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Research Article
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Morphological Identification of Foodborne Pathogens Colonizing Rice Grains in South Asia |
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K.R.N. Reddy,
N.I. Farhana,
A.R. Wardah
and
B. Salleh
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ABSTRACT
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The aim of this study was to identify the foodborne pathogens mainly, Aspergillus sp. colonizing rice grains using cultural and microscopic methods. Four differential media (Czapek Dox Agar (CZA), Czapek Yeast Agar (CYA), Malt Extract Agar (MEA) and Czapek yeast 20% sucrose agar (CYA20S)) were used for differentiation of five Aspergillus sp., colonizing rice grains comparing with standard cultures. We studied macroscopic (colony color and diameter, conidia color, exudates, sclerotia and colony texture) and microscopic (conidiophore color, length and breadth, conidia size, shape and surface texture, vesicle diameter and phialides length and breadth) characteristics for identification of 110 isolates of Aspergillus sp. isolated from 65 rice grain samples collected from various countries in South Asia (Cambodia, India, Indonesia, Malaysia and Thailand). According to morphological characters, all these isolates were belonging to Aspergillus flavus (45), A. fumigatus (8), A. ochraceus (7), A. niger (42) and A. tamarii (8). This is the first report on identification of large number of Aspergillus strains isolated from rice grains in South Asia.
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Received: March 16, 2010;
Accepted: May 23, 2010;
Published: August 18, 2010
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INTRODUCTION
Rice is one of the important staple foods in the world and in the South Asia
where high amounts of rice are consumed per capita per year. The main rice producing
countries are Bangladesh, China, India, Indonesia, Myanmar, Thailand and Vietnam
(Reiter et al., 2010). The environmental conditions
in South Asian countries are characterized by high temperatures (26-39°C)
coupled with high relative humidity (67-98%) and are hence conducive for the
growth of mycotoxigenic fungi and the production of mycotoxins in nearly all
agricultural crops throughout the year (Sales and Yoshizawa,
2005). Generally cereal crops usually are harvested during the rainy season
resulting in high moisture content of the grains, sub-optimal conditions for
processing and storage and potentially rapid accumulation of toxigenic fungi
(Sales and Yoshizawa, 2005). Mycotoxigenic fungi are
well-known to invade the rice grains under storage conditions and produce mycotoxins
(Reddy et al., 2009, 2010;).
Several reports are available on colonization of rice grains with Aspergillus
sp., from South Asia (Pitt et al., 1994; Sales
and Yoshizawa, 2005; Park et al., 2005; Makun
et al., 2007; Reddy et al., 2009;
Lampak et al., 2009).
Mycotoxigenic fungal contamination not only causes deterioration of foods, but
also causes food borne intoxicants in humans and animals as they may produce toxic
secondary metabolites called mycotoxin ( Murthy et al.,
2009). Most human diseases caused by Aspergilli (aspergilloses) are associated
with immunosupression. They are frequently fatal. As the number of immunosupressed
people in the population has risen, so has the importance of infection by Aspergillus.
A. fumigatus is involved in about 90% of human aspergilloses, followed
by A. flavus, A. terreus, A. niger, A. nidulans and
A. ochraceus ( Bertout et al., 2001). Interest
in Aspergillus sp., is increasing world wide due to the discovery of a
growing number of naturally occurring Aspergillus toxins that have proved
to be threat to the human and animal health ( Bhat et al.,
2010). Aspergillus sp. is known to produce aflatoxins, ochratoxins
and gliotoxin ( Reddy et al., 2009; Lanier
et al., 2009). Aflatoxin B 1 (AFB1) has been classified as
a class 1 human carcinogen and OTA as class 2B by the International Agency for
Research on Cancer ( IARC, 1993). Gliotoxin is one of the
most abundantly produced epithiodioxopiperazine metabolites from A. fumigatus.
Toxicological studies showed that gliotoxin can exacerbate the pathogenesis of
aspergillosis ( Sutton et al., 1996). A gardener
developed a fatal aspergillosis and died after inhalation of decayed plant matter
contaminated with A. fumigatus spores ( Russel et
al., 2008).
Generally identification of the Aspergillus sp. is based on the morphological
characteristics of the colony and microscopic examinations (McClenny,
2005; Diba et al., 2007). Though molecular
methods continue to improve and become more rapidly available, microscopy and
cultural methods remain commonly used and essential tools for identification
of Aspergillus sp. (Diba et al., 2007).
However, the aim of this study was to identify large number of Aspergillus
isolates through macroscopical and microscopical characters isolated from 65
rice grain samples collected from South Asian countries. As far as we know this
is the first study of its kind for identification of Aspergillus species
in South Asia through morphological characters.
MATERIALS AND METHODS
Collection of rice grain samples and isolation of Aspergillus sp.:
A total of 65 rice grain samples were randomly collected from sundry markets
and supermarkets in South Asia during the year 2009. The samples were mainly
from white rice, basmati, black glutinous rice, brown rice, fragrant rice and
white glutinous rice originating from Cambodia, India, Indonesia, Malaysia and
Thailand. All samples were analyzed for Aspergillus sp. using the agar-plate
method according to Reddy et al. (2009). Hundred
seeds were placed on one-half strength Potato Dextrose Agar (PDA) added with
rose bengal at a concentration of 50 ppm. The plates were incubated at 25±2°C
for 7 days and then the Aspergillus colonies emerging out from rice grains
were transferred onto fresh potato dextrose agar plates for further studies.
Purification of cultures through single spore isolation: All Aspergillus
sp. strains were purified through single spore isolation technique (Samapundo
et al., 2007). The single conidial isolates were maintained on low
nutrient medium for further studies.
Identification of Aspergillus sp. through morphological characters:
All Aspergillus sp. isolates were identified to the species level using
taxonomic systems of Aspergillus by Klich (2002).
All Aspergillus sp. were cultured on Czapeks yeast agar (CYA; Czapek
concentration 10.0 mL, K2HPO4 1.0 g, powdered yeast extract
5 g, sucrose 30 g, agar 15 g, distilled water 1 L), Czapeks yeast agar with
20% sucrose (CYA20S; Czapek concentration 10.0 mL, K2HPO4
1.0 g, powdered yeast extract 5 g, sucrose 200 g, agar 15 g, distilled water
1 L), Malt extract agar (MEA; powdered malt extract 20 g, peptone 10 g, glucose
20 g, agar 20 g, distilled water 1 L) and Czapeks Dox agar (CZA; Czapek concentration
10 mL, K2HPO4 1 g, sucrose 30 g, agar 17.5 g, distilled
water 1 L) at 25±2°C for 7 days. Some of the CYA plates were also
incubated at 37°C for 7 days. Macroscopical characters included colony color
and diameter, conidia color, exudates, sclerotia, colony texture and shape.
Microscopic characteristics for the identification were color and length of
conidial heads, stipes, vesicles shape and seriation, conidia size, shape and
roughness and phialides length and breadth (Diba et al.,
2007). To confirm our identification, we compared the morphological characteristics
of tested Aspergillus isolates with those of the standard species obtained
from Microbial Type Culture Collection (MTCC), Chandigarh, India.
RESULTS A total of 110 isolates of Aspergillus spp. which included A. flavus (45), A. fumigatus (8), A. ochraceus (7), A. niger (42) and A. tamari (8) were obtained from 65 rice samples collected from South Asian countries. Morphological characters were studied for identification of all these isolates along with standard cultures using four differential culture media.
Morphological characters of A. flavus isolates: Conidia of all
isolates were light sparse grey green to pale blue green or parrot green, mycelium
fluffy creamy white to dull white color and exudates were present on surface,
reverse uncolored to yellowish or orange and wrinkled mycelial growth; soluble
pigments were absent; very few sclerotia were present in wheat brown color (Fig.
1a-h). On reverse side of MEA plates no wrinkles were
observed. Conidia very sparse in dull blue green color reverse yellowish orange
to light peach on CZA (Table 1). Macroscopical and microscopical
characters of A. flavus are presented in Table 1.
Morphological characters of A. fumigatus isolates: Conidial colors on CYA25 grayish, mycelium white, inconspicuous to florescence; exudates were absent; reverse uncolored to yellowish, red brown or green, soluble pigments were absent; sclerotia were absent in all media (Table 2). On MEA, conidia colored as on CYA25, mycelium white, reverse uncolored to dull yellow or grey, soluble pigments were absent. Macroscopical and microscopical characters of A. fumigatus are presented in Table 2.
Morphological characters of A. niger isolates: Conidia are black
and densely packed on CYA; hyphae inconspicuous, white to dull yellow; exudates
were absent; reverse uncolored to florescent yellow and wrinkled mycelial growth;
soluble pigments were absent; sclerotia were absent in all media.
| Fig. 1: |
Macroscopical characters of A. flavus on different
agar media. (a-d) Front view and (e-h) Reverse view. (a, e) Mycelia growth
on CYA; (b, f) Growth on CYA20S; (c, g) Growth on CZA; (d, h) Growth on
MEA |
Table 1: |
Macroscopic and microscopic characters of A. flavus
isolates |
 |
± = Standard deviation |
On MEA, reverse uncolored to light brown and no wrinkles were present. On
CZA, reverse florescent yellow to light white, wrinkled. Macroscopical and microscopical
characters of A. niger are presented in Table 3.
Morphological characters of A. ochraceus isolates: Conidial color
on CYA25 near wheat to light yellow; fluffy mycelial growth, white or creamy
white, inconspicuous to florescence; exudates were absent; reverse dull yellow
to dark yellow or some times brown and wrinkled mycelial growth; soluble pigments
were absent; sclerotia were absent in all media.
Table 2: |
Macroscopic and microscopic characters of A. fumigatus
isolates |
 |
± = Standard deviation |
Table 3: |
Macroscopic and microscopic characters of A. niger
isolates |
 |
± = Standard deviation |
On MEA, conidia not dense usually pale to light yellow; reverse light yellow,
pale orange to grayish gold shades; and no wrinkles were present; colonies not
densely sporulating, variable in appearance (Table 4). Macroscopical
and microscopical characters of A. ochraceus are presented in Table
4.
Morphological characters of A. tamarii isolates: Colony and conidia color on CYA25 dark olive green; mycelium white to dull white, usually inconspicuous, reverse uncolored to red tinge with chocolate brown or brown and straight wrinkled mycelial growth; the outer surface of the colony was circular; colonies usually quite low, velutinous; sclerotia, exudates and soluble pigments were absent in all media (Table 5). On MEA, mycelium usually inconspicuous, reverse brown color with normal, sparse, loose, not dense and floccose growth and in all other media wrinkled growth; on CY20S generally puffy growth with slightly more yellow green than on CYA25; reverse reddish brown with wrinkled mycelial growth. On CZA, conidia very sparse in dark bluish green or olive green colors reverse reddish tinge to dark brown shade; on CYA37 dark blackish brown and concentric rings were observed on reverse (Table 5). Macroscopical and microscopical characters of A. tamarii are presented in Table 5.
Table 4: |
Macroscopic and microscopic characters of A. ochraceus
isolates |
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± = Standard deviation |
Table 5: |
Macroscopic and microscopic characters of A. tamarii isolates |
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± = Standard deviation |
DISCUSSION
When rice grains with moisture content higher than the desired level enter
the storage system, invasion of both field and storage fungi take place. The
harmful effects of such fungal invasion are glume or grain discoloration, loss
in viability, quality and toxin contamination (Reddy et
al., 2008). A. flavus is of ubiquitous occurrence in nature.
Since the discovery of aflatoxins, it has become the most widely reported food-borne
fungus, reflecting its economic and medical importance and ease of recognition,
as well as its universal occurrence. A. parasiticus is less common, but
the extent of its occurrence is obscured by the tendency for A. flavus
and A. parasiticus to be reported only as A. flavus (Reddy
et al., 2009). In this study we observed low frequency of A. fumigatus
in some of the rice grains. A. fumigatus is an airborne fungus and infection
occurs by inhaling conidia which may colonize airways prior to invasion. It
is an opportunistic fungal pathogen responsible for most cases of Invasive Aspergillosis
(IA), the most common systemic filamentous fungal infection worldwide (Bertout
et al., 2001).
Several reports are available on contamination of rice grains with Aspergillus
sp. A high incidence of A. flavus was found in the seed mycoflora of
rice (Reddy et al., 2008). The rice crop exposed
to frequent and heavy rainfall and flood is subjected to infection by Aspergillus
sp. (Reddy et al., 2009). Begum
and Samajpati (2000) isolated mycotoxin-producing fungi from contaminated
grains of rice sold in the local markets of Calcutta, India. Recently, Jayaraman
and Kalyanasundaram (2009) reported Aspergillus sp. contamination
of rice bran oils. Sales and Yoshizawa (2005) described
the incidence of A. flavus and A. parasiticus in rice bran (14%)
and rough rice (78%). Abdullah et al. (1998)
reported the incidence of aflatoxingenic fungi in rice grains from Malaysia.
Recently, Lampak et al. (2009) reported various
fungal mycoflora (Acremonium, Aspergillus, Bipolaris,
Colletotrichum, Curvularia, Drechslera, Fusarium,
Geotrichum, Nigrospora and Penicillium) in brown rice from
Thailand. A study by Purwoko et al. (1991) revealed
that A. flavus was the dominant fungi in broken rice and rice bran samples
in Indonesia. Still today there are no reports on occurrence of Aspergillus
sp. in rice from Cambodia. In this we made attempts to isolate and identify
Aspergillus sp. in rice samples collected from Cambodia.
Many Aspergillus strains are very close in their morphological characters
and chances are very high to misidentify them. Therefore, accurate identification
of Aspergillus sp. is important to develop proper management practices
to control these toxigenic fungi and their mycotoxins in food grains. Recently,
Kim et al. (2009) and Diba et al. (2007)
studied the morphological characters for identification of clinical Aspergillus
sp. isolates. Alwakeel (2007) identified Aspergillus
sp. isolated from kitchen samples in Riyadh, Saudi Arabia using morphological
methods. Morya et al. (2009) used morphology
based methods for identification of Aspergillus sp. isolated from soil
of teak forest. But none of them studied morphological characters for Aspergillus
isolates from rice grains. This is the first report on identification of huge
number of Aspergillus sp. isolates obtained from rice grain samples collected
from various countries in South Asia through morphological characters.
Askun (2006) used three (CZ, MEA and PDA) differential
media for identification of Aspergillus sp. using morphological characteristics
isolated from maize kernels. Similarly, Khosravi et al.
(2007) used only two differential media (PDA and CZ) for identification
of Aspergilli isolated from nut products in Iran. In this study we used morphological
method with four differential culture media for identification of five important
Aspergillus species isolated from rice grains. Using this method, all
standard strains were identified successfully. For the identification of Aspergilli
based on morphological methods requires adequate growth for evaluation of colony
characteristics and microscopic features. Diba et al.
(2007) reported that use of potato dextrose, potato flake, malt extract,
inhibitory mould agar, or similar sporulation agars as primary isolation media
for Aspergillus may accelerate growth rate and the production of conidia
(Diba et al., 2007). In our study, using four
differential media including CZA, CYA, CYA20S and MEA with macroscopic and microscopic
characteristics of fungal growth on this culture media enabled us to discriminate
five Aspergillus species isolated from rice grains. We preserved all
110 isolates of Aspergillus sp. at Universiti Sains Malaysia, Malaysia
culture collection centre for future studies.
CONCLUSION This study concludes that Aspergillus sp. can contaminate rice grains under storage conditions. Though molecular methods are well developed for identification of Aspergillus sp., the developing countries are still highly depending on morphological identification. In this study we have identified five Aspergillus sp. isolated from rice grains based on their morphological characters. Most important human pathogen, A. fumigatus were also observed in few rice grain samples destined for human consumption. In our view, morphological method using the differential media is the most reliable and sensitive assay to identify important Aspergillus sp. isolated from rice grains or some other sources. This study may be a basis for those who are interested to study morphological characters for Aspergillus sp. in developing countries. Mycotoxin profiles produced by these Aspergillus sp. isolates are under progress. ACKNOWLEDGMENTS Dr. K.R.N. Reddy acknowledges the Universiti Sains Malaysia, Penang, Malaysia for providing a postdoctoral fellowship. We are grateful for the research grants 1001/PBIOLOGI/811009 and 1001/PBIOLOGI/811147 provided by USM.
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