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State Key Laboratory of Grassland Agro-Ecosystems, College of Pastoral Agricultural Science and Technology,
Lanzhou University, Lanzhou 730000, P. R. China.
B
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
C
Institute of Agriculture, School of Earth and Environment, The University of Western Australia,
35 Stirling Highway Crawley, WA 6009, Australia.
D
Departments of Nurse and Chinese Medicine, Gansu University of Traditional Chinese Medicine, Lanzhou,
Gansu, 730000, P. R. China.
E
Department of Biology, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China.
F
Corresponding author. Email: jlzhang@lzu.edu.cn
Abstract. Bacillus subtilis strain GB03 enhances growth and photosynthesis in the model plant Arabidopsis thaliana and
several crop plants. In the present study, the effects of seed soaking with GB03 suspension culture and its volatile organic
compounds on seed germination of Codonopsis pilosula (Franch.) Nannf. were investigated, and soil-grown C. pilosula
seedlings were assayed to measure growth and photosynthetic capacity after soil inoculation with GB03. Both seed soaking
with GB03 suspension culture and the presence of volatile organic compounds enhanced seed germination, especially seed
germination vigour. GB03 signicantly improved shoot and root length, branching, plant biomass (whole plant fresh and dry
weight), leaf area and chlorophyll content in C. pilosula seedlings after 20, 40 and 60 days of soil inoculation. GB03
signicantly enhanced transpiration rate, stomatal conductance and net photosynthetic rate, but decreased intercellular CO2
concentration. This study provides insight for the application of selected bacteria to improve biomass in Chinese herbal crops.
Additional keywords: Bacillus subtilis, Codonopsis pilosula, growth promotion, photosynthesis, seed germination.
Received 1 April 2015, accepted 17 August 2015, published online 14 January 2016
Introduction
Plant growth-promoting rhizobacteria (PGPRs) are naturally
occurring soil microorganisms able to colonise roots and
stimulate plant growth (de Zelicourt et al. 2013; Gao et al.
2013). PGRPs have been applied to a wide range of
agricultural crops for the purpose of growth enhancement,
including improved seed germination and establishment, plant
biomass, yield, nutrient uptake efciency, and biotic and
abiotic stress tolerance (Kloepper et al. 1991; Zhang et al.
2008a; Harvey et al. 2009; de Zelicourt et al. 2013; Song and
Ryu 2013; Han et al. 2014). PGPR colonisation has also been
shown to activate bacterial synthesis of plant hormones, including
indole-3-acetic acid, cytokinin and gibberellin, which have
been correlated with PGPR-mediated plant-growth promotion
(MacDonald et al. 1986; Timmusk et al. 1999).
As an important species among PGPRs, Bacillus subtilis can
be isolated from many environments, terrestrial and aquatic, and
can adapt to grow in diverse conditions within the biosphere
*
www.publish.csiro.au/journals/cp
Y.-N. Wu et al.
120
90
(a)
b
80
ab
60
30
92
(b)
a
60
b
c
40
d
20
0
Control
10
20
Control
10
20
GB03
Fig. 1. (a) Germination rate and (b) germination vigour of C. pilosula seeds soaked in water control
or Bacillus subtilis GB03 suspension culture for various periods (5, 10 and 20 min). Capped vertical lines
are standard deviations (n = 6). Means with the same letter are not signicantly different at P = 0.05
(Duncans test).
5 mm
2 mm
GB03
Control
Fig. 2. Germinated C. pilosula seeds soaked in Bacillus subtilis GB03 suspension culture for 5 min (left) or in Luria broth
medium for 5 min (control, right).
100
(a)
60
a
b
80
60
40
20
0
120
(b)
a
40
b
bc
DH5
LB
Water
20
0
GB03
DH5
LB
Water
GB03
Treatments
Fig. 3. (a) Germination rate and (b) germination vigour of C. pilosula seeds with exposure to Bacillus subtilis
GB03, Escherichia coli DH5a, Luria broth (LB) and water control after 10 days. Capped vertical lines are
standard deviations (n = 6). Means with the same letter are not signicantly different at P = 0.05 (Duncans test).
93
Y.-N. Wu et al.
were separated and blotted gently with paper towel. Fresh weights
were determined immediately and samples were oven-dried at
808C for 2 days for dry weight measurement.
Photosynthetic capacity measurements
Leaf area, chlorophyll content, transpiration rate, stomatal
conductance, intercellular CO2 and net photosynthetic rate were
determined. Whole plant leaves were harvested to measure leaf
area per plant via a leaf area meter (Perfection 4870 Photo
scanner; Epson America Inc., Long Beach, CA, USA) for the
(c)
5 cm
GB03
(a)
Control
(b)
GB03
Control
GB03
Control
Fig. 4. Growth promotion of C. pilosula plants treated with Bacillus subtilis GB03 for various periods: (a) 20 days, (b) 40 days,
(c) 60 days. GB03 suspension culture is in Luria broth (LB) medium; control is LB medium without bacteria.
40
(a)
30
30
a
b
20
c
10
f
0
f
0
d e
20
40
60
94
(b)
a
b
20
c
d
e
10
f
0
f
0
20
40
60
Statistical analyses
Germination rate, germination vigour, shoot height, root length,
branching, fresh weight, dry weight, leaf area, chlorophyll
content, transpiration rate, stomatal conductance, intercellular
CO2 and net photosynthetic rate are presented as means with
standard deviations (n = 6). Data were subjected to analysis of
variance (ANOVA) and Duncans multiple range tests were
performed to detect differences among means at a signicance
level of P = 0.05 by using the software SPSS 13.0 (SPSS Inc.,
Chicago, IL, USA).
95
Results
GB03 augmented seed germination of C. pilosula
b
2
c
1
g g
0
20
40
60
Branch number
c
c
d
e
20
40
60
0.6
(a)
a
a
6
(b)
a
0.4
b
0.2
c
d e
20
40
60
Y.-N. Wu et al.
Discussion
60
(a)
a
b
40
c
d
20
e
g g
0
20
f
40
50
(b)
a
40
30
10
60
20
20
40
60
(a)
150
a
2
1
Stomatal conductance
(mmol m2 s1)
Transpiration rate
(g m2 h1)
500
Intercellular CO2
(ppm)
a
100
50
(c)
a
400
(b)
300
200
100
0
96
(d )
a
3
b
2
1
0
GB03
LB
GB03
LB
Fig. 9. (a) Transpiration rate, (b) stomatal conductance, (c) intercellular CO2 concentration and (d) net
photosynthetic rate of C. pilosula seedlings treated with Bacillus subtilis GB03 for 60 days. Shaded and
unshaded bars represent GB03 treatment and Luria broth control, respectively. Capped vertical lines are
standard deviations (n = 6). For each parameter, different letters indicate signicant difference at P = 0.05
(Duncan tests).
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