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Hydrogen Peroxide and Nitric Oxide mediated Disease Control of Bacterial Wilt in Tomato Plants

Plant Pathology Journal 2013 Dec; 29(4): 386–396.
doi:  10.5423/PPJ.OA.04.2013.0043

Reactive oxygen species (ROS) generation in tomato plants by Ralstonia solanacearum infection and the role of hydrogen peroxide (H2O2) and nitric oxide in tomato bacterial wilt control were demonstrated. During disease development of tomato bacterial wilt, accumulation of superoxide anion (O2−) and H2O2 was observed and lipid peroxidation also occurred in the tomato leaf tissues. High doses of H2O2and sodium nitroprusside (SNP) nitric oxide donor showed phytotoxicity to detached tomato leaves 1 day after petiole feeding showing reduced fresh weight. Both H2O2and SNP have in vitro antibacterial activities against R. solanacearum in a dose-dependent manner, as well as plant protection in detached tomato leaves against bacterial wilt by 106 and 107 cfu/ml of R. solanacearum. H2O2- and SNP-mediated protection was also evaluated in pots using soil-drench treatment with the bacterial inoculation, and relative ‘area under the disease progressive curve (AUDPC)’ was calculated to compare disease protection by H2O2 and/or SNP with untreated control. Neither H2O2 nor SNP protect the tomato seedlings from the bacterial wilt, but H2O2+ SNP mixture significantly decreased disease severity with reduced relative AUDPC. These results suggest that H2O2 and SNP could be used together to control bacterial wilt in tomato plants as bactericidal agents.

Bacterial wilt disease caused by Ralstonia solanacearum occurs in many plant species and leads to destructive and economical damages, especially, in tomato production in tropical, subtropical and warm temperate regions (Hayward, 1991). To invade and colonize host tissues successfully, soil-borne R. solanacearum has developed biochemical weapons; extracellular polysaccharides, cell-wall-degrading enzymes and type III secreted effectors (Macho et al., 2010; Saile et al., 1997; Valls et al., 2006). However, during infection process the bacterium experience reactive oxygen species (ROS) produced in host plants to arrest the bacterial growth (Flores-Cruz and Allen, 2009). Thus, the bacterium should overcome ROS-mediated host defenses by the activation of ROS-scavenging enzymes and the expression of oxidative stress tolerance gene (Colburn-Clifford et al., 2010; Flores-Cruz and Allen, 2011; Loprasert et al., 1996), and lead to compatible interactions between host plants and virulent bacterial strains. Virulence factors originated from R. solanacearum have been investigated to understand molecular machinery of the bacterial pathogenesis and to develop efficient disease control strategy in recent decades (Brown and Allen, 2004; Franks et al., 2008; Schell, 2000).

For the disease control of tomato bacterial wilt disease, multidirectional controls including cultural, biological and chemical methods have been applied so far. Chloropicrin was suggested as the most promising chemical for reducing tomato bacterial wilt when it was used as soil fumigant before transplanting (Enfinger et al., 1979). However, it can be irritating and phytotoxic under incomplete vaporization conditions in the treated soil. Antibiotic validamycin A inhibited in vitro growth of R. solanacearum and delayed symptom development of tomato bacterial wilt (Ishikawa et al., 1996). Commercial chemical pesticides containing copper hydroxide, copper hydroxide-oxadixyl, copper oxychloridedithianon and streptomycin-validamycin A, delayed tomato bacterial wilt when the bacterial pathogen was inoculated at the same time of pesticide application (Lee et al., 2012). Appearance of pesticide resistance has been concerned although there is no report on the resistance of R. solanacearum to antibiotics or chemical pesticides in tomato fields. In recent years, soil amendment, essential oils, antagonistic bacteria, plant growth-promoting rhizobacteria (PGPR) and plant defense-activating chemical agents have been tried to control tomato bacterial wilt as environmentally sustainable disease management (Anith et al., 2004; Ji et al., 2005; Nakaune et al., 2012; Nguyen and Ranamukhaarachichi, 2010; Park et al., 2007). More ecofriendly control methods need to be investigated for the integrated management of tomato bacterial wilt.

To minimize damages from pathogen infections, host plants have established sophisticated defense mechanisms including cell wall enforcement, accumulation of pathogenesis-related proteins and accelerated cell death (Deepak et al., 2010; Greenberg et al., 2004; van Loon et al., 2006). Plants can memorize prior pathogen infection and be ready for challenging pathogens. For the establishment of induced and systemic acquired resistance, a variety of plant defense signaling pathways are involved and cross-talked (Grant and Lamb, 2006; Koornneef and Pieterse, 2008; Liu et al., 2011; Pieterse et al., 2009). So far, several small chemicals are known to be de novo synthesized and accumulated during pathogenesis and hypersensitive defense responses in host plants. Salicylic acid, jasmonic acid and ethylene are among most distinguished defense signal molecules, but there is limited information whether these are mobile signals for systemic defense or not. More recently, H2O2 and nitric oxide have been demonstrated defense signal molecules which play roles in activation of plant resistance against pathogen attacks. H2O2 mediated plant defense responses by induced resistance against pathogen infection (Byun and Choi, 2004; Hafez et al., 2012). Elevated endogenous nitric oxide level in plants also resulted in enhanced disease resistance against viral, bacterial, oomycete and fungal pathogens (Chun et al., 2012; Fu et al., 2010; Guo et al., 2004).

Relatively high doses of H2O2 and nitric oxide can exert direct antimicrobial activities to kill microbes including plant pathogens. H2O2 showed direct antibacterial activity against Xanthomonas campestris pv. vignicola causing cowpea bacterial blight, and pretreatment of cowpea seeds and seedlings with H2O2 reduced the disease severity (Kotchoni et al., 2007). In vitro germination of sporangiospores of Peronospora tabacina causing tobacco blue mildew was gradually inhibited by increasing H2O2 concentration exogenously applied (Peng and Kuć, 1992). Endogenous generation of H2O2 in tobacco leaves mediated by peroxidase and cofactors simultaneous applications in the presence of NADH or NADPH significantly reduced disease severity of tobacco blue mold by P. tabacina infection (Peng and Kuć, 1992). Nitric oxide treatment inhibited in vitro spore germination, sporulation and mycelial growth of Aspergillus niger, Monilinia fructicola and Penicillium italicum, and fruits become rotten by these fungal infection during postharvest storage and marketing (Lazar et al., 2008). In vitro inhibition of spore germination and germ tube elongation was observed in grey mold fungus Botrytis cinerea treated with nitric oxide (Lai et al., 2011). Exogenous nitric oxide treatment of tomato fruits delayed the symptom development by direct antifungal activity as well as by indirectly enhancing resistance (Lai et al., 2011). These findings suggest that H2O2 and nitric oxide can be prevalently used for disease control including tomato bacterial wilt, although antibacterial activities of H2O2 and nitric oxide against R. solanacearum and phytotoxic effects on tomato plants have not been demonstrated yet. Genetically modified biocontrol bacterium, Pseudomonas fluorescens producing higher content of nitric oxide could confer much stronger disease suppression capability against tomato bacterial wilt (Wang et al., 2005). These studies promoted us to investigate whether H2O2 and nitric oxide can be applied directly in tomato plants to control bacterial wilt disease caused by R. solanacearum.

In this study, accumulation of ROS in tomato leaf tissues during the bacterial pathogenesis was investigated. In vitro antibacterial activity of H2O2 and nitric oxide, response of tomato plants to both chemicals and disease control of tomato bacterial wilt by these chemicals were evaluated.

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