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Original article

Reduction in dose of chemical fertilizers and growth enhancement of sesame

(Sesamum indicum  L.) with application of rhizospheric competent

Pseudomonas aeruginosa  LES4

Sandeep Kumar a, Piyush Pandey b, D.K. Maheshwari a,*

a Department of Botany and Microbiology, Gurukul Kangri University, Haridwar (U.A.) 249404, Indiab S.B.S.P.G. Institute of Biomedical Sciences and Research, Balawala, Dehradun (U.A.) 248161, India

a r t i c l e i n f o

 Article history:

Received 9 September 2008

Received in revised form

4 April 2009

Accepted 7 April 2009

Available online 19 April 2009

Handling editor:  Kristina Lindstro m

Keywords:

PGPR 

Chemical fertilizer

Macrophomina phaseolina

Fusarium oxysporum  oilseed crop

Sesame

a b s t r a c t

Pseudomonas aeruginosa LES4, an isolate of tomato rhizosphere was found to be positive for several plant

growth-promoting attributes like production of indole acetic acid, HCN and siderophore, solubilization of 

inorganic phosphate along with urease, chitinase and  b-1-3-glucanase activity. In addition, it showed

strong antagonistic effect against Macrophomina phaseolina  and  Fusarium oxysporum. P. aeruginosa  LES4

caused halo cell formation and other morphological deformities in mycelia of   M. phaseolina   and

F. oxysporum. Root colonization was studied with  Tn5  induced streptomycin resistant transconjugants of 

spontaneous tetracycline-resistant LES4 (designated LES4tetraþstrepþ) after different durations. The strain

was significantly rhizospheric competent, as 17.4% increase in its population was recorded in sesame

rhizosphere. Seed bacterization with LES4 resulted in significant increase in vegetative growth param-

eters and yield of sesame over non-bacterized seeds. However, application of LES4 with half dose of 

fertilizers resulted in growth equivalent to full dose treatment, without compromising with the growth

and yield of sesame. Moreover, the oil yield increased by 33.3%, while protein yield increased by 47.5%

with treatment of half dose of fertilizer along with LES 4 bacterized seeds, as compared to full dose of 

fertilizers.  2009 Elsevier Masson SAS. All rights reserved.

1. Introduction

Sesame (Sesamum indicum L.) is an important oilseed crop, next

to soybean and groundnut but has low yield potential  [35]. India

rank first in the world in area (about 2.47 m ha annually, 40% of the

world) and production (0.74 m tones, 27%of theworld). In intensive

cropping system, supplementing soil nutrients by use of chemical

fertilizer is considered inevitable for obtaining optimum yield of 

crops, however, their utilization efficiency remains low, due to loss

by volatization, denitrification, leaching and conversion into

unavailable forms. Continuous use of chemical fertilizers subvertsthe soil ecology, disrupt environment, degrade soil fertility and

consequently shows harmful effects on human health   [1]   and

contaminates ground water [15]. Presence of chemicals in sesame

had been major impediment in the promotion of sesame export.

Export consignments of sesame are sometimes unsuitable in the

international market due to the presence of pesticide residue,

resulting in loss of revenues [6].

Role of plant growth-promoting rhizobacteria (PGPR) in plant

growth promotion and biological control of soil borne pathogens

has been intensively investigated [17,19]. Integration of PGPR with

traditional inorganic fertilizers in the field may prove to be effective

means to increase the solubility of insoluble phosphorous ions and

other minerals to plants with simultaneous reduction in diseases

incidence. They take systemic and simultaneous account of envi-

ronmental aspects, quality of the produce and profitability of 

agriculture   [22]. Alternatively, substitution of chemicals withbacterial fertilizers and biopesticides, especially blending of 

chemical fertilizers with chemical adaptive PGPR is a promising

approach to obtain sustainable fertility of the soil and plant growth

[39]. There are several PGPR currently commercialized whose

growth-promoting activity in crop plants have been demonstrated

in several ways including production of iron-sequestering side-

rophores and antimicrobial compounds that hinder colonization of 

hosts by phytopathogens  [40],   induction of host systemic disease

resistance, solubilization of precipitated mineral nutrients and for

production of plant growth hormones, thereby enhancing the

plants ability to take up nutrients from soil and increasing yield [8].

*  Corresponding author. Tel.:  þ91 1334 246 767 (O), 265 469 (R),  þ91 983 730

8897 (M); fax:  þ91 1334 246 767 (O).

E-mail addresses:  piyushgkp@rediffmail.com   (P. Pandey),   maheshwaridk@

gmail.com  (D.K. Maheshwari).

Contents lists available at ScienceDirect

European Journal of Soil Biology

j o u r n a l h o m e p a g e :   h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / e j s o b i

1164-5563/$ – see front matter    2009 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.ejsobi.2009.04.002

European Journal of Soil Biology 45 (2009) 334–340

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Hence, the present work was aimed to blend chemical fertilizers

with effective PGPR to obtain the optimum benefits. This strategy

was designed to allow reduction in the dose of fertilizers, along

with an approach to increase productivity of sesame without

associated ecological harm.

2. Materials and methods

 2.1. Microorganisms

A number of bacterial strains were isolated using standard

microbiological technique from the rhizosphere of tomato

(Lycopersicon esculantum   L.) grown in nutrient deficient soil in

wasteland. Healthy and young tomato seedlings were gently

uprooted at Dehradun (Alt. 640 m, Lat. 303004000N, Long.

775201200E, Rainfall 216 cm) in India. The roots were cut into 2 cm

long segments and vortexed in 25 ml sterilized distilled water for

few minutes. Suitable dilution was plated by serial dilution plate

technique on nutrient agar medium (NAM). The bacterial colonies

were isolated and maintained on NAM slants at 4   C. The isolates

were characterized for direct and indirect plant growth-promoting

(PGP) activities including solubilization of inorganic phosphate, IAA

production, and HCN production along with antagonism against

two fungal pathogens. Seven isolates were selected and identified

on the basis of morphological, physiological and a biochemical

characteristic according to Bergey’s manual of determinative

bacteriology [14], and compared against  Pseudomonas aeruginosa

MTCC-1934,   Pseudomonas putida   MTCC-102 and   Pseudomonas

 fluorescens  MTCC-103 as standard strains. All isolates were main-

tained on tryptic soy agar medium (TSM) at 4   C for further use.

Macrophomina phaseolina   and   Fusarium oxysporum   were

procured from culture collection laboratory, Department of Botany

and Microbiology, Gurukul Kangri University, Haridwar, India. Pure

culture of fungal colonies was maintained on potato dextrose agar

(PDA) slants at 4   C for further use.

 2.2. Screening for plant growth-promoting attributes

Phosphate solubilization   [38], IAA production   [12], HCN

production   [23], siderophore production   [32], and chitinase and

b-1-3-glucanase activities   [7]   were determined as per standard

protocols. Antagonistic activity of isolates against phytopathogens

was determined according to Skidmore and Dickinson [36].

 2.3. In vitro antagonism

Antagonistic properties of bacterial strains were tested against

two fungal pathogens   M. phaseolina   and   F. oxysporum   causing

charcoal rot and wilt diseases on sesame using a dual culture

technique as described previously   [19]. Agar blocks (5 days old,

5 mm dia.) containing 5 days old mycelia were placed in four

corners of a Petri plate, and inoculated with loopful culture (24 h

old) of bacterial strain, spotted 2 cm apart from the fungus. These

plates were incubated at 28   C. Plates inoculated with only fungal

agar blocks served as control. Growth inhibition was calculated by

measuring the distance between the edge of bacterial and fungal

colonies, and percent inhibition was calculated by following

formula:

Growthinhibition   ¼ ½ðC   T Þ=C 100

where C  ¼  radial growth of fungus in control, T  ¼  radial growth of 

fungus in dual culture. Fungal hypha surrounding zone of inhibi-

tion, and from control plates were observed under the microscope

by standard procedure.

 2.4. Seed bacterization

The certified seeds of sesame (S. indicum   L. cv. ST-1) were

procured from Center for Biotechnology, Jamia Hamdard Univer-

sity, Hamdard Nagar, New Delhi, India. Seed bacterization was done

by the method of Weller and Cook [41]. Seeds were surface steril-

ized with 95% alcohol for 30 s, followed by 0.1% (w/v) HgCl2   for

1–2 min and then washed with sterile distilled water for 5–6 times.

These germinated seeds were dried under sterile air stream. 24 h

old culture of LES4 was centrifuged at 7000 rpm for 15 min at 4   C.

The pellets were retained and re-suspended in sterile distilled

water to obtain a population density of 108 cfu ml1 and the cell

suspension was mixed with 1% carboxymethyl cellulose (CMC)

solution in ratio of 1:0.5. Slurry thus obtained, was coated on the

surface of germinated seeds. The seeds coated with 1% CMC slurry

without LES4 served as control. The bacterized seeds were dipped

in known volume of sterile water and cfu were counted on TSM for

standardizing the inoculum. The population of LES4 was recorded

by dilution plate technique as 108 cfu seedling1.

 2.5. Root colonization

Root colonization of  P. aeruginosa  LES4 was studied by quanti-tative analysis of population dynamics in the rhizosphereof sesame

using antibiotic resistant marker strain. Tetracycline-resistant

strain of LES4 was isolated on TSM, containing 100 mg l1 of 

tetracycline (P. aeruginosa LES4tetraþ). LES4tetraþ was engineered for

streptomycin resistance (100 mg l1) (designated LES4tetraþstrepþ)

with Tn5  delivery suicide vector pGS9 [33] in donor Escherichia coli

strain WA803 (pGS9) as described   [18]. Sesame plants emerged

with bacterized seeds was sampled after 30, 60, 90 and 120 DAS,

and bacterial population on the roots were measured. Root

adhering soil particles were carefully removed and root was cut

into 1 cm long segments, which was vortexed in known volume of 

sterile water to release root-associated bacteria. Suitable dilutions

of the suspension were plated on TSM containing tetracycline and

streptomycin (100 mg l1 each) to enumerate the bacterialpopulation.

 2.6. Field trial

Field trials of sesame were carried in sandy loam soil (80.3%

sand, 6.5% silt, 7.7 clay, total organic C 0.0923%, pH 6.8 having 35%

water holding capacity). The recommended dose of chemical

fertilizer for sesame crop was 120 kg ha1 nitrogen, in three split

doses of urea, and 30 kg ha1 phosphate in the form of Dia-

mmonium phosphate (DAP) and 30 kg ha1 potassium in the form

of Murate of potash (MoP) in single doses. The combination of NPK

was N40þ40þ40P30K30. Seeds bacterized with P. aeruginosa LES4 and

non-bacterized seeds were sown on randomized block design

(RBD) in 7 sets of treatments with three replicates of each treat-ment, (i) seeds coated with   P. aeruginosa   LES4, (ii)   P. aeruginosa

LES4  þ  half dose of chemical fertilizers, (iii) half dose of chemical

fertilizers, (iv) full dose of chemical fertilizers, (v) seeds coated with

CMC slurry only without any fertilizer and bacteria (control). The

crop was irrigated three times (including one pre-sowing irriga-

tion) at different critical stages, i.e. at flowering/capsule formation

and seed filling. Seed germination rate (%) was noted on 15 days

after sowing (DAS). Vegetative growth parameters including

biomass accumulation, root and shoot lengths, leaf area were

recorded at 30, 60, 90 and yield attributes were recorded after 120

DAS on harvesting the crop. The experiment was conducted for two

consecutive years and data are presented as mean. The data were

analyzed statistically by using analysis of variance (ANOVA) to find

out significance at 1% and 5% levels  [9].

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 2.7. Estimation of oil and soluble protein content in sesame seeds

Oil content is defined as the whole of the substances extractable

by   n-hexane under specified conditions. The rapid gravimetric

determination of oil content was done by cold percolation as

described by Kartha and Sethi [16]. Theoil content was expressed in

percentage and yield in kg ha1. Soluble protein content of seeds

collected from field trials was estimated according to the method

given by Bradford [4], after precipitation with trichloroacetic acid

using bovine serum albumin (BSA) as standard.

3. Results

 3.1. Isolation of microorganisms

Among the seven fluorescent pseudomonads, LES4 was selected

for further studies on the basis of plant growth-promoting

attributes. The isolate LES4, identified as  P. aeruginosa  was Gram-

negative, non-spore forming rod, aerobic, motile and formed small,

round colonies with smooth margins on NAM plates after 24 h of 

incubation at 28 1 C. The strainwas positive forcatalase,oxidase,

urease and indole production and was negative for starch hydro-

lysis and mannose utilization (Table 3).

 3.2. Plant growth-promoting attributes

LES4 and LES7 were found to have excellent phosphate solubi-

lization activity. LES4 formed relatively large zone of clearing on

Pikovskaya’s medium. Most of the pseudomonads produced IAA

except LES2 and LES5. All pseudomonads produced siderophore

while LES3 and LES4 produced large halos around their colonies on

CAS agar medium. HCN production was evidenced by change in

colour of filter paper from yellow to reddish brown after 3 days of 

incubation in all the pseudomonads except LES2 and LES6. LES4 and

LES7 were positive for both chitinase and  b-1-3-glucanase activi-

ties. LES4 was more effective, showed excellent zone of inhibition

than that of LES7 in dual culture assay against  M. phaseolina and  F.oxysporum (Table 4).

 3.3. In vitro antifungal activities

P. aeruginosa LES4 strongly inhibited the growth of M. phaseolina

and F. oxysporum and growth inhibition was maximum after 5 days

of incubation. Increase in the incubation time corresponded with

the increase the zone of inhibition up to 5 days, thereafter the

growth of mycelia toward the interaction zone ceased, and the

mycelia gradually lost vigour.   P. aeruginosa   LES4 caused 85%

inhibition of  M. phaseolina and 67% of  F. oxysporum in their growth

respectively in comparison to control (Table 1). P. aeruginosa LES4

caused halo cell formation, mycelial deformities and hyphal tip

degradation of  M. phaseolina and  F. oxysporum (Figs. 1 and 2). Also,

sclerotial and conidial development was ceased to be arrested.

 3.4. Root colonization

The antibiotic resistant marker strain LES4tetraþstrepþ showed

positive root colonization of sesame. 17.44% increase in the pop-

ulation of LES4tetraþstrepþ in sesame rhizosphere was observed

between 30 and 120 DAS (Table 2). The final population of  P. aer-

uginosa LES4tetraþstrepþ was highest in treatment receiving half dose

of fertilizers, as compared to treatment of full dose of fertilizer.

Population of standard strain   P. aeruginosa   MTCC-1934 also

increased tenfold when applied with half dose of fertilizer between

30 and 120 DAS.

 3.5. Field trial experiment 

Seeds bacterized with  P. aeruginosa LES4 showed enhancement

in seed germination and seedling emergence, in all the treatments

as compared to control. 98% seed germination was recorded in

bacterized seeds with low dose of chemical fertilizers, which was

higher as compared to full dose of fertilizers (90.1%). All vegetative

plant growth parameters were also increased progressively after30, 60 and 90 DAS as compared to control. Most pronounced

positive interactive effect in all the vegetative parameters and yield

components was observed in treatment of bacterized seeds with

half dose of chemical fertilizers (i.e. N20þ20þ20P15þ15K15) where

30.6% increase in number of capsules per plant and 22.9% increase

in shoot fresh weight were obtained, as compared to half dose of 

fertilizer treatment (Table 5). Data of growth enhancement of 

sesame, obtained with application of full dose of fertilizer were

almost similar to treatment receiving bacterized seeds with half 

dose of fertilizers (Table 6).

 3.6. Oil and soluble protein content in sesame seeds

The percent oil content of seeds was almost similar in alltreatments; however, a small increase of about 10% oil content was

recorded with treatment of half dose of fertilizer plus LES4, as

compared to control (Table 6). However, the oil yield increase by

33.3% with treatment of half dose of fertilizer with LES bacterized

seeds, as well as full dose of fertilizers, compared to treatment of 

reduced dose of fertilizer only. The protein yield increased by 47.5%

in treatments where LES4 was applied in combination with half 

dose of chemical fertilizers as compared to full recommended dose

of chemical fertilizers.

4. Discussion

P. aeruginosa LES4 produced IAA, siderophore, HCN and solubi-

lized inorganic phosphate and showed strong antagonism againsttwo dreaded fungal pathogens, M. phaseolina and F. oxysporum. It is

well known that the beneficial effects of plant growth-promoting

rhizobacteria are attributed to the production of diverse metabo-

lites including siderophores, hydrocyanic acid (HCN), IAA and other

associated activities such as good phosphate solubilization and

competition in soil and root colonization [8]. In fact, similar to LES4,

there have been several reports of pseudomonads producing IAA

[2,3,10–12], HCN [3,10–12], siderophore [41,42], and solubilization

of inorganic phosphates [20,26,5]. Presence of wide array of attri-

butes made LES4 a suitable candidate as PGPR. LES4 also released

chitinase and b-1,3-glucanase in the medium, and inhibited growth

of  M. phaseolina  and  F. oxysporum. It is well known that chitin is

main component of cell wall of  M. phaseolina. Similarly, cell wall of 

the   F. oxysporum   composed of mostly of 47% chitin with 14%

 Table 1

Antagonistic effect of  P. aeruginosa   strain LES4 on dual culture plate at 28    1   C

against M. phaseolina and  F. oxysporum.

Fungal

pathogen

Incubation

(h)

Growth in dual

culture (mm)

Growth in

control (mm)

Growth

inhibition (%)

M. phaseolina   48 21.0   0.08 41.2   0.05 49.0

72 22.3   0.04 51.4   0.04 56.6

96 22.7   0.06 60.7   0.08 62.0

120 22.9   0.07 81.9   0.08 72.0

F. oxysporum   48 20.0   0.08 40.0   0.04 50.0

72 21.1   0.09 42.5   0.08 50.3

96 21.6   0.05 51.7   0.11 58.2

120 21.8   0.09 71.8   0.12 69.6

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laminarin. Therefore, their role in lysis of the cell wall and hence

biocontrol cannot be ruled out. This fact was supported by the

morphological changes in hypha, observed after interaction of 

M. phaseolina and  F. oxysporum with LES4.

An integrated approach wherein microbial inoculants applied

along with reduced level of fertilizers so as to obtain better

growth and yield is essentially required   [24,37]. The main

objective of this work was to access the efficiency of PGPR to

reduce the use of chemical fertilizers, without compromising with

the growth and yield of sesame. The results of this study suggest

that P. aeruginosa LES4 in combination with half dose of fertilizers

significantly influenced the yield viz. number of capsule per plant,

number of seeds per capsule as well as seed weight and oil yield

of  S. indicum   L. Earlier also, a strong positive correlation between

yield component and seed yield have been reported by several

investigators in other crops   [30]. Higher concentrations of 

chemical fertilizers have been reported as lethal for bacterial

growth, but found to stimulate its growth at low concentrations

[19]. This may be one of the reasons for growth enhancement of 

host plant by PGPR in presence of lower concentration of fertilizer

in soil.

The amount of protein concentration increased in all the

treatments of biofertilizers in combination with reduced dose of 

fertilizers in comparison to control. Application of  Azospirillum sp.

reduced the nitrogen requirement by 50% in  S .  indicum  [29]. This

was in accordance to earlier reports with crops like soybean where

significant increase in dry matter, seed protein, oil and yield of 

soybean on inoculation with rhizobia and phosphate solubilizing

Fig. 1.  Post-interaction morphological changes in  F. oxysporum  due to P. aeruginosa  LES4, (A) hyphal tip degradation, (B) halo cell formation, (C) cytoplasm coagulation, (D) hyphal

perforation, (E) digestion of fungal cell wall as compared to (F) control (bar   ¼ 10  mm).

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bacteria in combination with chemical fertilizers was reported

[21,43]. Contrary to this, Poul and Savithri  [28]   reported that thegrowth and yield of sesame with application of half dose of urea in

combination with biofertilizers was less as compared to dose of 

inorganic fertilizer. However, in present study,  P. aeruginosa   LES4

resulted equivalent yield as well as growth when applied with half 

dose of fertilizers, as compared to application of full dose of 

fertilizers, which may be because the difference in PGPR used.

Strong root colonization ability of LES4 lies in it being the

successful efficient colonizer in the rhizosphere of sesame. The use of 

genetic markers such as intrinsic levels of resistance to various anti-

bioticsis oneof the simple and rapid methods for strain identification

in rhizosphere [25,27], and hence utilized in this study. Combined

resistance against two broad range antibiotics in introduced marker

strain LES4tetraþstrepþ allowed no background population of other

strains, which was further assured by checking the phenotypic and

physiological characteristics. The LES4tetraþstrepþ showed efficient

root colonization after different time intervals of observation (i.e. 30,

60, and 90 days) and enhanced plant growth and growth yield. The

population density increased up to 60 days of inoculation, which

became almost stable thereafter. It has been suggested that bacteria

that attain CFU of aboutz

10

3

per gram orhigher on rootmasscan beconsidered as good colonizers [31], and we obtained population of 

LES4tetraþstrepþ of   z107. The continued presence of  P. aeruginosa

LES4tetraþstrepþ for 90 days in soil showed that it had reached

homeostasis after undergoing exchange with indigenous microflora

andis not affected by the active andpassive processes restricting soil

community. These results were similar to root colonization of 

P. aeruginosa NBRI2650 [25], however they studied the colonization

up to60 days only.Earlier, P. aeruginosa hasbeen reported as potential

biocontrol agentand simultaneously enhancedplant growth and root

colonization of various vegetables and cereals without showing any

deleterious effects of plants [13,27,34].

On the basis of present findings, it may be concluded that

P. aeruginosa LES4 contains large number of plant growth-promoting

attributes, and its application contributed in enhancement of 

sesame growth leading to better yield. In addition, its ability to

reduce the chemical requirement for obtaining optimum yield of 

sesame appears to be of great ecological and economic importance,

with possibilities of an efficient bioinoculant.

Fig. 2.  Post-interaction morphological changes in  M. phaseolina  due to P. aeruginosa  LES4, (A) sclerotial cease in  M. phaseolina, (B) halo cell formation, (C) mycelial deformities in

M. phaseolina  as compare to (D) control (bar   ¼ 10  mm).

 Table 2

Root colonization of  S. indicum L. by P. aeruginosa  LES4 and standard strain MTCC-1934.

Bacterial population (log10 cfu)

Treatments 30 DAS 60 DAS 90DAS 120 DAS

P. aeruginosa LES4 6.25   0.12 6.35   0.14 7.10   0.13 7.34   0.12

P. aeruginosa MTCC-1934 5.27   0.09 5.34   0.11 6.16   0.14 6.72   0.18

P. aeruginosa LES4 þ  half dose fertilizers 6.75   0.12 6.86   0.11 7.33   0.17 7.53   0.19

P. aeruginosa   MTCC-1934 þ  half dose of fertilizers 5.88   0.12 5.89   0.14 6.87   0.19 6.90   0.16

Data are mean   SD of two years.

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 Table 5

Interactive effect of Biofertilizers and Integrated Nutrient Management Protocol on the seed germination and growth and yield component of  S. indicum L. cv. ST-1 after 120 DAS.

Treatment Germination

(%)

Root Shoot

Length

(cm)

Fresh

weight (g)

Dry

weight (g)

Length

(cm)

Fresh

weight (g)

Dry

weight (g)

Leaf 

area (cm2)

Capsules/

plant

T-1   P. aeruginosa LES4 92.0 16.1 38.0 21.1 165 204 107.5 768 98.2

T-2   P. aeruginosa LES4 þ  half 

dose fertilizers

98.0 18.7 45.6 27.5 206 247 128.8 875 128

T-3 Half dose of fertilizers 89.3 14.0 38.6 20.2 163 201 97.5 759 98.7

T-4 Full dose of chemical

fertilizers

90.1 17.8 42.8 26.0 200 242 114.5 853 123

T-5 Control 76.2 10.2 27.6 14.2 130 146 74.1 569 48.2

SEM   0.732 0.437 0.756 0.247 0.563 1.168 0.611 0.939 1.185

CD at 1% 3.161 1.886 3.264 1.068 2.432 5.044 2.640 4.054 5.119

CD at 5% 2.255 1.346 2.329 0.762 1.735 3.599 1.883 2.893 3.653

Values are mean of 10 randomly selected plants from each set; ns, not significant; *significant at 5% LSD; **significant at 1% level of LSD as compared to control; full dose of 

chemical fertilizers N40þ40þ40, P30, K 30; half doses of chemical fertilizers N20þ20þ20, P15, K15.

 Table 4

Plant growth-promoting and antifungal properties of isolated fluorescent Pseudomonas species.

Strains IAAA Phosphate solubilizationB SiderophoreC HCND ChitinaseE b-1,3-glucanaseF Antagonism against

M. phaseolina

Antagonism against

F. oxysporum

LES1   þ þ þ þ

LES2   þ þ þ LES3   þ þþ þ þ

LES4   þ þþþ þþþ þþ þ þ þþþ þþ

LES5   þ þ þ þ

LES6   þ þ þþ

LES7   þ þþ þ þ þ þ þþ þ

MTCC-1934   þ þ

MTCC-103   þ þ þ þ

MTCC-102   þ þ

 Abbreviations: A, IAA negative;   þ, IAA positive; B, phosphate solubilization negative;   þ, phosphate solubilization positive; C, absence of halo formation;   þ, small

halos < 0.5 cm wide surrounding colonies; þþ, medium halos > 0.5 cm wide surrounding colonies; þþþ, large halos > 1.0 cm wide surrounding colonies; D, HCN negative;

þ, HCN positive; E, chitinase negative; þ, chitinase positive small halos < 0.5 cm wide surrounding colonies; þþ, medium halos > 0.5 cm wide surrounding colonies; F, b-

1,3-glucanase negative, þ, b-1,3-glucanase positive;G, no inhibition; þ, 0–25% inhibition; þþ, 26–50% inhibition; þþþ, 60–85% inhibition against M. phaseolina; P. aeruginosa

MTCC-1934;  P. fluorescens  MTCC-103; P. putida   MTCC-102.

 Table 3

Morphological, physiological and biochemical characters of fluorescent Pseudomonas strains with standard strains.

Characteristics LES1 LES2 LES3 LES4 LES5 LES6 LES7 MTCC-102 MTCC-103 MTCC-1934

Gram reaction  

Growth at

4   C   þ þ þ

41   2   C   þ þ þ þ þ þ þ

Generation time (h) 1.2 1.3 1.4 1.2 1.4 1.3 1.2 1.3 1.3 1.2

Fluorescent diffusible pigment   þ (bg)   þ (y)   þ (bg)   þ (bg)   þ (y)   þ (bg)   þ (bg)   þ (y)   þ (bg)Endospore  

Capsule  

Non-fluorescent non-diffusible pigment  

PHB accumulation  

Motility   þ þ þ þ þ þ þ þ þ þ

Urease   þ þ þ þ þ þ þ þ þ þ

Oxidase   þ þ þ þ þ þ þ þ þ þ

Catalase   þ þ þ þ þ þ þ þ þ þ

VP test  

MR test  

H2S production  

Gelatin hydrolysis   þ þ þ þ þ þ þ þ þ þ

Starch hydrolysis  

Arginine hydrolysis   þ þ þ þ þ þ þ þ þ þ

Citrate utilization   þ þ þ þ þ þ þ þ þ þ

Utilization of carbon as

Glucose   þ þ þ þ þ þ þ þ þ þ

Maltose   þ þ þ þ þ þ þ þ

Mannose  

Trehalose   þ þ

Mannitol   þ þ þ þ þ þ þ þ þ þ

Ribose   þ þ þ þ þ þ þ þ þ þ

Meso-Inositol   þ

 Abbreviations: þ, positive; , negative; y, yellow; bg, blue green; VP, Voges Proskauer, MR, methyl red; H 2S, hydrogen sulfide; PHB, poly-b-hydroxylbutyrate;  P. putida MTCC-

102; P. fluorescens MTCC-103 and P. aeruginosa  MTCC-1934.

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 Acknowledgments

The authors wish to thank the TMOP & M, CSIR, New Delhi for

financial assistance.

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 Table 6

Interactive effect of Biofertilizers and Integrated Nutrient Management Protocol on the yield and yield component of   S. indicum L. after 120 DAS.

Treatment Average seeds yield (kg/ha) 1000 seeds weight (g) Oil content

(%)

Oil yield

(kg/ha)

Protein

content (%)

Protein yield

(kg/ha)2004 2005 Mean 2004 2005 Mean

T-1   P. aeruginosa LES4 649 661 655 2.73 2.84 2.78 49.6 370 19.0 136

T-2   P. aeruginosa LES4 þ  half dose fertilizers 987 1011 999 3.01 3.04 3.02 50.4 496 20.2 208

T-3 Half dose of fertilizers 719 763 741 2.59 2.65 2.62 47.2 372 18.6 141

T-4 Full dose of chemical fertilizers 986 998 992 2.92 3.03 2.96 49.6 495 20.0 203

T-5 Control 462 476 469 2.40 2.48 2.44 45.8 215 18.3 97SEM   0.338 1.514 1.432 0.845 0.115 0.158 1.095 1.309 1.303 1.105

CD at 1% 1.459 6.539 6.183 0.365 0.500 0.685 4.729 5.654 5.626 4.773

CD at 5% 1.041 4.666 4.412 0.260 0.357 0.489 3.374 4.034 4.015 3.406

Data aremean of two years. Values of each yearare mean of 10randomlyselected plants from each set; ns,notsignificant; *significantat 5% LSD; **significantat 1% levelof LSD

as compared to control; full dose of chemical fertilizers N40þ40þ40, P30, K 30; half doses of chemical fertilizers N20þ20þ20, P15, K15.

S. Kumar et al. / European Journal of Soil Biology 45 (2009) 334–340340