Mycotoxigenic fungi isolated from corn, peanut, popcorn and sorghum samples were identified. Nine fungal species belonging to three genera were recovered from the tested samples. With the exception of the sorghum isolate, all tested A. flavus isolates were capable of producing variable amounts of G1 and B1 aflatoxin ranging from 1-6 parts per billion (ppb). The highest amount of aflatoxin B1 (8ppb) was produced by A. flavus isolated from corn grains. The sorghum isolate from P. oxalicum was the highest producer of citreoviridin (37ppb) while the F. subglutinans isolate from popcorn was capable of producing fumonisin B1, zearalenone and vomitoxin (DON). The corn isolate from F. proliferatum however, failed to produce fumonisin B1 and the sorghum isolate from F. verticillioides produced only fumonisin B1. The antifungal activity of yellow liquid fraction for Aloe vera against toxigenic fungi was tested. All tested concentrations were effective in inhibiting fungal growth.
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KEYWORDS: Anthraquinones, GC/MS analysis, seed-borne fungi, Mycotoxins
Seed-borne fungi are responsible for considerable grain and oilseed spoilage as well as bio-deterioration of other agriculture products worldwide . They can cause significant loss in seed quality and nutritive value of grains [1, 2]. Significant grain quality loss is also due to mycotoxins production by toxigenic fungi . The occurrence of toxigenic fungi and mycotoxins in peanut, corn and sorghum grains and its derived foods has been frequently reported worldwide [4-8].
Aspergillus, Fusarium and Penicillium are considered to be the most significant toxigenic fungi growing in pre and/or post-harvested and stored grains [9,10]. It was reported that more than 25% of the world cereals are contaminated with known mycotoxins . The detrimental health effects of mycotoxins on humans and animals has frequently been reported .
Several control methods could be used to control pre and post-harvest seed-borne fungi. The use of fungicides is common although it could lead to many ecological problems [13, 14]. Therefore, an alternative method without toxicity to either human or animals should be used to prevent grain deterioration by associated fungi during the storage.
Control of fungal diseases using different natural substances, such as essential oils and plant extracts has been widely investigated [15, 16]. The first report of Aloe vera antifungal activity against plant pathogenic fungi was reported by Saks and Barkai-Golan . They indicated that A. vera had antifungal properties against Fusarium oxysporum, Rhizoctonia solani and Colletotrichum coccodes.
Fungicidal activity of Aloe vera against plant pathogenic fungi have frequently been reported [18, 19]. Extracts of Aloe vera fresh leaves also showed in vitro inhibitory effects against B. gladiolorum, Fusarium oxysporum f. sp. gladioli, Heterosporium pruneti and Penicillium gladioli, isolated from ornamental plants . Thus the present study was undertaken to evaluate the antifungal potential of aqueous and organic solvent extracts of Aloe vera on in vitro growth of seed-borne fungi isolated from corn, popcorn, peanut and sorghum grains.
MATERIALS AND METHODS
Samples of corn, popcorn and peanut were collected from different locations of Riyadh, Kingdom of Saudi Arabia, and evaluated for frequency of seed-borne fungi. Fungi were isolated and cultured according to the method described by Hussaini et al. . Two sets of 10 gm of each sample were used either after surface sterilized (using 5% sodium hypochlorite solution and washed with three times by sterile distilled water) or without sterilization. Ten grains of each commodity were placed randomly on the surface of Petri-dish containing Potato Dextrose Agar (PDA) in triplicate. Petri dishes were incubated at 25°C and examined daily for five days, after which the colonies were counted. Isolates were purified either by single spore or hyphal tip methods and then transferred to PDA slants. The isolation frequencies of fungal species were calculated according to Gonzalez et al. . Identification of obtained fungal isolates was carried out based on morphological and microscopic characteristics according to the methods of Dugan .
Aspergillius isolates were grown in sterilized SMKY liquid medium [Sucrose- 200gm, magnesium sulphate- 0.5gm, potassium nitrate- 3gm, yeast extract-7gm and distilled water 1000 ml in 100 ml flasks for 10 days at 27±2°C with three replicates per isolate . After incubation, cultures were blended for 2 min using a high speed homogenizer and filtered through Whatman's filter paper 1. Aflatoxins were extracted from the homogenized filtrates using a mixture of chloroform-acetone 9/1 (v/v). Dried residues containing aflatoxin were dissolved in 1 ml of the same liquid mobile phase solution which contained methanol:acetic acid:water and was stored at -20°C in brown tubes.
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The method described by Christian  was used to detect and determine aflatoxin production. The extracts were passed through a 0.45 µm micro-filter. Analysis of compounds was performed on HPLC model PerkinElmer® Brownlee™ validated C18, with internal diameter of 100 mm - 4.6 mm, 3 micron. The HPLC was equipped with an UV detector and fluorescence detection with 365 nm excitation and 430 nm emission wavelengths. The liquid mobile phase yielded results of methanol:acetic acid:water (20:20:60 v/v/v). The total run time for the separation was approximately 25 min at a flow rate of 1 ml/min.
Test isolates were grown on sterilized liquid malt extract media prepared in 100 ml flasks and incubated for 7-10 days at 27±2°C with three replicates per isolate. Cultures were blended for 2 min using a high speed homogenizer and filtered using glass filter paper. Patulin was extracted from homogenized filtrate using acetonitrile:water (5:95 v/v) .
The solvent was then evaporated at 35°C under vacuum. The dried residues containing patulin were dissolved in 1 ml of the same liquid mobile phase. The method described by Christian  was used to determine patulin. The extract was passed through a 0.45 µm micro-filter. Analysis of compounds was performed on an HPLC model PerkinElmer® Brownlee™ validated C18, 250 mm with internal diameter; 2.1 or 3.2 mm. The HPLC was equipped with UV detector and the wave length was set at 280 nm. Patulin was completely resolved by using acetonitrile-water (95:5, v/v) as the mobile phase at a flow-rate of 1.0 ml/min for 25 min.
A reliable analytical quantitative method described by Stubblefield et al. . was used for citreoviridin determination. The toxin was extracted with 5 ml dichloromethane, and the extract was partially purified on silica and amino solid-phase extraction (SPE) columns. The extract was analyzed for citreoviridin by normal-phase liquid chromatography, using a mobile phase of ethyl acetate: hexane (75:25 v/v) at 1.5 ml/min and a fluorescence detector to measure the yellow fluorescence (388 nm excitation, 480 nm emission).
Fungal mats of each flask were blended with 5 gm sodium chloride and 100 ml of 80% methanol at high speed for one min, and then filtered through glass micro-fiber filter paper. Ten ml of the filtrate was diluted with 40 ml of wash buffer and filtered again through 1 μm micro-fiber filter. Ten ml of the diluted extract were passed through a fumontest column (Vicam Company) and the column was washed using 10 ml of the same dilute solution. Fumonisins were determined according to the method described by Mazzani et al. . Fumonisin was eluted by passing one ml of HPLC grade methanol through the column and then elutes were re-collected. One ml of each of developer A (Vicam product No. G5005) and developer B (Vicam product No. G5004) were added to the elute and placed in calibrated fluorometer (Series-4 /Vicam) for fumonisin toxin, but the dilution was made with 49 ml distilled water which were passed through a Zearatest and/or vomitoxin column (Vicam Company) and then measured in calibrated fluorometer model (Series-4/Vicam).
A. vera leaves were collected from the Botanical Garden of Botany and Microbiology Department, College of Science, King Saud University. Leaves weighing 1 kg or more were chosen and cut out from the whole plant at the basal point of attachment. The inner yellowish brown mucilage (glue-like substance) which is usually liquid in nature flowed from the cut leaves and was collected in dark sterilized bottles.
GC/MS analysis of Aloe vera extracts
Samples were methylated according to Vogel  and dissolved in ethyl ether. One ml of an ethyl ether solution was injected into the gas chromatograph. An HP-5 fused silica capillary column (30 - 0.25 mm), mass selective detector, Hydrogen carrier gas at 32 cm min-1 and split ratio 1:50 were used. Oven temperatures ranged from 75°C for 3 min. and ramp 15 °C/min to 270 °C, ending with an isothermal period of 10 minutes. Injector, transfer and detector temperatures were 260, 285 and 250°C, respectively while solvent delay was 2 min. Detection and identification of anthraquinones  was based on analysis of the corresponding mass spectrum and comparison with data of the Wiley-275 library and scan was from 50 to 500 Da.
Effect of A. vera on fungal growth
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To study the effect of A. vera on colony growth, concentrations of 1%, 2%, 4% and 8% from A. vera leaves were prepared. A. vera yellow liquid fraction was applied to PDA medium just before pouring in Petri plates. Five mm plugs cut from the margin of 10 days old fungal colonies were placed in the center of the plates. Cultures were incubated at 23°C and radial growth measured daily for five days. Inhibition of fungal growth in the plates containing tested crude was judged by comparison with growth in blank control plates. Maximal radial growth for some of the fungi was limited by dish diameter (80 mm). Three replicate plates were used for each treatment.
The isolation frequency (Fq) of genera was calculated according to Marassas et al. . A randomized complete block design was used in the present study. Analysis of variance (ANOVA) of the fungal isolation frequency was performed with the MSTAT-C statistical package, Michigan State Univ., USA). Least significant difference (LSD) was used to compare fungi means. Low frequency of contamination with B1, B 2, G1 and G2 did not permit model fit for inferential analysis of these toxins.
Analysis of variance of the fungal isolation frequency revealed that fungus and fungus - treatment (crop) interaction were highly significant sources of variation in frequencies of fungi isolated from tested grains (Table 1). The fungus - treatment (crop) interaction was the most importance as a source of variation in isolation frequency, while fungus was of second importance (Fig.1). Due to the significant effect of fungus - treatment interaction, a least significant difference was used to compare between fungi within each crop. Aspergillus spp. i.e., A. flavus and A. niger were recovered from all samples with the highest isolation frequencies from corn grains (Table 2). In addition, A. clavatus was recovered only from corn and popcorn samples. F. proliferatum (88.9%) was the most dominant fungus in corn samples followed by F. verticillioides (80.57%) and F. subglutinans (88.9%) was the most dominant in popcorn samples. In respect to Penicillium spp. P. funiculesum (100%) was the most dominant in sorghum samples and P. chrysogenum (74.07%) was the most dominant in peanut samples (Table 2).
Most Aspergillius isolates were capable of producing detectable levels of both B and G aflatoxins at variable levels. Three of the ten tested isolates failed to produce any detectable amount of aflatoxins (Table 3). All With the exception of the sorghum isolate, all A. flavus isolates were capable of producing variable amounts of B and G aflatoxin. The highest amount of aflatoxin B1 production (8ppb) was obtained from A. flavus isolated from corn grains. A. clavatus isolated from popcorn produced only aflatoxin B1 and B2.
All tested isolates were patulin producers except peanut and popcorn isolates from P. chrysogenum which did not produced any patulin (Table 4). A sorghum isolate from P. chrysogenum was the highest patulin (38ppb) producer. Citreovirdin however, was produced by all isolates except P. chrysogenum from popcorn and sorghum as well as the P. funiculosum isolate from sorghum. The sorghum isolate of P. oxalicum was the highest producer of citreoviridin (37ppb).
All isolates except the peanut isolate of F. subglutinans were toxin producers. Toxin-producing isolates varied in the type and concentrations of toxin produced. F. subglutinans isolate from popcorn was capable of producing fumonisin, zearalenone and vomitoxin F. proliferatum isolated from corn however, failed to produce fumonisin, and the sorghum isolate from F. verticillioides produced only fumonisin B1 (Table 5).
GC/MS analysis of Aloe vera material
Fractionation of Aloe vera juice using GC/Ms analysis resulted in four fractions with RT ranged from 3.242 to 15.871. According to the data of Wiley library which include in GC/Ms data these fractions are identified as shown in Table 7. Relative concentrations of theses fractions ranged from 1.576 to 30.489 (Fig. 2 and Table 7).
Effect of A. vera on fungal growth
Data analysis revealed that the A. vera concentration, and fungus x A. vera concentration interaction were highly significant sources of variation in linear growth of the tested fungi (Table 7). A. vera concentration was the most important source of variation as it accounted for 82.2% of the total variation in linear growth. Due to the significant of the interaction, a least significant difference was used to compare between concentrations within each fungus (Fig. 4). These comparisons showed that all the tested concentrations were effective in inhibiting the linear growth of fungi; however, the increase in concentration did not necessarily lead to an increase in the fungicidal activity of the extract.
Data obtained in this study indicate that samples of peanut seeds, corn, popcorn and sorghum grains were contaminated with a diverse group of seed-borne fungi. Aspergillus, Fusarium and Penicillium were the major genera frequently recovered from corn grains , peanut [7, 33] and sorghum grains [19, 34] from several regions of the world.
Diversity in isolation frequencies of recovered fungi in the present study could be attributed to the fungal colonizing ability which could be affected by (host) and origin of samples . Differences in genotype, sample location and storage environment could play critical role in fungi colonizing ability within the same host [36, 37].
Most of our tested isolates were capable of producing detectable levels of mycotoxins. Mycotoxins are mainly produce by Aspergillus, Fusarium and Penicillium species isolated from stored grains and have frequently been documented [38, 39]. Aflatoxins (AFs) are the most widespread toxins and are mainly produced by Aspergillus spp. and in this study were isolated from most of corn  and peanut isolates [7, 49].
Fusarial mycotoxins mainly fumonisins, vomitoxin and zearalenone  were also produced by corn and peanut isolates in this study . The sorghum isolate from F. verticillioides was capable of producing only fumonisin. This result agrees with those of [7, 43] and diifers with [21, 34] who stated that sorghum grain molding fusaria were zearalenone producers. Isolates of P. funiculosum and P. oxalicum recovered from corn, sorghum and peanut were patulin and citreoviridin producers . Plant pathogenic penicillia may produce these toxins in culture media, and in agricultural commodities as well as in food waste .
Some of the tested isolates failed to produce any detectable amounts of mycotoxins. Variation in mycotoxin productivity might be due to the probable genetic diversity among and within tested species.
In vitro screening of Aloe vera against seed borne fungi were effective in inhibiting the mycelial growth of test fungi. The antifungal potential of Aloe vera extracts has frequently evaluated . It was found that aqueous and organic extracts of Aloe vera shoots had inhibitory effects on hyphal growth, mycelium development, biomass production  and sporulation  of several phytopathogenic fungi. The fungicidal effects of Aloe vera might be due to its chemical composition . The yellowish exudate of Aloe vera contains several substances such as anthraquinone derivatives, glycosides, tannins and many active enzymes .
The current research demonstrated the susceptibility of some storage crops in Saudi to colonization with Aspergillus section Flavi, especially A. flavus isolates, Aspergillus section Nigri, Fusarium species and Pencillium species. Aqueous of A. vera were evaluated for antifungal activity against nine mycotoxigenic fungi isolated from corn, popcorn, peanut and sorghum grains. Results revealed the very toxic effect of the A. vera. Based on the antimycotic activity, crude plant extracts may be a commercial way of protecting storage crops against toxigenic fungi. Therefore, plant extract may become an alternative for the control of the harmful effects of mycotoxins on food products.
This work has received support from the King Saud University, College of Science, Research Center Project (Bot/2010/44). Thanks are due to Dr. Aly Abdel-Hady Aly for pre-submission reviews of this manuscript