screening plant growth-promoting rhizobacteria for improving growth and yield of wheat

Screening plant growth-promoting rhizobacteria for improvinggrowth and yield of wheat

A. Khalid, M. Arshad and Z.A. ZahirSoil Microbiology and Biochemistry Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan

2003/0311: received 14 April 2003, revised 14 October 2003 and accepted 18 October 2003

ABSTRACT

A. KHALID , M. ARSHAD AND Z.A . ZAHIR . 2004.

Aims: Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants for improving the growth

and yield of agricultural crops, however screening for the selection of effective PGPR strains is very critical. This

study focuses on the screening of effective PGPR strains on the basis of their potential for in vitro auxin production

and plant growth promoting activity under gnotobiotic conditions.

Methods and Results: A large number of bacteria were isolated from the rhizosphere soil of wheat plants grown at

different sites. Thirty isolates showing prolific growth on agar medium were selected and evaluated for their

potential to produce auxins in vitro. Colorimetric analysis showed variable amount of auxins (ranging from 1Æ1 to

12Æ1 mg l)1) produced by the rhizobacteria in vitro and amendment of the culture media with LL-tryptophan

(LL-TRP), further stimulated auxin biosynthesis (ranging from 1Æ8 to 24Æ8 mg l)1). HPLC analysis confirmed the

presence of indole acetic acid (IAA) and indole acetamide (IAM) as the major auxins in the culture filtrates of these

rhizobacteria. A series of laboratory experiments conducted on two cv. of wheat under gnotobiotic (axenic)

conditions demonstrated increases in root elongation (up to 17Æ3%), root dry weight (up to 13Æ5%), shoot elongation

(up to 37Æ7%) and shoot dry weight (up to 36Æ3%) of inoculated wheat seedlings. Linear positive correlation

(r ¼ 0Æ99) between in vitro auxin production and increase in growth parameters of inoculated seeds was found.

Based upon auxin biosynthesis and growth-promoting activity, four isolates were selected and designated as plant

growth-promoting rhizobacteria (PGPR). Auxin biosynthesis in sterilized vs nonsterilized soil inoculated with

selected PGPR was also monitored that revealed superiority of the selected PGPR over indigenous microflora. Peat-

based seed inoculation with selected PGPR isolates exhibited stimulatory effects on grain yields of tested wheat cv.

in pot (up to 14Æ7% increase over control) and field experiments (up to 27Æ5% increase over control); however, the

response varied with cv. and PGPR strains.

Conclusions: It was concluded that the strain, which produced the highest amount of auxins in nonsterilized soil,

also caused maximum increase in growth and yield of both the wheat cv.

Significance and Impact of Study: This study suggested that potential for auxin biosynthesis by rhizobacteria

could be used as a tool for the screening of effective PGPR strains.

Keywords: auxins, plant growth-promoting rhizobacteria, wheat growth.

Correspondence to: Zahir Ahmad Zahir, Soil Microbiology and Biochemistry, Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040, Pakistan

(e-mail: [email protected]).

ª 2004 The Society for Applied Microbiology

Journal of Applied Microbiology 2004, 96, 473–480 doi:10.1046/j.1365-2672.2003.02161.x

INTRODUCTION

Net effect of plant–microbe interactions on plant growth

could be either positive, neutral or negative. All those

bacteria inhabiting plant roots and influencing the plant

growth positively by any mechanism are referred to as

plant growth-promoting rhizobacteria (PGPR) (Kloepper

et al. 1986; Frankenberger and Arshad 1995; Arshad and

Frankenberger 1998). These bacteria significantly affect

plant growth by increasing nutrient cycling, suppressing

pathogens by producing antibiotics and siderophores or

bacterial and fungal antagonistic substances and/or by

producing biologically active substances such as auxins and

other plant hormones. A diverse array of bacteria including

species of Pseudomonas, Azospirillum, Azotobacter, Bacillus,

Klebsiella, Enterobacter, Xanthomonas and Serratia have been

shown to promote plant growth.

During the last couple of decades, the use of PGPR for

sustainable agriculture has increased tremendously in var-

ious parts of the world. Significant increases in growth and

yield of agronomically important crops in response to

inoculation with PGPR have been reported (Chen et al.

1994; Amara and Dahdoh 1997; Biswas et al. 2000a,b; Hilali

et al. 2001; Asghar et al. 2002). Studies have also shown that

the growth-promoting ability of some bacteria may be highly

specific to certain plant species, cv. and genotypes (Nowak

1998). Poi and Kabi (1979) reported that inoculation with

Azotobacter strains isolated from the rhizosphere soils of

Cucurbita maxima, wheat and jute improved the grain yields

but the strains were crop specific.

Like other phytohormones, auxins are also synthesized

endogenously by plants, however, their hormonal effects

have been elucidated by their exogenous applications. There

is also ample evidence that numerous soil micro-organisms

are actively involved in the synthesis of auxins in pure

culture and in soil (Arshad and Frankenberger 1998;

Barazani and Friedman 1999; Biswas et al. 2000a,b).

Generally, micro-organisms isolated from the rhizosphere

and rhizoplane of various crops have revealed more potential

of auxin production than those from the root free soil

(Sarwar and Kremer 1995a,b; Arshad and Frankenberger

1998). LL-tryptophan (LL-TRP), an amino acid, serves as a

physiological precursor for biosynthesis of auxins in plants

and in microbes (Frankenberger and Arshad 1995). Root

exudates are natural source of TRP for rhizosphere micro-

flora, which may enhance auxin biosynthesis in the rhizo-

sphere (Kravchenko et al. 1991; Martens and Frankenberger

1994). It is most likely that auxins of microbial origin in the

vicinity of plant roots could evoke a physiological response

in the host plant. Thus screening of the rhizobacteria for

their in vitro potential of auxin production could provide a

reliable base for selection of effective PGPR, particularly if

this approach is used in combination with screening of

rhizobacteria for their growth-promoting activity under

gnotobiotic conditions.

Thus, we used a combination of two approaches including

in vitro auxin production and growth-promoting activity

under gnotobiotic conditions to select effective PGPR

strains isolated from wheat rhizosphere. The selected PGPR

were tested for their growth and yield increasing potential

under natural soil (nonaxenic) conditions by conducting pot

and field experiments.

MATERIALS AND METHODS

Isolation and screening of PGPR

Several bacterial strains were isolated from the rhizosphere

of different varieties of wheat (LU-26S, Watan, Inqlab-91,

Pasban-90, Parvaaz) crop grown at different sites. Plants of

wheat were uprooted along with good amount of non-

rhizosphere soil, brought immediately to the laboratory in

polythene bags (2 kg size: 23 · 32 cm) and were air-dried

within 2 h. The nonrhizosphere soil was removed by gentle

shaking leaving behind the rhizosphere soil only (strongly

adhering to the roots). The rhizosphere soil was collected

from roots by dipping and gentle shaking in sterilized

water under aseptic conditions. The soil suspension

obtained was used to inoculate glucose peptone agar

medium (GPAM) and pure cultures were obtained by

streaking three to four times in the fresh medium

(Wollum-II 1982). Thirty bacterial isolates showing prolific

growth and having different morphological appearance on

agar medium were selected and stored for studying auxin

biosynthesis in vitro.

Auxin production by the rhizobacterial strains both in the

presence and absence of LL-TRP was determined by

colorimetry. For this purpose, 20 ml of GPAM broth were

added in 100-ml Erlenmeyer flasks, autoclaved and cooled.

Five millilitre of filter sterilized (0Æ2 lm membrane filter,

Whatmann) LL-TRP solution (5%) were added to the liquid

medium (GPAM) to achieve a final concentration of

1Æ0 g l)1. The flask contents were inoculated by adding

1Æ0 ml of 4-day-old bacterial broth adjusted to optical

density of 0Æ5 (107–108 CFU ml)1) measured at 550 nm by

spectrophotometer (ANA-720W; Tokyo Photo-electric

Company Limited, Tokyo, Japan). The flasks were plugged

and incubated at 28 ± 1�C for 48 h at 100 rev min)1

shaking. Noninoculated/untreated control was kept for

comparison. After incubation, the contents were filtered

through Whatmann filter paper no. 2. Auxin compounds

expressed as (IAA-equivalents) were determined by spec-

trophotometer (ANA-720W) using Salkowski colouring rea-

gent as described by Sarwar et al. (1992). To keep a check

on mutation due to subculturing, the ability of the isolates to

produce auxins in vitro was repeatedly confirmed prior to

474 A. KHALID ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 473–480, doi:10.1046/j.1365-2672.2003.02161.x

next experiment. Similarly, sterilized and nonsterilized soils

were inoculated with four selected PGPR and LL-TRP

(2Æ0 g kg)1 soil)-dependent auxin biosynthesis was monit-

ored. All auxin determinations were made in triplicate.

The qualitative production of auxin compounds in the

culture filtrates was further confirmed by HPLC-UV as

described by Martens and Frankenberger (1991). Filtrates

were examined on a reverse phase HPLC (10-A; Shimadzu

Corporation, Kyoto, Japan) by using mixture of methanol

and water (70 : 30 ratio) as a mobile phase (solvent) at

280 nm using the Shim-pack CLC-ODS C18 column

[4Æ6 mm internal diameter (ID), 250 mm long and 100 A

pore diameter].

Based upon in vitro auxin production, 30 isolates were

categorized into three major groups: (i) low (L) auxin

producers (a1–a10), (ii) medium (M) auxin producers

(a11–a20) and (iii) highly (H) effective auxin producers

(a21–a30). Plate and Leonard jar experiments were conduc-

ted on two cv. of wheat (Pasban 90 and Inqlab 91) under

gnotobiotic (axenic) conditions to see the effectiveness of

inoculation with these three groups of rhizobacteria on root

and shoot growth, respectively. Wheat seeds were surface

sterilized by momentarily exposing to 95% ethanol and

immersing in 0Æ2% HgCl2 solution for 3 min. The seeds

were then subjected to six washings with sterile distilled

water. Thoroughly washed seeds of the two cv. of wheat

were sown on sterilized filter paper sheets placed in Petri

plates. Six seeds were sown in each Petri plate with four

repeats. Two millilitre of 4-day-old broth of rhizobacteria

(107–108 CFU ml)1, 0Æ5 of O.D.550) were applied on seeds

present in each plate with the help of sterilized pipette.

Sterilized distilled water (10 ml) was added to each Petri

plate to wet the filter paper sheets and the seeds were

covered with another sterilized filter paper sheet. Sterilized

broth (free of bacterial population) was applied in case of

control. The plates were incubated in a growth room at

28 ± 2�C. After 2 weeks, the sheets were removed and

examined for root growth (root elongation and root weight).

For Leonard jar experiments, plastic glasses (383 cm3)

were filled with sand and 1/2 strength Hoagland solution

(Hoagland and Arnon 1950) was applied from jars (460 cm3)

through a wick to provide nutrition to the plants. The whole

apparatus was autoclaved (25 min at 121�C) prior to the

transplantation of seedlings. Surface-disinfected seeds were

sown on sterilized filter sheet in Petri plates. Uniformly

germinated seeds were transplanted to the glass containing

sand under aseptic conditions to eliminate the variation in

growth contributed by different endogenous germination

rate/potential of the seeds. Five millilitre of 4-day-old

inocula were applied to the seedlings growing in sand,

2 days after transplanting. The jars were incubated in

the growth room at 28 ± 2�C and 16 h of light

(1600 lmol m)2 s)1) were supplied daily. Two weeks after

transplanting, the plants were uprooted and length and

weight of the seedling shoots were measured.

Pot and field experiments

Based upon the performance of rhizobacteria in the Plate

and Leonard jar experiments, four effective auxin-producing

PGPR isolates (Ha21, Ha22, Ha23 and Ha30) were selected

and used in pot and field trials. Inocula were prepared by

growing the selected PGPR in GPAM broth and incubated

at 28 ± 1�C with 100 rev min)1 shaking. Four-day-old

inoculum (107–108 CFU ml)1, 0Æ5 of O.D.550) was injected

into sterile peat at 100 ml kg)1 peat and incubated for 24 h

at 28 ± 1�C prior to seed inoculation. Seeds were inocu-

lated by mixing with peat and 10% sugar solution at

100 ml kg)1 peat while controls consisted of the seeds

treated with peat having nutrient broth and sugar solution

without PGPR. Treated seeds were dried under shade

for 6–8 h.

For pot experiment, soil sample was collected, air-dried,

sieved (2-mm/10-mesh) and analysed for physico-chemical

characteristics before filling the pots. The soil was clay loam

having pH 7Æ9; electrical conductivity of saturated soil

extract (ECe), 1Æ6 dS m)1; cation exchange capacity

(CEC), 6Æ8 cmol(+) kg)1 and organic matter, 0Æ72%. Eight

inoculated and uninoculated seeds of two wheat cv. (Pasban-

90 and Inqlab-91) were sown in soil filled pots (12 kg soil

per pot) receiving nutrient inputs of NPK at 120, 75 and

50 kg ha)1 as urea, diammonium phosphate and muriate of

potash, respectively. All of PK and half of N were mixed

with soil at the time of sowing while remaining N was

applied in solution form at tillering. Four seedlings were

maintained in each pot after germination. The pots were

arranged randomly with four repeats at ambient light and

temperature in a wire house. Good quality canal water

[EC ¼ 0Æ03 dS m)1, sodium adsorption ratio (SAR) ¼ 0Æ26

(mmol l)1)1/2 and residual sodium carbonates (RSC) ¼ 0]

meeting the irrigation quality criteria for crops (Ayers and

Westcot 1985) was used for irrigation. The plants were

harvested after 5 months and data were recorded.

Two years replicated field trials were conducted with the

same treatments and agronomic practices as used in pot

experiment, however, whole dose of PK and half N was

broadcast at the time of soil preparation, and remaining half

N was applied at tillering. Seeds of wheat were sown with

single row seed drill keeping row to row distance of 25Æ0 cm.

Each experiment was conducted in randomized complete

block design (RCBD) with four repeats. The analysis of

composite soil (typic haplocambids) samples collected from

experimental field (ca 0–15 cm layer) revealed the following

characteristics: texture, clay loam; pH 7Æ7; ECe, 1Æ9 dS m)1;

CEC, 5Æ9 cmol(+) kg)1 and organic matter, 0Æ58%. Data on

plant height, number of tillers, spike length, spikelets per

SCREENING OF PGPR 475


spike, straw and grain yields were recorded on maturity after

5 months.

Statistical analysis

The experimental data were analysed statistically according

to Steel and Torrie (1980) and means were compared by

Duncan’s Multiple Range Test (Duncan 1955). Correlations

between in vitro auxin production by PGPR and effect on

growth parameter(s) were also calculated (Steel and Torrie

1980).

RESULTS

A series of laboratory, pot and field experiments were

conducted to assess the potential of various rhizobacterial

isolates for auxin biosynthesis and improving growth and

yield of two cv. of wheat (Triticum aestivum L.).

Biosynthesis of auxins

Results of colorimetric analysis indicated that different

isolates of rhizobacteria varied greatly in their efficiency for

producing auxins in the broth medium (GPAM), both in the

presence and absence of LL-TRP (Table 1). Among 30

isolates tested, 73% (22 isolates) produced auxins (ranging

from 0Æ6 to 12Æ1 mg l)1 IAA-equivalents) in the absence of

LL-TRP. Auxins produced by the 10 isolates belonging to

rhizobacterial group H (effective auxin producers; Ha21,…,

Ha30) ranged from 5Æ1 to 12Æ1 mg IAA-equivalents per litre,

with an average amount of ca 7Æ0 mg l)1. In the presence of

LL-TRP, bacterial efficiency for auxin synthesis was enhanced

by several folds (ranging from 1Æ8 to 24Æ8 mg IAA-equiv-

alents per litre). LL-TRP-derived auxin biosynthesis by group

H bacteria varied between 13Æ8 and 24Æ8 mg IAA-equiva-

lents per litre, which was almost two fold greater than the

LL-TRP unamended culture. All other isolates belonging to

group L (La1,…, La10) and M (Ma11, …, Ma20) were also

able to derive auxins from LL-TRP, however, they were

relatively less effective in auxin biosynthesis.

Auxin production in sterilized and nonsterilized soils,

amended with LL-TRP and inoculated with four selected

PGPR isolates was also monitored (Table 2). Data revealed

that inoculation with the PGPR substantially stimulated

auxin synthesis in soil. Inoculation with selected PGPR

strain Ha21 was found the most effective auxin producing

rhizobacterium in sterilized soil (27Æ5 mg IAA-equivalents

kg)1 soil). The magnitude of auxin synthesis was less in the

inoculated-sterilized soil than the uninoculated nonsterilized

soil both amended with LL-TRP at 2Æ0 g kg)1 soil (Table 2)

implying that the mixed indigenous microflora are compar-

atively more effective than the single strain inoculation.

However, inoculation of nonsterilized soil with PGPR

isolates further stimulated the auxin production indicating

that inoculation with specific micro-organisms is even

effective in the presence of indigenous soil microflora.

HPLC analysis demonstrated the presence of IAA and

indole-3-acetamide (IAM) as major LL-TRP-derived micro-

bial products both in broth medium and in soil. Presence of

IAM also indicated that TRP fi IAM fi IAA pathway

of auxin biosynthesis was active in these rhizobacteria.

All the isolates of rhizobacteria tested for auxin biosyn-

thesis in vitro were further screened for their growth-

promoting effects on wheat seedlings by conducting Plate

and Leonard jar experiments under controlled (axenic)

conditions. Different isolates of rhizobacteria had variable

effects (both negative and positive) on root elongation and

weight of roots in two wheat cv. tested (Table 3). Overall,

inoculation with the bacterial isolates of group H resulted in

maximum increase in root elongation (17Æ3%) and weight

(11Æ4%) of cv. Pasban-90 compared with uninoculated

control. Similarly in case of cv. Inqlab-91, the same group of

bacteria promoted root elongation and weight by 11Æ3 and

13Æ4%, respectively compared with uninoculated control.

Table 1 In vitro auxin production by low (L), medium (M) and high

(H) auxin producing rhizobacteria in glucose peptone agar medium.

Data are average of three replications

Group

of rhizobacteria

IAA-equivalents (mg l)1)

Range Mean ± S.ES.E.

Without

LL-TRP

With

LL-TRP

Without

LL-TRP

With

LL-TRP

L (a1–a10) 0Æ0–1Æ1 1Æ8–13Æ4 0Æ17 ± 0Æ014 6Æ2 ± 1Æ155

M (a11–a20) 1Æ2–5Æ0 2Æ7–14Æ3 3Æ1 ± 0Æ199 10Æ3 ± 1Æ857

H (a21–a30) 5Æ1–12Æ1 13Æ8–24Æ8 6Æ9 ± 0Æ581 17Æ7 ± 1Æ108

IAA, indole acetic acid; LL-TRP, LL-tryptophan; S.E.S.E., standard error.

Table 2 LL-tryptophan-derived auxin biosynthesis in soil inoculated

by four most active plant growth-promoting rhizobacterial isolates

belonging to group H (high auxin-producing rhizobacterial isolates).

The data are average of four replications

PGPR isolate

IAA-equivalents (mg kg)1 soil)

Sterilized soil Nonsterilized soil

Uninoculated 1Æ70 d 35Æ7 c

Ha21 27Æ5 a 45Æ2 b

Ha22 13Æ5 c 66Æ3 a

Ha23 17Æ6 b 41Æ5 b

Ha30 14Æ9 c 38Æ8 c

Mean values sharing similar letter do not differ significantly at

P < 0Æ05.

IAA, indole acetic acid.



Results of Leonard jar study revealed that shoot growth

(length and weight) of cv. Pasban-90 and Inqlab-91 was

significantly improved by bacterial inoculation (Table 4).

Once again, maximum increases in shoot length (24%) and

weight (47%) in cv. Pasban-90 were recorded in case of

inoculation with isolates belonging to H group. For cv.

Inqlab-91, increases in shoot length and weights observed in

response to inoculation with rhizobacteria of H group were

up to ca 37% in both parameters compared with uninoc-

ulated control.

When increases in root and shoot growth were regressed

against in vitro auxin production by the rhizobacteria,

significant linear correlations were found. Significant positive

linear correlation (r ¼ 0Æ99**) was observed between auxin

(IAA-equivalents) production in vitro in the absence of

LL-TRP by rhizobacteria and root elongation of cv. Pasban-90

and Inqlab-91. Also a significant correlation (r ¼ 0Æ99**) was

found between auxin production in the presence of LL-TRP

and root elongation of cv. Pasban-90. Similarly, a highly

significant correlation (r ¼ 0Æ99**) was found between

in vitro auxin production in the absence of LL-TRP and root

weight of both the cv. while in the presence of LL-TRP, only

root weight of cv. Inqlab-91 was significantly correlated. A

significant linear correlation (r ¼ 0Æ99**) was observed

between the shoot weight of both the cv. and production of

auxins (IAA) in vitro by rhizobacterial isolates in the absence

and in the presence of LL-TRP, however, in case of shoot

length, correlation was nonsignificant in both the cv.

Pot and field trials

Seed inoculation with four selected PGPR isolates signifi-

cantly affected the growth and yield of two wheat cv. under

wire house conditions (Table 5). PGPR inoculation signi-

ficantly enhanced number of tillers of cv. Pasban-90 while

response was nonsignificant in case of cv. Inqlab-91.

Maximum number of tillers were recorded in cv. Pasban-

90 in case of inoculation with isolate Ha30 (23Æ2% more than

uninoculated control). In case of cv. Inqlab-91, all the PGPR

isolates except Ha22 and Ha23 increased the tillers up to

6Æ1% over uninoculated control. Inoculation with PGPR

also caused significant increases in straw yields of both the

cv. compared with their respective uninoculated controls

(Table 5). In case of cv. Pasban-90, isolate Ha30 gave the

most promising results and caused an increase of 12Æ4% in

straw yield while in cv. Inqlab-91, straw yield was increased

up to 10Æ1% in response to inoculation with Ha23 compared

with respective uninoculated control. In case of cv. Pasban-

90, PGPR Ha23 and Ha30 significantly increased the grain

yield (by 13Æ7 and 14Æ7%, respectively) but none of the

PGPR significantly increased grain yield of cv. Inqlab-91

(Table 5).

Data of 2 years repeated field experiments indicated that

inoculation with selected PGPR isolates had significant but

variable effects on growth and yield of both the cv. of

wheat (Table 6). During the first year, PGPR inoculation

significantly increased number of tillers of two wheat cv.

Table 4 Effects of low (L), medium (M) and

high (H) auxin producing rhizobacterial iso-

lates on shoot growth of two wheat cv. grown

under gnotobiotic conditions in Leonard Jar

experiment (average of 40 values; four

repeats · 10 isolates)

Group

of rhizobacteria

Cultivar Pasban-90 Cultivar Inqlab-91

Shoot

elongation (cm)

Dry shoot weight

(gram per plant)

Shoot

elongation (cm)

Dry shoot weight

(gram per plant)

Uninoculated 15Æ7 c 0Æ113 c 14Æ6 d 0Æ113 c

L (a1–a10) 17Æ2 b 0Æ117 c 16Æ3 c 0Æ115 c

M (a11–a20) 18Æ8 a 0Æ140 b 17Æ2 b 0Æ131 b

H (a21–a30) 19Æ5 a 0Æ167 a 20Æ1 a 0Æ154 a

Values followed by different letters in a column were significantly different (P < 0Æ05), using

Duncan’s multiple range test.

Table 3 Effects of low (L), medium (M) and

high (H) auxin producing rhizobacterial iso-

lates on root growth of two wheat cv. grown

under gnotobiotic conditions in Plate experi-

ments (average of 40 values; four repeats · 10

isolates)

Group

of rhizobacteria

Cutivar Pasban-90 Cultivar Inqlab-91

Root

elongation (cm)

Dry root weight

(gram per plant)

Root

elongation (cm)

Dry root weight

(gram per plant)

Uninoculated 9Æ63 b 0Æ070 b 10Æ6 b 0Æ052 b

L (a1–a10) 8Æ16 c 0Æ064 c 10Æ4 b 0Æ048 c

M (a11–a20) 9Æ72 b 0Æ071 b 11Æ2 ab 0Æ052 b

H (a21–a30) 11Æ3 a 0Æ078 a 11Æ8 a 0Æ059 a

Values followed by different letters in a column were significantly different (P < 0Æ05), using

Duncan’s multiple range test.



tested, and isolate Ha30 produced the highest number of

tillers in case of cv. Pasban-90, which was 15Æ5% greater

than uninoculated control. Three isolates (Ha21, Ha22 and

Ha30) produced significantly more number of tillers

(ranging from 6Æ4 to 14Æ5%) in case of cv. Inqlab-91

compared with uninoculated control. Data regarding straw

yield revealed that all the four PGPR isolates significantly

increased straw yield of cv. Pasban-90 ranging from 13Æ6 to

17Æ2% over uninoculated control. In case of cv. Inqlab-91,

the isolate Ha22 gave the highest straw yield (10Æ2% over

uninoculated control). Inoculation also caused significant

increases in grain yield of cv. Pasban-90, which ranged

from 15Æ0 to 27Æ5%, over uninoculated control. In cv.

Inqlab-91, increases in grain yield were up to 18Æ9% over

uninoculated control in response to PGPR inoculation.

The most effective isolates in promoting grain yields of cv.

Pasban-90 and Inqlab-91 were Ha30 and Ha22, respect-

ively.

During the second year, there were significant differences

in tillering of both the cv. due to inoculation with PGPR

isolates. The most effective isolate was Ha21, which

produced the highest tillers (up to 37Æ0% increase over

uninoculated control) in cv. Pasban-90 and Inqlab-91.

Effects of inoculation on straw yields were also significant

in both cv. Cultivar Pasban-90 responded more promisingly

as all the isolates applied as inocula caused increase in straw

yields ranging from 22Æ3 to 34Æ7% over uninoculated

control. In case of cv. Inqlab-91, only one isolate (Ha21)

gave significantly higher straw yield (17Æ4%) than uninoc-

ulated control. PGPR isolate Ha21 caused maximum

enhancement in the grain yield of cv. Pasban-90 (19Æ6%),

while isolate Ha22 was the most effective promoter of grain

yield (12Æ2% greater than uninoculated control) in cv.

Inqlab-91.

DISCUSSION

In this study, two approaches were simultaneously employed

to select effective PGPR for wheat to be used as inocula in

pot and field trials. The approaches include screening of

rhizobacteria for in vitro auxin biosynthesis and for their

growth-promoting activity under gnotobiotic (axenic) con-

ditions. In general, it is observed that isolates belonging to

group H (more effective auxin producers) had more

promising effects on the inoculated plant seedlings, com-

pared with group L and M (least and medium auxin

Table 6 Effect of inoculation with selected plant growth-promoting rhizobacteria on growth and yield of two wheat cv. grown in field (average of

four replications)

PGPR Isolates

No. of tillers (m)2) Straw yield (t ha)1) Grain yield (t ha)1)

Pasban-90 Inqlab-91 Pasban-90 Inqlab-91 Pasban-90* Inqlab-91*

I-year II-year I-year II-year I-year II-year I-year II-year I-year II-year I-year II-year

Control 361 c 212 c 373 c 259 bc 6Æ98 c 5Æ97 c 8Æ11 bc 8Æ60 bc 3Æ60 d 2Æ91 c 4Æ18 cd 3Æ77 b

Ha21 396 b 292 a 399 b 294 a 8Æ18 a 7Æ94 a 8Æ12 bc 10Æ1 a 4Æ14 c 3Æ48 a 4Æ25 c 3Æ90 b

Ha22 387 b 288 a 427 a 255 bc 7Æ93 b 8Æ04 a 8Æ94 a 8Æ90 b 4Æ40 b 3Æ21 b 4Æ97 a 4Æ23 a

Ha23 398 b 234 b 368 c 265 b 8Æ16 a 7Æ30 b 7Æ58 c 8Æ50 bc 4Æ37 b 3Æ07 bc 3Æ97 d 3Æ67 b

Ha30 417 a 280 a 397 b 244 c 8Æ20 a 7Æ97 a 7Æ72 c 7Æ80 d 4Æ59 a 3Æ18 b 4Æ55 b 3Æ61 b

*Wheat cv.

Values followed by different letters in a column were significantly different (P < 0Æ05), using Duncan’s multiple range test.

Table 5 Effect of inoculation with selected

plant growth-promoting rhizobacteria

(PGPR) on growth and yield of two wheat cv.

grown in pots (average of six repeats)PGPR Isolaters

No. of tillers

per plant

Straw yield

(gram per plant)

Grain yield

(gram per plant)

Pasban-90 Inqlab-91 Pasban-90 Inqlab-91 Pasban-90 Inqlab-91*

Uninoculated 4Æ31 c 4Æ43 NS 5Æ73 bc 5Æ82 b 3Æ07 b 3Æ03 NS

Ha21 4Æ30 c 4Æ70 5Æ34 c 6Æ24 a 3Æ14 ab 3Æ13

Ha22 5Æ0 ab 4Æ10 6Æ09 ab 5Æ79 b 3Æ15 ab 2Æ96

Ha23 4Æ90 b 4Æ40 6Æ01 b 6Æ41 a 3Æ49 a 3Æ24

Ha30 5Æ31 a 4Æ50 6Æ44 a 6Æ18 ab 3Æ52 a 3Æ06

*Wheat cv.

NS, not significant. Values followed by different letters in a column were significantly different

(P < 0Æ05), using Duncan’s multiple range test.



producers, respectively). There was significant linear corre-

lation between auxins produced by rhizobacteria in vitro and

growth of wheat seedlings (particularly root and shoot

weights) under gnotobiotic conditions. This may imply that

auxins produced by PGPR isolates caused improvement in

root system, resulting in more biomass production. How-

ever, other mechanisms of action through which PGPR

influence plant growth can not be ruled out. Okon and

Vanderleyden (1997) suggested that the secretion of plant

growth-promoting substances by the bacteria could be

responsible for the beneficial effects of PGPR. Glick

(1995) also viewed that the mechanism most commonly

invoked to explain the various effects of PGPR on plants is

the production of phytohormones, and IAA may play the

most important role in plant growth promotion. Under

gnotobiotic conditions, Noel et al. (1996) demonstrated the

direct involvement of the plant growth regulators including,

IAA modifying the growth of canola and lettuce. The results

of our study suggest that simultaneous screening of

rhizobacteria for in vitro auxin production and growth

promotion under axenic conditions is a good tool to select

effective PGPR for bio-fertilizer development biotechno-

logy.

In our pot and field experiments, it was observed that

inoculation with selected isolates of PGPR significantly

promoted growth and yield of different cv. of wheat under

nonaxenic conditions. In general, inoculation resulted in

early seedling growth and development in pots. Our results

are in line with the findings of Dobbelaere et al. (2001) who

assessed the inoculation effect of PGPR Azospirillum

brasilense on growth of spring wheat. They observed that

inoculated plants resulted in better germination, early

development and flowering and an increase in dry weight

of both the root system and the upper plant parts. There was

a positive correlation between the increase in yield and the

improvement of root development. Similarly, promotion in

plant height, number of tillers, plant dry weight and grain

yields of various crop plants in response to inoculation with

PGPR were reported by other workers (Chen et al. 1994;

Khalid et al. 1997; Biswas et al. 2000a,b; Hilali et al. 2000,

2001). It is noteworthy that the strain, which produced

higher amount of auxin in nonsterilized soil also significantly

promoted yield of cv. Inqlab-91, in both the field trials. This

may imply that this strain had more competitive ability to

survive and affect the growth of inoculated plants in the

presence of indigenous microflora.

In some of the cases, rhizobacterial inoculation had

negative effects on different growth and yield parameters of

wheat. This might be due to production of some kind of

phytotoxins that inhibited the growth of inoculated plants

(Brown and Rovira 1999).

In this study, both the cv. tested responded differently to

inoculation with different rhizobacterial isolates. This

variation in response to inoculation might be due to genetic

make-up of different varieties/cv. or plant species. Different

crops and varieties or species might produce different types

of root exudates, which could support the activity of inocula

and/or serve as substrate(s) for the formation of biologically

active substances by the inocula (Frankenberger and Arshad

1995; Dazzo et al. 2000). Nowak (1998) reported that the

benefits of bacterization depended on plant species, cv. and

growth conditions. The degree to which the inoculation

imparts benefits to plant growth can vary with variety,

cultural conditions and PGPR strains. Work on identifica-

tion of selected PGPR is in progress.

As all the four selected PGPR had promising positive

effects on growth and yield parameters of wheat grown

under natural conditions, this supports the premises of

simultaneous use of both approaches including in vitro auxin

production and growth promotion under axenic conditions

for selection of effective PGPR.

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screening plant growth-promoting rhizobacteria for improving growth and yield of wheat

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