Ohcratoxin producing Aspergillus spp. isolated from tropical
soils in Sarawak, Malaysia
Jaya Seelan Sathiya Seelan 1 & Sepiah Muid 2
1 Institute for Tropical Biology and Conservation,
Locked bag 2073, Universiti Malaysia Sabah, 88999, Kota Kinabalu, Sabah,
Malaysia
2 Department of Plant Science and Environmental Ecology,
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300
Kota Samarahan, Sarawak, Malaysia
Email: 1 avinash80us@yahoo.com
Date
of publication (online): 26 March 2010
Date
of publication (print): 26 March 2010
ISSN
0974-7907 (online) | 0974-7893 (print)
Editor: V.B.
Hosagoudar
Manuscript details:
Ms
# o2247
Received
01 July 2009
Final
received 23 November 2009
Finally
accepted 09 January 2010
Citation:Seelan, J.S.S. & S. Muid (2010). Ohcratoxin producing Aspergillusspp. isolated from tropical soils in Sarawak, Malaysia . Journal of Threatened Taxa 2(3): 762-765.
Copyright: © Jaya
Seelan Sathiya Seelan & Sepiah Muid2010. Creative Commons
Attribution 3.0 Unported License. JoTT allows unrestricted use of this
article in any medium for non-profit purposes, reproduction and distribution by
providing adequate credit to the authors and the source of publication.
Acknowledgements: The
authors would like to thank Universiti Malaysia Sarawak (UNIMAS) for permission
granted to present these findings and MOSTI for providing financial support for
this project. The author is grateful
to Prof. Maren Klich of the United States Agricultural Department (USDA) for
confirming the indentification of the Aspergillusspecies.
For Figure, Images & Table – click here
Aspergillus spp.
has been widely studied for mycotoxin analysis. Mycotoxin is considered one of the chemical groups that causes serious side effects in humans and animals. Although Aspergillusspp. contains bioactive compounds, there is a need to screen for mycotoxin
metabolites since mycotoxin is hazardous to human health. Ochratoxin is a
mycotoxin with nephrotoxic, nephrocarcinogenic, teratogenic and
immunosuppressive properties, and has received growing interest in the
scientific community and food committees in the last few years (Battaglia et
al. 1996; Abarca et al. 2003). Only species belonging to the genera Aspergillus and Penicilliumhave been reported as capable of producing ochratoxins. They were initially described by Scott
(1965) in Aspergillus ochraceus but have also been found in other species of the section Circumdati: A.alliaceus, A. melleus,A. ostianus, A. petrakii,A. sclerotiorum, A.sulphureus (Hesseltine et al. 1972), A.albertensis, A. auricomus (Varga
et al. 1996); as well as in the black aspergilli of section Nigri: A. niger var. niger,A. carbonarius (Samson et al. 2004). In this study, eighteen species
of Aspergillus isolated from different habitats were selected to
screen for ochratoxin producing strains.
Material and Methods
Fungal isolates: Eighteen
isolates of Aspergillus species were selected (Table 1) and grown on potato
dextrose agar (PDA), Czapek’s yeast extract agar (CYA), and Malt extract agar
(MEA), incubated for 7 days at 25°C. The strains were identified based on Raper & Fennell (1965) and
Klich (2002). Colonies were
mounted in lactophenol blue and images were taken by using a NIKON digital
camera.
Extraction and immunochemical tests by using ELISA kit: Each fungus was cultured in a Czapek’s yeast extract
broth (CYB) medium for 7 days and incubated at 25°C, in shaker of 120rpm. Crude fermentation broth was blended
thoroughly and centrifuged at 4000rpm for 5 minutes. The supernatant was passed
through a filtration membrane (0.22µm, Minipore). Then the homogenized broth was extracted with
chloroform. The combined organic
extract was evaporated under reduced pressure yielding a crude semi-solid (Wang
2002). Then, the
extracts were tested by using enzyme-linked immunosorbent assays (ELISAs) test
for positive results. ELISA
positives were confirmed by HPLC technique.
OA detection by HPLC: The
samples were analysed by using a reverse phase HPLC equipped with a Jasco
FP-920 fluorescence detector (330nm excitation wavelength, 460nm emission
wavelength). Chromatographic
separations were performed on an analytical column (symmetry waters C18 ODS2,
150mm x 3.9mm, 5µm) fitted with a precolumn with the same stationary
phase. The mobile phase used was
pumped at 1.0ml/min and consisted of an isocratic program as follows: acetonitrile/water/acetic
acid (99:99:2, v/v). The injection
volume was 20µl. Samples were
taken as positive for OA presence if they yielded a peak at a retention time
similar to the OA standard peak (approximately 4 min), with a height five times
higher than the baseline noise.
Results and Discussion
ELISAs test: Altogether, 18Aspergillus strains were tested for OA production by the
immunochemical test. Among these,
only two strains of Aspergillus, namely, A. carbonarius and A. sulphureusproduced OA when tested with ELISA. The other sixteen strains of Aspergillus did
not produce this toxin (Table 1). In temperate regions, A. carbonarius and A. sulphureushave been widely studied on their OA production. These two species were earlier described as OA producing
species (Ciegler 1972; Hesseltine et al. 1972). In this study, we have showed that
these tropical strains produce a lower concentration of OA using the czapek’s
yeast extract broth (CYB). The OA production was influenced by the type of media and the
origin of the isolates (Harwig 1974). This was shown when two other A. carbonariuswere isolated from the peat soil in Bintulu and did not show any OA production
under the same liquid media. Thus,
not all strains of Aspergillus species are ochratoxin (OA) producers.
Morphological identification of OA producers
Aspergillus carbonarius (JS742): Colonies
on Czapek’s yeast extract agar (CYA) 57-58mm in diameter (7 days, 25°C),
wrinkled, dense and velutinous, exudates present, white at first and becomes dark
brown with forming of conidial heads, reverse dark yellow. Colonies on malt extract agar (MEA)
52-56mm in diameter (7 days, 25°C) similar to those on CYA but less dense and
conidia in duller colors, reverse dirty yellow. No growth at 5°C. Growth at 37°C is exceptionally rapid, colonies on CYA 38-40mm in
diameter in three days. This
strain can grow at 45°C. Conidial
apparatus develops as erect conidiophores. Tips of conidiophores enlarge and form vesicles with many
phialides producing conidia in long chains.
Conidial
heads are compactly columnar, 40-48µm in diameter, dark brown to black. Conidiophores are unbranched, smooth
and dark brown, stipes 200-300 x 3-4 µm. Vesicles are round to globose shaped, 20-30µm in diameter. Phialides crowded dark brown, 5-7µm
long. Conidia globose to
subglobose, roughened, hyaline, and often decidous spinules when young and
verruculose at maturity. Sclerotia
occasionally produced on CYA (Image 1).
Aspergillus sulphureus (JS500): Colonies
on Czapek’s yeast extract agar (CYA) 40-50mm in diameter (7 days, 25°C),
wrinkled, showing radial furrowing, sulphur yellow in color, forming white
conidial heads, reverse pale yellow, presence of abundant sclerotia shading
from white to cream to pale yellow. Colonies on malt extract agar (MEA) 60-70mm in diameter (7 days, 25°C),
similar to those on CYA but less dense and conidia dull yellow in color,
reverse pale yellow. No growth at
5°C. Growth at 37°C is
exceptionally rapid, colonies on CYA 55-60mm in diameter in three days. This strain did not grow at 45°C. Conidial apparatus develops as erect
conidiophores.
Conidial
heads are loosely radiate, with spore chains adherent into numerous narrow,
divergent and tangled columns, 400-450µm in diameter and white in color. Conidiophores up to 1mm long,
unbranched, smooth, colorless, stripes 500-650 x 5-7µm. Vesicles hyaline,
globose, 12-26µm in diameter. Sterigmata in two series (uniseriate or biseriate). Primaries (uniseriate) 4.5-7.5 x
3.0-4.5 µm, secondaries (biseriate) 6.5-8.0 x 2.0-2.5 µm. Conidia fusiforms and
slightly roughened when first formed, but quickly globose, smooth, 2.0-2.5 µm
in diameter (Image 2).
HPLC
analyses
For quantification of OA, an HPLC
apparatus equipped with a fluorescent detector was used. Extracts were considered positive if
they yielded a peak at a retention time identical to that of standard OA
(Figure 1). The amounts of OA
observed for A. carbonarius and A. sulphureus were
0.05 to 0.10 µg ml-1, respectively (Table
1). The OA concentration that was obtained in this study was lower in
concentration compared to the standard OA found in A. ochraceus(250 µg ml-1). These
two strains from our tropical region proved to be a low OA producer, similar toA. glaucus and the black Aspergillusstrains (Abarca et al. 1994). OA
production in A. carbonarius and A. sulphureus was
confirmed by HPLC comparing the UV spectra as recorded with a diode array
detector. The retention times (4.417 and 4.081) and UV spectra were similar to that
of the OA standard (Figure 1).
Conclusion
The
immunochemical method based on the application of a monoclonal antibody
preparation against OA proved to be a useful tool for the screening of
ochratoxin production among the Aspergilli. The present study helps to eliminate the toxic producing Aspergillusstrains. It also showed that only
minimum concentrations of ochratoxin producing Aspergillusoccurred compared to the temperate region countries.
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