Screening of Rhizosphere Bacteria from Clove (Syzygium aromaticum) in Tidore Island as Plant Growth Promoting Rhizobacteria

Tidore Island in North Maluku Province is one of the clove (Syzygium aromaticum) producing regions. Clove plant fertility is maintained even though it is not given organic and inorganic fertilizers. This study aims to explore bacteria in the rhizosphere of the clove trees planted in Tidore island as candidate of biofertilizer agents have potency in Indol-3Acetic Acid (IAA) production, phosphate solubilization and ammonium production. Bacteria were isolated from the rhizosphere of clove trees. Screening of the isolates was done according to qualitative and quantitative methods. Data collection of the qualitative method were based on medium color changes for each variable while that the quantitative method (i.e. the concentrations of produced IAA, solubilized phosphate, and produced ammonium) were obtained based on spectrophotometry. The results obtained 110 bacterial isolates, nineteen bacteria were potential as biofertilizer agents, of which isolate R11, R8P, and A1N showed the highest in IAA production (8.71 μgL-1), phosphate solubilization (8.37 μgL-1), and ammonium production (11.71 μgL-1), respectively. The three isolates respectively have similarities to the genus of Enterobacter, Burkholderia, and Stenotrophomonas.

INTRODUCTION 1  Total area of clove plantation in Tidore Island is 1,029 ha with the productivity of 500 kg.ha -1 [1].The clove trees on Tidore Island grow naturally and the soil has never been supplemented with either organic or chemical fertilizers.However, crop productivity is naturally preserved.Clove commodity is selected because the plantation area of this commodity estimates increase.In addition, increases of soil fertility without contamination by chemical fertilizers and the need of other perspectives in order to exploit the potential of spice crops also became reasons.It has been reported that the bacteria from the clove rhizosphere were tested in-vitro capable of producing Indole-3-acetic acid [2] but have not revealed the potency of bacteria in the production of ammonium and phosphate solubilization.On the other side of field observes no research report yet on the use of clove rhizosphere bacteria as a biofertilizer agent.
Generally, crop productivity is supported by external factors such as climate and soil microorganism, especially bacteria.The climate  Correspondence address: Ismat Ishak Email : ismatishak123@gmail.com Address : Dept.Biology, Faculty of Mathematics and Natural Science, University of Brawijaya, Veteran Malang, Malang 65145.
of Tidore is a humid tropical region influenced by sea breeze surrounded by the sea of Halmahera, Seram, and Maluku.Observations in this study recorded the type of soil in clove plantation commodity dominated by Inceptisol soil or Alluvial, although in some altitudes there is Mollisol soil.Bacteria found in the plant rhizosphere are called Plant Growth Promoting Rhizobacteria (PGPR).The bacteria use plant exudates as a source of nutrients for their growth [3].
The existence of PGPR on plant roots is beneficial for the plants.Some bacteria can support plant growth or act as agents of biocontrol for plant diseases [4].Bacteria in the rhizosphere can stimulate the growth of plants through direct or indirect mechanisms.Direct mechanisms involve bacteria as a biofertilizer to stimulate the growth of plants, and as an agent for preventing plant stress, while indirect mechanisms include reducing the intensity of plant diseases, producing antibiotics, inducing systemic resistance, and competing for nutrition and space [5].The main roles of PGPR are plant growth promoters including nitrogen fixation, production of plant growth hormones such as auxin and cytokinin, and solubilizing phosphate and iron from non-soluble form [6].
The use of PGPR as a biofertilizer agent was developed as an effort to maintain environmental health and prevent environmental damage due to the extensive use of synthetic chemical fertilizers.The use of PGPR has several advantages including 1) not polluting the environment, 2) not containing ingredients cause residues in the food chain; 3) not requiring repeated application as microbes can grow with a supportive environment; 4) as a companion for indigenous microbial plants, 5) can increase plant resistance against pathogens [7].Some special genera of microbes have been found to be useful as PGPR.Anandaraj and Dinesh [8] reported several genera of bacteria as PGPR including Acinetobacter, Alcaligenes, Azospirillium, Azotobacter, Bacillus, Beijerickia, Burkholderia, Enterobacter, Erwinia, Flavobacterium Rhizobium and Serratia.Several studies have reported the positive effect of PGPR on plant growth such as PGPR has a positive effect on wheat crops [9], fruits [10], and vegetables [11].
Due to the enormous potential of PGPR in extensive plantations and plant health, in this study, exploration of the potential bacteria in the rhizosphere of clove plantation in Tidore Islands, North Maluku as biofertilizer-functioning PGPR was carried out.

MATERIAL AND METHOD Soil Sampling
Soil sampling from a clove plantation on Tidore Island,was carried out at three location on different altitude.The rhizosphere soil samples were taken from several clove plant roots at a depth of 0-20 cm from the surface.Soil chemical compounds such as organic matter, C/N ratio, soluble phosphate, nitrogen content were analyzed in Faculty of agriculture, University of Brawijaya.

IAA Production Assay
Production of IAA by bacteria was tested using Tryptic Soy Broth (TSB) and Salkowski reagents (1.05 g FeCl3.6H2O;60 mL absolute H2SO4; 100 mL aquades).The bacteria were cultured in TSB medium containing L-tryptophan 200 mg.mL - and incubated with a rotary shaker at 120 rpm in the dark at room temperature for 3 days.Then the culture was centrifuged at 12,000 rpm for 15 min to pellet cells and two mL of supernatant were added to 3 mL Salkowsky reagent and incubated in the dark for 30 minutes at room temperature.The intensity of pink color, indicating positive IAA production, was measured using a UV-VIS spectrophotometer at a wavelength of 535 nm [14].From the standard curve prepared with known IAA concentrations, the quantity of IAA in the culture is determined and expressed as mg.mL -1 [15].

Phosphate Solubilization Assay
All bacterial isolates were first tested on the Pikovskaya agar medium to observe the phosphate solubilization index as described by Gaur [16].Quantitative analysis of phosphate solubilization was done in liquid medium as described by King [17].Isolates were cultured in 25 mL of Pikovskaya broth and incubated for 24 to 72 hours at room temperature 28 ± 2°C.Bacterial culture was centrifuged at 12,000 rpm for 15 min to pellet cells.One mL of the supernatant was mixed with 5 ml of chloromolybidic acid and added 0.25 mL clorostannous.The bluish gray absorbance was read at 690 nm.The amount of dissolved phosphorus was calculated based on the KH2PO4 standard curve.The pH of medium was measured to determine the correlation of phosphate concentration to the medium acidity.

Nitrogen Fixation (Ammonium Production) Assay
The concentration of ammonium was estimated using Serra Ammonium detection Kit.The isolates were cultured in 25 mL NFb broth medium.After centrifuging the liquid culture at 12,000 rpm for 10 min at room temperature, one mL of supernatant was transferred into a test tube.One drop of NH41, NH42, and NH43 was added to the sample and mix well and the sample was left at room temperature for 5 min.The ammonium concentration was estimated using the provided color chart [18].
Bacterial isolates were tested for the production of ammonia in peptone water.Bacterial isolates were inoculated in 20 mL Nfb broth medium without Bromothymol Blue in each tube and incubated for 48-72 hours at 25 ± 2 o C. One mL of the Nessler reagent was then added in each tube.Development of yellow color is a positive indication for ammonia production [19].The absorbance of the developing color was read at 425 nm.The quantity of ammonium production was calculated according to the NH4Cl standard curve.

Bacterial Identification
Identification was performed on bacterial isolates that produced the highest concentrations in IAA production, phosphate solubilization, and ammonia production (NH3).The extraction of genomic DNA from bacterial cells harvested from a 24-hour culture on NA medium was performed using the i-genomic DNA Extraction Kit (Intron) according to the manufacturer's protocol.
DNA amplification of 16S rDNA sequence was performed by Polymerase Chain Reaction (PCR) using primers 27F (5 '-GCC TAA CAC GTC ATG CAA GA-3') and 1496r (3 '-CGT AAC ATT CGG GGC TGC TGG TGC-5').The PCR process was performed according to Zarei et al. [20] with 35 cycles consisting of denaturation at 94°C 30 s annealing 55°C for 30 s and extension of 72°C for 65 s.The PCR products were visualized using 1% agarose gel to present the DNA band and then aligned using the O'genRulerTM Ladder Mix DNA.
The correct size of PCR products was sent to 1st BASE Malaysia for sequencing.The obtained sequences were aligned using the Nucleotide BLAST to find the similarity to GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
The results of BLAST and the reference strain were aligned and constructed by phylogenetic trees consisting of evolutionary relationships according to Saitou and Nei [21], Felsenstein's [22].While similarity percentage with Tamura and Nei [23].Evolutionary distances are shown using MEGA 6 [24].

Statistical Analysis
Quantitative data from each assay were subjected to analysis of variance (ANOVA) and means were compared by Tukey test α 0.05 using the SPSS ver.16.

RESULT AND DISCUSSION Soil Chemical Properties
Soil chemical properties of the sampling location (i.e.organic matter, total nitrogen, C/N ratio, available phosphate, organic matter, and acidity) are presented in Table 1.According to Table 1, Tdr1C soil samples contain low organic matter (1.98%), moderate total N content (0.21%) and very low dissolved phosphate content (2.25 mg.kg -1 ).Tdr2R contains moderate soil organic matter (4.08%), moderate total N content (0.4), and low dissolved phosphate (3.1 mg.kg -1 ).Tdr3A contains high soil organic matter (6.5%), very high total N content (0.77%) and very low dissolved phosphate content (0.81 mg.kg -1 ).Variation of chemical soil properties is influenced by soil nutrient cycle in nature, the process in this cycle includes physical, chemical and biology, soil formation, chemical elements that bind to soil molecules and immobilization of soil microorganisms in the process of soil nutrient formation [25].

Table 1. Chemical properties of soil sample from clove plantation
Note: *The categories according to CSR/FAO 1983 [26] Total Plate Count of Rhizosphere Bacteria Estimation of the rhizosphere bacterial population was done using total plate count (TPC) and MPN methods.The TPC and MPN results were analyzed using one way ANOVA with significance (α <0.05) according to the Tukey test.The MPN method used semisolid NFb medium to observe the activity of bacteria in nitrogen fixing.This activity is characterized by white ring-shaped on the surface of medium (Fig. 2c).The highest number of colonies shown on semisolid Nfb medium was in the range of 6.8 x 10 4 -7.03 x 10 7 cells.g - , TSA showed total bacteria in the range of 6.5 -6.7 x 10 5 CFU.g -1 and the Pikovskaya medium shows the number of colonies in the range of 6.1 -7.1 x 10 5 CFU.g -1 per gram (Fig. 1).The highest number of bacteria colonies were found in the Tdr3A soil samples which is correlated with the abundance of N total content.

Qualitative and Quantitative Assay of PGPR Bacteria as Biofertilizer IAA Production Assay
Qualitative assay of IAA were carried out using TSB medium containing L-tryptophan with a concentration of 200 mg.L -1 as a precursor of IAA synthesis.The Salkowsky reagent added to the supernatant of bacterial cultures resulting in red color indicated the production of IAA (Fig. 2a).The results of the screening, obtained 7 isolates able to produced IAA.

Isolation of PGPR
A total of 110 rhizospheric bacteria isolates have been obtained from all soil samples.In TSA medium was obtained 65 isolates, Pikovskaya medium was 37 isolates and Nfb medium was 9 isolates.Qualitative and quantitative selection resulted 7 potential isolates as IAA producing, 7 isolates as phosphate solubilization and 5 isolates as nitrogen fixation (Table 2).
The seven isolates were then tested quantitatively based on the standard curve.The results of the analysis obtained different concentrations of IAA for each isolate.The highest yielding IAA isolate was R11 with a concentration of 8.71 μg.L -1 , while the lowest in IAA production was A3 isolate with IAA concentration of 3.26 μg.L -1 (Fig. 3).Ahmad et al. [27] reported that there were different concentrations of IAA production in each type of bacteria with tryptophan at different concentrations.The using of 200 mg.L -1 concentration as the optimal precursor for IAA synthesis in this study, the presence of tryptophan consumption will affect the high levels of IAA concentrations produced by bacteria.Glickmann and Dessaux [15] reported the use of 2.5 mg.L -1 L-tryptophan which led to a decrease in IAA production.Kesulya et al also reported 100 mg.L -1 L-tryptophan obtained a maximum concentration of 5.82 mg.L -1 [28].Some microorganisms require tryptophan to produce IAA from the IAA biosynthesis mechanism [29].PGPR is known to affect plant growth because it can produce phytohormones, such as the auxin or in this study called IAA that has been known to stimulate cell lengthening and cell division differentiation [27] and gene regulation [30].The results of molecular identification using the 16S rDNA gene showed that the rhizosphere bacterium isolate R11 had similarities to the Enterobacter hormaechei The construction of phylogenetic trees showed a similarity of 98.5% (Fig. 4).It has been reported that bacteria from the soil of rhizosphere have similarities with Enterobacter sp. and capable of producing IAA (5.09 mg.L -1 ) as a biofertilizer agent [31].

Phosphate Solubilization Assay
The clear zone around bacterial colonies on Pikovskaya agar medium containing Tricalcium phosphate (Ca3PO4) shows the presence of phosphate dissolving activity.From qualitative selection, there were 7 isolates of bacteria showing solubilization activity of phosphate.Each isolate showed a phosphate solubilization index that varied in the range of 10 to 13 mm.Tukey test analysis with significance α 0.05 showed that each isolate showed significantly different in solubilization of phosphate (Table 3).The phosphate solubility index was obtained based on the clear zone around the bacterial colony.Clear zone formation is caused by the production of organic acids, the production of polysaccharides, or the activity of phosphatase enzyme from phosphate solubilizing bacteria [32].Quantitative tests with Pikovskaya broth medium at pH 7.0 were performed to measure the concentration of solubilized phosphate (PO4) based on a standard KH2PO4 curve.The assay results showed that pH medium varied in each isolate (Fig. 5).In the isolate R8 there was a correlation between the higher PO4 concentration (8.37 μg.L -1 ) followed by a decrease in the pH of the media to 4.09.Other isolates showed significant variations based on statistical analysis.Acid levels in bacterial media indicate the production of organic acids which are common mechanisms of phosphate dissolution [32,33].Molecular identification was performed on isolates with the Identification using a sequence of 16S rDNA genes showed that R8P isolate had similarities with Burkholderia multivorans at 95.5% (Fig. 6).B. multivorans WS-FJ9 has been known to dissolve phosphate and decrease pH from initial pH [34].

Nitrogen Fixation (Ammonium Production) Assay
The nitrogen fixation assay shows the change of media color from green to blue and has a white follicle on the sub-surface medium (Fig. 2c).The TPC results showed that nitrogen fixation bacteria had the highest number of cells in the range of 3.5 x 10 5 -6.5 x 10 6 cfu per gram of soil (Fig. 1).The result of the qualitative test using Serra ammonium test obtained 5 isolates of nitrogen fixation bacteria produce ammonium with varying concentration that is blue and dark green color showed high ammonium content while light green contains low ammonium concentration (Fig. 7).Qualitative test of ammonium concentration based on NH4Cl standard curve obtained 5 isolates with different concentration.The isolate that produces the highest concentration is A1N.
Results of molecular identification based on the sequence of 16s rDNA gene showed that A1N isolate had a similarity of 98.7% with Stenotrophomonas maltophilia species (figure 8).It is known that S. maltophilia bacterial strains are capable of producing ammonium with various carbon sources [18].Bacteria associated with clove plants in the rhizosphere are able to improve nitrogen fixation that has been widely reported as a plant growth promoter [35].The activity of nitrogen fixation organisms is generally active in plant roots.

CONCLUSION
This study obtained Enterobacter hormaechei, Burkholderia multivorans and Stenotrophomonas maltophilia that are potential for biofertilizer agents.The recommendation for further research is Application of synergism or antagonism then the isolates perform as consortium and prepare for biofertilizer and apply to plants of three isolates in a greenhouse to determine their effect on crops, without exception to commodity crops such as cloves.In addition, a biochemical and physiological characterization and identification of encoding genes are necessary to support the identified molecular data of bacterial species.

Figure 1 .
Figure 1.The bacteria population on the soil samples grouped by type of media TSA, PVK and Nfb.The same letter shows no significant different (P <0.05) based on Tukey's test.

Figure 2 .
Figure 2. Qualitative and Quantitative Test of PGR.a. Results of qualitative test of IAA Production b. qualitative test of Phosphate solubilization c. the indicator of Nitrogen-Fixing bacteria, and d. qualitative test of ammonium production.

Figure 3 .
Figure 3. IAA producing by Some Isolates from Rhizospheric Soil of Clove Plantation

Figure 4 .
Figure 4.The Phylogenetic Tree of R11 Isolate based on 16s rDNA Gene Sequence

Figure 5 .
Figure 5. Correlation of pH and PO4 Concentration

Figure 6 .
Figure 6.The phylogenetic tree of A1N isolate based on 16s rDNA gene sequence

Table 2 .
Number of Bacterial Isolates has Potentcy as Biofertilizer

Table 3 .
Phosphate Solubilization Index of Bacteria Isolates Isolates from Rhizospheric Soil of Clove