Screening and Optimization of IAA Production by PGPR Isolated from Rhizosphere of a Pterocarpus marsupium Roxb. and their Effect on Plant Growth

Indole Acetic Acid (IAA) production is important attribute of PGPR that promote plant growth and development. The rhizosphere is hotspot in the soil that harbors PGPR. The present study was aimed with isolation and screening of IAA producing bacteria from the rhizosphere of Pterocarpus marsupium Roxb. Optimum culture conditions (pH, temperature, incubation period and L-tryptophan concentration for IAA production were studied for selected isolates and their effect on wheat growth and root development was evaluated. Among twenty four IAA producing isolates five isolates (Et1, Rp1, Rp5, Rp6, and Rp9) produced maximum IAA in range of 50-70 μg/mL and was used in optimization studies. Maximum IAA was produced in 96 hours of incubation, at pH 7 and with 0.1mg/mL of L-tryptophan by all five isolates. 30 o C is the most suitable temperature for Et1,Rp1, Rp5, Rp9; whereas Rp6 produced nearly same amount of IAA at wide range of temperature 30-35 o C (77-84.12 μg/mL) and at pH 7-8 ( 73-74μg/mL). Out of the five isolates, Rp6 exhibits the highest potential, having a maximum IAA of 84.12 μg/mL at 35°C and pH 7. Although tryptophan influences IAA synthesis but at higher concentration of tryptophan inhibits IAA synthesis. To validate the production of IAA, crude extracts were analyzed using thin layer chromatography (TLC). A spot of standard IAA with the same Rf value (0.91) was found to match a specific spot from the crude IAA. All the five isolates significantly increase plant height and biomass. Rp6 has greater


Introduction
The rhizosphere is an intimate area of soil near the root system of the plant. 1 The rhizosphere consists of a micro-ecosystem that involves the root system of a plant, microorganisms, nematodes, etc. 2 The interaction between microorganisms and plants is special and beneficial to each other.Microbial components consist of bacteria and fungi, having multifarious plant growth-promoting potential such as nutrient acquisition (nitrogen fixation, phosphate/ zinc/ potassium solubilization), Siderophore production, ACC deaminase production, plant growth-promoting hormones(Auxins, Gibberellins) etc. 3 IAA is an important auxin that regulates plant growth and development, and also act an important signaling molecule.Ortiz-Castro R et al.(2012) 4 and Raheem et al. (2018)  5 have reported that IAAproducing bacteria viz, Bacillus amyloliquifacience significantly increased the length of the wheat plant and B. muralis D-5 and Enterobacter aerogenes S-10 had a positive impact on spike length and seed weight under drought stress.IAA-producing plant growth-promoting microorganisms (PGPM) include Pseudomonas, Bacillus, Azotobacter, Acinetobacter, Rhizobium, Bradyrhizobium, Burkholderia, Candida tropicalis, Ustilago esculanta etc. 6,7 Cultural conditions for IAA production is widely studied, major factor affecting IAA production are pH, temperature and tryptophan concentration, nitrogen source, days of incubation, etc. 8 Results of optimization studies in Pseudomonas putida UB1 revealed that addition of L-tryptophan at concentration of 0.2 mg/mL at pH 7.5 and 96 hrs of incubation produced maximum IAA. 9 Rhizobium spp.produced the highest IAA (166 μg/mL) at a temperature of 36°C, pH of 6.5, an incubation time of 24hrs.and respectively tryptophan and NaCl concentrations of 1 g/L and 0.1 g/L. 10 Wheat is one of the top three grains consumed worldwide.India is the world's second largest producer of wheat, although rising production per capita is quite low in contrast to current demand, 11,12 use of chemical fertiliser is significantly enhances output, but its impact on ecosystem and soil health cannot be neglected. 13Biofertilizers and compost application can reduce reliance on chemical fertilizers while also ensuring sustainable agriculture.According to reports, using compost, sludge, and Azotobacter has increased wheat production and nutritional content when compared to artificial fertiliser. 14icrobial community composition of rhizosphere is unique to plant and is affected by various biotic and abiotic factors such as plant genotype, age of the plant and environmental factors, various anthropogenic activities etc., respectively.
Research on Arabidopsis thaliana has revealed that, while grown under identical conditions, the seven cultivars exhibited distinct rhizodeposit compositions as well as the development of a genotype-specific rhizobacterial community. 15Moreover, the architecture of the root system is influenced by the rhizodeposits composition. 16Because native plants are confined to a specific region, their rhizosphere microflora must be distinct and may have agricultural significance, though such studies are meagre.Pterocarpus marsupium Roxb (Family: Fabaceae) is a large, indigenous, deciduous tree from Northern Western Ghats.Because of their great medicinal value, all the parts of this species have been used in homoeopathic, ayurvedic, and unani medical systems.Its rhizosphere could provide promising PGPR that is significant to agriculture.
The current study aims to isolate and screen IAAproducing PGPR from rhizosphere of Pterocarpus marsupium Roxb's.Optimization study for IAA production with selected isolates for parameters viz, pH, temperature, tryptophan concentration, and incubation period and their effect on root development of wheat plant was investigated.

Material and Methods
Soil sample Rhizosphere soil of Pterocarpus marsupium Roxb (Location: Paud Ghat, Dist-Pune, Maharashtra, India) was collected from 20-30 cm depth from the ground, soil firmly attached to the root was taken along with root hairs in a sterile container and was further processed in laboratory conditions.

Isolation and Screening of Bacteria from
Rhizospheric Soil 10 gm of rhizosphere soil was inoculated in the sterile nutrient broth and incubated at 28 o C for 48hrs.Serially diluted sample was plated on a sterile nutrient agar plate and incubated at 28 o C for 3-4 days, colony characteristics of all obtained isolates were noted.All isolates were purified and preserved on nutrient agar slants and stored at 4 o C for further use.

Quantitative screening of IAA Producing Bacteria
For quantitative screening of IAA production by bacterial isolates, Salkowsky reagent(0.5%FeCl3 in 70% perchloric acid) 8 was used.The sterile Luria broth with pH 7 and 0.1mg/mL of tryptophan was inoculated with the test bacterial cultures, and incubated at 28 o C for 7 days.The cultures were then centrifuged, 1ml of supernatant was added with 2ml of Salkwasky reagent and incubated at room temperature for 30 minutes in the dark reaction developed pink colour, intensity of colour was measured calorimetrically at 430 nm against blank.The standard curve of IAA, was prepared using the standard concentrations of IAA (Sigma Aldrich) in Luria broth, which was further used to calculate the concentration of IAA from test samples. 6,17

Detection of IAA by Thin layer chromatography (TLC)
The standard IAA (0.1 mg/mL in methanol) and crude IAA (produced in the previous experiment) were spotted on a 15 cm x 10 cm TLC plate (Silica gel Gf 254, thickness 0.25 mm) and separated in solvent system conating n-butanol, ethyl acetate, ethanol, and water in the ratio of 3:5:1:118.The plate was then developed with Ehmen reagent( 98 mL of 35% HClO 4 mixed with 2 mL of 0.5M FeCl 3 ) 15 The plate was heated at 90 o C for 3-4 minutes for visualization of spots.R f values of test samples and standard IAA were compared and those which coincide with standard IAA were identified. 16,17

Optimization of Culture Condition for Maximum IAAProduction for Selected Isolates
The effect of pH, incubation temperature, incubation period, and tryptophan concentration on IAA production was studied using classical method 6,8,10,18,19 for the five top IAA-producing isolates, the procedures for the same are listed below pH To study effect of pH on IAA production, Luria broth at pH 5, 6, 7, and 8 was supplemented with 0.1 mg/mL of tryptophan, inoculated individually with bacterial isolates, and incubated for 4 days at 28 o C. Concentration of IAA in test samples were determined by using Salkowsky reagent.

Incubation Temperature
Effect of temperature on IAA production was studied by inoculating test bacterial samples in Luria broth of pH 7 supplemented with 0.1mg/mL of tryptophan and incubated for 4 days at temperatures 25, 30, 35, 40 o C with continuous shaking at 100 rpm.IAA from test samples was determined by using Salkowsky reagent.

Incubation Period
Each test bacterial isolates was separately inoculated in Luria broth of pH 7 containing 0.1mg/ mL tryptophan and incubated at 28 o C in shaker incubator at 100 rpm.Sample from each flask was collected at 24hrs, 48hrs, 72hr, and 96hrs of incubation and cell free broth was assessed for IAA using Salkowsky reagent.

Tryptophan Concentration
Effect of tryptophan concentration on IAA production was determined by inoculating Luria broth of pH 7 supplemented with L-tryptophan at concentration of 0.05, 0.1, 0.5, and 1mg/mL with test bacterial isolates separately and incubated for 4 days at 28 o C, IAA from all tubes were determined by using Salkowsky reagent.

Effect of selected PGPR on growth of Wheat
Effect IAA producing PGPR was studied on growth and root architecture of Wheat (Triticum aestivum) MACS 6222 variety.MACS 6222 is high yielding, rust resistant and commonly grown variety in India especially Maharashtra.Wheat seeds were procured from MACS-Agharkar Research Institute (ARI), Hol farm, Dist-Pune Maharashtra India.Seeds were washed with few drops of Tween 20 and water thoroughly to remove detergent completely and further surface sterilized with 0.1% HgCl 2 for 1 minute, and washed with sterile distilled water five times.Surface sterilized seeds were treated with each test bacterial inoculum containing 10 8 cells/mL overnight, coated seed were dried and 20 seeds of each treatment were placed in a petri plate on moist paper towel.All the petri plates were incubated in at 25 o C for 10 days.Watering was done as per requirement.The physiological parameters viz., germination time and germination percentage, plant height, fresh biomass, root morphology were noted.Experiment was carried out in triplicate.

Statistical Analysis
The results of optimization of IAA are presented as means of three replicates ± standard deviation.Significance of PGPR treatment on plant growth and root development were statistically analysed using one-way analysis of variance (ANOVA) and means were compared using Dunnet multiple comparison test at p = 0.05 in Minitab18 statistical software.The graphs were plotted using MS excel and Minitab18 software.

Results and Discussion
A total of twenty four isolates were obtained from the rhizosphere of Pterocarpus marsupium Roxb., colony characteristics are mentioned in Table1.Isolates were further screened for quantitative IAA production using the Salkowsky reagent.All of the isolates displayed pink coloration, indicating that they are all capable of producing IAA (Fig1).Five bacterial isolates viz; Et1, Rp1, Rp5, Rp6, Rp9 with higher potential for IAA production (viz 50, 61,70, 76, 70 μg/mL respectively) were employed in further studies.( Table2).

Optimization Studies on IAA Production
Bacteria produce IAA by tryptophan-dependent and tryptophan-independent pathways, however majority of bacteria synthesize high amount of IAA in presence of L-tryptophan, as tryptophan is a precursor of IAA. 18,20The L-tryptophan content, temperature, incubation time, and medium pH were the factors that optimized for IAA production by top five producers using the classical method (One Factor at a Time).The current study's findings show that tryptophan supplementation in media raises IAA production.Mohite( 2013) 6 and Duca et al.( 2014) 21 support tryptophan's beneficial effects on IAA production.Maximum IAA was produced at 0.1mg/ mL of tryptophan by all five isolates i.e 54, 62, 73, 78, 72 mg/mL, respectively.Tryptophan concentrations between 0.05 and 0.1 mg/ml produced almost equal amount of IAA, although, IAA production is reduced at higher tryptophan concentrations.These results validate the findings of Shokri & Emtiazi(2010) 19 who found that higher tryptophan concentrations have a negative impact on IAA production.However, Bharucha et al.( 2013) 9 have also reported maximum IAA production by Pseudomonas putida UB1 at 0.2% tryptophan.(Fig 2a) Metabolic activities of microorganisms are highly affected by physiological factors such as pH, temperature, macro, and micronutrient sources and their concentration, etc.Every organism has a cardinal range of the above factors for growth.IAA production is also affected by factors viz., temperature, pH, tryptophan concentration, nitrogen source, NaCl, incubation period etc. 8,23,24 There is a diverse report on the temperature requirement and incubation period for maximum IAA production.In the current investigation four isolates (Et1, Rp1, Rp5, Rp9) have produced maximum IAA i.e 50, 61, 71,71μg/mL respectively at 30 o C and   2c).Results are in support of Patten&Glick(2002), 25 they have obtained maximum IAA production by Pseudomonas putida.GR12-2 in 96 hrs and beyond 96hrs IAA concentration was found to be decreased; B. siamensis also produces maximum IAA at 35 o C in 96 hrs. 26okri and Emtiaz(2010) 22 have reported 30 o C and 72 hrs as an optimum temperature and incubation period for Paenibacillus, and Rhizobium strains.IAA production was found to be increased with the incubation period as IAA production occurs at the stationary phase of growth. 10Rhodopseudomonas palustris produced maximum IAA (80.77± 2.13 μg/ mL) at 35 o C in 48hrs. 27that Rhizobium produces highest levels of IAA at pH 6.5, whereas Bharucha et al(2013) 9 reported that Pseudomonas putida UB19 has a pH of 7.5.

Detection of IAA by thin-layer chromatography (TLC)
The developed chromatogram showed pink colour spots with all bacterial samples and standard IAA.Rp6 appears to be most promising among all five as it causes approximately 50% increase in plant height and biomass followed by Rp5 (40%) and Et1 (30%) (Table3,Fig5a), these results are in agreement with studies performed by Dahmani et al.(2020), 29 in which plant growth promoting Bacillus megaterium  30 showed that rice rhizosphere isolates generated IAA and impacted plant height and root length of rice seedlings.
In our studies wheat seedlings treated with IAAproducing bacterial isolates Et1, Rp1, Rp5, Rp6, and Rp9 showed striking shift in the root system architecture, demonstrating significant expansion of lateral roots and root hairs in comparison to untreated control(Fig 5b).Several workers have previously described similar findings caused by inoculation with Bacillus altitudinis (strain FD48) in rice, 31 Phyllobacterium brassicacearum STM196 strain in Arabidopsis thaliana. 32Auxins and cytokinins plays significant role in lateral root and root hair development. 32,33g. 5

Conclusion
The current investigation's findings highlight the significance of the rhizosphere of the native plant Pterocarpus marsupium Roxb.as a source of IAA producing PGPR.Among five isolate Rp6 appears to be more promising as it produces high amount of IAA at diverse culture condition ( pH, temperature, incubation period etc.) hence it can enhance plant growth at diverse climatic conditions in the field.All the isolates under study enhanced wheat growth and development of lateral roots and root hairs.As a result, additional research is required to determine whether any of the chosen isolates have the potential to be a viable biofertilizer in the field settings.Therefore, it can be concluded that IAA producing isolates obtained from rhizosphere of Pterocarpus marsupium Roxb.species have a significant impact on plant growth and development and have prospective as a biofertilizer for sustainable agriculture.

Fig. 1 :
Fig.1 :IAA production by bacterial isolates obtained from rhizosphere of Pterocarpus marsupium Roxb, pink colour indicates presence of IAA.

Fig. 2 :
Fig. 2: Effect of different physiological parameter on IAA production (2a-Tryptophan concentration 2b-Incubation temperature 2c -Incubation period 2d-pH) The bacterial sample showed two spots each one with an Rf value of 0.91 which matches with standard IAA while another spot is of Rf value 0.23 which remains to be identified (Fig3).Our results of TLC of IAA are consistent with Shokri & Emtiazi(2010)22 and Kang et al(2019)28 studies.On TLC plates, one more component that has not yet been named was found in addition to IAA.

Table 2 :Quantitative IAA production by bacterial isolates obtained from rhizosphere of Pterocarpus marsupium Roxb. using Salkowsky reagent.
decreased further with increase in temperature; whereas RP6 produced maximum IAA (84.12 μg/mL ) at 35 o C (Fig2b).All the isolates produced highest IAA at 96 hrs. of incubation(Fig.

Table . 3: Effect of bioinoculation on height and biomass of wheat plant ( 10 days seedling) Treatment Avg height(m) Biomass(g)
strain (RmBm31) increases root biomass and positively develops root architecture of Arabidopsis thaliana.Similar to this,Ashrafuzzaman et al.(2009)