Rice is an important staple food crop. The global production of rice paddy was 746.9 million tons and 496 million tons milled rice (http://www.fao.org/economic/est/publications/rice-publications/rice-market-monitor-rmm/en/). The crop is widespread all over the world due to its wider adaptability under different environmental conditions. For this reason, Food and Agriculture Organization (FAO) regarded it as a strategic crop for food security in the world (Montano et al., 2014). Rice is used as a staple food in different areas of world especially in Asia (http://www.britannica.com/plant/rice). In Pakistan, beside wheat, rice is the 2nd major cereal crop, which is widely cultivated, consumed, and exported. Among various cultivated varieties, Super Basmati rice variety is the most sought variety, as it is very popular among consumers, farmers, traders, and exporters due to its high quality grain and aroma (http://reap.com.pk/news/news_detail.asp?newsid=3905). However, susceptibility of this variety to different diseases is a major problem. Among these, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight (BLB) is considered to cause severe yield losses (Arshad et al., 2015). This disease is widely prevalent among various rice varieties worldwide (Singh et al., 2015). Historically, BLB was initially reported in Japan during 1884–1885 and then reported in other rice growing countries (Gnanamanickam, 2009). Rice crop was severely affected by this disease in areas of tropica and Asia with heavy rainfall in monsoon. Initially, its occurrence in Pakistan was reported by Mew and Majid (1977) and Arshad et al. (2015). Later studies indicated an alarming increase in BLB incidence in Basmati rice growing areas of the country (Shah et al., 2009).

BLB occurs at different growth stages of rice and is manifested by either leaf blight or “Kresek” (acute wilting of young plants) symptoms. Xoo invades the plant through wounds or water pores. Lesions with wavy margins start from the tip of the leaf as the water pores are located at the margins of upper parts of the leaf. These water soaked lesions enlarge in size, turn yellow and ultimately lead to the death of plant (Nino-Liu et al., 2006).

In the past, various disease management strategies have been employed to reduce the yield losses and to avoid disease epidemics but use of chemicals has not been successful due to variation in sensitivity of pathogenic races toward applied chemicals. Development of mutation in pathogenic races is a major hindrance in developing a durable control (George et al., 1997). Moreover, due to toxic residues, usage of antibiotics and chemicals against rice BLB, has limitations (MacManus et al., 2002). Although, the use of host resistance genes seems to be practicable, single gene (Xa4) based breeding for BLB management has been shown to be ineffective due to evolution of sub-populations that overcome these resistance genes (Shanti et al., 2010). As a result, biological control seems to be a cost effective and environmentally friendly way to manage this serious threat (Gnanamanickam, 2009).

Most of the rhizospheric antagonistic bacteria such as Pseudomonas spp. can indirectly increase plant resistance by improving the plant growth. Responses of the host plant are due to root colonization of a plant by antagonistic rhizobacteria that play an important role in disease suppression. Lysobacter antibioticus have been documented as biocontrol agents against Xoo due to their rapid growth, easy application and effective leaf colonization (Ji et al., 2008). Plant growth promoting Bacillus spp. were found to suppress BLB in rice under greenhouse conditions (Chithrashree et al., 2011). Li et al. (2011) reported Streptomyces globisporus for the suppression of rice blast caused by Magnaporthe oryzae. The incidence of sheath blight was reduced by some biofilm forming and surfactant producing strains of Bacillus subtilis (Mousivand et al., 2012). Streptomyces philanthi and a commercial formulation of B. subtilis were found to be biologically active against rice sheath blight when integrated with chemical fungicides (Boukaew et al., 2013). Hydrogen cyanide (HCN) producing Pseudomonas chlororaphis significantly inhibited the growth of M. oryzae, showing its biocontrol properties against the causal agent of rice blast (Spence et al., 2014).

These antagonistic bacteria can directly suppress plant pathogens by producing antibiotics, enzymes like chitinases, glucanases, proteases, and siderophores or indirect mechanisms in which the antagonistic bacteria compete with the pathogen for a niche or nutrient sites (Bardin et al., 2015). These bacteria have been reported to reduce the disease incidence significantly under controlled as well as under natural field conditions. Bacillus and Pseudomonas spp. control the diseases caused by rice pathogens i.e., X. oryzae pv. oryzae, Rhizoctonia solani, and M. oryzae up to 90% depending on the bacteria used, pathogen and the rice variety (Montano et al., 2014). Induced systemic resistance (ISR) is an environmentally attractive option for disease control whereby plant defenses are enhanced as a result of their interaction with certain rhizobacteria. ISR contributes positively toward the biological control of plant pathogens and the defense related enzymes induced by the inoculated bacteria protect the host plants (Chithrashree et al., 2011). Systemic resistance was induced by Serratia sp. causing resistance against necrotrophic leaf pathogens in rice (Vleesschauwer et al., 2009).

However, most of the previous studies investigated either plant growth promoting activities or biocontrol activities of the bacterial isolates, exclusively. Previously, Kumar et al. (2005) and Fang et al. (2013) reported broad spectrum antifungal and biofertilizer activity of Pseudomonas aeroginosa and Pseudomonas aurantiaca. Similarly, different endophytic strains of B. subtilis have been reported to have plant growth promoting activity on cacao and biocontrol activity against phytopathogens like Moniliophthora perniciosa and Colletotrichum spp. but the antifungal metabolites were not investigated (Kumar et al., 2012; Nain et al., 2012; Falcao et al., 2014). The use of Pseudomonas and Bacillus strains have been reported for the biocontrol of rice pathogens such as Xoo, M. oryzae, and R. solani (Ji et al., 2008; Helene et al., 2011; Spence et al., 2014). However, again the major emphasis of these previous studies was on the biocontrol activity. Rhizospheric antagonistic Pseudomonas aeruginosa have been documented as beneficial biocontrol agents against Xoo (Yasmin et al., 2016) but the role of diverse secondary metabolites produced by different strains of P. aeruginosa in the suppression of BLB pathogen has not been reported earlier.

Members of the Pseudomonas spp., including pathogenic as well as non-pathogenic strains, are capable of producing various extracellular secondary metabolites. These metabolites exhibit diverse properties i.e., function as virulence factors, siderophores (having high-affinity of iron ions), biosurfactants, and antimicrobial agents as well as in cell-to-cell signaling etc. These metabolites enable Pseudomonas spp. to adapt in different environments, colonize different hosts and compete with other species. Pseudomonas metabolites constitute chemical entities such as N-acylhomoserine lactone (Cataldi et al., 2009), phenazine (Price-Whelan et al., 2006; Kumar et al., 2011; Jain and Pandey, 2016), pyochellin (Youard et al., 2007; Jayaseelan et al., 2014), phloroglucinol (Kidarsa et al., 2011), lahorenoic acid, diketopiperazines (Mehnaz et al., 2013), 4-hydroxy-2-alkylquinolines (Lepine et al., 2004), rhamnolipids (Soberon-Chavez et al., 2005; Grosso-Becerra et al., 2016), and cyclic lipopeptides (Mehnaz et al., 2013). Knowing the chemistries of these metabolites can help with characterization of their biological and physiological activities. However, most of the existing studies have been confined to the analysis of individual metabolite class analysis and its biological properties (Kumar et al., 2005). There are few studies where more than one class of metabolites have been analyzed simultaneously. Even during the study of one physiological aspect like bacterial quorum-sensing, analysis of multiclass metabolites [N-acyl-L-homoserine lactone and 2-alkyl 4-(1H)-quinolone] can enhance the scope of study (Ortori et al., 2011). In this study, detailed chemical characterization of the BRp3 supernatant has led to identification of a variety of previously known as well as novel metabolites of several chemical classes.

Despite the economic importance of BLB, complete resistance to this disease has not been reported. Furthermore, no local resistant varieties are commercially available in Pakistan. Therefore, to select bacteria with multiple beneficial applications for the improvement of rice crop, 512 rhizobacteria were isolated from different field sites of Punjab (Pakistan). After screening their plant growth promoting as well as biocontrol activities, the bacterium termed as BRp3, demonstrated the best results in rice yield improvement and biocontrol activity against the prevalent rice pathogen Xoo. This bacterium was subjected to detailed characterization of the secondary metabolites with ultimate aim to develop inocula of functionally well-characterized native antagonistic bacteria, having a “dual-purpose inoculum” with strong plant growth promoting and biocontrol aspects, for yield enhancement of “Super Basmati” rice variety.

Mass spectrometric analysis revealed the production of siderophores, rhamnolipids, a series of previously known and novel HAQs in the crude extract of BRp3. These results high-lighted the facts that P. aeroginosa BRp3, owing to its capability to produce a number of secondary metabolites in HAQ, rhamnolipids, and siderophores (phenazines and pyochellin) series, exhibited intense antimicrobial activity against Xoo pathogens. Hence, this bacterium could be used as an effective biocontrol agent against the rice pathogen Xoo.

Plant-associated beneficial bacteria are important growth promoters or biocontrol agents in modern agriculture where eco-friendly and sustainable approaches are growing and have more acceptance than ever before. Rice is grown on large area of Pakistan where a lot of chemical fertilizers as well as pesticides are applied. This study was carried out to evaluate the potential for rhizobacterial inoculum to promote growth and to control disease in rice.

The pathogenicity of X. oryzae strains to rice confirmed their identity as causal pathogen of BLB. Xoo1 and Xoo2 were used for further studies due to their aggressive behavior. In vitro plate assay for screening the rhizobacterial isolates (512) for inhibition of growth of Xoo led to the selection of P. aeruginosa BRp3 for further studies. In a previous study, only LB medium was used for isolation of antagonistic bacteria and the frequency of antagonistic bacteria among the total isolated bacterial population was low (Yasmin et al., 2016). A relatively higher number of antagonistic bacteria was obtained in the present study when different growth media (Nutrient Agar, King's B, and Gould's S1) were used for isolation of bacterial populations. As the biocontrol agents with broad spectrum antagonism are found to be more effective against phytopathogens in rhizosphere, therefore, the antagonistic bacteria isolated in the present study were also screened against different phytopathogens, other than the target pathogen (Table S1). Several rice varieties are infected by various fungal pathogens, including Fusarium spp. that cause bakanae disease in rice (Desjardins et al., 2000). The suppression of in vitro growth of the Fusarium spp. by the antagonistic bacteria showed its potential use as an inoculant for biocontrol of these fungal pathogens.

Phosphate solubilizing and IAA producing microbes are a vital fraction of the microbes that improve the development and growth of their host plant. Vassilev et al. (2006) reported that the solubilization of insoluble phosphates by microbial activity usually induce the secretion of certain metabolites mainly siderophores, lytic enzymes and phytohormones that are involved in the control of phytopathogens. The intrinsic resistance of strain BRp3 against different antibiotics may aid survival in the rhizosphere whenever used as a biological control agent (Dobereiner and Baldani, 1997). In our previous study, siderophore production and P-solubilization were found to be involved in growth promoting activities of antagonistic P. aeruginosa Rh323 (Yasmin et al., 2016) whereas the present study suggests that P solubilization accompanied with the production of IAA, siderophore and ACC may contribute to the growth promoting activities of BRp3, even in the presence of Xoo. ACC deaminase activity of this strain may help to lower the level of ACC caused by ethylene under stress conditions and protect the host plant. Bacterial ACC-deaminase is known to regulate plant growth under biotic and abiotic stress conditions (Singh and Jha, 2016).

It is necessary to understand the mechanisms involved in the suppression of pathogens by the application of biocontrol agents for effective disease management. The bio-antagonistic bacterium used in the present study was characterized for different biocontrol determinants i.e., HCN, chitinases, proteases, starch hydrolysis and siderophores production. Strain BRp3 produced volatile compounds such as HCN which is a known suppresser of phytopathogens (Brimecombe et al., 2001). Recent literature reported that cyanide producing bacteria can be considered as effective biocontrol agents because cyanide production by the bacterial strains induces resistance in the plant (Devi and Kothamasi, 2009; Spence et al., 2014). Gandhi et al. (2009) reported the production of an antifungal protease by rice rhizosphere associated Chryseobacterium aquaticum PUPC1 and its effects on the mycelial growth, germination of spores, and sclerotia of phytopathogenic fungi. It has been reported that chitinase can function in defense against many fungal pathogens and also correlated with induced resistance (Perez et al., 2002). Starch hydrolysing ability of the strains indicated their capability to produce amylase and to use a complex carbon source, which aids in the defensive mechanisms of bacterial strains (Marten et al., 2000).

There are many reports that highlight the importance of secondary metabolites in the biocontrol of plant pathogens (Heeb et al., 2011; Khare and Arora, 2011; Jayaseelan et al., 2014). P. aeruginosa BRp3 has the capability to produce a variety of metabolites, which include siderophores (1-hydroxy-phenazine, pyocyanin and pyochellin), rhamnolipids and a series of already characterized 4-hydroxy-2-alkylquinolines (HAQs) as well as novel 2,3,4-trihydroxy-2-alkylquinolines and 1,2,3,4-tetrahydroxy-2-alkylquinolines. Mass spectrometric analysis has confirmed their structures (Figures 1, 2, Table 2).

Siderophores have also been reported to induce systemic acquired resistance (Trivedi et al., 2008; Sulochana et al., 2014). P. aeruginosa BRp3 produces 1-hydroxy-phenazine, pyocyanin (Figure 1) and pyochelin (m/z 325, Figure 2). Phenazine produced by non-pathogenic strain of P. aeruginosa has been documented as an antifungal metabolite on the basis of NMR and MS analyses (Kumar et al., 2011). Pyochelin produced by P. aeruginosa showed antagonistic activity against Botrytis cinerea, the phytopathogen of groundnut (Khare and Arora, 2011). Purified pyocyanin produced by P. aeruginosa TO3 was reported for its inhibitory effect against Macrophomina phaseolina in tomato (Audenaert et al., 2002).

Rhamnolipids are widely reported to be produced by various microbes including the Pseudomonas spp. (Chong and Li, 2017). P. aeruginosa BRp3 demonstrated the production of mono- and di-rhamnolipids (ranged from m/z 500 to 650, Figure 2). LCMS analysis confirmed that the rhamnolipids produced by P. aeruginosa DR1 inhibited the growth of different plant pathogens like F. oxysporum, Sclerotium rolfsii, Phytophthora nicotianae, and M. phaseolina (Reddy et al., 2016).

P. aeruginosa BRp3 is capable of producing a large repertoire of HAQ and HAQ-related compounds in addition to the siderophores and rhamnolipids. Mass spectrometric analysis of cell free extract revealed the presence of nine analogs of 4-hydroxy-2-alkyl HAQs, having saturated and unsaturated alkyl carbon chains, varied from C₅ to C₁₃ chain length, exhibiting m/z values of 216–326 [M+H]⁺ (Table 2, Serial No. 1). Their fragmentation data, can confirm their structures, which is well correlated with previously reported literature data (Deziel et al., 2004; Lepine et al., 2004; Vial et al., 2008). Only one analog of the structure representing 3,4-dihydroxy-2-heptyl (HHAQ) was identified (Figure 3). HHAQ has demonstrated the highest peak intensity at m/z 258 (Figure 2), this compound also termed as “Pseudomonas Quinolone Signal” (PQS) and is involved in the mechanisms of cellular communication, to detect the pseudomonas cell density (Deziel et al., 2004).

Interestingly, putative polyhydroxy HAQ analogs, i.e., a 2,3,4-trihydroxy-2-alkylquinoline m/z 278 [M-H]⁻ and two analogs of 2,3,4-trihydroxy-2-alkylquinoline N-oxideat m/z 294 and 306 [M-H]⁻, respectively, were found to be present in crude extract (Series 5 and 6 compounds, Table 2). The fragmentation data correlated with the putative structures of these polyhydroxy HAQs (Figures 4, 5). Budzikiewicz and Kesselmeier (1979) and Neuenhaus et al. (1979) reported the putative 3-alkyl-3-hydroxy-2, 4-dioxo-1, 2, 3, 4-tetrahydroquinoline derivatives in Pseudomonas. To support the theme of 3-alkyl substitution, they further synthesized these compounds (Figure S5). Similarly, in a detailed study conducted by Lepine et al. (2004) on metabolic profiling of HAQs from Pseudomonas, polyhydroxy HAQs were spotted but the alkyl side chain was reported on 3-position. However, to the best of our knowledge the polyhydroxy HAQs representing alkyl groups at 2-position of the heterocyclic rings, have not been reported in Pseudomonas species before this study. To support our theory of alkyl substitution at 2-position, the proposed 2,3,4-trihydroxy-2-alkylquinoline at m/z 278 was fragmented through CID that generated predominant daughter ion at m/z 242, which on further fragmentation yielded similar finger prints of ions as produced by 4-hydroxy-2-heptylquinolines (Table 2, Serial 1; Figure S6). Finally, a 4-hydroxy-2-dodecenylquinoline N-oxide analog at m/z 340 [M-H]⁻ was identified, whose MS/MS data was correlated with the proposed structure (Table 2, Serial No. 7). The production of this HAQ by Pseudomonas, has also been reported by Lepine et al. (2004). Pseudomonas aeruginosa and other related bacteria produced 2-alkyl-4(1H)-quinolones, which exhibited antimicrobial activity (Heeb et al., 2011).

This work demonstrates that P. aeruginosa BRp3 is capable of producing a large repertoire of HAQ and HAQ-related compounds in addition to the siderophores and rhamnolipids. Identification of polyhydroxy HAQs may provide insight about the biosynthetic pathway of these interesting compounds. These HAQs along with siderophores in the presence of rhamnolipids (as emulsifying and bacterial cell membrane disrupting agents), may be collectively responsible for the profound antibacterial activity of BRp3 against Xoo. Further studies are needed to establish the individual role of each detected secondary metabolite entity as an active biocontrol agent against BLB pathogen. But the challenge to such study is to get these metabolites in measurable quantity in their purified form. Since these metabolites are being produced in nano grams concentration, the optimization of their production through fermentation would have to be optimized prior to their purification process. Secondly, their purification is challenging, owing to the slight variation in their structures, polarity, hydrophobicity etc. (especially the HAQs), and a routine silica column chromatography was failed to purify them. Although, Naureen et al. (2017) have successfully purified the secondary metabolites (2-pentyl-4-quinolinecarboxylic acid and 1- methylcyclohexene) from Lysinibacillus sphaericus ZA9 but the chemistry of these metabolites is significantly different from the HAQs identified in the present study. A high-quality preparatory HPLC system may be required to purify various analogs of HAQs (Wang et al., 2011).

Due to the aforementioned technical challenges, obtaining the sufficient quantities of purified metabolites for plant assays looks difficult. However, crude supernatant from pure cultures of BRp3 were shown to suppress the BLB pathogen (Figure S7). The extracted supernatant of antagonistic bacterial culture has been reported for the control of phytopathogens during in-planta evaluation (Simonetti et al., 2012). The extracellular filtrates of biocontrol bacteria having different secondary metabolites such as cyclic lipopeptides were found to be responsible for their antifungal activity (Petatan-Sagahon et al., 2011). Few reports have documented the significant pathogen suppression by supernatants of fermentation without extraction (Garcia et al., 2014). The mechanistic suppression of BLB can be supported by the fact that rice plants inoculated with the strain BRp3 and challenge inoculated with BLB pathogen resulted in an increased activity of defense related enzymes in the host plant. In a previous study, accumulation of defense related enzymes was observed in rice plant up to 48 h after foliar application of antagonistic bacteria Rh323 (Yasmin et al., 2016). Plants inoculated with BRp3 exhibited an increase in the activity of POD, CAT, and PAL after 24 and 48 h post inoculation while, PPO activity was observed even after 72 h and up-to 6 days after challenge inoculation. The production of plant defensive enzymes i.e., POD, PPO, CAT, and PAL in response to inoculated PGPR bacteria is associated with ISR in plants against the pathogen (Saikia et al., 2006; Liang et al., 2011).

A better evaluation of plant growth promoting efficiency of the bacterial agents under net house conditions is one of the prerequisites for transferring a strategy from the laboratory into the field. P. aeruginosa BRp3 showed effective and consistent pathogen suppression in different pot experiments conducted under net house conditions with different strains of BLB pathogen as indicated by significantly reduced diseased leaf area as compared to the respective non-inoculated control (infected control) as well as compared to positive control of streptocyclin treated plants. Velusamy et al. (2006) reported 58.8 and 64.5% BLB suppression by a DAPG producing P. fluorescens PTB9 in net house and field experiments, respectively. Literature showed that the application of P. fluorescens Pf1 treatment effectively controlled BLB and the treatment was found to be more effective than the standard streptocyclin in controlling the disease as well as in increasing the yield (Vidhyasekaran et al., 2001).

Field experiments conducted in the presence of Xoo indicated that inoculation with P. aeruginosa BRp3 significantly reduced diseased leaf area compared to respective non-inoculated control (infected control) but non-significantly compared to positive control of streptocyclin treated plants. Strain BRp3 showing suppression of BLB equivalent to the positive control under field conditions can be considered as a promising biological control agent to suppress Xoo in rice. Root or seed inoculation of rice plants with this bacterium provided protection to the plant against BLB at early growth stages while its foliar spray at later growth stage or maximum tillering stage protected the rice plant from subsequent disease incidence. Various secondary metabolites produced by this strain along with HCN, may be responsible for the suppression of phytopathogens. The production of increased biomass by inoculated BRp3 may be induced by the production of IAA or siderophores. Different studies reported enhanced growth of root-shoot length of cucumber, lettuce, potato and tomato due to inoculated Pseudomonas strains (Weller, 2007). Khare and Arora (2010) reported the role of IAA produced by P. aeruginosa in the suppression of charcoal rot disease of chickpea. Different types of siderophores such as pesudobactin and pyoverdine produced by rhizospheric bacteria chelate to the available form of iron present in the soil and suppress the pathogens by reducing the availability of iron for the phytopathogen (Wensing et al., 2010; Sulochana et al., 2014). Notably, the number of rhizosphere-associated Pseudomonas species involved in yield enhancement (Combes et al., 2011) and/or reduction in plant diseases, are increasing (Couillerot et al., 2009; Beneduzi et al., 2012).

The inoculated bacteria BRp3 supplemented with 80% of the recommended doses of N and P significantly enhanced the grain and straw yield with 51 and 55% increase, respectively as compared to the respective control. The single inoculated strain BRp3 showed yield increase comparable to that of commercial biofertilizer (BioPower). Application of strain BRp3 either with 80% of the recommended doses or at full/ recommended doses of N and P, significantly improved the growth parameters in rice variety Super Basmati in comparison with control plants without inoculation provided with the same doses of N and P. BioPower treatment also significantly increased the grain weight and straw weight even with 80% of the recommended doses of nitrogen and phosphorus (Figure 10). The inoculation effect of BRp3 on straw and grain yield with different levels of fertilization indicated that the inoculated bacteria may contribute equal to that of the 80% of the recommended doses of N and P indicating that it may save 20% of urea and DAP fertilizers during crop growth season. These results suggest that the rhizobacteria-inoculants can be applied after further evaluation for nutrient management programs. Adesemoye et al. (2009) reported that supplementing 75% of the recommended fertilizer rate with inoculants produced plant growth, yield, and nutrient (N and P) uptake that were statistically equivalent to the full fertilizer rate without inoculants. The use of fertilizer at rates below the recommended dose, in the absence of bioinoculants, resulted in inconsistent effects on the plant with significant reduction in nutrient uptake and yield. In a previous study by Yasmin et al. (2016), inoculation of Pseudomonas spp. and Serratia sp. had a significant effect on rice growth under net house conditions and the grain yield was increased in the field too when used in mixed consortia but the effect of field inoculation was non-significant compared to the respective control. In the present study, BRp3 as a single inoculum, not only suppressed the pathogen under field conditions but also improved the rice yield significantly. The factors contributed for higher grain yield due to strain BRp3 as compared to the previously isolated antagonistic strains may be due to its higher potential for IAA and siderophores production. The siderophores produced were also evidenced by LCMS analysis.

Several studies have demonstrated better plant protection when the inoculated bacteria with improved rhizosphere-competence were used (Bonaldi et al., 2015). Colonization studies of P. aeruginosa BRp3 under field conditions using viable count showed that the strain had the potential to colonize on rice roots as well as on shoots up to 60 days (Figures 12, 13). Green colored colonies and pigmentation were the main characteristics of P. aeruginosa BRp3 that facilitated its detection from the roots and shoots of rice among indigenous soil populations under field conditions using viable count method. Colonization studied by IF and CLSM was in accordance with the results of viable counts of strain BRp3. It appeared that strain BRp3 colonized the rhizoplane and was also found as a shoot endophyte of rice plant. The number of most of the biological control agents decreases with time after their application in the environment, which affect the synthesis of inhibitory metabolites produced by these bacteria (Ji et al., 2008). Efficacy of the biocontrol agent depends on the proportion of root-colonized bacterial cells of the antagonistic bacteria. The influence of the introduced strain BRp3 on indigenous microbiota was estimated by comparing the total culturable bacterial population levels with those of different treatments under field conditions. Detection of higher number of culturable bacterial population indicated that BRp3 inoculation did not decrease the native bacterial population, which may be involved to a certain extent for promoting the plant growth (Susana et al., 2007).

To study plant-interacting bacteria, such as phytopathogens, symbionts, endophytes, biocontrol agents, and rhizospheric bacteria requires demonstration of re-infection and establishment of the inoculated strain in or on field-grown plants (Berg et al., 2014). Efficient root colonization by fluorescent Pseudomonas spp. has been reported to play an important role in their biocontrol activity against various plant pathogens (Bonaldi et al., 2015).

The significance of this study is that functionally characterized antagonistic P. aeruginosa BRp3 may be used for biocontrol of BLB along with enhanced rice growth. Even though, Pseudomonas spp. are indigenous and present in various rhizomicrobiomes but some of these can grow above 37°C and may become opportunistic pathogens, hence suitable biosafety regulations are needed to practically implement this technology for field application (Vilchez et al., 2016). P. aeruginosa BRp3 will be subjected to acute toxicity tests on mice to study the biosafety of this novel bacterial biocontrol agent. Absence of hemolytic activity on blood agar plates (Unpublished data) indicated that rhizospheric P. aeruginosa BRp3 may be unlikely to be a human pathogen (Radhapriya et al., 2015) but whole genome of this bacterium will be sequenced and compared with non-pathogenic P. aeruginosa strains like ATCC 15442 (Wang et al., 2014) to better understand the pathogenicity for its safe application.

The study presents the detailed physiological characterization and effect of P. aeruginosa BRp3 inoculation on rice variety Super Basmati in the presence as well as in the absence of BLB pathogen. This bacterium produced a series of already characterized and novel analogs of HAQs, siderophores and rhamnolipids. The discovery of polyhydroxy HAQs may provide the insight about the biosynthesis pathway of these interesting compounds. The resistance was also induced in rice plants by BRp3 as the activity of defense related enzymes increased after pathogen inoculation. Collectively, the induction of defense related enzymes, HAQs along with siderophores in the presence of rhamnolipids and HCN, were responsible for the profound antibacterial activity of BRp3 against Xoo pathogen. The increased biomass production by strain BRp3 may be attributed to the production of IAA and siderophores. The colonization of the inoculated BRp3 and its re-isolation from rhizosphere indicated its better survival and rhizosphere-competence. On the basis of overall results achieved during this study, bacterial strain BRp3 may be an effective bio-inoculant for Super Basmati rice after ensuring its biosafety aspects. This is perhaps the first systematic effort to use functionally well-characterized beneficial Pseudomonas sp. capable of diverse secondary metabolite production for biocontrol of BLB pathogen in the country.

SY was involved in conducting whole research work, data analysis, and write up. FH gave the basic idea to use beneficial bacteria as biocontrol agent and supervised the study. MM helped in 16S rRNA gene sequencing and edited the manuscript. MR helped in lab/field experiments and data analysis. HA provided reference strains of pathogen and involved in conducting experiments at NIAB. MZ helped in LC-MS analysis. MI did LC-MS analysis and edited the manuscript.