A study of Fungis associated with maize and Zea mays everta samples, collected from markets throughout the Riyadh part of Saudi Arabia was investigated. Seventeen species from nine genera were recovered from maize grains, while 11 species from six genera were recovered from popcorn grains. Frequencies of the stray genera were statistically compared. Aspergillus flavus, A. Niger and Rhizopus stolonifer were most often isolated from non-sterilized grains, Aspergillus Niger, Fusarium proliferatum and F. verticillioides were dominant in sterilised maize grains, while Aspergillus clavatus, A. flavus volt-ampere. columnaris and Fusarium subglutinans were dominant in sterilised Zea mays everta grains Potential ability to bring forth ai¬‚atoxins ( AFs ) B1, B2, G1 and G2, was studied by isolate civilization followed by HPLC analysis of these mycotoxins in the civilization extracts. Most A. flavus isolates ( 75 % ) and some A. Niger isolates ( 25 % ) were toxin manufacturers. Sixty-seven per centum of A. flavus volt-ampere. 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 degrees of A. flavus inoculant in the environment and taint of maize with aflatoxins is valuable in the development of mycotoxin direction schemes.
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
Capital of saudi arabia 1145
Electronic mail: myassin @ ksu.edu.sa
Corn ( Zea maize L ) is one of the most of import cereal harvests for human ingestion and animate being provenders in the universe and is ranked in first in production degrees worldwide [ 5 ] . Saudi Arabia purchased about 79 million bushels of maize during 2008 and 2009. Imported maize may be exposed to pre-harvest fungous infection and such infection in the field may be continue and decline throughout the selling and storage periods. Infection by toxigenic Fungis affect the maize grain wellness regardless of the clip of infection [ 24 ] . For illustration, Fusarium species invade more than 50 % of maize grains before crop and bring forth mycotoxins [ 6 ] . Externally or internal seed-borne Fungis associated with the grains, may do seed impairment [ 34, 35 ] . In fact, Fungis are graded 2nd as the cause of impairment and loss of maize [ 31 ] . In add-on, maize grains are frequently colonized by a sequence of Fungi during storage, and this is dependent on temperature and wet degrees. More than 70 % of maize may be damaged during the storage period due to fungous activity [ 25 ] .
Corn grains may be colonized by a figure of fungous taxa and hence grains may be contaminated by several mycotoxins [ 30 ] . Although, most infections occur in the field, grain storage patterns can forestall postharvest development of mycotoxins. The of import genera normally encountered on maize in different parts are Aspergillus, Fusarium and Penicillium [ 32, 38 ] . Several other seed-borne Fungis e.g. Alternaria spp. , Curvularia spp. , Helminthosporium maydis, Monilia spp. , Rhizopus spp. and Trichoderma spp. are besides normally isolated from maize grains [ 6, 14 ] .
Infection by toxigenic Fungis affects grain quality and suitableness for homo and animate being ingestion. For illustration maize grain taints by Aspergillus flavus, which is known to bring forth aflatoxin B1, one of the most powerful carcinogenic and citotoxic compounds for human and animate beings, have often been recorded [ 43,44 ] . Chemical intervention to forestall seed putrefaction development consequences in environmental pollution ; wellness jeopardy and affects the natural ecological balance so should non be used. Therefore, early sensing of seed-borne Fungis would ensue in more extended grain putrefaction control and may be lead to improved storage. The present survey aimed ( 1 ) to look into the natural happening of seed-borne Fungis ( 2 ) to measure the ability of Aspergillus species to bring forth Aflatoxins in maize and Zea mays everta grains.
MATERIALS AND METHODS
Twenty-four maize grain samples and nine Zea mays everta samples, collected from different locations in Riyadh metropolis were used for isolation and sensing of seed-borne Fungis. Fungi were isolated and cultured harmonizing to the method described by Singh [ 40 ] . Two sets of 10 gram of grain samples were used either after surface sterilized ( utilizing 1 % Na hypochlorite solution and washed three times with unfertile distilled H2O ) or without sterilisation. Ten grains in each instance were placed indiscriminately on the surface of Petri-dish incorporating Potato Dextrose Agar ( PDA ) in triplicate. Plated grains were incubated at 25±2 & A ; deg ; C and examined daily for five yearss, after which the settlements developing from the grains were counted. Isolated Fungis were purified either by individual spore or hyphal tip methods and so transferred to slanted PDA. Isolates designation was carried out based on morphological and microscopic features in the Mycological Center, Assiut University, Egypt.
Determination of entire aflatoxins
Tested isolates were grown on sterilised SMKY liquid medium ( Sucrose- 200gm, Magnesium sulphate- 0.5gm, Potassium nitrate- 3gm, yeast extract agar and distilled H2O 1000ml [ 11 ] ) prepared in 100 milliliter flasks for 10 yearss at 27 ±2 & A ; deg ; C. Three replicate flasks were made per isolate. Resulting civilizations were blended for 2 min. utilizing high velocity homogenizer and filtered utilizing filter paper. Aflatoxins ( AFs ) were so extracted from such homogenized filtrates utilizing methanol solution ( 80:20 methanol/ isolate filtrate ) . Solvents were evaporated in vacuity pump at 35 & A ; deg ; C. , dried residues incorporating aflatoxin were dissolved in 1 milliliter of the same nomadic stage solution which consists of methyl alcohol: acetic acid: H2O ( 1:1:3 v/v ) and stored in dark phials.
Aflatoxin production was determined utilizing the method of Stroka et Al. [ 41 ] . Infusions were passed through a 0.45 µm micro-filter and analysis of compounds was performed on HPLC theoretical account PerkinElmer® Brownlee™ Validated C18, 100 mm – 4.6 millimeter, 3 micrometer. HPLC equipped with UV sensor and the moving ridge length in the UV sensor was 365nm. The nomadic stage yielded consequences of methyl alcohol: acetic acid: H2O ; 20/ 20/ 60 v/v/v ) . The entire tally clip for the separation was about 25 min at a flow rate of 1 ml/min.
The isolation frequence ( Fq ) of genera was calculated harmonizing to Marassas et Al. [ 29 ] . A randomised complete block design was used in the present survey. Analysis of discrepancy ( ANOVA ) of the fungous isolation frequence was performed with the MSTAT-C statistical bundle, Michigan State Univ. , USA ) . Least important difference ( LSD ) was used to compare fungous agencies. Cluster analysis by the unweighted pair-group method based on arithmetic mean ( UPGMA ) was performed utilizing SPSS6.0 package bundle. Low frequence of taint with B1, B 2, G1 and G2 did non allow theoretical account tantrum for illative analysis of these toxins.
Isolation frequences of Fungis recovered from non-sterilized and sterilised maize grains:
ANOVA of Table 1 revealed that fungus and fungus x intervention interaction were really extremely important beginnings of fluctuation in frequences of stray Fungis while intervention was undistinguished beginning of fluctuation ( Table 1 and Fig. 1 ) . The fungus was the first in importance as a beginning of fluctuation in isolation frequence, while fungus x intervention interaction was the 2nd importance ( Fig. 1 ) . Treatment of grains ( sterilized versus not sterilized ) significantly effected the fungous taxa recovered and hence LSD was used to compare the isolation frequence for each fungus ( Table 2 ) . Isolation frequences of A. flavus, A. Niger and R. stolonifer significantly decreased when grains were surface sterilized. These consequences may bespeak that these Fungis tended to colonise grain surface, therefore, their sensitiveness to come up sterilisation. Isolation frequences of A. terreus, F. proliferatum and F. verticillioides increased following surface sterilisation. It seems sensible to presume that these Fungis were subjected to less competition or hostility due to the cut down in the denseness of surface-colonizing Fungis when the grains were sterilized ; hence, the important addition in their isolation frequences. Other taxa were non significantly affected by surface sterilisation because they colonize the internal parts of the grains.
The average per centum of fungous species recovered from both sterilized and non-sterilized maize grains revealed 17 species from nine genera ( Table 2 ) . R. stolonifer, A. Niger and A. flavus were dominant Fungis on non-sterilized grains, with isolation frequences of 43.11 % , 23.44 % and 17.87 % severally. The dominant Fungis isolated from sterilized grains were F. verticillioides, A. Niger and F. proliferatum with isolation frequences of 37.85 % , 15.14 % and 15.05 % severally.
The phenogram in Fig. 2 indicated that Fungis isolated from non-sterilized maize grains appear to organize several distinguishable groups based on their distribution forms over samples. Within each group, Fungis were associated strongly and positively in their distribution forms over samples, whereas between groups, Fungis were associated weakly or negatively. This phenogram implies the possible being of sample ( environment ) related groups of Fungis.
Isolation frequences of Fungis recovered from non-sterilized and sterilised Zea mays everta grains:
Table 3 showed that fungus and fungus x intervention interaction were really extremely important beginnings of fluctuation in frequences of stray Fungis while intervention was non-significant beginning of fluctuation ( Table 3 and Fig. 1 ) . The interaction was the first in importance as a beginning of fluctuation in isolation frequence, while fungus was the 2nd importance ( Fig. 1 ) . Due to the significance of intervention x fungus interaction, LSD was used to compare between sterilized and non-sterilized grains in isolation frequence for each fungus ( Table 4 ) . These comparings showed that isolation frequences of A. flavus volt-ampere. columnaris, A. Niger and R. stolonifer were significantly decreased by surface sterilisation. F. subglutinans was the lone fungus which showed a important addition in isolation frequence from sterilized grains. Isolation frequences of the other Fungis were non affected by surface sterilisation. These consequences were inconsistent with those of maize grains.
The average per centum of fungous recoved from both sterilized and non-sterilized Zea mays everta grains indicate that 11 fungous species belonging 6 genera could be obtained ( Table 4 ) . A. Niger, A. flavus volt-ampere. columnaris and R. stolonifer were dominant on non-sterilized grains, with isolation frequences of 41.82 % , 25.43 % and 20.12 % severally. F. subglutinans was dominant in sterilised Zea mays everta grains with an isolation frequence of 64.69 % followed by A. clavatus and A. flavus volt-ampere. columnaris with isolation frequences of 7.41 % and 7.17 % severally. Other fungi occurred at frequences runing from 0.00-5.86 % . It is deserving observing that A. clavatus, A. Niger, P. oxalicum and R. stolonifer were besides isolated from maize grains.
The phenogram shown in Fig. 3 implies the possible being of sample ( environment ) related groups of Fungis.
Production of aflatoxins by Aspergillus species:
Several Aspergillus species were isolated in this survey and varied in their ability to bring forth aflatoxins. Therefore, although some isolates are toxigenic, others exhibited no noticeable toxin production ( Table 5 ) . Some isolates produced B-aflatoxin, while others produced both B and G-aflatoxins. The bulk ( 75 % ) of A. flavus isolates and fewer A. Niger isolates ( 25 % ) were Aflatoxin manufacturers while 67 % of A. flavus volt-ampere. columnaris isolates produced aflatoxins ( Table 6 ) .
This survey confirms documented informations that diverse seed-borne mycoflora are associated with maize grains [ 2, 18, 24, 39 ] . Speciess from several genera viz. , Aspergillus, Penicillium, Monilia, Drechslera, Mucor, Alternaria, Cladosporium, Fusarium, Acremonium and Rhizopus were recovered from maize grains and are similar to those antecedently reported [ 1 ] . Aspergillus, Fusarium and Penicillium were domanint genera recovered from maize grain samples in several surveies [ 3, 12, 15, 32 ] , while in this survey A. flavus, A. Niger and R. stolonifer were most often isolated from non-sterilized grains, A. Niger, F. proliferatum and F. verticillioides.
Isolation of diverse group of Fungis from non-sterilized maize and Zea mays everta grains in the present survey [ 16, 23, 32, 38 ] could be attributed to one or more of the undermentioned grounds: ( 1 ) long term storage of grains in mouldy inductive environmental conditions ( 2 ) ideal alimentary composing of maize grains, which make it a really good substrate for fungous growing compared with other grains such as millet and rice [ 4, 17, 45 ] . ( 3 ) Mechanical amendss occurred during harvest home and drying methods of maize grains [ 33, 36 ] every bit good as transit from other states.
The engagement of Aspergilli isolated in this survey in aflatoxin production agrees with the findings of Kang et Al. [ 22 ] , Calvo et Al. [ 10 ] , Kamei and Watanabe [ 21 ] and Kumar et Al. [ 26 ] . A. flavus, Fusarium spp. and other Fungis were besides detected in maize samples collected from assorted markets and small towns in Tanzania and Congo and their aflatoxin and fumonsin production was besides established [ 28 ] .
Some Fusarium isolates from the present survey were toxigenic, but the others were non. Despite of aflatoxins ( B and G ) production was by and large attributed to Aspergillus spp. [ 13, 27 ] , some of our isolates produced merely B-aflatoxin but the others produced both B and G-aflatoxins. These consequences agree with those of [ 19, 42 ] . Variation in aflatoxin productiveness every bit good as in the sorts of toxins produced might be explained by familial diverseness among and within tried species. The sort of mycotoxins produced by Fungi every bit good as the efficiency in bring forthing such toxins is under familial control [ 9, 13 ] . In add-on, the familial base for mycotoxin production, the familial signaling tracts and the maps of cistrons involved have been elucidated in several toxigenic species [ 7, 8, 13, 20 ] .
In drumhead, this research showed that A. flavus and A. Niger are a really frequent contaminations of corn imported into Saudi Arabia and may do taint of this nutrient by entire Aflatoxins. A. flavus volt-ampere. columnaris was the most efficient species in bring forthing aflotoxin B2, while A. flavus was most efficient in bring forthing B1, G1 and G2. A. Niger was the least efficient of species tested in bring forthing all toxins. The potent toxigenic fungous strains isolated from maize and Zea mays everta samples indicates that strict quarantine and healthy storage conditions should be adopted with importing trade goods to avoid taint with toxigenic Fungis, and prevent jeopardies to human and carnal wellness.
This work has received support from the KSU, College of Science, Research Center Project ( Bot/2010/06 ) . Thankss are due to Dr. Aly Abdel-Hady Aly for pre-submission reappraisals of this manuscript