Plant Soil (2015) 390:337–349
DOI 10.1007/s11104-015-2410-z
REGULAR ARTICLE
Soil inoculation with Burkholderia sp. LD-11 has positive effect
on water-use efficiency in inbred lines of maize
Xianwei Fan & Haiyang Hu & Guiyuan Huang &
Feiyan Huang & Youzhi Li & Jairo Palta
Received: 10 July 2014 / Accepted: 4 February 2015 / Published online: 12 February 2015
# Springer International Publishing Switzerland 2015
Abstract
Background and aims Plant-growth promoting
rhizobacteria (PGPR) can promote plant performance
under water deficit, but the physiology and biochemistry
of the promoting process induced by PGPR under dif-
ferent water deficits is not well known in maize (Zea
mays L).
Methods A glasshouse study was conducted to deter-
mine the effects of Burkholderia sp. LD-11 on
morphophysiological traits for plant growth and homeo-
stasis between reactive oxygen species (ROS) and
Responsible Editor: Jesus Mercado-Blanco.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-015-2410-z) contains supplementary
material, which is available to authorized users.
X. Fan (*) : H. Hu : G. Huang : F. Huang : Y. Li
State Key Laboratory for Conservation and Utilization of
Agricultural Bioresource in Subtropics, College of Life
Science and Technology, Guangxi University,
Nanning 530005, China
e-mail: xwfan001@163.com
X. Fan : Y. Li
Key Laboratory of Ministry of Education for Microbial and
Plant Genetic Engineering, Nanning 530005, China
J. Palta
CSIRO, Agriculture flagship, Private Bag No. 5, Wembley,
WA 6913, Australia
J. Palta
School of Plant Biology, The University of Western Australia,
35 Stirling Hwy, Crawley, WA 6009, Australia
antioxidant enzymes under five regimens in two maize
inbred lines.
Results Soil inoculation with Burkholderia sp. LD-11
promoted biomass accumulation and improved instan-
taneous water-use efficiency (WUEi), regardless of the
soil water availability. It also triggered production of
indole-3-acetic acid (IAA), decreasing the accumulation
of abscisic acid (ABA) induced by the water deficit,
alleviated ROS accumulation, and resulted in a reduc-
tion in lipid peroxidation induced by the water deficits.
Soil inoculation also enhanced the tolerance to water
deficit through reducing stomatal aperture by increasing
the sensitivity of stomatal conductance (gs) to small
changes in ABA concentration in the leaves.
Conclusions Soil inoculation with Burkholderia sp.
LD-11 enhanced root systems and WUEi, offering a
potential avenue for improving maize tolerance to water
deficit.
Keywords Burkholderia sp. LD-11 . Inbred lines of
maize . Water-use efficiency . Abscisic acid . Reactive
oxygen species
Abbreviations
ABA Abscisic acid
ACC 1-aminocyclopropane-1-carboxylic acid
CAT Catalase
gs Stomatal conductance
IAA Indole-3-acetic acid
LWP Leaf water potential
MDA Malondialdehyde
PFC Pot soil water capacity
338
PGPR Plant growth promoting rhizobacteria
Pn Net leaf photosynthetic rate
POD Peroxidase
PPFD Photosynthetic photon flux density
ROS Reactive oxygen species
SOD Superoxide dismutase
SWC Soil water content
Tr Transpiration rate
WUEi Instantaneous water-use efficiency
Introduction
Drought is one of the most common environmental
causes limiting crop growth, yield, and grain quality.
The development of drought-tolerant varieties has be-
come a priority in most crop breeding programs
(Cattivelli et al. 2008; Blum 1996). In each crop
environment, there is a diverse variety of soil
microorganisms surrounding and interacting with
the crop root system. It has been suggested that
some of these interactions can alleviate the severe
effects on growth and yield, and improve water
use and tolerance to drought (Yang et al. 2009;
Bresson et al. 2013; Naveed et al. 2014).
Climate change will potentially have severe im-
pacts on crop production over the next 50 years
(Tubiello et al. 2007). Maize is a crop that often
experiences water deficits during its vegetative and
reproductive stages (Çakir 2004), affecting its
growth, development, and grain yield (Sinclair
et al. 1990). Maize is likely to be more sensitive
to water deficits during seedling development than
at other stages, because the root system is still
under development (Steudle 2000).
The root system of maize grows in association with a
variety of microorganisms that exert beneficial effects
on plant growth and development (Kandeler et al. 2002;
Lugtenberg and Kamilova 2009; Lalande et al. 1989).
This includes a number of bacteria with an intrinsic
protective role that commences once they colonize the
root system. Plant growth-promoting rhizobacteria
(PGPR) is the name given to a number of bacteria which
colonize the root system, interact with plants and protect
against multiple stresses (Marulanda et al. 2006;
Bresson et al. 2013; Bharti et al. 2013; Huang et al.
2013; Lugtenberg and Kamilova 2009). Maize seed-
lings inoculated with Bacillus spp. can resist the severe
Plant Soil (2015) 390:337–349
effects of water stress on growth and development
(Vardharajula et al. 2011), although the level of allevia-
tion depends on the bacterial strain and genotype
(Naveed et al. 2014). The soil microbial community, as
a whole, can also interact with plants and influence plant
performance under drought stress (Zolla et al. 2013).
PGPR may increase stress tolerance by producing cer-
tain metabolites (i.e. trehalose and trehalase) and acti-
vating signal networks (Lopez et al. 2008; Friesen et al.
2011). For instance, bacteria containing 1-
aminocyclopropane-1-carboxylic acid (ACC) deami-
nase can cleave ACC to ammonia and α-ketobutyrate
(Glick 2004), and this alleviates plant stress and restarts
plant growth under water stress (Mayak et al. 2004;
Yang et al. 2009). Opportune production of indole-3-
acetic acid (IAA) by PGPR can also increase root length
(Xie et al. 1996; Yang et al. 2009), but the overproduc-
tion (>8 μg/mL) of IAA by Pseudomonas putida GR12-
2/aux3 can inhibit root elongation (Xie et al. 1996).
High cytokinin-producing PGPR, which can produce
zeatin at 201.56 ng/mL and trans-zeatin at 393.63 ng/
mL, can alleviate water stress and benefit plant survival
when the inoculated seedlings are transplanted into an
area of water shortage (Liu et al. 2013).
Burkholderia sp. LD-11 (LD-11) is a species of soil-
borne bacteria, rather than an endophytic bacterium
(Huang et al. 2013). It was chosen as a potential pro-
moter of plant growth mainly because of its ability to
produce indole-3-acetic acid (2.08 μg/mL), ACC deam-
inase (4–10 nm α-ketobutyrate /mL/h), and
siderophores (nearly 2.7 nm/mL) (Huang et al. 2013).
In addition, it has been suggested that soil inoculation
with LD-11 has the ability of decreasing ethylene pro-
duction, promoting the host root systems, and confer
plant tolerance to soil water shortages (Mayak et al.
2004; Patten and Glick 2002). However, after soil inoc-
ulation with LD-11, the physiology and biochemistry of
the process under different soil moisture regimens is not
well known in maize (Maiti et al. 1996). Most studies
have focused on a single water deficit, generally mod-
erate, or severe, but information on plant growth perfor-
mance under different water deficits is limited. The aim
of this study was to determine whether soil inoculation
has positive effects on water use efficiency under differ-
ent water regimes in maize. The hypothesis evaluated
was that soil inoculation could promote plant biomass
accumulation and instantaneous water-use efficiency
(WUEi) regardless of the water deficit experienced by
the maize seedlings.
Plant Soil (2015) 390:337–349
Material and methods
Bacteria and soil inoculation
The LD-11 strain used in this study was firstly reported
as a promoter of the accumulation of heavy metals in
plant tissues, which had been isolated from the soil of
mine tailings in a copper mine in Nanning, Guangxi
Province, China (Huang et al. 2013). The LD-11 was
long-term preserved at −80 °C in a glycerol solution
(30 %). To prepare inocula, single colonies of LD-11
were transferred to 250-mL flasks containing 100 mL
Luria-Bertani (LB) medium, and grown aerobically in
the flasks on a rotating shaker (120 rpm) at 37 °C for
18 h. Bacterial suspensions were centrifugated and the
pellets were resuspended in sterile deionized water. For
obtaining 5×107 colony-forming units (CFU) g−1 of
soil, the volume was adjusted based upon a correspon-
dence with the absorbance measured at 600 nm. In the
inoculation treatment, bacterial suspension in dH2O was
carefully added to the surface soil near the root–stem
transition zone of each seedling (10 ml per pot) at 4-leaf
stage. Control soil was treated by adding the same
volume of deionized water. Inoculation was conducted
once every 4 days for four times. Three replications
were used for each treatment. A high inoculum was
used in this study because larger pots (20 cm diameter
× 15 cm depth) and hence larger soil volume were used
than those used in previous studies with tomato seed-
lings (7 cm diameter × 10 cm depth) (Mayak et al.
2004). In addition, the soil of 5 seedlings per pot were
inoculated with 10 ml bacterial suspension in the dH2O,
following the successful inoculation rate of Mayak et al.
(2004). Multiple inoculations were also made to reduce
the effect of soil water deficit on the colonization of LD-
11 (Huang et al. 2013). Maize seedlings were grown
under an automatic rainout shelter with mean tempera-
tures of 28 °C in order to develop a fast and severe plant
water deficit (Kobata et al. 1992).
Plant material and growth conditions
We used two inbred lines of maize (Zea mays L) differ-
ing in the response to soil water shortage; the line
Huangzao4 (HZ4) is one of the foundation genotypes
for maize cross-breeding in China, and Chang 7–2 (C7-
2) is a genotype that is more tolerant to water deficiency
(Li et al. 2009). Eight seeds were sown in plastic pots
(20 cm diameter 15 cm depth) containing 3 kg of sieved
339
loamy clay and peat mixture (3:1, v/v) with a pH of 6.42,
electrical conductivity of 80 mS m−1, water-holding
capacity of 20 %, and a bulk density of approximately
0.64 g cm−3. At sowing on May 5, 2013, the equivalent
of 175 kg N ha−1 as urea, 27 kg P ha−1 as superphos-
phate, and 25 kg K ha−1 as potash were mixed into the
top 0.1 m of soil in each pot. After germination, seed-
lings were thinned to 5 per pot with 6-cm spacing
between each seedling, and the pots were grouped to-
gether to give a plant density of 30 plants m−2. The
plants were grown in an automatic rainout shelter, with
mean 28±2 °C temperature, 40–60 % relative humidity,
and a 14 h photoperiod, at the experimental farm station
of The Guangxi University, Nanning (22°49′12″N,
108°19′11″W), China. Plants were carefully watered
by hand approximately every second day to keep the
soil wet and avoid draining until the watering treatments
were applied.
Treatments
The experimental design was completely randomized
with three factors (inoculation, watering regimen, and
genotypes). Two inoculation treatment (LD-11 inoculat-
ed and non-inoculated), five watering regimens (80, 60,
45, 35 and 25 % of pot soil water capacity [PFC]), and
two inbred lines (C7-2 and HZ4) were used. The tops of
the pots were covered with a 3 cm uniform layer of
white plastic beads to reduce evaporative losses from the
substrate. All plants were kept well watered (80 % of
PFC) for 15 days, when the plants reached the 4-leaf
stage. Water deficit treatments began at the start of the
16th day. Soil water content (SWC) in each water reg-
imen was maintained at the target level; when SWC was
shifted 5 % off the target level, water was supplied (or
continued to withdraw by stopping watering) to main-
tain the target water content at 18:00 h each day (Luna
et al. 2005).
Sampling
Ten days after the application of the watering treatments,
plant height was recorded by measuring the length from
the soil surface to the base of the tallest leaf with a ruler.
Plants were then harvested by cutting the shoots from
the roots at the crown. Leaves were separated from
stems before being dried at 70 °C for 48 h and weighed.
The roots in each pot were recovered from the soil by
washing and sieving at 1.4 mm to produce a clean
340
sample, and then they were separated into coarse roots
(≥1 mm diameter), placed in paper bags, dried at 70 °C
for 48 h and weighed. Root shoot ratio was calculated as
the root dry weight/shoot biomass dry weight.
Leaf water potential and gas exchange measurements
Midday leaf water potential (Ψleaf) was measured be-
tween 1200 and 1400 h at and in three replicate leaves
per inbred line and inoculation and watering treatment.
Measurements of Ψleaf were made on three young fully
expanded leaves with a WP4 water potentiometer
(Decagon Devices, Inc., Pullman, WA, USA). The leaf
surface was abraded gently with a 5×2 cm piece of 600-
grit sandpaper, and dried thoroughly with a lint-free
tissue to remove any excess water. The leaf sample
was then excised from the plant. Ten discs (1.2 cm
diameter) per leaf (30 discs inbred line and inoculation
and watering treatment) were punched with punch pliers
and the samples were immediately sealed with a moist
towel in a plastic bag until they were transferred into the
WP4C sample cup and sealed in the chamber. Discs
were arranged such that the sample cup was fully cov-
ered and the measurement process was unaltered
(Martínez et al. 2013). Measurements were performed
at laboratory temperature.
Net photosynthetic rate (Pn), transpiration rate (Tr)
and stomatal conductance (gs) in the three uppermost
fully expanded leaves per inbred line and inoculation
and watering treatment was measured using a Li-
6400XT Portable Photosynthetic System (Li-Cor,
Lincoln, NE, USA). Measurements were made between
0900 and 1100 h on clear sunny days at 3-day intervals
during the development of water deficits when the pho-
tosynthetic photon flux density (PPFD) over the plant
canopies was 1200 μmol m−2 s−1. PPFD was measured
with a 6400-02B LED Red/Blue Light Source (Li-Cor,
Lincoln, NE, USA). Instantaneous water use efficiency
(WUEi) was calculated as the ratio of Pn/Tr.
Lipid peroxidation and reactive oxygen species (ROS)
The level of lipid peroxidation of the leaf tissue was
determined by malondialdehyde (MDA) accumulation
assay following the method described by Zhao et al.
(1994). The MDA content was calculated using the
following formula: C (μM) = 6.45 (OD532−OD600) −
0.56 OD450, and expressed as nanomoles per gram fresh
weight. O2− production was measured using nitrite
Plant Soil (2015) 390:337–349
formation from hydroxylamine in the presence of O2−
following the method by Elstner and Heupel (1976).
Hydrogen peroxide (H2O2) concentrations were deter-
mined as described by Ryan et al. (2009).
Antioxidant enzyme assays
A crude enzyme extract was prepared by homogenizing
0.5 g frozen leaf segments in 5 mL of ice-cold 50 mM
potassium phosphate buffer (pH 7.8) containing a little
polyvinyl pyrrolidone, using a chilled mortar and pestle.
The homogenate was centrifuged at 12,000×g for
20 min at 4 °C and the supernatant was used for the
following enzyme assays.
The method of Bradford (1976), which uses bovine
serum albumin as a standard, was used to measure total
soluble proteins because of its high reproducibility and
extreme sensitivity in the assays. Superoxide dismutase
(SOD, EC 1.15.1.1) activity was assayed by monitoring
the inhibition of the photochemical reduction of nitro
blue tetrazolium according to the method of
Giannopolitis and Ries (1977). Peroxidase (POD, EC
1.11.1.7) activity was determined by the guaiacol meth-
od according to the technique described by Hori et al.
(1997). Catalase (CAT, EC 1.11.1.6) activity was mea-
sured by the disappearance of H2O2 (extinction coeffi-
cient 39.4 mM−1 cm−1) at 240 nm for 180 s (Aebi 1984).
Abscisic acid (ABA)
The methods for extraction and purification of ABA
were modified from Mwange et al. (2003). ABA anal-
ysis was conducted on an Agilent 1260 Infinity (Agilent
Technologies, CA, USA). A Hypersil C18 column (4.6×
150 mm, 3 μm) and a detection wavelength of 265 nm
were used. Quantification was obtained by using co-
chromatography of exogenous standards of ABA
(Aladdin, Shanghai Corporation Ltd, China) in HPLC
conditions using retention time and peak area.
Statistical analysis
General linear models with ANOVA and post hoc
Tukey and Waller–Duncan tests were used to identify
significant differences in main effects, and to determine
all two- and three-way interactions between inoculation,
water deficit treatments, and genotypes of inbred lines
C7-2 and HZ4 (SPSS 13.0 Chicago, IL). Differences in
the inoculation, leaf gas exchange parameters, shoot
Plant Soil (2015) 390:337–349 341

height, shoot weight, root–shoot weight, root:shoot ra- ranged from 16.6 % at 60 % PFC to 83.4 % at 25 % PFC
tio, and leaf water potential between two the inbred lines (Fig. 1b). The decrease in gs in HZ4 showed a pattern
of maize were tested by ANOVA under well-watered similar to that of C7-2 (Fig. 1b).
and water deficit treatments. Data are presented as the
mean with standard deviation (±SD). Differences were -0.5 a) C7-2 NI
C7-2 I
considered significant if P<0.05. ABA, MDA, H2O2, -1.0 HZ4 NI
O2−, SOD, CAT, and POD were also measured at each -1.5
HZ4 I

water deficit level and control for the two inbred lines of
-2.0

Ψ leaf (MPa)
maize. All data were evaluated by two- and three-way
-2.5
ANOVA and means were compared by Duncan’s mul-
tiple range tests at P=0.05 using SPSS 13.0 (SPSS, -3.0
Chicago, IL). -3.5
-4.0
-4.5
Results 0.14 b)
0.12
Soil water content (SWC) and leaf water potential

gs (mol H2O m s )
(Ψleaf) 0.10
-2 -1
0.08
The soil inoculation experiment was conducted using
the two inbred lines of maize C7-2 and HZ4 under 0.06
different water regimes. There was a significant interac- 0.04
tion of inoculation × water regime × genotype for Ψleaf
0.02
(P<0.001). Ψleaf for both lines was higher in plants
grown in inoculated soil than in non-inoculated soil 0.00
under all water regimens (Fig. 1a; SI). This difference 16
c)
was significant across all water regimes treatments ex-
cept when the SWC was maintained at 45 % PFC.
Pn (μmol CO2 m s )
-2 -1

12

Stomatal conductance (gs), leaf photosynthesis

8
and transpiration rate

gs in C7-2 and HZ4 under well-watered conditions 4

(80 % PFC) was higher by 28.9 and 15.6 %, respective-
ly, when the soil had been inoculated with LD-11 0
1.8 d)
(Fig. 1b; SI). When the SWC was maintained below
80 % PFC, gs was lower regardless of the plants being 1.5
grown in inoculated and non-inoculated soil. The de-
Tr (mmol H2O m s )
-2 -1

crease in gs in plants of C7-2 grown in inoculated soil 1.2

0.9
Fig. 1 Effects of soil inoculation with Burkholderia sp. LD-11 b
and watering regime on leaf water potential (Ψleaf) and gas ex-
changes in the maize inbred lines C7-2 and HZ4. Data are the 0.6
mean ± SE of leaf water potential (a), stomatal conductance (gs, b),
leaf net photosynthetic rate (Pn, c) and transpiration rate (Tr, d) in 0.3
plants grown on inoculated (I) and non-inoculated (NI) soil under
well-watered (80 % PFC) and soil water deficit conditions (<80 % 0.0
PFC) during the seedling stage. The values of gs, Pn, Tr are the 80% 60% 45% 35% 25%
mean of three replicates per inoculation treatment and watering Pot soil water capacity
regime
342
Under SWC below 80 % PFC, net leaf photosynthet-
ic rate in C7-2 was slightly higher when grown in soils
inoculated with LD-11 (Fig. 1c; SI). Compared with C7-
2 grown in control soil, the net leaf photosynthetic rate in
C7-2 grown in inoculated soil was 24.1 % higher when
the SWC was maintained at 60 % PFC, 31.9 % at 45 %
PFC, 40.8 % at 35 % PFC, and 15.6 % at 25 % PFC
(Fig. 1c). Similar results were found for HZ4 (Fig. 1c).
Compared with plants in control soil, those in inoc-
ulated soil had significantly lower transpiration rate
(P<0.01) in plants grown on inoculated soil under
well-watered conditions and under water deficit condi-
tions (Fig. 1d; SI).
Instantaneous water-use efficiency (WUEi)
WUEi in plants grown in non-inoculated soil was sig-
nificantly lower (P<0.01) than those grown in inoculat-
ed soil under all the water regimens (Table 1, SI). In
plants grown in non-inoculated soil, WUEi was not
affected at 60 % or at 45 % PFC (Table 1), but was
significantly lower at 35 % and at 25 % PFC (Table 1).
Compared with plants grown in non-inoculated soil and
under well-watered conditions, WUEi in C7-2 grown in
inoculated soil was higher by 74.7 % when the SWC
was maintained at 60 % PFC, by 38.0 % at 45 % PFC,
and 15.3 % at 35 % PFC. The WUEi of inoculated C7-2
was also significantly higher compared with the non-
inoculated plants at 25 % PFC (Table 1). A similar effect
was found in HZ4 (Table 1).
Soil inoculation with LD-11 improves plant growth
and tolerance to water deficit
Plant height was higher by 11.5 % in C7-2 and by 4.3 %
in HZ4 when soil was inoculated with the LD-11 under
well-watered conditions (Fig. 2a). Plant height was also
higher in inoculated soil at SWC below 80 % PFC
Table 1 Effects of soil inoculation with Burkholderia sp. LD-11 under water deficit on instantaneous water-use efficiency (WUEi)
Lines Inoculation treatments 80 % PFC
C7-2 Non-inoculation 12.3±0.69 d
Inoculation 17.4±1.23 b
hi
HZ4 Non-inoculation 7.2±0.37
de
Inoculation 11.6±0.66
Data indicate the mean (±SE) for WUEi of plants grown in inoculated and non-inoculated soil under different watering regimens measured
during the seedling stage. Different letters indicate significant differences (P<0.05) following Dunn’s multiple comparison test
Plant Soil (2015) 390:337–349
(Fig. 2a). At 60 % PFC, plant height was 20 % higher
in C7-2 and 14.8 % higher in HZ4 in inoculated soil
compared with control soil. A similar difference was
observed in both lines at 45 and 35 % PFC (Fig. 2a). At
25 % PFC, the plant height was 42.1 % higher in C7-2
and 37.8 % higher in HZ4 in inoculated soil than in
control soil (Fig. 2a).
There was a significant interaction of inoculation ×
water regimen × genotype for shoot biomass (P<0.001)
(Electronic supplementary material). Under well-
watered conditions, shoot biomass in plants grown in
soil inoculated with LD-11 was 15.9 % higher in C7-2
and 10.4 % higher in HZ4 compared with plants in
control soil (Fig. 2b). At SWC below 80 % PFC, shoot
biomass was higher when the soil in the pots was
inoculated with LD-11 compared with control soil.
The difference in shoot biomass ranged from 2.5 to
13.9 % in C7-2 and 1.8 to 12.1 % in HZ4, depending
on the level of water deficit (Fig. 2b).
There was a significant interaction (P<0.001) of
inoculation × water regime × genotype for root biomass
(Fig. 2c; SI). Under well-watered conditions, root bio-
mass in plants grown in inoculated soil was 48.0 %
higher in C7-2 and 26.7 % higher in HZ4 (Fig. 2c; SI).
Root biomass was also higher with inoculation when the
SWC was maintained below 80 % PFC. The larger
difference in root biomass was 37.8 % in C7-2 at 60 %
PFC, and 27.1 % in HZ4 at 25 % PFC. (Fig. 2c; SI). The
root:shoot ratio in the two inbred lines was higher in
plants grown in inoculated soil compared with control
soil under well-watered and water deficit conditions
(Fig. 2d).
Effect of soil inoculation with LD-11 on lipid membrane
peroxide
MDA content was lower in plants grown in inoculated
soil, regardless of the watering treatment (Fig. 3a; SI).
60 % PFC 45 % PFC 35 % PFC 25 % PFC
11.1±0.56 e 11.6±0.66 de 7.7±0.70 gh 2.5±0.36 k
21.5±0.86 a 17.0±0.70 b 14.2±1.28 c 6.4±0.96 i
f fg j
8.6±0.32 8.3±0.92 4.4±1 .01 1.1±0.11 l
c c f
14.4±0.54 14.9±0.76 8.7±1.36 1.6±0.33 l
Plant Soil (2015) 390:337–349 343

Root biomass (g DW per plant)

36
a) C7-2 NI 1.6 c) C7-2 NI
C7-2 I C7-2 I
HZ4 NI 1.4 HZ4 NI
Shoot height (cm) 32 HZ4 I HZ4 I

1.2
28
1.0
24
0.8
20
0.6
16
0.4
12
Shoot biomass (g DW per plant)

5 b) 0.6
d)

Root shoot ratio

0.5

3
0.4

2
0.3

1
0.2
80% 60% 45% 35% 25% 80% 60% 45% 35% 25%
Pot soil water capacity Pot soil water capacity
Fig. 2 Effects of soil inoculation with Burkholderia sp. LD-11 grown on inoculated (I) and non-inoculated (NI) soil under well-
and water regime on plant height and shoot biomass in inbred lines watered (80 % PFC) and soil water deficit conditions (<80 % PFC)
of maize. Data are the mean ± SE of plant height (a), shoot during the seedling stage
biomass (b), root biomass (c) and root shoot ratio (d) of plants

Soil inoculation decreased MDA content between 6.7 At 35 % PFC, the difference between inoculated and
and 11.3 % under well-watered conditions (Fig. 3a), but non-inoculated soil increased sharply to 51.5 % in C7-2
the decrease was greater (6.7–9.1 %) when the PFC was and 42.8 % in HZ4 (Fig. 3c; SI).
maintained at 60 %. Further decreases in MDA content
occurred when the PFC was maintained lower than Changes in protective enzymes under water deficit
60 % (Fig. 3a). in the maize inbred lines inoculated with LD-11

Soil inoculation with LD-11 decreased the content Soil water deficit above 25 % SWC increased the
of O2− and H2O2 induced by water deficit activity of SOD and CAT enzymes (Fig. 4a and b;
SI), but it significantly decreased at 25 % SWC.
O2− and H2O2 contents in the leaves increased steadily SOD activities in inoculated plants at soil water
as the SWC decreased from 80 to 25 % PFC. Soil deficit above 25 % SWC were significantly higher
inoculation lowered the content of O2− and H2O2 re- in C7-2 than in d HZ4 (Fig. 4a). Soil inoculation
gardless of water deficit conditions (Fig. 3b and c; SI). increased the activity of the enzymes CAT and
In line C7-2, the content of O2− was 20.2 to 36.4 % POD under well-watered conditions (Fig. 4a).
lower in inoculated compared with non-inoculated soil, Under soil water deficit above 25 % SWC, leaf
depending on the level of water deficit. Similar differ- POD activities in C7-2 inoculated plants were
ences in the content of O2− (27.6 to 40.4 %) were seen in lower than in non-inoculated plants (Fig. 4c).
HZ4 (Fig. 3b; SI). As the SWC decreased from 60 % HZ4 inoculated plants had higher POD activities
PFC to 25 % PFC, soil inoculation decreased the content than in non-inoculated plants (Fig. 4c). CAT activ-
of H2O2 by 29.4 % in C7-2 and by 35.4 % in HZ4 at ities in inoculated plants followed a similar trend
60 % PFC, with similar differences at 45 and 25 % PFC. SOD (Fig. 4b).
344 Plant Soil (2015) 390:337–349

120 a) C7-2 NI 24
a) C7-2 NI
C7-2 I C7-2 I
110 HZ4 NI
HZ4 NI
100 HZ4 I HZ4 I
20
MDA (nmol g FW)

90

(U mg Protein)
SOD activity
-1

80
16
70

-1
60
12
50
40 8
30
O2 production rate (µmol g FW min )
-1

12
b) b)
50
10

(Δ A240 min mg Protein)

-1

40

CAT activity
8

-1
30
6

-1
20
4

10
18 c)
-

c)
0.20
(Δ A470 min mg Protein)
H2O2 content (mg g FW)

15
-1

POD activity

0.15
-1 -1

0.10 12

0.05
9
0.00 80% 60% 45% 35% 25%
80% 60% 45% 35% 25%
Pot soil water capacity
Pot soil water capacity
Fig. 4 Effects of soil inoculation with Burkholderia sp. LD-11
Fig. 3 Effects of soil inoculation with Burkholderia sp. LD-11 and water regimens on protective enzymes of maize inbred lines.
and water regime on MDA content and reactive oxygen species Data indicate the mean (± SE) of SOD activity (a), CAT activity
(ROS) in inbred lines of maize. Data are the mean ± SE of MDA (b) and POD activity in the leaf (c) of plants grown on inoculated
content (a), O2− production rate (b) and H2O2 content (c) of plants (I) and non-inoculated (NI) soil under well-watered (80 % PFC)
grown on inoculated (I) and non-inoculated (NI) soil under well- and soil water deficit conditions (<80 % PFC) during the seedling
watered (80 % PFC) and soil water deficit conditions (<80 % PFC) stage
during the seedling stage

significantly lower in inoculated plants at SWC below

Soil inoculation with LD-11 decreased ABA
accumulation in both inbred lines
Water deficit led to increase ABA accumulation signif-
icantly when SWC decreased from 80 to 25 % PFC. Soil
inoculation decreased ABA content in both inbred line
of maize regardless of water deficit regimes (Fig. 5a).
Compared with the control treatment, ABA content was
60 % PFC, with a difference of 21.9 % at 45 % PFC and
29.2 % at 25 % PFC in C7-2, respectively. Similar
reduction occurred in HZ4 grown in inoculation soil
(Fig. 5a). Changes in ABA content in the leaves of both
inbred lines of maize were apparent as SWC decreased
from 80 % PFC to 25 % PFC, but soil inoculation
decreased ABA accumulation in the leaves regardless
of the water deficit in both inbred lines (Fig. 5a).
Plant Soil (2015) 390:337–349 345

300 a) decline in gs as leaf ABA concentration increased was

faster in inoculated plants (slope=−0.3693, P<0.01)
ABA content (µg g FW)

250
compared with non-inoculated plants (slope=−0.3236,
-1

200 P<0.01) (Fig. 5b). However, The gs values ranged

between 125.67 and 13.48 mmol H2O m−2 s−1 in the
150 maize inbred lines, and were positively correlated with
100
leaf water potential (−0.87 to −3.23 MPa) (Fig. 5c).
Compared with control soil, soil inoculation showed a
50 swifter change in gs (slope=40.499, P<0.01) than the
increase in LWP (Fig. 5c).
0
80% 60% 45% 35% 25%
Pot soil water capacity
Discussion
b)
Ψleaf is generally accepted as the definitive parameter of
plant water status (Connor and Palta 1981). The data
presented in Fig. 1a demonstrated that Ψleaf in well-
watered plants (80 % PFC) was maintained high be-
tween −0.73 and −0.88 MPa. Ψleaf was lower as the
SWC in the pots was maintained below 80 % PFC. We
describe here water deficits as mild, moderate, severe,
and extreme at 60, 45, 35, and 25 % PFC, respectively.
There was evidence for a slower decrease in Ψleaf in
plants grown in inoculated soil (from −0.73 to
−3.37 MPa) than in non-inoculated soil (from −0.78 to
−3.93 MPa), and the impression is that soil inoculation
c) successfully maintained stable water status through the
stomatal response.
Plant growth-promoting bacteria exert beneficial ef-
fects on plant growth, development, and yield under soil
water deficits (Mayak et al. 2004; Belimov et al. 2009).
The present study showed that plants grown in inocu-
lated soil promoted shoot height, shoot biomass, and
root biomass compared with plants grown in non-
inoculated soil, but the effect depended on both the
intensity of the water deficits, and the plant genotype.
Soil inoculation with LD-11 promoted shoot and root
biomass accumulation under well-watered conditions,
Fig. 5 (a) changes in leaf ABA content with changes in soil water and ameliorated the effect of mild water deficits on
content, (b) the relationship between stomatal conductance (gs)
and leaf ABA content and (c) the relationship between leaf water shoot and root biomass accumulation (Huang et al.
potential (Ψleaf) and stomatal conductance (gs) measured in leaves 2013). Under moderate and severe water deficits, soil
from plants grown on inoculated (I) and non-inoculated (NI) soil inoculation slightly promoted shoot dry mass (by 2.5–
with Burkholderia sp. LD-11 and under well-watered (80 % PFC) 12.1 %), althought Pn was significantly increased (by
and water deficit conditions (<80 % PFC) during the seedling
stage. The line in b and c is the fitted linear regression. Data are 26.4–40.8 %). This indicates that the growth-promoting
the mean ± SE effect of LD-11 was superior in the root system than in
the shoot (Fig. 2d), which is consistent with the study by
The gs values in the inbred lines were negatively Bresson et al. (2013). This effect was more apparent
correlated with the concentration of leaf ABA regardless under the extreme water deficit, where more biomass
of soil inoculation or non-inoculation (Fig. 5b). The was allocated to root systems in inoculated soil,
346
resulting in the highest root:shoot ratio. Accumulated
evidence suggests that bacterial IAA acts as a major
molecular signal in the development of the host plant
root system (Patten and Glick 2002). Consequently,
IAA production by LD-11 in the soil could have been
responsible for larger root systems in inoculated soil.
Compared with plants in non-inoculated soil, plants
grown in inoculated soil had WUEi that were higher by
41.5–61.2 % under well-watered conditions. This arose
mainly from an increase in Pn and a reduction in Tr. It
was surprising that the increase in gs by inoculation with
LD-11 did not lead to a greater water loss as expected.
This was because the increase in gs, which is consistent
with findings from other studies with grapevines and
soybean (Zhang et al. 1997; Rolli et al. 2014) was not
coupled with an increase in Tr. In contrary, Tr decreased
with the increase in gs. The decrease in Tr was not
associated to a decrease in gs, likely because of an
increase in xylem cavitation which decreased the hy-
draulic conductance and partly override the direct re-
sponse of stomata to changes in evaporative demand
(Franks et al. 1997; Mencuccini et al. 2000). Another
possible reason was that gs did not only rely on the size
and frequency of stomata, but in the xylem conductance
(Aasamaa et al. 2001). In addition, it is also likely that
the capacity for osmotic adjustment could be stronger
because soil inoculation promoted the production of
osmotic regulation substances, such as choline and gly-
cine betaine and other osmolytes (Zhang et al. 2010;
Pérez-Montaño et al. 2014). The present study also
showed that soil inoculation with LD-11 led to signifi-
cantly higher plant WUEi in maize seedlings under mild
and moderate water deficit, but the effect differed be-
tween the two inbred maize lines. Indeed, the response
to water shortage varies among genotypes (Li et al.
2009), and the variety more tolerant to water shortages
(C7-2) reached higher WUEi under soil inoculation. The
sharp increase in WUEi in the line C7-2 inoculated soil
at 60 % PFC was partially associated with a reduction in
ABA content in the leaves, which resulted in a partial
stomatal closure, causing a decline in transpiration. Soil
inoculation with LD-11 ameliorated the effect of severe
and extreme water deficits on Pn, resulting in a signifi-
cant increase in WUEi.
Soil inoculation decreased ABA accumulation re-
gardless of the water regimens. This could be a result
of high Pn in the two inbred maize lines; these results are
in accordance with the literature reporting an increase of
Pn in Arabidopsis inoculated with Bacillus subtilis as
Plant Soil (2015) 390:337–349
being the result of decreased ABA concentrations in
plants (Zhang et al. 2008). A possible explanation for
the reduction in ABA accumulation could be that ACC
deaminase production by LD-11 in inoculated soil could
result in reduced ethylene formation in roots (Huang
et al. 2013; Glick 2004; Penrose and Glick 2001; Jiang
and Hartung 2008), inhibiting the biosynthesis of ABA
(Chiwocha et al. 2005). Interestingly, the reduced ABA
accumulation in the leaves of plants grown in inoculated
soil could be associated with reducing stomatal aperture
by increasing the sensitivity of gs to small changes in
ABA concentration in the leaves. This phenomenon
might be related to the high WUEi in plants grown in
inoculated soil.
Under well-watered conditions, plants grown in
soil inoculated with LD-11 produced less ROS than
plants grown in non-inoculated soil in both inbred
lines. The activities of CAT and POD were main-
tained high, but the activity of SOD was low. It is
likely that this change occurred to maintain the low
level of SOD in the balance between ROS and
protective enzymes induced by the application of
LD-11. Mild and moderate water deficits increased
ROS slightly in plants grown in inoculated soil,
probably as a result of the rapid increase in the
activities of both SOD and CAT, which resulted in
the low level of lipid peroxidation (Fan et al.
2009). The marked increase in the production of
ROS induced by the severe water deficit indicated
that the high activities of SOD and CAT could not
inhibit the production of ROS, leading to mem-
brane damage (Fan et al. 2009). However, soil
inoculation with LD-11 decreased the level of lipid
peroxidation through an increase in the level of
protective enzymes. Under the extreme water defi-
cit, there was an extremely high production of
ROS, causing a very high membrane lipid peroxi-
dation, presumably because the protective enzymes
started to degrade as the synthesis of SOD, CAT,
and POD could not cope with the collapse of anti-
oxidant enzymes (Fan et al. 2009). Soil inoculation
resulted in different effects in POD activity between
the two inbred lines of maize. It is likely that the
differences in POD activity were associated to dif-
ferences in the sensitivity of the two inbred lines to
soil water deficits (Li et al. 2009).
Genotypes with vigorous root systems may tolerate
water shortages better than non- vigorous root systems
(Palta and Watt 2009; Palta et al. 2011). In the present
Plant Soil (2015) 390:337–349
study, the inbred line C7-2 had a larger root system than
HZ4 as the SWC decreased from 80 to 25 % PFC,
reflecting its greater tolerance to water deficit (Li et al.
2009). The root systems in plants from both inbred lines
were larger in inoculated soil regardless of whether they
were well watered or under water deficit. In addition, the
tolerant line C7-2 had faster root growth than HZ4 when
plants were grown in inoculated soil under well-watered
conditions. The mild, moderate, and severe water defi-
cits did not change the root growth advantage of C7-2
over HZ4 under soil inoculation. Under the extreme
water deficit of this study, the total accumulation of root
dry matter was greater in C7-2 than in HZ4. The greater
tolerance of C7-2 may explain why this line had higher
Ψleaf, shoot biomass, net leaf photosynthetic rate, and
WUEi than HZ4 under soil inoculation.
Conclusions
Plants of two inbred lines of maize grown in soil inoc-
ulated with a Burkholderia sp. LD-11, an auxin and
ACC deaminase-producing PGP bacteria (Huang et al.
2013), showed higher plant growth and instantaneous
WUEi at the five-leafed stage, mainly resulting from
root systems growth. There is a potential to improve dry
matter accumulation by growing plants in inoculated
soil and regardless of water deficit. Plants grown in
inoculated soil also had lower ROS production, which
is induced by water deficit, and alleviated the detrimen-
tal effect of water deficit by increasing the antioxidant
capacity. Plants grown in inoculated soil had lower
ABA accumulation, but optimized the stomatal opening
by increasing the sensitivity of gs to ABA concentration,
leading to enhancement of WUEi regardless of the
plants being well-watered or under water deficit. The
inbred line with greater root system and faster rate of
root growth when grown in inoculated soil was more
tolerant to water deficit. We conclude that Burkholderia
sp. LD-11 may provide a new effective defense strategy
for crops to adapt to lack of water.
Acknowledgments The authors appreciate three anonymous
referees for their valuable comments. We are also thankful to Dr.
Yanlei Du for his comments on the manuscript and Mr. Dengfeng
Dong for his experimental assistance. This research was supported
by the NSFC project (31160287, 31360337), Guangxi Natural
Science Foundation (2014GXNSFAA118098), and Project of the
Special Basic Work of Ministry (2014FY120100).
347
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