Background: Materials and methods: In order to identify Aspergillus

Background: Aspergillus species not only cause damage to the color
of pistachio fruit but also reduce the value of this product since they produce
toxic secondary metabolites such as aflatoxins, ochratoxins, and fumonisin.

Materials and methods: In order to identify Aspergillus species associated with
pistachio, 300 samples of fresh fruits, dried fruits, leaves, and fresh old
wastages were collected from the main pistachio orchards of Iran (in Kerman,
Yazd, Semnan, and Ghazvin provinces). Fungal isolation was performed using single
conidial: 220 isolates were identified according
to morphological charachteristics. 22
species belonged to four subgenus and eight sections
were identified according to morphological characteristics. The OTA-producing
ability of the Aspergillus section Circumdati isolates was
determined by the thin layer chromatography (TLC) method.

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Results: The results showed that Aspegillus tubingensis
was the most prevalent species in pistachio orchards. Most isolates of A.
ochraceous and A. melleus are OTA producer in vitro, but
isolates of A. nakazawae and A. muricatus were not able to
produce OTA.

Conclusion: higher incidence of A. tubingensis as a
not-dangerous mycotoxins producer species can ecologically lead to competition
between this species and mycotoxigenic aspergillii and cause a decrease in
mycotoxins contamination of pistachio nut.


Keywords: Aspergillus, Pistachio orchards, Iran, Mycotoxins





Pistachio nuts (Pistacia
vera, Anacardiaceae) have long been
cherished as the symbol of health since ancient times. The kernels of pistachio
are enriched with many health-benefiting nutrients essential for optimum
health. Pistachio kernels are a rich source of many antioxidant phytochemical substances,
such as carotenes, vitamin-E, and polyphenolic antioxidant compounds. They are
the storehouse of minerals such as copper, manganese, potassium, calcium, iron,
magnesium, zinc, and selenium. Regular consumption of pistachio in the diet may
help lower total as well as bad LDL-cholesterol and increase good
HDL-cholesterol levels in the blood (1). Pistachio is
the only edible crop of 11 species in the genus Pistacia. The pistachio
is native to western Asia, where still found growing wildly in Turkey, Syria,
Iran, Iraq, India, Lebanon, Palestine, southern Europe, Asia, and Africa (2). Iran is the main producer of pistachio in
the world.  Most of this product is
exported to other countries. The European Union (EU) has traditionally been one
of the major destinations for Iran’s pistachio. Contamination of agricultural
products with mycotoxins including a?atoxins, ochratoxin, and fumonisin is one
of the major challenges encountered by producers (3). Species
of the Aspergillus are responsible for various plant and food secondary
rot, with the consequence of possible accumulation of mycotoxins (a secondary
metabolite that has powerful teratogenic, mutagenic and hepatocarcinogenic
effects (4). They can contaminate agricultural products at different stages,
including pre-harvest, harvest, processing, and handling. Changes due to
spoilage by Aspergillus species can be of
sensorial, nutritional, and qualitative nature, including pigmentation,
discoloration, rotting, development of off-odors, and off-flavors (4). This fungus is common all over the world as an
air and soil mycoflora found in live and dead animal and plant organisms. It is
particularly interested in colonizing nut kernels and oily cereals. Peanut,
corn, wheat, rice, pistachio, and almond are the major products infected by
this fungus. Iran has about 316,000 hectares of pistachio orchards and produces
about %57 of the world pistachio. More than %60 of the world pistachio export
is done from Iran to other countries, well showing the economic significance of
this product for the country. Iran is also recognized as the biggest and most
important producer and exporter of pistachio in the world, among other
pistachio producing countries (5). Contamination of pistachio nut by Aspergillus
species and their mycotoxins are the most serious challenge for pistachio
production, consumption, and exportation in the world. Factors influencing
infection of pistachio nuts to mycotoxin include cracking of pistachio nuts
(especially early hull splitting pistachios), environmental
factors, cultural practices, frequency and time of irrigation, animal
manures, and harvesting date (6). Aspergillus species not only cause physical damages to
pistachio nuts but also reduce the quality of pistachio since they produce
toxic secondary metabolites, such as aflatoxins, ochratoxins, and fumonisin
(6). Ochratoxin A
(OTA) was discovered as a secondary metabolite of a strain of Aspergillus
ochraceus. OTA exhibits intestinal fragility, nephrotoxicity,
immunosuppression, teratogenicity, carcinogenicity, and cytotoxicity in hepatic
cell lines (7). The OTA inhibits carboxypeptidase A, renal
phosphoenolpyruvate carboxykinase, phenylalanine tRNA synthetase, and
phenylalanine hydroxylase activity. Formation of free radicals has been
considered as one of the mechanisms for the carcinogenic/toxic effects of OTA (8). Fungi from two genera are known to produce
ochratoxins. In the genus Penicillium, OTA is produced by P.
verrucosum and P. nordicum, and in the genus Aspergillus by A.
ochraceus, A. melleus, A. auricomus, A. ostianus, A. petrakii, A. sclerotiorum,
and A. sulphureus, all in section Circumdati (formerly the A. ochraceus group). Aspergillus
alliaceus and Aspergillus albertensis, formerly placed in
section Circumdati but recently shown to be more closely related to
section Flavi, have also been described as OTA producers (9). Some members of Aspergillus section Nigri
(formerly the Aspergillus niger group) such as Aspergillus
niger var. niger and Aspergillus carbonarius have been
reported as ochratoxigenic fungi (7). Several food products worldwide face the
risk of OTA contamination, including coffee, cereal grains, processed foods,
beer, grapes, wine, cocoa, nuts, and dried fruits (10). A survey on contaminant fungal flora of some
nuts showed that 49% of market pistachio have fungal contamination, and Penucillium,
Mucor and Aspergillus with 26, 24, and 20% were found to be the
most dominant spoilage agents, respectively (11). This study was conducted to evaluate the
incidence and prevalence of Aspergillus species in the main pistachio
orchards of Iran and OTA production potential of Section circumdati
isolates in vitro.


Material and


300 Samples of fresh and dried pistachio,
pistachio fresh leaf (from various parts of the tree), fresh and old hull, and
hard skin were collected from pistachio orchards in Kerman, Yazd, Semnan and
Ghazvin provinces as the main pistachio production provinces. The number of samples was determined based on the cultivation area
of each province. All samples were brought to the laboratory for
further studies. Sampling was
conducted during August to November 2007. Samples were placed separately on sterile
filter papers and kept in a dark incubator at 25°C. After two to three weeks,
single conidial heads were chosen according to their phenotype and subculture
repeatedly on Malt Extract Agar (Sigma-Aldrich, Germany) media until complete
purification. Morphological identifications were carried out according to
following references (12-14).



15 isolates from Section Circumdati, of
which 10 isolates of A. ochraceus, two isolates of A. melleus,
three isolates of A. nakazaweae, and one isolate of A. muricatus, were
analyzed for OTA production. To assay ochratoxin production, isolates of
section Circumdati (most probable species for Ochratoxin production)
were grown in 100 ml flasks containing 50 ml (YES; yeast extract, sucrose)
liquid medium. Cultures were incubated without agitation for 14 days at 30°C in
the dark place. Ochratoxin A was extracted by adding 5 ml of chloroform.
OTA-producing ability of the Aspergillus isolates was determined by the Thin
Layer Chromatography (TLC) method. (15-19).



220 isolates were recovered from the pistachio and
related samples. Twenty two
species belonged to four subgenus and eight Sections were identified according
to morphological charachteristics.
The most common subgenus were Aspergillus subgenus Circumdati (74%)
(Fig. 1). Eight Sections of all the 22 sections of Aspergillus were
included in this study. These Sections are Flavi (27%),
Nigri (25%), Circumdati (18%), Aspergillus (12%),
Nidulantes (12%), Cremei (3%), Terrei (2%), and Fumigati (1%),
respectively (Fig. 1).

Altogether 22
species were identified in recovered isolates. They included A. alliaceus, A. flavus, A. parasiticu,s, A.
tamarii (Section Flavi),
A. japonicas, and A. tubingensis (Section
Nigri), A. ochraceus, A. nakazawae, A.
melleus, and Neopeteromyces muricatus, (section
Circumdati), A. dimorphicus and 
Chaetosartorya chrysella (section Cremei), A. terreus (section Terrei),
A. fumigatus (section Fumigati), A. sydowii, A. versicolor? Aspergillus sp. Emericella
quadrilineata, (section Nidulantes), E. repens, E. rubrum, E. chevalieri, and E. amstelodami (section Aspergillus). Of the identified species, A. tubingensis (24%), A.
flavus (21%), and A. ochraceous (12%) were the most prevalent
species, respectively (Fig. 2).

tubingensis and A.
flavus were isolated from all studied aeries, Aspergillus sp. A.
terreus, A. fumigatus, and A. alliaceus were isolated just
from Kerman province. A. dimorphicus and A. versicolor were
isolated only from Yazd province, and A. muricatus just from Semnan
province (Fig. 3).

Some species such as A. niger and A.
carbonarious (Section Nigri) have OTA production capability.
Fortunately, none of these species were isolated from pistachio orchards.
Therefore black aspergilli isolates were not analyzed for OTA production.
The results showed that 9 of the 10 isolates of A. ochraceus and all
isolates of A. melleus were capable of producing OTA. A. nakazaweae
and A. muricatus isolates are not OTA producers (Fig. 4). Severity of
OTA production is different among A. ochraceous
isolates. Qualitative data of TLC plate showed that most A. ochraceous
isolates have a high
potential for OTA production.



Our study shows that A. tubingensis
(formerly identified as A. niger) and A. flavus were the most
prevalent species in all pistachio production aeries of Iran. Many other
studies have shown that A. flavus and A. niger var. niger (20) are
the most prevalent species. Studies on the incidence of Aspergillus
in fig (Ficus carica) showed that, based on morphological
characteristics, A. niger var. niger is the most common Aspergillus
species (39.6%) in dried fig and fig orchards (21).  A.
niger and A. tubingensis are the most similar morphologic species,
and  it is often impossible to
distinguish them (22). Varga et al. (15) showed that some isolates of A. ochraaceus
are OTA producers and some others are not. Bayman et al. (19) analyzed 41 isolates of A. ochraceus
and 17 isolates of A. melleus recovered from fig, hazelnut, cottonseed,
walnut, and soil for OTA production. Their results showed that, except for one
isolate of A. ochraceus, the others were not OTA producers. Comparing
the results of these two studies is difficult. They concluded that some of
their isolates had the ability for OTA. 175 production at earlier studies but missed
it later. The isolates that were used in our studies were totally fresh.
Studies of the A. ochraceus group are complicated by difficulties in
distinguishing A. ochraceus from related species. It is possible that
the isolates they used were not a real A. ochraceus strain. Iamanaka
et al. (16) showed that, after black aspergili, A.
ochraceus is the most important OTA producer in dried fruits in Brazil. 87%
A. ochraceus were ochratoxigenic. El-Shayeb et al. (23) investigated that A. ochraceus and A.
alliaceus strains have the maximum quantities of ochratoxins produced on
yeast extract-sucrose (YES) medium.

Trace production of ochratoxin A in A.
melleus, A. ostianus, A. persii and A. petrakii was reported by
Frisvad et al. (24). They also concluded
that A. muricatus is ochratoxigenic, which is different from the results
we obtained. We analyzed just one isolate and it is not enough to make a
decision about their ability for OTA production.



Higher Prevalence of A. tubingensis than
A. flavus, in pistachio production area can be a hopeful finding. Since
the competition of these fungi for colonizing niches and substrate can lead to
suppression of A. flavus, the most common aflatoxins producer species in
many agricultural products is OTA production assay just performed In vitro,
and it should not be determined as contamination of pistachio to this
mycotoxin. So far, there has not been any studies on the contamination of Iranian
pistachio to OTA.