Plants develop an enhanced defence capacity against wide range of plant pathogens after root colonization by certain strains of microorganisms. Plant growth-promoting rhizobacteria (PGPR) occur naturally in the soil that colonizes the rhizosphere, resulting in improvement of plant growth and control of pathogens in several crops (Van Loon 2007). Various PGPRs and synthetic analogs of naturally occurring plant activators have been reported to elicit induced systemic resistance (ISR) in a number of crop plants against various kinds of diseases (Gupta et al. 2008). The majority of PGPRs are to colonize the root surfaces, closely adhere the rhizosphere soil (Kloepper et al. 1999). Root colonizing bacteria have been studied for the past many years as inducers for increasing plant productivity and controlling plant pathogens (Kloepper 1992). Certain root colonizing PGPR strains including Bacillus spp. have been used for the protection of plants from several diseases by soil or seed applications (Glick 1995; Kloepper et al. 2004).
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The bacterial biocontrol agents and the SAR chemical inducers in the plants have been reported for the control of plant diseases (Raupach and Kloepper 1998; Louws et al. 2001; Wilson et al. 2002). Systemic acquired resistance (SAR) is the phenomenon by which plant defence mechanisms are activated by a contact with a pathogen or its metabolites that are structurally unrelated compounds, including salicylic acid (SA), 2,6-dichloroisonicotinic acid (INA), β-aminobutyric acid (BABA), and BTH (Benhamou and Belanger 1998). Many of these are being commercialized, among which, BTH is a chemical compound known as 'Actigard', commercially being used to control several bacterial and fungal diseases on plants. Kloepper et al. (1999) for the first time derived the hypothesis that the elicitation of plant defence by PGPR is related to ISR in the treated plants. However, ISR is different from SAR that triggers systemically plant defence response following hypersensitive response after inoculation with plant pathogens (Durrant and Dong 2004). The induced resistance by chemical elicitors may increase the significant cost (Heil and Ian 2002), and this may overcome by combined use of SAR and ISR agents. Concern about excessive usage of pesticides and poor disease control in various crops, necessitated researchers in identifying alternative disease management approach by combining use of chemical pesticides and biocontrol agents (Abo-Elyousr et al. 2009).
However, till today there is only limited information available on enhancing systemic resistance and bio-control ability of introduced bacterial agent combining with chemical inducer. Looking into these aspects, the complex nature of mechanism of plant disease management by chemical elicitors necessitated us to study the role of BTH in enhancing the vitalities of bacterial cells, cell growth and their effect on suppression of the disease development, and triggering the GUS expression in the host plants. To develop eco-friendly and integrated strategies for the suppression of soft rot disease on tobacco, we investigated the combinations of chemical elicitor, BTH with PGPR agent, B4 strain under greenhouse condition in the present study.
In the present study, the ability of B4 strain individually or in combined with chemical elicitor BTH, was used in controlling soft rot disease on tobacco. This is one of the few reports of the Bacillus spp. with synergistic effect in combination with chemical inducer, BTH that shows significant biocontrol of plant diseases. Recently, some of the chemical inducers such as 2, 6 dichloro isonicotinic acid, benzothiadiazole, methyl jasmonate and probenazole are being used for systemic resistance in host defense (Von Rad et al. 2005). Host defense activation through the induction of chemical elicitors by their molecular mechanism of action has been reported (Fofana et al. 2002; Yang et al. 2002). Recently, the role of hyaluronic acid from Streptomyces sp. as potential ISR agent in cucumber and tomato against major economically important diseases has been established by Park et al (2008). The present study clearly demonstrated the superiority of combined application of B4 and BTH (0.1 mM) in disease suppression of soft rot of tobacco by increased bacterial populations. The host defense was triggered and significantly reduced the disease incidence on production of secondary metabolites by accumulation of SA in the presence of BTH to enhance the disease suppression through systemic resistance.
The treatment with B4 in combination of BTH that elicits systemic protection to the plant is not yet fully understood. Previous studies have found that SA- and ET-mediated systemic resistance during the interactions of bacterial pathogens in tomato (Block et al. 2005; van Loon et al. 2006). The results in our study, demonstrate that general pathways might be involved for B4+BTH mediated systemic protection. B4+BTH induced activation of PR-1a GUS expression in tobacco, suggesting that the potential of combined application for activating the defense responses within the plant. With application of BTH, the expression of PR-1a was highly prominent; seemingly BTH mediated induced protection responses mainly through SA dependent pathway. Similar findings were reported by BTH application in tobacco and Arabidopsis, where the induced expression of defense genes was noticed (Friedrich et al. 1996; Gorlach et al. 1996). Von Rad et al. (2005) have reported the activation of PR-1a and PDF1.2 upon treatment with BION, a commercial formulation of BTH in Arabidopsis, In addition, the systemic increase in activities of ï¢-1-3-glucanase, chitinase and peroxidase in the leaves and at infection sites have also been widely reported upon treatment with BTH (Ward et al. 1991; Yedidia et al. 1999). Herman et al (2008) reported the synergistic effect of BHT in combination with PGPR strain in which the reduction of bacterial speck disease caused by Pseudomonas syringae in tomato was more in combined application than either PGPR or BTH alone was used. Several reports have confirmed the existence of interactions among compounds signaling various types of induced resistance, that are triggered simultaneously (Bostock 2005).
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In most approaches of the biological control, single biocontrol agent was used as an antagonist to a single pathogen and has resulted often in inconsistent performance (Ryu et al. 2005). Similarly, some of the experiments demonstrated a range of BTH treatment rates reported a phytotoxic effect or reduced plant productivity to disease control (Asai et al. 2002). From the recent research by Gupta et al (2008), it has been documented that, various PGPR strains individually or in combination with synthetic analogs of naturally plant activators such as 4-amino-n-butyric acid (ABA) and acetyl-salicylic acid (ASA) have demonstrated to reduce the brown leaf spot (Cercospora moricola) or leaf rust (Cercospora fici) diseases in mulberry (Morus alba L). Recently, crown rot disease of banana fruit was reduced by integrated approach of Trichoderma harzianum DGA01 along with hot water (Dionisio and Miriam 2012). Our results demonstrate that the use of BTH even at lower concentration (0.01 to 0.1 mM) triggered the host defence in combination with PGPR strain B4, which helped greater reduction of disease incidence of SCC1 in tobacco than individual application. Hence, there is a scope for developing a new formulation with reduced dosage of chemical elicitor. Van Wees et al. (2000) quoted that combining SAR and ISR will be a promising strategy to improve plant disease control and growth promotion. Raupach and Kloepper (1998) suggested that combined application of biological and chemical control seem to be highly promising area for sustainable agriculture in the future. Conway (1997) reported that a foliar spray of Laetisaria arvalis and the fungicide CGA 173506, at reduced dosage was found to be greater disease suppression on rosemary than either with the biocontrol agent or fungicide alone. Our results are corroborative with the results obtained by Obradovic et al (2005) which reported that the application of BTH in combination with biocontrol agent was a successful strategy for the control of plant diseases with reduced foliage damage in tomato.
In a conclusion, the results of the present study suggest that the combined application of BTH and B4 as a synergistic effect could control soft rot disease in tobacco. Increased reduction of disease incidence of soft rot in tobacco upon pathogen challenge was through systemic resistance, and this was confirmed by enhancing PR-1a gene in tobacco by GUS expression analysis. The optimum dosage of BTH was found to be 0.1 mM in combination with B4 strain for bringing down the soft rot infection in tobacco plants. This synergistic application of BTH and B4 might be helpful as one of the best delivery methods in the biological control by production of secondary metabolites such as SA, enhancing the bacterial population. The technology is now under the progress for large scale field validation trials for improving the productivity and disease management in an eco-friendly manner.