA) i trihotecena tipa A (T-2 toksin i DAS) kod Fusarium kultura
izolovanih sa zrna kukuruza kontaminiranog mikotoksinima.
Istraÿivawem su bili obuhvaãeni uzorci kukuruza roda 2004/2005. god.
poreklom iz razliåitih regiona Baåke. Mikotoksikološka istraÿivawa datih
uzoraka su kod veãine pokazala znaåajnu kontaminaciju sa DON-om (200 — 2.460
mg/kg) i ZEA-om (520 — 1.680 mg/kg).
Izolacije i identifikacije fuzarija su pokazale iskquåivo prisustvo vr-
ste F. verticillioides. Ispitivawa toksigenosti ovih izolata su obavqena u teå-
nim semi-sintetiåkim podlogama sa glukozom (GPK) i saharozom (SPK), kao i
na vlaÿnom, sterilnom zrnu kukuruza.
U datim uslovima ispitani izolati nisu biosintetisali ZEA. Za razliku
od wih kontrolna kultura F. graminearum GZ-LES pokazala se kao veoma dobar
producent fuzariotoksina — proizvodila je maksimalno 465.900 mg/kg DON-a,
odnosno 4.416 mg/kg ZEA.
Na biosintezu T-2 toksina u laboratorijskim uslovima u velikoj meri su
uticali uslovi kultivirawa. U veãini sluåajeva su kod izolata F. verticillioides
dobijeni veãi prinosi u podlozi sa glukozom (80—240 mg/L). Za razliku od kon-
trolnog soja F. sporotrichioides KF-38/1m koji je u istim uslovima biosinteti-
sao pored T-2 toksina (4.000 mg/L) i DAS (240 mg/L), izolati F. verticillioides sa
zrna kukuruza nisu ispoqili tu sposobnost.
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