Journal of Plant and Pest Science (ISSN: )

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1 Open Access (ISSN: ) Vol. 1, Issue.1: (August 2014) journals.sfu.ca/jpps/index.php/jpps/index Original Research Article Received: 19 Jul 2014, Accepted: 02 Aug 2014, Published: 25 Aug 2014 Cultural, Morphological and Pathological Variability among Isolates of Aspergillus flavus in Maize Collected from Different Parts of Andhra Pradesh Kumar MR *, Sudhakar P, Santhoshi MVM, Krishna TG and KR Reddy Author Info Department of Plant Pathology, Institute of Frontier Technology, Regional Agricultural Research Station, Acharya N.G.Ranga Agricultural University, Tirupati , Chittoor (Dt), Andhrapradesh, India. *Corresponding author s reddi_kumar01@yahoo.com Key Words Maize, Aspergillus flavus, Aflatoxin, sclerotia, Culture media. Abstract Survey was conducted in maize growing areas of Andhra Pradesh to identify toxigenic isolates of Aspergillus flavus in maize samples and to study their cultural, morphological and pathological variability. In most of the samples, percent occurrence of A.flavus was high and interestingly Penicillium sp.incidence was more in samples collected from coastal districts. Clear demarcation was observed in cultural and morphological characters exhibited by aflatoxicogenic fungi grown on different solid media. Growth rate was fast, no pigmentation on reverse and no sclerotia formation was seen on PDA medium. Whereas, the growth rate was slow, pigment on reverse and sclerotia formation was observed on CDA medium. Introduction Maize ( Zea mays L.) is an important cereal crop in world after wheat and rice. The importance of maize lies in its wide industrial applications besides serving as human food and animal feed. It is the most versatile crop with wider adaptability in varied agro-ecologies and has highest genetic yield potential among the food grain crops In India, maize is grown in a wide range of environments, extending from extreme semi-arid to semi humid and humid regions. The crop is also very popular in low- and mid-hill areas of western and north eastern regions. Broadly maize cultivation can be classified into two production environments: (1) traditional maize growing areas, including Bihar, Madhya Pradesh, Rajasthan and Uttar Pradesh ( BIMARU), and (2) non-traditional maize areas, include Karnataka and Andhra Pradesh (KAP). In traditional areas, the crop is often grown in marginal ecoregions, primarily as a subsistence crop to meet food needs. In contrast, maize in the non- traditional areas is grown for commercial purposes i.e. mainly to meet food requirements of booming poultry sector. Maize grains are frequently contaminated by the mycotoxigenic fungi Aspergillus flavus, known to produce aflatoxin B1, one of the most potent carcinogenic and cytotoxic compounds for man and some animal species ( Juan et al.2008). Aflatoxins in maize are produced by Aspergillus flavus and the closely related species, A. parasiticus and A. nomius. These fungi, Aspergillus flavus and A.parasiticus can be recognized by olive green or gray green respectively on corn kernels, in the field or in storage. Although aflatoxins are not automatically produced whenever grain becomes moldy, the risk of aflatoxin contamination is greater in damaged, moldy corn than in corn with little mold ( Aycicek et al.2005). Aflatoxins are harmful or fatal to livestock and are considered carcinogenic to animals and humans ( Herrman 2002). Cite this article: Kumar MR, Sudhakar P, Santhoshi MVM, Krishna TG and KR Reddy (2014) Cultural, Morphological and Pathological Variability among Isolates of Aspergillus flavus in Maize,, 1 (1):

2 Corn is the cereal with the highest demand in India as being used in poultry feed. In consequence, many studies have been oriented towards the description of fungal mycoflora and mycotoxins associated to this agricultural commodity. There are several methods available for determining aflatoxin contamination in commodities such as HPLC, ELISA, TLC and fluorescence polarization assay, but these methods are expensive and time consuming. Therefore, the present study attempted to survey, isolation and characterization of aflatoxicogenic fungi from maize samples collected from different districts of Andhra Pradesh i.e Rayalaseema and coastal districts where the crop is grown throughout the year and the produce is mainly used for commercial purposes. Materials and Methods The experiments were conducted in the Department of Plant Pathology, Institute of Frontier Technology, Regional Agricultural Research Station, Tirupati. Seed mycoflora associated with Maize: Roving survey was conducted in maize growing areas of Rayalaseema districts viz; Chittoor, Ananthapur, YSR Kadapa and coastal districts viz; West Godavari, Guntur, Prakasam districts of Andhra Pradesh and Kolar district of Karnataka state during Rabi,2012 and Kharif,2013 and cobs and rhizospheric soil samples of maize during harvesting stage were collected ( Fig 1). A total of 100 soil samples and 150 seed samples were collected in the areas surveyed. All the samples were taken from freshly harvested lots in paper bags and stored in refrigerator and subjected to isolation of mycoflora. Agar plate method The maize seeds were surface sterilized to remove secondary growth of pathogens with 0.1% HgCl 2 for 60 seconds and subsequent three changes of distilled water and blot dried. Seeds were kept equidistantly on preplated PDA medium ( Hi media code No.M096)and incubated at 28 C for 5 days and observed the growth mycoflora. The isolated A.flavus strains were grown on coconut agar medium, prepared by adding 100 ml of coconut milk to one litre of PDA medium, autoclaved and used to screen for the detection of aflatoxin producing fungi and further subjected to UV radiation at 365 nm for presence of resultant fluorescence upon aflatoxin production (Davis et al; 1987). Isolation and Characterization of aflatoxin producing fungi Fungal colonies showing fluorescence upon exposure to UV radiation were isolated and pure cultures were maintained in slants ( Fig 2). Microscopic observation of the fungal morphology in case of each of the isolate was done using Magnus MLX-TR binocular microscope (400 X). The morphological and cultural characteristics of each of the aflatoxin producing fungal isolates were compared to known descriptions of fungi in different fungal identification manuals ( Domsch and Gams,1972) and identified. Growth rate of aflatoxicogenic fungi was studied using two solid media such as potato dextrose agar ( PDA) and Czapek Dox Agar ( CDA) medium. Results and discussion The areas surveyed for the collection of maize samples was given in the Table 1. From the samples collected, Aspergillus flavus was predominant in samples collected from Rayalaseema districts. But interestingly, Penicillium was predominant in samples collected from coastal districts. The mycoflora associated with maize seeds was isolated using Agar plate method. About 12 isolates of A.flavus were picked up based on the fluorescence of positive colonies upon exposure to UV radiation at 365 nm. Methodology for detection of aflatoxin by fluorescence of agar medium under ultra violet light provides a simple and reliable means of eliminating non-producing strains and also avoids difficulties encountered in extracting aflatoxin from complex natural substrates ( Abbas et al., 2004). The list of aflatoxicogenic fungi isolated from different areas was given in the Table (2). The prevalence of Penicillium sp. in coastal areas might be attributed to the climatic conditions 10

3 Table 1: Survey for collection of maize samples from different districts of Andhrapradesh S.no Name of the place Sample collected 1. Ananthapur Soil and ( Dist) seed Amadaguru 2. Karnataka Soil and Biluru seed 3. Chittoor ( Dist) Soil and B.Kothakota seed 4. Kadapa ( Dist) Seed Mydukur-1 5. Mydukur-2 Seed Isolates collected A. flavus isolate from the soil sample(adg-1) and seed sample(adg-2) A. flavus isolate from the soil sample(blr-1) and seed sample(blr-2) A. flavus isolate from the soil sample(bkk-1) and seed sample(bkk-2) A. flavus (MDKR-1) A. flavus (MDKR-2) 6. Mydukur-3 Seed A. flavus (MDKR-3) 7. Porumavilla Seed A. flavus (PRMA) 8. Prakasam Seed A. flavus (CBM) Cumbum 9. East Godavari ( Dist) Seed Trichoderma, Fusarium, Helmenthosporium Veyampadu 10. Uppalapadu Seed Trichoderma 11. Adamallu Seed Trichoderma, Fusarium, Pencillium 12. Venkatakrishnapuram Seed Trichoderma, A.niger,Curvularia,Fusarium. 13. Veerabhadrapuram Seed Trichoderma, A.flavus,Fusarium, Penicillium italicum 14. Kamavarapukota Seed Trichoderma,Fusarium 15. T.Narasapuram Seed Trichoderma, Fusarium 16. Jakkavaram Seed Trichoderma,A. niger,fusarium 17. Gandigudem Seed Trichoderma,Fusarium, A. niger 18. Tirumaladevipeta Seed Trichoderma,Fusarium 19. Guntur ( Dist) Seed Trichoderma, A.Flavus, A.niger, Pencillium species TNL TNL-2 Seed A.flavus, Fusarium 21. TNL-3 Seed A.flavus, A.niger, Pencillium, Fusarium 22. TNL-4 Seed A.flavus,Trichoderma harzianum 23. TNL-5 Seed A.flavus, Fusarium 24. TNL-6 Seed Trichoderma 25. TNL-7 Seed Fusarium, Trichoderma 26. TNL-8 Seed Fusarium,Trichoderma 27 Penumoodi Soil Trichoderma proliferatum, Aspergillus flavus, Aspergillus niger. 28 Nagaram Decomposi ng matter Trichoderma, Penicillum, Aspergillus flavus, Aspergillus niger. 29 Poodivaada Soil Trichoderma harzianum, Aspergillus flavus, Aspergillus niger, Pencillium. 30 Cherakupalli Soil Trichoderma, Rhizopus, Aspergillus flavus, Aspergillus niger, Penicillium. 31 Repalli Soil Trichoderma harzianum 32 Battiprolu Soil Trichoderma viride, Aspergillus niger 33 Angallakuduru Wet Trichoderma virence, Trchoderma flavus 11

4 mushrooms 34 Vaikuntapuram Soil Trichoderma aureoviride, Aspergillus flavus, Aspergillus niger, Penicillium. 35 Narsaraopeta Soil Trichoderma virence, Asperigillus flavus, Aspergillus niger. 36 Nagaram Decomposi ng matter Trichoderma, Penicillum, Aspergillus flavus, Aspergillus niger. 37 Krishna Soil Trichoderma viride, Aspergillus flavus, Aspergillus niger, Penicillium. Aswaraopalem 38 Modumoodi Soil Trichoderma viride, Aspergillus flavus, Aspergillus niger, penicillum. 39 Kottapeta Soil Trichoderma viride, Aspergillus flavus, Aspergillus niger, Penicillium. 40 K.kottapalem Soil Trichoderma viride, Aspergillus flavus, Aspergillus niger, Penicillium 41 Raamachandra puram Decompose d leaves Trichoderma virence, Aspergillus flavus, Aspergillus niger, Penicillium, Helmenthosporium. 42 Puligadda Decomposi Trichoderma harzianum, Aspergillus niger. ng organic matter 43 Mopidevi Soil Trichoderma harzianum, Pencillium, Aspergillus flavus. 44 Peddakallepalli Soil Trichoderma harzianum, Aspergillus flavus, Aspergillus niger, Penicillium, Rhizopus. 45 Viswanadhapalli Dry Trichoderma longibrachiatum. mushrooms 46 Pedana soil Trichoderma harzianum, Pencillium, Aspergillus flavus, Aspergillus niger, Pencillium. Table 2: List of Aflatoxicogenic fungi used for in vitro studies S.No. Isolate Location & District 1 BLR-1 Biluru, Karnataka 2 BLR- 2 Biluru, Karnataka 3 ADG-1 Amadaguru, Ananthapur 4 ADG- 2 Amadaguru, Ananthapur 5 BKK B.Kothakota, Chittoor 6 KBM Cumbum, Prakasam 7 MDKR Mydukuru, Kadapa 8 TNL Tenali, Guntur 9 UPP Uppalapadu, East Godavari 10 VKP Venkatakrishnapuram, E.Godavari 11 JKV Jakkavaram, East Godavari 12 VBP Veerabhadrapuram, East Godavari 12

5 Table: 3. Cultural, morphological and pathological characters of different isolates of Aspergillus flavus S.No. Isolate Cultural & Morphological characters on Potato Dextrose Agar ( PDA) Cultural & Morphological characters on Czapeck Dox Agar 1. BLR-1 Dull green in colour, No pigmentation on reverse, Sparse growth, No uniform radial growth, mycelium growth is wavy like, average radial growth is 8.2 cm after 10 days of incubation No sclerotia formed. moderate growth rate 2. BLR- 2 No pigmentation on reverse, sparse radial growth and scattered mycelium is light greenish in color. 3. ADG-1 Dull green colored mycelium. No pigmentation on reverse. No sclerotia formation is observed colonies are cushiony ( CDA) Green in color, Brown color pigmentation on reverse, very spars growth limited to 4.1 cm in 10 days. Sclerotia formation is observed, scattered sclerotial formation is at the margin of each circular ring. slow growth rate. No regular pattern of radial growth. Sclerotia formation is observed. Mycelium dark green colored and scattered. Mycelium growth is in clumps and progress in patterns. No sclerotia formation is observed. No pigmentation colonies are erumpent, dark green mycelium and formed in clumps. 4. ADG- 2 No pigmentation, light greenish mycelium, covered the plate in 10 days, progression in circular rings. 5. BKK Radial growth is fast. Dull green color. No pigmentation on reverse. No sclerotia formed. Mycelium growth is in concentric circles and sparse. 6. KBM Growth is faster rate covered the entire plate with dark green colored sporulation and formed in concentric circles. No sclerotia are formed. No pigmentation on its reverse. No sclerotia formation. No pigmentation on reverse side. Mycelium dark green. Radial growth is slow abundant sclerotia are formed growth is not uniform and formed in bunches colonies are dark green and formed in clumps. Rough textured, erumpent and powdery growth. Growth is slow dark green colored spore mass. No sclerotia formed. Mycelial growth is not in concentric circles. Dull brown pigmentation on its reverse. Abundant spore mass. 7. MDKR- 3 Moderate growth. Dull green colored mycelium. Progress in concentric circles. No sclerotia formed. No pigmentation on its reverse. Growth is very slow. Dark green colored mycelium. No sclerotia formed. Mycelium growth in concentric circles. 8. TNL Growth is not uniform, formed in bunches. Spore mass is dark green colored. No sclerotia formed. No pigmentation on its reverse. 9. UPP Radial growth is fast covered the plate dull green colored spore mass. Progression in the concentric circles. No sclerotia formed. No pigmentation formed in its reverse. Pink color pigmentation at 25DAI 10. VKP Growth rate is fast, covered the entire plate. Dull green colored spore mass in concentric circles. No sclerotia formed, light yellow colored pigmentation on reverse. 11. JKV Growth rate is fast dark green colored spore mass in concentric circles No sclerotia formed, no pigmentation on reverse. Abundant spore mass on clumps. 12 VBP Moderate growth rate. Dull green colored spore mass. No sclerotia formed. No pigmentation on its reverse. Mycelium is in concentric circles. Moderate growth mycelium formed in bunches. Growth is not progressed uniformly, in concentric circles. Sclerotia formed in the peripheral of concentric rings. Growth rate is slow, formed in clumps, dark green colored spore mass sclerotia formed. Slow growth rate. Dark green colored spore mass in concentric circles. Abundant spore mass, rough textured, erumpent. No sclerotia formed. No pigmentation formed on reverse. Turn to light brown color pigmentation. Growth rate is slow. Thick dark color spore mass like green mat on the surface of medium. Sclerotia is formed, no pigmentation. Snuff colored spore mass, growth rate is slow. 13

6 Table 4: Growth rate of Aspergillus flavus isolates on different solid media S. No Isolate name Growth Rate (cm) 2 days* 3 days* 4 days* 5 days* 7 days* 10 days* PDA CDA PDA CDA PDA CDA PDA CDA PDA CDA PDA CDA 1 BLR BLR ADG ADG BKK KBM MDKR TNL UPP VKP JKV VBP Fig. 1 fig.2.a fig.2.b Fig.2.c Fig : 2.d Fig : 2.e Fig : 2.f Fig : 3 Fig : 4 Fig : 4 Fig : 6 Fig : 7 Fig : 8 Fig: 9 14

7 Fig : 1 Maize kernels infested with Aspergillus flavus; Fig 2: Seed mycoflora associated with maize a) Aspergillus flavus, b) Penicillium spp.,c) Helmithosporium, d)curvularia.,e) Fusarium sp., f) Nigrospora; Fig 3: Pure cultures of aflatoxicogenic A.flavus isolates; Fig 4-9: Photomicrographs of different aflatoxicogenic fungi of isolates (4) BLR-1, 5 (ADG-1), 6 (BKK), 7 ( KBM), 8 (MDKR-3), 9 ( TNL). like high humidity associated with cool temperature. Cultural and morphological characters of aflatoxin producing fungi There is a clear demarcation exhibited by different isolates of A.flavus grown on Potato dextrose agar (PDA) and Czapek Dox Agar (CDA) medium. The growth of mycelium is fast, colonies are dull to light grayish coloured, scattered, no pigmentation on reverse and no sclerotia formation was observed in case of isolates grown on PDA medium ( Fig 3). Whereas, the isolates cultured on Czapek Dox Agar medium exhibited marked difference in their growth rate, pigmentation and sclerotia formation. Mycelium was dark green, slow growth rate, pigmentation on reverse and sclerotia formation was observed. Morphological, cultural characters exhibited by different isolates grown on two solid media were elucidated in tabular form ( Table 3). Photomicrographs of different isolates as observed under the trinocular microscope are shown in Fig 4. The growth of the pathogen was moderate to very fast in PDA medium, in contrast the growth of mycelium was very slow in CDA medium, this indicates the preferential utilization of carbohydrates and sugar available with PDA medium compared to CDA medium. Growth rate of aflatoxicogenic fungi on solid media The growth rate was fast almost in all isolates grown on PDA medium which could cover entire Petri plate in 10 days (8-9 cm) except two isolates MDKR and BLR-1 ( 5.5 to 6.5 cm) and hence the isolates were considered as fast growing. While, in the case of isolates cultured on CDA medium, the growth rate was slow almost in all isolates ( 3.75 to 5.0 cm) after 10 days except the two isolates, ADG-1 and ADG-2 collected from Amadaguru, Ananthapur district which growth rate of 6.2 and 6.0 cm respectively ( Table 4). The present study successfully attempted to detect aflatoxin producing fungi using cultural methods and differences exhibited when grown on different culture media. However, more work needs to be done with larger number and types of various samples in relation to their geographical origins. Molecular characterization of the fungal species involved followed by the development of database of aflatoxin producing fungi from maize, peanut, rice and spices may help us to accurately predict the possible levels of aflatoxins and other mycotoxins. Acknowledgement The authors of the research paper are grateful to University Grants Commission, New Delhi for providing financial support to carry the research work. References Aycicek H, Aksoy A, Saygi S. (2005). Determination of aflatoxin levels in some dairy and food products which consumed in Ankara, Turkey. Food Control. 16: Abbas HK, Shier WT, Horn BW, Weaver MA (2004) Cultural methods for aflatoxin detection, Journal of Toxicology, 23: Davis ND, Iyer SK, Diener UL (1987) Improved method of screening for aflatoxin with a coconut agar Medium, Applied and Environmental Microbiology, 53: Domir SC, Schreiber LR, Ichida JM, Eshita SM. (1992). Effect of elm selection, explants source and medium 15

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