Prevalence of fumonisin producing Fusarium verticillioides associated with cereals grown in Karnataka (India)

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1 Available online at ScienceDirect Food Science and Human Wellness 5 (2016) Prevalence of fumonisin producing Fusarium verticillioides associated with cereals grown in Karnataka (India) N. Deepa, H. Nagaraja, M.Y. Sreenivasa Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore , Karnataka, India Received 10 July 2015; received in revised form 13 June 2016; accepted 5 July 2016 Available online 13 July 2016 Abstract A total of 135 cereal samples were collected from different districts of Karnataka state, India in which 69 samples were infected with Fusarium species. Among these 51 samples were having Fusarium verticillioides infection and among them 42 samples were positive for fumonisin production. Per cent incidence and frequency were high in maize samples with 33.12% and 47.54%, respectively followed by paddy and sorghum, while pearl millet was free from F. verticillioides infection. Relative density of F. verticillioides association was 59.50% among the screened samples. A total of 326 Fusarium species were isolated by screening 135 cereal samples and among these 194 isolates of F. verticillioides scored positive for VERTF-1 and VERTR species-specific pair of primers. Further amplification with VERTF-1 and VERTF-2 pair of primers recorded 176 isolates of fumonisin producing F. verticillioides. The study revealed high incidence, frequency and relative density of fumonisin producing F. verticillioides and production of fumonisins in cereals. It was amplified using one forward and two reverse primers to discriminate fumonisin producing from fumonisin non-producing F. verticillioides which stresses the need for the development of managemental stratergies before they enter into the food chain Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( Keywords: Fumonisin; Fusarium; IGS; Cereal; Maize; Paddy; Sorghum 1. Introduction Cereals are the basic staple food of India and provide much of the energy and protein for majority of the population. They also have known to contain a range of micronutrients such as vitamin E, some of the B vitamins, sodium, magnesium and zinc. Contamination of cereals due to poor agricultural practices and intermittent rain at the time of harvest by fungal species of Aspergillus, Fusarium and Penicillium are often unavoidable and it is worldwide problem [1,2]. The most common mycotoxins present in cereals are aflatoxins, fumonisins, zearalenone, Corresponding author at: Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore , Karnataka, India. Tel.: address: sreenivasamy@gmail.com (M.Y. Sreenivasa). Peer review under responsibility of Beijing Academy of Food Sciences. ochratoxins, T2 toxin and deoxynivalenol [3]. Contaminantion of cereals and cereal based products with fumonisins poses threat to agriculture and food safety throughout the globe. Food and Agriculture Organization (FAO) estimated that each year 25% 50% of the world s food crops are contaminated by mycotoxins [4]. Of the fungi involved, the most common are Fusarium species which are associated with cereals all over the world. Totally 70 different Fusarium species were isolated and identified from many substrates throughout the world [2]. Fusarium verticillioides is an important fungal pathogen with a wide range of plant hosts such as maize, paddy, sorghum, etc. [5]. The risk of contamination by fumonisins is related to the association of F. verticillioides species with cereals [6,7]. Fumonisins are considered as agriculturally important environmental toxins produced by F. verticillioides and other Fusarium species in the field or during storage [8]. Fumonisins cause several diseases such as blind staggers and leukoencephalomalacia in horses [9], pulmonary edema in swine [10] and hepatic cancer in rats [11], esophageal cancer, liver cancer [12], skin lesions [13], wound [14], keratitis and polycystic kidney disease in humans. More than ten types of fumonisins have been characterized among / 2016 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (

2 N. Deepa et al. / Food Science and Human Wellness 5 (2016) which B1, B2 and B3 are the major types produced [15]. The International Agency for Research on Cancer (IARC) has indicated that FB1 is a possible carcinogen to humans. Rocha et al. [42] reported high frequency (96%) of F. verticillioides in maize grains collected from four different regions of Brazil. F. verticillioides and other Fusarium species are reported to cause ear rot in maize. F. proliferatum was reported along with F. verticillioides from Italy [16], Southern Europe [17] and Iran [18]. F. subglutinans was the species most frequently recovered from asymptomatic host tissue and was more frequent than F. verticillioides [19]. Many instances of asymptomatic infection of F. verticillioides in corn have been reported [20,21]. Levic et al., [39] reported dominance and frequency of Fusarium species isolated from corn kernels over years; F. subglutinans predominated in some years. High prevalence of F. verticilliodes associated with cereals consistently proved by molecular based study with species specific primers when compared to conventional methods. The most reliable method to distinguish between F. verticillioides and closely related species is DNA sequence comparison. DNA used included nuclear ribosomal DNA intergenic spacer (IGS), the nuclear ribosomal DNA internal transcribed spacer, genes encoding the translation elongation factor 1 (TEF), b-tubulin, calmodulin, cytochrome P450 reductase, and 28S ribosomal RNA [22,23]. One set of species specific primer VERTF-1 [24] and IGS based VERTR primer [25] have been used to differeniate Fusarium verticillioides from other Fusarium species. The other set of primer included VERTF-1 and VERTF-2 to discriminate fumonisin producing from non fumonisin producing isolates. Aim of the present work was to study the per cent incidence, frequency and relative density of F. verticillioides associated with maize, sorghum, paddy and pearl millet using conventional and PCR methods. Further, to know their ability to produce fumonisin by LC MS method. 2. Material and methods 2.1. Collection of samples A total of 135 cereal samples (61 maize, 42 paddy, 24 sorghum and 8 pearl millet) were collected from different districts of Karnataka state during November 2012 May All the collected samples (0.5 kg) were packed in sterile polythene bags, labeled appropriately and maintained at 4 C. They were subjected to mycological analysis Isolation of Fusarium species Sampling was done by hand halving method according to International Seed Testing Association (ISTA 2003). The incidence of Fusarium species was analyzed using both standard blotter and agar plating methods [26]. Two hundred grains from each sample were placed on moist blotting material as well as on agar media. Melachite Green Agar 2.5 (MGA-2.5) was used as the selective isolation medium [11]. The plates were incubated with alternating periods of 12 h darkness/light at 25 ± 2 C for seven days. After incubation, plates were visualized for Fusarium species by micro-morphological studies. Fusarium species were transferred onto Potato Dextrose Agar (PDA), (Himedia, India) to identify at the species level using fungal taxonomic keys [2,27]. All fusarium isolates were maintained on Czapek Dox Agar slants at 4 C for further studies. Percent incidence, frequency and relative density were calculated according to the following formula; Percent incidence (%) = No. of grains infected withfusarium sp. Total no. of grains plated Frequency (%) = Relative density (%) = 100 No. of samples withfusarium sp. Total no. of samples analyzed 100 No. offusarium sp. isolated Total no. offusarium plated DNA Isolation from Fusarium species Based on the morphological characters, a total of 372 Fusarium isolates were inoculated to 500 L of potato dextrose broth in 2 ml microfuge tubes and incubated with alternating periods of 12 h darkness/light at 25 ± 2 C for 4 days. From the resulting mycelium DNA was extracted [28]. The mycelial mat was pelleted by centrifugation at 5000 r per minute for 5 min. The pellet was ground in microfuge tubes with blunt ends of sterile disposable pipette tips in 500 L of lysis buffer (20% SDS, PVP, 0.1 mol/l EDTA, 2 mol/l Tris HCl, lithium chloride, ph 8.0) and incubated at 65 C for 15 min. During incubation, the mixture was briefly vortexed 2 3 times. The samples were then treated with 500 L of phenol: chloroform (1:1, v/v) and vortexed for 1 min and the supernatant was collected after centrifugation at 3000 r per minute for 5 min at 4 C. DNA was precipitated with an equal volume of ice-cold isopropanol, and incubated at 20 C for 60 min and centrifuged at 8000 r per minute for 8 min at 4 C. The pellet obtained was rinsed with 70% ethanol, air-dried, resuspended in 50 L of nuclease free water and for PCR Primers for PCR Isolates were confirmed as Fusarium verticillioides with the use of forward primer VERTF-1 (5 -GCG GGA ATT CAA AAG TGG CC-3 ) designed by Patino et al. [24] and the reverse primer VERT-R (5 -CGA CTC ACG GCC AGG AAA CC-3 ) designed by Sreenivasa et al. [29]. These were used to identify F. verticillioides strains at the species level. The isolates were tested using the PCR specific assay for fumonisin-producing F. verticillioides with primers VERTF-1 (5 -GCG GGA ATT CAA AAG TGG CC-3 ) and VERTF-2 (5 -GAG GGC GCG AAA CGG ATC GG-3 ) as described by Patino et al. [24]. The expected PCR amplicon sizes were 1016-bp and 400-bp for the primers VERTF-1/VERT-R and VERTF-1/VERTF-2, respectively.

3 158 N. Deepa et al. / Food Science and Human Wellness 5 (2016) PCR amplification The isolated 372 DNA samples were subjected to PCR (Sure cycler 8000 Agilent technologies) using VERTF-1 and VERT-R set of primers with the PCR conditions 95 C for 2 min of initial denaturation, 94 C for 30 s of denaturation, 61 C for 30 s of annealing, primer extension at 72 C for 1 min and final extension at 72 C for 13 min. VERTF-1 and VERTF-2 set of primers were used to discriminate fumonisin producing and non producing F. verticillioides with the PCR conditions 95 C for 2 min of initial denaturation, 94 C for 30 s of denaturation, 60 C for 30 s of annealing, primer extension 72 C for 45 s and final extension at 72 C for 13 min. PCR reaction included 12 L of master mix (Genei PCR Master mix), 1 L DNA, 0.6 L of each primer and 10.8 L of water in a total volume of 25 L reaction mixture. PCR products were analyzed in 1.5% agarose gel (50 TAE-Tris base, glacial acetic acid, 0.5 mol/l EDTA, ph 8) and image was documented with a gel documentation system (Vilber Lourmat-Lab India, Hyderabad) after staining with ethidium bromide Sequencing and construction of phylogenetic tree Randomly selected six PCR amplified products (four F. verticillioides isolates confirmed up to species level with VERTF-1 and VERT-R primer and two F. verticillioides isolates amplified with VERTF-1 and VERTF-2 primer for the presence of gene coding for the production of fumonisin) were sequenced (Amnion Sequencing, Bangalore, India). The sequences were deposited at NCBI, GenBank, USA. Phylogenetic tree was constructed by online software MEGA 5.1 using neighbor joining method for the sequenced isolates Analysis of fumonisin-producing ability of Fusarium isolates by LC/MS Randomly selected nine isolates of F. verticillioides which were identified up to species level as well as fumonisin producing F. verticillioides confirmed by PCR (three isolates each from maize, paddy and sorghum) were tested for their ability to synthesize fumonisin B1. Each isolate was artificially inoculated with the concentration of 1 ml of 10 6 spores/ml into 5 g autoclaved each cereal in culture tubes. The tubes after incubation for 27 days were finely ground using liquid nitrogen, with sterile pestle and mortar and used for fumonisin extraction. Ground sample weighing 0.4 g was taken in a sterile glass vial and suspended with 2 ml acetonitrile/water (1:1, v/v) extraction solvent and allowed for equilibration overnight in a gel rocker at 28 ± 2 C. The extracts were syringe filtered using 0.45 m nylon membrane filters and liquid chromatography/mass spectrometry (LC/MS Waters Acquity Synapt G2, United States) was performed for the sample extracts. A column C18 was used at 50 C with sample temperature being 24 C for a run time of 8 min and mobile phase was water/acetonitrile. MassLynx SCN781 software was used to validate the LC/MS results. All sample extractions were carried out in triplicate. Table 1 Cereal samples collected all over Karnataka, India. Sl. no. Cereal samples Number of samples Place of collection 1 Maize 61 Mandya, Madikeri, Mysore, Tumkur, Shimoga, Hospet, Chitradurga, Bagalkote, Dharwad, Hubli, Bellary, Chamrajnagar, Bangalore 2 Paddy 42 Mysore, Mandya, Hassan, Shimoga, Tumkur 3 Sorghum 24 Mysore, Mandya, Chamrajnagar, Dharwad, Tumkur, Davangere, Chitradurga, Hubli, Bellary, Hassan 4 Pearl millet 08 Chamrajnagar, Tumkur, Bellary, Hospet, Bagalkote Total samples Results Mycological analysis using standard blotter and agar plate methods showed that, among 135 cereal samples (maize-61, paddy-42, sorghum-24, pearl millet-8) collected (Table 1), 69 samples (maize-37, paddy-22, sorghum-9, pearl millet-1) were positive for Fusarium infection and 51 samples were having F. verticillioides contamination. Among these 42 samples were fumonisin producing F. verticillioides (Table 2). Among the samples, maize showed maximum incidence of F. verticillioides with 8.5% incidence in Mysore, 3.5% in Madikeri and 3.6% in Bellary samples. Further 2% incidence was in paddy from Hassan and 1.4% incidence was in sorghum from Mysore samples (Table 3). Pearl millet was free from infection. Frequency of F. verticillioides among screened samples was high (47.54%) with maize followed by paddy (42.85%) and least in sorghum (16.66%). Among the screened samples maize (40.98%), paddy (33.33%) and sorghum (12.5%) samples were found contaminated with fumonisin producing F. verticillioides (Table 2). Of the 326 Fusarium isolates obtained from 69 infected cereal samples, 194 were F. verticillioides which were isolated from 51 cereal samples. The relative density of the isolated F. verticillioides species was 59.50% whereas other Fusarium species screened had relative density of 40.5% (Fig. 1). Table 2 Frequency of cereal samples infected with Fusarium and its species with fumonisin production. Cereal samples screened Infected with Fusarium sp. % Infected with F. verticillioides % Maize Paddy Sorghum Pearl millet 12.5 ND ND ND, not detected. Infected with Fumonisin producing F. verticillioides %

4 Table 3 Percent incidence (%) of Fusarium verticillioides in cereal samples. Cereal samples Incidence (%) N. Deepa et al. / Food Science and Human Wellness 5 (2016) Maize Paddy Sorghum Pearl millet Note: 1, Mysore; 2, Mandya; 3, Hassan; 4, Shimoga; 5, Tumkur; 6, Madikeri; 7, Chitradurga; 8, Hospet; 9, Bagalkote; 10, Dharwad; 11, Hubli; 12, Bellary; 13, Chamrajnagara; 14, Bangalore, 15, Davangere;, no samples collected. Fig. 1. Relative density of the F. verticillioides isolated from cereal samples. When all the 326 Fusarium species isolates were subjected to PCR amplification with VERT primers, 194 isolates scored positive with species specific VERTF-1 and VERTR (1016 bp) primers, and 176 isolates scored positive for VERTF-1 and VERTF-2 (400 bp) primers (Fig. 2). Hence, among the 194 isolates, 176 isolates were fumonisin producing F. verticillioides and 18 isolates were non-fumonisin producing. F. verticillioides (MTCC 156) strain was used as positive control and F. graminerium (MTCC 1893) was used as negative control (Fig. 3). For reconfirmation of PCR results the randomly selected PCR amplified products of VERTF-1/VERTR and VERTF- 1/VERTF-2 were subjected to sequencing. Sequencing reads of PCR analyzed products were tested using nucleotide megablast. The sequences were 99% similar to Giberrella moniliformis strains. The same sequences were deposited at NCBI and obtained the accession numbers KJ410046, KJ410047, KR061317, KR with VERTF-1/VERTR primers and KJ410767, KJ with VERTF-1/VERTF-2. The sequenc- Fig. 2. Incidence of Fusarium and its species with fumonisin production based on PCR confirmation. Fig. 3. Agarose gel differentiating fumonisin and non-fumonisin producing isolates. Lane 1&7-Positive control (MTCC 156), Lane 2,3,4,5-Positive isolates of F. verticillioides amplified with VERTF-1 and VERTR primers, Lane6&12- Negative isolate (MTCC 1893), M-250bp ladder, Lane 8,9&11-Positive isolates of fumonisin producing F. verticillioides amplified with VERTF-1 and VERTF-2 primers, Lane 10-Negative isolate of fumonisin producing F. verticillioides. ing result provides as reconfirmation for PCR carried out for the F. verticillioides strains isolated from the cereals. The sequenced isolates are represented by phylogenetic tree analysis (Fig. 4). The fumonisin producing ability was reconfirmed through LCMS results and three randomly selected representative isolates from maize, paddy and sorghum were compared with toxin standard of fumonisin B1 with retention time of 1.76 min and molecular weight of g/mol. Six isolates (two each from maize, paddy, sorghum) recorded as fumonisin producing and three isolates were non fumonisin producers (one each from maize, paddy and sorghum) when compared to standard graph (Fig. 5). 4. Discussion During the last decade, fumonisin producing F. verticillioides and related species have received worldwide attention. The non-scientific methods of agricultural practices, poor storage facilities and unfavorable environmental conditions from the time of harvest to storage/marketing have led to colonization of fumonisin producing fungi [30]. Moulds, besides depleting the nutrients, also produce toxic substances that have potential health hazards to animals and in turn to humans [31,32]. In this study 29 maize samples exhibited the association of F. verticillioides out of 37 samples of maize screened for Fusarium species and this was followed by paddy and sorghum but pearl millet was devoid of F. verticillioides infection (Table 2). F. verticillioides was considered as predominant species on maize with signifi-

5 160 N. Deepa et al. / Food Science and Human Wellness 5 (2016) F. verticilliodes MYSA01 - KR F. verticilliodes MYSB04 - KR F. verticilliodess MYSG09 - KJ F. verticilliodes MYSB04 KJ F. verticilliodes MYSC04 KJ F. verticilliodes MYSB10 KJ Fig. 4. Represenation of phylogenetic tree for sequenced isolates of F. verticillioides. Fig. 5. Isolate representing production of fumonisin toxin with molecular weight of g/mol when compared to standard graph. cant levels of fumonisin [33] and G. fujikuroi was the dominant species that would probably be the main source for fumonisin production in cereals [34,35]. In the present study 194 F. verticillioides were isolated (maize-108, paddy-80, sorghum-6) from 326 Fusarium species (maize-158, paddy-137, sorghum- 29, pearl millet-2) screened from 135 cereal samples (Fig. 2). Even after certain measures taken for the control of fumonisin production, the association of fumonisin producing F. verticilloides is increasing day by day. One hundred and seventy six fumonisin producing F. verticillioides (maize-103, paddy-68, sorghum-5) were isolated from screened 326 Fusarium species in the present work (Fig. 2). Among the 45 maize samples collected from south Karnataka, 25 samples were found to be highly infected with FB1 producing F. verticillioides [36]. Among the 22 fumonisin producing isolates screened, 18 were F. verticillioides in which 17 (94.4%) isolates produced fumonisins (FB1 + FB2) at concentration range of g/g [37]. An epidemiological survey conducted in Karnataka and Andhra Pradesh during 1997 revealed that consumption of mouldy grains affected 1424 persons in 27 villages [38]. Greater attention is bestowed to investigate Fusarium species worldwide as they reduce the value of cereals used as food and feed. Cereals contaminated with toxigenic species cause acute and chronic poisoning and allergic symptoms to animals and human. Fungi causing deterioration of cereals especially in maize, paddy and sorghum are a major problem because they produce mycotoxins [40]. In the present study, the PCR assay used for the identification of F. verticillioides isolates was quick, accurate and more sensitive, as compared to conventional methods. It was shown that 194 out of 245 isolates were morphologically identified as F. verticillioides and this was further confirmed by VERTF1/VERT-R species-specific primers (Table 4). Among the remaining 51 isolates of Fusarium, 33 were identified as F. proliferatum and 18 as Fusarium species. F. proliferatum and F. verticillioides are having similar morphological characters. Fabrico Lanza et al. [41] did molecular identification of Fusarium isolates by PCR and species-specific primer pairs. Initially they identified five species as F. proliferatum based on morphological criteria but based on PCR test they were identified as F. verticillioides. Molecular analysis using species-specific PCR primers makes possible the precise identification of Fusarium species in such cases where morphological identification is not possible. PCR method also differentiates morphologically similar but toxigenically different F. verticillioides isolates. Present study revealed the association of 53.98% of fumonisin producing F. verticillioides with cereal samples collected from different regions of Karnataka state. In conclusion, data on the per cent incidence, frequency and relative density of F. verticillioides would be of great sig- Table 4 Conventional and Molecular identification of F. verticillioides from cereals. Cereal samples screened No. of F. verticillioides screened Conventional Maize Paddy Sorghum 9 6 Pearl millet ND ND Total ND, not detected. Molecular

6 N. Deepa et al. / Food Science and Human Wellness 5 (2016) nificance for predicting the extent of post-harvest infection, colonization and subsequent deterioration of cereals. Further, it also helps us to know the dry matter loss, nutritional changes and the extent of fumonisin levels during storage. Such a data is of immense value for assessing the possible health hazards in humans and animals upon consumption of such contaminated food grains. The high incidence of mycotoxigenic F. verticillioides is of primary concern for policy makers and food experts in this region to reduce the economic losses caused by these fungi and also to minimize the exposure of human and animal life to the potential risks of mycotoxins. Conflict of interest The authors declare that there are no conflicts of interest Acknowledgements We thank the Department of Science and Technology (DST-SERB) India, for providing grants through Young Scientist FAST TRAK research project (No. SR/FT/LS-176/2009; ) to Dr. M Y Sreenivasa, Principal Investigator. We thank the Institute of Excellence, University of Mysore, for their valuable support. References [1] S. Marin, V. Sanchis, D. Sanz, I. Castel, et al., Control of growth and fumonisin B 1 production by Fusarium verticillioides and Fusarium proliferatum isolates in moist maize with propionates preservatives, Food Addit. Contam. 16 (1999) [2] J.F. Leslie, B.A. Summerell, The Fusarium Laboratory Manual, Blackwell Publishing, Ames, Iowa, USA, [3] K.E. Akhande, M.M. Abubakar, T.A. Adegbola, et al., Nutritional and health implications of mycotoxins in animal feeds: a review, Pak. J. Nutr. 5 (5) (2006) [4] P. Fandohan, K. Hell, M.J. WFO Marasas, et al., Infection of maize by Fusarium species and contamination with fumonisins in Africa, Afr. J. Biotechnol. 2 (2003) [5] C.S. Nayaka, A.C. Udaya Shankar, S.R. Niranjana, G. Edner, et al., Detection and quantitation of fumonisins from Fusarium verticilloides in maize grown in southern India, World J. Microbiol. Biotechnol. 26 (2009) [6] H.H.L. González, E.J. Martínez, et al., Fungi associated with sorghum grain from Argentina, Mycopathology 139 (1997) (1997) [7] S.S. Navi, Fungi associated with sorghum grain in rural Indian storages, J. New Seeds 7 (2005) [8] P.K. Maheshwar, S. Ahmed Moharram, et al., Detection of fumonisin producing F. verticillioides in paddy (Oryza sativa L.) using polymerase chain reaction (PCR), Braz. J. Microbiol. 40 (2009) [9] W.F.O. Marasas, T.S. Kellerman, W.C.A. Gelderblom, J.A.W. Coetzer, et al., Leucoencephalomalacia in a horse induced by fumonisin B1 isolated form Fusarium moniliforme, Onderstepoort J. Vet. Res. 55 (1998) [10] L.R. Harrison, B.M. Colvin, J.T. Greene, L.E. Newman, et al., Pulmonary edema and hydrothorax in swine produced by Fumonisin B1, a toxic metabolic of Fusarium moniliforme, Vet. Diagn. Investig. 2 (1990) (1990) [11] M.R. Bragulat, E. Martinez, G. Castella, et al., Selective efficacy of culture media recommended for isolation and enumeration of Fusarium species, Food Prot. 67 (2004) [12] F.S. Chu, G.V. Li, Simultaneous occurrence of fumonisin B 1 and other mycotoxins in moldy corn collected from People s Republic of China in regions with high incidence of esophageal cancer, Appl. Environ. Microbiol. 60 (1994) [13] L. Ajello, A.A. Padhye, F.W. Chandler, M.R. Girmis, et al., Fusarium moniliforme a new mycetoma agent restudy of a European case, Eur. J. Epidemeol. 1 (1985) [14] J.W. Rippon, R.A. Larson, D.M. Rosenthal, et al., Disseminated cutaneous and peritoned hyalohyphomycosis caused by Fusarium species, Three Cases Rev. Lite 101 (1988) [15] K. Desai, M.C. Sullards, J. Allegood, E. Wang, et al., Fumonisins and fumonisin analogs as inhibitors of ceramide synthase and inducers of apoptosis, Biochem. Biophys. Acta 1585 (2002) [16] L. Covarelli, S. Stifano, G. Beccari, L. Raggi, et al., Characterization of Fusarium verticillioides strains isolated from maize in Italy: fumonisin production, pathogenecity and genetic variability, Food Microbiol. 31 (2012) [17] A. Logrieco, G. Mule, A. Moretri, et al., Toxigenic Fusarium species and mycotoxins associzted with maize ear rot in Europe, Eur. J. Plant Pathol. 108 (2002) [18] V. Rahjoo, J. Zad, M. Javan-Nikkhah, A.M. Gohari, et al., Morphological and molecular identification of Fusarium isolated from maize ears in Iran, J. Plant Pathol. 90 (2008) [19] L. Tamburic-Ilinicic, A.W. Schaafsma, The prevalence of Fusarium spp. colonizing seed corn stalks in southwestern Ontario, Canada, Can. J. Plant Sci. 89 (2008) (2008) [20] C.J. Kedera, J.F. Leslie, et al., Genetic diversity of Fusarium section Liseola (Gibberella fujikuroi) in individual maize stalks, Phytopathology 84 (1994) [21] M.D. Thomas, I.W. Buddenhagen, Incidence and oersistence of Fusarium moniliformie in symptomless maize kernels and seedlings in Nigeria, Mycologia 72 (1980) (1980) [22] K. O Donnell, H.I. Nirenberg, T. Aoki, et al., A multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species, Mycoscience 41 (2000) [23] E.A. Tsavkelova, C. Bomke, A.I. Netrusov, et al., Production of gibberellic acids by an orchid-associated Fusarium proliferatum strain, Fungal Genet. Biol. 45 (2008) [24] B. Patino, S. Mirete, T. Gonzalez-Jaen, G. Mule, et al., PCR detection assay of fumonisin producing F. verticillioides strains, Food Prot. 6 (2004) [25] M.Y. Sreenivasa, R.S. Dass, A.P. Charith Raj, et al., PCR method for the detection of genus Fusarium and Fumonisin producing isolates from freshly harvested sorghum grains grown in Karnataka, Indian J. Food Saf. 28 (2008 a) [26] S.B. Mathur, O. Kongsdal, Common Laboratory Seed Health Testing Methods for Detecting Fungi, International Seed Testing Association, Copenhagen, Denmark, [27] C. Booth, Fusarium Laboratory Guide to the Identification of Major Species, Common Wealth Mycological Institute, Ferry lae, Kew, Surrey, England, [28] M.Y. Sreenivasa, R.S. Dass, A.P. Charith Raj, et al., Molecular detection of fumonisin producing Fusarium species of freshly harvested maize kernels using polymerase chain reaction (PCR), Taiwania 51 (2006) [29] M.Y. Sreenivasa, M.T.G. Jaen, R.S. Dass, A.P. Charith Raj, et al., A PCR based for the detection and differentiation of potential fumonisin-producing Fusarium verticillioides isolated from Indian Maize kernels, Food Biotechnol. 22 (2) (2008) [30] R.V. Bhat, H.S. Prathapkumar, P.A. Rao, V.S. Rao, A food-borne disease outbreak due to the consumption of moldy sorghum and maize containing fumonisin mycotoxins, Clin. Toxicol. 35 (1997) [31] M.D. Thomas, I.W. Buddenhagen, Incidence and oersistence of Fusarium moniliformie in symptomless maize kernels and seedlings in Nigeria, Mycologia 72 (1980) [32] B. Fazekas, M. Kis, et al., Data on the contamination of maize with fumonisins B1 and other fusarial toxins in Hungary, Acta Vet. Hung. 44 (1996) [33] N. Mogen, S.K. Supreomiene, K.N. Linda, L. Irene, et al., Real time PCR for quantification of 11 individual Fusarium species in cereals, J. Microbiol. Methods 76 (2009)

7 162 N. Deepa et al. / Food Science and Human Wellness 5 (2016) [34] C.J. Kedera, R.D. Plattnes, et al., Incidence of Fusarium species and levels of fumonisin B1 in maize in western Kenya, Appl. Environ. Microbiol. 65 (1999) [35] C. Grim, R. Geisen, A PCR-ELISA for the detection of potential fumonisin producing Fusarium species, Lett. Appl. Microbiol. 26 (1998) [36] D.C. Mohana, S. Thippeswamy, R.U. Abhishek, et al., Natural occurrence of Aspergillus flavus and Fusarium verticillioides, and AFB1 and FB1 contamination in maize grown in Southern Karnataka (India), Canadi, J. Plant Prot. 2 (1) (2014) [37] M.Y. Sreenivasa, B.T. Diwakara, A.P. Charith Raj, et al., Toxigenic Fusarium species and Fumonisin B1 and B2 associated with freshly harvested sorghum and maize grains produced in Karnataka, India, Ann. Food Sci. Technol. 14 (1) (2013) [38] P.H. Shetty, R.V. Bhat, Natural occurrence of fumonisin B1 and its cooccurrence with aflatoxin B1 in Indian sorghum, maize and poultry feeds, J. Agric. Food Chem. 45 (1997) [39] J. Levic, L. Tamburic-Ilincic, et al., Maize kernel infection by Fusaria in the period , Cereal Res. Commun. 25 (1997) [40] International Seed Testing Association, in: S.R. Draper (Ed.), International Rules for Seed Testing, Rules, ISTA, Zurich, Switzerland, 2003, pp [41] F.E. Lanza, L. Zambolim, R. Veras da Costa, V.A.V. Queiroz, et al., Prevalence of fumonisin- producing Fusarium species in Brazilian corn grains, Crop Prot. 65 (2014) [42] L.O. Rocha, G.M. Reis, V.N. Silva, R. Braghini, et al., Molecular characterization and fumonisin production by F. verticillioides isolated from corn grains of different geographic origins in Brazil, Int. J. Food Microbiol. 145 (2011) 9 12.