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A survey of fungi associated with corn and popcorn samples, collected from markets throughout the Riyadh region of Saudi Arabia was investigated. Seventeen species from nine genera were recovered from corn grains, while 11 species from six genera were recovered from popcorn grains. Frequencies of the isolated genera were statistically compared. Aspergillus flavus, A. niger and Rhizopus stolonifer were most frequently isolated from non-sterilized grains, Aspergillus niger, Fusarium proliferatum and F. verticillioides were dominant in sterilized corn grains, while Aspergillus clavatus, A. flavus var. columnaris and Fusarium subglutinans were dominant in sterilized popcorn grains Potential ability to produce a¬‚atoxins (AFs) B1, B2, G1 and G2, was studied by isolate culture followed by HPLC analysis of these mycotoxins in the culture extracts. Most A. flavus isolates (75%) and some A. niger isolates (25%) were toxin producers. Sixty-seven percent of A. flavus var. columnaris isolates produced aflatoxinsand produced most B2 aflotoxin, while A. flavus produced most B1, G1 and G2 aflotoxins. A. niger produced the least aflatoxins. Information on the levels of A. flavus inoculum in the environment and contamination of corn with aflatoxins is valuable in the development of mycotoxin management strategies.
KEYWORDS: HPLC, Mycotoxins, seed-borne fungi, Zea mays,.
Address correspondence to:
Mohamed A. Yassin Ph.D.
Botany and Microbiology Department
Faculty of Science
King Saud University
P.O. Box 2455
E-mail: [email protected]
Corn (Zea maize L) is one of the most important cereal crops for human consumption and animal feeds in the world and is ranked in first in production levels worldwide . Saudi Arabia purchased about 79 million bushels of corn during 2008 and 2009. Imported corn may be exposed to pre-harvest fungal infection and such infection in the field may be continue and worsen throughout the marketing and storage periods. Infection by toxigenic fungi affect the corn grain health regardless of the time of infection . For example, Fusarium species invade more than 50% of corn grains before harvest and produce mycotoxins . Externally or internal seed-borne fungi associated with the grains, may cause seed deterioration [34, 35]. In fact, fungi are ranked second as the cause of deterioration and loss of corn . In addition, corn grains are often colonized by a succession of fungi during storage, and this is dependent on temperature and moisture levels. More than 70% of corn may be damaged during the storage period due to fungal activity .
Corn grains may be colonized by a number of fungal taxa and therefore grains may be contaminated by several mycotoxins . Although, most infections occur in the field, grain storage practices can prevent postharvest development of mycotoxins. The important genera commonly encountered on corn in different regions are Aspergillus, Fusarium and Penicillium [32, 38]. Several other seed-borne fungi e.g. Alternaria spp., Curvularia spp., Helminthosporium maydis, Monilia spp., Rhizopus spp. and Trichoderma spp. are also commonly isolated from corn grains [6, 14].
Infection by toxigenic fungi affects grain quality and suitability for human and animal consumption. For example corn grain contaminations by Aspergillus flavus, which is known to produce aflatoxin B1, one of the most potent carcinogenic and citotoxic compounds for human and animals, have frequently been recorded [43,44]. Chemical treatment to prevent seed rot development results in environmental pollution; health hazard and affects the natural ecological balance so should not be used. Thus, early detection of seed-borne fungi would result in more extensive grain rot control and may be lead to improved storage. The present study aimed (1) to investigate the natural occurrence of seed-borne fungi (2) to evaluate the ability of Aspergillus species to produce Aflatoxins in corn and popcorn grains.
MATERIALS AND METHODS
Twenty-four corn grain samples and nine popcorn samples, collected from different locations in Riyadh city were used for isolation and detection of seed-borne fungi. Fungi were isolated and cultured according to the method described by Singh . Two sets of 10 gm of grain samples were used either after surface sterilized (using 1% sodium hypochlorite solution and washed three times with sterile distilled water) or without sterilization. Ten grains in each case were placed randomly on the surface of Petri-dish containing Potato Dextrose Agar (PDA) in triplicate. Plated grains were incubated at 25±2°C and examined daily for five days, after which the colonies developing from the grains were counted. Isolated fungi were purified either by single spore or hyphal tip methods and then transferred to slanted PDA. Isolates identification was carried out based on morphological and microscopic characteristics in the Mycological Center, Assiut University, Egypt.
Determination of total aflatoxins
Tested isolates were grown on sterilized SMKY liquid medium (Sucrose- 200gm, Magnesium sulphate- 0.5gm, Potassium nitrate- 3gm, yeast extract agar and distilled water 1000ml ) prepared in 100 ml flasks for 10 days at 27 ±2°C. Three replicate flasks were made per isolate. Resulting cultures were blended for 2 min. using high speed homogenizer and filtered using filter paper. Aflatoxins (AFs) were then extracted from such homogenized filtrates using methanol solution (80:20 methanol/ isolate filtrate). Solvents were evaporated in vacuum pump at 35°C., dried residues containing aflatoxin were dissolved in 1 ml of the same mobile phase solution which consists of methanol: acetic acid: water (1:1:3 v/v) and stored in dark vials.
Aflatoxin production was determined using the method of Stroka et al. . Extracts were passed through a 0.45 µm micro-filter and analysis of compounds was performed on HPLC model PerkinElmer® Brownlee™ Validated C18, 100 mm - 4.6 mm, 3 micron. HPLC equipped with UV detector and the wave length in the UV detector was 365nm. The 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.
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 fungal means. Cluster analysis by the unweighted pair-group method based on arithmetic mean (UPGMA) was performed using SPSS6.0 software package. Low frequency of contamination with B1, B 2, G1 and G2 did not permit model fit for inferential analysis of these toxins.
Isolation frequencies of fungi recovered from non-sterilized and sterilized corn grains:
ANOVA of Table 1 revealed that fungus and fungus x treatment interaction were very highly significant sources of variation in frequencies of isolated fungi while treatment was insignificant source of variation (Table 1 and Fig. 1). The fungus was the first in importance as a source of variation in isolation frequency, while fungus x treatment interaction was the second importance (Fig. 1). Treatment of grains (sterilized versus non sterilized) significantly effected the fungal taxa recovered and therefore LSD was used to compare the isolation frequency for each fungus (Table 2). Isolation frequencies of A. flavus, A. niger and R. stolonifer significantly decreased when grains were surface sterilized. These results may indicate that these fungi tended to colonize grain surface, hence, their sensitivity to surface sterilization. Isolation frequencies of A. terreus, F. proliferatum and F. verticillioides increased following surface sterilization. It seems reasonable to assume that these fungi were subjected to less competition or antagonism due to the reduce in the density of surface-colonizing fungi when the grains were sterilized; hence, the significant increase in their isolation frequencies. Other taxa were not significantly affected by surface sterilization because they colonize the internal parts of the grains.
The mean percentage of fungal species recovered from both sterilized and non-sterilized corn grains revealed 17 species from nine genera (Table 2). R. stolonifer, A. niger and A. flavus were dominant fungi on non-sterilized grains, with isolation frequencies of 43.11%, 23.44% and 17.87% respectively. The dominant fungi isolated from sterilized grains were F. verticillioides, A. niger and F. proliferatum with isolation frequencies of 37.85%, 15.14% and 15.05% respectively.
The phenogram in Fig. 2 indicated that fungi isolated from non-sterilized corn grains appear to form several distinct groups based on their distribution patterns over samples. Within each group, fungi were associated strongly and positively in their distribution patterns over samples, whereas between groups, fungi were associated weakly or negatively. This phenogram implies the potential existence of sample (environment) related groups of fungi.
Isolation frequencies of fungi recovered from non-sterilized and sterilized popcorn grains:
Table 3 showed that fungus and fungus x treatment interaction were very highly significant sources of variation in frequencies of isolated fungi while treatment was non-significant source of variation (Table 3 and Fig. 1). The interaction was the first in importance as a source of variation in isolation frequency, while fungus was the second importance (Fig. 1). Due to the significance of treatment x fungus interaction, LSD was used to compare between sterilized and non-sterilized grains in isolation frequency for each fungus (Table 4). These comparisons showed that isolation frequencies of A. flavus var. columnaris, A. niger and R. stolonifer were significantly decreased by surface sterilization. F. subglutinans was the only fungus which showed a significant increase in isolation frequency from sterilized grains. Isolation frequencies of the other fungi were not affected by surface sterilization. These results were inconsistent with those of corn grains.
The mean percentage of fungal recoved from both sterilized and non-sterilized popcorn grains indicate that 11 fungal species belonging 6 genera could be obtained (Table 4). A. niger, A. flavus var. columnaris and R. stolonifer were dominant on non-sterilized grains, with isolation frequencies of 41.82%, 25.43% and 20.12% respectively. F. subglutinans was dominant in sterilized popcorn grains with an isolation frequency of 64.69% followed by A. clavatus and A. flavus var. columnaris with isolation frequencies of 7.41% and 7.17% respectively. Other fungi occurred at frequencies ranging from 0.00-5.86%. It is worth noting that A. clavatus, A. niger, P. oxalicum and R. stolonifer were also isolated from corn grains.
The phenogram shown in Fig. 3 implies the potential existence of sample (environment) related groups of fungi.
Production of aflatoxins by Aspergillus species:
Several Aspergillus species were isolated in this study and varied in their ability to produce aflatoxins. Thus, although some isolates are toxigenic, others exhibited no detectable toxin production (Table 5). Some isolates produced B-aflatoxin, while others produced both B and G-aflatoxins. The majority (75%) of A. flavus isolates and fewer A. niger isolates (25%) were Aflatoxin producers while 67% of A. flavus var. columnaris isolates produced aflatoxins (Table 6).
This study confirms documented data that diverse seed-borne mycoflora are associated with corn grains [2, 18, 24, 39]. Species from several genera viz., Aspergillus, Penicillium, Monilia, Drechslera, Mucor, Alternaria, Cladosporium, Fusarium, Acremonium and Rhizopus were recovered from corn grains and are similar to those previously reported . Aspergillus, Fusarium and Penicillium were domanint genera recovered from corn grain samples in several studies [3, 12, 15, 32], while in this study A. flavus, A. niger and R. stolonifer were most frequently isolated from non-sterilized grains, A. niger, F. proliferatum and F. verticillioides.
Isolation of diverse group of fungi from non-sterilized corn and popcorn grains in the present study [16, 23, 32, 38] could be attributed to one or more of the following reasons: (1) long term storage of grains in moldy inductive environmental conditions (2) ideal nutrient composition of corn grains, which make it a very good substrate for fungal growth compared with other grains such as millet and rice [4, 17, 45]. (3) Mechanical damages occurred during harvesting and drying methods of corn grains [33, 36] as well as transportation from other countries.
The involvement of Aspergilli isolated in this study in aflatoxin production agrees with the findings of Kang et al. , Calvo et al. , Kamei and Watanabe  and Kumar et al. . A. flavus, Fusarium spp. and other fungi were also detected in corn samples collected from various markets and villages in Tanzania and Congo and their aflatoxin and fumonsin production was also established .
Some Fusarium isolates from the present study were toxigenic, but the others were not. Despite of aflatoxins (B and G) production was generally attributed to Aspergillus spp. [13, 27], some of our isolates produced only B-aflatoxin but the others produced both B and G-aflatoxins. These results agree with those of [19, 42]. Variation in aflatoxin productivity as well as in the kinds of toxins produced might be explained by genetic diversity among and within tested species. The kind of mycotoxins produced by fungi as well as the efficiency in producing such toxins is under genetic control [9, 13]. In addition, the genetic base for mycotoxin production, the genetic signaling pathways and the functions of genes involved have been elucidated in several toxigenic species [7, 8, 13, 20].
In summary, this research showed that A. flavus and A. niger are a very frequent contaminants of maize imported into Saudi Arabia and may cause contamination of this food by total Aflatoxins. A. flavus var. columnaris was the most efficient species in producing aflotoxin B2, while A. flavus was most efficient in producing B1, G1 and G2. A. niger was the least efficient of species tested in producing all toxins. The potent toxigenic fungal strains isolated from corn and popcorn samples indicates that rigorous quarantine and healthy storage conditions should be adopted with importing commodities to avoid contamination with toxigenic fungi, and prevent hazards to human and animal health.
This work has received support from the KSU, College of Science, Research Center Project (Bot/2010/06). Thanks are due to Dr. Aly Abdel-Hady Aly for pre-submission reviews of this manuscript