Improvement in biocontrol activity of Bacillus subtilis UTB1 against Aspergillus flavus using gamma-irradiation Hamideh Afsharmanesh a, b, * , Masoud Ahmadzadeh b , Mohammad Javan-Nikkhah b , Keivan Behboudi b a Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NARS-NSTRI), Karaj 31485/498, Iran b Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj 31587-77871, Iran article info Article history: Received 4 August 2013 Received in revised form 17 February 2014 Accepted 18 February 2014 Keywords: Biological control Iturin Random mutagenesis Swarming Aflatoxin abstract Bacillus subtilis UTB1, a biocontrol bacterium isolated from Iranian pistachio nuts, has revealed to have antagonistic activity against aflatoxin-producing Aspergillus flavus R5. The strain UTB1 produces lip- opeptide compounds and is able to degrade aflatoxin B1. In this study, a random mutagenesis generated using different doses of gamma irradiation (0.1e3 KGy) was applied on B. subtilis UTB1 to improve its antagonistic activity against A. flavus R5. Five hundred bacterial colonies were selected randomly after irradiation, and their effects against A. flavus R5 were assessed in a plate assay. Forty-five colonies (9%) exhibited higher inhibition activity as compared to the non-irradiated wild type. Eight colonies out of the 45 were selected based on different polymorphism patterns obtained by repetitive element sequence polymorphism-PCR (ERIC and BOX) analyses; six of which could significantly inhibit the fungal growth utilizing washed cells and cell-free supernatants as compared to the parental strain. According to thin- layer chromatograms, the production of lipopeptides including surfactin, fengycin and iturin families increased in these six mutants. A considerable inhibition of the fungal growth was observed using bioautography analysis, which associated with iturins production. A. flavus sporulation and aflatoxin content decreased significantly in pistachio nuts treated with mutants M419 and M464 as compared to the strain UTB1. These results suggest that both mutants M419 and M464 could be promising biocontrol candidates against A. flavus in pistachio nuts. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Aspergillus flavus is one of the major food spoilage fungi due to the production of secondary metabolites, aflatoxins that are extremely toxic chemicals with carcinogenic, mutagenic and tera- togenic effects (Zhang et al., 2008). Aflatoxin production occurs when A. flavus or Aspergillus parasiticus invades nuts, cottonseed and corn under favorable conditions of temperature and relative humidity (Diener and Davis, 1967; Koehler et al., 1985; Laciakova et al., 2008). Pistachio nuts are among the commodities with the highest risk of aflatoxin contamination (Pittet, 1998). Iran is the largest pistachio nut producer (FAO, 2001; Cheraghali and Yazdanpanah, 2010) and aflatoxin contamination has been re- ported in Iranian pistachio nuts (Mojtahedi et al., 1979) especially during the harvest/postharvest period and storage stage. The traditional chemical control methods are neither economical nor effective at all times. Moreover, fumigation as well as other chemical control methods may have risks to human health and the environment (Droby, 2006). These disadvantages encouraged emphasis on biocontrol methods in recent years (Droby, 2006). In nature, aflatoxin-producing molds share the same habitat with other microorganisms such as lactic acid bacteria, Bacillus subtilis, Bacillus pumilus, Burkholderia cepacia and many molds, which can inhibit aflatoxin production. This inhibition may result from many factors including competition for space and nutrients in general, competition for nutrients required for aflatoxin production, and production of antiaflatoxigenic metabolites by co-existing micro- organisms (Munimbazi and Bullerman, 1998). The biocontrol potential of B. subtilis strains is based on their ability to produce antibiotic compounds such as lipopeptides including the fengycin, iturin and surfactin families (Stein, 2005; * Corresponding author. Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NARS-NSTRI), Karaj 31485/498, Iran. Tel.: þ98 9128050197. E-mail addresses: afsharmnh@alumni.ut.ac.ir, hafsharmanesh@nrcam.org (H. Afsharmanesh). Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro http://dx.doi.org/10.1016/j.cropro.2014.02.013 0261-2194/Ó 2014 Elsevier Ltd. All rights reserved. Crop Protection 60 (2014) 83e92