1

Molecular Characterization of Ochratoxigenic Fungi

Associated with Raisins

Rukaia M. Gashgari1 , Yassmin M. Shebany

2 and Youssuf A.

Gherbawy*2,3

1 Science College, Girls Branch, King Abdulaziz University, Jeddah ,

Saudi Arabia

2Botany Department, Faculty of Science, South Valley University, Qena,

Egypt

3 Biological Sciences Department, Faculty of Science, Taif University, Taif,

Saudi Arabia

2

Abstract

Dried grapes (raisin) may carry a significant mycological load

acquired via cultivation, post-harvest processing practices and drying

processing. The contamination of raisin with fungi can cause accelerated

spoilage or illness, if pathogens are present. Since raisins are used as food

additives in many dishes in Saudi kitchen, there are healthy concerns on the

safety of raisins consumed. In this paper, the mycological profile of raisins

sold in different markets at Jeddah (KSA) was studied. The black raisin

samples showed higher fungal load in comparison with the white samples.

Aspergillus, Alternaria, Cladosporium, Epicoccum Fusarium and Rhizopus

were the most prevalent genera isolated from raisin samples. Among 6

Aspergillus species isolated in this study, A. carbonarius and A. niger were

the most frequently isolated species. Ochartoxins production in raisins was

also investigated. Ochratoxin A was detected in 70% of the raisin samples in

this study. Also, A. carbonarius (14 out of 19 isolates) and A. niger (2

isolates out of 9) were potential producers for OTA. This work applies a

combination of chromatographical (UUUUU) and molecular (yyyy)

techniques for detecting of ochratoxin A contaminating raisin. The study

showed the prevalence of ochratoxin A in different raisin samples. Also,

some molecular markers for detecting the contamination of raisin samples

with ochratoxin A directly without isolating the producers were tested.

Keywords: Raisins, TLC, RAPD-PCR, Multiplex PCR, Saudi Arabia.

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Introduction

Mycobiota and ochratoxin of dried grapes

Dried vine fruits (raisins, sultanas, currants) are regarded as ‘healthy

foods’ and are also ingredients in muesli, biscuits, cakes and other foods.

Fructose and glucose concentrations in grapes can be increased even more

by sun drying, and pressed when sugar levels reaches 300 g l-1

.

Contamination by different moulds can occur during preharvest, harvest

and grape processing. Botrytis, Alternaria, Cladosporium, Aspergillus,

Eurotium and Rhizopus are regarded as the main natural contaminants in

this sort of foodstuffs (Sage et al. 2002, Magnoli et al. 2003). Mycological

studies have reported black aspergilli as the predominant fungi in raisins

(ElHalouat and Debevere 1997, Abarca et al., 2003, Magnoli et al. 2004),

and within them, A. carbonarius and A. niger have been recorded of

vineyards in a study carried out in Australia (Leong et al. 2004). Black

aspergilli are the most common fungi responsible for post-harvest decay of

fresh fruits and are found on the surface of healthy grapes at all stages of

development (Pitt and Hocking 1997, Zahavi et al. 2000) , and considered

as opportunistic pathogens of grape and may cause bunch rot (sour rot) or

berry rots, and raisin mold rot (Varga et al. 2004). This disease is

particularly severe in warmer grape-growing areas including southern parts

of Spain, Portugal, France, Italy, Greece, Morocco and Egypt (Logrieco et

al. 2003).

Ochratoxin A (OTA), a kind of toxin produced by Aspergillus

ochraceus and Penicillium verrucosum, is one of the most abundant food-

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contaminating mycotoxins in the world which is potentially carcinogenic to

humans. Many fungal species isolated from grapes, e.g., A. carbonarius, A.

fumigatus, A. niger, A. ochraceus, P. verrucosum, and P. pinophilum, are

capable of producing OTA in grapes. However, among the known

producers of OTA in grapes, A. carbonarius has been proved to be the main

one, therefore, this fungus is considered as the main source of OTA in

wines and vine fruits, especially in regions with hot and dry climates (Pitt

2000, Battilani and Pietri 2002, Da et al. 2002, Abarca et al. 2003, Magnoli

et al. 2004, Serra et al. 2003, 2005, 2006, Sage et al. 2004, Bejaoui et al.

2006, Bellı´ et al. 2006, Gomez et al. 2006, Guzev et al. 2006, Tjamos et

al. 2006, Leong et al. 2007). 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 of isolates having the ability to produce OTA in culture

(Heenan et al. 1998). It has been proposed that A. carbonarius would be the

main source of OTA production in grapes and derivatives (Pitt 2000)

particularly in Mediterranean region (Serra et al. 2003), while A. ochraceus

would be the main source of OTA in coffee (Logrieco et al. 2003, Taniwaki

et al. 2003). Due to the ability of A. carbonarius and A. niger to produce

OTA at a wide range of temperatures, OTA can be continuously produced

in the field. This fact has to be taken into account in commodities such as

grapes, raisins and wine, where A. carbonarius and members of the A. niger

aggregate are considered to be the main sources of the OTA contamination.

5

Ochratoxin A contamination of dried vine fruits was also found to be due to

the action of black aspergilli in Europe including Spain (Abarca et al.

2003), the Czech Republic (Ostry et al. 2002a), Hungary (Varga et al.

2006) and in other parts of the world including Argentina (Magnoli et al.

2004, Romero et al. 2005) and Australia (Leong et al. 2004).

Processing of grapes into dried fruit products has been shown to

have a substantial effect on OTA levels in the final consumer products.

Fruits to be used for drying must be of high quality and the process of

drying must start immediately after harvest (Magan and Aldred 2005).

Fruits are generally sun-dried for 7-14 days and turned regularly to control

moisture loss. During the process, sugars in the fruit become more

concentrated resulting in a selective medium for black aspergilli. The

occurrence of rain during the drying process increases the risk of OTA

contamination since drying becomes uneven. Consequently, the drying

process should be done as rapidly as possible and the product packaged and

stored only after proper moisture levels have been reached (Drusch and

Ragab 2003).

Molecular detection of ochratoxin

The traditional schemes for the isolation and identification of

ochratoxigenic fungi from food samples are time-consuming and require a

high knowledge of fungal taxonomy. Even with taxonomic expertise,

identification is commonly difficult in some genera of fungi that contain a

large number of closely related species. Aspergillus Section Nigri includes

several species difficult to be identified with traditional methods because

differences are mainly based on the uniseriate and biseriate condition of the

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sterigmata of conidial heads, and on the size and roughness of the conidia

(Battilani et al., 2008) . Hence, it is imperative to develop methodologies

that are relatively rapid, highly specific and as an alternative to the existing

methods. The application of molecular biology techniques can help to

overcome these problems because it can reduce the time for identification

from days to hours and also allow precise species identification (Sartori et

al. 2010). PCR-based methods that target DNA are considered a good

alternative for rapid diagnosis because of their high specificity and

sensitivity (Accensi et al. 1999, Perrone et al. 2004), especially when

multi-copy sequences are used to develop species specific primers (Bluhm

et al. 2002). Patino et al. (2005) developed two PCR assays to detect

Aspergillus carbonarius and Aspergillus ochraceus, considered the main

sources of ochratoxin A (OTA) contaminating commodities, particularly

grapes, coffee and derivatives, in warm climates. Random amplified

polymorphic DNA (RAPD) or amplified fragment length polymorphism

(AFLP) have been applied successfully for revealing specific marker

sequences, such sequences have been used to design species-specific

primers that allow the identification and detection of some ochratoxigenic

species in food samples (Schmidt et al.2003, 2004, Fungaro et al. 2004,

Sartori et al., 2006).

Saudi Arabia is one of the most important raisin markets in the

world with an annual consumption of many tones. However, the

mycological studies inspecting the fungal contaminants and safety of the

related products are limited. Therefore, the aims of this work were : To

determine the occurrence and load of fungi, the important food borne

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pathogens in particular, in raisins offered for sale to consumers in the retail

stores at Jeddah region in Saudi Arabia; to analyze specifically the

occurrence of Black aspergilli group in these samples; to investigate the

occurrence of ochratoxin A using chromatographically and molecularly

techniques; and to testing more reliable methods for detecting ochratoxin A

in the samples of raisin directly.

Materials and methods

Mycobiota determination

Fifty raisin samples of different origin were collected from different

markets in Jeddah City. The samples were surface sterilized, and placed on

malt extract and dichloran-rose bengal medium (King et al. 1979). The plates

were incubated at 25 °C for 7 days, and fungal colonies growing on these

plates were purified and identified by classical taxonomic methods. From

media plates, only colonies belonging to black Aspergilli were transferred

to slants to ensure precise counting and for identification to species level

(Klich and Pitt 1988). These isolates were preserved at –80ºC for later

studies.

Determination of ochratoxin production (TLC)

For the determination of ochratoxin production by black aspergilla

strains, method of Davis et al. (1969) was used.

Detection of natural occurrence of ochratoxin A in raisin samples

Raisin samples which show high contamination with black aspergilla

went for ochratoxin A detection . Each sample mixed, homogenized and

placed in glass bottle and stored at 2–8°C until further analysis. For the

qualitative analysis of ochratoxin TLC (thin layer chromatography)

8

technique used. Ochratoxin A detected in the sample extracts after thin-

layer chromatography (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 R r (0.55) as authentic ochratoxin A. The fluorescence turned blue on

treatment with ammonia (Shotwell et al. 1969).

DNA isolation from fungal isolates

The isolation of DNA from mycelia of A. carbonarius and A. niger

performed according to the method described by Accensi et al. (2001).

DNA extraction from fungal-contaminated raisins

The contaminated raisins were immersed in a 0.4% hypochlorite

solution for 2 min. Then they were rinsed with sterilized distilled water for 1

min. This washing procedure was repeated three times. To extract the DNA,

a CTAB protocol (Doyle and Doyle 1987) was used. The raisins were first

freeze dried and ground using a blender to a fine powder macerated with the

addition of liquid nitrogen and protocol of Sartori et al. (2006) was used.

Multiplex PCR reaction using artificially contaminated raisins

The total DNA obtained from one raisins was serially diluted 101,

102, and 10

3, in distilled water and used as a template DNA in the multiplex

PCR reaction. The PCR reaction mixture contained 3.0 μl DNA of each

template, 0.5 μl MgCl2 (50 mM), 2.5 μl Taq DNA polymerase buffer, 1.0x,

1.0 μl primer (10 pmol/μl each), 10.9 μl H2O, 0.1 μl Taq polymerase (5

U/μl), and 2.0 μl dNTP (2.5 mM). Details of cycling conditions and primers

were shown in the work of Sartori et al. (2006).

Multiplex PCR assays in naturally contaminated raisins

9

Raisins obtained from markets were immersed in a 0.4% hypochlorite

solution for 2 min and rinsed with sterilized distilled water for 1 min. The

samples were put onto soaked filter paper disks inside Petri dishes and

incubated at 28°C for 24 h. DNA extraction was performed as described

above.

Genetic diversity of A. carbonarius isolates

For molecular analysis 10-mers were used as random primers in the

RAPD. Amplifications carried out as described by Fungaro et al. (1996).

Computer assisted analysis with the TREECON for Windows v.1.3b

The results went through the computer-assisted analysis and the

produced matrices will be analyzed by TREECON for Windows (version

1.3b, 1998) whereof evolutionary tree – dendrogram - constructed and

produced (Van de Peer and Wachter 1994).

Results and Discussion

Mycobiota and potential OTA producing fungi in raisin samples

Table 1 showd the mean values of total fungal counts and the

Aspergillus spp. counts in MEA and DRBC culture media using direct

plating technique. Mean total fungal counts and Aspergillus spp. counts

obtained with DRBC culture medium were higher than the mean values

obtained with the MEA culture medium. This results may be attributed to

the presence of dichloran in DRBC culture medium, which suppressed

zygomycetes fungi and thus give the chance for other fungi to appear. Varga

et al. (2006) reported that seven samples were heavily contaminated by

zygomycetes fungi which overgrew other filamentous fungi on malt extract

10

agar plates. The dichloran-rose Bengal medium (King et al. 1979) was

successfully used to identify other contaminants even in these samples.

The black fruit samples showed high contamination in comparison with

white fruit samples. Mean values of total fungal and Aspergillus spp.

counts from black and white fruit were 658.5 and 519; and 525.3 and

390.8 CFU/g, respectively (Table 1). This results came in agreement with

results reported by Magnoli et al. (2004) during their work in dried vine

fruit from Argentina.

The results of Table (2) showed predominant mycobiota of raisin

samples, taking into account their occurrences and abundance,

respectively, belonged to Aspergillus spp. (72% and 45.73%), Alternaria

alternata (52% and 11.40%), Cladosporium cladosporioides ( 48% and

25.35%), Epicoccum purpurascens ( 36% and 1.69%), Fusarium

oxysporum (36% and 1.74), Rhizopus stolonifer (36% and 0.87% ) and in a

lesser extent Dreschlera spicifera (26% and 2.12), Penicillium spp. (24%

and 6.52%), Humicola gresia (20% and 0.72%), Trichoderma harzianum

(16% and 0.48%), Ulocladium atrum (12% and 0.92%). Magnoli et al.

(2003) studied mycoflora of 50 samples of wine grape from Argentina.

Their results indicated presence of 7 genera of filamentous fungi and

Alternaria alternate was the dominant species and it was found in 80% of

the samples, while Aspergillus species were isolated from 70% of the

samples. Magnoli et al. (2003) and Sage et al. (2002) indicated that,

Botrytis, Alternaria, Cladosporium, Aspergillus, Eurotium and Rhizopus

are regarded as the main natural contaminants in this sort of foodstuffs.

Alternaria, Aspergillus, Cladosporium and Penicillium species have been

11

reported as the predominant mycobiota in harvested grapes from

Argentina and Brazil (Da et al. 2002, Magnoli et al. 2003), from France

(Sage et al. 2002), and also during ripening of grapes from Spain (Bau et

al. 2005, Bellí et al. 2004). Serra et al. (2005) reported that the most

frequent genera isolated from grapes from Portugal were Cladosporium

(25%), Alternaria (24%), Botrytis (15%), Penicillium (9%) and

Aspergillus (8%). Medina et al. (2005) isolated 8 fungal genera

(Aspergillus, Alternaria, Acremonium, Penicillium, Cladosporium,

Fusarium, Rhizopus, and Phoma ) from the Spanish grape samples. They

reported also, that the most frequently isolated fungi were Alternaria spp.

and Cladosporium spp.

Six species of Aspergillus spp. isolated from MEA and DRBC

were identified and are listed in Table 2. The occurrence and abundance of

each Aspergillus spp. are summarized in the same table. Each Aspergillus

spp. abundance is expressed as the average of percentage counts for each

species in the total of samples. Most frequent isolated species were

Aspergillus carbonarius and A. niger. The occurrences and abundance of

Aspergillus carbonarius and A. niger were (38% and 31.4%) and (18%

and 11.59%), respectively (Table 2). The high rate of appearance of A.

carbonarius in hot and dry regions is attributed to the resistance of the

black Aspergilli to sunlight and ultraviolet light (Benford et al., 2001).

Previous mycological studies have reported black aspergilli as the

predominant fungi with occurrences between 33 and 100% in raisins

(Abarca et al. 2003, ElHalouat and Debevere 1997, Magnoli et al. 2004),

and within them, A. carbonarius and A. niger have been recorded between

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69 and 100% of vineyards in a study carried out in Australia (Leong et al.,

2004). Magnoli et al. (2004) studied 50 samples of dried vine fruit (31

black,19 white) from markets in Mendoza and San Juan Provinces,

Argentina. Their results showed that Aspergillus spp. were predominant.

The frequency of isolation from black and white dried vine fruits samples

were 95% and 41% on DRBC (Dichloran Rose Bengal Chloramphenicol

medium, Oxoid Ltd.) and 75% and 33% on DG18 (Dichloran 18%

Glycerol medium, Oxoid Ltd.), respectively. Also, they reported that the

predominant species isolated were A. niger var. niger (75% and 65%), A.

niger var. awamori (80% and 55%) and A. carbonarious (45% and 15%)

from black dried vine fruits samples in DRBC and DG18, respectively.

These species from white dried vine fruit were isolated in lower

frequency. In a survey of six vineyards in Australia, soil and vine

remnants on soil were the primary sources of A. carbonarius and A. niger

(Leong et al. 2006a,b). These fungi were also isolated occasionally from

fallen dried berries, dead canes, vine bark, dried bunch stems and dead

cover crop trash, but were seldom isolated from leaves (green and/or

senescent), tendrils and green cover crop plants (Hocking et al. 2007). In

contrast, in Argentina, weeds are thought to represent an important

inoculum source for A. niger in vineyards (Chulze et al. 2006). Varga et

al. (2006) studied the mycobiota of raisins purchased in Hungary. Their

results showed that , most raisins were heavily contaminated with black

aspergilli.

The mycotoxin analysis showed that OTA was found in 70% of the

raisin samples used in this study (Table 1). From black and white raisins

13

samples OTA was detected in 42 and 28% of the total samples. In

comparison, previous UK surveys have reported incidences of OTA in

raisins of 85% (MacDonald et al. 1999) and 97% (Maff 1999), while a

German survey (Engel, 2000) reported a 95% overall incidence of OTA in

raisins and currants. Data from Finland and France have indicated

incidences of 71 and 46%, respectively (Miraglia and Brera 2002).

Ochratoxin A was detected in 74% of dried vine fruit samples from

Argentinean, and was detected in 68% and 88% of the black and white

fruit (Magnoli et al. 2004). Varga et al. (2006) used 20 dried vine fruit

collected from Turkey, Greece and Iran , for studying the naturally

occurrence of OTA. Their results showed that Ochratoxin A was detected

in all samples and the highest OTA contamination was measured in a

raisin sample deriving from Iran. Ochratoxin A was present, in 67 (79%)

of 85 samples of raisins from the Canadian retail market (Lombaert et al.,

2004). The percentage of contaminated samples with OTA in our study is

similar to those observed in other European countries including Greece,

France, Finland, Germany and the Czech Republic (Miraglia and Brera

2002, Ostry et al. 2002a,b, Stefanaki et al. 2003).

Aspergillus carbonarius (31.40%), A. niger (11.59) and A.

ochraceus (1.83%) were potential producers for OTA among the

mycobiota isolated in this study. Fourteen isolates of Aspergillus

carbonarius (73.7% of total A. carbonarius isolates) and two isolates of A.

niger (22.2% of A. niger isolates ) were ochratoxingenic isolates (Table

3). Eighty-eight (96.7%) A. carbonarius isolates and one (0.6%) A. niger

var. niger isolate from dried vine fruits from the Spanish market were

14

found to be OTA producers (Abarca et al. 2003). From the A. carbonarius

strains isolated from grapes in Europe and Israel, 96.7% were OTA

producers (Bau et al., 2005). A very small percentage of A. niger

aggregate isolated from grapes and sun-dried grapes samples in Spain

showed being OTA producers (1.4%) whereas OTA production was

detected in 72.6% of A. carbonarius isolates (Valero et al. 2005). Serra et

al. (2005) recorded that 6.0% of isolated fungi from grape in Portugal

were OTA producers. Of 205 Aspergillus section Nigri isolates from

Spanish Grape that were tested for OTA production, 92 (44.9%) produced

this toxin (Medina et al. 2005). Also, they indicated that 89 of the

ochratoxinogenic isolates were classified as A. carbonarius (74.2% of the

120 tested isolates). Chulze et al. (2006) reported that 83% of A.

carbonarius strains, isolated from grapes and dried vine fruits in South

America were OTA producers.

Aspergillus carbonarius molecular characterization and OTA

production

Nineteen strains of A. carbonarius were used in the part to study the

genetic diversity among A. carbonarius population isolated from raisin

samples. RAPD-PCR technique was employed using two different primers

OPX7 and V6 (Figs.1&2). All carbonarius isolates were repeatedly tested.

The polymorphic fragment patterns were reproducible with slight variations

in the intensity and occasionally in the banding pattern. With the used

primers , each isolate yielded 8 to 16 bands. Therefore under each primer all

amplification products were scored for each isolate according to molecular

weight. The band pattern obtained from agarose gel electrophoresis from

15

each primer was digitalized by hand to a two-discretecharacter- matrix (0 and

1 for absence and presence of RAPD bands, respectively) and the data of two

primers were combined. The analysis data was based on the Nei and Li

coefficient (Nei and Li 1979). A dendrogram was constructed by the

unweighed paired group method of arithmetic average (UPGMA) based

Jaccard’s similarity coefficient. The dengrogram constructed from the

combined results of two primers (Fig. 3) showed that the percentage of

similarity between tested isolates fluctuated from 45 to 87%. This results

support that, this population has high genetic diversity. Non ochratoxigenic

isolates (KAUAcar 2, KAUAcar 4, KAUAcar 10, KAUAcar 16 and

KAUAcar 19) were scattered in all clusters of dendrogram. So, the

constructed dendrogram showed no correlation between DNA banding

patterns and the ability of toxin production. Similar results have been

described in other studies with Aspergillus spp., showing no correlation

between DNA band profiles and production or non production of mycotoxins

(Bayman and Cotty 1993, Jovita and Bainbridge 1996, Lourenço et al, 2007,

Gashgari et al. 2010,). Fungaro et al. (2004) compared RAPD patterns of

toxigenic and non-toxigenic strains of A. carbonarius isolated from coffee

beans. Although the PCR-based assay described by the authors was

successfully employed to detect A. carbonarius in coffee samples, no

association was found between the RAPD genotype and the ability to

produce OTA of the strains. Since RAPD-PCR technique amplified random

fragments of the fungal genome, so the fragment that containd gene

regulating toxin production may not be amplified using this techniques with

the used primers as recorded by Gashgari et al. (2010).

16

Using the PCR conditions described in materials and methods section ,

the primer pair designed by Sartori et al. (2006) yielded a product of 372 bp

from all A. niger strains tested. No amplified product was observed in a

reaction using DNA from A. carbonarius, A. ochraceus or A. flavus (Fig.

4A). Using the primer pair OPX7F372 and OPX7R372 (Fungaro et al., 2004)

all A. carbonarius strains tested yielded a product of 809 bp. Also, no

amplified product was observed in a reaction using DNA from A. nige, A.

ochraceus or A. flavus (Fig. 4B). The PCR assays for A. carbonarius and A.

niger identification in pure culture were also successfully applied for

detecting an amplicon of 809 and 372 pb, respectively, when using DNA

from inoculated raisins (Fig.5 ). The multiplex PCR assay for detecting A.

carbonarius and A. niger simultaneously was first analyzed using DNA from

raisin inoculated with these fungal species. Fig. 5 shows the amplification

profiles using, simultaneously, the primer pairs designed for A. carbonarius

(Fungaro et al. 2004) and A. niger (Sartori et al. 2006). Amplification

products of 372 and 809 bp, in a single PCR reaction confirmed the presence

of A. niger and A. carbonarius, respectively. The applicability of the

multiplex PCR assay in quality control of raisins was also analyzed using

naturally contaminated samples. This method was previously used by Sartori

et al. (2006) for detection of potential ochratoxin-producing Aspergillus

species in coffee beans. As shown in Fig. 5 this methodology successfully

allowed the detection of amplification products from naturally occurring

fungi in raisins. The method described in this study represents a much

quicker and more reliable detection procedure for the presence of

ochratoxigenic fungi found in raisins.

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Acknowledgment

This work was supported by a grant (Contract No. 18-011-430) from

King Abdulaziz University, Kingdom of Saudi Arabia. So, authors are very

grateful for the deanship of scientific research at king Abdulaziz for the

financial support.

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Table 1

Mean values of total fungal counts and Aspergillus spp. counts from two

types of raisin samples collected from Jeddah city using the direct plating

technique and occurrence of ochratoxin A.

27

Samples Culture

media

Total fungal

counts

(CFU/g)

Mean value

Aspergillus spp.

counts (CFU/g)

Mean value

Ochratoxin A

(number of

positive samples)

Black

fruits

MEA

DRBC

Mean value

548

769

658.5

423

615

519

21

White

fruits

MEA

DRBC

Mean value

243

541

392

156

369

262.5

14

Total 525.3 390.8

Table 2

28

Occurrence , average total counts and abundance of the predominant species

isolated from raisin samples collected from Jeddah city using two different types

of media collectively.

Species Occurrence

(%)

Average total

counts

Abundance

Alternaria alternate

Aspergillus

A. candidus

A. carbonarius

A . flavus

A. niger

A. ochraceus

A. terreus

Botrytis cinerea

Cladosporium

C. herbarium

C. cladosporioides

Dreschlera spicifera

Epicoccum purpurascens

Eurotium

E. amstelodami

E. chevalieri

Fusarium oxysporum

Humicola gresia

Penicillium

P. chrysogenum

P. decumbens

P. funiculosum

Rhizopus stolonifer

Trichoderma harzianum

Ulocladium atrum

26 ( 52)

36 (72)

1 (2)

19 (38)

3 (6)

9 (18)

2 (4)

3 (6)

5 (10)

24 (48)

6 (12)

20 (40)

13 (26)

18 (36)

9 (18)

6 (12)

2 (4)

18 (36)

10 (20)

12 (24)

5 (10)

6 (12)

2 (4)

18 (36)

8 (16)

6 (12)

236

947

4

651

8

240

38

6

32

525

45

480

44

35

19

15

4

36

15

135

60

65

10

18

10

19

11.40

45.73

0.19

31.4

0.39

11.59

1.83

0.29

1.55

25.35

2.17

23.18

2.12

1.69

0.92

0.72

0.19

1.74

0.72

6.52

2.90

3.14

0.48

0.87

0.48

0.92

29

Table 3

Summary data of ochratoxin produced by the different Aspergillus

carbonarius and A. niger strains recovered from raisin samples.

Aspergillus

carbonarius

strains

OTA*

Aspergillus

niger

strains

OTA

KAUAcar 1

KAUAcar 2

KAUAcar 3

KAUAcar 4

KAUAcar 5

KAUAcar 6

KAUAcar 7

KAUAcar 8

KAUAcar 9

KAUAcar 10

KAUAcar 11

KAUAcar 12

KAUAcar 13

KAUAcar 14

KAUAcar 15

KAUAcar 16

KAUAcar 17

KAUAcar 18

KAUAcar 19

+

-

+

-

+

+

+

+

+

-

+

+

+

+

+

-

+

+

-

KAUAnig 1

KAUAnig 2

KAUAnig 3

KAUAnig 4

KAUAnig 5

KAUAnig 6

KAUAnig 7

KAUAnig 8

KAUAnig 9

-

+

-

-

-

-

-

+

-

*Detection limit was 0.5 μg/kg

30

Fig. 1. DNA banding patterns from random amplified polymorphic DNA

analysis of Aspergillus carbonarius isolates primed by OPX7. Lane M is

a 100 bp DNA ladder .

31

Fig. 2. DNA banding patterns from random amplified polymorphic DNA

analysis of Aspergillus carbonarius isolates primed by V6 . Lane M is a

100 bp DNA ladder .

32

Fig. 3. Dendrogram showing relationships among 19 isolates of

Aspergillus carbonarius isolated from different raisin samples. Genetic

distances were obtained by random amplified polymorphic DNA analysis

using combining results of OPX7 and V6 primers. Non-ochratoxigenic

isolates were marked with stars.

33

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Fig. 4. (A) Specific detection of A. niger using DNA obtained from culture

and the primer pair OPX7F372 and OPX7R372. Lane 1: DNA marker

lanes 2–10: A. niger isolates collected from raisin samples from Jeddah;

lane 11 : A. carbonarius; lane 12: A. ocharaceus; lane 13: negative

control; lane 14: positive control of PCR reaction (using A. niger

TUAniger).

(B) Specific detection of A. carbonarius by using the primer pair

OPX7F372 and OPX7R372. Lane 1: DNA marker; lane 2-20 A.

carbonarius isolates collected from raisin samples from Jeddah; lane 21:

A. niger; lane 22: A ocharaceus; Lane 23: negative control; lane 24:

positive control of PCR reaction (using A. carbonarius TUAcar ).

34

1 2 3 4 5 6 7 8 9 10

Fig. 5. Amplification products obtained by using DNA from inoculated

raisins. Lane1: DNA marker; lane 2: negative control ; lane 3 : DNA from

raisins inoculated with A. niger by using OPX7F372 and OPX7R372

primers; lane 4: DNA from raisins inoculated with A. carbonarius by

using OPX7F809 and OPX7R809 primers; lane 5 : multiplex PCR using

DNA from raisins inoculated with A. carbonarius and A. niger and the 2

sets of primer pairs; lane 6 : multiplex PCR using DNA from type strains

(TUAniger and TUAcar). Multiplex PCR obtained from raisin naturally

contaminated: lane 7 : no fungi detection; lane 8: detection of A.

carbonarius; lane 9 : detection of A. niger ; lane 10 : detection of A.

carbonarius and A. niger .