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The present study identified Bacillus subtilis strain 21-1 as one of the potent plant growth-promoting rhizobacteria strains that promote growth of red-pepper (Capsicum annum L.) and suppress the phytophthora blight infection caused by Phytophthora capsici on red-pepper in two different soil conditions. BS21-1 was significantly effective in promoting the growth by supporting maximum plant height and leaf width in organic soil, and also enhanced the percentage of seed germination, compared to seed bed soil in red-pepper. There was only a minimum level of phytophthora blight infection on red-pepper in organic soil by BS21-1 treatment, while there was 30.5% disease incidence in seed bed soil after pathogen inoculation, and this reduced infection in organic soil was significantly greater than in seed bed soil by BS21-1 or BTH (0.1 mM) when compared to water treated control. Additionally, the plants treated with methanol extract of BS21-1 at 10-7 dilution, reduced soft rot incidence in red-pepper, determining the elicitors for induced systemic resistance (ISR). Based on these results, gene expression analysis using RT-PCR was carried out on organic soil for pathogenesis-related (PR) genes upon challenge inoculation with P. capsici on red-pepper. BS21-1 treatment enhanced the gene expression of PR-1 and β-1,3-gulcanase (GUS), while there was no expression of these two genes in BTH and water treated control. This resulted in enhanced defense activation against P. capsici. The overall results of this study suggest that BS21-1 might possibly be used as one of the biocontrol agents for disease suppression through induced systemic resistance against P. capsici, and for plant growth promotion.
Key words: Phytophthora blight, Growth enhancement, organic and seed bed soil, Induced systemic resistance, Pathogenesis-related gene expression,
Phytophthora blight, caused by the oomycete Phytophthora capsici, which is wide-spread globally, is one of the most devasting diseases affecting many crop plants including red-pepper, which causes a high level of damage to the crop (Lee et al. 2003; Hausbek and Lamour 2004). P. capsici is a soilborne pathogen and survives as oospores for many years in the soil. Zoospores of P. capsici can be readily dispersed across a field by rain and irrigation. P. capsici can infect more than 50 species of the plants (Tian and Babadoost 2004). In modern agriculture, the relative thrust is on the role of rhizosphere microorganisms that stimulate plant growth termed as plant growth-promoting rhizomicroorganisms (PGPR). Rhizobacteria positively affect plant growth and prevent plant diseases either directly or indirectly (Hall et al. 1996; Raupach and Kloepper 1998; van Loon and Glick 2004). The antifungal activities include competition for habitat (Demoz and Korsten 2006), inducing plant resistance (Kloepper et al. 2004), suppressing plant diseases (Compant et al. 2005), and production of antibiotics (Kloepper et al. 1989).
Most of the PGPR strains protect the plants through direct mechanisms by production of bacterial allelochemicals, including iron-chelating siderophores, antibiotics, biocidal volatiles, lytic enzymes and detoxification enzymes (Raupach et al. 1996). The rising popularity of organic farming has spurred efforts to raise red-pepper in an environment-friendly way. Responding to this, farmers have explored the use of microbes as a means to suppress P. capsici growth (Lim and Kim 2010). The varieties of microbial pesticides that have been explored over the past several decades included the use of rhizobacteria (Raupach and Kloepper 1998). PGPR have been studied extensively for promoting plant growth and for inducing systemic resistance as well. PGPR-mediated induced systemic resistance (ISR) has been shown to effectively suppress several fungal, bacterial and viral pathogens in a number of crops both in greenhouse and field trials (Kloepper et al. 2004). Sustainable agriculture employs many approaches and techniques to reduce the negative effects of conventional agricultural practices on the environment. One of these strategies is the utilization of soil microorganisms for the promotion of plant growth and control of plant diseases (Botelho and Hagler 2006). The widespread use of chemical pesticides and fertilizers has been a concern of the public and environmental protection agencies. The harmful chemicals may affect human health; contaminate the environment (Zaki et al. 1998; Heydari and Misaghi 2003).
The soil-inhabiting bacteria indigenous to the plant rhizosphere have been shown not only to improve plant growth, but also to trigger host defense mechanisms (Reitz et al. 2000; Ryu et al. 2004) which may vary in depending upon the soil types. Therefore, the two different soil types such as organic and seed bed soils were selected for the present study. Organic soil is mostly environmental friendly used, as it improves the soil structure and feeds the microorganisms which are beneficial for plant growth promotion. The seed bed soil usually amended with minimum amount of chemical fertilizers required by the plants which do not supports the growth of other beneficial microbes. Under organic cultivation, agro-chemicals and chemical fertilizers cannot be used for crop cultivation. The main objective of the present investigation was to study the efficacy of BS21-1 for plant growth promotion and disease suppression on red-pepper plants in Korea under organic and seed bed soil conditions.
Materials and Methods
Isolation of bacterial strains, culture conditions, and primary screening for biocontrol activity
Rhizobacteria were collected from different regions of Korea from red-pepper rhizosphere soils. After shaking the excised roots of red-pepper to remove all but tightly-adhering soil, root segments (1 cm) were agitated in 50 ml of sterilized phosphate-buffered saline (pH 7.3) for 5 min. Diluted soil samples (106 and 105) were placed on nutrient agar (0.3% beef extract, 0.5% peptone and 1.5% agar). A total of 62 bacterial isolates from soil and 33 from roots were selected based on the differences in colony morphology and pigment production. Among them, only 22 strains were selected based on their antagonistic screening test against major plant fungal pathogens in vitro (data not shown), and from these results, BS21-1 strain was selected based on root colonization assay (data not shown). This strain was used to test for biocontrol efficacy on red-pepper plants against P. capsici and growth promotion as well. The strain was maintained at -80oC in trypticase soy broth (TSB) with glycerol (20%) for long-term storage. For preparing bacterial suspensions, cultures from -80oC were grown on trypticase soy agar (TSA) for 24 h at 28oC, and single colonies were transferred to TSB and incubated at 28oC 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 treatment.
Organic and seed bed soils
Organic soil was obtained from Seoul Bio company product OHM1 (South Korea), which amended with compost from organic sources, and manure from organically fed animals. The ingredients of organic soil contains peat moss (60%), vermiculite (15%), perite (10%), peolite (5%), polomite (5%), guano (2%), humic acid (1%), wood (1%) and eucca extract (1%). Seed bed soil was obtained from TKS2 Flora Gard Ltd (Germany), which contains 10% perite and inorganic fertilizers.
Sources of plant pathogen and seed material, surface sterilization and germination of seeds
The pathogen P. capsici, used in the present study was obtained from plant pathology laboratory, National Academy of Agricultural Sciences (NAAS), RDA, Korea. The seeds of red-pepper (Capsicum annum L.) var. Manita, were obtained from Hungnong seed company Ltd, Jeonbuk, Korea. Seeds were surface sterilized by immersion in 1% aqueous sodium hypochlorite for 60 min and thoroughly rinsed in SDW prior to germination in Petri dishes containing sterile moist cotton. For seed germination test, 100 seeds were used in each of the soil. Plates were incubated at 25oC in the dark for 48 h. Germinated seeds were carefully removed from the cotton and sown in a mixture of peat-perlite-vermiculite (1:1:1) at four seedlings per pot (6 cm diameter). Plants were fertilized every 2 days with a nutrient solution containing (in nM) NO3 (12), PO4 (1.0), K (1.7), Mg (1.5), Ca (2.8), S (0.5), Fe (70), Mn (18), Zn (7.7), Cu (1.5), B (27.5), and Mo (0.5). Seedlings were grown on a greenhouse bench at 24 to 26oC with 16 h of light provided by high-pressure sodium lamps (100 micro Einstein m-2s-1) in two different soil types treated with BS21-1 or distilled water (negative control) by soil drench. After 7 to 10 days, the percentage of germination was recorded.
Preparation of spore suspensions of P. capsici
P. capsici inoculum was prepared as described by Ploetz et al. (2002). A 5 mm diameter mycelial plug of an isolate was transferred to a V8 agar plate. After one week of incubation at 25oC, V8 agar plugs with mycelia were placed onto Petri dishes containing V8 broth, and allowed to grow for another week at 25oC. V8 broth was then drained and plates containing mycelia were washed twice with SDW. The SDW was added to cover the mycelia on each plate, and then the plates were placed under wide-spectrum light at room temperature for 48 h to induce sporangial development. The sporangia were chilled at 4oC for 45 min to induce the release of zoospores. The spores were adjusted to a final concentration of 1-105 spores/ml by haemocytometer before using for challenge inoculation under greenhouse conditions.
Disease suppression and growth promotion by BS21-1 in red-pepper under two different soil conditions
Three-week-old red-pepper seedlings were treated with BS21-1 bacterial suspensions (1-108 cfu/ml) of 30 ml per plant by soil drench. Benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester BTH (0.1 mM) and SDW served as positive control and negative control, respectively, in both organic and seed bed soils. After four days, the disease assessment of P. capsici was performed on red-pepper by square plate (24-24 cm) assay using whole leaves. The leaves from BS21-1 or BTH or SDW treated plants were brought to the lab and placed in square plates containing moist tissue paper. The mycelial disks with P. capsici were placed on the leaves and then the plates were incubated for 2 days at 28oC. The percentages of disease affected leaves were recorded. One set of plants were maintained with the same conditions to measure the plant height and leaf width for plant growth promotion.
Induced disease suppression of P. carotovorum SCC1 by methanol extract of BS21-1
The extract from BS21-1 was obtained from cultures grown on TSA plates. The cells of 48 h old cultured on TSA plates were harvested and dissolved in 80% of methanol with water and concentrated using rotary vacuum evaporator. The residue of 10 ml was stored at 4oC until further use. The extract was diluted to 10-6 - 10-8 concentrations and three-week-old red-pepper plants were soil drenched with diluted methanol extract. BTH and SDW served as positive and negative control, respectively. After 7 days, the leaves were brought to the laboratory and disks (8 mm diameter) were made with sterile cork borer and then ISR activity against soft rot disease was performed in 24 well plates containing the bacterial suspensions of P. carotovorum SCC1 (1-106 cfu/ml). After 24 h of incubation at 28oC, the percentage of disease infected lesions per disk in each treatment was calculated.
Reverse transcriptase (RT)-PCR analysis for pathogenesis-related (PR) gene expression in red-pepper leaves against P. capsici
For ISR analysis, 30-day-old red-pepper seedlings were soil drenched with 30 ml BS21-1 cell suspensions or distilled water (negative control) or 0.1 mM BTH (positive control) per pot containing organic soil. After 7 days, third leaves of the treated plants were inoculated with mycelial pathogenic disks of P. capsici. The inoculated leaves were sampled after 24 h to analyze the expression of pathogenesis-related (PR) genes, and the tissues were frozen in liquid nitrogen until use. Defense-related β-1,3-glucanase, chitinase, PR-4, peroxidase, and PR-10 gene expressions were analyzed by RT-PCR using specific primers (Table 1). Leaf tissue samples were homogenized using a mortar and pestle, and the total RNA was isolated using easy-spinTM IIP Total RNA Extraction Kit (iNtRON Biotechnology, South Korea). RT-PCR was performed according to Kishimoto et al. (2005) with Ex Taq polymerase (Takara Biomedicals, Otsu, Japan). The reaction mixture contained 0.1µg of cDNA, 10 pMol each of forward and reverse primers, 250nM dNTPs, and 0.5U of Ex Taq polymerase in 20µl of buffer solution. The PCR was carried out in a MJ Research thermal cycler (PTC-100, USA) with the following conditions; 94oC for 5 min followed by 94oC for 1 min, 58oC for 1 min for 25 cycles, followed by 72oC for 10 min for final extension. PCR products were separated by electrophoresis on 1% agarose gel in 0.5 x TAE buffer at 80 V for 60 min. All RT-PCR experiments were conducted twice.
Data were analyzed (mean±SE) with SAS JMP software (SAS Institute, USA) (SAS, 1995) using LSD at P=0.05. Two independent experiments were performed with 12 replications (plants) per treatment. For each experiment, data were analyzed separately. Results of one representative experiment are shown.
Treatment of the red-pepper plants with cell suspensions of BS21-1 by soil drench enhanced plant growth and reduced P. capsici infection on red-pepper plants in one of the two soil conditions demonstrated. BS21-1 was isolated from red-pepper rhizosphere soils, and the isolate was further characterized by sequencing the 16S rRNA gene. The isolate, BS21-1 was effective to increase the percentage of seed germination in organic soil when compared to seed bed soil (Fig. 1a). For growth promotion of red-pepper under greenhouse conditions, BS21-1 treatment enhanced plant growth by supporting the maximum plant height (12.38 cm) and leaf width (2.96 cm) in organic soil. While, there were 7.78 cm and 1.74 cm of plant height and leaf width respectively, in seed bed soil (Fig. 1b), in which BS21-1 did not perform greater with respect to plant height and leaf width when compared to organic soil.
The strain, BS21-1 was effective in preventing P. capsici infection by systemic resistance. The induced suppression of disease development was observed in red-pepper plants in addition to plant growth promotion under greenhouse conditions. It was an interesting to note that under greenhouse conditions in organic soil, there was only a minor level (5%) of infection of phytophthora blight of red-pepper caused by P. capsici in BS21-1 treated plants compared to BTH (15.5%) and water treated control (45.33%), and there was an increased percentage of disease incidence in seed bed soil (Fig. 2). Further, to study the effect of metabolites from BS21-1, the methanol extract obtained from BS21-1 was used to induce suppression of disease against SCC1 in red-pepper plants (Fig. 3). Methanol extract at 10-7 dilution reduced the disease incidence to 28.36% compared to water treated control (85.2%) and BTH (37.57%). Compared to seed bed soil, the disease incidence was greatly reduced in organic soil treated with BS21-1. This data is supportive for the induction of systemic resistance by BS21-1, as the disease suppression might be due to the presence of ISR elicitor compounds in BS21-1.
In order to ascertain the enhancement of disease suppression through induced systemic resistance by BS21-1, pathogenesis-related (PR) gene expression by RT-PCR analysis for defense gene expression in red-pepper plants against P. capsici was studied only in organic soil, it was due to the effective results in BS21-1 treated organic soil rather than seed bed soil. Upon challenge inoculation with pathogen, BS21-1 treatment enhanced the gene expression of PR-1 and β-1,3-gulcanase (GUS), while there was no expression of these two genes in BTH and water treated control, respectively (Fig. 4). However, compared to the water treated control or BTH or BS21-1 without pathogen, all these three treatments after pathogen inoculation, enhanced expressions of CAChi2, CaPR-4, and CaPR-10. The overall results of this study suggest that BS21-1 might possibly be used as one of the biocontrol agents for disease suppression through induced systemic resistance against P. capsici, and for plant growth promotion in red-pepper plants.
From the overall results of this study show that Bacillus subtilis strain 21-1 (BS21-1) not only promoted growth in terms of plant height and leaf width, and increased seed germination percentage, but also significantly reduced the disease development caused by P. capsici relatively greater in organic soil compared to seed bed soil. The results of this study reveal the potential of BS21-1 for increasing growth, and protection from P. capsici infection through induced systemic resistance upon pathogen inoculation. Research reports have demonstrated that the effects of ISR are sometimes variable, both inter and intra-specifically (Heil 1999). Therefore, the stability of crop disease control attributed to ISR needs to be tested across different types and cultivars of plants. The beneficial effects of PGPR and PGPR-mediated disease resistance have been demonstrated under greenhouse and field conditions (Ramamoorthy et al. 2001). Several biocontrol agents alone or in combination have been employed to control phytophthora blight caused by P. capsici (Sid Ahmed et al. 2003; Ezziyyani et al. 2007). In the context of our present investigation, we studied the effect of introduced PGPR agent, BS21-1 in two different soil conditions for growth promotion and enhancement of disease suppression under greenhouse conditions.
A positive correlation between plant growth promotion and, disease suppression through ISR against P. capsici in red-pepper by BS21-1 treatment in organic soil was confirmed by increased plant height and leaf width as well as seed germination. The induced suppression of disease in red-pepper by BS21-1 under greenhouse conditions clearly demonstrated that BS21-1 had greater ability to induce systemic resistance in organic soil than seed bed soil. The level of biocontrol may vary with different parameters in the environment, and to some extent this explains why some biocontrol agents do not work under field conditions, while, they are effective in greenhouse conditions (Zhang et al. 2010). Our study also demonstrated the role of BS21-1 on disease suppression of P. capsici in red-pepper through ISR. These positive aspects brought down the importance and scope for the use of BS21-1 as one of the broad spectrum PGPR and ISR agents in red-pepper which has been evidenced by PR gene expression. Previously, Mendoz Garcia et al. (2003) reported that it is an important to determine to what extent environmental factors such as temperature, moisture, soil types and other parameters affect biocontrol performance.
Earlier, the role of wide varieties of other microbial fauna and flora coexist with PGPR in the natural environment, and have been shown to vary between soil and plant types due to crop rotations and addition of organic soil amendments, nutritional status of the plant, and rhizosphere conditions (Bent 2006). However, in the present study, we investigated the role of bacterial strain, BS21-1 as potential elicitor of ISR against P. capsici disease as well as of plant growth promotion in red-pepper. Members of the genus Bacillus are often considered as microbial factories for the production of a vast array of biologically active molecules against phytopathogens and beneficial interaction of Bacillus spp with plants by stimulating host defense mechanisms (Emmert and Handelsman 1999; Ongena and Jacques 2007). Molecular and physiological evidence of the PR gene expression demonstrated that root-treatment with BS21-1 was effective to establish ISR in leaves of the red-pepper plants against P. capsici infection. BS21-1 enhanced the expression of the defense related genes such as, PR-1, β-1,3-glucanase, chitinase, PR-4, peroxidase, and PR-10 only after pathogen challenge. Among these genes, PR-1 and β-1,3-glucanase were expressed only in BS21-1 treated plants which were not observed in BTH water treated controls. Lee and Hwang (2005) demonstrated that the indication of defense-related genes such as CABPR1 was essential for establishing the local and systemic acquired resistance (SAR) in red-pepper plants. Expression of PR-1 gene has been known to be triggered through a SA-dependent signaling pathway (Cameron et al. 1999). This gives us a wider scope for large scale delivery of BS21-1 as PGPR and ISR elicitor in different soil type regimes and also augmentation of these ISR elicitors will help in sustainable management of P. capsici disease in Korea.
More recently, the role of introduced bacilli strains for ISR and as PGPR was also reviewed by Kloepper et al. (2004) in different crops, and the role of PGPR agents in combined effect as ISR and SAR has been highlighted in tobacco by Venkatesan (2008). Our study demonstrated that the application of BS21-1 was greater in effective for growth promotion and disease protection in organic soil compared to seed bed soil under greenhouse conditions. Interestingly, when the inoculation of pathogen on red-pepper leaves after soil drench with BS21-1 bacterial cell suspensions, the PR genes were expressed strongly when compared to the plants without challenge inoculation with pathogen. These results are corroborative with the results of potentiation of defense activation by elicitor (Heil and Bostoc 2002). Thus, this provides great opportunity for the use of BS21-1 as one of the effective PGPR agents in suppressing P. capsici in red-pepper through induced systemic resistance at different soil conditions for sustainable cultivation in Korea.
The study was supported in part by a grant (Project code: PJ0067412012) from National Academy of Agricultural Sciences (NAAS), RDA, South Korea, 441-707.