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1 Available online at Life Science Archives Volume 1; Issue - 1; Year 2015; Page: Research Article BIOCONTROL OF Aspergillus flavus IN GROUNDNUT R. Sanskriti, Vijayalakshmi, R. Neelam, P. Ezilrani and J. Godwin Christopher*, School of Biosciences and Technology, VIT University, Vellore , Tamil Nadu, India. Abstract The groundnut is particularly valued for its protein content. Groundnuts contain more protein than meat and about two and a half times more than eggs. Aspergillus flavus is a predominant fungus which causes yellow mold of groundnut seedlings. Aspergillus flavus cause extensive economic losses either by destroying the plant or by contaminating peanut kernels with the aflatoxins. Groundnuts grown under drought stress may also be predisposed to subsequent aflatoxin in contamination during harvest, handling, or storage. Pseudomonas fluorescens work as a antagonist and inhibits the pathogenicity of A. flavus on groundnut. P. fluorescens were isolated from garden soil samples and selected for their growth inhibition of A. flavus. In this study, the antifungal activity of Pseudomonas fluorescens against A. flavus in groundnut was examined in laboratory and field trials. Inoculation of Pseudomonas fluorescens on groundnut resulted in significant reduction of seed infection by A. flavus, it also reduces the yellowish black spot from the leaves of groundnut. This results suggest that application of this bacteria is effective in reducing fungal population in groundnut. By the utilization of P. fluorescens as the antagonist of A. flavus, health of the groundnut plant can be maintained. So disease free healthy seeds can be produced and disease propagation can be prevented. Hence this investigation will be eco-friendly and useful in increasing productivity as well as storability of Groundnut seeds Published by JPS Scientific Publications Ltd. All right reserved ARTICLE INFO Article History: Received: ; Revised: ; Accepted: Keywords: Groundnut, Aspergillums flavus Pathogen) and Pseudomonas fluorescence (antagonist). 1. Introduction The cultivation of groundnut has its origin from South America and spread to Brazil, Southern Bolivia and North-western Argentina. Groundnut was introduced by the Portuguese from Brazil to West Africa and then to south-western India. India is the second largest producer of groundnuts (6.25 million metric tonnes ) after China (14.30 million metric tonnes ). Groundnut is the largest oilseed in India in terms of production, it accounted for per cent of the oilseeds production of the country during * Corresponding author : J. Godwin Christopher Tel.: ; address: godwinj@vit.ac.in Gujarat is the largest producer by contributing 25 per cent of the total production followed by Tamil Nadu (22.48%), Andhra Pradesh (18.81%), Karnataka (12.64%) and Maharashtra (10.09%) during The other important states where it is grown are Madhya Pradesh, Rajasthan, Uttar Pradesh and Punjab. Groundnut contains on an average 40.1 per cent of fat and 25.3 per cent of protein and is a rich source of calcium, iron and vitamin B complex like thiamine, riboflavin, niacin and vitamin. It is used not only as a major cooking medium for various food items but also for manufacture of soaps, cosmetics, shaving creams

2 J. Godwin Christopher /Life Science Archives 29 and lubricants. Important pathogens of the groundnut are Fusarium solani, Fusarium oxysporum, Macrophomina phaseolina, Rhizoctonia solani, Sclerotium rolfsii, Aspergillus niger, A. flavus etc (Singh and Oswalt, 1992). During May 1984, a seedling disease of groundnut was observed in a commercial groundnut farm in Atascosa County, Texas (Subrahmanyam et al., 1987). The A. flavus groups of fungi are facultative parasites. They invade plant tissues directly or attack tissues that have been predisposed by environmental stresses such as dry weather or damages caused by insects, nematodes, natural cracking, and harvest equipment (Pettit, 1984). A. flavus produces a toxin called aflatoxin which is carcinogenic to humans and animals (Thirumala Devi et al., 2002). The health of the consumers as well as the enhancing the economy of the farmers the groundnut breeding program should be carried out for aflatoxin resistance and thereby increasing the quality of the groundnut. According to Liang et al. (2006) and Cotty and Jaime (2007) derived that removal of contamination by aflatoxin is a multi-stage process. Pseudomonas fluorescens: They are obligate aerobes, motile, non- pathogenic saprophytes. They are readily available in soil, water and plant surface and have simple nutritional requirement. They are readily amenable to various genetic engineering technique. P. fluorescens have been extensively employed for control of various pathogen like.a flavus, Fusarium, Helminthosporium etc. They are very useful in controlling the disease due to the release of various secondary metabolites like antibiotics, siderophore and hydrogen cyanide (Trujillo et al., 2007). Aspergillus flavus: The A. flavus is a facultative fungus. They invade plant tissues directly or attack tissues that have been predisposed by environmental stresses such as dry weather or damages caused by insects, nematodes, natural cracking, and harvest equipment (Pettit, 1984). The yellow mold fungus, Aspergillus flavus, is commonly found in the seed of both rotten and apparently healthy pods of groundnut. Many strains of this fungus are capable of producing aflatoxins that render the seed unacceptable due to high toxicity for human or animal consumption (Reddy and McDonald, 1983). Aflatoxin contamination in groundnut can occur in the stems of seedlings, pods, and seeds. The fungus is capable of invading groundnut seeds before harvest, during postharvest drying, and during storage. Aflatoxins are carcinogenic and produced by the Aspergillus group of fungi that have been identified as B1, B2, G1, and G2 (Pettit, 1984). Yellow mold first appears on groundnut cotyledons after the emergence of seedlings. Necrotic spots become covered with masses of yellow-group spore heads of the A. flavus group of fungi. Fungus toxins are translocated throughout the seedling in the transpiration stream. Infected plants generally become stunted with symptoms of vein clearing chlorosis on the leaflets. Such seedlings lack a secondary root system condition known as "aflaroot." Yellow-green Aspergillus colonies develop on over mature and damaged seeds and pods (Singh and Oswalt, 1992). This study was focused on the antagonistic property of Pseudomonas fluorescens against A. flavus. The first part of this study was done in laboratory. Then the next part is in the field. The result of this study proves that P. fluorescens inhibit the growth of A. flavus. Thus it can be used as a biocontrol agent against yellow mold in groundnut. 2. Methodology 2.1. Isolation of Aspergillus flavus from groundnut seeds Groundnut seeds were taken, washed in running tap water for 10 min., surface disinfected by immersion in sodium hypochloride solution containing trace of tween 80 for 5 min. and placed on Czapez-Dox agar (Difeo laboratories, Detroit, MI) supplemented with Rose Bengal and streptomycin (bacterial inhibitor). The plates were incubated at 25 ºC in dark for 4 days. Then LPCB staining of isolated fungi was performed. After that the isolated fungi was subcultured on PDA slant for mass propagation.

3 J. Godwin Christopher /Life Science Archives Isolation of Pseudomonas fluorescens from soil Soil samples were taken in a sterile polythene bag from VIT garden 5 gm of Soil sample was serially diluted till 10-7 dilution. Spread plate was done in the dilutions of 10-5, 10-6 and The plates were incubated at room temperature for hr. The colony morphologies were noted and preliminary tests were performed. The isolate was sub-cultured on media and following tests were performed. Preliminary tests of Gram staining, capsular staining and hanging drop technique was done. Followed by different biochemical tests which include Indole production, Citrate utilization, Catalase test, Urease test, Arginine hydrolysis, Esculin hydrolysis, Mannitol motility test (As per the methodology of (Rajan and Christy, 2010) Antifungal activity of Pseudomonas fluorescens Well Diffusion Method PDA plates were prepared and suspension of A. flavus is mixed in PDA media and was poured in the plates. After solidification well were punched with the help of sterile borer. P. fluorescens was inoculated in nutrient broth and kept for incubation at room temperature for hrs and was centrifuged at 6000 rpm for 20 min. High concentrated cells were pipetted and was poured into the well. These plates were incubated at room temperature for hrs. (Rajan and Christy, 2010) Field Trials For the field study two different methods were adopted to infect the groundnut plant with A. flavus: Method 1: A. flavus was grown on PDA slants for weeks at 25 ºC in the dark and conidial suspension were prepared in sterile distilled water containing trace of tween 80. Seeds were surface sterilized with sodium hypochlorite (0.52%) for 20 seconds. Then the seeds were coated with A. flavus suspension for 5min, air dried for a period of overnight and planted in plastic bags. Method 2: Suspension of A. flavus was mixed with soil, then groundnut seeds were planted in soil Control of Aspergillus flavus Groundnut plant which is infected by A. flavus is directly treated with suspension of P. fluorescens. 3. Results 3.1. Isolation of Aspergillus flavus A. flavus which were grow and isolated from groundnut seeds are confirmed by LPCB staining. The growth on 4 th day is shown in Fig - 1. A. flavus was the predominant fungus followed by A. niger and A. fumigatus. In general, more fungi were isolated from surface sterilized seed then from unsterilized seed. The process of surface sterilization could have removed or diluted the fungicide present on the seed. Thus, enabling more fungi to grow out of the seed. A. flavus was isolated from seed surface in higher frequencies then other fungi, suggesting that A. flavus infection was deeply embedded in the seed (Subrahmanyam et al., 1987). Fig 1: Growth of A. flavus from surface disinfected seeds on Czapek dox agar

4 J. Godwin Christopher /Life Science Archives 31 Fig 2: LPCB staining shows conidiophores of Aspergillus flavus 3.2. Isolation of Pseudomonas fluorescens Pseudomonas fluorescens were isolated from garden soil were confirmed by performing several preliminary tests and biochemical tests. The morphological characteristics of the strain by preliminary test is shown in Table - 1 and result obtained by various biochemical parameters is shown in Table - 2. The growth of P. fluorescens on King s B medium is shown in Fig.3. The aim was to isolate P. fluorescens which showed antifungal activity against A. flavus. Subsurface garden soil was used for the isolation of the bacterium. Serial dilution was prepared till 10-7, thereby decreasing the bacterial load with each subsequent dilution. Selective medium for the growth of Pseudomonas spp., King s B medium was used. Spread plate technique, was employed for the isolation of the bacteria. The incubation temperature is ºC. The colony morphology were noted down, after the incubation period was over. Preliminary test comprising the various staining technique such as Gram staining and Capsular staining were carried out. Hanging drop experiment was carried out to check for the motility of the organism. The Gram negative rod shape, motile bacterial colonies obtained, were sub-cultured on Pseudomonas agar medium and were subjected to a series of biochemical tests. Fig - 3: Showing colonies of Pseudomonas spp on King s B medium Experiment Gram staining Capsular staining Hanging drop motility test Table - 1: Preliminary test Observation Pink colour, gram negative rod shape White colours rod against dark black background Movement of the organism was observed. Table - 2: The isolate was observed for the various biochemical parameters. The results obtained were as summarized below S.No. Test Result 1 Indole production (-) 2 Citrate utilization (+) 3 Catalase activity (+) 4 Urease activity (+) 5 Arginine hydrolysis (+) 6 Esculin hydrolysis (+) 7 Mannitol motility test (+)

5 J. Godwin Christopher /Life Science Archives Antifungal activity The result of the well diffusion method showed zone of inhibition (Fig.4). The Pseudomonas fluorescens shows inhibitory activity against Aspergillus flavus. The inhibition is thought to be because of the production of secondary metabolites or some antimicrobial compounds (Anjaiah et al., 2005). The process of centrifugation helped in breaking open the bacterial cells and the release of antimicrobial compound (Gulfeshan, 2010). for invasion. A. flavus invasion of pods are generally high when the crop was subjected to drought stress during pod development. The disease incidence was higher when the fungus was inoculated into the soil then when the fungus was applied to the seeds. Yellow mold first appears on groundnut cotyledons after the emergence of seedlings. Necrotic spots become covered with masses of yellow green spore heads of A. flavus group of fungi. Fungal toxins are translocated throughout the seedlings in the transpiration stream. Infected plants generally become stuned with symptoms of vein clearing chlorosis on the leaflets. Such seedlings lack a secondary root system, a condition known as aflaroot. Yellowgreen Aspergillus colonies develop on over mature and damaged seeds and pods Fig - 4: Showing Zone of inhibition by Pseudomonas fluorescens against Aspergillus flavus 3.4. Field study Groundnut seeds that were grown over the soil mixed with A. flavus positively infected all the plants with yellow mold disease (Fig. 5). The symptoms were seen right from the cotyledon stage. Necrotic lesions were seen (Fig. 6). The lesions on the cotyledons were covered with conidia of pathogen. Plants were stunted and chlorotic. Leaflets were small, had pointed tips, and vein-clearing. Development of the root system was also poor. Some of the infected plants eventually recovered from the disease and produced normal foliage. Similar symptoms were reported by El-Khadem (1968). Yellowish black spots (Fig.7) were observed on the leaves of groundnut plant. These symptoms developed after 2 weeks. The reason for such symptoms of yellow mold was attributed due to aflatoxin produced by the fungus at the infection site (Chohan and Gupta, 1968). A. flavus can invade groundnut pods at any stage of crop development but the post harvest conditions are often specially favourable Fig - 5: Showing positive infection of A. flavus on groundnut plant Fig - 6: Showing necrotic lesions on the cotyledons of groundnut seed.

6 J. Godwin Christopher /Life Science Archives References Fig 7: Showing yellowish black spot on leaves of groundnut plant 3.5. Biocontrol by Pseudomonas fluorescens After treatment with P. fluorescens, diameter of yellowish black spot which was developed on the groundnut leaves was found to be reduced shown in Table - 3. Biological control is a recent trend in disease management. P. fluorescens have been found to be the most important in the area of biological control. P. fluorescens has the ability to solubilize phosphate in vitro and to produce several antibiotics with high specificity against several microorganism. Table - 3: Reduction in the diameter of spot present on groundnut leaves S. No Day Diameter of the spot (mm) 1 1 st day th day No change 3 14 th day st day 4.3 The mechanism behind the biocontrol of disease needs more understanding. It was assumed to be because of the production of secondary metabolites. Hence, P. fluorescens act as biocontrolling agent which inhibits the growth and reduces the activity of A. flavus. Acknowledgment We thank VIT University for the help and support for extending necessary facility to carry out this research work. 1) Anjaiah, V., Thakur, R. P., & Koedam, N Evalution of bacteria and Trichoderma for biocontrol pre-harvest seed infection by Aspergillus flavas in groundnut. Biocontrol Science and Technology, 16(4), ) Chohan, J. S., & Gupta, V. K Aflaroot, a new disease of groundnut caused by Aspergillus flavus link ex fries. The Indian Journal of Agricultural Sciences, ) Cotty, P., & Jaime-Garcia, R Influences of climate on aflatoxin producing fungi and aflatoxin contamination. International Journal of Food Microbiology, 119, ) El-Khadem, M Die Bedeutung von Aflatoxinen fur die durch Aspergillus flavus verursachte keimlingskrankheit der erdnub. Journal of phytopathology, ) Gulfeshan, R Isolation and characterization of a biocontrol agent from soil and testing its efficacy against various pathogens. M.Sc. Thesis, VIT University, ) Liang, X. Q., Luo, M., Guo, B. Z Resistance mechanisms to A. flavusinfection and aflatoxin contamination in peanut (Arachis hypogaea). The Plant Pathology Journal, 5, ) Pettit, R. E Yellow mold and aflatoxin. In Compendium of Peanut Diseases ) Rajan, S., & Christy, R. S Experimental procedures in life sciences. First Edition, ) Reddy, D. V. R., & McDonald, D National seminar on management of disease of oilseed crops. Agricultural college and Research Institute, ) Singh, F., & Oswalt, D. L Major diseases of groundnuts. International Crops Research Institute for the Semi-Arid Tropics Patancheru, Andhra Pradesh 5, India, ) Subrahmanyam, P., Smith, D. H., Raber, R. A., & Stepherd, E An outbreak of yellow mold of peanut. Seedlings in Texas, 100, ) Thirumala Devi, K., Mayo, M. A., Hall, A.J., Craufurd, P. Q., Wheller, T. R., Waliyar, F.,

7 Subrahmanyam, A., Reddy, D. V. R Development and application of an indirect competitive enzyme linked immunoassay for aflatoxin M1 in milk and milk-based confectionery. Journal of Agricultural and Food Chemistry, 50, ) Trujillo, M., Velazquez, E., Miguelez, S., Jimenez, M., Mateos, P., & Martinez - Molina1 E. (2007). Characterization of a strain of Pseudomonas fluorescens that solubilizes phosphates in vitro and produces high antibiotic activity against several microorganisms. Biomedical and Life Sciences, 102, J. Godwin Christopher /Life Science Archives 34