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Evaluation of Pseudomonas aeruginosa

Z5 for biocontrol of cotton seedling
disease caused by Fusarium oxysporum
a

Sumera Yasmin , Fauzia Y. Hafeez

ab

& Ghulam Rasul

National Institute for Biotechnology and Genetic Engineering

(NIBGE), Faisalabad, Pakistan
b

Department of Biosciences, COMSATS Institute of Information

Technology, Islamabad, Pakistan
Accepted author version posted online: 10 Jun 2014.

To cite this article: Sumera Yasmin, Fauzia Y. Hafeez & Ghulam Rasul (2014) Evaluation
of Pseudomonas aeruginosa Z5 for biocontrol of cotton seedling disease caused by
Fusarium oxysporum , Biocontrol Science and Technology, 24:11, 1227-1242, DOI:
10.1080/09583157.2014.932754
To link to this article: http://dx.doi.org/10.1080/09583157.2014.932754

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Biocontrol Science and Technology, 2014

Vol. 24, No. 11, 12271242, http://dx.doi.org/10.1080/09583157.2014.932754

RESEARCH ARTICLE
Evaluation of Pseudomonas aeruginosa Z5 for biocontrol of cotton
seedling disease caused by Fusarium oxysporum
Sumera Yasmina*, Fauzia Y. Hafeeza,b and Ghulam Rasula

Downloaded by [National Library of Biological Sciences] at 20:16 25 August 2014

National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan;
b
Department of Biosciences, COMSATS Institute of Information Technology, Islamabad,
Pakistan
(Received 21 February 2014; returned 16 May 2014; accepted 4 June 2014)
Plant growth-promoting bacteria-mediated biocontrol of plant pathogens is
renowned to enhance the growth of the plants using different direct or indirect
mechanisms. The goal of the present investigation was the evaluation of
Pseudomonas aeruginosa Z5 isolated from cotton grown in Pakistani soils for
the suppression of Fusarium oxysporum associated with cotton seedling disease. In
dual culturing techniques, four bacterial strains inhibited fungal pathogens, i.e.
F. oxysporum, Fusarium moniliforme, Fusarium solani and Rhizoctonia solani,
significantly with percent inhibition ranging from 25% to 91.5%. P. aeruginosa Z5
showed maximum suppression of all the tested pathogens. Net-house experiments
showed that the application of P. aeruginosa Z5 both separately and in
combination with Bacillus fusiformis S10 significantly reduced the disease
incidence by suppressing F. oxysporum (the causal agent of cotton seedling
disease) up to 6465% and improved the percent germination as compared to the
infected control plants. The production of antibiotics, proteases and siderophores
may be the contributing factors for its antagonistic properties. Highest bacterial
population (8.9 CFU/g root) observed on roots of cotton plants inoculated with
P. aeruginosa Z5 showed its good colonisation aptitudes even in the presence of
high inoculation of soil with F. oxysporum. Confocal laser scanning microscopy
supported the root colonisation of cotton plants with fluorescently labelled
P. aeruginosa Z5. Because of innate fungicidal potential, growth promoting
P. aeruginosa Z5 can be used as a bioinoculant and an antagonist to suppress the
growth of cotton root-associated fungal pathogen.
Keywords: Pseudomonas aeruginosa; biocontrol; cotton seedling disease; Fusarium
oxysporum

Introduction
Cotton production all over the world is susceptible to seedling diseases, which can lead
to stand losses when the disease is not managed or environmental conditions are
conducive for the disease. Seedling disease of cotton is one of the important diseases
that cause reduction in seed production and cotton lint (Howell, 2002). In cotton
seedling disease, seeds or seedlings are killed by the pathogen during the period from
seed sowing to about one month after the seedlings emergence. Seedlings of important
crop plants are damaged by different pathogens like Rhizoctonia solani, Colletotrichum

*Corresponding author. Email: sumeraimran2012@gmail.com

2014 Taylor & Francis

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1228

S. Yasmin et al.

gossypii, Fusarium oxysporum, Fusarium moniliforme, Fusarium solani and Verticillium

dahliae (Chauhan, Wadhwa, Vasudeva, & Narula, 2012). The use of fungicides for
seeds, seedlings and soil is the most common practice for the suppression of these
pathogens. The application of fungicides is not only costly but also causes the
environmental hazards. The sustainable agriculture provides eco-friendly approaches
to decrease the environmental hazards. One of these strategies is the application of
naturally occurring plant growth-promoting bacteria (PGPR) for enhanced plant
growth and suppression of plant pathogens (Pane, Villecco, Campanile, & Zaccardelli,
2012; Xue, Li, Zheng, Ding, & Guo, 2013). Different species of Pseudomonas that are
abundantly present in the soil have been regarded as a substitute of agrochemicals for
controlling plant diseases and improving the plant growth (Hofte & Altier, 2010;
Santoyo, Orozco-Mosqueda, & Govindappa, 2012). The fluorescent pseudomonads
have been reported to suppress cotton seedling disease successfully caused by Fusarium
spp., Pythium ultimum or R. solani (Weller, 2007). The primary mechanisms of
biocontrol for most of the pseudomonads are the production of secondary metabolites
like siderophores, enzymes, antibiotics and hydrogen cyanide (HCN; Bouizgarne,
2013). Plant-associated P. aeruginosa acts as both PGPR and biocontrol agents
(Hofte & Altier, 2010; Minaxi, 2010; Silva et al., 2014). P. aeruginosa has been
documented in the literature as a biocontrol agent of numerous plant diseases caused
by different pathogens like R. solani, Pythium, Fusarium, Macrophomina phaseolina,
Colletotrichum truncatum, etc. (Meon & Azadeh, 2009).
We reported in our previous study that the seed treatment of cotton plants with
P. aeruginosa Z5 and B. fusiformis S10 improved the boll mass, lint and seed yield
compared to uninoculated controls in field experiments (Yasmin, Hafeez, Schmid, &
Hartmann, 2013). In the present work, a native growth-promoting P. aeruginosa Z5
was explored for its antagonistic properties, colonisation and biocontrol potentials
against F. oxysporum, the causal agent of cotton seedling disease.

Materials and methods

Micro-organisms and growth conditions
Nine cotton rhizosphere-associated bacterial strains, i.e. B. fusiformis Z1 (AY548950),
Pseudomonas putida Z2 (AY548951), P. aeruginosa Z5 (AY548952), Bacillus sp. Z7,
P. aeruginosa Z11 (AY548953), isolate Z12, B. fusiformis S3 (AY548954), Bacillus
pumilus S9 (AY548955) and B. fusiformis S10 (AY548956), were obtained from
BRCEN Culture Collection, National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad. The bacterial strains were grown at 37 2C on
Luria Bertani agar routinely and preserved in 20% glycerol at 80C. Potato dextrose
agar (PDA) was used for culturing of fungal pathogens, i.e. F. oxysporum,
F. moniliforme, F. solani and R. solani obtained from first Fungal Culture Bank
of Pakistan, Department of Mycology & Plant Pathology, University of Punjab,
Pakistan.
Antifungal activity in plate assay
Nine cotton rhizosphere-associated bacterial strains were studied for their antagonistic activity against fungal pathogens, i.e. F. oxysporum, F. moniliforme, F. solani

Biocontrol Science and Technology

1229

and R. solani using dual culture technique (Montealgro et al., 2003). Following
formula was used to calculate the percent inhibition:

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% inhibition 1  fungal growth=control growth  100

Detection of antifungal metabolites
The production of siderophores by the antagonistic bacteria was detected and
quantified as described by Tariq, Yasmin, and Hafeez (2010). Burkholderia
vietnamiensis LA3 and Burkholderia cepecia LA8 obtained from the Department of
MicrobePlant Interactions, Helmholtz Zentrum Munchen, German Research
Center for Environment Health (GmbH), Neuherberg/Munich, Germany were
used as positive control for siderophore production.
Production of HCN by antagonistic bacterial strains was studied as described by
Lork (1948). Production of proteases and chitinases was detected on skim milk agar
(Denizci, Kazan, & Erarslan, 2004) and chitin agar media (Brien & Colwall, 1987),
respectively. A minimal medium supplemented with glucan source, i.e. Laminarin at
0.5% yeast extract, was used to assay the glucanolytic activity of the bacterial strains
(Qing, Shiping, Haibo, & Yong, 2002). Production of volatile and diffusible
antibiotics by antagonistic bacteria was determined according to the method
described by Kazempour (2004). Starch hydrolysis was studied by plating the
bacterial culture on nutrient agar (Mark), containing 2% starch (Merck) as
mentioned by Marten, Smalla, and Berg (2000).
Net-house experiments to study the biocontrol potentials of P. aeruginosa Z5
Experiment 1
P. aeruginosa Z5 was further studied at NIBGE Net House to evaluate its ability to
inhibit the cotton seedling pathogen, i.e. F. oxysporum. P. aeruginosa Z5 was grown in
liquid LB medium up to 1 109 CFU/mL, centrifuged at 8000g for 5 min, washed
with 0.85% saline and diluted to 1 108 CFU/mL with saline. Seeds of cotton variety
Bt. 121 susceptible to cotton seedling diseases used in this study were obtained from
Agricultural Biotechnology Division, NIBGE. Acid delinted cotton seeds were
surface sterilised with 0.1% HgCl2 solution for 3 min, washed repeatedly with sterile
distilled water and then dipped in bacterial suspension (1 108 CFU/mL) for 1 h. Seeds
were planted in four plastic trays of 30 cm length and 25 cm width filled with 7.5 kg
sterilised soil. The plants were watered as required and supplemented with recommended doses of N:P fertilisers. The inoculum of F. oxysporum was prepared as
mentioned by Abo-Elyousr, Hashem, and Ali (2009). Healthy control was uninoculated plants grown in trays filled with non-infested soil. Cotton seeds treated with
fungicide, i.e. Confidor WS 70 Bayer (imidacloprid 70% W/W) at 5g/kg seed, were
grown in infested soil and used as positive control. Twenty-five-day-old plants were
harvested, washed and studied for disease index (DI) percentage, and dry weights.
Root Image Analysis programme (Washington State University, USA) was used to
measure the root area of the harvested plants. DI percentage was estimated using the
following formula (Abo-Elyousr et al., 2009):

DI%

X 1A 2B 3C 4D
 100
4T

1230

S. Yasmin et al.

where, A, B, C and D are the number of plants corresponding to the numerical grade
1, 2, 3 and 4, respectively, and 4T is the total number of plants (T) multiplied by the
maximum discoloration grade 4, where T = A + B + C + D. To detect the different
degrees of disease, plants were classified into four categories: (1) discoloration of
roots up to 5%, (2) discoloration of roots up to 625%, (3) discoloration of roots up
to 2650% and (4) discoloration of roots up to 51100%.

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Experiment 2
Two bacterial strains, i.e. P. aeruginosa Z5 with highest inhibition in in vitro and
chitinase producing B. fusiformis S10 with good growth-promoting properties, were
evaluated in NIBGE net-house using earthen pots of 28 cm diameter filled with
12 kg sterilised soil/pot. The seeds of cotton variety Nuclear Institute for Biology and
Agriculture (NIAB) 846 were obtained from NIAB. Three blocks (1 cm diameter) of
F. oxysporum, grown on PDA, were homogenised thoroughly with the soil one week
before planting. Six seeds were grown initially in each pot and each treatment had
four replicates. The experiment was carried out using completely randomised design
(CRD). Healthy and positive controls were the same as mentioned in previous
experiment. Bacterial strains, i.e. P. aeruginosa Z5 and B. fusiformis S10, were
applied separately as well as in combination using seed pelleting technique. The
bacterial strains were applied at a concentration of 0.5 ml inoculum/seed containing
106107 bacterial cells/mL. Percent germination was determined seven days after
planting. Forty-five-day-old plants were harvested to record DI percentage and
studied for dry weight, plant height, root length and root area as described above.
Statistical analysis
Analysis of variance was used to study the results of measurement and the
significance was tested at 0.05% level to detect differences among treatments
by using MSTATCprogramme. Mean values and standard deviation were
determined. The pot data were analysed by CRD.
Root colonisation of P. aeruginosa Z5
Fluorescent antibodies were raised against P. aeruginosa Z5 for colonisation studies
according to the method reported by Somasegaran and Hoben (1994). The
separation of immunoglobulins, the conjugation with fluorescein isothiocyanate
(FITC) and the separation of unconjugated dye using a Sephadex G-25 column
chromatography were carried out as described by Bilal, Rasul, Arshad, and Malik
(1993). Controls were used to check the quality and the specificity of the fluorescent
antibody (FA) following the method as reported by Schmidt (1974). Pre-immune
serum processed in the same way was used as control.
For pot assays, surface-sterilised seeds of cotton variety Bt. 121 were grown in
small plastic pots containing 50 g air dried, sieved autoclaved sand under net-house
conditions. The pots with uninoculated plants were treated separately as control.
P. aeruginosa Z5 was seed-inoculated as described in Experiment 1. The plants were
kept under net-house conditions during cotton growing season and were harvested
21 days after seed germination. The intact roots were taken from inoculated and
uninoculated control plants, and the adhering soil was removed by shaking the roots
gently in sterile distilled water, separately. The roots were cut into small pieces,

Biocontrol Science and Technology

1231

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placed on a glass slide and covered with gelatin-rhodamine isothiocynate (RhITC)

conjugate. The RhITC conjugate was used to suppress non-specific adsorption of
stain. The staining of roots with specific FA was carried following the method of Bilal
et al. (1993). Fluorescent bacteria were detected by a confocal laser scanning
microscope (Olympus FV 1000, Japan). An Argon-Ion laser facilitated the wavelength of 488 nm and 525 nm for absorption and emission of FITC, respectively.
FV10-ASW 1.7 software was used for imaging.

Results
Antifungal activity in plate assay
Out of nine tested cotton rhizosphere associated bacteria, four bacterial strains, i.e.
P. aeruginosa Z5, Bacillus sp. Z7, P. aeruginosa Z11 and an isolate Z12, inhibited the
growth of Fusarium spp. and R. solani significantly on PDA up to seven days at 24
2C showing the percent inhibition ranged from 25% to 82.5%. Bacterial strains, i.e.
Z1, Z2, S3, S9 and S10, did not inhibit the fungal growth. Maximum growth
inhibition was recorded in vitro by P. aeruginosa Z5 (82.5%) followed by
P. aeruginosa Z11 with percent inhibition of 75.7. As a result of the further
screening, two strains, i.e. Z5 and Z11, produced detectable zone of inhibition
against F. oxysporum, F. moniliforme, F. solani and R. solani on agar (Table 1).
Detection of antifungal metabolites
Among all the tested cotton rhizosphere-associated bacteria (Table 1), two
P. aeruginosa strains Z5 and Z11 produced red halos around their colonies indicating
siderophore production by these bacterial strains. Quantification of siderophores
using standard succinate medium indicated maximum siderophore production
(19 mg/L) by strain Z5 (Table 1). Only P. aeruginosa Z11 and B. fusiformis S10
showed production of chitinases. All the tested strains showed the production of
proteases except isolate Z12 while all the strains did not produce glucanases. All the
tested strains showed the hydrolysis of starch but did not produce HCN. Volatile and
diffusible antibiotics produced by antagonistic bacteria inhibited the fungal pathogen
ranged from 10% to 42% and from 12% to 40%, respectively. Antibiotics produced
by P. aeruginosa Z5 showed the maximum fungal inhibition, i.e. 42% (Table 1).
Net-house experiments to study the biocontrol potentials of P. aeruginosa Z5
Experiment 1
P. aeruginosa Z5 was evaluated in plastic trays under net-house conditions compared
with fungicide, i.e. Confidor WS 70 Bayer (imidacloprid 70% W/W) and non-treated
soil. The effects of P. aeruginosa Z5 were studied for percent seed germination in the
soil infested with F. oxysporum. Fungicide provided the best seed germination
(72.5%) and maximum disease control (34% DI) followed by Z5 treatment showing
50% seed germination and 36% DI compared with infected control (Figure 1a).
Highest bacterial population (8.9 CFU/g root) was observed on roots of cotton
plants inoculated with P. aeruginosa Z5 followed by the plants treated with fungicide
(6.5 Log10 CFU/g root). Minimum bacterial population (4.8 Log10 CFU/g root) was
observed in infected control plants (Figure 1a). Overall growth-promoting effects of

% inhibition of F. oxysporum by
antibiotics produced by
antagonistic bacteriab
Strain
P. aeruginosa Z5
Bacillus sp. Z7
P. aeruginosa Z11
Isolate Z12

Siderophore productiona
(mg/L)
19
1.0
0.6
0.8

2.1
0.3
0.3
0.9

Volatile
antibiotics
42
11
26
10

Diffusible
antibiotics

3.2
3.1
2.1
1.9

Note: The values after the symbol denote standard deviation.

a
Siderophore production was measured using spectrophotometer.
b
Dual culture assay was performed in vitro on PDA plates against F. oxysporum.

40
12
24
12

2.1
2.2
1.7
1.5

% inhibition of fungal pathogens

F. oxysporum
82.5
35
75.7
25

2.1
1.1
3.2
1.7

F. moniliforme
55 4.8
0
0
0

F. solani

R. solani

40.7 3.7 86.5 3.1

0
86.5 4.1
26.7 3.5
74 2.5
0
91.5 5.1

S. Yasmin et al.

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1232

Table 1. Biocontrol determinants of cotton rhizosphere associated antagonistic bacteria.

100

% Germination

90

80

10.0

Log10 CFU

9.0
8.0

70

7.0

60

6.0

50

5.0

40

30

10

4.0
3.0

20

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% DI

1233

Log10 CFU/ g root.

% Germination/ DI

Biocontrol Science and Technology

2.0
1.0

0.0
Healthy control Infected control

Fungicide

P. aeruginosa Z5

Treatments

Figure 1. (Colour online) (a) Effect of P. aeruginosa Z5 on percent (%) germination and
percent disease index (% DI) of cotton seedlings using soil articially infested by a cotton
seedling pathogen, i.e. F. oxysporum in plastic trays under net-house conditions. Twenty-veday-old plants were used for % germination, % DI and viable bacterial cells. Means are the
average of four replicates. Means followed by the same letter differ non-signicantly at p =
0.05 according to DMRT. Healthy control, untreated control; infected control, inoculated
with pathogen only; fungicide, Condor WS 70 Bayer (imidacloprid 70% W/W) inoculated
with pathogen; and P. aeruginosa Z5, inoculated with antagonistic P. aeruginosa Z5 and
pathogen. (b) Effect of P. aeruginosa Z5 on different growth parameters of cotton seedlings
using soil articially infested by a cotton seedling pathogen, i.e. F. oxysporum in plastic trays
under net-house conditions. Twenty-ve-day-old plants were used to study plant height, root
length, root area and plant dry weight. Means are the average of four replicates. Means
followed by the same letter differ non-signicantly at p = 0.05 according to DMRT. Bars
represent the standard deviation.

Z5 on plant dry weight, plant height, root length and diameter were higher or
equivalent to those of the fungicide treatment (Figure 1b).
Experiment 2
Two bacterial isolates one with high antagonistic activity to F. oxysporum, i.e.
P. aeruginosa Z5, and the other one, i.e. B. fusiformis S10, with growth-promoting
potential (Yasmin et al., 2013) were selected for evaluation as compared to a
fungicide, i.e. Confidor WS 70 Bayer and a non-treated soil under net-house
conditions. Mixed treatment of P. aeruginosa Z5 and B. fusiformis S10 gave the
highest reduction in disease incidence (35% DI) followed by Z5 treatment (39% DI)
as compared to the infected control (Figure 2a). Maximum increase in the
percentage of germination (62.5%) was observed with the uninoculated healthy
control plants followed by fungicide (55%) and Z5 (52%) treatments as compared to
the infected control. Highest bacterial population (9.8 Log10 CFU/g root) was
observed on roots of cotton plants inoculated with P. aeruginosa Z5 followed by the
plants with mixed inocula of Z5 and S10 strains, i.e. 8.9 Log10 CFU/g root.
Minimum bacterial population (4.6 Log10 CFU/g root) was observed in infected
control plants (Figure 2a). Dry weight of seedlings was enhanced as compared to the
infected control plants in response to mixed treatment of Z5 and S10 as well as of
fungicide application (Figure 2b).

1234

S. Yasmin et al.

(cm) Plant height

25

20

B
15

10

Root Length

A
-5

Healthy control

Infected control

Plant height (cm)

Plant dry weight (g)

Fungicide

P. aeruginosa Z5

Root length (cm)

0.40
0.35

B
BC

0.30
0.25

0.20
0.15
0.10
0.05
0.00

C
1.4

A
AB

1.2

Root area (mm)

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BC

1.0
0.8

0.6
0.4
0.2
0.0
Healthy control

Figure 1. Continued

Infected control

Fungicide

P. aeruginosa Z5

Biocontrol Science and Technology

% Germination

% DI

1235

Log10 CFU
9

% Germination/ DI

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90

80
70

A
B

60

B
C

50

C
C

40

20
10

4
3

30

Log10 CFU/ g roots .

10

100

1
0

Healthy
control

Infected
control

Fungicide Pseudomonas Bacillus sp.

sp. Z5
S10

Mixed

Treatments
Figure 2. (Colour online) (a) The effect of P. aeruginosa Z5 (as a single inoculant or in mixed
inocula) on percent (%) germination and percent disease index (% DI) of cotton seedlings
using soil articially infested by a cotton seedling pathogen, i.e. F. oxysporum, in a pot
experiment under net-house conditions. Forty-ve-day-old plants were used for % germination, % DI and viable bacterial cells. Means are the average of four replicates. Means followed
by the same letter differ non-signicantly at p = 0.05 according to DMRT. Bars represent the
standard deviation. Healthy control, untreated control; infected control, inoculated with
pathogen only; fungicide, Condor WS 70 Bayer (imidacloprid 70% W/W) inoculated with
pathogen; P. aeruginosa Z5, inoculated with antagonistic P. aeruginosa Z5 and pathogen;
Bacillus sp., inoculated with B. fusiformis S10 and pathogen; and mixed, inoculated with
P. aeruginosa Z5 + B. fusiformis S10 + pathogen. (b) The effect of P. aeruginosa Z5 (as a
single inoculant or in mixed inocula) on different growth parameters of cotton seedlings using
soil articially infested by a cotton seedling pathogen, i.e. F. oxysporum in a pot experiment
under net-house conditions. Forty-ve-day-old plants were used to study growth parameters.
Bars represent the standard deviation.

Root colonisation of P. aeruginosa Z5

Using immunofluorescence microscopy and confocal laser scanning microscopy
(CLSM), attachment of P. aeruginosa Z5 on roots was directly examined.
P. aeruginosa Z5 was found to colonise all over the cotton roots when inoculated
separately, indicating colonisation. Fluorescently labelled bacterial cells were
apparently localised in intercellular spaces as well as on the root cells at 100
resolution. Roots of uninoculated control plants did not produce any fluorescence.
Bacterial cells were observed as single cells associated with the surface of roots as
well as doublets on the root in the form of a chain. Colonisation was observed
around the whole surface of few rhizodermal cells and in the form of microcolonies
(Figure 3).

Discussion
Rhizobacteria-mediated promotion of plant growth has been the main focus of
research because of its practical importance. Field application of these bacteria has

1236

S. Yasmin et al.
A

(cm) Plant height

AB

45

BC
35

25

Root length

-5

Healthy
control

Infected
control

Fungicide

Pseudomonas
sp. Z5

Bacillus sp.
S10

Mixed

Plant height (cm)

Root length (cm)

Plant dry weight (g)

3.0

2.5

2.0

BC

1.5
1.0

0.5
0.0
5.0

Root area (mm)

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15

4.0

3.0

CD
2.0

BC

B
D

1.0
0.0
Healthy
control

Infected
control

Fungicide

Pseudomonas
sp. Z5

Bacillus sp.
S10

Mixed

Figure 2. Continued

tremendous potential for plant growth and biocontrol of plant pathogens. Previous
studies reported the occurrence of Fusarium spp. associated with cotton seedling
diseases in Pakistan. Several fungicides like carbendazin, thiophanate methyl, thiovit

Downloaded by [National Library of Biological Sciences] at 20:16 25 August 2014

Biocontrol Science and Technology

1237

Figure 3. (Colour online) Colonisation of 21-day-old cotton roots by antagonistic

P. aeruginosa using FA staining and CLSM. Fluorescence image was obtained from CLSM
of whole root after preparation and staining by FA technique. Plant assay was carried out in
sterile sand under net-house conditions.

and dithanewere, etc., reported to reduce the Fusarium infection in cotton to

different extent. Confidor WS 70 Bayer (imidacloprid 70%) is being applied as
insecticide and seed treatment of cotton in Pakistan (Javed, Hanif, Niaz, & Ali,
2008). Application of these fungicides is expensive and has the environmental
hazards too. Vigorous growth can help the seedlings to survive and compete under
stress condition (Hafeez, Safdar, Chaudhary, & Malik, 2004). Biological control by
antagonistic bacteria may be one of indirect mechanisms of growth promotion that
cause the suppression of disease by reducing the time in which a plant is in the
susceptible state thereby decreasing the incidence of cotton seedling disease.
Different species of fluorescent Pseudomonas such as P. aureofaciens, P. aeruginosa,
P. fluorescence, P. putida and P. pyrrocinia were reported to have antifungal activity
with varying degrees of antagonism (Bouizgarne, 2013; Hassanein, Awny, ElMougith, & El-Dien, 2009). Therefore, this research was directed at basic and
applied aspects of using P. aeruginosa as microbial inoculant to provide biological
control of F. oxysporum causing cotton seedling disease. In vitro and in vivo
evaluations were conducted to provide biological support for more basic
investigations.
P. aeruginosa Z5 isolated from cotton grown in Pakistani soils showed the
suppression of all the tested fungal pathogens of F. oxysporum, F. moniliforme,
F. solani and R. solani. Various fungal pathogens like P. ultimum, R. solani and
Fusarium spp., i.e. F. oxysporum, F. moniliforme, F. solani attack cotton seedlings.
Pre- or post-emergence cotton seedling damping-off caused by these well-known
phytopathogens often results in a substantial stand loss. Fluorescent Pseudomonas

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S. Yasmin et al.

spp. including P. aeruginosa have been reported to protect the crop plants with
enhanced inoculation effects (Fang et al., 2013; Hofte & Altier, 2010). Pseudomonas
fluorescens CS85, isolated from cotton seedlings rhizosphere, reported to promote
the plant growth as well as acted as a biocontrol agent for fungal pathogens of
cotton, e.g. F. oxysporum, V. dahliae, R. solani and C. gossypii (Wang, Wang, &
Zhou, 2004).
P. aeruginosa Z5 showed maximum siderophores production (Table 1) which
indicated the potential of this bacterium for its application in protecting the plants
from pathogens. Disease reduction due to siderophores decreases the available iron
required for the germination of fungal spores and the hyphal growth of the fungal
pathogens. This ultimately results in the suppression of pathogen from the plant
(Bouizgarne, 2013; Sadeghi et al., 2012). Previous studies also identified the
siderophore production by antagonistic bacteria as their main biocontrol mechanism
(Naureen, Hafeez, & Roberts, 2011).
Production of proteases, chitinases and glucanases was studied as some of the
important mechanisms for the inhibition of fungal pathogens. Strains Z5, Z7 and
Z11 showed the production of proteases while Z11 also showed the production of
chitinases. The role of proteolytic enzymes in biocontrol can often be assigned to
antibiosis and parasitism. Although B. fusiformis S10 did not suppress the tested
fungal pathogens in in vitro tests, it showed the production of chitinases. The use of
lytic enzymes-producing bacteria in combination with other biological agents was
documented to have synergistic effects for the suppression of plant diseases
(Bouizgarne, 2013).
Volatile and diffusible antibiotics produced by P. aeruginosa Z5 showed the
maximum fungal inhibition in in vitro test (Table 1). Most of the antagonistic
Pseudomonas spp. was reported to produce one or more antibiotics with antifungal
properties in vitro. The production of different antibiotics is a plant growthpromoting property that is usually considered for the antagonistic activity of the
bacteria to inhibit the growth of plant pathogens (Glick, 2012). The bacterial strains
under study were able to secrete defensive metabolites involved in biocontrol.
Presence of biocontrol traits such as production of siderophores, antibiotics and
proteases without HCN enhances the potential use of P. aeruginosa Z5 as an
effective biocontrol agent for cotton.
Net-house experiment to study the biocontrol potential of antagonistic bacteria
Z5 against F. oxysporum on cotton indicated that the application of Z5 either
separately or in combination with a chitinase-producing B. fusiformis S10 significantly decreased the incidence of cotton seedling disease caused by F. oxysporum and
improved the percent germination as compared to the infected control plants.
Percent germination was not significantly different between Z5 (52%) and fungicide,
i.e. Confidor WS 70 (55%) treatments. In these trials, bacterial strain Z5 showed the
suppression of F. oxysporum up to 6465%. This effect was equivalent to that of used
fungicide. B. fusiformis S10 showed more suppression of disease when applied in
combination with Z5 as compared to the treatment where S10 was inoculated
separately. Previous reports also showed that a mixture of P. fluorescens and
B. subtilis was effective against the causal pathogens of seedling disease in cotton
(Chauhan et al., 2012).
Mixed inoculation of Z5 and S10 showed significant effects on plant growth
parameters like dry weights, plant height, root area and length as compared to the

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1239

infected control plants. These results were found to be in accordance with the
previous studies showing that indole acetic acid (IAA)-producing B. fusiformis S10
used either singly or in combination with P. aeruginosa Z5, i.e. a phosphate (P) and
zinc (Zn) mobilizer with the production of IAA and Aminocyclopropane-1carboxylate (ACC) deaminase, was an efficient bacterium promoting the yield of
cotton with reduced chemical fertilisers (Yasmin et al., 2013). The direct role of IAA
production by the bacterial strains was reported for suppressing the charcoal rot of
chickpea by improving root system, nutrient uptake and protected the plants from
Microphomina phaseolina (Khare & Arora, 2010). Highest bacterial population
observed on roots of cotton plants inoculated with P. aeruginosa Z5 showed its
survival and good colonisation aptitudes even in the presence of inoculated pathogen
(Figure 2a).
It was usually an established concept that only those growth promoting bacteria
are beneficial which effectively and successfully colonise the roots and persist in the
plant rhizosphere (Benizri, Baudoin, & Guckert, 2001; Bouizgarne, 2013; Compant,
van der Heijden, & Sessitsch, 2010). Therefore, a study of root colonisation by
P. aeruginosa Z5 was conducted by tagging the strain with fluorescent antibodies
observed with CLSM. Cells of strain Z5 were detected on the roots as strings of
bacterial cells as well as microcolonies. The findings of root colonisation by
P. aeruginosa Z5 also support its biological control and plant growth enhancement
abilities (Dutta & Podile, 2010). Immunofluorescence technique was successfully
used with viable counts to monitor the colonisation of bacteria in soil or on roots
(Gamalero, Lingua, Berta, & Lemanceau, 2003). Previous studies in literature
showed that the efficient colonisation of the bacterial strains might play an
important role to inhibit fungal infection of cotton plants by F. oxysporum
(Compant et al., 2010). Biocontrol activity of fluorescent Pseudomonas spp. against
different phytopathogens has been attributed to their efficient root colonisation
(Gamalero, Berta, Massa, Glick, & Lingua, 2010).
Present study reported the antagonistic traits of a native strain of P. aeruginosa
Z5. Because of its innate fungicidal potential and good colonisation aptitudes,
P. aeruginosa Z5 can be used as a bio-inoculant and an antagonist against
phytopathogenic fungi that cause seedling diseases in cotton. The production of
antibiotics, proteases and siderophores was found to be the contributing factors for
its antagonistic properties against phytopathogens. P. aeruginosa Z5 can play an
essential role in helping plants to establish and grow even in the presence of
phytopathogenic fungi associated with cotton seedlings. Previous reports also
documented Pseudomonas spp. as a biocontrol agent with plant growth promotion
(Bhattacharyya & Jha, 2012; Hofte & Altier, 2010; Ramadasappa, Ashwani, Jaat,
Singh, & Rai, 2012). Growth promotion and enhanced yield of cotton due to
inoculation of Z5 under field conditions have already been reported (Yasmin et al.,
2013), but further evaluation for suppression of cotton seedling disease caused by
F. oxysporum has to be carried out in field trials. The present study helps us in the
selection of environmentally novel inoculants for cotton with dual traits, i.e.
biofertilizer with biocontrol potential. However, further studies should be carried
out to clarify the growth temperature of P. aeruginosa Z5 since these bacteria can
grow above 32C and may become a human pathogen. P. aeruginosa has been
reported as an important toxin producer and it should be tested for the pathogenic
potential before its use in agriculture. Moreover, B. fusiformis is known for its

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S. Yasmin et al.

growth-promoting traits but the present study indicated its potential use along with
other biocontrol agent for enhanced plant growth and protection. Because of
important capabilities of practical utility, a novel growth-promoting antagonistic
P. aeruginosa Z5 was deposited to DSMZ German Culture Collection with accession
no. DSM16519.
Acknowledgement

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We are thankful to Mr. Tariq Mehmood, Mr. Naveed and Mr. Tariq Shah (Technical
Assistant, NIBGE) for their assistance.

Funding
This work was supported by IAEA TC Project No. PAK/5/037 and BIRCEN project funded
by IDB.

References
Abo-Elyousr, K. A. M., Hashem, M., & Ali, E. H. (2009). Integrated control of cotton root
rot disease by mixing fungal biocontrol agents and resistance inducers. Crop Protection, 28,
295301. doi:10.1016/j.cropro.2008.11.004
Benizri, E., Baudoin, E., & Guckert, A. (2001). Root colonization by inoculated plant growth
promoting rhizobacteria. Biocontrol Science and Technology, 11, 557574. doi:10.1080/
09583150120076120
Bhattacharyya, P. N., & Jha, D. K. (2012). Plant growth-promoting rhizobacteria (PGPR):
Emergence in agriculture. World Journal of Microbiology and Biotechnology, 28, 13271350.
doi:10.1007/s11274-011-0979-9
Bilal, R., Rasul, G., Arshad, M., & Malik, K. A. (1993). Attachment, colonization and
proliferation of Azospirillum brasilense and Enterobacter spp. on root surfaces of grasses.
World Journal of Microbiology and Biotechnology, 9, 6369. doi:10.1007/BF00656519
Bouizgarne, B. (2013). Bacteria for plant growth promotion and disease management. In
D. K. Maheshwari (Ed.), Bacteria in agrobiology: Disease management (pp. 1547). Berlin
Heidelberg: Springer-Verlag.
Brien, M. O., & Colwall, R. R. (1987). A rapid test for chitinase activity that uses 4methylumbelliferyl N-acetyl-beta D-Glucosamine. Applied Environmental Microbiology, 53,
17181720.
Chauhan, S., Wadhwa, K., Vasudeva, M., & Narula, N. (2012). Potential of Azotobacter spp.
as biocontrol agents against Rhizoctonia solani and Fusarium oxysporum in cotton
(Gossypium hirsutum), guar (Cyamopsis tetragonoloba) and tomato (Lycopersicum esculentum). Archives of Agronomy and Soil Science, 58, 121.
Compant, S., van der Heijden, M. G., & Sessitsch, A. (2010). Climate change effects on
beneficial plant-microorganism interactions. FEMS Microbiology and Ecology, 73, 197214.
Denizci, D., Kazan, E. C. A., & Erarslan, A. A. (2004). Newly isolated Bacillus clausii
GMBAE42, an alkaline protease producer capable to grow under highly-alkaline conditions. Journal of Applied Microbiology, 96, 320327. doi:10.1046/j.1365-2672.2003.02153.x
Dutta, S., & Podile, A. R. (2010). Plant growth promoting rhizobacteria (PGPR): The bugs to
debug the root zone. Critical Review of Microbiology, 36, 232244. doi:10.3109/
10408411003766806
Fang, R., Lin, J., Yao, S., Wang, Y., Wang, J., Zhou, C., Xiao, M. (2013). Promotion of
plant growth, biological control and induced systemic resistance in maize by Pseudomonas
aurantiaca JD37. Annals of Microbiology, 63, 11771185. doi:10.1007/s13213-012-0576-7
Gamalero, E., Berta, G., Massa, N., Glick, B. R., & Lingua, G. (2010). Interactions between
Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences on the
growth cucumber under salt stress conditions. Journal of Applied Microbiology, 108, 236
245. doi:10.1111/j.1365-2672.2009.04414.x

Downloaded by [National Library of Biological Sciences] at 20:16 25 August 2014

Biocontrol Science and Technology

1241

Gamalero, E., Lingua, G., Berta, G., & Lemanceau, P. (2003). Methods for studying root
colonization by introduced beneficial bacteria. Agronomie, 23, 407418. doi:10.1051/
agro:2003014
Glick, B. R. (2012). Plant growth-promoting bacteria: Mechanisms and applications.
Scientifica, 2012, 115. doi:10.6064/2012/963401
Hafeez, F. Y., Safdar, M. E., Chaudhary, A. U., & Malik, K. A. (2004). Rhizobial inoculation
improves seedling emergence, nutrient uptake and growth of cotton. Australian Journal of
Experimental Agriculture, 44, 16. doi:10.1071/EA03074
Hassanein, W. A., Awny, N. M., El-Mougith, A. A., & El-Dien, S. H. S. (2009).
Characterization and antagonistic activities of metabolite produced by Pseudomonas
aeruginosa Sha8. Journal of Applied Science Research, 5, 404414.
Hofte, M., & Altier, N. (2010). Fluorescent pseudomonads as biocontrol agents for
sustainable agricultural systems. Research in Microbiology, 161, 464471. doi:10.1016/j.
resmic.2010.04.007
Howell, C. R. (2002). Cotton seedling pre-emergence damping-off incited by Rhizopus oryzae
and Pythium spp. and its biological control with Trichoderma spp. Phytopathology, 92, 177
180. doi:10.1094/PHYTO.2002.92.2.177
Javed, M. S., Hanif, M., Niaz, M., & Ali, I. (2008). Impact of storage period and temperature
on the pathogenic behaviour of Fusarium solani on cotton (Gossypium hirsutum L.) seeds.
Mycopath, 6, 711.
Kazempour, M. N. (2004). Biological control of Rhizoctonia solani, the causal agent of rice
sheath blight by antagonistic bacteria in greenhouse and field conditions. Plant Pathology
Journal, 3, 8896. doi:10.3923/ppj.2004.88.96
Khare, E., & Arora, K. N. (2010). Effect of indole-3-acetic acid (IAA) produced by
Pseudomonas aeruginosa in suppression of charcoal rot disease of chickpea. Current
Microbiology, 61, 6468. doi:10.1007/s00284-009-9577-6
Lork, H. (1948). Production of hydrocyanic acid by bacteria. Physiology Plant, 1, 142146.
doi:10.1111/j.1399-3054.1948.tb07118.x
Marten, P., Smalla, K., & Berg, G. (2000). Genotypic and phenotypic differentiation of an
antifungal biocontrol strain belonging to Bacillus subtilis. Journal of Applied Microbiology,
89, 463471. doi:10.1046/j.1365-2672.2000.01136.x
Meon, S., & Azadeh, B. F. (2009). Molecular characterization of Pseudomonas aeruginosa
UPM P3 from oil palm rhizosphere. American Journal of Applied Science, 6, 19151919.
doi:10.3844/ajassp.2009.1915.1919
Minaxi, Saxena J. (2010). Characterization of Pseudomonas aeruginosa RM-3 as a potential
biocontrol agent. Mycopathologia, 170, 181193. doi:10.1007/s11046-010-9307-4
Montealgro, J. R., Reyes, R., Perez, L. M., Herrera, R., Silva, P., & Besoain, X. (2003).
Selection of bioantagonistic bacteria to be used in biological control of Rhizoctonia solani in
tomato. Electronic Journal of Biotechnology, 6, 115127.
Naureen, Z., Hafeez, F. Y., & Roberts, M. (2011). Biological control of sheath blight disease
of rice by siderophore producing rhizobacterial strains and their role in efficient
mobilisation of micronutrients from soil. Current Opinion in Biotechnology, 22(Suppl),
S48S48. ISSN 0958-1669. doi:10.1016/j.copbio.2011.05.124
Pane, C., Villecco, D., Campanile, F., & Zaccardelli, M. (2012). Novel strains of Bacillus,
isolated from compost and compost-amended soils, as biological control agents against soilborne phytopathogenic fungi. Biocontrol Science and Technology, 22, 13731388.
doi:10.1080/09583157.2012.729143
Qing, F., Shiping, T., Haibo, L., & Yong, X. (2002). Production of -1,3-glucanase and
chitinase of two biocontrol agents and their possible modes of action. Chinese Science
Bulletin, 47, 292296. doi:10.1360/02tb9070
Ramadasappa, S., Ashwani, K. R., Jaat, R. S., Singh, A., & Rai, R. (2012). Isolation and
screening of phlD+ plant growth promoting rhizobacteria antagonistic to Ralstonia
solanacearum. World Journal of Microbiology and Biotechnology, 28, 16811690.
doi:10.1007/s11274-011-0975-0
Sadeghi, A., Karimi, E., Dahaji, P. A., Javid, M. G., Dalvand, Y., & Askari, H. (2012). Plant
growth promoting activity of an auxin and siderophore producing isolate of Streptomyces

Downloaded by [National Library of Biological Sciences] at 20:16 25 August 2014

1242

S. Yasmin et al.

under saline soil conditions. World Journal of Microbiology and Biotechnology, 28, 1503
1509. doi:10.1007/s11274-011-0952-7
Santoyo, G., Orozco-Mosqueda, M. C., & Govindappa, M. (2012). Mechanisms of biocontrol
and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas:
A review. Biocontrol Science and Technology, 22, 855872. doi:10.1080/09583157.2012.
694413
Schmidt, E. L. (1974). Qualitative autecological study of microorganisms in soil by
immunofluorescence. Soil Science, 118, 141149. doi:10.1097/00010694-197409000-00002
Silva, Vasconcellos F., de Oliveira, A., Lopes-Santos, L., Beranger, O., Cely, T. M.,
Simionato, A., Andrade, G. (2014). Evaluation of antibiotic activity produced by
Pseudomonas aeruginosa LV strain against Xanthomonas arboricola pv. pruni. Agricultural
Sciences, 5, 7176. doi:10.4236/as.2014.51008
Somasegaran, P., & Hoben, H. J. (1994). Producing and applying fluorescent antibodies. In
Hand book for rhizobia: Methods in legume-rhizobium technology (pp. 120130). New York:
Springer-Verlag.
Tariq, M., Yasmin, S., & Hafeez, F. Y. (2010). Biological control of potato black scurf by
rhizosphere associated bacteria. Brazilian Journal of Microbiology, 41, 439451. doi:10.1590/
S1517-83822010000200026
Wang, C., Wang, D., & Zhou, Q. (2004). Colonization and persistence of a plant growthpromoting bacterium Pseudomonas fluorescens strain CS85, on roots of cotton seedlings.
Canadian Journal of Microbiology, 50, 475481. doi:10.1139/w04-040
Weller, D. M. (2007). Pseudomonas biocontrol agents of soil borne pathogens: Looking back
over 30 years. Phytopathology, 97, 250256. doi:10.1094/PHYTO-97-2-0250
Xue, Q. Y., Li, J. Q., Zheng, Y., Ding, X. Y., & Guo, J. H. (2013). Screening tomatoassociated bacteria for biological control of grey mold on tomato. Biocontrol Science and
Technology, 23, 245259. doi:10.1080/09583157.2012.755612
Yasmin, S., Hafeez, F. Y., Schmid, M., & Hartmann, A. (2013). Plant-beneficial rhizobacteria
for sustainable increased yield of cotton with reduced level of chemical fertilizers. Pakistan
Journal of Botany, 45, 655662.