Natural occurrence of Nivalenol and mycotoxigenic potential of Fusarium graminearum strains in wheat affected by Head Blight in Argentina

Abstract

The principal agents of Fusarium head blight in the main cropping area of Argentina were investigated in heavily infected samples. The ability of the isolates to produce trichothecenes was determined by GC and HPLC. Fusarium graminearum was the predominant species and of 33 isolates, 10 produced deoxinivalenol (DON) (0.1- 29 mg kg(-1)), 13 produced both deoxinivalenol (1.0- 708 mg kg(-1)) and nivalenol (0.1- 6.2mg kg(-1)), 12 produced 3-acetyldeoxinivalenol (0.1- 14 mg kg(-1)), 13 produced 15-acetyldeoxinivalenol (0.1- 1.9 mg kg(-1)), 10 produced Fusarenone X (0.1- 2.4 mg kg(-1)) and 7 produced zearalenone (0.1- 0.6 mg kg(-1)). These results suggest that F. graminearum strains isolated from the wheat growing regions in Argentina belong to DON chemotype. Although some strains produced both deoxinivalenol and nivalenol, nivalenol was produced in lower levels. The natural occurrence of nivalenol in wheat affected by head-blight collected in the main production area during two years (2001-2002) was also determined. From 19 samples 13 were contaminated with deoxinivalenol in a range of 0.3 to 70 mg kg(-1)and 2 samples with both deoxinivalenol (7.5 and 6.7 mg kg(-1)) and nivalenol (0.05 and 0.1 mg kg(-1)), respectively. This is the first report of natural occurrence of nivalenol in wheat cultivate in Argentina.

Full text

Brazilian Journal of Microbiology (2008) 39:157-162
ISSN 1517-8382
157
NATURAL OCCURRENCE OF NIVALENOL AND MYCOTOXIGENIC POTENTIAL OF FUSARIUM
GRAMINEARUM STRAINS IN WHEAT AFFECTED BY HEAD BLIGHT IN ARGENTINA
V.E. Fernandez Pinto
1
*; L.A. Terminiello
2
; J.C. Basilico
3
; A. Ritieni
4
1
Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad
Universitaria, Buenos Aires, Argentina;
2
Cátedra de Industrias Agrícolas de Lechería-Agroindustrias, Facultad de Ciencias
Agrarias y Forestales Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina;
3
Ingeniería en Alimentos, Facultad
de Ingeniería Química, Universidad Nacional del Litoral, Sta. Fe, Argentina;
4
Dipartamento di Scienza degli Alimenti, Università
di Napoli “Federico II”, Portici, Napoli, Italia.
Submitted: July 11, 2007; Returned to authors for corrections: August 15, 2007; Approved: November 16, 2007.
ABSTRACT
The principal agents of Fusarium head blight in the main cropping area of Argentina were investigated in
heavily infected samples. The ability of the isolates to produce trichothecenes was determined by GC and
HPLC. Fusarium graminearum was the predominant species and of 33 isolates, 10 produced deoxinivalenol
(DON) (0.1- 29 mg kg
-1
), 13 produced both deoxinivalenol (1.0- 708 mg kg
-1
) and nivalenol (0.1- 6.2mg kg
-1
), 12
produced 3-acetyldeoxinivalenol (0.1- 14 mg kg
-1
), 13 produced 15-acetyldeoxinivalenol (0.1- 1.9 mg kg
-1
), 10
produced Fusarenone X (0.1- 2.4 mg kg
-1
) and 7 produced zearalenone (0.1- 0.6 mg kg
-1
). These results
suggest that F. graminearum strains isolated from the wheat growing regions in Argentina belong to DON
chemotype. Although some strains produced both deoxinivalenol and nivalenol, nivalenol was produced in
lower levels. The natural occurrence of nivalenol in wheat affected by head-blight collected in the main
production area during two years (2001-2002) was also determined. From 19 samples 13 were contaminated
with deoxinivalenol in a range of 0.3 to 70 mg kg
-1
and 2 samples with both deoxinivalenol (7.5 and 6.7 mg
kg
-1
) and nivalenol (0.05 and 0.1 mg kg
-1
), respectively. This is the first report of natural occurrence of
nivalenol in wheat cultivate in Argentina.
Key words: Fusarium, nivalenol, toxicogenic potential, trichotecenes.
INTRODUCTION
Wheat is one of the most important cereal crops in Argentina
both for local consumption and as an export commodity, with
yields over 12.000.000 tons (1). The cultivated area of 6.000.000
ha is distributed according to agrometeorological conditions
into five zones, with a main production area on the Buenos Aires
province (Zones IIS, IV and VS), East of La Pampa province
(Zone VS) and South of Santa Fe province (Zone IIN). This
extended area presents very different conditions of temperature
and humidity. The incidence of toxigenic Fusarium spp. and
their toxins overall this wide cultivated area has been scarcely
studied. F. graminearum Schwabe [teleomorph Gibberella zeae
(Schw.) Petch] is the most common causal agent of Fusarium
head blight (FHB) in many parts of the world. Recent outbreaks
had been reported in USA, Asia, Canada, Europe and South
America. This destructive disease affects wheat, barley and other
small grains in temperate and semitropical areas. The disease
has the capacity to destroy a potentially high– yielding crop
within a few weeks of harvest, causing economic looses due to
reduced seed quality. Additionally, infected grains may contain
significant levels of mycotoxins like trichothecenes and
zearalenone. Trichothecenes toxins are sesquiterpenoids that
are potent inhibitors of eucaryotic protein biosynthesis. Adverse
Departamento de Química Orgánica. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Ciudad Universitaria. Pabellón II. 3
er
Piso
(1428), Buenos Aires, Argentina. Tel./Fax: +54 11 4576-3346. E-mail: virginia@qo.fcen.uba.ar

158
Pinto, V.E.F. et al.
effects of the toxins in animals are food refusal, diarrhoea, emesis,
alimentary haemorrhaging, contact dermatitis and
immunosuppression. Zearalenone is an estrogenic mycotoxin
(11). Some earlier works have shown that F. graminearum was
the main occurring species and deoxinivalenol (DON) was the
main toxin contaminant detected in wheat and wheat-products
in Argentina (10,18,19,20). Two main chemotypes were reported
in G. zea from rice in Japan: the “NIV chemotype” which produces
nivalenol (NIV) and Fusarenone X (Fus-X) and the “DON
Chemotype” which produces deoxinivalenol (DON) and its
acetylated forms (12). Differences in the geographical distribution
of DON and NIV chemotypes of F. gramineraum have been
reported. Only the DON chemotype has been found in the United
States and Canada (16), while both chemotypes were isolated in
Japan (26), Italy (14), South Africa (23), and Australia (2).
Successively, Sugiura et al. (22) demonstrated that some G. zeae
isolated from rice stubble in Japan produced both NIV and DON.
This capability was also confirmed in some strains in Hungary
(24) and in strains isolated from maize and wheat in Nepal (6).
The ability of Argentinean Fusarium isolates to produce
trichothecenes is controversial and the pattern of micotoxin
production is not already well defined. Faifer et al. (8) reported
that F. graminearum isolated from wheat in Argentina
produced DON, 15- acetyldeoxinivalenol (15AcDON), 3-
acetyldeoxinivalenol (3AcDON) and zearalenone (ZEA) while
Ramirez et al (20) found only DON and 3-AcDON producers. On
the other hand, Lori (15) studied the capability of 76 F.
graminearum strains isolated from Argentinean wheat and 56%
produced DON, 3AcDON, 10% NIV, FUS-X and 34% both DON
and NIV. All those studies were carried out in crops heavily
affected by FHB.
The purposes of this study were to determine:
a) the pattern of trichothecene production by the isolates
of the principal FHB agents
b) the presence of both NIV and DON producing strains
c) the natural occurrence of NIV, in Argentinean wheat
affected by FHB
MATERIALS AND METHODS
Wheat samples
Nineteen wheat samples affected by head blight were
collected from four regions in the major wheat production area
in Argentina, 5 in the 2000- 2001 harvest and 14 in the 2001-2002
one. In both harvests the meteorological conditions favour the
infection. The regions were: Region II North (IIN) and Region II
South (IIS) (North of Buenos Aires province), Region IV (South
East of Buenos Aires province) and Region V (South West of
Buenos Aires and La Pampa province). Regions IIN and IIS
have a temperate humid climate while Regions IV and V have a
more continental semiarid one. Samples were also collected from
farms in 15 districts of Bs. As. and La Pampa provinces. At least
0.5 kg was collected per sample, selected from heads with
spikelets with visible symptoms of FHB. All samples were from
locally grown cultivars.
Mycological analyses
For the isolation of the internal micoflora, 100 wheat kernels
of each sample were surface disinfected in a 2% aqueous solution
of commercial sodium hypochlorite for 1 minute and rinsed twice
with sterile distilled water. The kernels were placed, 10 kernels
per plate, on potato dextrose agar (PDA) with 2% of pentachloro
nitrobenzene (PCNB) plates and incubated for 5 days a 28ºC
under fluorescent and black light lamps (2700 lux; 12 h
photoperiod) to stimulate conidial formation and the percentage
of infected kernels was calculated. Representative cultures of
the species isolated were grown from single conidia for 7 days
on petri dishes with carnation leaves agar (CLA) and with potato
dextrose agar (PDA) and incubated a 28ºC under fluorescent
light. The identification of colonies of Fusarium species was
made according to the criteria and synoptic keys of Leslie and
Summerell (13)
Toxin analysis
Sample analysis
A subsample of 25g of wheat was milled at 2500 rpm in a
commercial grinder. The extraction of deoxynivalenol (DON),
nivalenol (NIV), was done with 125 ml of acetonitrile: ethyl
acetate: water (50:41:9) as extraction solvent per 1 hour at 300
rpm. The clean-up was made with a column packed with charcoal:
alumina: celite (0.7:0.5:0.3) (19); and the extract was dried in a
rotary evaporator. The dry residue was re-dissolved in 500 ml
ethyl acetate: metanol (95:5) and transferred to a vial.
Evaluation of toxicogenic capacity of Fusarium isolates
To test for trichothecenes and zearalenone production,
Fusarium isolates were placed in duplicate on 25g of sterilized
rice in 500 ml Erlenmeyer flasks. The rice was added with 15 ml
of sterile distilled water before sterilization by autoclaving al
121ºC for 15 min. Each flask was inoculated with a suspension
containing 10
6
conidia ml
-1
. The conidial suspension was
performed by adding 5,0ml of sterile distilled water to a slant of
a 7day culture at 28ºC on PDA and gently scraping the agar
surface with a wire loop to give a turbid suspension.
Toxin extraction
DON, NIV, 15AcDON, 3AcDON, FUSX, and ZEA were
extracted with acetonitrile: ethyl acetate: water (50: 41: 9) as
extraction solvent, and the clean up was made with a column
packed with charcoal: alumina: celite (0.7: 0.5: 0.3) as was
previously described (19).
Standards of DON, NIV, 15AcDON, 3AcDON, FUSX, and
ZEA were purchased from SIGMA chemical Company (St. Louis,
MO, USA).

Nivalenol and mycotoxigenic Fusarium graminearum
159
Toxin detection
Toxins detection for both samples and isolates production
was made by gas liquid chromatography (CGL) and the NIV
presence was confirmed in all cases by HPLC.
DON, 3-AcDON, 15 AcDON, FUSX and ZEA were also
confirmed by TLC (15)
CGL
Trichothecenes and ZEA were detected and quantified by
gas liquid chromatography with Ni
63
electron capture detection
Shimadzu Model GC17, equipped with split/splitless injector
and fitted with a RX-5MS capillary column (25 m x 0, 2 mm i.d.).
The injector temperature was 250ºC and the detector one 300ºC.
Separation was achieved with a temperature program consisting
of 1min at 80ºC, then increase 80ºC - 140ºC at 30ºC per min,
followed by 140ºC-280ºC at 5ºC per min. Both the carrier and
auxiliary gas were nitrogen. The derivatization was carried out
with heptafluorbutyric anhydride with the method reported by
Croteau et al. (4). The detection limit for DON and its acetyl
derivatives was 20 µg kg
-1
and for NIV, FUS and ZEA 50 µg kg
-1
respectively
HPLC
NIV was confirmed with Shimadzu HPLC (2 pumps LC-10
ADvp, 1 DAD SPD- M10Avp, 1 System controller SCL-10 Avp)
with precolumn Phenomenex Widepore C18 (ODS) (4x3 mm)
and column Phenomenex Jupiter 5u C18 (300A, 20x 4,6 mm).
Each analysis was done with a 20 ul loop, water (A) + acetonitrile
(B) as eluent solvents (gradient: 0 min 10%B, 10min 100% B, 13
min 100%B, 16 min 10%B, 20 min 10%B), total flow 1ml per min
at wavelength: 219nm. The detection limit was 50 µg kg
-1
.
RESULTS
The percentage of infected kernels in the samples is
illustrated in Table 1. In 13 of the 19 samples the infection was
of 100%. In the other samples the level of infection was always
high (69 to 95%). Fusarium graminearum was found as the
main occurring species and it was found in 17 samples in levels
from 4 to 88%. Fusarium poae and F. verticillioides were also
rarely encountered (less of 2%).
All the samples were assayed from the presence of DON
and NIV. The results were presented in Table 1, where it is also
shown the geographical origin of the samples.
Fifteen of the 19 samples were contaminated with DON
and the levels ranged from 0.3 to 70 mg kg
-1
. In this study 2
samples (Arrecifes from North of Buenos Aires province and
Conhelo from La Pampa province) were contaminated with
DON (7.5 and 6.7 mg kg
-1
) and NIV (0.05 and 0.1 mg kg
-1
)
respectively. From the samples of wheat most heavily
contaminated were isolated 33 strains of F. graminearum and
their toxicogenic potential was determined in autoclaved rice.
Table 1. Natural occurrence of DON and NIV in Argentinean wheat affected by head blight.
Zone Sample Infected. kernels % F. graminearum% DON mg kg
-1
NIV mg kg
-1
IIN 13-Pergamino 100 50 7.8 ND
14-San Pedro 100 82 70 ND
1-Arrecifes 100 74 7.5 0.05
IIS 9- Salto 100 88 16 ND
15- Villegas 100 40 3.0 ND
19- Lincoln 100 6 3.3 ND
10 -Saladillo 100 85 14 ND
2- Olavarria 100 - - ND
3- Olavarria 100 21 1.3 ND
IV 4- Olavarria 83 - 0.8 ND
7- Gral.Pueyrredon 100 12 0.3 ND
11- Gral. Pueyrredon 100 4 0.4 ND
5- Cnel Suarez 85 39 5.4 ND
8- Conhelo 95 67 6.7 0.1
12- Conhelo 100 43 5.8 ND
V 6- Mt. Nievas 100 39 5.8 ND
16- Luiggi 86 4 ND ND
17- Winifreda 69 7 ND ND
18- Castex 79 6 ND ND

160
Pinto, V.E.F. et al.
Concentration of trichothecenes and zearalenone produced
are illustrated in Table 2.
In this study, 10 strains produced DON ranging between 0.1
and 29mg kg
-1
.
Of these strains 3 produced also 3 AcDON
ranging between 0.7 and 3.0 mg kg
-1
and 4 produced 15 AcDON
ranging between 0.1 and 0.9 mg kg
-1
. The results also
demonstrated the production of both DON (ranging between
1.0 and 708 mg kg
-1
) and NIV (ranging between 0.1 and 6.2 mg
kg
-1
) by 13 of 33 F. graminearum strains tested. In DON and
NIV producers, 9 strains produced FUS X (up to 2.4 mg kg
-1
), 8
produced 3AcDON (up to 14 mg kg
-1
) and 9 produced 15 AcDON
(up to 1.9 mg kg
-1
). Of these, 5 strains produced 3 AcDON and
15 AcDON simultaneously. Only one strain produced FUS X
(0.2 mg kg
-1
), DON (20 mg kg
-1
) and 3 AcDON (4.1 mg kg
-1
).
Zearalenone was produced by 7 strains in low amounts, ranging
between 0.1 and 0.6 mg kg
-1
and no exclusive relationship could
be observed between ZEA production and the DON and NIV
production. Finally, we did not detect trichothecene production
from eight strains.
DISCUSSION
These results agree with other reports carried out in Argentina
where F. graminearum proved to be the main toxigenic Fusarium
species (10,15,20). All the samples collected in the zones IIN
and IIS are 100% infected. On the contrary, zone V can be
considered less contaminated because 71.42%
of samples don’t reach 100% of infection. In
fact, F. graminearum is the most frequent fungi
in the zone IIN and IIS while the percentages
are lower in the zones IV and V.
The levels of toxin contamination found
in samples in this study are similar to those
reported earlier in heavily infected wheat in
Argentina (10,19). This is the first report of
the natural occurrence of NIV in wheat
cultivated in Argentina. DON and NIV are
very similar in their chemical structure, but
differ markedly in their toxicological
properties with NIV considered more
mycotoxic than DON (25). Therefore the
presence of NIV in Argentinean wheat may
have a significant impact on the food safety
aspects because its presence can synergize
the toxicity of DON. By this study and other
reports (5,10,19) Argentina prove to have a
high risk of DON wheat contamination due
to the high incidence of FHB, particularly in
the area with temperate humid climate. This
risk seems also to be present in South of Brazil
(9) and Uruguay (18).
Overall, the DON production by strains
proved to be higher that NIV. All the data
analyzed in this study support the fact that
the variation among the toxigenic potential of
F. graminerum strains isolated from
Argentinean wheat is quite complex and may
contribute to a wide range of mycotoxin
content in this cereal. The introduction of
novel FHB chemotypes via global trade in
agricultural products should be assessed
because could have the potential to exacerbate
FHB problem (17). In particular the relative
frequency of DON and NIV is of concern
because recent reports suggest that strains of
Table 2. Production of trichothecenes and zearalenone by F. graminearum
isolates (mg kg
-1
).
Strain Nº Sample Region NIV FUSX DON 3 ADON 15 ADON ZEA
4620 13 IIN ND ND 25 ND ND ND
4614 10 IIS ND ND 19 0.9 ND ND
4601 1 IIS ND ND 29 0.7 0.1 ND
4602 1 IIS ND ND 8.3 3.0 0.9 ND
4604 1 IIS ND ND 10 ND ND ND
4622 14 IIS ND ND 2.5 ND 0.2 ND
4626 14 IIS ND ND 25 ND 0.1 ND
4629 15 IIS ND ND 16 ND ND ND
4632 9 IIS ND ND 6.0 ND ND 0.1
4617 13 IIN ND ND 0.1 ND ND
4619 13 IIN 0.8 2.4 120 2.9 0.3 0.1
4615 10 IIS 1.5 2 95 ND 1.8 ND
4616 10 IIS 0.4 0.1 38 2 0.2 0.6
4600 1 IIS 6.2 0.7 708 14 ND ND
4603 1 IIS 0.2 ND 11 ND ND ND
4623 14 IIS 0.1 ND 1.0 0.3 ND 0.1
4634 9 IIS 1.1 ND 14 1.0 0.3 ND
4635 9 IIS 2.1 ND 21 ND 1.9 0.1
4636 9 IIS 0.6 0.1 37 0.3 0.1 ND
4637 9 IIS 0.2 0.1 98 0.1 ND ND
4638 9 IIS 2.2 0.1 17 ND 0.7 0.1
4640 15 IIS 0.5 0.1 8.5 ND 0.3 ND
4606 7 IV 0.2 0.1 58 1.0 0.4 0.4
4609 8 VS ND 0.2 20 4.1 ND ND
4618 13 IIN ND ND ND ND ND ND
4624 14 IIS ND ND ND ND ND ND
4625 14 IIS ND ND ND ND ND ND
4627 14 IIS ND ND ND ND ND ND
4628 14 IIS ND ND ND ND ND ND
4631 9 IIS ND ND ND ND ND ND
4633 9 IIS ND ND ND ND ND ND
4605 7 IV ND ND ND ND ND ND

Nivalenol and mycotoxigenic Fusarium graminearum
161
the F. graminearum complex that produce NIV may be more
aggressive towards corn but less aggressive to wheat than
DON producing strains. These results indicate that there may
be important consequences for the fitness and aggressiveness
of FHB pathogens of different chemotype on particular hosts
(3,7,11). Further studies will be necessary in order to characterize
the genetic diversity, chemotype and virulence of F.
graminearum populations from Argentina, a country where this
species is widespread and wheat is cultivated under different
agro-meteorological conditions.
RESUMO
Ocorrência natural de nivalenol e potencial
micotoxigênico de cepas de Fusarium graminearum em
trigo afetado por giberela na Argentina
O principal causador de giberela no trigo na Argentina e sua
capacidade de produzir tricotecenos foram estudados por GC e
HPLC em amostras altamente infectadas. A espécie
predominante foi Fusarium graminearum, sendo que de um
total de 33 isolados, 10 produziram deoxinivalenol (0,1-29 mg
kg
-1
), 13 produziram deoxinivalenol (1,0-708 mg kg
-1
) e nivalenol
(0,1-6,2 mg kg
-1
), 12 produziram 3-acetildeoxinivalenol (0,1-14
mg kg
-1
), 13 produziram 15-acetildeoxinivalenol (0,1-1,9 mg kg
-
1
), 10 produziram fusarenona X (0,1- 2,4 mg kg
-1
) e 7 produziram
zearalenona (0,1- 0,6 mg kg
-1
). Esses resultados sugerem que as
cepas de F. graminearum isoladas de trigo cultivado na
Argentina pertencem ao quimiotipo DON. Embora algumas
cepas tenham produzido tanto DON quanto NIV, NIV foi
produzido em quantidade inferior ao DON. A ocorrência natural
de nivalenol em trigo afetado pela giberela coletado na principal
área de produção durante dois anos (2001-2002) foi também
determinada. De 19 amostras, 13 estavam contaminadas com
deoxinivalenol na faixa de 0,3 a 70 mg kg
-1
e 2amostras continham
tanto deoxinivalenol (7,5 e 6,7 mg kg
-1
) quanto nivalenol (0,05 e
0,1 mg kg
-1
), respectivamente. Esse é o primeiro relato da
ocorrência de nivalenol em trigo cultivado na Argentina.
Palavras-chave: Fusarium, nivalenol, potencial toxicogênico,
tricotecenos.
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