406 Abdjah (Selection and Characterization).cdr SELECTION AND CHARACTERIZATION OF SIDEROPHORE-PRODUCING RHIZOBACTERIA AND POTENTIAL ANTAGONISTIC ACTIVITY TOWARD Ralstonia solanacearum ABDJAD ASIH NAWANGSIH , IDA PARIDA , SURYO WIYONO and 1* 1 1 JUANG GEMA KARTIKA 2 1 aDepartment of Plant Protection, Faculty of Agriculture, Institut Pertanian Bogor, Bogor 16680, Indonesi 2 aDepartment of Agronomy and Horticulture, Faculty of Agriculture, Institut Pertanian Bogor, Bogor 16680, Indonesi Received 24 June 2014/Accepted 9 November 2016 ABSTRACT Ralstonia solanacearum is an important disease of tomato. An alternative method to control the disease is the application of biocontrol agents. Plant Growth-Promoting Rhizobacteria (PGPR) could be used as potential biocontrol agents. PGPR with siderophores is among compounds having important role in disease suppression. This experiment was conducted to select and characterize the siderophore-producing rhizobacteria from tomato and to determine their potential as antagonistic agents for R. solanacearum. Candidates of the PGPR were isolated from tomato grown in West Java Province, Indonesia. The isolates were detected as siderophore-producing bacteria using CAS medium. Among 29 isolates producing siderophore and having negative result on hypersensitivity reaction, two isolates provided the widest diameter of inhibition zone toward R. solanacearum. Both isolates were CP1C and CP2D with diameter of inhibition zone up to 3.6 and 7.0 mm, respectively. Based on the sequence of 16S rDNA, isolate CP1C was identified as Brevundimonas sp., while isolate CP2D was identified as Enterobacter sp. Both bacteria did not cause negative effect on the increasing plant height and dry weight of the plants, compared with control. Keywords: Bacterial wilt, biocontrol, Brevundimonas, Enterobacter, PGPR BIOTROPIA 4 2 7 85 93 Vol. 2 No. , 201 : - DOI: 10.11598/btb.201 .2 . .7 4 2 406 * Corresponding author: asnawangsih@yahoo.com 85 INTRODUCTION Bacterial wilt of tomato caused by Ralstonia solanacearum is one of important diseases of tomato in tropics and subtropics area (Jeung et al. 2007). The bacteria survive for a long time in the soil (Wang & Lin 2005). The bacteria also multiply in the xylem as well as attacking the xylem and affect the water and nutrient translocation, causing wilting and death of the plant (Agrios 2005). Among the control methods to control the disease, is the application of biological control (biocontrol) agents (Yuliar et al. 2015). According to Jenifer et al. (2013), one of the most important mechanisms responsible for suppressing Pseudomonas sp. (a plant pathogen) is siderophore- mediated competitions for iron. Siderophores (from the Greek “iron carriers”) are small ferric-ion-specific chelating agents produced by bacteria and fungi which grow in low iron condition causing them to scavenge iron from the environment and to make iron available to the microbial cell (Neilands 1995; Pal & Gokarn 2010). Siderophores are also known to bind molybdenum and lead (Pal & Gokarn 2010). Sayyed et al. (2005) reported that siderophore- producing Pseudomonas sp. plays vital role in stimulating plant growth and in controlling several plant diseases. In Pseudomonas fluorescens the pigment produced is a siderophore. Pigment production is increased in the presence of sodium, potassium, lead, molybdenum, cadmium a n d a m m o n i u m s u l p h a t e ( ( N H ) S O ) 4 2 4 (Bhattacharya 2010; Sayyed et al. 2005). Adding exogenous amino acid did not significantly increase the production of siderophore (Hu & Xu 86 BIOTROPIA Vol. 24 No. 2, 2017 Java Province, Indonesia. From each village, 1,500 g of rhizosphere soil samples (with tomato roots) were collected from five different plots (300 g per plot as subsample) and thoroughly mixed using trowel, until becoming a composite sample. From each composite sample, 10 g of rhizosphere soil was immersed in 90 mL 0.85% NaCl. After a serial dilution, 0.1 mL of each dilution was inoculated on Chrome Azurol Sulphate (CAS) medium (Gross 1990; Louden et al. 2011). Inoculation was repeated three times. Plates containing the inoculation were incubated at room temperature (±28 C). Numbers of o colony of siderophore-producing rhizobacteria were calculated at 24 - 48 hours after incubation, based on the production of orange zone around colony of the bacteria. Successfully isolated bacteria were transferred to King's B agar medium and separated from each other to obtain pure culture. Each isolate was preserved in Nutrient Broth (NB) with 20% glycerol and kept in -20 C environment. For daily maintenance, o isolates were preserved in NB and kept at room temperature (±28 C) o . Hypersensitive Reaction Test Hypersensitive Reaction (HR) test was conducted to select the nonpathogenic bacteria as candidates of biocontrol agents. The test was 8 9 conducted by inoculating 10 – 10 cfu/mL of rhizobacteria on tobacco leaves. One milliliter of suspension was injected into tobacco leaf using 5 mL sterile syringe. Successful injection was indicated by water soaked zone around the point of injection. Inoculation of each isolate was conducted in duplo. Inoculated leaves were o incubated at room temperature (±28 C) for 24 hours. Leaves showing necrotic symptoms before 24 hours were counted as having positive reaction to the hypersensitive reaction and thus, be eliminated. Antagonistic Activities of Siderophore- producing Rhizobacteria against In vitro Ralstonia solanacearum Bacteria isolates having negative reaction on the previous hypersensitive test were tested on their antagonistic activity toward R. solanacearum. Both bacteria, i.e. R. solanacear um and rhizobacteria, were grown on a King's B agar 2011). Sayyed et al. (2005) also reported that Mn, Hg and Co showed inhibitory effect on siderophores growth and production. Presence of potassium, magnesium and calcium had little inhibitory effect on siderophores production compared to controls (Battacharya 2010). Sayyed (2005) reported that production of et al. siderophores by bacteria was affected by growing media. In growing media such as Nutrient Broth (NB) and MacKonkey's Broth (MB), siderophores were not produced because both media are luxurious media having high content of Fe. Fe- free succinic acid medium (SM) was found to provide maximum (92.25%) siderophores production in comparison to 88.00% in Barbhayya Rao broth (BR), 79.00% in Cassamino Acid broth (CAA) and 48.00% in Enrichment Medium (EM) (Sayyed 2010). A study on pH et al. effect to the production of siderophores by Rhizobium sp. showed that the growth and production of siderophores started at pH 4.5, reaching maximum at neutral pH; at pH 10.0, there was no siderophores production (Sridevi et al. 2008). Study on genetic diversity of tobacco rhizosphere which produces siderophore demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium; some of them are , Pseudomonas Enterobacter Serratia Pantoea Erwinia , , , and Stenotrophomonas γ-Proteobacteria which belong to (Tian 2009).et al. This experiment was conducted to select and ch a r a c t e r i z e t h e s i d e r o p h o r e - p r o d u c i n g rhizobacteria from tomato and to determine their potential as antagonistic agents for R. solanacearum. MATERIALS AND METHODS Isolation and Quantification of Siderophore- producing Rhizobacteria Siderophore-producing rhizobacteria were isolated from rhizosphere samples taken from healthy tomato plants, collected from tomato field in Cipanas Sub-district (Cianjur District) and Lembang Sub-district (West-Bandung District). In Cipanas Sub-district, samples were collected from three villages, while in Lembang Sub-district samples were collected from two villages. Both sub-districts are the center of tomato field in West 87 Siderophore-producing Rhizobacteria and potential antagonistic activity toward Ralstonia solanacearum – Nawangsih et al. plate. Five hundred micro liter suspension of R. solanacearum (10 - 10 cfu/mL) was spread on the 8 9 surface of the King's B agar plate. After being air dried, three sterilized filter papers having 0.5 cm diameter were placed besides the agar plate with 2 cm distance from each other. Filter paper in the center was inoculated with 50 µL of sterilized distilled water and designated as control. The two other filter papers were each inoculated with 50 µL suspension of one isolate of rhizobacteria. Each treatment was repeated three times. Inoculated plates were incubated at room temperature (±28 C). The antagonistic activity o was observed at 24 hours after incubation. Antagonistic activity was indicated by the production of inhibition zone around the filter paper inoculated with siderophore-producing rhizobacteria. E f f e c t o f S i d e r o p h o r e - p r o d u c i n g Rhizobacteria on the Viability of Tomato Seeds Tomato seeds (Arthaloka and Ratna varieties) were dipped in 10 - 10 cfu/mL suspension of 7 8 siderophore-producing rhizobacteria for 16 hours before being planted on sterilized mixture of soil and compost (1 : 1 ratio). The soil mixture was put in 30 - 50 cm polyethylene pot tray having 128 holes. The experiment was arranged as completely randomized factorial design with 7 isolates of biocontrol agents and one control (without bacteria) as the first factor and two tomato varieties (Arthaloka and Ratna) as the second factor. Thus, 16 treatments were applied in this experiment. Each treatment was replicated three times. There were 48 units in total; each unit contained 15 seeds. One tomato seed was grown in one hole of polyethylene pot tray. The isolates of biocontrol agents used in this experiment were CP1C (code of one isolate of bacteria), CP2B, CP2D, CP3E, LB1A, LB1C and LB1L. Seeds dipped in sterilized distilled water were used as control. Layout of the treatments was shown in Table 1. Total of the emerging seedlings were calculated every day. Seed viability (SV) was calculated using the following formula: Total normal seedlings SV (%) = x 100% Total seeds sown E f f e c t o f S i d e r o p h o r e - p r o d u c i n g Rhizobacteria on the Height, Fresh and Dry Weight of Tomato Plants An experiment to test the effect of siderophore-producing rhizobacteria toward the height, fresh and dry weight of tomato plants was conducted in a green house. Tomato seeds of Arthaloka and Ratna varieties were dipped in suspension of siderophore-producing rhizobacteria (10 - 10 cfu/mL) for 16 hours 7 8 before being planted in 10 x 15 cm polybag filled with 2.5 – 3 kg of sterilized mixture of soil and compost (1 : 1 ratio). The experimental design applied was completely randomized factorial design having similar layout with the one presented in Table 1. The only difference was that each unit contained 5 seeds. Plant height was measured every 5 days starting from the day when the first leaf was fully opened. Total area under height of plant growth cur ve (AUHPGC) was calculated using Table 1 Layout of experiment to test the effect of siderophore-producing rhizobacteria toward the viability of tomato seeds Tomato Varieties Isolate’s code CP1C CP2B CP2D CP3E LB1A LB1C LB1L Control Arthaloka Repl 1 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds Repl 2 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds Repl 3 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds Ratna Repl 1 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds Repl 2 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds Repl 3 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 88 BIOTROPIA Vol. 24 No. 2, 2017 modification of formula reported by Van der Plank (1963): where: y = increasing of plant height at the next i+1 observation y = increasing of plant height at the time of i observation t = the next observation (II, III, …, VI) i+1 t = time of observation (I, II, …, V)i Two months after planting, all plants (five plants) from each replication of each treatment were rooted and weighted using digital balance. The average of the five plants represented data of fresh weight for each replication. Dry weight of plant was determined by drying the fresh plants in the oven at 100 C. The weight of the sample was o checked periodically until the weight of the sample was constant. Characterization and Identification of the Siderophore-producing Rhizobacteria Seven isolates of the siderophore-producing rhizobacteria were characterized based on microscopic and colony appearances, as well as on physiological and biochemical properties, following the methods of Klement . (1990) et al and Schaad . (2001). Two isolates of the et al siderophore-producing bacteria having potential as biocontrol agents were genetically identified by sequencing the 16S rDNA gene. DNA was extracted from log phase culture using phenolchlorofor m extraction procedure (Sambrook & Russel 2001). The 16S rDNA gene was amplified using universal primer for prokaryotes which were the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'GGTTACCTTACG- ACTT-3'). Total volume reaction for Polymerase Chain Reaction (PCR) was 25 µL consisted of 1 µL of DNA template; 12.5 µL of 1x Ready mix PCR, 1.5 mM MgCl , 0.2 mM dNTPs and Taq 2 polymerase 5 units/reaction; 9.5 µL of ddH O; 1 2 µL of 120 pmol Primer 27F; and 1 µL of 120 pmol Primer 1492R. PCR was performed under the following conditions: one cycle of pre- o denaturation at 95 C for 5 minutes, followed by 35 o cycles of denaturation at 95 C for 1 minute, o annealing at 55 C for 1 minute and extension at o 72 C for 2 minutes. The reaction was terminated o with a final extension at 72 C for 10 minutes. The PCR products were sent to the First Base Laboratory, Malaysia for sequencing. BLAST searches were performed for sequences obtained to find the similarity with sequence data in GeneBank. Data Analysis Data of the bacterial population was analyzed using t test of Minitab program version 13.3. Effects of the bacteria to the plant growth were statistically analyzed using ANOVA for completely randomized factorial design with isolates of biocontrol agents as the first factor and tomato varieties as the second factor. Treatment means were compared using the DMRT test at 5% level of significance. SAS Program version 9.1 was used for performing statistical analyses for completely randomized factorial design and for the DMRT test. RESULTS AND DISCUSSION Abundance and Antagonistic Activities of Siderophore-producing Bacteria Based on the t test, the average of siderophore- producing bacteria isolated from Cipanas District 7 was 1.98 10 cfu/g, which was not significantly different with those from Lembang District 7 having average of 5.3 x 10 cfu/g. Colonies of the siderophore-producing bacteria on CAS medium were shown in Figure 1. Siderophore production was indicated by the orange color around the colony of the bacteria. Figure 1 shows that each colony produced different amount of siderophores, indicated by the diameter of orange area around each colony of bacteria. Among the 60 isolates of siderophore- producing rhizobacteria, 31 isolates positively showed hypersensitive reaction on tobacco, while 29 others showed negative reaction. Bacteria were also tested further for their antagonistic activities against . Based on the Ralstonia solanacearum antagonistic test, 16 isolates positively produced inhibition zone (Fig. 2) having diameter between 0.5 to 7.0 mm. Among those 16 isolates, 9 isolates were HR positive which had to be eliminated from being candidate of biocontrol agents. The AUHPGC n – 1 y y i i+ +1 2 t i +1 – tiS( (( ( 89 other 7 isolates were HR negative, having respective diameter of the inhibition zone of CP1C (3.6 mm), CP2B (2.3 mm), CP2D (7.0 mm), CP3E (5.0 mm), LB1A (1.6 mm), LB1C (1.6 mm) and LB1L (0.5 mm). The widest diameter of inhibition zone was produced by isolate CP2D which was isolated from Cipanas (Table 2). Microorganisms growing under aerobic conditions need iron for a variety of functions, including reduction of oxygen for ATP synthesis, reduction of ribotide precursors of DNA, for formation of heme and for other essential purposes. A level of at least one micromolar iron is needed for optimum growth (Neilands 1995). Figure 1 Production of siderophores by tomato rhizobacteria on CAS agar was indicated by yellow-orange color around colony of bacteria Figure 2 Production of inhibition zone (arrow sign) by the isolate of siderophore-producing rhizobacteria (Note: The inset shows magnification of the inhibition zone) Table 2 Isolates of siderophore-producing rhizobacteria which produced inhibition zone against R. solanacearum on Kings’s B agar Isolate code1) Diameter of inhibition zone (mm) Isolate code Diameter of inhibition zone (mm) Isolate code Diameter of inhibition zone (mm) CP1B2) CP1C CP2B CP2C CP2D CP2H 6.8 3.6 2.3 0.6 7.0 4.5 CP2L CP2S CP3E CP3M CP3T LB1A 1.6 2.6 5.0 2.2 4.0 1.6 LB1C LB1D LB1E LB1L 1.6 0.5 0.5 0.5 Note: 1) CP = isolates from Cipanas; LB = isolates from Lembang 2) Isolates written in bold were positively causing Hypersensitive Reaction (HR) Siderophore-producing Rhizobacteria and potential antagonistic activity toward Ralstonia solanacearum – Nawangsih et al. Table 3 Effect of siderophore-producing rhizobacteria on tomato seed viability of Arthaloka and Ratna varieties Treatment Seed viability (%)*) Var. Arthaloka Control CP1C CP2B CP2D CP3E LB1A LB1C LB1L Var. Ratna Control CP1C CP2B CP2D CP3E LB1A LB1C LB1L 93.33 ab 86.63 abc 88.83 abc 84.40 abc 88.87 abc 75.50 abc 100.00 a 95.53 a 66.60 c 75.53 abc 79.97 abc 84.43 abc 75.53 abc 68.83 bc 66.63 c 84.30 abc Note: *) Means in the same column followed by the same letter are not significantly different according to Duncan Multiple Range Test (p < 0.05) 90 BIOTROPIA Vol. 24 No. 2, 2017 Table 4 Effect of siderophore-producing rhizobacteria on the increase of tomato plant height of Arthaloka and Ratna varieties Treatment Plant height increase (cm)1) AUHPGC3) (cm days) I 5 DAP2) II 10 DAP III 15 DAP IV 20 DAP V 25 DAP VI 30 DAP Var. Arthaloka Control CP1C CP2B CP2D CP3E LB1A LB1C LB1L Var. Ratna 1.04 abcde 0.99 bcde 1.51 abc 1.69 ab 1.23 abcd 1.66 ab 1.71 ab 1.87 a 3.57 a 1.42 a 2.30 a 2.32 a 2.78 a 2.48 a 2.43 a 2.69 a 2.03 b 3.83 a 2.04 b 2.24 b 2.00 b 2.25 b 1.81 b 1.81 b 2.86 a 2.49 a 2.65 a 2.61 a 2.58 a 3.05 a 2.70 a 2.61 a 4.34 a 4.53 a 4.74 a 4.66 a 3.93 a 4.61 a 4.44 a 4.19 a 6.85 a 7.50 a 7.20 a 7.81 a 6.43 a 6.98 a 6.63 a 6.65 a 83.75 a 82.56 a 80.40 a 82.90 a 75.53 a 83.53 a 77.78 a 77.78 a Control CP1C CP2B CP2D CP3E LB1A LB1C LB1L 0.79 cde 1.09 abcde 0.29 e 0.48 de 0.87 bcde 0.91 bcde 0.53 de 0.40 de 1.99 a 0.99 a 1.51 a 1.91 a 1.23 a 1.78 a 1.45 a 2.00 a 1.89 b 3.73 a 1.89 b 2.03 b 1.87 b 1.46 b 2.0 b 1.81 b 1.97 a 2.11 a 1.31 a 1.85 a 1.86 a 1.57 a 1.44 a 1.35 a 3.19 a 3.98 a 2.85 a 2.97 a 2.89 a 2.29 a 2.57 a 2.27 a 5.09 a 6.47 a 3.81 a 4.37 a 4.23 a 3.67 a 3.77 a 3.55 a 59.90 a 72.92 a 48.05 a 56.00 a 52.00 a 46.97 a 48.17 a 47.09 a Note: 1) Means in the same column followed by the same letter are not significantly different according to Duncan Multiple Range Test (p < 0.05) 2) DAP = Days After Planting 3) AUHPGC = Area Under Height of Plant Growth Curve Seven isolates of the bacteria were tested for their effects on seed viability, plant height and the fresh and dry weight of two varieties of tomato plants, i.e. Arthaloka and Ratna, as presented in Table 3, 4, and 5, respectively. Data in Table 3, 4, and 5 show not only that the isolates of bacteria did not significantly increase the seed viability, plant height, fresh and dry weight of tomato compared to control, but also they did not have harmful effects to the tomato plants. The characteristics of colony morphology, physiology and biochemistry aspects of the seven isolates were presented in Table 6 and 7, respectively. 91 Table 6 Morphological characteristic of colony of the seven isolates of siderophore-producing rhizobacteria on King's B Agar Isolate code1) Colony characteristic Diameter Color Elevation Edge Form CP1C CP2B CP2D CP3E LB1A LB1C LB1L ± 1 mm ± 1 mm ± 1 mm ± 1 mm ± 1 mm ± 3 mm ± 1 mm White Broken white Broken white Dark yellow Greenish white Pale white Broken white Convex Domed Convex Convex Convex Umbonate Convex Wavy Entire Entire Entire Entire Curly Wavy Circular Circular Circular Circular Circular Irregular Circular Note: 1) CP = isolates from Cipanas; LB = isolates from Lembang Table 7 Physiological and biochemistry characteristics of siderophore-producing rhizobacteria having potential as antagonist for R. solanacearum Isolate code1) Fluorescence Gram reaction Phosphate solubilization Resistance to 80 oC CP1C - - + + CP2B +++ - + - CP2D - - + + CP3E - - - - LB1A + - + - LB1C - + + + LB1L - - + + Note: 1) CP = isolates from Cipanas; LB = isolates from Lembang Table 5 Effect of siderophore-producing rhizobacteria on fresh and dry weight of tomato plants Treatment1) Fresh weight (g/plant)2) Dry weight (g/plant) Var. Arthaloka Control CP1C1) CP2B CP2D CP3E LB1A LB1C LB1L Var. Ratna Control CP1C CP2B CP2D CP3E LB1A LB1C LB1L 15.439 a 14.266 a 15.070 a 15.265 a 9.515 a 12.557 a 12.572 a 11.335 a 10.109 a 8.748 a 6.887 a 10.744 a 9.566 a 7.880 a 6.887 a 5.319 a 3.381 a 2.587 a 2.900 a 2.986 a 2.381 a 2.721 a 2.579 a 2.742 a 2.360 a 1.954 a 1.801 a 2.281 a 2.006 a 1.547 a 1.436 a 1.535 a Note: 1) CP = isolates from Cipanas; LB = isolates from Lembang 2) Means in the same column followed by the same letter are not significantly different according to Duncan Multiple Range Test (p < 0.05) Based on the inhibition zone production, effect on seed viability, effect on plant height increase, and effect on fresh and dry weight of tomato product, two isolates of siderophore- producing bacteria having the best effects were selected. The two isolates were CP1C and CP2D. The sequence of 16S rDNA of those isolates were referred to the gene bank. Using the BLAST program the isolate of CP1C was identified as Brevundimonas sp., while the isolate CP2D was Siderophore-producing Rhizobacteria and potential antagonistic activity toward Ralstonia solanacearum – Nawangsih et al. 92 BIOTROPIA Vol. 24 No. 2, 2017 · = Enterobacter sp. CP2D = Brevundimonas sp. CP1C Figure 3 Phylogenetic tree of isolates Enterobacter sp. CP2D and Brevundimonas sp. CP1C Enterobacter sp. MTH17 MTH17 gi323218729 Enterobacter sp. b49 b49 gi389827949 Enterobacter ludwigii B-5 carrot gi444438257 Enterobacter sp. ACC2 ACC2 gi399936203 Enterobacter sp. M.D.E.NA4-3 M.D.E.NA4-3 gi326635001 Enterobacter sp. E6-PCAi-T2P21 E6-PCAi-T2P21 gi358365187 Enterobacter sp. enrichment culture clone gi355343574 CP2D Brevundimonas sp. 13630G 13630G gi206581409 Brevundimonas nasdae 13636E gi206581436 Brevundimonas sp. MC8-1 MC8-1 gi300253164 CP1D Brevundimonas vesicularis L17 gi375004753 blackwater bioreactor bacterium BW23 BW23 gi16589030 uncultured bacterium gi253770558 uncultured bacterium gi253770269 100 84 100 100 80 100 73 100 69 100 41 46 75 0.05 Table 8 Maximum score, E value and percentage of similarities of siderophore-producing bacteria Isolate Species homolog Identity Max score Query cover E value Accession number CP1C Brevundimonas sp. 13630 G 16S ribosomal RNA gene, partial sequence 95% 2021 92% 0.0 EU741063.1 CP2D Enterobacter sp. enrichment culture clone DWSR 106 16S ribosomal RNA gene, partial sequence 88% 998 69% 0.0 JN944751.1 identified as Enterobacter sp. with percentage of similarity of 92% and 93%, respectively. Maximum score, E value and the percentage of similarities of siderophore-producing bacteria were presented in Table 8. Phylogenetic tree of the related bacteria is presented in Figure 3. Tian et al. (2009) reported that Enterobacter and Pseudomonas were dominant in the rhizosphere of tobacco, with 44.5% and 24.7% total frequency, respectively. Siderophores are produced by various bacteria and fungi, usually classified by the ligands used to chelate the ferric iron. The major groups of s i d e r o p h o r e s i n c l u d e t h e c a t e c h o l a t e s (phenolates), hydroxamates and carboxylates (e.g. derivatives of citric acid) (Saharan & Nehra 2011). Rachid & Ahmed (2005) reported that streptomycin and penicillin added to succinate medium acts differently on the siderophores production. Streptomycin reduced siderophores production below 10 µM in different iron concentrations, while penicillin increased the production of siderophores in the presence of excess iron (above 100 µg/mL). The growth of P. fluorescens and siderophore production were inhibited by the occurrence of heavy metals (lead, mercury and cadmium), especially in iron-limited condition . 93 CONCLUSIONS The average of siderophore-producing bacteria isolated from Cipanas District (1.98 x 10 7 cfu/g) was not significantly different from those isolated from Lembang District (5.3 x 10 cfu/g). 7 The highest diameter of inhibition zone to Ralstonia solanacearum was 7.0 mm, produced by isolate CP2D. The selected bacteria producing the inhibition zone did not significantly affect seed viability, plant growth, fresh weight and dry weight of tomato compared to control. Based on the characteristics of colony morphology, physiology, biochemistry and partial sequence of 16S rDNA, two selected isolates, i.e. CP1C and CP2D were identified as sp. and sp., Brevundimonas Enterobacter respectively. ACKNOWLEDGEMENTS This research was funded by DIPA IPB with scheme of Decentralized Research Program (Program Penelitian Desentralisasi: Hibah Bersaing), Contract No. 27/I3.24.4/SPK/ PD/2010, on 5 March 2010. The authors also thank Dr Kikin Hamzah Mutaqin for the guidance in several molecular tests activities. REFERENCES Agrios GN. 2005. Plant pathology. Fifth Edition. New York (US): Academic Press. 992 p. Bhattacharya A. 2010. Siderophore mediated metal uptake by Pseudomonas fluorescens and its comparison to iron (III) chelation. Cey J Sci (Bio. Sci.) 39(2):147-55. Gross M. 1990. Siderophores and fluorescent pigments. In: Klement Z, Rudolph K, Sands DC, editors. Methods in phytobacteriology. Budapest (HU): Akadémiai Kiadó. 568 p. Hu QP, Xu JG. 2011. A simple double-layered chrome azurol S agar (SD-CASA) plate assay to optimize the production of siderophores by a potential biocontrol agent . Afr J Microbiol Res Bacillus 5(25):4321-7. 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