Rice, Aspergillus niger, A. ochraceus, Czapek Yeast Extract (CYA) medium, ochratoxin A (OTA).

Rice (Oryza sativa L.) is the most important cereal crop and millions of small farmers; landless labourers depend on rice cultivation for their income generation. Among total rice production, human consumption accounts for 85% compared to wheat and maize (Mazaheri, 2023). The major rice producing states in India are West Bengal, Uttar Pradesh, Andhra Pradesh, Punjab, Tamil Nadu, Bihar, and Odisha which contribute 72% of total rice output. In Tamil Nadu, cultivable area was around 2.04 million hectares, with production of 7.98 million metric tons contributing 6% of India's total rice production. Globally, India was the largest rice exporter of 16.5 million metric tonnes and Bangladesh, United Arab Emirates were the major importers of Indian rice. In India, about 10% of the agricultural GDP comes from rice sector (Singh et al., 2021).

Rice which is cultivated commonly under flood irrigation and prevailing high moisture level promote the fungal growth. The fungal contamination may occur at pre harvest stage in the field or during post-harvest such as transport, inadequate drying and improper storage condition (Reddy et al., 2008). The predominant mycotoxigenic fungi in rice belong to the genera are Aspergillus sp., Fusarium sp and Penicillium sp. In rice samples, 80% of the fungal isolates belonged to the genera Aspergillus and 20% were Penicillium (Laut et al., 2023). As per the report of Trung et al. (2001) 43.8% Aspergillus sp., 21.9% Fusarium sp., 10.9% Penicllium sp., and 23.4% of other fungi were found in rice samples.

Ochratoxin A producing fungi that can contaminate rice primarily belongs to the genera Aspergillus and Penicillium (Lee and Ryu 2015). The major producers of OTA belonging to the genera Aspergillus, Penicillium including species such as A. ochraceus, A. carbonarius, A. niger, and P. verrucosum (Ben Miri et al., 2024). Several authors reported the investigation of isolates of A. niger in rice samples at different countries. Accordingly, Trung et al. (2001) in Vietnam; Shanakht et al. (2014) in Lahore; Ranjbar et al. (2019) in Khuzestan. The ochratoxigenic fungi which was isolated from Korean polished rice was A. niger, A. ochraceus and P. verrucosum (Park et al., 2005). Hafez et al. (2022) identified the prevalence of A. ochraceus in rice samples of Egypt. 

The fungi A. niger and its closely related species (A. carbonarius, A. awamori, A. aculeatus and A. japonicus) all belong to Aspergillus Section Nigri and the colonies of A. niger are black or dark brown in colour (Perrone et al., 2007). The predominant OTA producer in Aspergillus Section Circumdatiis A. ochraceus produces yellow brown (ochre) colonies (Visagie et al., 2014). Further molecular techniques are developed to distinguish Aspergillus spp (Esteban et al., 2006). The species-specific PCR-based assays to differentiate ochratoxigenic species were developed based on ITS region for A. ochraceus (Patino et al., 2005). In A. ochraceus, the sequence of AoLC35-12 encode for acyl transferase domain of a polyketide synthase, which is involved in the OTA biosynthesis pathway (Atoui et al., 2006).

Under favourable condition, the ochratoxigenic fungi produce ochratoxin A (OTA) well-known fungal mycotoxins of worldwide concern. It was first isolated from Aspergillus ochraceus in south Africa by Van der Merwe et al. (1965), hence the name ochratoxin A (Li et al., 2021). Gonclaves et al. (2019) documented the prevalence of A. ochraceus in rice and its ability to produce OTA. OTA exhibits neurotoxic, hepatotoxic, immunotoxic, mutagenic, and teratogenic effects. OTA is also classified as possibly human carcinogen (group 2B) by IARC (International Agency for Research on Cancer) (Humans et al., 1993). The impact of Aspergillus infection and ochratoxin A production cause economic loss in rice production by reducing the market value of rice (Pandey et al., 2023). Further it decreases the nutritional quality of rice and cause harmful effects on animal and human health. Hence the present investigation was undertaken to assess the prevalence of fungal contaminants in rice and identification of ochratoxigenic fungi particularly A. niger and A. ochraceus.

Chemicals used. For culture maintenance, sterile disposable plates (Himedia, Mumbai) were used and they were autoclaved twice at 121 °C for 15 minutes each before being discarded. Glasswares used for the experiments such as funnels, beakers, and Erlenmeyer flask was cleaned by immersing it in 1% NaOCl for two hours and then in 5% acetone for 30 minutes.

Collection of samples. The samples were collected from different rice growing districts of Tamil Nadu, India during 2022-23. A total of ninety samples including nine paddy and eighty-one brown rice, polished rice samples were obtained to assess the fungal contaminants and to isolate, identify the ochratoxigenic fungi particularly Aspergillus spp. The samples were brought randomly from petty shop, retail stores and whole sale markets of various districts viz., Coimbatore, Erode, Salem, Trichy, Thanjavur, Kanyakumari, Namakkal, Madurai, Dharmapuri, Dindigul, Karur, Tirupur, Theni, Kangayam, Nilgiris, Thoothukudito enumerate the fungal population and for identification of ochratoxin A producing fungi. The sampling size of 2 kg was obtained by pooling 10 cumulative samples of 200 g each. The samples were grounded to fine particle size using hand mill (BTC, India), labelled and stored at 4 °C for further experiments.

Assessment of fungal contaminants in paddy, brown rice and polished rice samples. The fungal population associated with paddy, brown rice and polished rice samples were enumerated using dilution plating technique as documented by Pitt and Hocking (1997). The finely grounded sample of one gram was taken in test tube containing ten ml of sterile distilled water. The test tubes were shaken for 30 min and were serially diluted with sterile water. Then, one ml of solution was plated on Rose-Bengal Chloramphenicol Agar medium and the plates were incubated at 30 ºC for three days to promote the growth of fungi. The fungal population on plates were expressed as colony forming units (CFU/g). Pure culture of fungal colony was obtained by single colony isolation method on PDA medium and maintained at 4 °C for further studies.

Identification of ochratoxigenic fungi Aspergillus spp

Morphological characterization. To identify the fungal genera, single hyphae were transferred to PDA amended Petri plates and incubated for 7 days at 30ºC. Subsequently, fungal colonies were transferred to Czapek Yeast Extract (CYA) (Sucrose 30 g, Yeast Extract 5 g, NaNO3 2 g, K2HPO4 1 g, KCl 0.5 g, MgSO4 0.5 g, FeSO4 0.01 g, ZnSO4.7H2O 0.1g, CuSO4.5H2O 0.005 g Agar 15 g per 1 L)agar medium and incubated for 15 days at 25 ºC under dark.  After the incubation period, colony morphology of fungi was observed and characteristics like colour (observe and reverse), shape, sporulation are used as a key description factor for identification (Pitt, 2009). The ochratoxigenic fungal isolates were characterized morphologically at 20x and 40x magnification with the help of Phase contrast microscope (Leica DM 2000 LED). The vegetative and sexual morphology was observed and imaged using software (LAS version 4.11.0) by Leica Microsystems (Switzerland) ltd.

Species confirmation of A. ochraceus using polyketide synthase gene (pks). The morphologically identified Aspergillus isolates were subjected to molecular characterization through the sequence analysis of 18S rDNA using ITS 1 and ITS 4 primers. Further Aspergillus ochraceus isolates were confirmed using  AoLc35F, AoLc35R primer by the amplification of polyketide synthase gene (pks).

DNA extraction. DNA extraction was carried out from the fungal mycelial mat by adopting CTAB method prescribed by Priyadharshini et al. (2019). Five mm mycelial disc from 10days old Aspergillus colony was inoculated into 100 ml sterile potato dextrose broth and incubated at room temperature 28±2°C for seven days. After incubation, the mycelialm at was harvested and dried on filter paper. The mycelialm at was then ground finely with 3-5ml of CTAB buffer. The slurry (700µl) was then transferred to 1.5 ml centrifuge tube. The samples were incubated in water bath at 65 °C for 30 min and cooled at room temperature. To the extract 750 µl of phenol: chloroform: isoamyl alcohol (25:24:1) were added and centrifuged at 13,000 rpm for 15min.Thesupernatantwastransferredtonew1.5mlsterilecentrifugetube and 2/3 volume of ice-cold isopropanol was added and mixed gently. The samples were incubated at -20 °C overnight for the precipitation of DNA. After the incubation, the mixture was centrifuged at 13,000 rpm for 15 min at 4°C and the supernatant was discarded. The pellet was then washed thrice with 70 percent ethanol and centrifuged at 13,000 rpm for 5 m into remove impurities. The ethanol was discarded and the pellet was air dried for 30 min to remove traces of ethanol. Finally, the pellet containing DNA was suspended in 50 µl of 1X TE buffer and stored at -20 °C. The purity and the concentration of the DNA was determined by spectrophotometrically at 260/230 nm using a nanodrop spectrophotometer (ND-1000, Wilmington, DE) and the total genomic DNA was checked by agarose gel electrophoresis.

Confirmation by PCR analysis using AoLc35F, AoLc35R primer. For species confirmation, A. ochraceus isolates were subjected to PCR amplification of pks gene using AoLc35F, AoLc35R primer. The PCR cycle followed for each reaction contains 35 cycles of initial denaturation at 94 °C for 4 min followed by denaturation at 94°C for 40S, primer annealing at 58°C for 40S, extension at 72 °C for 40 S.A final extension step was carried out at 72°C for 10min (Algammal et al., 2021). The amplified PCR products were sequenced by using sanger dideoxy sequencing method at Biokart, Bangalore. The identification of isolates was determined by comparing the acquired DNA sequences with the National Centre for Biotechnology and Information's (NCBI) database.

Enumeration of fungal contaminants in paddy, brown rice and polished rice samples. The mycobiota study revealed different fungal genera belong to Aspergillus, Penicillium, Fusarium, Alternaria, Rhizopus, Cladosporium, Mucor, Curvalaria, Trichoderma and Bipolaris were documented in paddy, brown rice and polished rice samples. In line with our study, Pinciroli et al. (2013) documented the presence of Alternaria, Bipolaris, Curvularia, Cladosporium, Penicillium and Fusarium species in paddy, brown and polished rice samples. Egbuta et al. (2015), reported the rice samples were contaminated with fungus belonging to Aspergillus, Penicillium, Fusarium, Alternaria, Rhizopus, and Cladosporium. Similarly, Moharram et al. (2019) observed the prevalence of Aspergillus, Penicillium, Alternaria, Cladosporium and Fusarium in rice collected from the market at different localities of Egypt. The major fungi found in rice samples are Aspergillus, Penicillium, Fusarium, Alternaria, Mucor, Rhizopus, Trichoderma, Curvularia, Helminthosporium and Cladosporia (Makun et al., 2007). The occurrence of Aspergillus spp in rice was documented by Qi et al. (2022). Several authors reported the occurrence of common mycoflora associated with rice including Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Alternaria spp., Mucor spp (Phan et al., 2021); Bipolaris spp., Fusarium spp., Aspergillus spp., Curvularia spp., Botryodiplodia spp. (Ackaah et al., 2023); and Alternaria alternata, F. moniliforme, Rhizopus nigricans (Kumar et al., 2023).

In our current study, the highest fungal population in paddy (Table 1, Fig. 1a) was recorded by Aspergillus spp with a range of 8.24×103–6.03×106 CFU/g followed by Penicillium which showed 4.82×103–2.76×105 CFU/g and Fusarium with a range of 1.9×103–0.7×105 CFU/g. Further, the present study investigated that Aspergillus, Penicillium and Fusarium were the most dominant fungal genera recorded in paddy samples. In agreement with our study, Reddy et al. (2009) reported the paddy samples from India found to be infected with Aspergillus sp mainly by A. flavus, A. niger, A. ochraceus and A. parasiticus. The incidence of fungal genera investigated in brown rice were elucidated in Table 2, Fig. 1b. Aspergillus and Penicillium were the most predominant genera recorded with fungal population lies in the range of 5.4 ×103–3.8 ×106 CFU/g, 2.6 × 103 –1.5 × 105 CFU/g followed by Fusarium showed 0.8×103 – 0.5×105 CFU/g respectively. In polished rice, the findings recorded highest fungal population of 1.8×103–0.5×106 CFU/g by Aspergillus followed by Penicillium documented 1.4 ×103–0.9 ×105 CFU/g (Table 3, Fig. 1c). 

The study revealed that among the isolated fungal genera, Aspergillus spp were the most and predominant fungal contaminant with an isolation frequency of 69.56% followed by Penicillium 20.24% and the other genera (10.20%) respectively. In agreement with our findings, Katsurayama et al. (2020) documented the Aspergillus, Penicillium and Fusarium were the dominant fungal species found in rice samples marketed in Brazil. Rice in tropical Asia is mostly infected with Aspergillus fungi due to the prevailing condition occur during pre-harvest, harvest and postharvest stages (Santos et al., 2022). Laut et al. (2023) revealed the analysis of rice samples documented the major fungal isolates belong to Aspergillus (80%) and Penicillium (20%). In India, Begum and Samajpati (2000) isolated Aspergillus sp. and Penicillium sp.  from contaminated rice sold in the local markets of Calcutta. Gautam et al. (2012) documented the prevalence of Aspergillus, Penicillium, Fusarium, Curvularia Cladosporium and Alternaria in rice samples.

Morphological characterisation of Aspergillus niger. The macroscopic and microscopic characters of A. niger isolates were studied on CYA medium (Fig. 2). The colour of the colony was white at initial stage which later turned into brown to dark black due to production of conidial spore. Initially, there was slight yellowish tinge on white mycelia which occurred due to radial cracks in mycelia. Conidial heads are globose, biseriate, dark brown, radiating, and prone to splitting into several loose columns as they age. Conidiophores have smooth walls, hyaline or turn dark towards the vesicle and the conidia are globose to sub globose, black in colour. The morphological characters of A. niger was similar with the descriptions given by Klich (2002) reported that the A. niger of section Nigri characteristically produces dark-brown to black spores. Likewise, Silva et al. (2011); Karlton-Senaye et al. (2015); Zulkifli and Zakaria 2017; Abdallah et al. (2022) documented the growth characteristics of A. niger cultured on CYA and MEA (Malt Extract Agar) medium. Diaz et al. (2021) reported A. niger showed dark brown colour conidia at initial stages on CYA medium which later turns to characteristic black colour. The colour of the  colony was dark brown to black on the reverse side of Petri plate. El-Sayed et al. (2019) documented the occurrence of A. niger in polished rice and found to be OTA producer.

Morphological characterisation of Aspergillus ochraceus. The vegetative mycelium of Aspergillus ochraceus was white and mostly submerged, while the conidial heads are typically arranged in zones and are closely packed. The colour of the colony was yellow and on the reverse side, pale to brownish in colour (Fig. 3). The colony characters are consistent with the descriptions by Frisvad et al. (2004); Visagie et al. (2014). By visually under naked eye, conidiophore appear as a powdery mass with chalky yellow colour. The conidial head appeared as globose initially but with age, splitting into two or three divergent columns with smooth wall spherical conidia. Similar colony characters of A. ochraceus were documented by Pandit et al. (2014) in CYA medium. Abdul-kareem et al. (2021) studied the macroscopic and microscopic characters of A. ochraceus and reported A. ochraceus produced yellow colour colony on PDA medium. Some colonies may form pinkish to purple, pebble-like sclerotia measuring up to 1 mm in diameter and the findings were in line with the report of Borges et al. (2011) reported pink to purple sclerotia formation by A. ochraceus is the main distinguishable feature for the species.

Species confirmation of A. ochraceus. The A. ochraceus isolates were subjected to PCR amplification of pks gene using AoLc35F, AoLc35R primer and resulted with the expected amplicon size of 520 bp (Fig. 4). Similarly, Dao et al. (2005) used AoLc35F, AoLc35R primer for the specific identification of A. ochraceus. Algammal et al. (2021) documented the confirmation of A. ochraceus isolates was performed by the amplification of polyketide synthase gene (pks) using AoLc35F, AoLc35R primer pair. The amplification of pks gene confirmed that the isolates of A. ochraceus are ochratoxigenic and involved in the biosynthesis of ochratoxin A. The present findings were in agreement with the report of Moghadam et al. (2019) documented the confirmation of A. ochraceus as OTA producer using AoLc35F, AoLc35R primer pair.

Table 1: Assessment of fungal contaminants in paddy samples.

Table 2: Assessment of fungal contaminants in brown rice.

Table 3: Assessment of fungal contaminants in polished rice.

Fig. 1a. Assessment of fungal contaminants in paddy using dilution plate technique.

Fig. 1b. Assessment of fungal contaminants in brown rice using dilution plate technique.

Fig. 1c. Assessment of fungal contaminants in polished rice using dilution plate technique.

Fig. 2. Morphological characterization of Aspergillus niger isolate.

Fig. 3. Morphological characterization of Aspergillus ochraceus isolate.

M-100 bp ladder; Lane 1-AO1; Lane 2-AO2; Lane 3-AO3; Lane 4-AO4; Lane 5-AO5;  Lane 6-AO6; Lane 7-AO7;

Lane 8-AO8; Lane 9-AO9; Lane 10-AO10; Lane 11- Negative control

Fig. 4. Molecular confirmation of Aspergillusochraceus isolates by amplification of pks gene using AoLc35F, AoLc35R primers.

Fungal infection in rice and subsequent ochratoxin A production showcase the importance of adoption of proper agricultural and management practices to prevent and mitigate the fungal contamination. Further, the study emphasis on the need of good storage facilities along with strict monitoring programme for the betterment of safe and healthy food for human livelihood.

Nandinidevi S. and Paranidharan  V.  (2024). Enumeration of Fungal Contaminants in Rice and characterization of Ochratoxigenic Fungi Aspergillus spp. Biological Forum – An International Journal, 16(11): 56-62.