Validation and Evaluation of Plant Growth Promoting Potential of Rhizobacteria Towards Paddy Plants

This study aimed to characterize, validate, and evaluate the plant growth potential of bacterial isolates (E-2, T-2

because of their complexity.2][23][24][25] .In addition, PGPR may improve plant growth by producing phytohormones and iron chelating compounds. 21,26Iron chelating compounds, such as bacterial siderophores, which help in efficient iron acquisition, can be easily taken up by plants for their own growth.1][32][33][34][35] Many bacteria, such as Agrobacterium, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Caulobacter, Chromobacterium, Enterobacter, Erwinia, Flavobacterium, Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum s e r o p e d i c a e L M G 6 5 1 3 , M i c r o c o c c o u s , Pseudomonas, Rhizobium and Serratia have been found to enhance plant growth. 36,37,15Identification of efficient PGPR as potentially useful microbes for application in agriculture to improve plant growth, health, and crop yields has received attention over the past several years, and research has frequently been carried out successfully in field experiments. 30,38,39Many biofertilizers are in application across the globe, but their selection is subjective to their performance, which may be influenced by various environmental factors (temperature, salinity, pH, and many others that influence the survival and growth of microorganisms) that vary across geographically distinct locations and with time. 40,41Genetic variation is an ongoing process, which may be spontaneous or induced, leading to the emergence of efficient and new PGPR genera or species.Thus, the isolation of efficient PGPR strains at par application environments is a prime concern for eco-friendly and sustainable agriculture.Safety issues are of utmost importance, especially when studies must be carried out under field conditions to explore the in-field performance of PGPR isolates.The present work has been undertaken to validate and evaluate the plant growth-promoting potential, tolerance to saline environment, and preliminary concern about safety issues of rhizospheric bacteria in lab conditions, which can be further investigated under field conditions for application as a biofertilizer/plant-biostimulant.

Source and information of the microbial isolates
Three bacterial isolates (E-2, T-2, and T-1) were obtained from Kumar's laboratory at Central University of Jharkhand, India. 9,10All three isolates exhibited four plant growth-promoting characteristics: (1) nitrogen fixation, (2) phosphate solubilization, (3) siderophore production, and (4) Indole acetic acid production. 9,10These three isolates were also tolerant and grew in a broad pH range (pH 3.5-10). 10These three isolates were used in this study.

Surface sterilization of paddy seeds
All experiments were performed in the Department of Life Sciences, Central University of Jharkhand, India.Plant inoculation assays were performed according to the modified method of Majeed et al. 28 A total of 200 paddy seeds of variety Gorakhnath-509 (hybrid paddy, minimum germination 80%, minimum genetic purity 95%, minimum physical purity 98%, recommended area of cultivation including Jharkhand, produced and marketed by Nath Bio-Genes (I) Ltd.Nath House-India) with an average weight of 16.6 ± 0.2 mg per seed were used in this study.][44] Briefly, paddy seeds were washed thrice with 50 mL autoclaved double-distilled water (DDW) with vigorous shaking at 250 rpm at room temperature for 5 min.Water was then decanted, and the seeds were treated with 70% ethanol with shaking at 250 rpm for 2 min.The seeds were then rinsed thrice with autoclaved DDW for 2, 5, and 15 min.Steps of ethanol treatment followed by rinsing with autoclaved DDW were repeated.Seeds were then treated with 0.1% mercuric chloride (HgCl 2 ) for 2 min and 1% sodium hypochlorite for 15 min, respectively, with each treatment followed by rinsing thrice with autoclaved DDW.Five surfacesterilized seeds and 100µL of water from the last wash step were plated on sterile Luria-Bertani (LB) agar media plates separately to check for sterility.
After the microbial sterility check, the remaining seeds were soaked aseptically overnight in 50 mL autoclaved DDW.Seed germination was assessed prior to the hydroponic experiment.Most of the seeds germinated (86-88% germination) 3 d after sowing in the sand matrix flooded with Hoagland media.

Hydroponics study: Growth of paddy plants under different nutritional and inoculant control
Overnight-soaked seeds were sown (buried approximately 1 cm deep) aseptically in six beakers (volume-250 mL, height 95 mm, outer diameter 70 mm, inner diameter 63 mm).Each beaker contained sterilized sand-medium (red sand-repeatedly washed and autoclaved, 300 g of sand attained height of 74 mm and volume 183.78 cm 3 , added with 85 mL respective growth media) and 15 paddy seeds.The beakers were labeled 1-6:1-nitrate deficient Hoagland media with bacterial inoculum as H-NO 3 ¯(IN), 2-nitrate deficient Hoagland media without bacterial inoculum as H-NO 3 ¯(UNIN), 3-Hoagland media without bacterial inoculum as H(UNIN), 4-Hoagland media with bacterial inoculum as H(IN), 5-phosphate deficient but tri-calcium phosphate (TCP)-supplemented Hoagland media without bacterial inoculum as H-PO 4 ¯+TCP(UNIN), and 6-phosphate-deficient but TCP supplemented Hoagland media with bacterial inoculum as H-PO 4 ¯+TCP(IN).All the beakers containing seeds were kept under similar growth conditions of light, temperature, and humidity and kept in the same chamber of an enclosed light rack, and hydroponics experiments were performed in months of April-July for germination and growth.Following germination of seeds, 3 days after sowing, 10 mL of bacterial cells (O.D. 1 in normal saline) was added to beakers labeled as (IN), whereas others labeled as (UNIN) received 10 mL of normal saline.A 10 mL (approximate volume determined equivalent to volume of water loss) autoclaved DDW was added on alternate days to the plant growth-supporting matrix in every beaker to keep the sand media flooded (~ 10 mm).Various phenotypic parameters of plant growth were recorded over 21 days following inoculation.Plants were uprooted on the 21 st day, and the length of root (longest root in the plant) and shoot (length with longest leaf in the plant), number of leaves and number of root fibers/ branches, fresh weight of roots, and fresh weight of shoots, as used by Souza et al 30 Kumar et al 38 and Majeed et al 28 were measured separately for each plant except for fresh weight (Supplementary file).The root weights of individual plants in some treatment sets were very low.Therefore, the shoots and roots of all plants were cut and separated.Fresh weight of shoot/root of all plants in a treatment set, at a time, was measured and the average was calculated by dividing it with the number of plants in the respective treatment set.

Data analysis
One-way analysis of variance (ANOVA) was performed to determine the effects of different treatment/test conditions on plant growth.Plant growth promoting potential of all the three isolates E2, T2 and T1 was evaluated under the mentioned conditions except 'Hoagland media without bacterial inoculum' condition which was omitted for bacterial isolate T1.For each trait (shoot length, root length, ratio of shoot length to root length, number of leaves in each plant, and number of roots in each plant), data were analyzed using GraphPad Prism Software 5.0 (GraphPad, San Diego, CA, USA) through one-way analysis of variance (ANOVA) (Tukey's post-hoc test: compared all pairs of columns).The results are expressed as mean ± standard deviation (SD).Replicates of plants (individual plants in each treatment set) were taken and ANOVA (a = 0.05) was performed using the measured values of the plant traits.

Growth response of isolates on different concentrations of salt
Qualitative growth response of PGPR isolates to different concentrations of salt (NaCl in LB agar media: 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%) was performed according to the method described by Swarupa and Kumar 9 .The plates were checked by observing the relative visible growth density and opacity against the light source of the bacterial culture spot on the culture plates, and the image was captured as a record.

Antibiotic sensitivity profiling of the isolates
An antibiotic (antimicrobial agents) sensitivity assay was performed on Mueller Hinton agar by the standard disc diffusion method 45 using an antibiotic sensitivity teaching kit (HTM002-15PR; HiMedia Laboratories Pvt.Ltd, India).The bacterial isolates were grown overnight in LB broth at 37 °C with shaking at 160 rpm.Bacterial cultures were centrifuged (MIKRO 200R, Hettich ZENTRIFUGEN) at 10000xg for 2 min at room temperature.The pellet was washed and resuspended in sterile normal saline (0.85% NaCl).Bacterial suspensions in normal saline were spread on sterile Mueller-Hinton agar using sterile cotton swabs.The discs of the respective antibiotics, as given in Table 1, were placed on the bacterial lawn spread on Mueller-Hinton agar.The diameters of the inhibition zones were measured in millimeters after 24 h and 48 h of incubation at 37°C.The sensitivity and resistance of the bacteria to the respective antibiotics were inferred from the standard reference zone of inhibition (Table 1).

Identification of isolates by 16S-rRNA
Phylogenetic analysis of bacterial isolates E-2, T-2, and T-1 was done using the 16S-rRNA gene sequence.Phylogenetic analysis services were procured from Xcelris Labs Ltd. (India).The genomic DNA of E-2, T-2, and T-1 isolates was subjected to 16S-rRNA PCR in    The neighbor-joining method was used to establish the evolutionary history of the bacteria. 46The bootstrap consensus tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed. 47ranches corresponding to partitions reproduced in less than 50% of bootstrap replicates were collapsed.Kimura 2 parameter method was used to infer evolutionary distance in the units of base substitution per site. 48Overall, 16 nucleotide sequences, including our query sequence, were used in this analysis, and evolutionary analyses were conducted in MEGA7. 49

Effect of isolates on the growth of paddy plants
Paddy plants grown hydroponically under identical conditions with and without inoculation plants growing in nitrate-or phosphate-deficient media and without bacterial inoculum (Fig. 1: a-2, a-5, c-2, c-5, e-2, e-5) were yellowish-green and chlorotic, respectively, compared to plants grown in nitrate-or phosphate-deficient media but inoculated with bacteria (Fig. 1: a-1, a-6, c-1, c-6, e-1, e-6), where the leaves of plants were dark green.
Statistical analyses of the plant growth responses to isolates E-2, T-2, and T-1 under different test conditions are shown in Fig. 2-3.The contribution of the bacterial inoculum was mainly focused on nutrient (nitrate/phosphate)-deficient treatment sets.With all the isolates, the length of shoots of plants growing in 'treatment sets with bacterial inoculum' were more (significant except E-2 under nitrate deficient condition) than shoot length of plants grown in 'treatment sets without inoculum' (Fig. 2a-c).With all the isolates, the length of roots of plants growing in 'treatment sets with bacterial inoculum' were shorter (significant except T-2 under phosphate deficient condition and T-1 under nitrate deficient condition) than the roots of plants grown in 'treatment sets without inoculum' (Fig. 2d-f).The ratios of the shoot-length to root-length (SL/RL) of plants in 'treatment sets with bacterial inoculum' were significantly higher than the ratios SL/RL of plants in 'treatment sets without inoculum' (Fig. 2g-i).Increases in the number of leaves were recorded under all test conditions when augmented by bacterial inoculum, although the increases were insignificant except for T-2 under nitrate-deficient conditions (Fig. 2j-l).The augmentation of media by bacterial inoculum resulted in a significant increase in the number of roots per plant under phosphate-deficient conditions in the case of E-2, the control (without nutrient deficiency in Hoagland medium), and nitrate-deficient conditions in the case of T-2 (Fig. 2m-o).In the other cases, the number of roots either increased or decreased insignificantly (Fig. 2m-o).
All isolates contributed to increased average fresh weight of shoots under all growth media conditions when inoculated with bacterial inocula E-2, T-2, or T-1 (Fig. 3).Although a definite trend was not seen, the observations on root biomass were represented in Fig. 3. Fresh biomass of roots increased in 'control' or 'phosphate deficient media' when inoculated with bacteria E-2 or T-2.Fresh biomass of roots was found to be either slightly decreased or comparable in nitratedeficient media when inoculated with bacteria E-2 or T-2, respectively.The fresh biomass of roots was found to increase seven-fold when the control medium was inoculated with bacteria T-2.The fresh biomass of roots increased in nitratedeficient media when inoculated with bacteria T-1, whereas it decreased in phosphate-deficient media when inoculated with the same bacteria.Similarly, the shoot biomass to root biomass ratio did not follow a definite trend, except when inoculated with E-2.

DISCuSSIoN
Plant growth-promoting bacteria comprise a heterogeneous mix of bacteria that can colonize plants or around the root system and also provide beneficial effects to the plants by many different mechanisms, including nitrogen fixation, phosphate solubilization, siderophore production, and IAA production among others. 11,51,20,8,15Three bacterial isolates were used in this study, which had been tested biochemically for their potential for nitrogen fixation, phosphate solubilization, siderophore production and IAA production. 10hese isolates exhibited a positive influence on the growth parameters of the plants when inoculated under hydroponic conditions (Fig. 1-3).The three isolates, E-2, T-2, and T-1, were identified by 16S-rRNA gene sequencing as Klebsiella sp.strain BAB-6433, Citrobacter freundii strain R2A5, and Citrobacter sp.DY1981, respectively (Fig. 5).Other studies also reported similar findings, where Klebsiella pneumoniae type strain (KPY17657) and Citrobacter were found to be associated with plant roots of rice, wheat, and fruit plants, and also exhibited plant growthpromoting activities, such as nitrogen fixation, phosphate solubilization, siderophore production and IAA production. 52,53Klebsiella and Citrobacter freundii have also been associated with other plant species, including Zea mays, 54 Triticum aestivum, 55 Saccharum officinarum, 56,57 and Glycine max. 58trobacter freundii and Klebsiella have been used for biocontrol, bioremediation, and plant growth promotion in tomato, 59 maize, 60,61 and sugarcane. 62owever, in the present study, these bacteria were isolated from different plants such as eggplant (E-2) and tomato-plant (T-2 and T-1) but exhibited positive influence by enhancing the growth of the tested paddy plants.It also substantiated that these bacteria might not show a strict association with a particular plant, but can also be applied to improve the growth of other plants.
Paddy plants grown under nitrate-or phosphate-deficient media were short, had yellowish-green leaves, were narrow, and had fewer leaves.Application of bacterial isolates resulted in improved growth of plants with better plant height, green leaves, and an increased number of leaves, revealing that these isolates are fit to be called, as stated by Vessey, 21 PGPR as a biofertilizer.Paddy growth was significantly enhanced following inoculation with E-2, T-2, or T-1, although these isolates manifested improved growth in different ways (Fig. 1-3).Isolates E-2, T-2, and T-1 increased the shoot length of plants compared to un-inoculated sets, indicating that these isolates promoted plant growth, as reported by Souza et al., (2013). 30On the other hand, root length decreased following inoculation with E-2, T-2, or T-1, which is corroborated by the interpretation of Fageria and Moreira (2011) 63 that "when there is deficiency of a determined nutrient, roots try to grow longer to take nutrients from lower soil depths".5][66][67] Increased root length under nitrogen-or phosphorus-deficient uninoculated plants indicated that the plants are experiencing nutrient deficiency, and hence, roots grow faster to acquire more nutrients.In contrast, roots were short under conditions of nitrogen or phosphorus deficient media but were inoculated with isolates E-2, T-2, or T-1, which substantiated that these isolates supplemented deficiency of nitrogen or phosphorus by nitrogen fixation or phosphate solubilization, respectively.Similarly, fresh shoot biomass increased when plants were inoculated with isolates E-2, T-2, or T-1.The increase in shoot length to root-length ratio under inoculated nitrogen-or phosphorusdeficient media compared to its respective uninoculated sets further evidenced the growthpromoting characteristics of isolates E-2, T-2, and T-1.5][66][67] Thus, isolates E-2, T-2, and T-1 led to phenotypic and statistically improved plant growth.9][70][71] Growth-promoting effects on paddy plants were also observed when isolates E-2 and T-2 were inoculated into plants growing in Hoagland media, where both 'N' and 'P' are available.The present setup seems insufficient to explain this, but nutrient enrichment by isolates may be one of the reasons especially, during the late stages when plants start experiencing nutrient deficiency.
Due to irregular rainfall patterns, human activities, extreme climatic changes, improper drainage, and inadequate leaching of mineral salts, soil salinity has increased at an alarming rate. 72,73Increased levels of soil salinity lead to physiological, molecular, and biochemical changes and reduce crop productivity and yield. 74,75he ability of these isolates E-2, T-2, and T-1 to grow at a wide range of salt concentrations (E-2: up to 7%, T-2 and T-1: up to 6% of NaCl) is of additional significance for their application to alleviate the abiotic stresses experienced by most agricultural plants, which are sensitive to high salt concentrations in agricultural fields.E-2, T-2, and T-1 showed moderately halo-tolerant character. 76][79][80][81] The isolates were identified as Klebsiella sp.strain BAB-6433, Citrobacter freundii strain R2A5, and Citrobacter sp.DY1981 annotated the risk status of the isolates E-2, T-2 and T-1, respectively.3][84] In view of the safety aspects of the field application of isolates T-2 and T-1, the above-mentioned studies have clearly mentioned that these opportunistic pathogens are commensal to the human gut and are not the primary cause of diseases.Therefore, the pathogenesis of these bacteria is not due to the inherent virulence of Citrobacter.Despite the emergence of antibiotic resistance and the risk factor bla TEM-1 resistance gene of these bacteria, there are still reliable antimicrobial agents such as cephalosporins, amikacin, and quinolones available for controlling these resistant strains. 84lebsiella spp.][87][88][89][90] Klebsiella infection is generally nosocomial in nature and generally affects individuals who are either immunocompromised or have underlying diseases such as type 2 diabetes, cancer, and pulmonary infections. 88,89In this study, Klebsiella spp.were isolated from rhizospheric soil; hence, its probability of being pathogenic to humans may be very low.In fact, some strains of Klebsiella pneumonia and Klebsiella oxytoca have been used as effective bioinoculants in some studies, [91][92][93] which themselves belong to one of the most pathogenic species of Klebsiella genera. 89herefore, based on the above facts, isolates E-2, T-2 and T-1 may be assigned Risk Group 2 (with moderate individual risk and limited community risk, includes opportunistic pathogens). 94Besides susceptibility to some tested antibiotics, E-2, T-2, and T-1 showed resistance to ampicillin, penicillin, and vancomycin, indicating the emergence of drug resistance among a number of culturable PGPR or soil microbes, which is also supported by previous finding. 95Ferjani et al., (2018)  98 also found a low rate of antibiotic resistance among isolated PGPR isolates, which could be used for the development of microbial inoculums to enhance plant growth while considering human health.The sensitivity of isolates E-2, T-2, and T-1 to broadspectrum antibiotics such as chloramphenicol, kanamycin, and gentamicin makes them suitable for safe (readily available antibiotics may help immediate prevention of infection or disease outbreak) industrial production and formulation of biofertilizers for field application.Although antibiotic resistance of PGPR has been sporadically investigated by others, 98 the risk status of PGPR has also been explored in this study by literature search and 16S-rDNA based identification.
The cited studies from the literature and the antibiotic sensitivity study of this study are suggestive of the safe application of E-2, T-2, and T-1.However, safety parameters must be evaluated experimentally in detail or caution should be taken prior to the release of antibiotic-resistant bacteria, especially if such resistance genes are borne on mobile genetic elements.Further field studies using these isolates may be required to evaluate their response under different prevailing environmental conditions, including the influence of other biotic communities that influence the growth of PGPR microbes. 99,9,10

Fig. 3 .
Fig. 3. Effect of isolates E-2, T-2 and T-1 on biomass of the plants: average fresh weight of shoot and root, fresh weight shoot to root ratio; average was calculated as (to avoid measurement errors): total fresh weight of shoots/ roots of surviving plants in a treatment set divided by total number of surviving plants of respective treatment set pot; legend symbols of the graph: H-NO 3 ¯(IN), H-NO 3 ¯(UNIN), H(UNIN), H(IN), H-PO 4 ¯+TCP(UNIN), H-PO 4 ¯+TCP(IN); where H-Hoagland, IN-Inoculated with bacterial isolate, UNIN-Uninoculated, TCP-Tricalcium phosphate, H -NO 3nitrate deficient Hoagland medium, H-PO 4 --phosphate deficient Hoagland medium, +TCP-supplemented with TCP.

Fig. 4 .Fig. 5 .
Fig. 4. Growth of isolates E-2, T-2 and T-1 on different concentration of salt (0-8% of NaCl) constituted in LB agar plates.White spots on the plates represent growing bacterial lawn at 24 h of incubation following spot inoculation of 5µL of respective bacterial suspension at ~1.0 optical density. 9

Table 2 .
Antibiotic sensitivity profile of different bacterial isolates