Evaluation of endophytic bacteria for enhancing plant growth and its effect on the growth and productivity of betel leaf plants

doi:10.21203/rs.3.rs-5001253/v1

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Evaluation of endophytic bacteria for enhancing plant growth and its effect on the growth and productivity of betel leaf plants

https://doi.org/10.21203/rs.3.rs-5001253/v1

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The aim of this research is to identify potent strains of endophytic bacteria from Piper betle L. that exhibit a wide range of in vitro plant growth-promoting (PGP) characteristics and to assess their effectiveness in promoting plant growth in vivo through greenhouse experiments. A total of 27 endophytic bacteria isolated from betel leaves were screened for PGP traits using both qualitative and quantitative methods. All bacterial isolates demonstrated positive results for IAA production, phosphate solubilization, ammonia production, siderophore production, pectinase activity, and HCN production. Specifically, the bacteria produced IAA in the range of 1.7 to 224.7 µg/ml, solubilized phosphate between 17.8 and 35.17 µg/ml, produced ammonia in concentrations of 0.14 to 0.51 µmol/ml, and exhibited siderophore production ranging from 38.92–71.9%. Four bacterial isolates, selected for their superior PGP traits, were identified through 16S rRNA gene analysis as Pseudomonas aeruginosa, Bacillus velezensis, Enterobacter cloacae, and Serratia marcescens. Among these, Pseudomonas aeruginosa showed the most significant enhancement of all growth parameters in betel plants under greenhouse conditions.

Ammonia production

Betel leaves

Endophytic bacteria

IAA production

Phosphate solubilization

Siderophore production

Piper betle L., a vital and widely cultivated asexually propagated cash crop, belongs to the Piperaceae family. It is a shade-loving perennial creeper with broad, heart-shaped leaves that vary in color from pale green to bright green or yellowish-green (Guha and Nandi, 2019). These large leaves are aromatic, offering a range of flavors from sweet to spicy (Ravindran et al., 2004). Endophytes, including bacteria and fungi, are microorganisms that form endosymbiotic relationships with various plants and are found throughout different plant parts, such as roots, stems, leaves, bark, floral organs, and seeds (Hallmann et al., 1997; Compant et al., 2011; Dudeja et al., 2012). These endophytes have been extensively studied for their ability to promote plant growth, and their presence is well-documented in various plant species. Plant growth-promoting bacteria (PGPB) play a crucial role in aiding nutrient acquisition through mechanisms like phosphate solubilization, nitrogen fixation, ammonia production, and siderophore production. Additionally, plant-associated microorganisms improve nutrient uptake by making minerals and other macro/micronutrients more accessible from the soil (Caldwell et al., 2000; Barrow, 2003). As a result, there is a great deal of promise for enhancing plant development in the isolation and characterisation of endophytic bacteria with a variety of characteristics from unexplored host plants. (Patten and Glick, 2002; Sergeeva et al., 2007). One effective approach for promoting plant growth and modifying plant structure involves the use of endophytic bacteria that influence phytohormones. These bacteria have demonstrated significant potential in agricultural applications due to their distinctive properties (Hallmann et al., 1997; Sturz et al., 2000).

Continuous agricultural practices, such as fertilization, have the potential to cause significant environmental harm on a global scale. These practices can lead to unintended negative consequences, including the accumulation of chemical residues and the disruption of ecosystems (Shenoy et al., 2001; Adesemoye et al., 2009; Hazell et al., 2007). In response to these environmental concerns, biofertilizers have emerged as a sustainable alternative to conventional farming methods. These biofertilizers contain beneficial microbes that form symbiotic relationships with plants, thereby enhancing plant health and growth. This results in improved agricultural outcomes, including increased root and shoot length, greater fresh and dry weight of shoots and roots, higher yield production, and enhanced soil quality and nutrient cycling (Soliman et al., 2019; Elkelish et al., 2020). The objective of this study was to evaluate the effectiveness of four plant growth-promoting bacteria treatments on betel leaf cultivation by measuring plant growth and yield.

The plant samples of betel leaves were obtained from Nongtrai village, East Khasi Hills District, Meghalaya, India (25.2207’ N latitude and 91.6116’ E longitude) during the study period 2019–2021. The plant sample were plucked from healthy plant parts were kept in autoclaved polythene bags and those with injury were excluded. The plant sample carried to the laboratory were washed in running water. For isolation, 1 g of each plant parts including leaves, stems and roots were surface sterilized with 1% sodium hypochlorite for 3 min, followed by 70% ethanol followed by washing with distilled water for 3 times and dried on sterilized filter paper. The plant parts were crushed separately in a sterile mortar and pestle and sap was streaked on nutrient agar (NA) plate. Dilutions of 10^–1 was made. Each isolation procedure was done in triplicate. 100 µl of 10^–1 dilution was spread on nutrient agar plates and incubated at 28°C for 48 h. Bacterial cultures were maintained by streaking on nutrient agar (NA) plates. However, no microbial growth was observed on control nutrient agar medium plates inoculated with the final wash solution of surface sterilization procedure which proved the satisfactory of surface sterilization methods (De Araújo et al., 2002) with some minor modification.

Plant growth promoting (PGP) traits of endophytic bacteria

Siderophore production

Qualitatively the bacteria isolates were spot inoculated on CAS agar plates and were incubated at 28 ± 2°C for 4 days. After incubation, an orange halo around the colonies indicates positives for the production of siderophores while no change in colour indicate negative results. Quantitative analysis on siderophore production by using Schwyn and Neilands (1987). The cultures were grown in Luria broth and incubated at a 28 ± 2°C in rotatory shaker at 150 rpm. After 4 days, centrifugation was carried out for 15 min at 7000 rpm and estimated by CAS-Shuttle assay in which 0.5 ml of culture supernatant was mixed with 0.5 ml of CAS reagent and the absorbance was recorded at 630 nm against a reference consisting of 0.5 ml of uninoculated broth and 0.5 ml of CAS reagent.To assess the loss of blue colour to orange, the optical density (630 nm) was changed in contrast to the blank. Units of siderophore production were calculated as

Siderophore production %= A_reference-A_sample/A_reference x 100

Where, Ar = Absorbance of blank containing media, CAS assay solution and shuttle 44 solution. As = Absorption of test sample containing supernatant, CAS assay solution and shuttle solution at 630 nm

Indole-3-acetic-acid (IAA) production

supernatant, CAS assay solution and shuttle solution at 630 nm

Indole-3-acetic-acid (IAA) production

IAA estimation of endophytic bacteria was done using method given by Brick et al., (1991). Bacterial isolates were grown in Luria Broth (LB) supplemented with tryptophan) with 0.1% L-tryptophan in triplicates and incubated at 28°C for 48 hours in a shaker incubator at 150 rpm. The bacterial culture were centrifuged at 10,000 rpm for 15 minutes. 1 ml supernatant was taken into a fresh microcentrifuge tube and added 2 ml Salkowaski reagent (49ml 0f 35% perchloric acid, 1ml of 0.5M FeCl3 solution) in the ratio of 1:2. To the test tube 2–3 drops of concentrated orthophosphoric acid was added. The intensity of the colour was measured against the standard curve after 30 minutes using the Lambda 35 UV–VIS spectrophotometer at 530 nm. Development of Pink colour in media indicated the presence of IAA.

Phosphate solubilization

Qualitatively all the bacteria cultures were spot inoculated on Pikovskaya’s plates (Pikovskaya1948). After incubation 28 ± 2°C of 4–5 days, development of clear zone around the growth indicates positive for phosphate ability. Quantitative estimation of tricalcium phosphate {Ca3(PO4)2} solubilization in broth was carried out using Erlenmeyer flasks (250 ml) containing 100 ml of Pikovskaya’s broth inoculated with 1 ml of bacterial suspension (3 x 105 cells/ml). After 5 days incubation the bacterial culture were centrifuged at 7000 rpm for 15 min and the supernatant was used to estimate soluble phosphate (P) concentration and uninoculated brothused as a control. Phosphate solubilisation was determined by 45 ammonium molybdate-ascorbic acid method (Lowry and Lopez,1946). Reagent for colour development was prepared by combining H2SO4, potassium antimony tartrate solution, ammonium molybdate and ascorbic acid solution and the absorbance was recorded at 880 nm using a Lambda 35 UV-Vis Spectrophotometer. The experiments were conducted in triplicates and data were expressed as the mean value ± standard error and were expressed as µg/mL.

Ammonia production

Detection of ammonia production was done by the method described by Cappuccino and Sherman (1992). The bacteria isolates were inoculated in 10ml peptone broth for 48 h at 28 ± 2°C. After incubation period, the bacteria culture was centrifuged at 12,000 rpm for 15 mins and 1 ml of Nessler’s reagent was added to the 1ml supernatant in each tube. The formation of faint yellow color indicated minimal ammonia production, while the deep yellow color to brownish precipitate indicated the highest ammonia production was measured spectrophotometrically at 450 nm using the Lambda 35 UV–VIS spectrophotometer and the amount of ammonia produced was estimated from the standard curve of ammonium sulfate..

HCN Production

Production of hydrogen cyanide (HCN) was detected with Lorck’s method (Lorck 1948). Bacteria isolates after 48 hours of growth were treak on nutrient agar (NA) plates ammended with glycine and Whatman filter paper (No.1) dipped in 0.5% picric acid the plates are sealed using a paraffin. Production of cyanogen is confirmed by discoloration of the filter paper form orange to brown after incubation of 3 days.

Pectinase

Pectinase activity was done by spot inoculation of bacterial isolates on nutrient agar supplemented by 0.5% pectin (Sigma) and incubated at 30 ± 1°C for 5 days. After incubation, the plates were flooded with lugal iodine) solution for 15–20 minutes. Pectinase activity was detected by the presence of a clear halo zone surrounding the colony.

Lipase

Lipase activity were examined using tributyrin agar medium (Ahmed et al., 2010) were loopful of each pure culture bacterial isolates were spot inoculated and the plates were then incubated for 24 hours at 37°C. Following incubation, lipase activity was demonstrated by the distinct hydrolysis zone surrounding the colonies and were considered positive.

Bacterial DNA Extraction and Amplification

Molecular identification of potential bacterial strains was performed by growing a pure culture of bacteria isolates in nutrient broth (NB) at 30 + 2°C for 48 hours, and DNA was extracted using the Bacterial Genomic DNA Purification Kit (Himedia) to extract bacterial isolates. Molecular identification of the isolates was performed through amplification of the 16S rRNA region using primers 27F (5′ AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) (Lane, 1991) in a thermal cycler (Thermo Fisher, United States). The PCR reaction mixture consist of 1X PCR buffer with 1.5Mm MgCl2, 200 µM dNTPs, 0.2 µM primers and 50 ng template DNA. The following cycling conditions were used: inital denaturation step at 95°C for 5 min, followed by 35 cycles (denaturation at 95°C for 1 min, annealing at 55°C for 1min and extension at 72°C for 2 mins and final extension step at 72°C for 5 min) using a Bio-Rad C1000 touch, USA. The amplified PCR products was sent to Agrigenome Labs Pvt Ltd, Kerala, India.

Preparation of Bacterial Inocula for betelvine cutting treatment

Bacterial strains were cultured in 500 ml conical flasks containing 300 ml Luria Bertani broth on an orbital shaker at 120 rpm for 72 h at 28 ± 2°C. Bacterial cells pellet was collected via centrifugation at 7000 rpm for 15 min at 4°C, and each pellet was washed twice with autoclave water. The bacterial pellets were suspended in 0.6 ml autoclave distilled water, vortexed and used for soil treatment. The number of bacterial cells was determined via serial dilutions, approximately 10⁷ CFU/soil. In order to test the ability of isolates to promote growth in betelvine plants. The soil from the study site described above was used as potting medium. After autoclaving at intervals at 121°C and 15 psi for 90 min. 200 g of the sterilized soil was placed in each sterilized plastic pot (20 cm × 30 cm size). Cutting is prepared with 4 nodes for crop propagation, and the cutting is normally picked from healthy vines and soaked in test bacteria suspension 1× 10⁷ CFU/ml for 20 minutes and grown separately in soil. After acclamatisation, one months old betelvine were inoculated again with 1 × 10⁷ CFU/soil of each test bacterial isolate suspension. The uninoculated cuttings served as control plant. A completely randomized design (CRD) was used with three replications. The pots were maintained under greenhouse conditions for 4 months. After harvest, the growth parameters such as root and shoot lengths fresh weight, dry weight of the plants were measured. The shoots and roots were separated and dried in an oven at 50 ± 2°C for 48 h.

Statistical Analysis

The statistical analysis was performed using graphpad prism 9 and MS-Excel.The data obtained were subjected to one way analysis of variance (ANOVA) and Tukey’s test at p B 0.05 using replicates.

Ammonia production

All the endophytic bacteria were tested for ammonia production using the Nesslerization reaction. A positive reaction was indicated by the appearance of a golden to dark brown color. All isolates demonstrated both ammonia production and growth. The results underscored the capability of the isolated bacterial endophytes to produce ammonia in the media after the addition of Nessler’s reagent. Among the tested strains, NL8 exhibited the highest ammonia production, with a value of 0.51 ± 1.32 µmol/ml ( Fig. 1 & Table 1).

Table 1

Screening of endophytic bacterial isolates for their plant growth promoting traits
Bacteria Isolates	Ammonia production (µgmol/ml^− 1)	Siderophore production (%)	Indole acetic acid (IAA) (µg /ml-1)	Phosphate solubilisation (µg /ml-1)	Lipase	HCN	Pectinase
NL1	0.31 ± 0.01	58.45 ± 0.12	190 ± 0.51	33.6 ± 0.26	+	+	+
NL2	0.45 ± 0.15	71.9 ± 0.15	86.03 ± 1.85	28.53 ± 0.1	+	+	+
NR3	0.37 ± 0.23	63.84 ± 0.19	82.9 ± 0.4	17.8 ± 0.0	+	+	+
NL4	0.33 ± 1.64	59.02 ± 0.13	68.2 ± 0.5	35.97 ± 0.21	+	+	+
NL5	0.29 ± 0.65	57.97 ± 0.77	56.07 ± 1.52	28.97 ± 2.22	+	+	+
NR6	0.35 ± 0.28	68.61 ± 0.03	62.07 ± 0.65	34.1 ± 0.11	+	+	+
NL7	0.36 ± 0.03	65.9 ± 0.03	131.97 ± 0.28	36.07 ± 0.11	+	+	+
NL8	0.51 ± 1.32	47.1 ± 0.29	173.57 ± 1.15	35.06 ± 0.21	+	+	+
NS9	0.26 ± 0.72	66.03 ± 0.05	111 ± 1.85	34.6 ± 0.36	+	+	+
NL10	0.47 ± 0.01	60.65 ± 0.11	71.23 ± 2.00	34.13 ± 0.15	+	+	+
NL11	0.64 ± 0.01	55.23 ± 0.51	163.47 ± 0.81	33.83 ± 0.06	+	+	+
UL12	0.21 ± 0.06	68.61 ± 0.03	111.93 ± 1.43	40 ± 0.15	+	+	+
US13	0.20 ± 0.02	63.34 ± 0.24	76.8 ± 0.4	34.73 ± 0.06	+	+	+
NL14	0.51 ± 0.04	43.52 ± 0.22	224.7 ± 0.55	34.46 ± 0.11	+	+	+
US15	0.35 ± 0.01	69.12 ± 0.16	175.57 ± 0.5	32.03 ± 0.06	+	+	+
NS16	0.30 ± 0.01	59.21 ± 0.04	122.2 ± 0.62	29.17 ± 0.11	+	+	+
US17	0.25 ± 0.59	57.15 ± 0.21	54.6 ± 0.3	34.5 ± 0.11	+	+	+
UR18	0.37 ± 0.65	55.37 ± 0.33	76.8 ± 0.02	34.46 ± 0.06	+	+	+
UL19	0.28 ± 0.08	65.14 ± 0.16	1.7 ± 1.03	14.8 ± 0.51	+	+	+
UR20	0.49 ± 0.01	57.61 ± 0.15	65.57 ± 0.87	30.96 ± 0.06	+	+	+
NL21	0.29 ± 0.05	60.82 ± 0.15	108.33 ± 0.92	30.27 ± 0.25	+	+	+
NS22	0.17 ± 0.62	69.56 ± 0.26	77.2 ± 0.36	27.6 ± 0.34	+	+	+
UR23	0.24 ± 0.02	58.74 ± 0.22	161.53 ± 1.02	31.6 ± 0.1	+	+	+
Ul24	0.32 ± 0.02	65.53 ± 0.07	33.83 ± 0.20	35.36 ± 0.37	+	+	+
NL25	0.14 ± 0.02	38.92 ± 0.07	76.87 ± 1.90	30.37 ± 0.06	+	+	+
NL26	0.25 ± 0.06	53.17 ± 0.10	177.43 ± 0.92	35.17 ± 0.21	+	+	+
UR27	0.45 ± 0.15	47.1 ± 0.29	73.3 ± 1.55	32.56 ± 0.20	+	+	+
Values are means of three replicates ± SD

Siderophore production

Screening for siderophore production in all bacterial isolates was conducted using the method described by Schwyn and Neilands (1987). Qualitatively, the formation of a yellow to orange halo surrounding the bacterial growth indicated the initiation of iron chelation by siderophores. Isolates that did not exhibit any color change around the growth were considered negative for siderophore production. The color change to yellowish-orange is due to the siderophores' ability to remove iron from the dye complex.

Quantitatively, all 27 bacterial endophytes tested positive for siderophore production, with varying potentials ranging from low to high ammonia production, between 38.92% and 71.9%. Among them, isolate NL2 demonstrated the highest siderophore production, with 71.9% siderophore units (Fig. 1 & Table 1).

Indole Acetic Acid (IAA) production

The Salkowski colorimetric assay was used to evaluate IAA production, which was quantified using a standard curve. A total of 27 bacterial endophytes were screened for IAA production after 48 hours of incubation in a growth medium supplemented with 0.1% tryptophan as a precursor. The results revealed that all 27 bacterial isolates were capable of synthesizing IAA, as indicated by a color change from light to dark pink. The IAA concentration ranged from 1.7 to 224.7 µg ml⁻¹, with isolate NL14 showing the highest production rate (Fig. 1 & Table 1).

Phosphate solubilization

The ability of bacterial isolates to solubilize phosphate was qualitatively indicated by the presence of a clear halo zone around the bacterial colony on Pikovskaya’s agar media supplemented with Ca₃(PO₄)₂. These isolates were then quantitatively assessed for their phosphate-solubilizing capacity. The results showed that all isolates were capable of solubilizing phosphate, with solubilization levels ranging from 17.8 to 35.17 with NL14 showing the highest (Fig. 1 & Table 1).

Pectinase, lipase and HCN production

All endophytic bacteria were able to produce pectinase, lipase and HCN production. Notably, all bacterial isolates show positive for pectinase, lipase while all bacterial isolates show positive except 3 show negative results for HCN production (Fig. 1 & Table 1).

Sequencing and accession numbers of strains

The genomic DNA sequences of these strains, after sequencing at Agrigenome Labs, Kerala, were searched for homologous sequences using BLAST analysis at NCBI and sequences were submitted to NCBI GenBank. Accession numbers of the bacterial antagonists and the fungal pathogens were obtained and identified as Pseudomonas aeruginosa NL8 (OR347773) and Bacillus velezensis NL7 (OR400705), Enterobacter cloacea NL14 (OR400686) and Serratia marcescens NL2 (OR401335) (Table 2).

Table 2

Characterisation of 16S rRNA gene of selected endophytic bacteria with their sequence accession numberof *Piper betle* L.
Strain Code	Query cover (%)	Similarity (%)	Closest sequence	Genbank accession no.
NL2	100%	99%	Serratia marcescens	OR401335
NL7	100%	100%	Pseudomonas aeruginosa	OR347773
NL8	100%	99.04%	Bacillus velezensis	OR400704
NL14	100%	99.34%	Enterobacter cloacea	OR400686

Growth characteristics of betel leaves plant treated with selected bacteria isolates

All four bacterial isolates, NL2, NL7, NL8, and NL14, significantly enhanced all growth parameters. Among these, NL8 exhibited the most substantial increase across all growth metrics, including shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight and root dry weight, compared to the un-inoculated plants, which demonstrated stunted growth (Fig. 3 & Table 3).

Table 3

Plant growth promoting effect of endophytic bacteria on various growth traits of *P. betle* L.
Treatment	Shoot height (cm)	Shoot fresh weight (g)	Shoot dry weight (g)	Root length (cm)	Roots fresh weight (g)	Roots dry weight (g)
Control	14.33 ± 1.5	14.65 ± 1.17	2.54 ± 0.39	11.33 ± 0.57	2.67 ± 0.68	0.48 ± 0.04
NL2	17.67 ± 1.5 ^ns	16.58 ± 0.96 ^ns	3.42 ± 0.19^ns	14 ± 1^ns	4.91 ± 0.59^ns	1.21 ± 0.14^****
NL7	28.33 ± 1.5^***	35.81 ± 1.25^****	5.67 ± 0.34^****	20.7 ± 1.15^****	8.72 ± 0.81^***	1.87 ± 0.09^****
NL8	19.33 ± 1.5^**	32.98 ± 1.79^****	4.51 ± 4.7^***	16 ± 0.58^**	7.66 ± 0.38^**	1.52 ± 0.05^****
NL14	18.67 ± 1.5^*	27.38 ± 0.87^****	4.12 ± 0.57^**	15.33 ± 1.15^*	6.82 ± 0.78^**	1.35 ± 0.08 ^****
Data are means of three replicates ± SD. Data with the same sign within each column are signifcantly different at p ≤ 0.05 as determined by Tukey’s HSD tests statistically significant differences at p < 0.0001, p < 0.001, and p < 0.05 are identified by ***, , , and , respectively

In this study, all four bacterial isolates demonstrated significant growth-promoting capabilities. Among them, Pseudomonas aeruginosa (NL7) exhibited the highest phosphate production, consistent with the findings of Fernández et al. (2012). Pseudomonas strains are known for their ability to produce organic acids during solubilization of inorganic phosphates (Vyas et al., 2009). Additionally, Bacillus velezensis and Enterobacter cloacae showed the highest ammonia production, B. velezensis BAC03, has been shown to stimulate plant development by secreting various substances, including ammonia and indole-3-acetic acid (Meng et al., 2016) while Serratia marcescens produces significant amounts of indole acetic acid (IAA), which promote host plant development. This suggests its potential as a plant biofertilizer or bioenhancer, offering a less harmful alternative to artificial fertilizers and pesticides (Devi et al., 2016) and Enterobacter cloacae exhibit highest siderophores production. The secretion of siderophores by bacteria can promote plant growth by either enhancing nutrient availability or inhibiting the establishment of competitors through the sequestration of iron from the environment. Plants are generally not harmed by the bacterial-mediated depletion of iron and some plants are capable of capturing and utilizing Fe3+-siderophore complexes produced by bacteria (Dimkpa et al., 2009b).

In greenhouse experiments, Pseudomonas aeruginosa (NL8) significantly improved plant growth parameters in betelvine, including increased plant length and leaf number. These results are consistent with Linu et al. (2019), who reported enhanced growth in chili plants due to Pseudomonas aeruginosa isolates, and with Qessaoui et al. (2019), which found that Pseudomonas sp. isolates could colonize tomato plant roots, solubilize phosphate, and produce siderophores, ammonia, and IAA, highlighting their potential as effective plant growth-promoting bacteria.

Four potent endophytic bacteria isolated from Piper betle have shown the ability to promote plant growth, but Pseudomonas aeruginosa (NL8) stands out due to its exceptional production of IAA, ammonia, solubilized inorganic phosphate, and siderophores. When Piper betle was inoculated with Pseudomonas aeruginosa (NL8), it significantly outperformed the non-inoculated control, other inoculated bacteria plants in all growth measures. This beneficial effect on the inoculated plants may be attributed to Pseudomonas aeruginosa (NL8) growth-promoting abilities. Therefore, further research is needed to explore its potential in sustainable agriculture as a plant growth promoter and as an alternative to chemical fertilizers, which deplete soil microbiomes and negatively impact soil health.

Acknowledgements

The authors are thankful to the Head of Department of Botany, North-Eastern Hill University, for providing laboratory facilities. The first author is also grateful to the UGC Non-Net Fellowship, New Delhi, for financial support in the form of a research fellowship.

Competing Interests

The authors have no relevant financial or non-financial interest to disclose.

Author contributions

All authors contributed to the study conception and design. KS carried out material preparation, data collection and analysis. KS wrote the manuscript. HK reviewed and edited manuscript.

Data availability

All relevant data have been included within the article

Conflict of interest

The authors declare that there is no conflict

Ethical statement

Ethical approval was not needed for this study .

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No competing interests reported.

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Evaluation of endophytic bacteria for enhancing plant growth and its effect on the growth and productivity of betel leaf plants

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Abstract

Figures

Introduction

Sample collection and isolation of endophytic bacteria

Plant growth promoting (PGP) traits of endophytic bacteria

Siderophore production

Indole-3-acetic-acid (IAA) production

Indole-3-acetic-acid (IAA) production

Phosphate solubilization

Ammonia production

Bacterial DNA Extraction and Amplification

Preparation of Bacterial Inocula for betelvine cutting treatment

Statistical Analysis

Results

Ammonia production

Siderophore production

Indole Acetic Acid (IAA) production

Phosphate solubilization

Pectinase, lipase and HCN production

Sequencing and accession numbers of strains

Growth characteristics of betel leaves plant treated with selected bacteria isolates

Discussion

Conclusion

Declarations

References

Additional Declarations

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