Potential Role of Rice Plant Growth Promoting Phylloplane and Rhizospheric Bacteria in Controlling Xanthomonas oryzae pv. oryzae

Rice is an important cereal worldwide and it occupies the top position among the cereals in Bangladesh. Rice plant suffers from around 32 diseases of which ten are major in Bangladesh at present. Among the diseases, Bacterial Blight (BB) caused byX. oryzae pv. oryzae (Xoo) considered as a most destructive disease occurs in both rainfed and irrigated seasons of Bangladesh. BB causes considerable yield loss varies from 30 to 50% depending on the outbreak. It is also an important disease in most of the South and Southeast Asian countries. To develop environment-friendly sustainable management approach against BB of rice, in total sixty three plant growth promoting bacteria were identified from rice phylloplane and rhizosphere that are antagonistic toX. oryzae pv. oryzae during boro and aman seasons 2018 and 2019.These bacterial species inhibited the growth of X. oryzae pv. oryzae in vitro by 20.83 to 76.19%. These bacterial isolates were identified by sequencing of PCR products of 16SrDNA belonging to the genera mostly Pseudomonas, Bacillus and Serratia. Out of these bacterial species, 48 bacterial species were found as positive for IAA production, all 63 bacterial species were found positive for siderophore production and 48 were found capable to solubilize insoluble phosphate. Based on growth inhibition of X. oryzae pv. oryzae in in vitro, thirty two bacterial species were selected for plant growth promotion assessment and evaluation of net house and field efficacy in controlling BB of rice. These bacterial species were formulated using talcum powder which was viable for at least three months post formulation. Assessment of plant growth promoting determinants revealed that all 32 bacterial species identified in this study enhance the growth of rice plants as measured by root and shoot length and and reduced the BB severity in susceptible rice cultivar significantly as compared with untreated control.


Introduction
Rice (O. sativa L.) suffers from 32 diseases of which in Bangladesh 10 has been known as dreadful diseases [1]. Among the diseases three bacterial diseases are frequently occurred in Bangladesh. Among these three diseases, Bacterial Blight (BB) caused by X. oryzae pv. oryzae (Xoo) considered as a most destructive disease occurs in all Agro Ecological Zones (AEZs) of Bangladesh and mostly in two rice growing seasons namely viz. raifed and irrigated [2][3][4] and cause severe yield loss. In Japan, India and Bangladesh due to this devastating disease around 50%, 60% and 30% yield loss was observed [5], respectively in the highly infected rice fields. It is also a crucial disease in most of the South and Southeast Asian countries [6]. Bacterial blight (BB) is disease associated with several growth phases of rice plant showing either "Kresek" (acute wilting of young plants) symptoms and "leaf blight" (straw color blighted area with weavy margin) symtoms [7]. Excess amount of nitrogenous fertilizer in rice varieties (HYV) facilitates the emergence of this disease and its severity in the field [8][9][10][11][12]. In Bangladesh different pathogenic [13,14] and genetic variability [15] have been detected and those were excessively perilous for rice [16].
Chemical fungicides (copper compounds, other chemicals and antibiotics) are not effective in controlling this disease [17]. However, control measures are including chemical, cultural, host resistance, genetic modification methods, among them cultural practices are not also effective in all circumstances as well as no fruitful chemical control and commercial product was found in this tropical climatic area which can be suppressed this disease nicely [18,19]. Moreover, using antibiotics, toxic residues and chemicals have several limitations against BB of rice [20]. Apart from that, the uses of host resistance genes are used, in case of breeding single gene (Xa4) are manifested ineffective BLB management due to sub-populations [21].
Thus, biological control alleviates costs and it also serves as an environment friendly approach to mitigate this devastating threat [22], besides, the application of biological strains of PGPB would be the fullest alternative way of minimizing chemical pesticides, fertilizer and environmental pollution [23]. PGPB plays a crucial role in developing immunization in plants body, ISR is triggered by PGPB which is a signaling pathway while SAR mainly dependent on salicylic acid triggering a induced resistance by a particular infection, However, it is observed that ISR requires salicylic acid (SA) and ISR demands ethylene (ET) and jasmonic acid (JA) signal pathways [24] and both of these are triggered latent resistance mechanism subsequently after inoculation [25]. In recent years, application of PGPB in the field has been evaluated as an inducer showing systematic resistance [26,27,38]. Due to fruitful leaf colonization, quick growth, normal application procedure of L. antibioticus have been utilized as a bio control agents against Xoo [28]. Bacillus spp. also found effective in quelling BLB of rice under greenhouse condition [29]. According to [30], S. globisporus have been effective against rice blast. Sheath blight disease was alleviated by using a few biofilm and surfactant delivering strains of Bacillus subtilis [31]. Amalgamation of B. subtilis and Streptomyces philanthi were biologically effective againstrice sheath blight adding with chemical fungicides [32]. HCN (Hydrogen cyanide) played an effective role inhibiting the surges of M. oryzae as well as developing its bio control agents against blast of rice [33]. These antagonistic bacteria have the ability to subvert plant pathogens by releasing chemicals such as glucanases, proteases and chitinases, siderophores [34]. Rice disease can be controlled by the antagonistic strains of Bacillus and Pseudomonas spp. up to 90% based on what kind of strains are used [35]. When systemic resistance is exposed is called as ISR, and conversely, by other phenomenon is called SAR [36]. No necrosis manifested while ISR developed by PGPB [36]. Last few decades, PGPB have been showing as a systematic resistance in the field [26,27,37,38]. ISR demands three systematic pathway which are jasmonic acid (JA), ethylene (ET), salicylic acid (SA) signaling pathways [24]. PGPB can induce priming by the release of volatiles. For instance, Bacillus subtilis GBO3 induces a signaling pathway that is independent of salicylic acid (SA), jasmonic acid (JA) and the Npr1 gene (SA insensitive or nonexpresser of PR genes), yet it requires ethylene [39]. Priming offers an energy cost efficient strategy, enabling the plant to react more effectively to any invader encountered by boosting infection induced cellular defense responses [40,41]. The increased levels of defense related enzymes during ISR are known to play a crucial role in host resistance [42,43], reported that Pseudomonas fluorescens have been used as a bacterial antagonists against BLB of rice. A plentiful of bacterial strains B. cereus, B. pasteurii, pumilus, Bacillusmycoides, B. amyloliquefaciens, B. sphaericus, B. pumilus, B. cereus have been effective in reducing disease resistance upon using ASM (acibenzolar-S-methyl) [39,43].
Species such as Bacillus spp. which showed ISR are radically linked to plant growth modification promotion [39] and this strains have been manifested resistance activity against a number of plant diseases studied by several researcher [44][45][46][47][48][49][50]. In rice, limited number of studies found discussing on induced resistance, the main theme of PGPB also includes production of growth hormones such as IAA and IA (inorganic phosphate) (Khan et al., 1997 and [51]), and zinc solubilization [52], atmospheric nitrogen [53]. Plant health also maintained by PGPB by producing ISR, siderophores and competition [54] as well as mitigate plant pathogens by developing enzymes such as antibiotics, proteases, glucanases and chitinases [34]. In both lab and field conditions PGPB bacteria are significantly reduced plant disease incidence, among them Bacillus and Pseudomonas spp. suppressed diseases up to 90% based on rice variety and types of pathogens [35]. ISR (Induced systemic resistance) is an environment friendly option for plant disease control because it initiates defense related genes and enzymes in host plant through inoculated bacteria to reduce disease incidence [29]. Bacterial Blight pathogen, however, radical information on rice PGPB which can be used as both biopesticide and biofertilizer is not disclosed in Bangladesh. Besides, more investigation needs to be executed from other dimension to completely minimize this deadly disease.

Plant sample collection
To isolate the bacteria from rice phylloplane and rhizosphere, the healthy rice plants with root system and soils of different rice cultivars were collected from 40 districts representing 30 Agroecological Zones (AEZs) of Bangladesh from the vicinity of BB infected rice plants during boro and aman season, 2018 and 2019 at maximum tillering stage to pre-ripening stage. Then the rice plant samples were brought into the laboratory in labeled polybags.

Isolation and purification of bacteria
The phylloplane bacteria were isolated using washing method. Freshly harvested 2nd, 3rd, 4th leaves were vortexed in sterile saline solution for 12 minutes with two or three brief intervals. Then 100 μl solution was placed at the center of Luria Bartani (LB) or King's B agar plate and the solution was spread with glass spreader. The inoculated plates were incubated for 3-5 days at room temperature. After incubation of the inoculated plates, bacterial colonies appeared with various types of colors. Then the bacterial colonies were selected and isolated depending on their color and were streaked on LB media separately. Again the streaked LB plates were incubated at room temperature for 2 days. For isolation of antagonistic bacteria from rhizosphere, 1 g roots with rhizospheric soils were taken and then it was shaken with 100 ml sterile water for about 10-15 min to obtain soil suspension. Isolation of bacteria were carried out from rhizospheric soil by serial dilution technique up to 10 À5 to 10 À6 using LB (Luria Bertani) medium. Then the solution was placed at the center of Luria Bartani (LB) or King's B agar plate and the solution was spread with glass spreader. The inoculated plates were incubated for 3-5 days at room temperature. After incubation of the inoculated plates, bacterial colonies appeared with various types of colors. Then the bacterial colonies were selected and isolated depending on their color and were streaked on LB media separately. Again the streaked LB plates were incubated at room temperature for 2 days.

Assay of antagonism of bacterial spp. to X. oryzae pv. oryzae by dual culture method
Antimicrobial activity of antagonistic strains of Pseudomonas spp./Bacillus spp. were determined by agar diffusion technique method [55] with some modifications. Antagonistic bacterial suspension was spot inoculated (5 μlof10 8 CFU/ml) at three places on the NBY plates that were prior inoculated with X. oryzae pv. oryzae cell suspension (10 8 CFU/ml $ optical density: 0.3). The plates were incubated for 7 days post inoculation at 28°C. Then X. oryzae pv. oryzae growth inhibition by the antagonistic bacterial isolates indicated by clear halo zones were measured with a ruler in mm. The percent growth inhibition of X. oryzae pv. oryzae by bacterial isolates was calculated as follows:

Assessment of plant growth promoting determinants of bacteria antagonistic to X. oryzae pv. oryzae
Active isolates with antagonistic potential against X. oryzae pv. oryzae were further evaluated for their ability to produce plant growth promoting determinants viz. siderophore production, Indole acetic acid (IAA) production and phosphate solubilization capability as follows:

Assay for siderophore production
Siderophore productions by antagonistic bacterial isolates were tested qualitatively as described by Alexander and Zuberer [56]. 5 μl of antagonistic bacterial cell suspension (5 Â 108 CFU/mL) was spot inoculated on Chrome azurol S (CAS) agar plate. The plates were then incubated at 30°C for 5 days. Development of yelloworange halo zone around the bacterial growth was considered as positive for siderophore production. Experiment was performed with a completely randomized design with 3 replications. CAS agar was prepared from 4 solutions. Solution 1 (Fe-CAS indicator solution) was prepared by mixing 10 mL of 1 mmol L À 1 FeCl3.6H2O (in 10 mmol L À 1 HCl) with 50 mL of an aqueous solution of CAS (1.21 g L À 1). The resulting dark purple mixture was added slowly with constant stirring to 40 mL of aqueous solution of hexadecyl trimethyl ammonium bromide (1.821 g L À 1). The yielded of dark blue solution which was autoclaved, then cooled to 50°C. The entire reagent was freshly prepared for each batch CAS agar. Solution 2 (buffer solution) was prepared by dissolving 30.24 g of piperazine-N, N-bis (2-ethane sufonic acid) (PIPES) in 750 mL of salt solution containing 0.3 g K 2 PO 4 ,0.5gNaCland1.0g NH 4 Cl. The pH was adjusted to 6.8 with 50% (w/v) KOH, and water was added to bring the volume 800 mL. The solution was autoclaved after adding 15 g of agar then cooled to 50°C. Solution 3 contained 2 g glucose, 2 g mannitol, 493 mg MgSO 4

Assay for indole acetic acid (IAA) production
IAA production of antagonistic bacterial isolates were carried out as per the procedure described by Patten and Glick [57]. Every isolate was grown in LB media supplemented with (0.005%) L-tryptophan and incubated in shaker at 30°C with 160 rpm for 48 h. Then bacterial culture was centrifuged at 8000 rpm for 15 min and 1 mL culture filtrate was mixed with 4 mL salkowski's reagent (1.5 mL FeCl3.6H2O 0.5 M solution in 80 mL 60% H2SO4) and the mixture was incubated at room temperature for 30 min, presence of pink color indicate qualitatively that isolate produced IAA. Formation of pink color indicated the presence of indoles [58].

Identification of selected plant growth promoting antagonistic bacterial
isolates by sequence analyses of 16SrDNA

Extraction of genomic DNA
Bacterial culture from NA media was transferred in LB broth and shaken for 18 h at 28°C. Then genomic DNA of antagonistic bacteria was extracted according to Wizard® Genomic DNA purification Kit (Promega, Madison, USA). Obtaining the DNA pellet was rehydrated by adding 25 μL DNA rehydration solution and kept it overnight at 4°C. Finally the genomic DNA samples of the isolates were preserved at À20°C for further use.

Primers and PCR conditions
To identify the antagonistic bacterial isolates, the primer sets 27F (5 0 -AGA GTT TGATCM TGG CTC AG-3 0 ) and 1518R (5 0 -AAG GAG GTG ATC CAN CCR CA-3 0 ) specific to 16SrDNA were used for amplification of 16SrDNA from the prepared genomic DNA template [61]. The PCR conditions were as follows: initial denaturation at 95°C for 5 min, 35 cycles denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and finally a 7 min extension at 72°C. PCR products were visualized by electrophoresis on 1.0% agarose gel containing 0.5% of ethidium bromide using a Gel documentation System after separating the PCR products in the agarose gel for 50 min at 80 volt.

Sequencing of PCR products
A partial nucleotide sequencing of 16SrDNA was performed from amplified PCR products using primers 27F (5 0 -AGA GTT TGATCM TGG CTC AG-3 0 ) in the Macrogen Lab, South Korea via Biotech Concern Bangladesh. The sequencing was done directly from PCR products according to the standard protocols for the ABI 3730xl DNA genetic analyzer (Applied Biosystems, Foster City, CA, USA) with BigDye® Terminator v1.1 and 3.1 Cycle Sequencing Kits.

Processing of sequence data
The sequencing data were processed and nucleotide sequence data was exported using Chromas software version 2.6.4.The quality of nucleic acid sequences was evaluated using Chromas (Version 2.6) software to avoid the use of low quality bases.

Analyses of nucleotide sequences
The nucleotide sequences were analyzed using online bioinformatics tools. The DNA sequences of 16Sr DNA of the bacterial isolates were compared with 16Sr DNA of the bacterial spp. and the sequences of ITS region of the fungal isolates were compared with ITS region of the fungal spp. that were available in the NCBI database using Basic Local Alignment Search Tool (BLAST) algorithm to identify closely related sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Formulation of plant growth promoting antagonistic bacterial species
The pure cultures of thirty two selected potential bacterial antagonists were grown on LB agar medium for 24 hrs. Then the bacterial isolates were transferred in LB broth for about six hours by taking a loopful of bacteria from the LB agar plate. After that the liquid culture was then centrifuged and resuspended the pellet in previously prepared 200 ml peptone broth aimed to fortify the carrier materials. This culture broth was then grown for another two hours with shaking. After that 5 ml of sterile 100% glycerol was added to this 200 ml culture. Then the cultures of the bacterial antagonists (200 ml fortified with 1% peptone and 1% glycerol) were added to the mixture of 500 g talcum powder amended with 5 g carboxy methyl cellulose (CMC) and 7.5 g Calcium carbonate which were autoclaved for two consecutive days at 121°C under 15PSI pressure for 30 min each. The formulations were then being dried overnight in the tray. After that the formulations were powdered with hand, the formulated bacterial antagonists were packed in plastic bags. The formulated bacterial antagonists were then kept at both room and 4-8°C temperature in the refrigerator.

Assessment of viability of the formulated fungal and bacterial antagonists
The viability of the bacterial and fungal antagonists were checked by drawing 1 g of the formulated products in sterile water in every 30 days after formulation and diluted serially up to 10 À4 or 10 À5 . The numbers of viable cells (colony forming unit) were counted per gram formulations kept at both room temperature and 4-8°C temperature in the refrigerator.

Assessment of plant growth promotion induced by antagonistic bacterial and fungal isolates
Rice seeds (cv.IR24) were surface sterilized and dried. Then the sterilized rice seeds were treated with formulated bacterial and fungal antagonists (10 g/kg seeds) and the treated seeds were left for 1 h under shade. The rice seeds were then sown in the plastic pots previously filled with sterile soils. Fifty seeds were sown in each pot and three replications were maintained. Then the germination of seeds were recorded at 7DAS. The seedlings were uprooted at 7 DAS, 14 DAS and 28 DAS to measure the root length, shoot length and to calculate the vigor index [= (root length + shoot length) Â germination percentage] were measured.

Seed priming, raising of seedlings and transplanting
Seeds of IR24 were treated with 32 selected formulated PGP antagonistic bacterial isolates. The treated sees were left for 1 hr. for adherence of the bacterial and fungal isolates with the treated seed surface. The treated seeds were then sown in the plastic pots filled with sterilized soils. One month old seedlings were then transplanted in the plastic pots filled with puddle soils.

Foliar spray of formulated PGP bacterial and fungal isolates
Formulated PGP antagonistic bacterial isolates were sprayed two times (at 50 and 55 DAS) before inoculation and two times after inoculation i.e. 65 and 70 DAS.

Inoculation of the rice plant with X. oryzae pv. oryzae
Rice plants were inoculated with a strain of X. oryzae pv. oryzae by Scissor clip method at 60 DAS.

Isolation and identification of antagonistic bacteria against X. oryzae pv. oryzae
Rice plant samples were collected from 40 districts of Bangladesh representing 30 AEZs during boro seasons 2018-2019 and aman seasons 2018-2019. In total 300 bacterial isolates and 100 fungal isolates were isolated and purified from rice plant samples during boro season, 2018. Some selected representative bacterial species were shown in Figure 1. Out of 300 bacterial isolates, eighteen were identified as antagonist against X. oryzae pv. oryzae and inhibited the growth of X. oryzae pv. oryzae in vitro which was ranged by 28.39-76.19% (Table 1 and Figure 2). The maximum (76.14%) growth inhibition of X. oryzae pv. oryzae in vitro was recorded by BDISOB05P while the minimum (28.59) growth inhibition was exhibited by BDISOB272R. These antagonistic bacterial isolates were identified by sequencing of PCR products of 16SrDNA gene ( Figure 3A). The identified bacterial species were BDISOB04P (P. putida), BDISOB05P (P. putida), BDISOB98P (Stenotrophomonas maltophilia), BDISOB241P (Burkholderia sp.), BDISOB242P (B. gladioli), Table 1). In total 400 bacterial isolates and 40 fungal isolates were isolated and purified from rice plant samples collected in aman season, 2018. Seventeen bacterial isolates were identified as antagonist against X. oryzae pv. oryzae and inhibited the growth of X. oryzae pv. oryzae in vitro which was ranged by 38.33-60.66% ( Table 2). The highest (60.66%) growth inhibition of X. oryzae pv. oryzae was exhibited by BDISO147Pand the lowest (38.33%) growth inhibition was shown by BDISO135P.These antagonistic bacterial isolates were identified by sequencing of PCR products of 16SrDNA gene ( Figure 3B  In boro season 2019, 300 bacterial isolates were isolated and purified. In boro season 2019, out of 400 bacterial isolates fourteen were identified as antagonist against X. oryzae pv. oryzae and inhibited the growth of X. oryzae pv. oryzae in vitro which was ranged by 20.83-60.87% (Table 3 and Figure 3C). The maximum (60.87%) growth inhibition of X. oryzae pv. oryzae in vitro was recorded by BDISOB37R while the minimum (20.83%) growth inhibition was exhibited by BDISOB14R.  Table 4). The maximum (61.54%%) growth inhibition of X. oryzae pv. oryzae in vitro was recorded by BDISOB54R while the minimum (50.93%) growth inhibition was exhibited by BDISOB12R. These antagonistic bacterial isolates were identified by sequencing of 16SrDNA gene ( Figure 3D)

Phosphate solubilization
Among all bacterial isolates three of them those were BDISOB05MymP (P. putida), BDISOB45PanP (Bacillus paramycoides) and BDISOB01MymR (B. amyloliquefaciens) manifestedsupreme amount of phosphate solubilization activity. Whereas, another three of them which wereBDISOB186KusR (Pseudomonas sp.), BDISOB258GaiR (P. putida) and BDISOB70KusR (Seratia marcescens) showed lowest phosphate solubilization activity. Except highest and lowest phosphate solubilization producing isolates rest of them were showed moderate type activity. In this case, a noticeable differences were observed that except two isolates BDISOB221GaiR and BDISOB222GaiR all other isolates are statistically similar. The others bacteria in case of phosphate solubilization were statistically similar ( Table 5 and Figure 4).

Plant growth promotion by bacterial isolates antagonistic to X. oryzae pv. oryzae
Based on the growth inhibition of X. oryzae pv. oryzae by these antagonistic bacterial species, 32 bacterial isolates were selected for plant growth promotion assay and for subsequent assessment of their net house and field performances. Different plant growth promoting bacterial antagonists enhanced the root length, shoot length and vigor index at 14, 21 and 28 DAS ( Table 6). Among 32 bacterial

Plant growth promotion by different bacterial isolates antagonistic to
Xanthomonasoryzae pv. oryzae

Fresh shoot weight
At 28 days after sowing the highest shoot weight (2260 mg) was recorded in plants raised from the seed treated with the bacterial isolate BDISOB01MymR followed by the bacterial isolates BDISOB05MymP (2250 mg), BDISOB45PanP (2173 mg), BDISOB04DinP (2033 mg), BDISOB86FarR (2033 mg), BDISOB07FarR, (2033 mg) BDISOB283KisR (1950 mg). But the lowest shoot weight was observed in control (untreated seed) (933 mg) Rest of the isolates were showed moderate fresh shoot weight. Among all bacterial isolates seventeen were statistically similar and others denoted statistically dissimilar ( Table 7).

Effect of some selected antagonistic bacterial isolates on the reduction of lesion length in susceptible check cultivar (IR24) caused by X. oryzae pv. oryzae
To evaluate the mechanisms of BB severity reductionby plant growth promoting antagonistic bacteria, susceptible check variety IR24 was used. The results of plant inoculation showed a significant reduction of lesion length in plants sprayed with formulated bacterial bioagents as compared with untreated control.

Discussion
Antagonistic bacterial isolates were identified mostly as different species of Pseudomonas, Bacillus,Serratia and Delftia. In a previous study, frequency of antagonistic bacteria on LB medium was low [62], but another study revealed that using different growth media such as King's B, and Gould's S1 and Nutrient Agar were effective for the isolation of higher number of antagonistic bacteria [63]. It was reported that some antagonistic bacteria such as B. subtilis, B. amyloliquefaciens, B. valismortis, Streptomyces sp., P. chlororaphis and Acinetobacter baumannii were identified based on 16S rRNA sequence analysis [64]. A number of bacteria from species Alcaligens, Arthobacter, Burkholderia, Alcaligens, Arthobacter, Burkholderia, Bacillus, Azospirillum, Azotobacter, Klebsiella, Enterobacter and Serratia have been observed to develop plant growth. However, as bio control agents, isolates of fluorescens, Pseudomonas, and Bacillus have been the most exploited and studied [65][66][67][68]. Nowadays, antagonistic bacteria were also used for plant roots as a biological control infecting by numerous plant pathogens [26,69]. Out of 300 bacterial isolates sixteen isolates of several species were evaluated in vitro and they exposed antagonistic activity to X. oryzae pv. oryzae. It was observed that 54.03% to 96.56% of lesion length was diminished when treating with antagonistic bacteria. These findings were identical to the reported by Monteiro et al. [63] because they also showed that BB pathogen was suppressed by antagonistic bacteria. According to Ranjbariyan et al. [70] who also experimented that three bacterial isolates significantly acted higher growth inhibition of X. oryzae pv. oryzae. Antibiotics, enzymes like chitinases, glucanases, proteases, and siderophore produce directly or indirect mechanisms in which the antagonistic bacteria compete with the pathogen for a niche or nutrient sites [34].
Out of the 63 bacterial isolates, 48 bacterial species were found as positive for IAA (Indole Acetic Acid) production, all 63 bacterial species were found positive for siderophore production and 48 were found capable to solubilize insoluble phosphate. IAA also has been speculated to fasten the overall fitness of plant-microbe associations [57]. It was proved that numerous plant-associated bacteria have the ability to produce IAA by stimulating plant roots development and improving absorption of water and nutrients from soil [71,72]. The IAA producing bacteria encouraged adventitious root formation, produced the greatest roots and shoots weight [73]. All 63 bacterial isolates were found to produce siderophore. When iron availability is in stress microorganism those who produce siderophore supplied Fe nutrition to enhance plant growth [74]. Siderophore also assists when it comes to the growth condition of shoots, roots as well as nutrition in plants [75]. Siderophore plays a crucial role in selecting a potential bioagent [76], besides, it has been considered as an alternative to ruinous pesticides effects [77]. The biological control mechanism depended on the role of siderophore as competitors for Fe in order to reduce Fe availability for the phytopathogen [78]. Siderophores produced by numerous bacteria had a significant role in the biocontrol and negatively affected the growth of several pathogens [78,79]. Forty eight bacterial isolates showed the capability of phosphate solubilization. It has been also experimented that phosphate solubilizing bacteria (PSB) can also triggered plant growth promotion [80]. This PSB inoculants have been exploited as a possible alternative for phosphate fertilizers which is inorganic [81] and it also influences phosphate uptake and plant growth [82,83]. It has also been documented that the application rates of phosphate fertilizers reduced to 50% by inoculating phosphate solubilizing microbes (PSM) added phosphate fertilizers reduced the disease incidence up to 50% [84].
Among the bacterial isolates, 32 were selected based on their antagonistic capability and growth promoting determinants. PGPB have significant impact in surging root length, vigor index and shoot length. Sakthivel et al. [85] and Mishra and Sinha [86] reported to enhance growth of rice seedling with bioagent application. Van Peer and Schippers [87] stated that shoot, root and fresh weight was raised for cucumber, lettuce, tomatoand potato as a result of bacterization with Pseudomonas strains. The results of the present study depicts that the effect of plant growth promoting bacterial isolates on growth and vigor of rice plants was significantly higher than control. It has been reported that P. fluorescens and other plant growth promoting rhizobacteria can show antagonisms to potentially harmful bacterial pathogens and eventually those bacteria contribute to enhance plant growth [88]. Biological agents like plant growth promoting bacteria (PGPB) can be used as bio-fertilizer [89].
Forty eight bacterial species were found positive for phosphate solubilization out of 63 antagonistic bacterial species identified in this study. It has been reported that phosphate solubilizing bacteria (PSB) induced plant growth promotion [80]. Plant roots-associated PSB have been considered as one of the possible alternatives for inorganic phosphate fertilizers for promoting plant growth and yield [81]. Plant growth and phosphate uptake have increased in many crop species due to the results of PSB inoculants [82,83]. It has also been documented that the application rates of phosphate fertilizers reduced to 50% by inoculating phosphate solubilizing microbes (PSM) in crops without significantly reducing crop yield [84]. In sustainable agriculture, specific plant pathogens can be supressed by biological agents such as plant growth promoting bacteria (PGPB) which can also be used as bio-fertilizer [89]. There are a plenty of PGPB strains that reported to suppress numerous of 25 Potential Role of Rice Plant Growth Promoting Phylloplane and Rhizospheric Bacteria… DOI: http://dx.doi.org/10.5772/intechopen.99854 plant pathogen, reduced disease incidence, triggered plant growth factor and provides nutrition for the growth of the plant [63,90]. Thus, it has been considerable research interest in the potential use of antagonistic bacteria as PGPB [91,92]. To evaluate plant-interaction with bacteria, such as endophytes, biocontrol agents, phytopathogens, and symbionts needs to be re-infection and development of those experimental strains in or on field grown plants [93]. Effective root colonization by fluorescent Pseudomonas spp. has been manifested to take an inevitable part in controlling plant pathogens as a biocontrol agent [94]. The significance ofthis study is that functionally characterized all antagonistic bacteria may be used for biocontrol of BB along with enhanced rice growth. Even though, Pseudomonas spp. are indigenous and involve in various rhizomicrobiomes but few of them have the ability to grow above 37°C and become opportunistic pathogens, thuspredictable biosafety regulations are needed to implement this technology practically for field application [95]. In a nutshell, based on all results achieved from during this study, bacterial strains may be an effective bio-inoculant for controlling BB of rice by ensuring its biosafety aspects.

Conclusion
Thirty two potential bacterial isolates were identified belong to the genera mostly Pseudomonas, Bacillus and Serratia from rice phylloplane and rhizosphere among sixty three that inhibited the growth of X. oryzae pv. oryzae in in vitro significantly and were found positive for enhancing plant growth promotion by the production of plant growth promoting determinants viz. IAA, siderophore and phosphate solubilization. Formulated bacterial isolates can be viable in talcum powder for at least three months post formulation. Reduction of lesion length caused by X. oryzae pv. oryzae on susceptible cultivar IR24 by the formulated bacterial isolates primarily indicates their potentiality in controlling BB of rice. Patenting, registration, large scale formulation and commercilization of these PGP bacteria would be the next step of this work.