300 Circumdati (most probable species for Ochratoxin production)

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).

 

1.1.             
Ochratoxin
analysis

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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).

 

2.      
Results

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).

Aspergillus
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.

 

3.      
Discussion

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.

 

4.      
Conclusions

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.