Article

1. Introduction

Plant microbiomes consist of a huge diversity of micro-organisms which inhabit different plant tissues and organs such as seeds, roots, stems, leaves, flowers and fruits [3]. These microbial communities are present externally as epiphytes and in internal tissues and organs of a plant as endophytes, influencing plant vigor and health [5]. Among these microbial communities, bacterial endophytes colonize plant tissues without any apparent pathogenic symptoms and establish beneficial associations with their plant host [6]. Endophytic bacteria play an important role in the functioning of the host plant by influencing their physiology and developmental processes [10]. The prominent abilities of endophytic bacteria to improve plant growth and minimize pathogen infestation via direct and indirect mechanisms are attracting scientists globally. Direct mechanisms are set up to apply to those bacterial traits that directly promote plant growth. They include the production of phytohormones production such as indole acetic acid (IAA) gibberellin and cytokinin [10]. Endophytic bacteria promote plant growth by facilitating the acquisition of mineral resources such as phosphorus, potassium, zinc and iron which are usually less bioavailable [11-12].  Additionally, strains of endophytic bacteria are capable of producing ACC deaminase that converts 1-aminocyclopropane-1-carboxylic (ACC) acid, the precursor of ethylene biosynthesis, into ammonia and αketobutyrate. This decreases the ethylene level and prevents overproduction of ethylene that leads to growth reduction in plants [9; 7]. A report showed that enhancement of salt tolerance in maize was achieved by inoculating the plants with ACC-deaminase producing endophytic bacteria [4]. Under indirect mechanisms, endophytic bacteria improve plant growth by acting as biocontrol agents. In addition, many endophytic bacteria have been reported to produce compounds classified as secondary metabolites for inhibition of pathogens, and they also adopt different mechanisms in order to produce cell wall degrading enzymes, antibiotics, competition and induced systemic resistance (ISR) [16; 6; 17]. Studies on plant-endophytic microbe interactions are essential due to the application of beneficial microbes as plant growth-promoting agents and biocontrol agents for controlling phytopathogens [3; 17-18]

Rice (Oryza sativa L.) is the most widely important grown cereal crop and is consumed by more than 50% of the world’s population [1-2]. The ever-increasing world population demands sustainable agriculture production to feed 7.3 billion people; that population may become 9.7 billion up to 2064 (estimated by the United Nations (UN). An increasing population needs a high yield of rice to compete for this competition, but it must be achieved without, or by minimizing, the application of synthetic products due to high concern about environmental protection. Still, the main source of crop improvement in the agriculture sector is the practice of applying synthetic products, such as commercial fertilizers, nutrient supplements, insecticides, and pesticides. This action has hazardous effects on the environment and human and livestock health [15; 1; 4].

The farmer communities are convinced to change their old practices by using alternative products that could be environmentally friendly. The bio-fertilizers and bio-pesticides consist of non-pathogenic microorganisms; this uniqueness in nature facilitates attaining sustainable agriculture and environmental protection from chemical hazards. Additionally, bio-formulations might be the best alternative option to reduce environmental degradation and the threat to human health [13].In the last few decades, a large array of endophytic bacteria have been found to possess plant growth-promoting properties, including species of Pseudomonas, Azospirillum, Bacillus, Klebsiella, Stenotrophomonas, Burkholderia, Serratia, Microbacterium, Enterobacter, Alcaligenes faecalis etc., [21-22]. Previous research reported that inoculation with plant growth promoting endophytic bacteria can enhance growth attribute, yield and nutrient content of rice crops [19; 15; 20]. The objectives of the present study were to evaluate the effect of endophytic bacteria on plant growth attributes of rice under pot experiment.

2. Materials and Methods

2.1. Sample location, plant material and isolation of endophytic bacteria

The present research was conducted on the isolation of endophytic bacterial strains from rice roots. Samples of rice roots were collected from healthy rice (var. MTU1010) cultivated in fields at Indian Institute of Rice Research Rajendranagar, Telangana, India (IIRR-ICAR). The plants were removed from the soil with the entire root system, collected in sterile polythene bags, kept in a cool container until isolating the endophytic bacteria. The root samples were completely removed from the soil under running tap water. The root samples were then, soaked in 70% ethanol for 2 min before being soaked in a 2.5% NaOCl solution for 5 min with the addition of 2–3 drops of Tween 20 [29-31]. Finally, the root samples were washed 5 times with sterile distilled water. The disinfected root samples were then crushed with the help of autoclaved pestle and mortar using 2 mL of phosphate saline buffer (PSB) solution. The ground suspension (1 ml) was then transferred to 9 ml water blank and further dilutions were produced until reaching a dilution of 10^-7. A volume of 0.1 ml of this suspension was evenly distributed on nutrient agar media plates, which were then placed in an incubator at a temperature of 30 °C for a period of 3 days. The colonies from plates were selected and separated based on morphology and color, then were streaked on fresh plates to purify and finally stored at -80 °C in nutrient broth containing 30% glycerol.

2.2. Plant growth promoting (PGP) traits of isolated endophytic bacteria

Isolated endophytic bacteria were tested for plant growth promoting characteristics such as indole acetic acid (IAA) production, phosphate solubilization, siderophore production and ammonia synthesis under in vitro conditions.

2.2.1. Test for indole acetic acid (IAA)

Production of IAA was carried out by inoculating isolated endophytic bacteria in nutrient broth enriched with 5 mM L-Tryptophan (Hi-media), incubated at 28±2^oC for 48-72 h. After incubation, Salkowski reagent was added to tubes and then tubes were incubated in dark condition for 30 minutes. Development of pink colour or reddish color indicated positive result [23].  

2.2.2. Qualitative estimation of phosphate solubilization

Endophytic bacterial isolates were qualitatively tested for phosphate solubilization by spot inoculating on National Botanical Research Institute’s (NBRI) Phosphate agar medium. A clear zone around bacterial colony was considered as positive for phosphate solubilization.

2.2.3. Siderophore and ammonia production by endophytic bacteria

Isolated endophytic bacteria were tested for siderophore production using chrome azural S (CAS) agar medium as per the method of [33]. Actively grown bacterial cultures were spot inoculated onto CAS agar medium and incubated at 30⁰ C for 3 to 3 days. Development of yellow to orange halo around bacterial colonies was considered as positive for siderophore production. The bacterial isolates were examined for their ability to produce ammonia in peptone water broth, using the method described by [34]. Actively grown bacterial cultures was introduced into a 5 mL solution of peptone water and subjected to incubation for duration of four days at a temperature of 30 degrees Celsius and a speed of 120 rpm in an incubator shaker. Following the incubation period, Nessler’s reagent was introduced into each tube. The occurrence of a deep yellow to brown tint was regarded as a positive indication of ammonia production.

2.3. Evaluation of selected potential bacterial endophyte on plant growth parameters of rice

Selected potential bacterial endophytes were further evaluated for their PGP potential in greenhouse conditions on rice (var. MTU1010). The rice seeds were surface sterilized using 95% ethanol for 1 minute and 0.2% HgCl₂ for 3 minutes. Afterward, they were washed three times with sterile distilled water. Bacterial culture that was actively growing was introduced into 100 ml of sterilized nutrient broth and placed in an incubator at a speed of 150 revolutions per minute and a temperature of 30±2ºC for duration of 48 hours. Subsequently, the mixture was subjected to centrifugation at a speed of 8,000 revolutions per minute for duration of 15 minutes. The pellet was rinsed twice using sterile saline solution and then utilized for seed bacterization. The rice seeds, which had been sterilized, were immersed in the corresponding suspension of cell pellets for the entire night. Following the process of seed bacterization, a total of 3 seeds were planted in plastic pots measuring 5 inches in diameter. The pots were filled with an optimal amount of sterile soil, reaching a level of three-fourths capacity. Subsequently, the pots were placed in the greenhouse and left to incubate for a period of 30 days. The control group consisted of seeds treated with distilled water. Two treatments (IIRR-F2, and un-inoculated control) were applied, each with three replications. Measurements of shoot length, root length, and dry weight (total biomass) were documented and compared to plants in the control group.

2.4. Statistical analysis

All experiments were performed twice by three replications each. Statistical analysis was done and mean, standard error and standard deviation were calculated.

3. Results

3.1. Isolation and PGP traits of isolated endophytic bacteria In this study, a total of 4 morphologically distinct endophytic bacteria were isolated (labelled as IIRR-F1, IIRR-F2, IIRR-F3 and IIRR-F4) from the roots of rice (Table 1). These bacteria were then tested for various plant growth promoting (PGP) traits, including the production of indole acetic acid (IAA), siderophore, ammonia synthesis, and phosphate solubilization under in vitro conditions. Among the 4 bacterial isolates, 2 isolates (IIRR-F1 and IIRR-F2) exhibited the ability to produce indole-3-acetic acid (IAA). Further out of these two isolates, IIRR-F2 showed high intensity (+++) of pink color indicated higher production of plant growth promoting hormone indole acetic acid. The isolate IIRR-F1, showed a slight intensity (+) of the pink color, indicating lower production of plant growth-promoting, indole acetic acid (Table 1). Among the 4 bacterial isolates, 2 isolates (IIRR-F2 and IIRR-F4) exhibited a zone of solubilization on NBRIP medium, demonstrating their ability to solubilize phosphate (Fig.1B and Table 1). Among the 4 bacterial isolates, 2 isolates, namely IIRR-F2 and IIRR-F3, exhibited the ability to produce ammonia. Furthermore, out of 2 bacterial isolate,s IIRR-F2 showed strong activity (+++) for ammonia production, while isolate IIRR-F3 exhibited moderate activity (++) for ammonia production (Fig.1A and Table 1). Out of 4 bacterial isolates, only bacterial isolate IIRR-F2 was able to produce siderophore, and this was verified by the appearance of an orange halo zone around the colony (Table 1).
3.2. Effects of potential endophytic bacterial isolate on growth parameters of rice

One potential bacterial isolates, namely IIRR-F2 (Bacillus sp.,) which showed all the plant growth-promoting traits (Table 1) was selected and evaluated for the growth promotion of rice under greenhouse conditions. The findings indicated that rice seeds treated with bacterial isolate IIRR-F2 exhibited enhanced root length, shoot length, and dry weight in comparison to the control group. Treatment of rice seeds with bacterial isolate IIRR-F2 significantly improved shoot length by (42.5%), root length (48%) and dry weight by (31%) as compared to uninoculated control plants (Fig. 1C and Table 2).

Fig.1. (A) Ammonia production by endophytic bacteria (B) Halo zone on NBRIP media, indicating phosphate solubilization (C) Impact of endophytic bacterial isolate IIRR-F2 on growth parameters of rice (D) Purified culture of bacterial endophyte IIRR-F2 (Bacillus sp.).

4. Discussion

Plants live in diverse environments, interacting with a plethora of microbes. The realization that plants and microbes function together as a dynamic entity to produce an amalgamated response to environmental challenges, led to the holobiont concept. Among the microbes that are associated with plants, endophytes are the ones that can dwell within plant tissues without any external sign of infection or other harmful effects on the host plants. Endophytes are present in all known plant species and can inhabit different compartments of a plant, such as the leaf, stem, seeds, root, and flower. Some endophytes are beneficial to the plants by promoting plant growth, fixing nitrogen and suppressing diseases. Plant endophytes are predominantly bacteria and fungi, though archaebacteria, algae, protozoa, and nematodes are rarely found to be living as endophytes; however, they exert significant effects on the plant [24]. Endophytic bacteria have been shown to have several beneficial effects on their host plant. Plant growth is promoted through improved nutrient acquisition, including nitrogen fixation and production of plant growth-enhancing substances such as indole acetic acid (IAA), cytokinins. In addition to enhanced growth properties, modulation of plant metabolism and phytohormone signalling by the endophytic bacteria enhances adaptation to environmental abiotic or biotic stress. Endophytic bacteria present a special interest for improved crop adaptation to stress as they have the advantage of being relatively protected from the harsh environment of the soil under drought, high salt or other stress conditions [26; 11-12].

Agricultural intensification is an important condition for the food security of the population of the world. However, the use of chemical fertilizers to improve soil fertility and increase crop yields poses a threat to both ecosystems and human health. Against this background, the use of biofertilizers consisting of bacteria that are naturally associated with plant roots may be a useful and promising alternative to the widespread application of agricultural chemicals. Biofertilizer application may be utilized in the agricultural bio-economy to maximally ensure food production, and incorporation into the crop-breeding programs [21; 27]. In this work, a total of 4 endophytic bacteria were recovered from the roots of rice. The bacterial cultures were screened for various plant growth-promoting traits in vitro. This involved assessing features such as the production of indole acetic acid, solubilization of phosphate, production of ammonia (NH3), and generation of siderophores. In the present work, one potential bacterium namely IIRR-F2 exhibited most of the PGP traits which was identified based on morphological and biochemical characteristic as Bacillus sp. Indole-3-acetic acid (IAA) is the most prevalent and widely recognized kind of auxin in plants. It has a crucial function in processes such as cell division, growth, and specialization, as well as in promoting the germination of seeds and tubers, initiating the growth of lateral and adventitious roots, and facilitating the production of various metabolites [15]. Production of IAA is considered one of the main ways in which plant growth is promoted by PGPB. During the current study, Bacillus sp., strain IIRR-F2 exhibited the highest level of indole-3-acetic acid (IAA) synthesis (Table 1). Several studies have documented the synthesis of indole-3-acetic acid (IAA) by different strains of Bacillus [13; 28; 23]. A significant proportion of phosphorus (P) in soil exists in the form of insoluble inorganic compounds, making it inaccessible to plants. Phosphate-solubilizing bacteria (PSB) are microorganisms that can solubilize phosphate. They can provide plants with a readily available form of phosphorus, which might potentially replace inorganic phosphatic fertilizers [13]. During the current study, Bacillus sp., strain IIRR-F2, exhibited the highest level of phosphate solubilization, which is evident by the formation of a halo zone on NBRIP media (Table 1). The findings of the previous study conducted by [30-31] align with the results of the current investigations, which showed that Bacillus sp., are capable of solubilizing inorganic phosphate. Similarly, [13] documented the ability of bacteria from the genera Bacillus, Stenotrophomonas, Enterobacter, and Pseudomonas, isolated from the rhizosphere of rice, to solubilize phosphate.

The findings of the current investigation shown that bacterial isolates namely IIRR-F2 (Bacillus sp.,) significantly enhanced the root length, shoot length, and dry weight in rice plants comparison to the control group (Table 2). The results of this study are consistent with the earlier studies conducted by [13; 32] which found that the growth parameters of rice increased when it was inoculated with PGP Bacillus sp. Similarly, [29] observed an augmentation in the growth characteristics of rice when it was inoculated with endophytic bacteria Bacillus subtilis. The increase in root length and shoot length of rice after being exposed to the aforementioned potent bacterial isolates may be attributed to the numerous plant growth regulator (PGR) characteristics exhibited by our bacterial inoculants.

5. Conclusion

At a global scale, the effects of continuous and heavy use of agrochemicals for improving agricultural productivity can cause serious damage on the soil fertility, life of living organisms, and as well as their environment. Use plant growth-promoting endophytic bacteria as agricultural crop inoculums are costly reasonable and environmentally-friendly approach to increase crop production on a sustainable way. The present study concludes that one plant growth-promoting endophytic bacteria IIRR-F2 (Bacillus sp.,)can be employed as bio-inoculants in the cultivation of rice in a sustainable manner. In future studies suitable PCR-based genotypic techniques (16S rRNA sequence) can be employed to confirm its identity at strain level and the isolate can be subjected to field trials to improve the yield and available nutrients in the rice crop.

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