Isolation and characterization of plant growth-promoting rhizobacteria from the rhizosphere of common bean ( Phaseolus vulgaris L.) in high-altitude regions of Kashmir

Introduction

The common bean (Phaseolus vulgaris L.) is one of the world's most consumed leguminous crops (Castro Guerrero et al., 2016) [7] . In Latin America, it is the main staple food in people's diet because of its high nutritional quality (Torres et al., 2017) [23] , with a protein range of ~16%-33% depending on different landraces (Luna-Vital et al., 2015) [16] . As a legume, it is connected with microbes that fix nitrogen. It minimizes the usage of synthetic fertilizers, which is crucial for sustainable agriculture. Common beans originated in Mexico, Central America and South America and were domesticated approximately 8, 000 years ago. This has resulted in adaptation to Mediterranean conditions in two key gene pools, Mesoamerican and Andean (Castro-Guerrero et al., 2016) [7] . Common bean (Phaseolus vulgaris L.) is a significant pulse crop in the Fabaceae family that has grown in favor among Indian farmers because to its high profitability, short growth cycle and adaptability to a wide range of agroclimatic conditions. As a result, increasing output appears to be one of the options for meeting the demand for this pulse crop, which can be accomplished either by the use of chemical fertilizers or the application of rhizosphere competent plant growth boosting bacteria. However, the usage of chemical fertilizers is not environmentally beneficial because it harms microorganisms, plants and soil. Microorganisms play an important role in agriculture by increasing the supply of plant nutrients and reducing the need of chemical fertilizers. Biofertilizers based on microorganisms could be a highly effective microbial stimulant in agriculture due to their various plant development activities (Josephand Lawrence, 2007) [14] . The rhizosphere is a unique location inhabited by numerous microorganisms capable of influencing plant growth. These favorable plant-microbe interactions in the rhizosphere can improve soil fertility (Dastager et al 2011) [9] . Microbial community-plant root interactions are complicated, allowing plants to absorb nutrients from the soil via a variety of methods. However, in the rhizosphere, just 1% to 2% of bacteria can boost plant development (Antoun et al 2001) [3] . Plant growth boosting rhizobacteria are bacteria that inhabit the rhizosphere and encourage plant development, either directly or indirectly. Root secreted flavonoids are important signals for legume-rhizobial and legume-mycorrhizal interactions and their establishment. Rhizobia, along with other PGPRs, colonize legume roots and can directly boost plant growth by influencing the host's physiological and biochemical properties. Bacteria are common microbes in the rhizosphere, and many of them have PGPRs, which promote plant growth. Their method of action involves the synthesis of indole-3-acetic acid (IAA), soil phosphorus solubilization and nutrient delivery. Various soil microorganisms create naturally occurring auxins, including indole-3-acetic acid (IAA), as a direct mechanism for promoting plant development (Spaepen and Vanderleyden 2011) [22] . IAA specifically affects plant growth promotion and root nodulation. Bacterial IAA improves root surface area and length, giving the plant better access to soil nutrients. Furthermore, bacterial IAA loosens plant cell walls, allowing for greater root exudation, which supplies additional nutrients to encourage the proliferation of beneficial rhizosphere bacteria (Glick, B.R.2012) [12] Phosphate solubilizing bacteria (PSB) are known to solubilize phosphate in soil by acidity, chelation, or enzymatic processing, as well as motility or adhesion of bacteria in the root, all of which aid in legume-Rhizobium symbiosis. PGPR can also be used as biocontrol agents against plant diseases, either by stimulating the plant's natural defensive systems, antagonism, or antibiosis (El kinany S et al., 2017) [11] . This is achieved through a variety of methods, including the formation of hydrogen cyanide (HCN) (Goswami D et al 2015) [13] and direct competition for nutrients and space inside the rhizosphere (Laslo É et al 2010) [15] .

Materials and Methods

Soil Sample Collection

The common bean rhizosphere soil samples were gathered from a healthy common bean field in Kupwara, Kashmir. Bacterial strains were isolated from either the rhizoplane or the roots of healthy common bean plants. The rhizosphere soil samples were immediately taken to the laboratory and stored at 4 °C for future isolation and species diversity studies.

Isolation of Bacteria from Soil Samples

The microflora were isolated from the collected common bean rhizospheric soil using King's B media and the serial dilution process and then cultured for 24 hours at 28 °C until bacterial colonies formed. Individual colonies were selected for further purification and streaked on King's B plates.

Characterization of isolates

Pure culture generated thirty rhizobacterial isolates, which were identified as KB1, KB2, KB3, KB4, KB5, KB6, KB7, KB8, KB9, KB10, KB11, KB12, KB13, KB14, KB15, KB16, KB19, KB20, KB21, KB22, KB23, KB24, KB25, KB26, KB27, KB29, and KB30. The morphological properties of each isolated colony were investigated using King's B plates. Following three days of incubation, numerous colony features were detected, including size, shape, color, elevation, and the presence and location of endospores. Each rhizobacterial isolate was biochemically characterized using Gram's reaction (Dubey and Maheshwari 2006) [10] .

Indole Acetic Acid (IAA) Determination

According to Brick et al., (1991) [5] , indole acetic acid production was discovered. A loopful of each isolated culture was mixed with 20 milliliters of Luria Bertani (LB) broth (tryptone 10 g, yeast extract 5.0 g, NaCl 10 g, distilled water 1 lit., pH 7.0), which contained 0.2% L-tryptophan. The broth was then cultured for 24 hours on a rotary shaker at 28°C before being centrifuged for 15 minutes at 10, 000 rpm. Salkowski's reagent (100 ml 35% perchloric acid and 2 ml freshly prepared 0.5 M FeCl2 solution) was mixed with 2 ml of supernatant and 2-3 drops of o-phosphoric acid.The sample was then incubated at room temperature for 30 minutes in the dark. The appearance of a pink colour indicated the formation of IAA. On a different day, the quantitative analytical method was utilized to quantify IAA generation. The samples were examined with varied amounts of tryptone in nutritious broth, and absorbance at 530 nm was measured using a visible spectrophotometer. The spectrophotometry results were used to produce a standard IAA solution curve that illustrated the relationship between the standard IAA solution and its absorbance.

Assay for Phosphate Solubilization

The method of (Pikovaskaya, 1948) was employed to detect phosphate solubilization. Bacterial isolates were grown on Pikovskaya's agar (HIMEDIA) plates for 2-3 days at 28 ± 2 °C. Following that, the plates were examined for the creation of halo zones around the colonies.

Hydrogen Cyanide.

According to Cappuccino and Sherman (1992) [6] , each bacterial isolate underwent a qualitative ammonia production test. Nutrient broth was used to quantify ammonia generation over a 24-hour period at 30 degrees Celsius and 150 rpm. Nutrient broth supernatants devoid of cells were supplied with 5% Nessler's reagent, whereas nutrient broth that had not been inoculated served as the control. For positive isolates, the supernatant color shifted from pale to deep yellow. The ammonium sulfate standard curve with concentrations ranging from 0 to 100 mM was used to cultivate the quantity of ammonia produced, and absorbance was measured at 425 nm (Chrouqi et al., 2017) [8] .

Results and Discussion

The study's purpose is to develop effective rhizobacterial isolates for promoting common bean plant growth. Thirty rhizobacteria isolates were collected and identified using morphological and biochemical characteristics. Table 1 lists the morphological traits of all 30 PGPR isolates, which mostly included colony features and Gram's nature. Each rhizobacterial isolate has unique biochemical properties, which are given in Table 2.

IAA Production

Plant hormones may be natural or synthetic. There are various categories of phytohormones, the most well-known being the auxin group. Microorganisms that live in the rhizosphere of diverse plants are likely to synthesize and release auxin as secondary metabolites. IAA is the primary auxin involved in a wide range of plant physiological functions. The amount of IAA produced by rhizobacteria is quantitatively measured using the Salkowski method (Dubey and Maheshwari, 2006) [10] . IAA, a phytohormone, is widely regarded the most important natural auxin. IAA may play a key role in regulating plant development.

Approximately 80% of rhizospheric bacteria are capable of producing IAA. When L-tryptophan is added to bacterial growth media, it is regarded a precursor to IAA synthesis. Plant root exudates are a natural source of L-tryptophan that enhances auxin production in the plant root zone, while rhizosphere micro flora is blocked (Ahmadloo et al., 2014) [2] . Several PGPRs from the genera Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Pantoea, and Pseudomonas have been found to produce IAA in the presence of tryptophan as a precursor (Tsavkelova et al., 2007) [24] . Out of 30 isolates, four (KB-14, KB-19, KB-12 and KB-29) were found to produce significant levels of IAA, while the remaining isolates produced moderate amounts.

HCN Production

It was originally believed that HCN production play its role in plant growth promotion by suppressing the plant pathogens (Voisard, et a., 1989) [25] . However, this concept has recently been changed. It has been believed that HCN production indirectly increases phosphorus availability by chelation and sequestration of metals and indirectly increases the nutrient availability to the rhizobacteria and host plants (Rijavec et al., 2016) [20] . The production of HCN by PGPR are independent of their genus, thus they are used as biofertilizers or biocontrol to enhance crop production and these are being used as biofertilizer in growth promotion and yield enhancement. Bacteria Micrococcus sp. and Bacillus sp. can produce HCN. HCN is a secondary metabolite produced by bacteria engaged in plant pathogen biocontrol, and it acts as a potential antifungal. HCN inhibits the function of enzymes that have a cofactor in the form of metal ions, such as Cu 2+ , on cytochrome C oxidase. Species of rhizobacteria that create cyanide, particularly Pseudomonas and Bacillus. The production and control of HCN have the potential to be exploited in reducing infections to enhance sustainable agriculture (Sehrawat et al., 2022) [21] . Out of 30 isolates only 5 were found to produce significant level of HCN Production while the remaining isolates produce moderate amount.

Phosphate Solubalization

Phosphorus is the second most important nutrient for plant growth, behind nitrogen (N). The majority of phosphorus in soil is in the form of insoluble phosphates, which plants cannot absorb directly. Despite a substantial P reservoir, the number of accessible forms for plants is often minimal. The majority of soil phosphorus is contained in insoluble forms, while plants absorb it only in two soluble forms, monobasic and dibasic ions (Bhattacharyya and Jha, 2012) [4] . To combat P deficit in soils, phosphatic fertilizers are frequently applied in agricultural fields. Plants absorb a little quantity of applied phosphatic nutrients, and the remainder is quickly transformed into insoluble complexes in the soil (Bruto et al., 2014) [17] . However, using phosphate fertilizers on a regular basis is not only expensive but also harmful to the environment. This has prompted a search for an environmentally safe and economically viable approach for increasing crop output in low P soils. In this setting, organisms coupled with phosphate-solubilizing activity, sometimes termed phosphate-solubilizing microorganisms (PSM), can offer the accessible fertilizers. Phosphate-solubilizing bacteria (PSB) are considered promising biofertilizers among the many PSM(s) that live in the rhizosphere because they may feed plants with P from sources that would otherwise be unavailable due to multiple processes (M.S. Khan et al., 2006) [18] . Bacterial taxa such as Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Microbacterium, Pseudomonas, Rhizobium, and Serratia have been identified as the most important phosphate-solubilizing bacteria (A. Zaidi, et al 2006) [1] . In this investigation, only five of the 30 isolates had the highest phosphate solabilization.

Conclusion

In this study, plant growth-promoting rhizobacteria were isolated from the rhizosphere of common beans. Based on the findings, it was concluded that all of the isolates connected with common bean root soil had plant growth-promoting properties such as phosphate solubilization, phytohormone production (IAA production) and HCN production. The results of this study clearly show that rhizobacterial isolates can be employed as bioinoculants, either alone or in combination in the sustainable production of agricultural crops.

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