ORIGINAL ARTICLE Efficient colonization and harpins mediated enhancement in growth and biocontrol of wilt disease in tomato by Bacillus subtilis S. Gao, H. Wu, W. Wang, Y. Yang, S. Xie, Y. Xie and X. Gao Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Ministry of Education, Nanjing Agricultural University, Nanjing, China Significance and Impact of the Study: Bacillus subtilis and harpins are biological control agents with respective advantages. In this study, combinations of the both were applied to tomato in the form of hpaG Xooc -expressing B. subtilis, showed much better effects on resistance to wilt disease, and equivalent effects on plant growth promotion compared with the progenitor strain have a great potential in agri- cultural use. Keywords Bacillus subtilis, biocontrol, colonization, defence-related genes, HpaG xooc . Correspondence Xuewen Gao, Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China. E-mail: gaoxw@njau.edu.cn 2013/0829: received 27 April 2013, revised 2 August 2013 and accepted 3 August 2013 doi:10.1111/lam.12144 Abstract Both Bacillus subtilis and harpins stimulate plant growth and defence against various plant pathogens. In this study, B. subtilis 168 and two derivatives, surfactin producer OKB105 and combined surfactin and HpaG Xooc producer OKBHF, were applied to tomato plants to investigate the mechanisms underlying this effect. To evaluate colonization ability, strains were labelled with green fluorescent protein (GFP). Although biofilm distribution of the three strains was similar on root surfaces, Colonization populations of the two surfactin producers were approximately 2- to 3-fold higher than that of strain 168, and this was accompanied by significantly increased tomato growth. These results suggest that efficient colonization, possibly facilitated by surfactin production, enhanced the efficiency of plant growth promotion by B. subtilis. All three B. subtilis treatments caused plants to have less severe disease symptoms after inoculation with Ralstonia solanacearum, with plants treated with OKBHF being the most resistant, suggesting that hpaG Xooc improves biocontrol efficiency of B. subtilis. Analysis of defence-related genes showed a synergistic effect of HpaG Xooc on B. subtilis enhancement of the expression of the pathogenesis-related genes PR1b1 and PR-P2. In contrast, expression of the defence-related genes PINI and PINII was suppressed. Introduction Bacterial wilt of tomato caused by Ralstonia solanacearum is a destructive disease and responsible for severe economic losses in tomato production throughout the world (Elphin- stone et al. 2005). Although substantial research effort has been devoted to control this disease, no completely immune cultivars and effective chemical pesticides are available currently (Elphinstone et al. 2005). Recently, bio- logical control agents (BCAs) have been effectively applied in tomato cultivation. Various BCAs have been reported so far, including many antagonistic rhizobacteria such as Bacillus spp. (Wang et al. 2011; Xia et al. 2011), beneficial fungi and natural products such as harpins (Kim and Beer 2000; Peng et al. 2003). As typical representatives of plant growth-promoting rhi- zobacteria (PGPR), the Bacillus genus are often considered the best candidates for developing efficient biopesticide products because of the production of a vast array of bioac- tive compounds as well as highly adversity-resistant endosp- ores (Ongena and Jacques 2008). Bacillus-based products represent about half of the commercially available BCAs (Fravel 2005). Within the Bacillus genus, members of the B. subtilis species have been well studied and widely used as Letters in Applied Microbiology 57, 526--533 © 2013 The Society for Applied Microbiology 526 Letters in Applied Microbiology ISSN 0266-8254