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Asiatic Journal of Biotechnology Resources; 2011; 2 (04) 392-402

Asiatic
journal of
BIOTECHNOLOGY
RESOURCES
International Quarterly
Journal of Bioscience

Research communication

Natural incidence of fusarial mycotoxins in Finger millet

(Eleusine coracana L.) of Andhra Pradesh, India
Shilpa Penugonda, V. Koteswara Rao, Girisham. S. and Reddy.S.M
Department of Microbiology, Kakatiya University, Warangal- A.P. India
Correspondance: Shilpa Penugonda, 101 Pinnacle CT, Apt. 40, Frankfort, Kentucky - 40601
Email -shilpa.penugonda1@gmail.com, Contact No. 502 498 4949
Received 23 Feb 2011; accepted19 March 2011
Available online: 31 March 2011

Keywords: Finger millet, Fusarial toxins, Fusarium spp.

Abstract
An extensive and intensive investigation
was carried to determine the natural
contamination of important fusarial
mycotoxins in finger millet of different
regions of A.P. A total of 110 market
samples were investigated for these
mycotoxins and contamination was
detected in 70 percent sample. A high
percentage of isolates of Fusarium were
mycotoxigenic and produced one or
more mycotoxins. Zearalenone (ZEA),
T2 toxin, nivalenol (NIV) and
Deoxynivalenol
(DON)
and
Deoxyscripenol (DAS) were some of the
mycotoxins detected. Nevertheless these
results indicate the need to establish, a
continuous monitoring program to
prevent and manage the occurrence of
these contaminants. Copyright 2011
Pacific Publishers International. All rights
reserved.

Introduction
In the current temperate climatic conditions, Fusarium fungi are
significant in the cereal food chain, being capable of reducing crop
yield and contaminating grain with mycotoxins. Moulds, besides
depleting the nutrients, may also produce toxic substances that
are potential health hazards to animals and, in turn to humans
(Fazekas et.al., 1996; Trucksess, 2001). Majority of the Fusarium
species produce trichothecene mycotoxins. Trichothecenes are
esters of sisquiterpenoid alcohols containing the trichothecene
tricyclic ring system (Pestka and Smolinski, 2005). Deoxynivalenol
(DON) and nivalenol (NIV) are a group of closely related
secondary fungal metabolites, that are produced predominantly,
although not exclusively, by several species of the genus Fusarium,
especially F.graminearum (Marasas, 1991). In recent times
fumonisins contamination has been reported more frequently from
different parts of the World (Kim et.al., 1993; Xie et.al., 1997;
Castella et.al., 1999; Martins et.al., 2001; Nikiema et.al., 2004;
Massimo et.al., 2009; Ana et.al., 2009).These mycotoxins can be
very stable to food processing (Malin-ieet.al., 2005) and be present
in the final products. Finger millet are known as ragi in India is
important staple food for people belonging to the low socioSubmitted online at submit@pacificjournals.com
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393

S. Penugonda et al.

economic group, several reports have shown that millet (Pathak

et.al., 2000) are inexpensive and nutritionally comparable or even
superior to major cereals. Regular consumption is known to reduce
the risk of diabetes mellitus (Gopalan, 1981) and gastro intestinal
tract disorders (Tovey, 1994). These seeds are vulnerable to the
huge diversity of opportunistic microbes especially the Fusarial
species, further it is anticipated that seeds are more vulnerable to
mycotoxin contamination. There are several studies dealing with
the mycoflora of cereal foods and mycotoxicoses of live stock
from different countries (Phillips et.al, 1996; Kubena et.al, 1997;
Magnoli et.al, 1999; Bennett et.al, 2003; Dalcero. A et.al, 2004).
Such reports are also available from India (Rajan et.al, 2006; Vinod
Kumar et.al, 2008; K. R. N. Reddy et.al, 2009; Narshima Rao et.al,
2009). However, little or no studies on the incidence of Fusaria
in Finger millet and Fusarial mycotoxin contamination are available.
In this context, the purpose of this study was to obtain some data
on mycotoxin production in Finger millet destined to human
consumption.

Materials and Methods

Source of materials
A total of 110 pre-packaged samples were selected from
local retail commerce in Andhra Pradesh of India. No particular
preference was used in selecting samples or locations. The sample
size was 250gm were analyzed. The mycoflora of seed as well as
the mycotoxins were assayed.

Isolation of Fungi
Mycoflora of Finger millet was analyzed by Blotter
technique (ISTA, 1985). Three layers of blotters equivalent to
Petri dish size were sterilized and soaked in distilled water, excess
of water was drained. Place 35-40 of seeds at equidistance in
each Petri dish, incubated at 27+2C for 7 days. The fungi were
isolated from the third day until the seventh day and identified by
standard monographs (Ellis 1976; Samson et.al., 1984). Special
attention was paid to the isolation and identification of different
species of Fusarium (Nelson et.al., 1983). The percentage of
incidence of individual fungi was calculated using the following
formula% of incidence =

No. of colonies of species in all the plates

x 100.
Total no. of colonies of all the species
in all the plates

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394

Mycotoxin analysis
Fusarial mycotoxins were analyzed using thin layer
chromatography (TLC). For this purpose, Fusarial culture filtrates
were extracted twice with 100ml of ethyl acetate. The combined
extracts were passed through an anhydrous Na 2So4 bed to remove
moisture and then evaporated to dryness before dissolving in 1ml
of methanol and spotting onto the TLC plates. The toxins were
identified by spraying the plates with different spray reagents (Table
1) as suggested by Kamimura et.al (1981) and Rao et.al (1985),
and the compounds thus separated were identified based on the
color of the fluorescence of the spot and by the Rf values, as
compared with standards. The Rf was calculated by using formula.

Rf =

Distance traveled by the compound

Distance traveled by the solvent

Results and Discussion

Incidence of Fusarial contamination was found to be 70%
of the Finger millet samples (Table-2), 13 species of Fusarium
was recorde4d with varying percentages of incidence. Sample
collected from Warangal were comparatively more infested by
Fusaria, while from Nizamabad contained low infestation. Samples
from Ongole, Khamman and Vijayawada come next with regard
to this degree of Fusarial infestation. Of all the Fusarium species,
F.moniliforme was dominant and could be isolated from all the
samples collected. F.oxysporum was also isolated from all the samples
with a high percentage of incidences, except in samples from
Nizamabad. F.equiseti could be isolated only in samples of Warangal
and Vijayawada. F.solani, F.latertium and F. poae could not be isolated
from samples of Khamman, Nalgonda, Adilabad and Nizamabad,
and its incidence in samples of other places was low. F.proliferatum,
F.subglutinans, F.sporotrichoides and F.heterosporum could be isolated
from samples of Nalgonda, Khamman and Nizamabad. The very
low incidence of Fusarial species collected from Nizamabad may
be attributed to the prevailing dry climate. In comparison, the
observed higher incidence of different Fusarium species in Finger
millet samples of Vijayawada may be due to the warm humid
climate prevailing in that region. However, more samples, during
different times of the year and from different regions, should be
collected and analyzed before a definitive conclusion can be
reached.
F.graminearum could be recorded in samples of Warangal,
Khammam, Nizamabad and Vijayawada with a low percentage of
Asiatic J. Biotech Res. 2011; 2(04)392-402

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S. Penugonda et al.

395

Table 1. Detection of trichothecences and other mycotoxins produced by fusarium

Name of the toxin

Solvent system

Spray reagent
UV

Dection
Visible

Reference

Deoxynivalenol ( DON)

Ea: T:F ( 5:4:1)

4,7,8

Ch,bl

Y,-,-

Diacetoxy Scirpenol (DAS)

C: M ( 97: 3)

6,9

bg

-,br

Ramakrishna and Bhatt ( 1987)

Kamimura et al., ( 1981) Ramakrishna and
Bhatt ( 1987)

Fusarinone -X

C: M ( 97: 3)

bl

Kamimura et al., ( 1981) Ramakrishna and

Bhatt ( 1987)

HT-2 toxin

C: M ( 97: 3)

bg,-

Kamimura et al., ( 1981) Ramakrishna and

Bhatt ( 1987)

Nivalenol (NIV)

Ea: T:F ( 5:4:1)

4,7,8

-,Chl,bl

y,-,-

Kamimura et al., ( 1981) Ramakrishna and

Bhatt ( 1987)

T-2 toxin

C: M ( 97: 3)

6,9

bg,-

-,P

Kamimura et al., ( 1981) Ramakrishna and

Bhatt ( 1987)

Zearalenone

C: M ( 97: 3)

1,2,3,4,5,7,8

br,Ch,bl

Gorst -Allman and steyn (1979) Miroche et

br,do,ip,- - - al., ( 1974) Pathre et.al ( 1979)

Fumonisins

W:M ( 3:)

4,11

br

Solvent systems
Spary reagents

:
:

Detection colours :

Kamimura et al.,( 1981)

C= Chloroform ; H= Methanol; W- Water

1= Ce (SO4)2 1% 6N H2 SO4 ; 2=2,4-DNP; 3=FeCl3 3% in ethanol ;
4= P- anisal dehyde ; 5=50% H2SO4 in methanol ; 6=20% H2SO4;
7= H2SO4; 8=20% AICI3; 9= Chromatropic acid
bl=Blue ; Ch=Charring ; Y =Yellow ; bg= blue green ; br= Brown ;
P=purple do= Dark organe ,ip-light purple ; bb=bright blue.

incidences. F.culmorum and F.chlamydosporum could not be recorded

in the samples of Nalgonda, Adilabad and Nizamabad. However,
incidence was high only in samples of Warangal, followed by
Vijayawada. F.graminearum could be recorded with a high percentage
in samples of Khamman and Vijayawada. The incidence of
F.moniliforme was high in samples collected from Warangal and
Vijayawada.
In all, the thirteen Fusarium species could be recorded in
samples collected from different regions of A.P, India. Some strains
of F.culmorum and F.graminearum are able to produce type B
trichothecenes, such as DON (deoxynivalenol) and NIV
(nivalenol), while other species are not (Marasas et.al., 1984).
Table 3 reveals that of the 708 Fusarium isolates, 207 were
mycotoxigenic. Many of the strains could produce more than one
mycotoxin. Screening of 131 strains of F.moniliforme indicated that
45 were mycotoxigenic, of which 11, 5 and 4 strains produced
Zearalenone (ZEA), T2 toxin and DAS respectively. Only 5 strains
could produce DON, while HT2 toxin was produced by four strains
of F.moniliforme. Screening of 77 strains of F.oxysporum indicated
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396

Table 2. Mycotoxigenic potential of different species of Fusarium isolated strains from Finger millet of A.P
Name of the fungi

Warangal
A
B

Khammam
A
B

Nalgonda
A
B

Ongole
A
B

Nizamabad
A
B

Adilabad
A
B

Vijayawada Name of the toxin

A
B

F.chlamydosporum

21

13

--

--

17

12

--

--

14

ZEA, T2, DAS, DON

F.culmorum

17

14

12

16

--

--

11

ZEA, DON,NIV, Niosolanil

F. graminarum

17

--

--

--

--

11

--

--

DON,NIV,ZET,T2

F. oxysporum

14

13

11

14

--

--

14

11

ZEA,T2,FB,DON

F.moniliforme

28

11

23

24

14

12

16

14

ZEA,HT2,FB,DON

F. equiseti

--

--

--

--

--

--

--

--

--

--

--

--

F.solani

--

--

11

ZEA,NIV,T2,DON

F. latertium

--

--

--

--

--

--

--

--

--

NIV

F. sporotrichoides

14

18

--

--

--

--

12

11

ZEA,T2,NIV

F.poae

18

--

--

--

--

--

--

14

ZEA, DAS

F. heterosporum

13

--

--

11

NIV,DAS,T2,HT2

F. proliferatum

--

--

--

ZEA,T2

F. subglutinans

11

--

--

12

--

ZEA,T2,DAS,FUX

A-No. of strains screened : B-No of toxin producing strains ZEA- Zearalenone ; T2-T2-toxin ;
DAS- Diacetoxscirpenol; DON- Deoxynivalenol; NIV-Nivalenol; HT2-HT2-toxin;
FB1-Fumonisin B1; FUX-Fusarinone X.

that 21 were mycotoxigenic and produced one or more mycotoxins.

Of these strains 2, 2, 6 and 3 strains respectively, produced
Zearalenone, T2 toxin, HT2 and DAS. NIV was not produced by
F.oxysporum. Of the 42 strains of F.solani screened, 24 were toxigenic
and produced Zearalenone, T2, DON and NIV. Screening of 38
strains of F.graminearum indicated that 8 were mycotoxigenic and
produced T2 toxin, NIV, ZEA and DON. Out of 61 strains of
F.sporotrichoides, indicated that 21 were mycotoxigenic and produced
ZEA, T-2 and NIV. F.latertium produced NIV in 2 strains were
mycotoxigenic. F.subglutinans in which out of 48 strains, indicated
that 13 were mycotoxigenic and produced ZEA, T-2, DAS and
FUX respectively. Screening of 78 strains of F.culmorum indicated
that 25 were mycotoxigenic and produced ZEA, DON, NIV and
Niosalanil. F.equseti failed to produce mycotoxin in all the samples
of Finger millet. Remaining all the Fusarial isolates produced ZEA,
T-2 toxin, DAS and NIV. According to the findings of ODonnell
et.al (1998) working with Fusarium, strains can be distinguished on
the basis of DNA polymorphism in the -tubulin gene as well as
in the large ribosomal subunit or the internal transcribed spacer
(Mule et.al., 1997; ODonnell et.al., 1998).According to
trichothecene production, F.culmorum and F.graminearum strains have
been divided into two chemo types: the NIV chemo type, which
includes isolates producing niavlenol, and the DON chemo types,
which includes isolates producing DON and acetyl deoxynivalenol
(Bakan et.al., 2001; Syndenham et.al., 1991). However, the
Asiatic J. Biotech Res. 2011; 2(04)392-402

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397

Table 3. Incidence of mycotoxigenic fusaria in Finger millet from different regions of A.P.

14.6

% of
frequency
7.8

%of
abundance
8.6

9.2

9.2

15.6

--

12.3

6.1

24.8

--

4.6

14.0

9.2

17.2

30.0

40.3

39.0

46.0

10.8

31.8

--

--

--

2.8

3.1

3.7

2.3

0.5

2.0

2.0

12.4

9.2

8.1

0.9

--

0.8

--

--

--

3.1

4.3

3.1

2.3

--

1.2

--

2.3

2.3

7.8

5.9

F. Poae

4.1

--

--

6.4

--

1.9

5.2

6.1

10.2

F. heterrosporum

2.8

2.3

3.0

0.8

--

2.8

2.3

9.2

6.4

F. proliferatum

3.6

0.8

3.0

4.8

2.1

1.9

6.2

9.2

5.4

F.subglutinans

2.3

--

5.5

1.2

4.6

1.9

2.3

9.2

9.1

Name of the fungus

Warangal

Khammam

Nalgonda

Ongole

Nizamabad

F.chalmydosporum

1.2

F.culmorum

4.3

F.graminearum

Adilabad Vijayawada

1.1

--

8.3

4.8

--

4.8

1.2

2.1

--

1.9

3.6

10.3

--

--

6.7

F.oxysporum

8.2

1.3

6.8

5.5

F. moniliforme

46.8

27.5

38.1

F.equiseti

2.2

--

--

F. solani

2.7

--

F. latertium

--

F.sporotrichoides

substrate on which a Fusarium strain is grown could influence mycotoxin

production (ONeill et.al., 1993).
In addition, it has been demonstrated that, with in the same species
and in the same culture conditions, toxin production by Fusarium strains
may vary sharply; some strains produce large amounts of trichothecenes,
where as others produce small or undetectable amounts of trichothecenes
(Syndenham et.al.,1991; Muthomi et.al.,2000; Walker et.al., 2001; Llorens
et.al., 2006). Doohan et.al, (2003) showed that production of trichothecenes
by F.culmorum and F.graminearum is favored by warm and humid conditions.
The main mycotoxins produced by Fusarium include the trichothecenes DON,
NIV, 3-acetyl deoxynivalenol and acetyl T2 toxin, ZEA and Fuasrins
(Demeke et.al, 2005; Llorens et.al, 2006). The trichothecenes including
DON, acetyl deoxynivalenol, NIV and fusarenone X, are common fungal
contaminants of millets (Jennings et.al, 2000; Magan and Olsen, 2004) and
occur naturally World wide on millets (Dalcero et.al., 1997).Consumption
of these toxins is a potential problem for humans and farm animals (Rotter
et.al., 1996; Eriksen et.al., 1998). In general, about 25-34.8% of the strains
of different species of Fusarium isolated from samples of different regions
of A.P was mycotoxigenic and produced toxic substances that are health
hazards to animals, and, in turn to humans.

Acknowledgement:
Our thanks are due to Head, Department of Microbiology, Kakatiya
University, A.P, India for providing necessary facilities and financial support
by JNMF, New Delhi, India.

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