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Stress tolerance and plant growth promotion potential of Enterobacter

ludwigii PS1 isolated from Seabuckthorn rhizosphere

Article  in  Biocatalysis and Agricultural Biotechnology · April 2018

DOI: 10.1016/j.bcab.2018.04.012

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Author’s Accepted Manuscript

Stress tolerance and plant growth promotion

potential of Enterobacter ludwigii PS1 isolated
from Seabuckthorn rhizosphere

Diskit Dolkar, Phuntsog Dolkar, Stanzin Angmo,

OP Chaurasia, Tsering Stobdan
www.elsevier.com/locate/bab

PII: S1878-8181(17)30261-X
DOI: https://doi.org/10.1016/j.bcab.2018.04.012
Reference: BCAB748
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 6 May 2017
Revised date: 7 April 2018
Accepted date: 17 April 2018
Cite this article as: Diskit Dolkar, Phuntsog Dolkar, Stanzin Angmo, OP
Chaurasia and Tsering Stobdan, Stress tolerance and plant growth promotion
potential of Enterobacter ludwigii PS1 isolated from Seabuckthorn rhizosphere,
Biocatalysis and Agricultural Biotechnology,
https://doi.org/10.1016/j.bcab.2018.04.012
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 Stress tolerance and plant growth promotion potential of Enterobacter ludwigii PS1 isolated

from Seabuckthorn rhizosphere

Diskit Dolkara, Phuntsog Dolkara, Stanzin Angmoa, OP Chaurasiaa, Tsering Stobdana*

a
Defence Institute of High Altitude Research, DRDO, Leh, Jammu and Kashmir, 194101, India

*Tel.: +91-9419176057; Fax: +91-1982-252096

E-mail address: ts_mbb@yahoo.com

ABSTRACT

Plant growth promotion by microbial inoculants is affected by environmental factors.

The study was therefore aimed at developing microbial inoculants for promoting tomato

growth in regions experiencing temperature, pH and salt stressed conditions. Enterobacter

ludwigii PS1 capable of solubilizing insoluble inorganic phosphate was isolated from

Seabuckthorn rhizosphere growing in the Indian trans-Himalaya. PS1 showed ability to

solubilize insoluble phosphate under different stress conditions viz. 4–44°C temperature, 1–

5% salt concentration and 4–12 pH range. The isolate exhibited multiple plant growth

promoting traits viz. auxin (24.3 mg L-1), siderophore (79%) and hydrogen cyanide (0.21 O.D

at 625nm) production. Tomato seed bacterization resulted in 25% and 37% increase in shoot

and root length. Inoculation of tomato seedling with PS1 promoted plant growth in pot trial

experiments in trans-Himalayan (34º08ʹ 0.2ʺ N, 77º34ʹ 0.3ʺ E, 3340m asl) condition.

Increase in fruit yield was 15% in open and 27% in shade net condition. E. ludwigii PS1 with

phosphate-solubilizing ability under stress conditions appears to be attractive for exploring its

plant growth-promoting activities towards development of microbial inoculants in stressed

regions.

Keywords Biofertilizer, Enterobacter, PGPR, Seabuckthorn, Stress, Tomato

1
1. Introduction

Phosphorus (P) is one of the most important macronutrients for plants. It plays crucial

role in photosynthesis, seed formation and other vital physiological function making

application of phosphatic fertilizer unavoidable in intensive farming (Fernandez et al., 2007).

However, a large portion of soluble inorganic phosphate applied to the soil as chemical

fertilizer is immobilized rapidly into insoluble forms-CaHPO4, Ca3(PO4)2, FePO4 and AlPO4-

and are not efficiently taken up by the plants, which lead to an excess application of P

fertilizer to crop land (Goldstein,1986; Omar, 1998). The excessive fertilization of P leads to

pollution due to soil erosion and runoff water and cause environmental and economical

problems (Brady, 1990).

Microorganisms are involved in a range of processes that affect the transformation of

soil P and are thus an integral part of the soil P cycle. In particular, soil microorganisms are

effective in releasing P from inorganic and organic pools of total soil P through solubilization

and mineralization (Hilda and Fraga, 1999). Phosphate solubilization occurs by carboxylic

acids synthesized and released by microorganisms that results in decrease in pH (Puente et

al., 2004; Rodriguez et al., 2006). Currently, the main purpose in managing soil P is to

optimize crop production and minimize P loss from soils. Recently, phosphate solubilizing

bacteria (PSBs) have attracted the attention of agriculturists as soil inoculums to improve

plant growth and yield (Young et al., 1998; Goldstein et al., 1999; Fasim et al., 2002).

Tolerance to different temperature, pH and high salt concentration stress may be

important factor in the establishment, multiplication and spread of microbes in soil (Zahran,

1999). The information regarding the stress-tolerant agriculturally important strains is limited

to some microorganisms, including Rhizobium strains tolerant to high salt, pH, desiccation

and temperature (Kulkarni and Nautiyal, 2000; Rehman and Nautiyal, 2002; Arun and

Sridhar, 2005; Mnasri et al., 2007), Bacillus sp. tolerant to low pH (Pal, 1998), Pseudomonas
2
pseudoalcaligenes and P. alcaligenes tolerant to salinity (Rangarajan et al., 2002), Pantoea

dispersa 1A tolerant to low temperature (Selvakumar et al., 2008), Xanthomonas campestris

RMLU-26 and Pantoea agglomerans R-42 tolerant to high salinity, pH, and temperature (Son

et al., 2006; Sharan et al., 2008), Eupenicillium parvum tolerant to high desiccation, salinity,

acidity, aluminum and iron (Vyas et al., 2007) and Rhodotorula sp. tolerant to temperature,

salinity and pH (Mundra et al., 2011).

The insoluble phosphate solubilizing activity of PSBs is severely affected by

environmental factors, especially under stress conditions (Leyval and Barthelin, 1989). Little

information is available regarding isolation and characterization of temperature-, pH- and

salt- tolerating microbes, which could enhance production of food and forage in high altitude

regions where temperature is usually low. Phosphate-solubilizing fluorescent Pseudomonads,

plant growth promoting Acinetobacter rhizosphaerae strain BIHB 723 and Rhodotorula sp.

have been isolated from the rhizosphere of Seabuckthorn (Hippophae rhamnoides L.)

growing in Indian trans-Himalayas (Gulati et al., 2008, 2009; Mundra et al., 2011). However,

presence of plant growth promoting Enterobacter sp. with multiple plant growth promotion

traits (PGPTs) is largely unknown. Therefore, in the present work, we report isolation of

stress tolerating Enterobacter ludwigii PS1, which possess multiple PGPTs like phosphate

solubilization, Indole acetic acid (IAA), siderophore production and Hydrogen cyanide

(HCN) production. Plant growth promotion potential of the isolate was established in open

and shade net house conditions.

2. Materials and methods

2.1. Soil sampling

3
 Seabuckthorn (Hippophae rhamnoides L.) plants growing at experimental field of

Defence Institute of High Altitude Research in trans-Himalaya (latitude 34º08ʹ 0.2ʺ N,

longitude 77º34ʹ 0.3ʺ E, altitude 3340m amsl) were selected. Soil around the Seabuckthorn

plants was dug and the root and adhered soil were collected in plastic bags and stored at 4ºC

until analysis and isolation. Bulk soil associated near rhizosphere was used to study physico-

chemical properties of the soil. The pH and electrical conductivity of soil sample was 7.8 and

415 µS cm-1. Soil test using a soil testing kit (Lamotte Combination Soil Outfit 5010-01,

Maryland, USA) revealed presence of nitrate nitrogen 60 lb/a, phosphorus 75 lb/a, potassium

160 lb/a, calcium 2800 ppm, ferric iron 5 lb/a, nitrite nitrogen 1 ppm, sulfate 50 ppm, low

magnesium and manganese.

2.2. Isolation and characterization of phosphate solubilizing microbes

Two gram of rhizospheric soil was added in 250 ml flask with 100 ml sterile saline

(0.85% NaCl). The flask was agitated on a rotary shaker (INNOVA 4230, New Brunswick

Scientific Edison, USA) at 200 rpm for 30 min at 20ºC. Ten-fold dilutions were made in

phosphate buffer and serially diluted (10−1 to 10−9) and plated on Pikovaskaya agar medium

(pH 6.8–7.0), which contained (per liter): 0.5g yeast extract, 10g dextrose, 5g Ca3(PO4)2, 0.5g

(NH4)2SO4, 0.2g KCl, 0.1g MgSO4.7H2O, 0.0001g MnSO4.H2O, 0.0001g FeSO4.7H2O and

15g agar (Pikovskaya, 1948). This medium contained tricalcium phosphate as a sole source

of phosphorus for selective screening of microorganisms, which have the ability to release

inorganic phosphate from tricalcium phosphate. Uninoculated plates served as control. Plates

were incubated at 4oC and 32oC for 5 days and examined for presence of colonies developing

clear zone. PSBs could be easily identified because they develop clear zone around their

colonies (Gyaneshwar et al., 1999). Colonies with conspicuous clear zones around them were

picked up and further purified by repeating on Pikovaskaya agar media. Pure cultures were

maintained as a glycerol suspension (25% v/v) at -80ºC. Bacterial isolate PS1, which had a
4
marked phosphate solubilizing activity on Pikovaskaya agar as visualized by the clear zone

developed around the colony, was identified as Enterobacter ludwigii using biochemical and

16S rRNA sequencing approach by Microbial Type Culture Collection (MTCC), Institute of

Microbial Technology, India.

2.3. Insoluble inorganic phosphate solubilization in broth culture under stress conditions

Isolate PS1 was screened in Pikovaskaya liquid medium containing Ca3(PO4)2 at a

concentration of 5g L-1 as insoluble phosphate source. The phosphate solubilization potential

of PS1 under stress conditions was determined at 4-44ºC and pH 4-12 conditions. One

variable was changed at a time while maintaining the other factors as constant. pH 7.0 was

maintained for studying growth and phosphate solublization at different temperature, while

28ºC was maintained for studying growth and solubilization at different pH conditions. Effect

of individual salt viz KCl, NaCl and CaCl2 (1-5% concentration) on growth and phosphate

solubilization was determined at 28ºC and pH 7.0. The isolate was also tested for the capacity

to solubilize two other common types of insoluble phosphates viz. FePO4 and AlPO4 with the

concentration range of 0.4%-1.6%. The assay was done in two hundred and fifty milliliter-

Erlenmayer flask containing 50 ml of Pikovaskaya broth medium inoculated with 1 ml of

culture (109 CFU ml-1). Uninoculated flasks served as a control. The flasks were incubated at

a range of temperature and pH with shaking (200 rpm) on a rotary shaker. The culture was

harvested by centrifugation at 11,400g for 30 min. Supernatant was used to assess phosphate

released into the solution.

2.4. Analytical Method

The cell growth was determined by measuring absorbance at 600 nm recorded in a

micro-plate reader (SpectroMax M2 e, Molecular Devices, Sunnyvale, CA, United States).

5
Samples from culture grown in medium with insoluble phosphate were diluted with 1N HCl

(1:1 v/v) to dissolve the residual insoluble phosphate and measured against a blank

identically treated (Rodriguez et al., 2000). Solubilized phosphate content of the culture

filtrate was determined by the molybdenum blue method (Murphy and Riley, 1962). Change

in pH of the culture broth was recorded using a pH meter (SensION+ PH3, HACH,

Barcelona) equipped with a glass electrode in supernatant after centrifugation. IAA

production was studied according to Patten and Glick (2002) with some modification, where

DF salt minimal media was amembed with filter sterilized 50 μg/ml L-Tryptophan and

appearance of pink auxin complex on addition of Salkosky reagent was measured at 535 nm

and result expressed in mg L-1. Siderophore production was estimated in liquid medium by

the chrome azurol-S assay in iron free media broth at pH 7.0 (Schwyn and Neilands, 1987)

and HCN production was determined by the qualitative method of Bakker and Schipper

(1987) in King’s B broth medium (pH 7.0).

2.5. Plant growth promotion bioassay on tomato

2.5.1. Paper towel method

Plant growth promotion ability of the isolate PS1 was determined independently by

paper towel method. Tomato (var. Tolstoi) seeds were surface sterilised with 1% sodium

hypochlorite and washed 5 times with sterile distilled water. Seeds were coated with peat

based inoclum (108-109 CFU ml-1) using 1% carboxylmethylcellulose (CMC) as adhesive;

dried in air and the cell count was 108-109 CFU per seed as determined after reisolation from

bacterized seed. After bacterisation, 20 seeds were placed in each germination paper and

incubate in plant growth chamber (Adaptis A1000, Conviron, Canada) at 25°C and 60%

relative humidity. Seeds treated only with sterilized peat served as control. Five replication

6
for each treatment were maintained and repeated thrice. Shoot and root length of 5 seedlings

from each treatments were recorded after 10 days.

2.5.2. Pot culture assay under open and shade net condition

Pot culture assay for plant growth promotion ability of the isolate PS1 was determined

in non-sterile soil mixed with farm yard manure (10:1) in a completely randomized design in

open and green shade net conditions. A preplanting soil test revealed nitrate nitrogen 150

lb/a, phosphorus 75 lb/a, potassium 220 lb/a, ferric iron 5 lb/a, calcium 2800 ppm, nitrite

nitrogen 1 ppm and sulfate 50 ppm. Soil pH and electrical conductivity was 7.5 and 419 μS

cm-1. Urea (0.2g/pot) was applied in two split doses at vegetative and reproductive growth

stage. The mean maximum temperature during the crop growing period was 21.0±2.6ºC

while the mean minimum temperature was 8.3±2.0ºC. Root of 30 days old tomato (var.

Tolstoi) seedlings raised in a passive greenhouse without microbial treatment were washed

with sterile distilled water and soaked in peat based bacterial suspension at the concentration

of 109 cells ml-1 about 30 min prior to plantation. Number of leaf, number of flower bud and

fruit, and stem diameter were recorded at 30, 60 and 90 days after transplant (DAT). At the

end of the experimental period (90 DAT), plants were uprooted, washed under running water,

and root/shoot biomass and fruit yield were determined.

2.6. Statistical analysis

All statistical analysis was performed using SPSS for Windows 17.0 version. One-

way ANOVA was performed with the help of 2-sided Tukey’s HSD at P ≤ 0.05. Independent

sample T-test was performed with 95% confidence level.

3. Results

3.1. Isolation of phosphate solubilizing rhizobacteria

7
 A total of 152 isolates of phosphate solubilizing microbes were screened for in vitro

phosphate solubilizing activity at 4°C and 32ºC. Bacterial isolate PS1, which had a marked

phosphate solubilizing activity (solubilization index 2.9±0.1 mm) on Pikovaskaya agar as

visualized by clear zone developed around the colony was selected and later checked for

temperature, salt and pH stress tolerance. PS1 was a Gram negative bacterium. The isolate

showed negative for casein hydrolysis, oxidase and urease tests, and positive for catalase and

citrate utilization tests. PS1 was identified as Enterobacter ludwigii using biochemical and

16S rRNA sequencing approach.

3.2. Effect of different stress conditions on phosphate solubilization

Effect of incubation temperatures on Ca3(PO4)2 solubilization by PS1, concomitant

microbial growth and media pH are given in Table 1. Different incubation temperatures had

significant effect on phosphate solubilization, growth of culture and resultant media pH. The

highest growth and phosphate solubilizing activity was obtained at 28°C at which soluble

phosphate concentrations was 327 mg L-1 and maximum drop in pH was observed.

Effect of initial pH on phosphate solubilization and growth of PS1 are shown in Table

2. Initial pH of the medium had significant effect on phosphate solubilization and microbial

growth. Isolate PS1 was able to solubilize Ca3(PO4)2 at pH range of 4 to 12 and the phosphate

solubilization ranged from 118.4 to 363 mg L-1. Highest soluble phosphate and microbial

growth was observed at initial pH 7.

Effects of different salts with varying concentrations from 1-5% on phosphate

solubilization are presented in Table 3. Results showed that amount of soluble phosphate

production was less affected by presence of KCl as compared to NaCl and CaCl2 in varying

concentrations tested. The growth and amount of phosphate solubilized decreased

significantly with increased concentration of KCl, NaCl and KCl. Therefore, the isolate is not
8
very resistant to high salt concentration. The isolate solubilized Ca3(PO4) to a greater extent

than AlPO4 and FePO4 (Table 4).

3.3. Plant growth promoting traits

E. ludwigii PS1 was tested for PGPTs at 28°C at different time intervals upto 5 days.

IAA, siderophore and HCN production are presented in Table 5. IAA production was

maximum (21.4±0.5 mg L-1) at 60 h and then showed a declining trend. Siderophore

production ranged from 24 to 79.2% at different time interval and maximum value was

recorded at 60 h. PS1 also showed HCN production with increasing trend upto 48 h followed

by a declining trend.

3.4. Plant growth promotion bioassay on tomato

PS1 showed plant growth promotion activity as determined by paper towel method.

The mean shoot and root length of tomato seedling germinated from bacterized seed after 10

days was 4.5±0.7 cm and 7.8±1.2 cm, as compare to 3.6±0.4 cm and 5.7±0.8 cm in case of

untreated seed. Therefore, increase in shoot and root length due to PS1 treatment was 25%

and 37% over control.

Results of pot culture assay for plant growth promotion ability of the isolate PS1 is

presented in Table 6. Significantly higher vegetative growth (at 30, 60 and 90 DAT) and fruit

yield (at 90 DAT) was observed in PS1 treated seedlings in open field condition. However,

under green shade net condition, enhanced vegetative growth in PS1 treated plants was more

conspicuous at 60 and 90 DAT. The mean shoot and root biomass of PS1 treated tomato

plants in shade net after 90 days was 144±22 g and 9±5 g, as compare to 128±26 g and

4.2±0.3 g in case of uninoculated seedlings. Therefore, increase in shoot and root biomass

9
due to PS1 treatment was 12.5% and 114% over control. Increase in fruit yield due to PS1

treatment was 15.2 and 27.0% in open and shade net conditions.

4. Discussion

The effectiveness of plant growth promoting microbes depends largely on

environmental conditions. It is suggested that the antistress effect of diazotrophs on the plants

is an important mechanism of their interaction and mutual resistance to unfavorable stressed

conditions (Belimov et al., 1994). The bacterial isolate PS1 was able to grow and solubilize

inorganic phosphates at wide temperatures ranging from 4 to 44°C. The survival and

inorganic phosphate solubilizing activity of the isolate under wide range of temperature is of

significance in area experiencing low temperature such as the trans-Himalayan region. Even

though the growth and inorganic phosphate solubilizing activity of PS1 was highest at pH 7,

the isolate was able to grow and produce soluble phosphate at wide initial media pH ranging

from of 4 to 12. Similar results was observed by Son et al. (2006) in Pantoea agglomerans R-

42 and Mundra et al. (2011) in Rhodotorula sp. PS4, which are also capable of solubilizing

insoluble inorganic phosphate at a wide range of temperature, pH and varying salt

concentrations. However, changing the pH to extreme values significantly affected growth

and phosphate solubilizing activities. Soil of trans-Himalayan region is alkaline and villages

adjoining rivers are salt affected. Therefore, PS1 with characteristics to grow at wide pH

range and in presence of salt seems promising for the region.

Although PS1 was isolated from low temperature environment, the result showed that

its growth and activity was highest at 20ºC. Similarly, even though the bacterium was

isolated from alkaline soil (pH 7.8), its growth and phosphate solubilizing activity was

10
highest at neutral pH. The results suggested that optimal conditions for microbial growth and

activity are not necessarily that of its inhabiting environmental conditions.

Inorganic phosphate solubilizing activity of PS1 was recorded highest (334 mg L-1) at

28°C. In comparison the values are 334 mg L-1 in case of Acinetobacter strain at 26°C

(Chaiharn and Lumyong, 2009), 184 mg L-1 at 28°C for mutant Pseudomonas flurorescens

(Katiyar and Goel, 2003), 900 mg L-1 by Pantoea agglomerans at 30°C (Son et al., 2006), 80

mg L-1 at 20°C by mutant Pseudomonas corrugata (Trivedi and Sa, 2008). Therefore, the

phosphate solubilising activity of PS1 was moderate at optimum temperature as compared to

other reported species. Similarly, the inorganic phosphate solubilizing activity of PS1 was

recorded highest (363 mg L-1) at pH 7. In comparison the values are 215 mg L-1 in case of

Pantonea aggrolmerans at pH 7.5 (Son et al., 2006) and 278 mg L-1 at pH 7 for Rhodotorula

PS4 (Mundra et al., 2011).

E. ludwigii PS1 showed solubilizing activity in presence of 1 to 5% of varying salt

(KCl, NaCl and CaCl2) concentration. However, a decrease in phosphate solubilization with

increasing salt concentration was observed, which is in agreement with earlier reports

(Sharan et al., 2008; Mundra et al., 2011). The amount of phosphate solubilized gradually

decrease with increased concentration of NaCl and KCl. On the contrary, there was a large

decrease in solubilization of Ca3(PO4)2 with increasing CaCl2 concentration. Son et al. (2006)

and Mundra et al. (2011) also observed similar phenomenon with increasing concentration of

CaCl2 on inorganic phosphate solubilizing activity by Pantoea agglomerans R-42 and

Rhodotorula PS4.

E. ludwigii PS1 showed solubilization of different inorganic phosphate sources. The

production of soluble phosphate from Ca3(PO4)2 was significantly higher compared to AlPO4

and FePO4. The result is in agreement with earlier observations in Pantoea agglomerans R-42

(Son et al., 2006) and Rhodotorula sp. PS4 (Mundra et al., 2011). Preference for Ca3(PO4)2
11
over AlPO4 and FePO4 has also been reported in Pseudomonads (Gulati et al., 2008), Pantoea

and Enterobacter (Sharon et al., 2016).

PGPR with multifaceted PGPTs are more beneficial to crop than biofertilizers with

only phosphate solubilizing ability (Hariprasad and Niranjana, 2009; Mehta et al., 2014).

Phytohormone production by plant associated bacteria is an important aspect of PGPTs. Role

of auxin in plant growth and development has been extensively reviewed (Zaharan, 1999).

Auxin production by Pseudomonas putida has been shown to associate with enhanced host

plant root system, improved mineral and water uptake by the root (Patten and Glick, 2002).

Microbially secreted siderophores interact with insoluble ferric (Fe3+) complexes and reduced

to biologically relevant ferrous (Fe2+) form (Dimkpa, 2016). HCN production is known to be

associated with antagonistic activity against pathogenic microorganisms. PS1 possessed

multiple PGPTs, like phosphate solubilization, IAA, siderophore and HCN production.

Seed bacterization and treatment of tomato seedlings with PGPR belonging to the

genus Bacillus (Mena-Violante et al., 2007; Hariprasad and Niranjana, 2009; Mehta et al.,

2014), Pseudomonas, Azotobacter, Serratia (Hariprasad and Niranjana, 2009) have been

reported to increase plant growth and yield. In this study, an increase in the plant growth by

seed bacterization and seedling treatment with E. ludwigii PS1 was demonstrated. The

increased growth and fruit yield may be attributed to the cumulative effect of phosphate

solubilization, IAA, siderophore and HCN production, which promote plant growth directly,

indirectly, or synergistically.

In conclusion, solubilization of inorganic phosphate has been previously reported for

Enterobacter (Hwangho et al., 2003; Walpola et al., 2013). However, the present

investigation on E. ludwigii PS1 is the first report on phosphate solubilizing activity of the

genus under varying temperature, pH and salt stressed conditions. The isolate possessed

multiple PGPTs, like phosphate solubilization, IAA, siderophore and HCN production. The
12
result of this study provides a strong basis for further development of this isolate as a

bioinoculants to attain the desired plant growth-promoting activity in horticultural crops in

high altitude regions. However, long term studies in actual field conditions are required.

Acknowledgements

The study was supported by Defence Research and Development Organisation (DRDO),

Ministry of Defence, Government of India. DD, PD and SA are grateful to DRDO for

providing Senior Research Fellowship. The authors also acknowledge the Microbial Type

Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India for

identification of the isolate.

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Table 1 Effect of different incubation temperatures on Ca3(PO4)2 solubilization by
Enterobacter ludwigii PS1 and concomitant microbial growth and final media pH

Temperature Period Soluble P Growth (A600) Final pH

(°C) (days)* (mg L-1)
4 8 167.6±4.4f 1.8±0.008f 4.1±0.03e
8 8 176.0±0.6g 1.8±0.003f 3.6±0.02c
12 7 197.8±0.9h 1.9±0.006g 3.9±0.02d
16 7 203.7±1.1i 1.9±0.015h 3.7±0.03c
20 6 314.5±0.9j 1.7±0.006e 3.5±0.05b
24 6 313.7±2.5j 2.3±0.025i 3.2±0.03b
28 5 327.1±2.2k 2.5±0.003j 3.0±0.02a
32 2 92.1±0.2e 1.7±0.013e 4.3±0.01e
36 2 70.9±0.2d 1.2±0.000d 4.5±0.03f
40 1 29.6±0.7c 0.9±0.004c 4.7±0.02g
42 1 17.0±0.1b 0.8±0.007b 4.9±0.03h
44 1 4.1±0.3a 0.01±0.006a 5.6±0.02i
Different superscripts in a column differ significantly (P<0.05)
* Incubation period (days) at which maximum phosphate solubilization was observed

Table 2 Effect of initial pH on Ca3(PO4)2 solubilization by E. ludwigii PS1 at 28°C

temperature on 5th day after incubation

Initial pH Soluble P (mg L-1) Growth (A600)

4.0 124.0±0.6a 1.7±0.03c
d
5.0 181.9±7.1 1.8±0.05d
6.0 335.0±1.7g 1.9±0.01d
h
7.0 363.3±0.7 2.0±0.03e
8.0 235.0±1.0f 1.8±0.01c
e
9.0 195.9±1.8 1.7±0.02c
10.0 149.1±2.3c 1.6±0.01b
b
11.0 140.0±1.9 1.5±0.03b
12.0 118.4±1.3a 0.2±0.01a
Different superscripts in a column differ significantly (P<0.05)

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 Table 3 Effect of different salts and their concentrations on Ca3(PO4)2 solubilization by E.
ludwigii PS1

Salt Conc. Growth (A600) Soluble P (mg L-1)

KCl NaCl CaCl2 KCl NaCl CaCl2
1% 1.32±0.001ze 0.19±0.005xd 0.22±0.003yd 355.6±6.12xd 324.5±1.82xe 317.2±4.04xc
2% 1.24±0.002zd 0.17±0.005yc 0.16±0.004xc 233.4±1.37yc 273.7±4.16zd 193.0±3.46xb
3% 1.19±0.004zc 0.15±0.002yb 0.14±0.002xb 221.4±2.83zb 247.3±3.21yc 16.3±1.00xa
4% 1.17±0.001zb 0.15±0.004yb 0.12±0.003xa 213.5±1.85yab 216.9±1.80yb 12.6±0.10xa
5% 1.16±0.006ya 0.12±0.004xa 0.12±0.003xa 205.2±0.90ya 202.0±2.60ya 12.8±1.32za
Growth and phosphate solubilization was 2.0±0.03 (A600) and 363.3±0.7 mg L-1, respectively
in absence of salt
a, b, c, d, e
different superscripts in a column differ significantly (P<0.05)
x, y, z
different subscripts in a row differ significantly (P<0.05)

Table 4 Effect of different insoluble phosphate and their concentration by E. ludwigii PS1 in
modified Pikovaskaya broth media containing various insoluble phosphate sources
Conc. Growth(A600) Soluble P (mg L-1)
AlPO4 FePO4 Ca3(PO4)2 AlPO4 FePO4 Ca3(PO4)2
0.4% 1.4±0.018yd 1.3±0.008xd 1.3±0.004xa 56.6±1.3xd 65.4±0.4yd 327.7±1.5za
0.8% 0.8±0.003yc 0.7±0.042xc 1.4±0.004zb 48.1±1.4xc 50.1±0.5xc 348.2±8.1yb
1.2% 0.6±0.006yb 0.5±0.015xb 1.6±0.015zc 36.6±6.4xb 48.6±0.5yb 439.5±2.9zc
1.6% 0.5±0.007ya 0.5±0.006xa 1.9±0.012xd 13.7±0.8xa 43.6±0.4ya 480.2±9.4zd
a, b, c, d
different superscripts in a column differ significantly (P<0.05)
x, y, z
different subscripts in a row differ significantly (P<0.05)

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Table 5 Plant growth promoting traits of E. ludwigii PS1 at 28°C

Time interval (h) Plant growth promoting traits

HCN (A625) IAA (mg L-1) Siderophore (%)
0 0.03±0.00a 5.05±0.19a 28.8±9.2a
b
12 0.06±0.00 12.6±1.2b 32.7±1.2a
c
24 0.09±0.00 14.4±0.7b 61.2±1.2bc
e
36 0.15±0.02 17.8±0.7cd 60.6±3.9bc
f
48 0.21±0.01 17.4±0.8cd 68.2±1.6c
d
60 0.13±0.01 21.4±0.5g 79.3±0.8d
b
72 0.07±0.00 19.2±0.4ef 68.7±1.2cd
a
84 0.02±0.00 19.0±0.2ef 51.2±1.0b
a
96 0.01±0.01 15.5±0.7bc 32.3±4.2a
a
108 0.09±0.06 6.5±2.2a 24.1±3.6a
Different superscripts in a column differ significantly (P<0.05)

Table 6 Effect of inoculation of E. ludwigii PS1 on growth and fruit yield of tomato (var.
Tolstoi)

DAT# Growth parameter Open field condition Shade net condition

PS1 Control PS1 Control
30 Stem dia. (mm) 5.8±1.4 ⃰ ⃰ 3.7±1.0 4.5±1.5 ⃰ 3.0±1.1
⃰ ⃰
No. of leaf 7.1±1.7 ⃰ ⃰ 4.9±1.2 6.1±0.9 5.2±1.3
60 Stem dia. (mm) 14.9±3.6 ⃰ 12.3±2.6 14.8±3.1 12.4±2.7
No. of leaf 52.0±5.3 ⃰ ⃰ 29.3±7.1 40.6±11.0 ⃰ 29.6±7.4
⃰
No. of bud 34.7±3.9 ⃰ ⃰ 21.8±9.8 36.2±13.8⃰ 22.5±10.0
⃰
90 Plant height (cm) 58.1±3.3 ⃰ 53.3±5.1 64.0±9.1⃰ 55.82±4.5
No. of fruit 30.2±3.7 ⃰ 26.9±3.8 29.8±5.7 ⃰ 24.45±5.1
Yield/plant (gm) 916.5±111.2 795.0±123.6 926.4±209.5 729.09±226.2
⃰ ⃰ ⃰
#
Days after transplanting
Values are the mean of ten replicates.
Values are significantly different between PS1 treatment and untreated within open field/ shade net conditions
by Independent T test; ⃰ significant at (P≤0.05), ⃰ ⃰ significant at (P≤0.01), ⃰ ⃰ ⃰ significant at (P≤0.001)

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 Highlights

 Temperature-, pH- and salt-tolerant Enterobacter ludwigii PS1was isolated

 PS1 exhibit plant growth promoting traits (IAA, siderophore and HCN production)
 Seed bacterization and seedling treatment was studied on tomato in trans-Himalaya
 Seed bacterization resulted in 25% increased in shoot and 37% in root length.
 PS1 treatment resulted in 15% more fruit yield in open and 27% in shade net condition

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