1204 Plant Disease / Vol. 96 No. 8 Utilization of Filamentous Phage φRSM3 to Control Bacterial Wilt Caused by Ralstonia solanacearum Hardian S. Addy, Ahmed Askora, Takeru Kawasaki, Makoto Fujie, and Takashi Yamada, Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan Abstract Addy, H. S., Askora, A., Kawasaki, T., Fujie, M., and Yamada, T. 2012. Utilization of filamentous phage φRSM3 to control bacterial wilt caused by Ralstonia solanacearum. Plant Dis. 96:1204-1209. The wide host range of Ralstonia solanacearum, causal agent of bacte- rial wilt, and its ability to survive for long periods in the environment restrict the effectiveness of cultural and chemical control measures. The use of phages for disease control is a fast-expanding trend of plant protection with great potential to replace chemical measures. The fila- mentous phage φRSM3 that infects R. solanacearum strains and inacti- vates virulence on plants is a potential agent for controlling bacterial wilt in tomato. We demonstrated that inoculation of φRSM3-infected cells into tomato plants did not cause bacterial wilt. Instead, φRSM3- infected cells enhanced the expression of pathogenesis-related (PR) genes, including PR-1a, PR-2b, and PR7, in tomato plants. Moreover, pretreatment with φRSM-infected cells protect tomato plants from infection by virulent R. solanacearum strains. The effective dose of φRSM3-infected cells for disease prevention was determined to be approximately 10 5 CFU/ml. Because the φRSM3-infected cells can grow and continue to produce infectious phage particles under appropriate conditions, φRSM phages may serve as an efficient tool to control bacterial wilt in crops. Bacterial wilt, caused by Ralstonia solanacearum, is an im- portant plant disease of many crops, damaging more than 200 spe- cies in 50 botanical families, occurring widely in the world, and persisting in the environment (24). Some management strategies are currently employed to control this disease such as chemical control, soil treatment, crop rotation, and resistant plants (14). Although soil treatments such as modification of soil pH or heat have occasionally been effective in suppressing the pathogen, they are limited to small-scale agriculture and are unfriendly to the environment (8). Crop rotation has not been effective, because R. solanacearum has a wide host range and survives for long periods in the soil (2). Although the use of resistant plant cultivars has been reported to be the most reliable method to control bacterial wilt, it is not completely effective because cultivars exhibit reduction in yield and plant quality, often lacking in stability or durability (7,24). Thus, alternative control methods for bacterial wilt, which are more effective, safer to applicators, and have lower envi- ronmental impact, are still needed. Various studies have indicated that biological control of bacterial wilt could be achieved using antagonistic bacteria (10,27). Rhizo- bacteria like Bacillus spp. (31), Pseudomonas spp. (23), and Strep- tomyces spp. (17) are examples of bacteria with efficacy. Another potential biological agent to control bacterial wilt caused by R. solanacearum is avirulent mutants of R. solanacearum (15) through spontaneous or genetic mutation. Recently, we reported that filamentous φRSM-type phages (φRSM3 is a typical phage of this group) changed host bacterial cells to be avirulent after infec- tion (5). Numerous studies reported that the use of avirulent strains could reduce disease severity. Avirulent strains of Erwinia amylo- vora and E. chrysanthemi reduced fire blight disease on apple (34) and soft rot disease on saintpaulia plants (Saintpaulia ionantha) (30), respectively. Ciardi et al. (11) reported that an avirulent strain of Xanthomonas campestris pv. vesicatoria increased the tolerance of tomato plants against bacterial spot disease. Similar effects were also demonstrated by avirulent strains of R. solanacearum against bacterial wilt in some plants (32,35). Mechanisms underlying the control of bacterial diseases by avirulent strains are thought to involve production of bacteriocins and induction of plant resistance (4,9). Stem inoculation in tomato by an avirulent strain of the bacteria Clavibacter michiganensis subsp. michiganensis induced long-lasting, high-level-protection against the virulent bacterial strain (22). An avirulent strain of Pseudomonas syringae pv. pisi was shown to induce systemic acquired resistant (SAR) in pea (13). Moreover, Edreva (16) de- scribed a detailed mechanism by which an avirulent strain induces plant resistance via SAR through a salicylate acid signaling path- way. The aim of this research was to demonstrate the application of filamentous phage φRSM to control bacterial wilt in tomato caused by R. solanacearum. Materials and Methods Bacterial strains and bacteriophage. R. solanacearum strain MAFF (The Ministry of Agriculture, Forestry, and Fisheries of Japan) 106603 (race 1, biovar 3, and phylotype I) was from the National Institute of Agrobiological Sciences (Japan). Avirulent strain M4S (race 1, biovar 3, and phylotype 1) was from the Leaf Tobacco Research Center, Japan Tobacco Inc. (33). The bacterial cells were cultured in CPG medium containing 0.1% casamino acids, 1% peptone, and 0.5% glucose at 28°C with shaking at 200 to 300 rpm (25). Strain MAFF 106603 carrying a green fluorescent protein (GFP)-expressing plasmid pRSS12 was described previ- ously (26) and was cultivated in CPG containing kanamycin (50 μg/ml). In some cases, bacterial cells were cultivated in minimal medium (MM) containing 1.75 g of K 2 HPO 4 , 0.75 g of KH 2 PO 4 , 0.15 g of Na-citrate, 0.25 g of MgSO 4 , and 1.25 g of (NH 4 ) 2 SO 4 per liter (6). Bacteriophage φRSM3 (a member of filamentous φRSM-type phages belonging to the family Inoviridae) was de- scribed previously by Askora et al. (5). φRSM3 was routinely propagated using strain MAFF 106603 as the host. To collect suffi- cient phage particles, a total of 2 liters of bacterial culture was grown. When the cultures reached 0.1 unit at an optical density at 600 nm (OD 600 ), the phage was added at a dose of 0.01 to 0.05 Corresponding author: T. Yamada, E-mail: tayamad@hiroshima-u.ac.jp * The e -Xtra logo stands for “electronic extra” and indicates that Figures 1, 2, and 4 appear in color online. Accepted for publication 13 March 2012. http://dx.doi.org/10.1094 / PDIS-12-11-1023-RE © 2012 The American Phytopathological Society e - Xt ra *