Several fungi are associated with corn during pre- and post-harvest periods, of which Fusarium verticillioides represents the most important fungal species (Marasas et al., 1984; Fandohan et al., 2005). In addition, F.

Biological and chemical detection of fumonisins produced on agar medium by Fusarium

verticillioides isolates collected from corn in Sohag, Egypt

M. B. Aboul-Nasr and M. R. A. Obied-Allah

Correspondence M. B. Aboul-Nasr [email protected]

Received 4 May 2013 Accepted 6 June 2013

Department of Botany, Faculty of Science, University of Sohag, Sohag, Egypt

Fusarium verticillioides(Sacc.) Nirenberg is among the most commonFusariumspecies corn pathogens worldwide, and has been recognized as a fumonisin B1(FB1) and fumonisin B2(FB2) producer. In the present work, extracts of 58F. verticillioidesisolates from corn samples collected from Sohag Governorate, Egypt, were tested for their biotoxicity and production of fumonisin toxins. Forty-fourFusarium verticillioidesisolates out of 58 tested produced FB1or FB1and FB2

(15 and 29 isolates, respectively) on potato–sucrose agar medium, detected by TLC, whereas the other 14 isolates did not produce fumonisin toxins. HPLC crude extract analysis confirmed the results from TLC plates. Brine shrimp larvae as well as the Gram-negative bacteriaPseudomonas aeuroginosashowed low bio-sensitivity towards theF. verticillioidescrude extract toxicity, whereas the Gram-positive bacteriaBacillus cereusandBacillus subtilis, especiallyB.subtilis, showed higher sensitivity towards the testedFusariumcrude extracts. These results enabled us to bio-evaluate and chemically detect fumonisin mycotoxins using a simple agar medium technique.

INTRODUCTION

Several fungi are associated with corn during pre- and post-harvest periods, of which Fusarium verticillioides represents the most important fungal species (Marasas et al., 1984; Fandohan et al., 2005). In addition, F.

verticillioides has a wide geographical distribution and many researchers have reported fumonisins being impli- cated in animal and human toxicoses all over the world (Shephard et al., 1996; Munkvold & Desjardins, 1997;

Marasas, 2001; Taligoola et al., 2004). Wilson & Maronpot (1971) identified F. verticillioides as the predominant contaminant of mouldy corn that had caused cases of equine leukoencephalomalacia (ELEM) in Egypt, and reproduced ELEM by feeding culture material of the fungus on corn. Also, fumonisin B

(FB

) has a postulated role in human oesophageal cancer and neural tube defects (Marasas et al., 2004).

Fumonisins are a group of mycotoxins isolated initially from corn culture of Fusarium moniliforme (syn. F.

verticillioides) (Gelderblom et al., 1988; Fadl, 1997).

Because of the great effect of Fusarium toxins, fumonisins, on human and animal health, the aim of the present work was to find an easy, reliable and inexpensive biological and chemical assay to detect them. The protocols developed in this study are helpful for the rapid and small-scale detection of fumonisins from fungal mycelia occurring

on contaminated corn samples, rather than using compli- cated or expensive tools. Many researchers depend on genomic tools such as PCR analysis, which is no doubt one of the most accurate methods of detection; however, it is too complicated to be achieved in developing countries.

METHODS

F. verticillioidesisolate collection.A total of 58F. verticillioides isolates were collected by the Laboratory of Fungal Physiology, Department of Botany, Faculty of Science, University of Sohag, Egypt, from corn samples using the direct plate method to isolate exogenous Fusariumisolates (Pittet al., 1992) and the surface disinfection plate method to isolate endogenousFusariumisolates (Mu¨llenbornet al., 2008) on DRBC (dichloran-Rose-Bengal-chloromphenicol) agar medium (Tournaset al., 2006) and PSA medium (potato–sucrose agar) (Booth, 1971, 1977). After single-spore isolation, each isolate was identified on the basis of both macroscopic and microscopic characteristics using identification keys. Collected cultures were inoculated onto sterile PSA medium slants and incubated for 1 week at 25uC and stored at 5uC for further chemical and biological analysis.

Cultivation ofF. verticillioidesisolates on PSA medium and extraction of toxins.Fifty-eight different F. verticillioides isolates were examined for the production of fumonisins. Each isolate was cultivated on PSA medium under aseptic conditions, using 15 cm Petri dishes (Baillyet al., 2005), and incubated at 20±2uC for 5 days.

At the end of the incubation period, all plates were transferred into a refrigerator for 10 days. The agar media with mycelium were cut into small pieces (1 cm²), transferred into 250 ml Erlenmeyer flasks Abbreviations:FB₁, fumonisin B₁; FB₂, fumonisin B₂.

Downloaded from www.microbiologyresearch.org by containing 50 ml 96 % methanol, and the contents were shaken on a

rotary shaker (200 r.p.m., 24 h) and filtered through filter paper (Whatman no.1). The residue was then washed twice with the same solvent (25 ml each). The methanol extracts were combined, dried over anhydrous sodium sulphate and then evaporated to near dryness under vacuum. The residue was transferred quantitatively to a small dram vial with methanol and evaporated to near dryness under a stream of nitrogen.

Biological assays.Brine shrimp (Artemia salinaLinnaeus) larvae (Korpinen, 1974; Dinget al., 2008) and antibacterial activity using the disc diffusion method (Sleigh & Timburg, 1981) withBacillus cereus and Bacillus subtilis (Gram-positive), and Pseudomonas aeruginosa (Gram-negative) were used to evaluate the bioactivities of the toxin extracts from allF. verticillioidesisolates tested.

Chromatographic analysis of the crude extracts ofF. verticil- lioidesisolates.

TLC analysisThe thin-layer chromatographic technique adopted by El-Kady & Moubasher (1982) was employed. The crude extracts (10ml) were spotted onto a TLC plate (aluminium sheet, silica gel;

Merck) along with 5mg FB1 standard (purchased from Sigma- Aldrich). The spots were dried during application with a flow of warm air and developed using 96 % methanol/water (80 : 20, v/v). The developed plates were viewed after spraying with 50 % concentrated sulphuric acid in methanol under short-wave (254 nm) and long- wave (365 nm) UV irradiation. FB1and fumonisin B2(FB2) appear as slight bluish-green spots under both near and far UV (Mubatanhema et al., 1999) at Rf0.7 and Rf0.61, respectively.

HPLC analysisFive filtered fungal extracts (four fumonisin producers and one non-producer) according to TLC analysis were analysed with a Waters Binary model 1525 HPLC equipped with a Waters 2475 multi-wavelength fluorescence detector and the data workstation software Breeze 2. A phenomenex C18 column (25064.6 mm internal diameter, 5mm particle size) from Waters was used along with a mobile phase of methanol/0.1 M NaH2PO4 (75 : 25, v/v), adjusted to pH 3.35 by the addition of phosphoric acid and filtered through a membrane filter, in an isocratic system. The separation was performed at ambient temperature at a flow rate of 1.0 ml min²¹. The injection volume was 20ml for both standard solutions and sample extracts. The fluorescence detector was operated at 335 nm (excision) and 440 nm (emission) wavelengths.

Standard solution was prepared from stock standard solution by transferring 25ml to a vial and then adding 975ml acetonitrile/H2O (1 : 1, v/v). The obtained standard solution contained 25mg ml²¹FB1. Fifty microlitres of FB1working standard solution was transferred to the base of a small vial and then mixed with 225mlo-phthaldialde- hyde (OPA) reagent and 10ml of reacted mixture was injected into the HPLC machine within 1 min.

The purified dry film residue of sample extract was dissolved in 200ml methanol. Fifty microlitres of this extract was transferred to the base of a small vial and then 225ml OPA reagent was added, mixed and 10ml derivative was injected into the HPLC machine within 1 min of adding OPA reagent. The mixed solutions of standard as well as sample extracts after derivatization were filtered through a 0.22 nm membrane filter. FB1contents in the sample were calculated from chromatographic peak areas (AOAC International, 2007).

RESULTS AND DISCUSSION

Fifty-eight F. verticillioides isolates (10 endogenous isolates and 48 exogenous isolates) were collected from maize samples from Sohag Governorate in Egypt and tested for

fumonisin production. Results of the biological assays are summarized in Tables 1 and 2. Results of the TLC analysis of the crude culture extracts of F. verticillioides isolates tested are also shown. Out of 48 exogenous F. verticillioides isolates, two were strongly toxic, 11 isolates showed moderate toxicity, whereas the rest of the tested isolates ranged from non-toxic to low toxicity. In the case of endogenous F. verticillioides isolates, one isolate showed high toxicity and two isolates had moderate toxicity, whereas the rest had low or non-toxic bioactivity in the brine shrimp (A. salina) bioassay. Hartl & Humpf (2000) found that the brine shrimp assay proved to be a convenient and rapid system for toxicity assessment of this group of mycotoxins. Mexı´a-Salazar et al. (2008) also used a white shrimp bioassay to evaluate the biotoxicity of FB

and reported that at all FB

levels tested, the shrimps were affected. Other researchers have used the brine shrimp bioassay to evaluate the biotoxicity of FB

(Hlywka et al., 1997; Moretti et al., 2007). However, the lower sensitivity of the Artemia species to several chemical or physical agents in comparison to other invertebrate test organisms and the decreased solubility of some chemical substances in saline or seawater medium, in addition to the various conditions that control the test, such as temperature, pH, chemical composition of the medium, oxygen, photo- period, nutrients, some population effects, type of growth stage, etc. may affect the toxicity results (Sorgeloos et al., 1978; Song & Brown, 1998; Soares et al., 1992; Okamura et al., 2000; Guerra, 2001; Nałecz-Jawecki et al., 2003;

George-Ares et al., 2003; Mayorga et al., 2010).

The Gram-positive bacteria B. cereus and B. subtilis and the Gram-negative bacterium P. aeruginosa were used to test the antibacterial activity of the F. verticillioides isolate extracts. None of the 58 F. verticillioides crude extracts tested had any antibacterial activity against Gram-negative bacteria. In contast, 50 (40 exogenous and 10 endogenous) F. verticillioides isolate extracts showed antibacterial activity against B. subtilis and 42 (32 exogenous and 10 endogen- ous) extracts showed activity against B. cereus. Larger growth inhibition zones were observed with B. subtilis, indicating greater sensitivity toward the crude extracts of the F. verticillioides isolates tested (Tables 1 and 2). These results are in full agreement with those of Hugo & Russell (1983), Farag et al. (1989), Zohri et al. (1995), Eftekhar et al. (2005) and Murthy et al. (2006). The tolerance shown by Gram-negative bacteria could be due to the permeability barrier provided by the cell wall or the membrane accumulation mechanism (Adwan & Abu-Hasan, 1998) or related to lipopolysaccharides in their outer membranes (Sawer et al., 1997; Gao et al., 1999).

TLC analysis of F. verticillioides culture extracts confirmed the results of the bioassays, showing that, of the 58 tested isolates, 15 isolates (12 exogenous and 3 endogenous) produced FB

whereas 29 other isolates (22 exogenous and 7 endogenous) produced both FB

and FB

. Several other studies dectected FB

and FB

by TLC as well (Ross et al., 1990; Pepeljnjak et al., 2003; Bailly et al., 2005; Benedetti et al., 2006).

http://mic.sgmjournals.org 1721

Table 1.Biotoxicity of fumonisins produced by exogenousF. verticillioidesisolates grown on PSA medium and TLC analysis of their crude extracts

Isolate Toxicity tests Mycotoxins detected on TLC

A. salina* BacteriaD Fumonisinsd

B. subtilis B. cereus P. aeuroginosa FB1 FB2

1 N – – – 2 2

2 L 1.9 1 – + +

3 N – – – 2 2

4 L 1.3 – – + +

5 N – – – 2 2

6 L 1.6 1.4 – + +

7 N 1.2 1.2 – + +

8 N – – – 2 2

9 N 1 – – + 2

10 N 1.5 1.4 – 2 2

11 L 1.2 – – + 2

12 L 1.3 1.3 – + 2

13 L 1.2 – – + 2

14 N 1.1 – – 2 2

15 L 1.7 1 – + +

16 L 1.5 1.3 – + 2

17 L 1.2 – – + +

18 L 1.2 1.2 – + +

19 L 1.2 – – + 2

20 L 1 1 – 2 2

21 M 1.1 – – + +

22 L 1.2 1.5 – + 2

23 L 1.3 1.3 – + 2

24 L 1.7 2 – + +

25 L 1.7 2 – + +

26 M 2.3 2 – + +

27 M 0.7 1.1 – + +

28 M 2.2 1.9 – + +

29 M 2.6 2 – + +

30 N 1.3 1.3 – 2 2

31 L 2 1.8 – + +

32 L 1.3 1.3 – 2 2

33 N 1.3 1.1 – + +

34 M 1.3 1.5 – + +

35 L 1.6 1.3 – 2 2

36 M 1.4 1.5 – + +

37 M 1.4 1.4 – + 2

38 N – – – 2 2

39 H 1.1 1.5 – + 2

40 H 1.3 1.5 – + +

41 M 1.8 1.9 – + +

42 N – – – 2 2

43 M 1.4 1.1 – + +

44 N 1.3 1.1 – 2 2

45 L 1.8 1.3 – + 2

46 M 1.3 1.3 – + +

47 L – – – + 2

48 N – – – 2 2

*H, High toxicity (more than 75 % dead larvae); M, moderate toxicity (50–75 % dead larvae); L, low toxicity (25–50 % dead larvae); N, non-toxic (0 dead larvae).

DClear inhibition zone diameter (cm).

d–, Not detected;+, detected.

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The HPLC analysis of five cleaned extracts of F.

verticilloides isolates (four fumonisin producers and one non-producer according to TLC analysis) detected FB

ranging from 0.037 to 1.0

on agar medium, in the case of the fumonisin producers, and no detectable amounts of fumonisins in the case of the non-toxic isolate. These results confirmed the production of fumonisins by the tested F. verticilloides isolates. This level of production was lower than those reported in Ghana, where the tested F.

verticillioides strains produced all three types of fumonisins (B

, B

and B

) and the total fumonisin levels ranged from 127 to 11 052

on maize substrates, not agar medium (Kpodo et al., 2000). Also, Sydenham et al. (1993) reported higher levels of fumonisin produced by a strain of F.

verticillioides isolated from Argentinean maize and also grown on natural medium. Consistent with our results, Visconti &

Doko (1994) and Castella´ et al. (1996) showed low fumonisin production for F. verticillioides isolates from maize and mixed poultry feeds. Also, a few F. verticillioides isolates from Nepal (Nelson et al., 1991) did not produce any fumonisin and several isolates of this species from south-east Asia (Miller et al., 1993) produced fumonisins at low levels, whereas some isolates from Australia (Nelson et al., 1991) produced only trace quantities of FB

. Moreover, Fadl (1997) screened 18 isolates of F. verticillioides from corn samples at Minia Governorate in Egypt for their ability to produce fumonisins on polished rice grains. Based on TLC analyses, it was found that 14 of the 18 isolates tested produced FB

and FB

, whereas four isolates were not able to produce fumonisins.

Conclusion

In this study, we could bio-evaluate and chemically detect fumonisins produced by F. verticilloides using an agar

medium technique, which is an easy, inexpensive, time- saving and reliable method.

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