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Plant growth-promoting rhizobacteria (PGPR) are being used as potential agents for plant growth promotion and biological control of plant pathogens. The combined use of PGPR with commercial fungicide, benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH), a systemic acquired resistance (SAS) might increase their efficacy and broaden the disease control spectrum. In the present study, the effect of Bacillus subtilis strain B4 (B4), PGPR applied individually or in combined with BTH for plant growth promotion and control of anthracnose infection by Colletotrichum orbiculare on cucumber plants was evaluated. There was significant growth promotion in terms of plant height, leaf length, and leaf width in combined application when compared to individual application of B4 (106 cfu/ml) or BTH (0.1 mM). Anthracnose disease was significantly suppressed by combined application of B4 and BTH when compared to individual application in cucumber plants. Additionally, the population of bacterial cells of B4 strain was found to be greater when cultured in growth medium amended with BTH compared to the growth of B4 when alone was cultured. The spectrophotometer analysis revealed that there were increased secondary metabolites of salicylic acid (SA) in the culture filtrate of B4+BTH compared to individual B4 strain. The increased L-Tryptophan utilization by B4 strain when combined with BTH for the production of indole acetic acid (IAA) was to enhance the growth promotion. Thus the integrated use of BTH and B4 strain may complement each other as an alternative management strategy against anthracnose disease on cucumber plants.
Key words: Bacterial agent, Fungicide, Growth enhancement, Systemic resistance, disease control, Colletotrichum orbiculare
Plant growth-promoting rhizobacteria (PGPR) are naturally occurring beneficial soil bacteria that colonize the plant roots, resulting in enhancement of plant growth and protection against certain pathogens in various crops (Van Loon, 2007). Biocontrol of plant pathogens achieved by PGPR strains can be mediated through several mechanisms, including induced systemic resistance (ISR), production of antimicrobial compounds and competition for nutrients and colonization sites with pathogens (Kloepper et al., 2004). Application of several PGPR strains by treatment with soil drench or seed soaking have been used to enhance the growth and for the protection of plants from various diseases (Glick, 1995; Kloepper et al., 2004). Use of bacterial biocontrol agents and compounds that induce systemic acquired resistance (SAR) in the plant have been reported as effective alternative tools for disease control (Louws et al., 2001). 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). Among them, BTH is a synthetic compound known as 'Actigard' that is commercially available, used to control several plan diseases caused by bacteria and fungal pathogens. The mechanisms for plant growth promotion and systemic resistance by PGPR have been extensively studied (Kloepper et al., 1999).
Previous works demonstrated that several bacterial determinants such as siderophores, SA and lipopolysaccharides (LPS) contributed to SAR (Van Loon et al., 1998). As a result of poor disease control and concern about excessive use of pesticides, facilitated researchers in identifying alternative disease management approach by combination of biocontrol agents with resistance inducers in order to achieve a better control of plant pathogens (Abo-Elyousr et al., 2009). It has been well documented in previous reports about the various ways of usage of integrated disease management such as the addition of organic manures and opposed chemical fertilizers increased growth, yield and fruit quality of vegetable crops (Yousef et al., 2001; Poudel et al., 2002). Ozores-Hampton et al., (1994) proved that there was an increased yield on squash plants when grown in municipal solid waste compost amended soil in spite of application of chemical fertilizers.
The use of alternative disease control methods can effectively replace the usage of chemical fungicides. Application of safety chemicals to activate SAR-type reaction provides novel alternatives to control diseases in agronomic systems. SA is the only plant-derived substance that has been demonstrated to be an inducer of SAR (Ward et al., 1991). There is only limited information available on enhancing systemic resistance and bio-control ability of introduced bacterial agent in combination with chemical inducer. From this view, the complex nature of mechanism of plant disease management by chemical elicitors in some cases necessitated us to study the role of BTH in enhancing the vitalities of bacterial cells, cell growth and their effect on disease suppression, and triggering the GUS expression in the host plant. To develop sustainable and integrated strategies to control anthracnose disease on cucumber, we investigated the combinations of SAR inducer, BTH with biocontrol agent, B4 in greenhouse experiments in this study.
MATERIALS AND METHODS
Isolation of bacterial strains and culture conditions. Rhizobacteria were isolated from cucumber rhizosphere soils in Korea. After shaking the excised roots to remove all but tightly-adhering soil particles to root segments (1.0 cm) were agitated in 50 ml of sterilized phosphate-buffered saline (PBS, pH 7.3) for 5 min. Diluted soil samples (106 and 105) were spread on tryptic soy agar (TSA) plates. A total of 130 bacterial isolates were selected based on differences in their colony morphology and pigment colour by visual observation. Purified strains were stored on nutrient medium at 4°C for 10-15 days. Among them, only one strain was selected based on in vitro ISR activity using 24-well plate assay upon challenge inoculation with SCC1 suspensions to PGPR treated seedlings (data not shown). This strain was tentatively identified by 16S rDNA analysis and maintained at -80oC in tryptic soy broth (TSB) with glycerol (20%) for long-term storage. For preparing bacterial suspensions, culture from -80°C was grown on TSA plates at 28°C for 24 h, and single colonies were transferred to TSB and incubated at 28°C for 24 h with shaking at 150 rpm. Bacteria were pelleted after centrifugation for 5 min at 8,000-g and resuspended in sterile distilled water (SDW) to a final concentration of 1-108 cfu/ml before application.
Effect of the combined use of BTH and B4 strain on cell viability, population dynamics, production of secondary metabolites and production of IAA by L-Tryptophan utilization. Among different bacillus strains, the most potential B4 strain isolated from cucumber rhizosphere was selected for further studies and incubated in TSB with different concentrations of BTH or without BTH at 28°C and different duration of time (0 to 100 h). The absorbance was measured at 600 nm using spectrophotometer (UV-2101PC, Japan). Secondary metabolites were measured by spectrophotometer. The increase in cell number and cell growth of B4 in TSB by the addition of BTH or without BTH was computed (Pattern and Glick, 2002). L-Tryptophan utilization study with BTH or without BTH was carried out according to Thakuria et al. (2004). The pure colonies from 24 h culture were inoculated into nutrient broth with 2% tryptophan or in the absence of tryptophan, and incubated at room temperature for 48 h. Five milliliters of culture was removed from each tube and centrifuged at 12000-g for 15 min. Two milliliter aliquot of the supernatant was transferred to a fresh tube and washed with ethyl acetate to extract free IAA like substance. This was then treated with 4 ml salkowsky reagent (1 ml 0.5 M FeCl in 50 ml HClO4) and incubated at room temperature for 25 min. the absorbance of the solution (pink colour development) was read at 530 nm. Sterile nutrient broth was used for blank. The concentration of IAA in the culture supernatant was determined using a calibration curve of pure IAA as a standard.
Preparation of C. orbiculare spore suspensions. The fungal pathogen, C. orbiculare isolated from the infected leaves of cucumber plants was cultured on potato dextrose agar (PDA) plates in the darkness for 10 days at 28oC. The conidia of C. orbiculare were harvested with SDW. The resulting conidial suspensions were filtered through two layers of cheesecloth into a flask to remove hyphal debris. The suspensions were adjusted to a final concentration of 1-105 conidia/ml using heamocytometer before challenge inoculation.
Plant growth promotion and suppression of anthracnose disease in cucumber. Cucumber plants were soil drenched with bacterial suspensions of B4 strain or BTH or in combination of both BTH (0.1 mM) and B4 stain at cotyledonary stage. Later, they were challenge inoculated with C. orbiculare suspensions seven days after treatment. The plants were transferred to greenhouse conditions after incubating them at 24oC for 24 h in humidity chamber. The number of anthracnose lesions per leaf was recorded after 7 days. Another set of plants were maintained for growth promotion. The growth promotion in terms of plant height, leaf length and leaf width were measured 14 days after treatment. For each treatment six replicates were used in a complete randomized block design and the experiment was repeated at least once.
Effect of combined use of BTH and B4 strain on GUS activity in PR-1a labeled tobacco. Seeds of Nicotiana tabacum L. cv. Xanthi-nc, genetically engineered with GUS reporter gene fused to PR-1a were kindly provided by J. Ryals (Novartis Agricultural Biotechnology Research Unit, Research Triangle Park, NC, USA). The seeds were germinated in the plastic trays (20-15cm) containing soilless potting mixture, commercially known as Flora Guard (TKS 2 INSTANT, Kultur substrate), Each germinated seedling was transplanted to plastic pots (10 cm diameter) containing the same potting mixture described as above, and kept in the greenhouse with daily watering. Six-week-old tobacco plants were soil drenched with 50 ml suspensions of B4 strain (1-108 cfu/ml) or BTH (0.1mM) or in combination of BTH (0.1mM) and B4 strain. Seven days after treatment, three leaves from each plant were collected and carried to lab aseptically for quantitative determinations of GUS activity using fluorometric GUS assay (Jefferson et al. 1987; Park and Kloepper 2000). GUS activity was expressed as nM of methylumbelliferone (MU)/10 mg of sample fresh weight/hour.
Statistical analysis. Data were analyzed with SAS JMP software, SAS Institute, USA (SAS, 1995). All the experiments were repeated at least once with similar results. For each experiment, data were analyzed separately, and the results of one representative experiment are shown. Significant differences in treatment means (mean±SE) on each sample data were determined using LSD at P=0.05.
Effect of BTH on population survival of B4 strain. In the present study, we evaluated identified the vitality of combined application of B4 stain with the chemical inducer BTH, and in imparting disease suppression of anthracnose in cucumber plants. The results indicated progressive increase in the bacterial population of B4 strain when amended with BTH, compared to the population of B4 stain without BTH (Fig. 1). The cell number was found to be relatively greater in combined application of B4+BTH (0.1 mM) compared to individual growth of B4 stain, which signifies the potentiality of BTH in promoting systemic resistance in host along with PGPR agent (B4 strain) at lower dosage (0.1 mM). The growth curve at B4+BTH (0.1mM) differed from the rest of other treatments. Furthermore, the number of bacterial cells of B4 stain increased to 16-105 cfu/ml at 0.1mM BTH, followed by 9-105 cfu/ml of B4 stain with 1.0 mM BTH and 10-105 cfu/ml with 0.01 mM BTH. There was a least cell number (3-105 cfu/ml) when B4 alone was used. The results clearly showed that the optimum concentration of BTH was found to be 0.1 mM for greater efficient growth of B4 than other concentrations. In the case of growth curve of B4 strain, there was increased cell viability when B4 strain was grown in combination with BTH rather than B4 strain alone was grown (Fig. 2). The results on time taken for bacterial cell growth of B4 stain revealed that the highest cell growth was observed in 0.1 mM BTH at 50th h of incubation and remained constant for further incubation, the next highest cell growth was observed at 1.0 mM BTH, and followed by B4 alone, indicating the enhancement of cell growth and faster cell multiplication at lower dosages of chemical elicitors than at higher concentrations.
Secondary metabolites and IAA production. The increased production of secondary metabolites such as SA and IAA were observed in combined application of B4 strain and BTH (0.1mM), when compared to the individual application of B4 strain (Fig. 3). Tryptophan mediated cell growth forms a basis for plant growth promotion. To ascertain this aspect on increased production of IAA, the optimum concentrations (100 and 300 ïg/ml) of tryptophan were used to increase the vitality of cells in the presence of BTH (0.1mM). The results showed progressive increase in cell growth with IAA production at higher concentration of tryptophan (Fig. 4). However, the IAA production by B4 strain when amended with BTH (0.1mM) was relatively greater effect than the IAA production by individual B4 strain. There was increased production of IAA when tryptophan at 300µg/ml was used and found to be the highest production of IAA compared to other concentration. Thus, the results clearly indicated that there was better utilization of L-Tryptophan in combined application of B4+BTH than B4 strain individually.
Plant growth promotion and suppression of anthracnose disease in cucumber. Plant growth promotion was observed in cucumber plants with combined application of B4 strain and BTH. The results indicated that there was an enhanced plant height in combined application of B4 strain and BTH (0.1mM) compared to the application of B4 strain alone (Fig. 5). Maximum plant height (27 cm) was recorded in B4+BTH (0.1mM) treated plants followed by B4 strain (20 cm) and BTH (20 cm) treated plants individually and 16.5 cm in untreated control plants (Fig. 5). On the other hand, the greater leaf length and leaf width were observed in B4 alone treated plants when compared to combined application of B4 strain and BTH. The role of combined application of B4+BTH was also assessed in controlling anthracnose of cucumber plants by soil drench (Fig. 6). The least number of lesions (<20 lesions) per leaf was recorded in combined application of B4 strain and BTH when compared to the individual application of B4 (40 lesions/leaf) or BTH (20 lesions/leaf). Whereas, the disease incidence was drastically increased by 160 lesions/leaf in untreated control. These results clearly showed that enhanced vitality of B4 strain application in combined with BTH at lower concentration (0.1 mM) resulted the significant change in plant growth parameters, and also ability to bring down the anthracnose disease incidence in cucumber plants.
Effect of combined use of B4 strain and BTH on GUS expression analysis in tobacco leaves. In order to ascertain the enhancement of defense gene activity by combined application of B4 strain and BTH, we studied PR-1a gene expression in tobacco plants (Table 1). The results clearly demonstrated that there was strong GUS expression in combined application of B4+BTH (0.1mM) followed by treatment with BTH (0.1mM) alone, and the least activity was observed in water treated control plants.
In the present study, the ability of B4 strain individually or in combined application with chemical inducer, BTH, was demonstrated to control anthracnose disease in cucumber plants under greenhouse conditions. 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 innate improvement in host defense (Von Rad et al., 2005). Activation of host defense by induction of chemical elicitors proved 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 strain and BTH (0.1 mM), and also supported to increase the bacterial populations. The host defense was triggered and significantly reduced anthracnose incidence on production of secondary metabolites, plant hormone IAA, by tryptophan utilization and accumulation of SA in the presence of BTH to enhance the plant growth parameters and disease suppression through systemic resistance, respectively. There are several determinants for mechanisms of growth promotion that include bacterial synthesis of the plant hormones such as indole-3-acetic acid (IAA), cytokinin, gibberellin, break down of plant-produced ethylene by bacterial production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and increased mineral and nitrogen availability in the soil (Timmusk et al., 1999).
The treatment with B4 strain in combination of BTH that elicits systemic protection to the plant is not yet fully understood. Previous studies have reported that SA- and ET-mediated responses increase during compatible 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 responsible 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 greater 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 defence 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 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 convergence and interactions among compounds signaling various types of induced resistance, that are triggered simultaneously with the functional outcome of either positive or negative, or neutral interactions (Bostock, 2005). It is not clear the exact mechanism which responses to stimuli from pathogenic or non-pathogenic microorganisms. In particular, SA-dependent SAR and JA/ET-dependent ISR can interact either synergistically or antagonistically (Pieterse and Van Loon, 1999).
Upon evaluation of integrated application of PGPR strain, B4 and plant activator BTH, a highly significant (P=0.05) suppression of anthracnose disease in cucumber was observed during its growth under greenhouse conditions upon pathogen challenge. The PGPR stain B4 and BTH (0.1 mM) were significantly effective relatively when compared to water treated control. Further, combined application of the B4 strain and BTH was highly effective to protect the plants from anthracnose compared to their individual application. Earlier, individual applications of PGPR strain and amino salicylic acid (ASA) have been reported to induce systemic resistance in susceptible mulberry cultivars against the infection of either brown leaf sport or leaf rust disease (Gupta et al., 2002). In the present study, the integration of B4 stain and chemical elicitor, BTH might have enhanced their potential to elicit ISR in cucumber against anthracnose infection. Previously, it has been reported that, the mixtures of different compatible PGPR strains may enhance the combinations of plant defense mechanisms more than that of individual PGPR strains (Raupach and Kloepper, 1998; Jetiyanon and Kloepper, 2002). The same may hold good for the integrated application of compatible PGPR stain B4 and chemical elicitor BTH as demonstrated in the present study. In biological control, most of the approaches used single biocontrol agent as an antagonist to a single pathogen and has resulted often in inconsistent performance (Ryu et al., 2005).
The results from the present study suggest that the use of BTH even at lower concentration (0.01 to 0.1 mM) triggered the host defense in combination with PGPR strain B4, which helped greater reduction of disease incidences of C. orbiculare in cucumber 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 an attractive tool for the improvement of disease control and plant growth promotion. Raupach and Kloepper (1998) suggested that, for conventional agriculture, combinations of biological and chemical control seem to be the most promising area for the future. Conway (1997) reported that Laetisaria arvalis and a foliar spray of the experimental fungicide CGA 173506, at reduced dosage of the recommended rate suppressed the disease on rosemary more than treatment with either 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 would have provided for successful disease control with reduced foliage damage in tomato. Earlier studies also showed that the integration of bio-agents and fungicides were found to be effective against several diseases caused by Phytophthora spp such as stem and root rot (Phytophthora vignae) of cow pea (Fernando and Linderman, 1994), crown and root rot (Phytophthora cactorum) of apple (Utkhede, 1987) and damping off (Phythium aphanidermatum) of tobacco (Mukhopadhyay et al., 1986).
It has been documented that colonization of plant roots by rhizobacteria is essential for growth promotion and biological control of plant pathogens (Kleopper et al., 1992). In the present investigation, the ability of individual B4 strain or in combined application with commercial pesticide, BTH to enhance the plant growth and increase the efficacy of biocontrol activity. The population dynamics of B4 strain into the culture media with or without BTH was also determined. Similarly, Yan et al. (Yan et al., 2003) reported that, when the PGPR strain was incorporated into a soilless medium at several densities, these remained stable for 4 weeks after planting. Kinsella et al. (2009) found that formulated B. subtilis strain in the rhizosphere of cucumber presented a small decline in logscale population with time. It is important to note that root colonization levels in sterilized conditions may differ from those in natural conditions. The present results showed that the commercially available BTH is also at a par with a combined application in respect of efficacy against anthracnose, but the integrated application could be better for eco-friendly management of anthracnose of cucumber.
In a conclusion, the results of the present study suggest that integrated control of anthracnose and growth promotion in cucumber can be achieved by combined use of PGPR strain, B4 and commercial fungicide, BTH by production of secondary metabolites, enhancing the bacterial population, and triggering the biosynthesis of plant hormones such as IAA. The optimum dosage of BTH was found to be 0.1 mM in combination with B4 strain for supporting plant growth and bringing down the anthracnose infection in cucumber plants. Increased reduction of disease incidence of anthracnose in cucumber by systemic resistance was confirmed by enhancing PR-1a gene in tobacco by GUS expression. 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.