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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
[email protected], 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-ie’et.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 “
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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+2°C 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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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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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)
8
bl
-
Kamimura et al., ( 1981) Ramakrishna and Bhatt ( 1987)
HT-2 toxin
C: M ( 97: 3)
6
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 Copyright © 2010 Pacific Publishers
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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
5
13
1
--
--
17
3
12
4
--
--
14
5
ZEA, T2, DAS, DON
F.culmorum
17
5
14
3
12
4
16
7
--
--
11
4
8
2
ZEA, DON,NIV, Niosolanil
F. graminarum
17
3
6
2
--
--
--
--
11
2
--
--
4
1
DON,NIV,ZET,T2
F. oxysporum
14
2
13
2
11
3
14
6
--
--
14
4
11
3
ZEA,T2,FB,DON
F.moniliforme
28
11
23
5
24
8
14
5
12
4
16
7
14
5
ZEA,HT2,FB,DON
F. equiseti
8
3
--
--
--
--
--
--
--
--
--
--
--
--
F.solani
8
1
--
--
4
1
2
9
3
11
2
8
2
ZEA,NIV,T2,DON
F. latertium
--
--
5
--
--
--
4
1
--
--
--
--
3
1
NIV
F. sporotrichoides
14
5
18
7
--
--
6
1
--
--
12
4
11
4
ZEA,T2,NIV
F.poae
18
7
--
--
--
--
8
3
--
--
14
6
6
1
ZEA, DAS
F. heterosporum
13
2
7
2
9
4
8
5
--
--
11
2
6
2
NIV,DAS,T2,HT2
F. proliferatum
5
1
4
--
--
--
9
2
6
3
4
1
8
1
ZEA,T2
F. subglutinans
11
4
--
--
12
4
9
3
5
1
6
1
5
--
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 O’Donnell et.al (1998) working with Fusarium, strains can be distinguished on the basis of DNA polymorphism in the â-tubulin gene as well as in the large ribosomal subunit or the internal transcribed spacer (Mule et.al., 1997; O’Donnell 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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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 (O’Neill 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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