Species and Functional Diversity of Rhizobacteria of Rice Plant in the Coastal Soils of Indonesia

Rhizobacteria are important components of soil and directly or indirectly influence the soils quality and plant growth for maintaining adequate plant nutrition and reducing the negative environmental effects of fertilizers. Applying high dose of chemical fertilizers in most of rice fields in the coastal areas could reduce the quality of the soil in the long time. There are few studies addressed to verify the species and functional diversity of cultivable rhizobacteria associated with rice plant in the coastal soils. The objective of the study was to verify the species and functional diversity of rhizobacteria isolated from the coastal soils of two rice production areas of Subang and Indramayu, West Java. Special focus was given to verify phosphate solubilization, nitrogen fixation, IAA and cellulase production of the selected 78 strains of rice rhizobacteria isolated from the coastal paddy field, as well as taxonomical analyses based on 16S rRNA. The results showed that among 78 bacterial isolates from the coastal paddy field, mostly were belonging to the Firmicutes, most of them affiliated with genera Bacillus, 75 strains produced IAA, 32 strains fixed nitrogen, 37 strains solubilized phosphate and 33 strains produced cellulase. Several strains of the rhizobacteria were capable of producing plant growth promoting substances (PGPR), alone or in combination, such as IAA, fixing nitrogen, solubilizing phosphate, and producing cellulase. Taking all of these diverse PGPR characteristics into account, it is clear that the 78 identified isolates have great potential for improving saline soils of the coastal paddy fields in Indonesia.


ABSTRACT
Rhizobacteria are important components of soil and directly or indirectly influence the soils quality and plant growth for maintaining adequate plant nutrition and reducing the negative environmental effects of fertilizers. Applying high dose of chemical fertilizers in most of rice fields in the coastal areas could reduce the quality of the soil in the long time. There are few studies addressed to verify the species and functional diversity of cultivable rhizobacteria associated with rice plant in the coastal soils. The objective of the study was to verify the species and functional diversity of rhizobacteria isolated from the coastal soils of two rice production areas of Subang and Indramayu, West Java. Special focus was given to verify phosphate solubilization, nitrogen fixation, IAA and cellulase production of the selected 78 strains of rice rhizobacteria isolated from the coastal paddy field, as well as taxonomical analyses based on 16S rRNA. The results showed that among 78 bacterial isolates from the coastal paddy field, mostly were belonging to the Firmicutes, most of them affiliated with genera Bacillus, 75 strains produced IAA, 32 strains fixed nitrogen, 37 strains solubilized phosphate and 33 strains produced cellulase. Several strains of the rhizobacteria were capable of producing plant growth promoting substances (PGPR), alone or in combination, such as IAA, fixing nitrogen, solubilizing phosphate, and producing cellulase. Taking all of these diverse PGPR characteristics into account, it is clear that the 78 identified isolates have great potential for improving saline soils of the coastal paddy fields in Indonesia.

INTRODUCTION
Indonesia has approximately 8.1 million ha of rice fields; 3.25 million ha or 40.3% of these are spread over the northern coastal areas of Java Island. Some of the rice production centres in the northern coastal area of West Java, such as Subang and Indramayu, contributed significantly to the national rice production (Central Bureau of Statistics 2011). The lowland areas of these regions, however, have different soil salinity as a result of the seawater intrusion.
Rice plants are highly sensitive to salinity, especially during the germination period and at the beginning of vegetative growth. Therefore, the presence of seawater intrusion continuously would be a serious threat which leads to plant toxicity, poor growth, and reduced yield (Suriyan and Chalermpol 2009). At the long time, this may affect agroecosystem sustainability.
In general, farmers apply high dose of chemical fertilizers in most of rice fields in the coastal areas of Subang and Indramayu, West Java. Such practices could inhibit the interaction of microorganisms and their host plants. In the long term, it would reduce the quality of the soil due to saturation of certain soil elements hence not available for plant growth. There are very few studies addressed to verify the species and functional diversity of cultivable rhizobacteria associated with rice plant in the coastal soils.
Plant Growth Promoting Rhizobacteria (PGPR) are important components of soil and directly or indirectly influence soil quality and plant growth for maintaining adequate plant nutrition and reducing the negative environmental effects of chemical fertilizers. These microbes mediate soil processes, such as nitrogen fixation, nutrient mobilization, mineralization, denitrification, and decomposition. Rhizosphere bacteria also produce phytohormones, such as indole acetic acid (IAA), cytokinin, and gibberellin (Madhaiyan et al. 2006;Kang et al. 2009). The activity of cellulase acts as a key enzyme for the invasion and colonization of plant roots (Hallmann et al. 1997;Reinhold-Hurek and Hurek 1998).
The objective of this study was to verify the species and functional diversity of rhizobacteria isolated from coastal soils of two rice production areas of Subang and Indramayu, West Java. Special focus was given on the verification of phosphate solubilization, nitrogen fixation, IAA and cellulase production of selected 78 strains isolated from the coastal paddy field. The study is expected to understand the diversity of rhizobacteria and their potential role in improving saline soil of the coastal paddy field in Indonesia.

Location and Soil Sampling
Three replicated samples each of rice rhizosphere soils were collected from paddy field near coastal area in Subang (06°14'59"S, 107°54'31"E) and Indramayu (06°18'48", 108°02'15"E) during December 2012 and January 2013. The physicochemical properties of the soil samples were analysed at the Indonesian Soil Research Institute, Bogor. The microbiological analysis of the soil samples was carried out immediately after sampling to minimize the storage effects.

Source of Rhizosphere Bacterial Isolates
The rhizosphere soil was obtained by taking the whole rice plant with soil on it to the laboratory. The rhizosphere soil was shaken manually to remove the soil from the roots, while the fine layer of soil firmly attached to the roots was immersed into 100 mL sterile distilled water to remove them. The resulting mixed solution was referred as rhizosphere soil. Soil microbes were isolated using a standard dilution plating technique on a Plate Count Agar (PCA) on Soil Extract Agar (SEA) consisted of 0.1% glucose, 0.05% dipotassium phosphate, and 1.775% yeast extract. The incubation was done at 28 o C for 7 days. All isolates had already been maintained in the Indonesian Culture Collection (InaCC) of the Indonesian Institute of Sciences (LIPI) and the Biogen Culture Collection (BiogenCC) of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD). The isolates were routinely cultured on SEA for the functional characterizations.

DNA Extraction
Bacterial DNA was extracted from all isolates of the rhizosphere bacteria. Each isolate was grown in a liquid medium 1/10 strength of Luria Bertani Broth composed of tripton 1%, 0.5%, yeast extract and 1% NaCl. Overnight cultures were centrifuged at 13,000 rpm for 2 minutes to pellet the cells and remove the supernatant. Genomic DNA of each isolate was then extracted from bacterial cell pellet by using the Wizard DNA genomic extraction kits (Promega). Quality and quantity of the isolated DNA were checked by using nanodrops and gel electrophoresis on a 0.8% agarose gel.

PO 4 Solubilization
Rhizobacterial ability to solubilize inorganic phosphate (tricalcium phosphate) was assessed using phosphate-solubilizing medium (Pikovskaya agar medium) and incubated at 28°C for 7 days. Colonies with clearing zones were scored as positive for phosphate solubilization (Pikovskaya 1948). The experiments were performed with three replicates for each bacterial strain.

Cellulolytic Activity
Screening of the cellulolytic isolates was performed on carboxy methyl Cellulose (CMC) agar media containing 0.5% caboxymethyl cellulose, 0.1% NaNO 3 , 0.1% K 2 HPO 4 , 0.1% KCl, 0.05% MgSO 4 .7H 2 O, 0.05% yeast extract, 1.5% bacto agar, pH 8.0. Five days after incubation at 28°C, a solution of Congo red was poured on the surface of the agar to detect the cellulolytic enzyme activity as described by Teather and Wood (1982). The cellulolytic activity was indicated by a clear zone around the colony of bacteria.

IAA Producer
IAA was detected calorimetrically in the supernatants of the bacterial cultures using Gordon and Weber's reagent (1 ml 8.12% FeCl 3 .6H 2 O, 50 ml 35% HClO 4 in dark bottle). The isolates were grown overnight in a modified nutrient broth M26 (0.5% NaCl, 1% peptone, 1% beef extract). As many as 100 µl of the overnight culture was added to 10 ml of minimal salt medium (0.136% KH 2 PO 4 , 0.213% Na 2 HPO 4 , 0.02% MgSO 4 .7H 2 O, and 10 ml trace element After further incubation for 48 hours, IAA production was assessed as follows. Bacterial cells were removed from the culture medium by centrifugation (8,000 rpm, 4°C, 10 minutes), and then 2 ml of the above reagent was mixed with 1 ml of culture supernatant, followed by incubation at room temperature for 25 minuters. The optical density of the culture grown in the minimal salt medium supplemented with tryptophan was measured using a spectrophotometer at 530 nm. The concentration of IAA in each culture medium was determined by comparison to a standard curve generated from known concentration of IAA (Gordon and Weber 1951). The readings were performed with three replicates for each bacterial strain.

RESULTS AND DISCUSSION
The physicochemical properties of the two soil types collected from Subang and Indramayu coastal soils varied in terms of their textures, pH, salinity (EC or TDS), total carbon, and total nitrogen (Table 1). Microbiological properties of the two sites showed that population number in Indramayu was greater than that in Subang.

Functional Characterization of Cultivable Rhizosphere Bacteria from Coastal Soil
The functional groups of rhizosphere microbes are difficult or even impossible to assess directly. Therefore, this study was based on dilution plating, which is a quick, inexpensive and reliable technique.
Seventy eight bacterial taxes collected from rhizosphere of rice plant grown in coastal soil were able to grow on SEA medium. Most of the rhizobacteria were Gram-positive (66.7%) mostly belonging to the Firmicutes, most of them were affiliated with genera Bacillus. Different morphotypes of the rhizobacteria from Subang coastal soil were purified, which belonged to 12 genera and 19 species. Rhizobacteria from Indramayu consisted of 18 genera and 34 species (Table 2). Among the isolates obtained in this study, the most often genera identified was Bacillus. Members of Bacillus are ubiquitous bacteria that include both free-living rhizobacteria and pathogenic species. Bacillus in the rhizosphere of rice plant in this study had the proportion of 32/78. It suggests they are highly competent in the rhizosphere compared with other genera. Rhizobacteria of the genera Bacillus have been reported to enhance the growth of several plants such as Pinus, wheat, tomato, sugar beet, sorghum, peanut and onion (Caceres et al. 1996;Probanza et al. 2001;Yan et al. 2003). Previous studies reported that numerous PGPR belonging to Bacillus were mostly restricted to certain species. However, this study indicated a high diversity of Bacillus PGPR species, such as Bacillus stratosphericus, B. amyloliquefaciens, B. cereus, B. methylotrophicus, B. pumilus, B. marisflavi, B. megaterium and B. flexus. Lysinibacillus and Microbacterium were the second largest PGPR group found in this study. The four isolates in each of these genera (Lysinibacillus and Microbacterium) had high plant growthpromoting properties. Two Lysinibacillus species were identified; among them were Lysinibacillus sphaericus (three isolates), the most dominant, followed by Lysinibacillus boronitolerans (one isolate). The other genera was Microbacterium, commonly found in the soil samples. Microbacterium species identified as PGPR in this study were M. arborescens, M. fluvii, M. xylanilyticum, and M. awajiense. Some Microbacterium species have function in phytoextraction by assimilating heavy metals, such as Pb and Ni. They have been reported to increase the growth of apple and rape (Karlidag et al. 2007;Sheng et al. 2008).
The third group of PGPR belonged to the genera Aeromonas, Arthrobacter, Enterobacter, Pantoea, Pseudomonas and Streptomyces. The Pseudomonas species identified as PGPR in this study were P. mosselii and P. knackmussii (one isolate each). The finding of both bacteria as PGPR is the first time, as we known, therefore, lending to the significance of our study. Interestingly, previous studies showed that Pseudomonas PGPR are highly resistant to various environmental stresses. For instance, the effects of the PGPR Pseudomonas fluorescens MSP-393 are not altered in highly saline soils such as those on coastline (Paul and Nair 2008). Furthermore, production of 1 aminocyclopropane-1-carboxylic acid (ACC) deaminase by P. fluorescens increases the resistance of plants to salt stress (Saravanan et al. 2007).
PGPR members of the genera Arthrobacter are usually found in soil; they role in bioremediation. For instance, Arthrobacter chlorophenolicus has the ability to degrade high concentrations of 4chlorophenol, which is a recalcitrant toxic compound in contaminated soils (Westerberg et al. 2000). A PGPR Pantoea was shown to promote the growth of pepper plants (Kang et al. 2007).
The study identified a large variation among isolates of these species with respect to different combinations of PGP traits that they carried. Other  (Kumar et al. 2011;Sharma et al. 2011;Timmusk et al. 2011).

) and
Acinetobacter soli (Er II B1.5) were obtained from moderate saline soil from the Indramayu coastal soil. Our study confirmed 16 taxa of rhizosphere bacteria being positive for nitrogen fixation, IAA production and P solubilization. It found that only 20.5% (16 out of 78) of strains tested had the ability to produce IAA, fix nitrogen and solubilize P. These included seven isolates from Subang and nine isolates from Indramayu coastal soils. Among them, Providencia rettgeri (Er I B1.9 and Er I B2.10) had the ability to fix nitrogen, solubilize phosphate and produce high amount of IAA (50 ppm) (Fig 1; Table 3).

Diversity in IAA Production Traits of Rhizobacteria
The rhizobacteria were functionally diversed and some possessed more than one PGP trait. The bacteria from Subang coastal soil were relatively poor in IAA producer trait. Among the rhizobacteria, a total of 97.9% isolates from Indramayu coastal soil showed IAA producer as compared to only 93.5% of the isolates from Subang coastal soil (Table 3). It is reported that 80% of microbial isolates from the rhizosphere of various crops possess the ability to synthesize and release auxins as secondary metabolites (Pattern and Glick 1996).
High levels of bacterial indolic compounds stimulate the formation of lateral and adventitious roots (Patten and Glick 1996), which could increase the absorption of nutrients including phosphate. Generally, IAA secreted by rhizobacteria interferes with the many plant developmental processes because the endogenous pool of plant IAA may be altered by the acquisition of IAA secreted by bacteria (Spaepen et al. 2007;Glick 2012).
Several species of the genera Bacillus could produce IAA, but with variable efficiency. Bacillus amyloliquefaciens Ptb I B2.9 produced IAA of more than 50 ppm, but B.amyloliquefaciens (Er I B3.8) produced IAA of less than 50 ppm. Other species such as B. cereus (Er I B2.1) also produced much higher IAA concentration than B. cereus (Er II B2.14, Er II B3.11), and B. megaterium (Er I B1.2) produced   higher level of IAA than B. megaterium (Er I B2.12, Er I B3.4). Thus, these strains varied in their potential to produce IAA, and even strains belonging to the same genera or same species. Mirza et al. (2001) showed that IAA production by PGPR can vary among different species and strains, and it is also influenced by culture condition, growth stage and substrate availability.

Diversity in Phosphate Solubilizing Traits of the Rhizobacteria
Phosphorus is the second major nutrient for plants and exists in nature in a variety of organic and inorganic forms, however, it is the least soluble in soil either by adsorption, chemical precipitation or both (Paul and Clark 1996). Several soil microorganisms known as PSB have the ability to solubilize insoluble phosphate mineral by producing various organic acids, siderophores, mineral acids, protons, humic substances, CO 2 and H 2 S (Ivanova et al. 2006). This results in acidification of the surrounding soil, releasing soluble orthophosphate ions (H 2 PO 4 -1 , HPO 3 -2 and PO 4 -3 ) which can be readily taken up by plants.

Diversity in CMC-ase (Cellulolytic Degradation) Traits of the Rhizobacteria
A key role in the decomposition and transformation of organic matter such as plant residues in the soil ecosystem is attributed to aerobic microorganisms, especially cellulolytic bacteria that are ubiquitous in soils (Mullings and Parish 1984;Szegi 1988). Cellulolytic bacterial isolates obtained from this study belong to the genera of Streptomyces, Bacillus, Rhodococcus, Pseudomonas, Arthrobacter, Stenotrophomonas, Staphylococcus, Acinetobacter, Rhodobacter, Pantoea, Sinomonas, Microbacterium and Citrobacter (Fig 3). These results correspond to the findings of Eriksson et al. (1992), who discussed the striking role of Bacillus, Cellulomonas, Cytophaga, Pseudomonas, Sporocytophaga and Streptomyces in cellulose decomposition in soil.
Among all the bacteria isolated from the rice rhizosphere, isolates belonging to the Bacillus   (Fig. 3).

Diversity in N 2 Fixing Traits of the Rhizobacteria
Nitrogen is one of the most limiting essential nutrients into the plant. Prevalence of nitrogen-fixing ability among bacteria colonizing rhizosphere might ensure their survival and multiplication. A high proportion of nitrogen-fixing bacteria in rhizosphere may also be attributed to the host-associated factors. However, the contribution of rhizobacteria is yet to be estimated (Beneduzi et al. 2008). A total of 78 rhizosphere bacteria obtained were selectively isolated from two rice field soil samples with three different levels of salinity. The putative nitrogen fixing bacteria were selected based on their growth on selective semi-solid media without nitrogen (NFb). Twelve bacterial isolates capable of fixing N 2 were obtained from the coastal soil of Subang and 20 isolates were found from Indramayu. The partial sequencing of 16S rRNA gene of nitrogen fixation classified 12 different genera. Strains belong to Bacillus, Brevibacterium, Streptomyces, Rhizobium, Aeromonas, Enterobacter, Pantoea, Staphylococcus, Acinetobacter, Pseudomonas, Providencia and Sinomonas were the N 2 fixers obtained from this study (Fig 4). This is consistent with the result obtained by Farina et al. (2012) that Agrobacterium, Enterobacter, Pseudomonas, Acinetobacter and Streptomyces were the most abundant PGPR found as nitrogen fixers associated with canola plants.
Our results showed that Bacillus is the most dominant as nitrogen fixer, phosphate and cellulose solubilizer, and IAA producer. This might be due to its ability to efficiently use nutrients provided by the plant through exudates, and to inhibit the growth of other strains. Many strains of Bacillus have been reported to produce substances that act as growth inhibitors for other microorganisms (Lilinares et al. 1994).
It might be interesting to test in the future, the resistance of the selected PGPR of this recent study to various environmental stresses and also the functional characterization of PGPR for practical applications in the rice field in coastal areas.

CONCLUSION
Taking all of these diverse PGPR characteristics into account, it is clear that the 78 isolates of rhizobacteria identified have great potential for future research. Several strains of the rhizobacteria produced plant growth promoting activities, alone or in combination, including IAA production, nitrogen fixation, phosphate solubilization and cellulase production. Our study on the species and functional diversity of rhizobacteria associated with rice plant from coastal soils may be useful for selecting proper PGPR strains for improving saline soils.