Mycobiota and Mycotoxins (Aflatoxins and Ochratoxin) Associated with Some Saudi Date Palm Fruits

Abstract

This study aimed to determine the mycological profile of the retail date fruits distributed in different markets at Taif, Saudi Arabia. The presence of aflatoxins and ochratoxin A was also measured. Twenty-two fungal species belonging to 12 genera were isolated from 50 different date samples. Aspergillus flavus, A. niger, Penicillium chrysogenum, and Rhizopus stolonifer were the most prevalent species among isolated fungi. Eighty isolates of A. flavus and 36 of A. niger were detected for their aflatoxins and ochratoxin production potentials using thin layer chromatography. Toxicity test using Artimia larvae indicated that seven out of 18 A. flavus isolates had aflatoxins potentials, while nine out of 36 isolates of A. niger were ochratoxigenic. The quadruplex polymerase chain reaction using specific primers demonstrated the presence of four genes: nor A, ver 1, omt A, and avf A in seven A. flavus toxigenic isolates. Nine A. niger toxigenic isolates showed positive results for the presence of the PKS gene. In conclusion, the present study highlighted the potential hazards of mycotoxins on human health from consuming raw dates. Rapid molecular detection methods described here might help the food authorities to assure the safety of raw dates distributed in local markets.

Introduction

T he Kingdom of Saudi Arabia is considered one of the major date producing zones in the world, contributing 13% of the total world production. Therefore, the Kingdom of Saudi Arabia exerts great effort to maximize date production and maintain this market share through product quality and safety assurance. In Saudi Arabia, date fruits brought a benefit of $688 per ton to the producers in 1990, whereas in 2000 it was $767 per ton. During the 2002 season, date fruit production was more than 130,000 tons, and approximately 1,092 tons were used for exportation (Saudi Ministry of Agriculture and Water, 2003). Most of the dates are delivered to local markets and consumed by Saudi people without any treatment to minimize the microbial contaminants.

Fresh fruits are prone to fungal contamination in the field; during harvest, transport, and marketing; and with the consumer. Fungi play a substantial role in spoilage of fruits and vegetables, because of their pathogenicity to the harvested products. Fungi (Aspergillus, Alternaria, and Penicillium spp.) may grow on high-moisture dates, especially when harvested following rain or high humidity periods (Kader and Hussein, 2009). Mycological profiles for date palm fruits were studied previously in several places around the world, including Saudi Arabia (Salik et al., 1999; Gherbawy, 2001; Ragab et al., 2001; Shenasi et al., 2002; Aba Alkhail et al., 2004; Alghalibi and Shater, 2004; Iamanaka et al., 2005; Al-Sheikh, 2009; Bokhary, 2010).

During the various stages of pathogenesis, however, some of these fungi may generate different mycotoxins—secondary metabolites that are toxic to humans and animals who consume the products. Aflatoxins are secondary metabolites produced by aflatoxigenic fungi Aspergillus flavus Link and Aspergillus parasiticus Speare (Roehuck and Maxuitenko, 1994). Several researchers documented aflatoxin production in date palm fruits (Mahjoub et al., 1989; Ahmed and Robinson, 1997; Abdel-Sater and Saber,1999; Ragab et al., 2001; Shenasi et al., 2002; Alghalibi and Shater, 2004). Ochratoxin A (OTA) is a nephrotoxic mycotoxin naturally found in a wide range of food commodities throughout the world. In addition to the other toxic effects, OTA is carcinogenic, teratogenic, genotoxic, and immunosuppressive (Petzinger and Weidenbach, 2002). Two Aspergillus sections are known to produce OTA: the section Circumdati (also called the Aspergillus ochraceus group) and the section Nigri (Aspergillus carbonarius and Aspergillus niger) (Varga et al., 1996; Heenan et al., 1998). Among the species of the section Nigri, A. carbonarius shows high ochratoxigenic potential, with most isolates having the ability to produce OTA in culture (Heenan et al., 1998). Sartori et al. (2010) reviewed and discussed the detection of microorganisms capable of producing OTA, prior to ochratoxin production and accumulation. However, very limited information is available in detecting ochratoxin in date fruit (Abdel-Sater and Saber, 1999; Ferracin et al., 2009).

Polymerase chain reaction (PCR)–based methods are considered a good alternative for rapid diagnosis of fungi because of their high specificity and sensitivity (Schmidt et al., 2003), especially when multi-copy sequences are used to develop species-specific primers (Bluhm et al., 2002). The biosynthetic pathway for aflatoxin production by A. flavus has been deciphered, and genes in the aflatoxin biosynthetic pathway have been identified (Payne et al., 1993; Trail et al., 1995; Yu et al., 1995, 2004). The AflR gene plays an important role in the aflatoxin biosynthetic pathway by regulating the activity of other structural genes such as omt-A, ver-1, and nor-1 (Woloshuk et al., 1994; Chang et al., 1999). In particular, aflR gene integrity has been considered a good candidate for diagnostic purposes in discriminating between aflatoxins producer and nonproducer strains of A. flavus (Chang et al., 1995). Reports on the detection of aflatoxigenic fungi using PCR are rather limited (Shapira et al., 1996; Farber et al., 1997; Gashgari et al., 2010, 2011). The Aspergillus niger group is well known as an ochratoxin producer, and several studies have been conducted to explore this group of fungi. Patino et al. (2005) developed two PCR assays to detect A. carbonarius and A. ochraceus, two main sources of OTA contamination, particularly to grapes, coffee, and derivatives in warm climates. Selma et al. (2008) developed a multiplex real- time PCR system for the simultaneous detection of A. carbonarius and A. niger aggregate species. They designed a primer pair and a probe specific for the A. niger aggregate species derived from a conserved region in the acyltransferase domain sequences of PKS genes after comparison with different Aspergillus and Penicillium species. Their method allowed the detection of all the species included in the aggregate, without differentiating between ochratoxigenic and nonochratoxigenic strains. Castellá and Cabañes (2011) designed a primer pair from a PKS gene described in A. niger CBS 513.88 genome that has a strong similarity to the PKS gene of A. ochraceus involved in OTA biosynthesis. This primer pair produced a fragment of 951 bp specific for ochratoxigenic strains of the A. niger aggregate.

The present study aimed to (i) determine the occurrence and load of fungi (the important foodborne pathogens) in date fruits offered for sale to consumers in retail stores at the Taif region in Saudi Arabia; (ii) analyze specifically the occurrence of the Aspergillus niger group and the Aspergillus flavus group in the collected samples; and (iii) investigate the occurrence of OTA and aflatoxins, a public health concern, using chromatographic and molecular techniques.

Methods

Mycobiota determination

Fifty replicated samples of date fruits were collected from different local supermarkets in the Taif region. Fungi were isolated using the dilution plate method as described by Johnson and Curl (1972). Twenty-five grams of each dried fruit sample were comminuted for 2 min in 250 mL of 0.12% sterile plain agar. Further dilutions were made, and 1 mL of an appropriate final dilution was placed on 1% glucose–Czapek's agar plates. The plates were incubated at 28°C for 10 days, and fungal colonies growing on these plates were purified and identified by classical taxonomic methods (Pitt and Hocking, 1997). From media plates, only colonies belonging to A. niger and A. flavus groups were transferred to slants to ensure precise counting and for identification at the species level (Klich and Pitt, 1988). These isolates were preserved at −80°C for further studies.

Mycotoxin detection using thin layer chromatography

Aflatoxins

The isolates identified as Aspergillus species were tested for the production of aflatoxins based on the thin layer chromatography (TLC) method (Rahimi et al., 2007).

OTA

Each isolate from black aspergilli was grown as stationary cultivation and incubated at 28°C for 15 days. Liquid medium of the following composition was used: sucrose, 30 g; peptone, 10 g; NaNO₃, 2 g; K₂HPO₄, 1 g; yeast extract, 1 g; KCL, 0.5 g; MgSO₄. 7H₂O, 0.5 g; FeSO₄. 7H₂O, 0.01 g; 1 L of distilled water. The cultivation was made in 250-mL Erlenmeyer flasks. The content of each flask (medium + mycelium) was homogenized for 3 min in a high-speed blender (16,000 rpm) with 75 mL of chloroform. The extraction procedure was repeated three times. The combined chloroform extracts were washed with equal volumes of distilled water, dried over anhydrous sodium sulphate, filtered and then concentrated under a vacuum or a stream of nitrogen to near dryness (El-Kady and Moubasher, 1982). OTA was detected in the sample extracts after TLC on 0.3-mm layers of Adsorbosil 5 silica gel, using tolueneethyl acetate–90% formic acid (6:3:1 vIvIv) for development. Each extract formed a green fluorescent spot under ultraviolet light with the same Rf (0.55) as authentic OTA. The fluorescence turned blue on treatment with ammonia (Nesheim, 1976).

Molecular detection of mycotoxin producing genes

The isolation of DNA from mycelia was performed according to the method described by Farber et al. (1997). Specific primers used in the current study are shown in Table 1. For aflatoxins, PCR assays were performed in 25 μL of a reaction mixture that contained the following: 2.5 μL buffer, 1.5 μL MgCl₂, 0.5 μL dNTPs, 0.5 μL Taq polymerase, 0.5 μL of each primer (aflR-1, aflR-2; omt-1, omt-2; ver-1, ver-2; and nor-1, nor-2), 0.5 μL template DNA, 1.5 μL deionized water; 14.5 μL was added to make the volume up to 25 μL. PCR technique was as follows: one step for denaturation at 96°C for 5 min followed by 35 cycles of: 95°C for 1 min; 65°C for 30 s; 72°C for 30 s; for the first cycle; and 94°C, 30 s; variable (56–62°C), 30 s; 72°C, 30 s for the next 34 cycles (Rashid et al., 2008).

Table 1.

List of Primers Used for Detecting Fungi from Date Fruits in Saudi Arabia

Primer name	Primers sequence	PCR product (bp)	Reference
aflR-1	5′-TATCTCCCCCCGGGCATCTCCCGG-3′	1000	Rashid et al. (2008)
aflR-2	5′-CCGTCAGACAGCCACTGGACACGG-3′
omt-1	5′-GTGGACGGACCTAGTCCGACATCAC-3′	797	Geisen (1996)
omt-2	5′-GTCGGCGCCACGCACTGGGTTGGGG-3
ver-1	5′-GCCGCAGGCCGCGGAGAAAGTGGT-3′	600	Rashid et al. (2008)
ver-2	5′-GGGGATATACTCCCGCGACACAGCC-3′
nor-1	5′-ACCCTACGCCGGCACTCTCGGCAC-3′	400	Farber et al. (1997)
nor-2	5′-TTGGCCGCCAGCTTCGACACTCCG-3
ANPKS-1	5′-CGACGACTAATCGCAAGTCA-3′	951	Castellá and Cabañes (2011)
ANPKS-2	5′-CATCGTTGCTCATAGGGGTT-3′

For OTA assay, PCR reactions were performed in 50 μL as final volume, containing 5.0 μL of template DNA, 5.0 μL of 10×PCR buffer, 0.2 μM of each dNTP, 1.5 μM MgCl2, 0.3 μM each primer (ANPKS 1, 5′-CGACGACTAATCGCAAGTCA-3′ and ANPKS 2, 5′-CATCGTTGCTCATAGGGGTT-3′), and 2.5 U of Taq polymerase. The amplification process consisted of a pre-denaturation step at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C/30 s, annealing at 51°C/1 min and extension at 72°C/1 min, and a final extension of 7 min at 72°C (Castellá and Cabañes, 2011). Twelve microliters of the PCR products with positive and negative controls were loaded in the gel wells after mixing with appropriate amount of 6.7 μL of 6×loading dye. Also, 7.5 μL of 100-bp ladder mixed in 6×loading orange dye was loaded. PCR products were electrophoresed in a 1.2% agarose gel prepared in 1×TBE stained with ethidium bromide (1.5 μg/mL) for 2–3 h. DNA bands were visualized on a UV transilluminator at a wave length of 540 nm (Rashid et al., 2008).

Bioassay of toxins

The toxicity of A. flavus and A. niger strains were compared by bioassay against Artemia salina (Biji et al., 1981). The chloroform extract (0.05 mL) of fungal strains was placed in each test tube, the chloroform was evaporated, and about 10–20 shrimp larvae in 1 mL of salt water were transferred into tubes. The tubes were kept at room temperature (22–24°C). Control tubes with 0.05 mL of chloroform were also made. After 24 h, mortality was determined.

Results and Discussion

Date mycobiota

Data in Table 2 showed that 22 species belonging to 12 genera were collected from date fruits on 1% glucose–Czapek's agar at 28°C. The gross total counts of glucophilic fungi in dates samples were 1108 colonies per gram dry weight (Table 2). Most of the recovered fungi had been isolated previously from date fruits in many countries of the world (Nassar, 1986; Salik et al., 1999; Ragab et al., 2001; Gherbawy, 2001; Shenasi et al., 2002; Aba Alkhail et al., 2004; Alghalibi and Shater, 2004; Al-Sheikh, 2009; Bokhary, 2010), but almost all with different frequencies.

Table 2.

Average Total Counts (ATC), Number of Cases of Isolations (NCI), and Frequencies (% F) of Various Fungal Genera and Species Isolated from 50 Date Fruit Samples on 1% Glucose-Czapek's Agar at 28°C for 10 Days

Genera and species	ATC ^a	NCI	% F	% Total fungi
Alternaria alternate	65	12	24	5.9
Aspergillus	337	45	90	30.4
A. flavus	44	18	36	4.0
A. fumigatus	6	2	4	0.5
A. niger	230	36	72	20.8
A. terreus	50	8	16	4.5
A. versicolor	7	2	4	0.6
Cladosporium	63	8	16	5.7
C. cladosporioides	28	5	10	2.5
C. herbarum	35	8	16	3.2
Dreschlera spicifera	189	10	20	17.1
Eurotium	10	4	8	0.9
E. amstelodami	7	3	6	0.6
E. chevalieri	3	1	2	0.3
Fusarium	49	12	24	4.4
F. solani	32	10	20	2.9
F. oxysporum	7	2	4	0.6
F. moniliforme	10	3	6	0.9
Mucor racemosus	18	6	12	1.6
Myrothecium verrucaria	15	4	8	1.4
Penicillium	156	27	54	14.1
P. corylophilum	36	5	10	3.2
P. chrysogenum	120	12	24	10.8
P. rubrum	6	4	8	0.5
Rhizopus stolonifer	175	32	64	15.8
Trichoderma harazinum	23	2	4	2.1
Uolcladium atrum	8	3	6	0.7
Total count	1729

Calculated per gram dry weight of fruits.

Aspergillus, represented by five species, was the most frequently isolated genus. It was found in 45% of the samples comprising 30.4% of total fungi. In Egypt, Abdel-Sater and Saber (1999) found that Aspergillus was the predominant genus isolated from dates (100% of the samples). A. niger and A. flavus were the most prevalent species among aspergilla. They occurred in 36% and 72% of the samples, matching 4% and 20.8% of all cultured clones (Table 2). Abu-Zinada and Ali (1982) found that A. flavus was generally associated with some date fruit varieties from Saudi Arabia when the relative humidity (RH) was increased to 90% at 30°C. Nassar (1986) isolated Aspergillus represented by three species, A. niger and two species from Aspergillus (namely A. ruber and A. amstelodami) from dates in Aswan, Egypt. Shenasi et al. (2002) recorded A. flavus in 10 date fruit varieties from the United Arab Emirates (UAE) at the first stage of maturation. Alghalibi and Shater (2004) reported that Aspergillus was the most frequently isolated genus from date samples collected from Yemen and occurred in 75% of the samples comprising 71.4% of total fungi in dates. Also, they reported that Aspergillus niger was the most common Aspergillus species and isolated from 13 date samples out of 20.

Rhizopus stolonifer was the second most common fungus isolated in the present study. It occurred in 64% of the samples comprising 15.8% of total fungi (Table 2). Alghalibi and Shater (2004) isolated Rhizopus stolonifer from date palm fruits in Yemen, and it occurred in 25% of the samples comprising 11.7% of total fungi.

Penicillium was isolated from 27 samples of dates (54%). Similar findings were reported by Gherbawy (2001) in Egypt, who found Penicillium species isolated in high frequency from date fruit (53.3%). In contrast to those findings, Abdel-Sater and Saber (1999) found Penicillium species isolated in low frequency from dates (30%).

The remaining genera and species were isolated with low frequency and their total counts as represented in Table 2. Most of these fungi were isolated previously from date fruits in many parts of the world as reported by several researchers (Nassar, 1986; Salik et al., 1999; Ragab et al., 2001; Shenasi et al., 2002; Aba Alkhail et al., 2004; Al-Sheikh, 2009; Bokhary, 2010).

Aflatoxins and OTA assessment

Eighty isolates of A. flavus were subjected for detecting their aflatoxigenic potentials. Analysis of the toxigenic potentials of the tested isolates revealed that they varied in their abilities to produce total aflatoxins. Thus, although some isolates were toxigenic, others exhibited no detectable toxin production (Table 3). Seven out of 18 isolates showed aflatoxigenic potential, represented 38.88% of isolates collected. All the seven isolates showed aflatoxin B1 production; six isolates out of seven positive isolates showed aflatoxin B2 production, and only one isolate produced G1 and G2 (Table 3). The difference in production of diverse types of aflatoxins might be due to an inhibition by one (or all) of the parameters which alternatively were favorable for the production of aflatoxins. Contrary to those results, Mahjoub et al. (1989) reported that molds isolated from fresh and cold-stored Tunisian date variety Deglet Nour did not produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin, penicillic acid, patulin, or citrinin. Toxigenic A. flavus and occasionally A. parasiticus were isolated from the surface of 12 Emirati date fruits at three different stages of maturation (Ahmed and Robinson, 1997). Abdel-Sater and Saber (1999) reported that aflatoxin B1 was found in two samples out of 20 tested samples of dry date in Egypt. Using TLC analysis of 40 different date samples, Ragab et al. (2001) revealed that two out of five samples of pitted date fruits stuffed with peanut were contaminated by aflatoxin B1. Alghalibi and Shater (2004) reported that two samples of dates in Yemen out of 20 samples were naturally contaminated with aflatoxin B1. All varieties of date fruits in Saudi Arabia tested by Aba Alkhail et al. (2004) were able to support the production of different forms of aflatoxins (B1, B2, G1, and G2), and the content of Aflatoxin G1 produced by either A. flavus or A. parasiticus were higher than the other forms.

Table 3.

Toxicity Potentials, Total Aflatoxins, and Aflatoxigenic Genes Detected in Aspergillus flavus Isolates Collected from Date Fruit Samples

		Total aflatoxins				Aflatoxin genes specific primers
Strains code	Toxicity test ^a	B1	B2	G1	G2	aflr-1 aflr-2	omt-1 omt-2	ver-1 ver-2	nor-1 nor-2
TUAflavus1	T	+	+	−	−	+	+	+	+
TUAflavus2	N.T	−	−	−	−	−	−	−	+
TUAflavus3	N.T	−	−	−	−	−	−	−	−
TUAflavus4	T	+	+	+	+	+	+	+	+
TUAflavus5	T	+	+	−	−	+	+	+	+
TUAflavus6	N.T	−	−	−	−	−	−	−	−
TUAflavus7	N.T	−	−	−	−	−	−	−	−
TUAflavus8	N.T	−	−	−	−	+	−	−	−
TUAflavus9	N.T	−	−	−	−	−	−	−	−
TUAflavus10	T	+	+	−	−	+	+	+	+
TUAflavus11	T	+	+	−	−	+	+	+	+
TUAflavus12	T	+	+	−	−	+	+	+	+
TUAflavus13	N.T	−	−	−	−	−	−	−	−
TUAflavus14	T	+	−	−	−	+	+	+	+
TUAflavus15	N.T	−	−	−	−	−	−	−	−
TUAflavus16	N.T	−	−	−	−	−	−	−	−
TUAflavus17	N.T	−	−	−	−	−	+	−	−
TUAflavus18	N.T	−	−	−	−	−	−	−	−

T and NT, toxic and nontoxic for more than 50% mortality of larvae tested.

Thirty-six isolates of Aspergillus niger were tested for ochratoxin production (Table 4). The tested isolates showed variable abilities to produce ochratoxin. Only nine isolates (25%) were reported as ochratoxin producers (Table 4). Few previous investigations were interested in detection of ochratoxin producers in date fruits. In this respect, Abdel-Sater and Saber (1999) recorded a detectable amount of OTA in two samples of dates in Assiut, Egypt (360–450 μg/kg). Ferracin et al. (2009) tested 20 isolates from A. niger isolated from date fruits in Brazil for their ochratoxin potential. Their results indicated that only nine out of 20 isolates were ochratoxigenic.

Table 4.

Toxicity Potentials, Ochratoxin A, and Ochratoxingenic Gene (PKS) Detected in Aspergillus niger Isolates Collected from Date Fruit Samples

Strains code	Toxicity test ^a	Ochratoxin A ^b	Specific primers ANPKS1 and ANPKS2
TUAniger1	N.T	ND	−
TUAniger2	T	+	+
TUAniger3	N.T	ND	−
TUAniger4	T	ND	−
TUAniger5	T	+	+
TUAniger6	T	+	+
TUAniger7	N.T	+	+
TUAniger8	N.T	ND	−
TUAniger9	N.T	ND	−
TUAniger10	T	ND	−
TUAniger11	T	+	+
TUAniger12	T	+	+
TUAniger13	N.T	+	+
TUAniger14	T	ND	−
TUAniger15	N.T	+	+
TUAniger16	N.T	ND	−
TUAniger17	N.T	ND	−
TUAniger18	N.T	ND	−
TUAniger19	N.T	ND	−
TUAniger20	N.T	ND	−
TUAniger22	N.T	ND	−
TUAniger23	N.T	ND	−
TUAniger24	N.T	ND	−
TUAniger25	N.T	ND	−
TUAniger26	N.T	ND	−
TUAniger27	T	+	+
TUAniger28	N.T	ND	−
TUAniger29	N.T	ND	−
TUAniger30	N.T	ND	−
TUAniger31	N.T	ND	−
TUAniger32	N.T	ND	−
TUAniger33	N.T	ND	−
TUAniger34	N.T	ND	−
TUAniger35	N.T	ND	−
TUAniger36	N.T	ND	−

T and NT, toxic and nontoxic for more than 50% mortality of larvae tested.

ND, not detectable by the TLC method; +, detectable by the TLC method.

Detection of the polymerase chain reaction (PCR) product on 1% agarose gel: −, no product detected; +, PCR product detected.

PCR assay

The results indicated that all aflatoxingenic strains of A. flavus (7 isolates) contained the four tested genes: norA, ver1, omtA, and avfA (aflR) genes (Fig. 1 and Table 3). These results complement the results of toxicity test and aflatoxin detection using TLC. Such results were in agreement with the findings obtained by Rashid et al. (2008), who stated that aflatoxigenic isolates from A. flavus and A. parasiticus had all four norA, ver1, omtA, and avfA (aflR) genes.

FIG. 1.

Amplification products obtained by quadruplex polymerase chain reaction (PCR) with the extracted DNA from the Aspergillus flavus isolates. Lane 1, DNA marker. Lanes 2–8 (TUAflavus1, TUAflavus4, TUAflavus5, TUAflavus10, TUAflavus11, TUAflavus12, and TUAflavus14) are toxigenic isolates. Lanes 9–11 (TUAflavus2, TUAflavus8, and TUAflavus17) are atoxigenic isolates. Lane 12, negative control.

Some of the atoxigenic isolates showed the presence of only one gene. For example, the three atoxigenic isolates TUAflavus2, TUAflavus8, and TUAflavus17 had norA, avfA, and omtA, respectively (Table 3). The aflatoxinogenic strains had a quadruplet pattern, indicating the presence of the four genes in the aflatoxin biosynthetic pathway, whereas nonaflatoxinogenic strains presented different patterns (Criseo et al., 2001). On the other hand, Yang et al. (2004) reported different results by multiplex-PCR and enzyme-linked immunosorbent assay (ELISA). They stated that, although norA, ver1, omtA, and avfA (aflR) genes had been detected in all tested strains, some of these samples were negative for aflatoxin detection. Gashgar et al. (2010) reported the presence of aflR1 gene in seven isolates of A. flavus isolated from wheat flour in Saudi Arabia.

Using the primer pairs of Castellá and Cabañes (2011), the ochratoxigenic isolates of A. niger isolated from date fruit samples were easily differentiated from nonochratoxigenic isolates (Table 4). The ochratoxigenic isolates produced a 951-bp DNA fragment with those primers, while no amplification was reported with nonochratoxigenic isolates (Fig. 2). Castellá and Cabañes (2011), using various PCR reactions, demonstrated that the identified fragment was very specific for OTA-producing strains of the A. niger aggregate. Also, the nonochratoxigenic strains of the A. niger aggregate gave negative results. Moreover, no PCR products were obtained with other ochratoxigenic species of Aspergillus section Nigri, such as A. carbonarius or A. sclerotioniger. All other fungi tested, even ochratoxin A–producing species from Aspergillus and Penicillium, proved to be negative.

FIG. 2.

Specific detection of ochratoxigenic Aspergillus niger isolates using the primer pair ANPKS1and ANPKS2. Lane 1, DNA marker. Lanes 2–10, ochratoxigenic isolates (TUAniger2, TUAniger4, TUAniger5, TUAniger6, TUAniger10, TUAniger11, TUAniger12, TUAniger14, and TUAniger27). Lane 11, atoxigenic isolates (TUAniger1). Lane 12, negative control. Lane 13, positive control (KAUAnig2).

Footnotes

Acknowledgments

This work was supported by a grant (Contract No. 1024-432-1) from Taif University, Kingdom of Saudi Arabia.

Disclosure Statement

No competing financial interests exist.

References

Aba Alkhail

ARA

, Ibrahim

, Al-Rehiayani

et al. Susceptibility of some varieties of date fruits to support the production of aflatoxins: Analysis by high performance liquid chromatography. Pak J Biol Sci, 2004; 7:1937–1941.

Abdel-Sater

, Saber

. Mycoflora and mycotoxins of some Egyptian dried fruits. Bull Fac Sci Assiut Univ, 1999; 28:91–107.

Abu-Zinada

, Ali

. Fungi associated with dates in Saudi Arabia. J Food Prot, 1982; 45:842–844.

Ahmed

, Robinson

. Incidence of Aspergillus flavus and Aspergillus parasiticus on date fruits. Agric Eng Int, 1997; 49:136–139.

Alghalibi

SMS

, Shater

ARM

. Mycoflora and mycotoxin contamination of some dried fruits in Yemen republic. Assiut Univ Bull Environ Res, 2004; 7:19–27.

Al-Sheikh

. Date-Palm fruit spoilage and seed-borne fungi of Saudi Arabia. Res J Microbiol, 2009; 4:208–213.

Biji

, Dive

, Van Pelegheim

. Comparison of some bioassay methods for mycotoxin studies. Pollut A Ecol Biol, 1981; 26:173–182.

Bluhm

, Flaherty

, Cousin

et al. Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal. J Food Prot, 2002; 65:1955–1961.

Bokhary

. Seed-borne fungi of date-palm, Phoenix dactylifera L. from Saudi Arabia. Saudi J Biol Sci, 2010; 17:327–329.

10.

Castellá

, Cabañes

. Development of a real time PCR system for detection of ochratoxin A–producing strains of the Aspergillus niger aggregate. Food Cont, 2011; 8:1367–1372.

11.

Chang

, Ehrlich

, Bhatnagar

et al. Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section flavi. Appl Environ Microbiol, 1995; 61:40–43.

12.

Chang

, Yu

, Bhatnagar

et al. The carboxy-terminal portion of the afatoxin pathway regulatory protein AFLR of Aspergillus parasiticus activates GAL1:lacZ gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol, 1999; 65:2508–2512.

13.

Criseo

, Bagnara

, Bisignano

. Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group. Lett Appl Microbiol, 2001; 33:291–295.

14.

El-Kady

, Moubasher

. Toxigenicity and toxins of Stachybotrys chartarum isolated from wheat strain samples in Egypt. Exp Mycol, 1982; 6:25–30.

15.

Farber

, Geisen

, Holzapfel

. Detection of aflatoxinogenic fungi in figs by a PCR reaction. Int J Food Microbiol, 1997; 36:215–220.

16.

Ferracin

, Frisvad

, Taniwaki

et al. Genetic relationships among strains of the Aspergillus niger aggregate. Braz Arch Biol Technol, 2009; 52:241–248.

17.

Gashgari

, Shebany

, Gherbawy

. Molecular characterization of mycobiota and aflatoxin contamination of retail wheat flours from Jeddah markets. Foodborne Pathog Dis, 2010; 7:1047–1054.

18.

Gashgari

, Shebany

, Gherbawy

. Molecular characterization of ochratoxigenic fungi associated with raisins. Foodborne Pathog Dis, 2011; 11:1221–1227.

19.

Geisen

. Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. Syst Appl Microbiol, 1996; 19:388–392.

20.

Gherbawy

YAMH

. Use of RAPD-PCR to characterize Eurotium strains isolated from date fruits. Cytologia, 2001; 66:349–356.

21.

Heenan

, Shaw

, Pitt

. Ochratoxin A production by Aspergillus carbonarius and A. niger isolates and detection using coconut cream agar. J Food Mycol, 1998; 1:67–72.

22.

Iamanaka

, Taniwaki

, Menezes

et al. Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil. Food Addit Contam, 2005; 22:1258–1263.

23.

Kader

, Hussein

. Harvesting and Postharvest Handling of Dates. Aleppo, Syria: ICARDA, 2009.

24.

Johnson

, Curl

. Methods for Research on Ecology of Soil-Borne Pathogens. Minneapolis, MN: Burgess Publishing Co., 1972.

25.

Klich

, Pitt

. A Laboratory Guide to Common Aspergillus Species and Their Teleomorphs. North Ryde, Australia: CSIRO Division of Food Research, 1988.

26.

Mahjoub

, Glenza

, Bullerman

. Microbiology of dates. Proceedings of the Second Symposium on the Date Palm in Saudi Arabia

Makki

Riyadh, Saudi Arabia. Mars Publishing House, 1989; 165–170.

27.

Nassar

MSM

. Mycoflora associated with dates in Aswan area [M.S. thesis] Assiut, Egypt: Botany Department, Faculty of Science, Assiut University, 1986.

28.

Nesheim

. The ochratoxins and other related compounds. Adv Chem Ser, 1976; 149:276–295.

29.

Patino

, Gonzalez-Salgado

, Gonzalez-Jaen

et al. PCR detection assays for the ochratoxin. Int J Food Microbiol, 2005; 104:207–214.

30.

Payne

, Nystrum

, Bhatnagar

et al. Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl Environ Microbiol, 1993; 59:156–162.

31.

Petzinger

, Weidenbach

. Mycotoxins in the food chain: The role of ochratoxins. Livestock Sci, 2002; 76:245–250.

32.

Pitt

, Hocking

. Fungi and Food Spoilage, 2 ^nd. New York: Springer, 1997.

33.

Ragab

WSM

, Ramadan

, Abdel-Sater

. Mycoflora and aflatoxins associated with saidy date as affected by technological processes. Presented at the 2nd International Conference on Date Palms, Al-Ain, United Arab Emirates, 2001.

34.

Rahimi

, Sharifnabi

, Bahar

. Detection of aflatoxin in Aspergillus species isolated from pistachio in Iran. J Phytopath, 2007; 156:15–20.

35.

Rashid

, Khalil

, Ayub

et al. Categorization of Aspergillus flavus and Aspergillus parasiticus isolates of stored wheat grains in to aflatoxinogenics and non-aflatoxinogenics. Pak J Bot, 2008; 40:2177–2192.

36.

Roehuck

, Maxuitenko

. Biochemical mechanisms and biological implication of the toxicity of aflatoxins as related to aflatoxin carcinogenesis. The Toxicology of Aflatoxins: Human Health, Veternary and Agricultural Significance. Eaton

, Groopman

. San Diego, CA: Academic Press, 1994; 27–41.

37.

Salik

, Rosen

, Kopelman

. Microbial aspect and the deterioration process of soft dates. Lebensm Wiss Technol, 1999; 12:85–87.

38.

Sartori

, Taniwaki

, Iamanaka

, Fungaro

MHP

. Molecular diagnosis of ochratoxigenic fungi. Molecular Identification of Fungi. Gherbawy

, Voigt

. New York: Springer Verlag, 2010; 195–212.

39.

Saudi Ministry of Agriculture and Water. Statistical Predictions About Agriculture in Al-Qassem Region, 5. Al-Qassen, Saudi Arabia: Department of Statistical and Economic Research Agriculture Administration, 2003.

40.

Schmidt

, Ehrmann

, Vogel

et al. Molecular typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP. Syst Appl Microbiol, 2003; 26:138–146.

41.

Selma

, Martínez-Culebras

, Aznar

. Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes. Int J Food Microbiol, 2008; 122:126–134.

42.

Shapira

, Pasti

, Fyai

et al. Detection of aflatoxigenic moulds in grains by PCR. Appl Environ Microbiol, 1996; 62:3270–3273.

43.

Shenasi

, Aidoo

, Candlish

AAG

. Microflora of date fruits and production of aflatoxins at various stages of maturation. Int J Food Microb, 2002; 79:113–119.

44.

Trail

, Muhami

, Mehigh

et al. Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in the aflatoxin pathway. Appl Environ Microbiol, 1995; 61:2665–2673.

45.

Varga

, Kevei

, Rinyu

et al. Ochratoxin production by Aspergillus species. Appl Environ Microbiol, 1996; 62:4461–4464.

46.

Woloshuk

, Foutz

, Brewer

et al. Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis. Appl Environ Microbiol, 1994; 60:2408–2414.

47.

Yang

, Shim

, Wonbo

et al. Detection of aflatoxin producing moulds in Korean fermented foods and grains by multiplex PCR. J Food Prot, 2004; 67:2622–2626.

48.

, Chang

, Cary

et al. Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus. Appl Environ Microbiol, 1995; 61:2365–2371.

49.

, Chang

, Ehrlich

et al. Clustered pathway genes in aflatoxin biosynthesis. Appl Environ Microbiol, 2004; 70:1253–1262.