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Journal of Microbiology Research 2012, 2(2): 26-35
DOI: 10.5923/j.microbiology.20120202.05
Effectiveness of Rhizosphere Bacteria for Control of Root
Rot Disease and Improving Plant Growth of Wheat
(Triticum aestivum L)
Seema Dua, S. S. Sindhu*
Department of Microbiology, CCS Haryana Agricultural University, Hisar, 125004, India
Abstract Pathogenic fungus Rhizoctonia solani causes root rot disease in wheat leading to collapsing of the aerial part of
the plant. To characterize antagonistic bacteria, one hundred and thirty bacterial isolates were obtained from the rhizosphere
soil of wheat and these rhizobacterial isolates alongwith 72 reference strains were screened for their antagonistic interactions
against R. solani under cultural conditions. Sixteen bacterial isolates inhibited the growth of R. solani and growth inhibition
zone varied from 6-15 mm by different rhizobacterial isolates. Two isolates WPS3 and WPS90 caused maximum growth
inhibition of the fungi. Growth inhibiton of the pathogenic fungi was also observed using culture filterates of antagonistic
rhizobacterial isolates. The protein estimation of the culture filterates showed that the amount of protein excreted by different
rhizobacterial isolates varied from 3.6 to 33.0 mg ml-1 of the supernatant. The loss of antagonistic activity after treatment with
proteinase K and high temperature treatment indicated that excreted proteins are responsible for the antagonism. Pot house
studies showed that inoculation of R. solani in wheat caused 85-90% root rot disease incidence at 60 to 90 days of plant
growth. The single inoculation of rhizobacterial isolate WPS3 resulted in 131% increase of plant dry weight as compared to
uninoculated control plants. The coinoculation of isolate WPS3 with R. solani enhanced 115% plant dry weight whereas
coinoculation of Pseudomonas isolate WPS90 caused 98% increase in plant dry weight in comparison to control uninoculated
plants at 90 days of plant growth. Coinoculation of Pseudomonas isolates WPS3 and WPS90 with R. solani also caused 88.9
and 66.7% disease control, respectively at 90 days of plant growth. Thus, Pseudomonas isolate WPS3 could be further ex-
ploited for plant growth improvement under field conditions.
Keywords Rhizosphere bacteria, Rhizoctonia solani, Biological control, Pseudomonas sp., Bacillus sp., Root rot disease,
plant dry weight
1. Introduction
The plant rhizosphere is an important ecological envi-
ronment in soil for plant-microbe interactions. These inter-
actions with plants could be beneficial, neutral or with det-
rimental effects resulting in plant diseases[1-3]. The patho-
genic microorganisms cause various plant diseases that
usually weaken or destroy plant tissues and reduce crop
yields varying from 25% to 100%. Root diseases are esti-
mated to cause 10-15% yield losses annually in the world.
These plant diseases are mostly controlled by application of
chemical pesticides. However, the widespread use of
chemical pesticides has been a subject of public concern due
to potential harmful effects on the environment, their unde-
sirable effect on non-target organisms and the possible car-
cinogenicity effect of some chemicals. Moreover, the patho-
* Corresponding author:
sindhuss58@gmail.com (S. S. Sindhu)
Published online at http://journal.sapub.org/microbiology
Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved
gens develop resistance against the pesticides applied.
Therefore, biological control offers an alternative approach
to the use of costly and harmful chemicals, and provides low
cost, environmental friendly control measures to reduce the
activity of plant pathogens[4-5]. Antagonistic rhizosphere
microorganisms inhibit the growth of pathogenic microor-
ganisms without disrupting the ecological balance and thus,
biological control strategies are highly compatible with the
sustainable agriculture.
The saprophytic, pathogenic and plant growth promoting
strains of bacteria have been found to colonize the plant
rhizosphere[6]. Field application of some rhizosphere bac-
teria has resulted in significant promotion of root biomass,
plant growth and yield of different crops[2, 7-9]. These
beneficial bacteria are generally referred as plant growth
promoting rhizobacteria (PGPR)[10]. The beneficial effects
of PGPR have been correlated with increased recycling,
solubilization and uptake of mineral nutrients[11], synthesis
of vitamins, amino acids, auxins and gibberellins[12, 13],
and by antagonism of potential plant pathogens[14-16]. The
antagonistic microorganisms by their interactions with
Journal of Microbiology Research 2012, 2(2): 26-35 27
various soil-borne plant pathogens play a major role in bio-
logical disease control[5, 17]. Therefore, Bacillus and
Pseudomonas species that can establish in rhizosphere are
ideal candidates for use as inoculant to enhance plant growth
and as biocontrol agents for suppression of plant diseases
under pot house and field conditions[7, 18].
Wheat is the second most important grain crop and is a
source of staple food in many countries of the world. Though
the production of wheat has increased after green revolution
but the attack of various diseases like head blight, powdery
mildew, root rots, rusts, smuts, take-all and Karnal bunt of
wheat has greatly affected its yield and quality[19-21]. Root
rot disease in wheat is caused by fungus Rhizoctonia solani
that produces reddish brown lesions on the root surface just
below the soil line. It causes hindrance to absorption of water
and minerals through the roots leading to collapsing of the
aerial part of the plant. In this study, bacterial isolates ob-
tained from wheat rhizosphere were tested for growth inhi-
bition of pathogenic fungi R. solani on medium plates and
the antagonistic Pseudomonas sp. were inoculated onto
wheat for plant growth improvement and control of root rot
disease under pot house conditions.
2. Materials and Methods
2.1. Isolation of Bacterial Cultures from the Rhizosphere
Soil
One hundred and thirty rhizobacterial isolates were ob-
tained from the rhizosphere soil of wheat by serial dilution
plate method using King’s B medium. Soil samples were
collected randomly from the rhizosphere of wheat at 60 and
90 days of plant growth from 3 different locations of CCS
Haryana Agricultural University, Hisar farm. From each
location, samples were collected from six different sites. The
serial dilutions of the soil samples were made up to 10-5 and
0.1 ml of diluted soil suspension was plated on King’s B (KB)
medium plates[22]. The plates were incubated at 28+2°C in
BOD incubator for 3-4 days. Pseudomonas, Bacillus and
other rhizobacterial colonies were selected based on typical
morphological and pigment production characteristics.
Seventy two reference strains were procured from the De-
partment of Microbiology, CCS Haryana Agricultural Uni-
versity, Hisar. The rhizobacterial strains/isolates were
maintained by periodic transfer on Luria Bertani agar
slants[23]. Rhizobacterial isolates showing zone of inhibi-
tion were screened for oxidase test, catalase test, spore
staining and Gram staining[24]. These bacterial cultures
were stored at 4°C in refrigerator for further use.
2.2. Host Species
Seeds of wheat (Triticum aestivum L.) variety WH542
were obtained from Department of Seed Science and Tech-
nology, CCS Haryana Agricultural University, Hisar.
2.3. Screening of Rhizobacterial Isolates for Antagonistic
Activity Using Fungal Pathogens
The interaction of rhizobacterial isolates with Rhizoctonia
solani was studied by the spot test method on PDA medium
plates[25]. The fungi R. solani was grown on PDA slants and
spore suspension of fungi was prepared in 3 ml sterilized
water. Two ml of fungal spore suspension (containing
5.2-6.0 x 108 spores ml-1 ) were incorporated into molten
PDA medium and plates were prepared. Growth suspension
of 48 h old rhizobacterial isolate (2.0 µl) was spotted on
preseeded plates. Plates were incubated for 48 h at 28+2°C
and growth inhibition of fungi R. solani was recorded.
Rhizobacterial isolates showing zone of inhibition were
selected.
2.4. Determination of location of Antimicrobial Sub-
stance
Bacterial isolates were grown in LB medium broth for 2, 5
and 10 days at 28+2°C in the incubator with a rotary
shaker (100 rpm speed). Bacterial growth suspension (1.0
ml) was centrifuged at 10,000 rpm for 20 minutes to separate
cells and cell free culture filterate (supernatant). Seven mm
diameter wells were made into the PDA agar medium plates
with the help of sterile cork borer, preseeded with spore
suspension of the fungi. Cell free culture filtrate (50 µl)
obtained from selected bacterial isolates was loaded in the
wells and plates were incubated at 28+2°C. Observations
for antifungal activity against R. solani were scored by
measuring growth inhibition zone on PDA medium plates
after 2 days.
2.5. Determination of Nature of Antimicrobial Substance
To determine nature of antimicrobial substance, the cul-
ture supernatants were analysed for production of specific
proteins. Culture filterates of rhizobacterial isolates were
precipitated with concentrated trichloro-acetate solution
(100%). The precipitated proteins were solubilized in
phosphate buffer saline (PBS) (pH 7.0) and used for study of
growth inhibition against R. solani by spot test method on
PDA medium plates. Protein concentrations in the total
exoproteins were determined by the method of Lowry et
al.[26]. using bovine serum albumin (BSA) as standard.
Absorbance at 690 nm (A690) of different samples
wells was measured in a spectrophotometer. A stan-
dard curve of BSA concentration (µg ml- 1) versus A690
was drawn and the regression equation was obtained
using MS Excel programme and the protein concen-
tration in the test samples was determined.
Protein obtained from culture supernatants of
rhizobacterial isolates were treated with proteinase K
enzyme. Stock solution of proteinase K was prepared
by dissolving 3.0 mg of proteinase K into 600 µl
Tris-EDTA buffer. Protein samples mixed in Laemmli
buffer were treated with proteinase K (50 µg ml-1) and
the mixture was incubated for 90 minutes at 370C. The
suspension was incubated for 10 min at 1000C and
after cooling Proteinase K was again added to a final
concentration of 100 µg ml-1 . The preparation was then
incubated for another 60 minutes at 370C. Samples
28 Seema Dua et al.: Effectiveness of Rhizosphere Bacteria for Control of Root Rot
Disease and Improving Plant Growth of Wheat (Triticum aestivum L)
were also treated with high temperature by incubating
for 45 minutes at 850C. Samples treated with pro-
teinase K and high temperature were tested for an-
tagonistic activity.
2.6. Inoculation with Pseudomonas Strains for Control of
Root Rot Disease in Wheat
Two selected Pseudomonas isolates i.e., WPS3 or WPS90
were tested for disease control and plant growth promotion
of wheat under pot house conditions. Six treatments were
made and each treatment had three replications as described
below.
(i). T1: Soil (control, uninoculated)
(ii). T2: Soil + Pseudomonas isolate WPS3
(iii). T3: Soil + Pseudomonas isolate WPS90
(iv). T4: Soil + R. solani
(v). T5: Coinoculation of R. solani + WPS3
(vi). T6 : Coinoculation of R. solani + WPS90
Sandy loam soil was collected from CCS Haryana Ag-
ricultural University farm dry-land area. The earthen pots
of 10 kg capacity were filled with sandy loam soil and
washed river sand, mixed in 70:30 ratio. The cultures of
Pseudomonas isolates were grown on LB medium slants for
2 days. About 3 ml of sterilized water was added to Pseu-
domonas cultures. The bacterial growth was scrapped and
vertexed on rotary shaker to get uniform suspension. The
growth suspension of Pseudomonas culture WPS3 (5.2 × 108
cells ml-1 ) was inoculated on the roots of wheat plants after
germination in the T2 and T5 treatments only and isolate
WPS90 (5.7 × 108 cells ml-1 ) was inoculated in the T3 and
T6 treatments.
The seeds of wheat (Triticum aestivum L.) variety WH542
were surface sterilized with acidic alcohol (70:30) for three
minutes and 4-5 washings were given with sterilized water.
Surface sterilized seeds were inoculated with broth culture of
Pseudomonas isolates. The viable count in the broth was
kept 108-109 cells ml-1 and 10 g seeds were inoculated with 2
ml of bacterial growth suspension[27]. Growth of R. solani
(4 days old) was harvested from PDA plates with the help of
inoculation needle and then sterilized saline water was added
to get uniform fungal growth suspension. Fungal growth
suspension (50 ml) was mixed in soil: sand mixture in
earthern pots in treatments T4, T5 and T6. The growth sus-
pension of fungus was also inoculated on the roots of wheat
plants after germination in the R. solani treatments only as
control treatment. The plants were grown in the pot house
under day light conditions during November 2008 - March
2009. The plants were uprooted at 60, 75 and 90 days of
growth and observations were taken for the dry weight of
root and shoot and control of plant disease.
2.6.1. Plant Fresh and Dry Weight
Shoot and root portions of the plants after uprooting were
weighed first. Then they were dried in oven at 900C for 24h
and weighed again.
2.6.2. Disease Index and Reduction in Disease
On the basis of symptoms observed percent disease index,
percent final stand and percent disease control were calcu-
lated by the formulae.
( )
Total no. of disease plants
% Disease incidence DI 100
Total no. of plants
= ×
Final stand = 100 - % Disease incidence
100 DI in treatment
% Disease control 100
Disease incidence in control
−
= ×
Disease control and disease incidence were recorded at 60,
75 and 90 days of sowing. It was calculated on the average of
five plants grown per pot.
3. Results
Certain bacteria isolated from rhizosphere soils possess
properties that allow them to exert beneficial effects on
plants either by enhancing crop nutrition or by reducing
damages caused by pathogens or pests. Some of these
rhizosphere bacteria, such as Pseudomonas and Bacillus
have emerged as important biological inputs of agricultural
soils. During the present investigation, the effect of bacterial
cultures on the inhibition of growth of fungal pathogen
Rhizoctonia solani (causal agent of root rot of wheat) was
tested under culture conditions. The control of root rot dis-
ease of wheat was examined using rhizobacterial isolates
under pot house conditions.
3.1. Isolation of Rhizobacteria from the Rhizosphere Soil
Bacterial isolates were obtained from soil samples col-
lected from rhizosphere of wheat by dilution plate method
using King’s B medium. Both fluorescent and
non-fluorescent Pseudomonas and Bacillus isolates were
obtained. Originally, one hundred thirty bacterial colonies
were selected based on morphological and pigment produc-
tion characteristics. For oxidase test, different rhizobacterial
isolates were grown on KB medium plates for 2 days at
28+2°C. One percent solution of tetramethyl-p-
phenyl-diamine dihydrochloride was added to cover surface
of plates. Seventy four isolates were found oxidase positive
and these isolates belong to Pseudomonas species. Fifty six
isolates showed Gram positive staining reactions, catalase
positive and formed spores indicating that these isolates
belong to Bacillus species.
3.2. Screening of Bacterial Cultures for Growth Inhibi-
tion of Fungi
All the 202 rhizobacterial isolates/strains were screened
for their antagonistic interaction against the fungi i.e., R.
solani on PDA medium plates using spot test method[25].
Detection of antagonistic activity for rhizobacterial isolates
depended on the ability of bacteria to inhibit fungal growth
under cultural conditions. Out of 202 rhizobacterial iso-
lates tested, 16 strains were found to inhibit the growth
of R. solani (Table 1; Fig. 1). The fungal growth inhibition
zone varied from 6-15 mm with different strains/isolates
Journal of Microbiology Research 2012, 2(2): 26-35 29
tested. Eight rhizobacterial isolates i.e., WPS1, WPS3,
WPS73, NNY60, CBS14, WPS90, SB153 and SB155
showed 12-15 mm growth inhibition zone. Rhizobac-
terial isolate WPS3 showed maximum inhibition zone
(15 mm) whereas, isolate WPS90 showed 14 mm in-
hibition zone against R. solani under cultural condi-
tions. Out of 16 antagonistic isolates, 12 rhizobacterial
isolates belonged to Pseudomonas sp. and 4 cultures
were found Bacillus sp.
Tab l e 1. Inhibition of R. solani growth on PDA medium plates by Pseu-
domonas and Bacillus isolates
Rhizobacterial
isolates
Inhibition of
R. solani
(Halo zone
size)
Rhizobacterial
isolates
Inhibition of
R. solani
(Halo zone
size)
Pseudomonas isolates
Pseudomonas isolates
WPS1
13 mm
WSF300
10 mm
WPS3
15 mm
NNY19
10 mm
WPS59
-
NNY60
13 mm
WPS72
-
KNY47
9 mm
WPS73
13 mm
Bacillus isolates
WPS90
14 mm
CBS14
12 mm
WPS106
-
CBS16
-
CPS8
-
CBS25
-
CPS39
-
CBS28
10 mm
P17
6 mm
SB153
13 mm
SNY2
6 mm
SB155
13 mm
SNY3
9 mm
SYB105
-
WSF53
10 mm
Antagonistic activity of rhizobacterial isolates was tested
on the basis of growth inhibition of fungal pathogens on
PDA medium plates by spot test method[27]. The cultures
with – sign did not inhibit the growth of the fungi.
Figure 1. Antagonistic activity of different rhizobacterial isolates on PDA
medium plates. The fungi Rhizoctonia solani was presedded in the agar
medium and seven rhizobacterial isolates were spotted on the medium plates
3.3. Effect of Cell Free Culture Filtrate on Growth Inhi-
bition of R. solani
Culture filtrate of selected rhizobacterial isolates were
tested for inhibition against fungal pathogens by spot test
method. The cell free culture filtrate obtained from all the
antagonistic isolates, inhibited the fungal growth of R. solani
and the zone of inhibition varied from 6-15 mm (Table 2; Fig.
2). Cell free culture filtrate of WPS3 and WPS90 caused
maximum inhibition of the fungi. More inhibition of fungi
was observed with culture filtrate obtained from 10 day-old
growth of bacterial culture. Rhizobacterial isolate WPS3
showed 15 mm inhibition zone against R. solani, whereas
isolate WPS90 showed 14 mm inhibition zone. The cell free
culture filtrates obtained from bacterial culture NNY19
showed maximum inhibitory activity at 5th day but did not
inhibit fungal growth at 10th day of growth. Thus, cell free
culture filtrate studies of the antagonistic cultures showed
that antagonistic substance is extracellular.
Tab l e 2. Inhibition of R. solani growth by culture filterate of selected
antagonistic rhizobacterial isolates at 5 days of growth
Rhizobacterial
isolates
Inhibition of R.
solani
(Halo zone
size)
Rhizobacterial
isolates
Inhibition of R.
solani
(Halo zone
size)
Pseudomonas isolates
Pseudomonas isolates
WPS1
12 mm
WSF300
10 mm
WPS3
15 mm
NNY19
8 mm
WPS59
-
NNY60
13 mm
WPS72
-
KNY47
10 mm
WPS73
13 mm
Bacillus isolates
WPS90
14 mm
CBS14
13 mm
WPS106
-
CBS16
-
CPS8
-
CBS25
-
CPS39
-
CBS28
10 mm
P17
6 mm
SB153
13 mm
SNY2
6 mm
SB155
11 mm
SNY3
8 mm
SYB105
-
WSF53
10 mm
Antagonistic interactions of the antimicrobial substance
were determined by loading cell free culture filtrate of se-
lected rhizobacterial isolates in well made in PDA medium
plates. The cultures with – sign did not inhibit the growth of
the fungi.
Figure 2. Antagonistic activity of cultural filterates obtained from rhizo-
bacterial isolates WPS 3 and WPS 106 (control) against R. solani seeded on
PDA medium plates
3.4. Nature of Antimicrobial Substance
To determine the nature of antimicrobial substance, cul-
ture filtrate of bacterial isolates were analysed for production
30 Seema Dua et al.: Effectiveness of Rhizosphere Bacteria for Control of Root Rot
Disease and Improving Plant Growth of Wheat (Triticum aestivum L)
of specific proteins. Culture filtrate of selected rhizobacterial
isolates i.e., WPS1, WPS3, WPS73, WPS90, NNY19,
NNY60, WSF300, SNY3, CBS14, CBS25, CBS28, SB153
and SB155 were treated with concentrated trichloroacetate
(TCA) and resultant protein precipitates were solubilized in
PBS buffer. Proteins (50 µl) were loaded in wells made in
PDA medium plates that were preseeded with spore sus-
pension of R. solani. The concentration of loaded proteins
varied from 0.31-0.66 µg 50 µl-1 of sample. The fungal
growth inhibition zone varied from 6-12 mm after 48 h of
incubation (Table 3; Fig. 3). Maximum inhibition zone was
shown by proteins obtained from Pseudomonas cultures
WPS3 (12 mm) and WPS90 (11 mm). Presence of inhibition
zone by loading of precipitated proteins on PDA medium
plates showed that the antimicrobial substance could be of
proteinaceous nature.
Tab l e 3. Screening of proteins obtained from antagonistic cultures for
growth inhibition of R. solani against on PDA medium plates
Microbial culture
Protein concentration
(µg 50 µl
-1
)
Inhibition zone size
(mm, diameter)
Pseudomonas isolates
WPS1
WPS3
WPS73
WPS90
SNY3
NNY19
NNY60
WSF300
Bacillus isolates
CBS14
CBS25
CBS28
SB153
SB155
0.45
0.53
0.47
0.46
0.44
0.66
0.51
0.49
0.46
0.43
0.43
0.49
0.31
10 mm
12 mm
10 mm
11 mm
10 mm
10 mm
9 mm
6 mm
10 mm
-
6 mm
8 mm
5 mm
Figure 3. Antagonistic activity of precipitated proteins obtained from
cultural filterates of rhizobacterial isolates against R. solani seeded on PDA
medium plates. Rhizobacterial isolates used are: 1. WPS 106 (control); 2.
WPS 3; 3. SNY2; 4. WPS73; 5. CBS25, and 6. WPS90
Proteins obtained from Pseudomonas isolates WPS3 and
WPS90 were treated with proteinase K and high temperature.
The treated proteins were analysed for residual antagonistic
activity against R. solani growth on PDA medium plates.
Antagonistic activity was lost on treatment with proteinase K
and by incubation at high temperature indicating that the
antimicrobial compound is of proteinaceous nature (Fig. 4).
SDS-PAGE analysis of the total proteins from selected
antagonistic cultures followed by Coomassie blue
staining showed the presence of four common pro-
teins/polypeptides with molecular weight of 22, 25, 45
and 86 kDa in all antagonistic bacterial isolates and
none of these proteins was found in control bacterial
culture (data not shown).
Figure 4. Antagonistic activity of precipitated proteins obtained from
cultural filterates of rhizobacterial isolates WPS3 and WPS90 against R.
solani seeded on PDA medium plates. The precipitated proteins were treated
with proteinase K and high temperature before loading in the wells
3.5. Effect of Inoculation of Pseudomonas and R. Solani
on Root Rot Disease Control and Plant Growth of
Wheat
Wheat seeds inoculated either singly with Pseudomonas
isolate WPS3 or WPS90 and/or with R. solani fungi were
grown in pots containing 10 kg soil and sand mixture. Fungal
growth suspension (50 ml) was mixed in the pot containing
soil. Inoculated plants were grown under day light conditions
in the pot house during the month of November 2008 to
February 2009. Single inoculation with Pseudomonas strain
WPS3 resulted in 76.3% increase of plant dry weight at 60
days of plant growth in comparison to uninoculated control
plants. Inoculation of R. solani caused root rot disease in
85.0% of the inoculated plants (Table 4). Coinoculation of
WPS3 with R. solani enhanced 70.2% plant dry weight as
compared to uninoculated control plants. Single inoculation
with WPS90 resulted in 46.3% increase of plant dry weight,
whereas its coinoculation with R. solani enhanced 34.4%
plant dry weight as compared to uninoculated control plants.
The coinoculation of Pseudomonas strain WPS90 with R.
solani showed 58.8% disease reduction whereas 82.3%
disease reduction was observed with Pseudomonas strain
WPS3.
At 75 days of plant growth, single inoculation with
Pseudomonas isolate WPS3 caused 256% increase in root
dry weight and 249% increase in shoot dry weight (Table 4)
whereas inoculation with isolate WPS90 resulted in 140%
increase in root dry weight and only 116% increase in shoot
dry weight as compared to control uninoculated plants.
Coinoculation of isolates WPS3 and WPS90 with the fungi
showed 238% and 156% gain in shoot dry weight, respec-
tively. Maximum 88.2% disease control was observed on
coinoculation of Pseudomonas isolate WPS3 with R. solani
and only 58.8% disease reduction was observed on coin-
oculation of Pseudomonas isolate WPS90.
Journal of Microbiology Research 2012, 2(2): 26-35 31
Tab l e 4. Effect of single and mixed inoculation of Pseudomonas cultures and R. solani on root, shoot fresh and dry weight in wheat cultivar WH542 at 60
and 75 days of plant growth
Treatments
Days of plant
growth
Root fresh
wt (g)
Root dry
wt (g)
Shoot fresh
wt (g)
Shoot dry
wt (g)
Disease
incidence %
Disease con-
trol %
Control
60
75
0.222
0.268
0.031
0.142
1.924
3.734
0.296
1.051
-
-
-
-
Control +
WPS3
60
75
0.636
1.282
0.249
0.506
3.100
8.044
0.522
3.770
-
-
-
-
Control +
WPS90
60
75
0.504
0.832
0.155
0.341
2.594
5.990
0.433
2.268
-
-
-
-
Control + R.
solani
60
75
0.038
0.236
0.016
0.124
0.382
3.090
0.084
0.780
85
85
-
-
WPS3 + R.
solani
60
75
0.592
0.862
0.162
0.361
2.902
6.936
0.504
2.639
15
10
82.3
88.2
WPS90 + R.
solani
60
75
0.448
0.718
0.149
0.296
2.698
5.696
0.398
1.998
35
35
58.8
58.8
CD at 5%
60
75
0.110
0.281
0.021
0.151
0.812
1.655
0.123
0.782
Tab l e 5. Effect of single and mixed inoculation of Pseudomonas cultures and R. solani on root, shoot fresh and dry weight in wheat cultivar WH542 at 90
days of plant growth
Treatments
Root fresh wt (g)
Root dry wt (g)
Shoot fresh wt (g)
Shoot dry wt (g)
Disease incidence %
Disease control %
Control
0.320
0.180
4.322
2.494
-
-
Control + WPS3
1.472
0.692
9.378
5.754
-
-
Control + WPS90
0.696
0.358
7.268
3.655
-
-
Control + R. solani
0.270
0.167
3.734
2.016
90
-
WPS3 + R. solani
0.942
0.534
8.980
4.332
10
88.9
WPS90 + R. solani
0.814
0.368
8.286
3.996
30
66.7
CD at 5 %
0.339
0.174
3.247
0.989
The values given are average value of 5 plants. Disease
incidence is the % of plants infected and disease control is
the % reduction of diseased plants after inoculation with
bacteria. The values of plant fresh and dry weight are cal-
culated as per plant basis.
Figure 5. Effect of inoculation of isolate WPS3 and R. solani on wheat at
90 days of plant growth under pot house conditions. 1. Control; 2. Pseu-
domonas isolate WPS3; 3. R. solani; and 4. Pseudomonas isolate WPS3 + R.
solani
At 90 days of plant growth, single inoculation with
Pseudomonas strain WPS3 resulted in 131% increase of
plant dry weight as compared to uninoculated control plants
(Table 5). Single inoculation with R. solani caused 90%
disease incidence at this stage of plant growth. Coinoculation
of isolate WPS3 with R. solani enhanced 115% plant dry
weight as compared to uninoculated control plants and
caused 88.9% disease control (Fig. 5). Single inoculation
with Pseudomonas isolate WPS90 resulted in 47% gains in
plant dry weight in comparison to uninoculated control
plants and its coinoculation with R. solani caused 98% in-
crease in plant dry weight. Coinoculation of Pseudomonas
strain WPS90 with R. solani caused 66.7% disease control
(Table 5). The values given are average value of 5 plants.
Disease incidence is the % of plants infected and disease
control is the % reduction of diseased plants after inoculation
with bacteria. The values of plant fresh and dry weight are
calculated as per plant basis.
4. Discussion
Microbial populations having pathogenic, saprophytic and
plant-growth promoting ability colonize the same ecological
niche rhizosphere[6] and interact with each other as well as
with the plant through symbiotic, associative, neutralist or
antagonistic effects[1, 28, 29]. Several rhizosphere bacteria
have been reported with the potential to control various root
and foliage diseases of agricultural crops[15, 30-32].
One hundred and thirty bacterial isolates were obtained
from the wheat rhizosphere based on morphological and
pigment production characteristics. Seventy four isolates
belonged to Pseudomonas species whereas fifty six
Gram positive isolates belonged to Bacillus species.
Gupta et al.[33] isolated rhizobacteria from the rhizotic
zones of green gram using 7 selective and 4 non-selective
media. Gram negative bacteria accounted for 65% out of 121
bacteria isolated and dominant genera were Pseudomonas,
Bacillus, Enterobacter, Proteus and Klebsiella. Similarly,
Pseudomonas was found most predominant (42%) followed
by Bacillus (28%) and Enterobacter (21%) in rhizosphere
and rhizoplane of groundnut[34].
Screening of rhizobacterial isolates for fungal growth
inhibition on PDA plates showed that only 7.92% cul-
32 Seema Dua et al.: Effectiveness of Rhizosphere Bacteria for Control of Root Rot
Disease and Improving Plant Growth of Wheat (Triticum aestivum L)
tures possess the ability to inhibit pathogenic fungi R.
solani in cultural conditions (Table 1). Results of oxidase
test, catalase test, Gram and spore staining showed that
12 antagonistic rhizobacterial isolates belonged to
Pseudomonas sp. and 4 isolates were found Bacillus.
Growth inhibition of the fungi by different rhizobacterial
isolates varied from 6-15 mm (Table 1; Fig. 1). Pseu-
domonas isolate WPS3 showed maximum inhibition
zone (15 mm) followed by isolate WPS90 that showed
14 mm inhibition zone against R. solani. Khot et al.[35]
isolated 36 rhizobacteria from rhizosphere of chickpea and
five bacteria were found to inhibit the growth of Fusarium
oxysporum and Rhizoctonia bataticola. Siddiqui et al.[8]
showed that Pseudomonas aeruginosa and Bacillus
subtilis strains produced inhibition zones by inhibiting
the radial growth of Macrophomina phaseolina,
Fusarium oxysporium and Rhizoctonia solani. Le-
messa and Zeller[36] found that six strains of rhizo-
bacteria i.e., RP87, B2G, APF1, APF2, APF3 and
APF4 showed good inhibitory activity against
Rhizoctonia solanacearum out of 118 strains tested.
Ahmad et al.[2] reported that siderophore production and
antifungal activity was exhibited by 10 to 12.77% of
Azotobacter and Pseudomonas isolates. Pseudomonas Ps5
and Bacillus B1 isolates showed broad-spectrum antifungal
activity on Muller-Hinton medium against Aspergillus,
Fusarium and Rhizoctonia bataticola. Similarly, Karuppiah
and Rajaram[16] showed that eight Bacillus sp. out of
63 different Bacillus isolates exhibited plant growth
promoting activities and six of these Bacillus isolates
also inhibited the growth of Penicillium sp., Cerco-
spora sp. and Fusarium oxysporum.
The cell free culture filtrate obtained from antago-
nistic rhizobacterial isolates showed inhibition of R.
solani growth on medium plates and inhibition zone
varied from 6-15 mm (Table 2; Fig 2). Cell free culture
filtrate obtained from WPS3 and WPS90 caused
maximum inhibition of the fungi. Inhibitory effect
observed with culture filterate of the antagonistic
rhizobacterial isolates indicated that antifungal com-
pound is of extracellular nature. Maximum fungal
growth inhibition zone was obtained from the super-
natants of 10 day-old growth cultures suggesting that
the secondary metabolites may be produced in more
quantity at this growth phase of the antagonistic cul-
ture. Various studies have shown that secondary me-
tabolites produced by rhizobacterial cultures are in-
volved in antagonism of fungal pathogens[37, 38].
Nagarajkumar et al.[39] found that oxalic acid (OA) de-
toxifying fluorescent P. fluorescens strain PfMDU2 was
most effective in inhibiting the mycelial growth of R.
solani in vitro. Several proteins were detected in the
culture filtrate of P. fluorescens strain PfMDU2 when
it was grown in medium containing oxalic acid. The
plasmid-deficient strain (PfMDU2P−) failed to grow in
medium containing OA and did not inhibit the growth
of R. solani. Similarly, fluorescent Pseudomonas
isolates PGC1 and PGC2 were found to produce
chitinase and β-1, 3-glucanase that inhibited the
growth of R. solani and Phytophthora capsici[40].
Chitinase and β-1, 3-glucanase were involved in the
inhibition of R. solani, whereas antifungal metabolites
of non-enzymatic nature were found responsible for
inhibition of P. capsici.
Extracellular proteins secreted by antagonistic
cultures were found to inhibit the fungal growth on
PDA medium plates that was preseeded with R. solani
and growth inhibition zone varied from 6-12 mm
(Table 3; Fig. 3). Maximum inhibition zone was ob-
served from the proteins obtained from Pseudomonas
cultures WPS3 (12 mm) and WPS90 (11 mm). The
results suggested that excreted proteins obtained from
culture filtrates of antagonistic bacteria are responsi-
ble for the inhibition of fungi under cultural conditions.
The loss of antagonistic activity after treatment with
proteinase K and high temperature incubation indi-
cated that antifungal compound was of proteinaceous
nature. Similar high temperature treatment given to
broth culture of Lysobacter enzymogenes strain 3,
inactivated the cells and lytic enzymes did not show
inhibition zone against F. graminearum under in vitro
conditions[41]. The SDS-PAGE analysis of total
proteins obtained from different selected antagonistic
isolates followed by Coomassie blue staining showed
that four protein/polypeptide bands, i.e., 22, 25, 45 and
86 KDa are common in all the antagonistic isolates
(data not shown). It showed that any or all of the four
protein/polypeptides could be responsible for an-
tagonism of R. solani. Grover et al.[42] reported that
antifungal compound produced by Bacillus subtilis
RP24 was proteinaceous in nature. Partially purified
methanol fractions on SDS-PAGE showed one pro-
tein/peptide band with molecular weight between
1.0-1.5 kDa whereas no band was observed for the
negative mutant establishing the proteinaceous nature
of the compound. The extracellular, methanol soluble,
thermostable and pH-stable antifungal metabolites
were characterized as cyclic lipopeptides belonging to
the iturin group of peptide antibiotics. Disease sup-
pressive pseudomonads were found to produce an
antifungal polyketide (2, 3-deepoxy-2, 3-didehy-
drorhizoxin)[43]. A significant relationship between
the antagonistic potential of P. fluoresecens strain
MDU2 against Rhizoctonia solani and its production
level of extracellular β-1, 3-glucanase, chitinase,
salicyclic acid and hydrogen cyanide was ob-
served[44]. They also reported that extracellular
chitinase and laminarinase produced by P. stutzeri had
marked effect on mycelial growth inhibition rather
than spore germination.
Inoculation of wheat with Pseudomonas isolates WPS3 or
WPS90 resulted in significant increase in plant dry weight as
compared to control uninoculated plants at all the three
stages of plant growth (Table 4, 5). Maximum increase of
Journal of Microbiology Research 2012, 2(2): 26-35 33
plant dry weight (135.8%) was observed with single inocu-
lation of Pseudomonas strain WPS3 in comparison to 46.6%
increase in plant dry weight due to single inoculation of
WPS90. Inoculation with R. solani caused root rot disease in
85% of inoculated plants. However, the coinoculation of
Pseudomonas isolate WPS3 with R. solani lowered the dis-
ease incidence and 88.2% disease reduction was ob-
served (Table 4). Whereas, inoculation of Pseudomonas
isolate WPS90 with R. solani caused 58.8% disease
reduction at 60 days of plant growth. At 75 days of plant
growth, single inoculation with Pseudomonas isolate
WPS3 caused 249% increase in shoot dry weight
whereas inoculation with isolate WPS90 resulted in
only 116% increase in shoot dry weight as compared to
control uninoculated plants (Table 4). Coinoculation
of isolates WPS3 and WPS90 with R. solani showed
238% and 156% gains in shoot dry weight, respec-
tively. Maximum 88.2% disease control was observed
on coinoculation of Pseudomonas isolate WPS3 with R.
solani and only 58.8% disease reduction was observed
on coinoculation of isolate WPS90. At 90 days of plant
growth, single inoculation with Pseudomonas isolates
WPS3 and WPS90 resulted in 131% and 47% increase
of plant dry weight as compared to uninoculated con-
trol plants (Table 5) The coinoculation of Pseudomonas
isolate WPS3 with R. solani showed only 115% increase in
plant dry weight whereas coinoculation of Pseudomonas
isolate WPS90 resulted in 98% increase in plant dry weight.
Only 66.7% disease control was observed on coinoculation
of Pseudomonas culture WPS90 with R. solani whereas
coinoculation of Pseudomonas isolate WPS3 with R. solani
caused 88.9% disease control (Table 5; Fig. 5). Thus,
Pseudomonas strain WPS3 was found more effective in
controlling the root rot disease caused by R. solani.
Different microbial antagonists have been found to control
the root rot caused by Rhizoctonia solani in different
crops[45, 46]. Similarly, inoculation with P. aeruginosa
and B. subtilis was found to significantly suppress root
rot infection under green house as well as field con-
ditions and enhanced the plant growth and yield in
moongbean, wheat and maize[8]. Baig et al.[34] also
reported that effects of bacterial inoculation on plant growth
varied and plants showed stunted growth, root and shoot
elongation or a neutral response. Three bacterial isolates
increased root length while 14 isolates increased shoot length
over the uninoculated control. An increase in fresh and dry
matter was recorded by 16 bacterial strains. Sindhu et al.[14]
reported plant growth promoting effects of fluorescent
Pseudomonas sp. on coinoculation with Mesorhizobium sp.
Cicer strain under sterile and “wilt sick” soil conditions in
chick pea. The coinoculation resulted in enhanced nodula-
tion by Mesorhizobium sp. and shoot dry weight was in-
creased by 3.92 to 4.20 times in comparison to uninoculated
controls. Recently, coinoculation of siderophore-producing
Pseudomonas strain CP56 with Bradyrhizobium strain and R.
solani showed maximum 275.8% increase in plant dry
weight of green gram (Vigna radiata) at 60 days in com-
parison to control plants and also completely suppressed the
root rot disease under pot house conditions[47]. In view of
the potential application of these rhizosphere bacteria
as biocontrol agents leading to suppression of plant
diseases and due to their plant growth-promoting ef-
fects, the inoculation of plants with antagonistic
rhizobacteria is a promising area of research to achieve
maximum benefits in improvement of crop produc-
tivity.
5. Conclusions
Pseudomonads and bacilli are predominantly found in the
rhizosphere of cereal and legume crops. These rhizobacteria
have immense potential for use as biofertilizer, biocontrol
agent and/or in bioremediation due to their plant
growth-promoting ability and antagonistic activity[9, 18, 29].
During these investigations, Pseudomonas and Bacillus
isolates obtained from the rhizosphere of wheat were tested
for antagonistic effect against pathogenic fungi R. solani and
sixteen rhizobacterial isolates were found to inhibit the
growth of R. solani. Growth inhibition zone varied from 6-15
mm by different rhizobacterial isolates. The culture filterate
of selected antagonistic rhizobacterial isolates also showed
growth inhibiton of the pathogenic fungi. Single inoculation
with Pseudomonas isolates WPS3 and WPS90 resulted in
131% and 47% increase of plant dry weight as compared to
uninoculated control plants, respectively at 90 days of plant
growth. Whereas, coinoculation of Pseudomonas strain
WPS3 with R. solani showed only 115% increase in plant
dry weight and coinoculation of Pseudomonas strain WPS90
caused 98% increase in plant dry weight. Coinoculation of
Pseudomonas isolate WPS3 with R. solani also caused
88.9% disease control. Thus, complex tripartite interactions
between the rhizosphere bacteria, plant and pathogenic fungi
showed variability in disease suppression and plant growth
promotion in different Pseudomonas-inoculated treatments.
The performance of these PGPR strains has to be tested
under field conditions before their application as biocontrol
agent in commercial agriculture.
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