Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Drought degree constrains the beneficial effects of a
fungal endophyte on Atractylodes lancea
T. Yang1,2,3, S. Ma1 and C.C. Dai1
1 Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of
Microbial Resources, College of Life Sciences, Nanjing Normal University, Nanjing, China
2 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
3 University of Chinese Academy of Sciences, Beijing, China
Keywords
abscisic acid, Acremonium strictum,
antioxidant enzyme, Atractylodes lancea,
drought treatments, fungal endophytes.
Correspondence
Chuan-Chao Dai, Jiangsu Key Laboratory for
Microbes and Functional Genomics, Jiangsu
Engineering and Technology Research Center
for Industrialization of Microbial Resources,
College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
E-mail: [email protected]
2014/0927: received 7 May 2014, revised 22
July 2014 and accepted 28 July 2014
doi:10.1111/jam.12615
Abstract
Aims: Plants, fungal endophytes (FEs) and the changing environment interact
with each other forming an interlaced network. This study evaluates
nonadditive and interactive effects of the FE Acremonium strictum and drought
treatment on Atractylodes lancea plantlets.
Methods and Results: By applying FEs (meristem cultures of At. lancea, fungal
inoculation of Ac. strictum and plantlet acclimatization) and drought treatment
(regular watering, mild drought, severe drought), a research system of
At. lancea ramets under different treatments was established. During 12 days of
drought treatment, the plantlets’ physiological responses and basic growth
traits were measured and analysed. Although drought and FE presence affected
plantlet traits to differing degrees, the interactive effects of the two were more
pronounced. In particular under mild drought treatment, the FE conferred
drought tolerance to plantlets by enhancing leaf soluble sugars, proteins,
proline and antioxidant enzyme activity; decreasing the degree of plasmalemma
oxidation; and increasing the host’s abscisic acid level and root:shoot ratio.
When exposed to regular watering or severe drought, these effects were not
significant.
Conclusions: Plant traits plasticity was conferred by dual effects of drought
stress and FEs, and these factors are interactive. Although FEs can help plants
cope with drought stress, the beneficial effects are strictly constrained by
drought degree.
Significance and Impact of the Study: During finite environmental stress, FEs
can benefit plants, and for this reason, they may alleviate the effects of climate
change on plants. However, because the benefits of FEs are highly context
dependent, the role of FEs in a changing background should be re-assessed.
Introduction
Drought is a meteorological term for a scarcity of water.
According to statistics, dry lands, including arid, semiarid and dry subhumid ecosystems, cover approx. 41% of
Earth’s land surface and support over 38% of the global
human population (Reynolds et al. 2007). Recently, with
climate change, fire interference and land use conversion,
drought is becoming more severe and widespread (Austin
2011; Saumel et al. 2011; Li et al. 2013). As far as plants
are concerned, in addition to soil water deficit, drought
brings a range of other stresses with which they must
cope, for example, high vapour pressure, oxidative stress,
increased soil salinity, decreased nutrient availability and
mechanical impedance to root growth (Wilkinson and
Davies 2010). Nevertheless, plants also provide some vital
feedbacks to the changing environment and global ecosystem (Zhao et al. 2011; Zhou et al. 2012). Maestre
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
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Drought degree constrains FEs’ benefits on plants
T. Yang et al.
et al. (2012) found a positive and significant relationship
between plant species richness and ecosystem multifunctionality in global dry lands. Hence, how to help plants
cope with drought is already a hot issue and critical concern in parts of the world (Pennisi 2008).
Interestingly, currently, it is generally accepted that
plants are not alone but instead are a bit like a large
‘Trojan Horse’ filled with symbiotic microbes, especially
due to their associations with fungi (Rodriguez et al.
2009; Porras-Alfaro and Bayman 2011; Yang et al.
2013a). There is a substantial body of literature describing and assessing the impacts of fungal endophytes (FEs)
on plant resistance to drought (Bae et al. 2009; Kane
2011; Worchel et al. 2013; Oberhofer et al. 2014). Due to
either reductionist tendencies or the urgency of finding
certain FEs that can save plants from drought, many
experiments have been designed (Malinowski and Belesky
2000). The only variables are the things that one wants to
test. Other factors are controlled carefully, and if they
cannot be controlled, often they are neglected. This
pointed focus is necessary to test the hypothesis, but it
seems to make us forget the tremendous complexity of
our research systems (Porras-Alfaro and Bayman 2011).
Plants, symbiotic fungi and the changing environment
should be an interlaced network instead of one-way
arrows (Rudgers and Swafford 2009; Ren et al. 2011; Kivlin et al. 2013; Pineda et al. 2013). However, few studies
have deliberately focused on the dual effects of an environmental factor (drought) and a biofactor (symbiotic
fungi) on plant physiology and growth (Emery and Rudgers 2013).
Atractylodes lancea is an economically important Chinese medicinal herb endemic to East Asia (Hasada et al.
2010; Meng et al. 2010). This perennial aster relatively
holds some promise for modulating the intestinal
immune system in humans. Wild At. lancea is inhabited
by various opportunistic FEs (Chen et al. 2008). Some
among them have balanced symbiosis with the host and
play vital roles in the modification of medicinal components and the inducement of host plant defences (Wang
et al. 2011, 2012; Ren and Dai 2012, 2013; Yang et al.
2013b). However, whether they can help plantlets adapt
to drought stress has not been investigated. In addition
to having numerous FE partners, wild At. lancea are
highly genetically diverse, like their relative Atractylodes
macrocephala (Zheng et al. 2012). Therefore, wild or
adult plantlets of At. lancea are not suitable for experimentation. We reported previously (Yang et al. 2013b)
about a system (meristem cultures, fungal inoculation
and plantlet acclimatization) that avoids the above disadvantages effectively. By applying drought and FE treatment to ramets of At. lancea simultaneously, we
addressed the following questions: (i) Can the FE Acre1436
monium strictum of At. lancea ameliorate responses of
host plantlets to drought? (ii) Does drought degree constrain the beneficial effects of FEs on At. lancea, just as
with the nutrient or nitrogen level (Ren et al. 2009,
2011)? (iii) Do environmental factors (drought degree)
and biofactors (FEs yes/no) have interactive, nonadditive
effects on plant traits (Emery and Rudgers 2013; Kivlin
et al. 2013)?
Materials and methods
Plant and fungal material
Meristem cultures of At. lancea (collected in Maoshan, Jiangsu Province, China, 31°140 – 31°470 N, 119°040 –
119°260 E) were established as previously described (Wang
et al. 2011; Ren and Dai 2012, 2013). The explants were
surface-sterilized and grown in Murashige and Skoog
(MS) medium supplemented with 03 mg l1 naphthaleneacetic acid (NAA), 20 mg l1 6-benzyladenine,
30 g l1 sucrose, and 10% agar in 150-ml Erlenmeyer
flasks. Rooting medium (1/2 MS) contained 025 mg l1
NAA, 30 g l1 sucrose and 10% agar. All media were
adjusted to pH 60 before being autoclaved. Cultures
were maintained in a growth chamber (25/18°C day/
night, with a light intensity of 3400 lm m2 and a photoperiod of 12 h) and were subcultured every 4 weeks.
Thirty-day-old rooting plantlets were used for subsequent
treatments.
The fungal endophyte AL16 was isolated from leaves of
wild At. lancea collected in Maoshan, Jiangsu Province,
China. This fungal material was also used in Wang et al.’s
research (Wang et al. 2013). It was identified as Ac. strictum based on molecular phylogeny and morphological
characteristics (Figures S1 and S2). The GenBank Accession Number is JQ339308.
In vitro inoculation, plantlet acclimatization and
examination of endophytes
The FE AL16 (Ac. strictum) was cultured on potato dextrose agar and incubated at 28°C for 5 day. Thirty-dayold plantlets were inoculated with 5-mm AL16 mycelial
discs. An equal-sized disc of potato dextrose agar was
used as a control. All treatments were conducted in a
sterile environment (Ren and Dai 2013).
A total of 160 rooting plantlets of At. lancea were
chosen for acclimatization (Yang et al. 2013b). One-half
were inoculated with AL16 Ac. strictum and designated
the E+ group. The other half were free from any
microbes and designated the Egroup. After acclimatization of the At. lancea plantlets, the survival rates of the
E+ group and Egroup were summarized. The specific
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
T. Yang et al.
acclimatization procedure was described previously (Yang
et al. 2013b).
Endophytes were detected via re-isolation from surface-sterilized leaves of the E+ group (Yang and Dai
2013). After acclimatization, roots and leaves of At. lancea were collected for microscopic observation. The
leaves were surface-sterilized and then immersed in a
2% KOH solution for 3 days at 58°C. After decolouration, the leaves were washed repeatedly in sterile water,
bleached with 3% H2O2 for 30 s, washed twice with 2%
HCl and stained with 005% trypan blue. After staining,
a dehydration gradient of 60, 80, 95 and 100% ethanol
was used with 6 min for each step. Finally, the leaves
were rinsed once with xylene and placed on a coverslip.
For the roots, 1- to 2-cm root segments from plantlets
were collected and surface-sterilized. The roots were
immersed in a 10% KOH solution and kept in a boiling
water bath for 30 min. After that, the root segments
were neutralized with 2% HCl for 5 min and washed
repeatedly in sterile double-distilled water. Finally, the
roots were stained using a 001% lactic acid magenta
solution in a 90°C water bath and decoloured with lactic
acid–glycerol (1 : 1). Permount was used for mounting,
and photographs were taking using a Nikon TIFL542942 (Melville, NY). The imaging software used was
NIS-Elements D3.0.
Drought degree constrains FEs’ benefits on plants
measured the basic agronomical indexes, including root
branching number, tiller number, total tiller length, root
fresh weight, shoot fresh weight and root:shoot ratio.
Measurement of soluble sugar, protein, malondialdehyde
and proline
Leaf soluble sugars were extracted with distilled water
and heated in boiling water for 30 min. Sugars were measured spectrophotometrically at 630 nm by the anthrone
colorimetric method (Zhou 2001).
The total soluble protein concentration was measured
by a dye-binding assay as described by Bradford (Bradford and Williams 1976).
MDA levels indicating the level of lipid peroxidation in
leaf tissue were measured by a thiobarbituric acid reaction using the method of Health and Packer (Heath and
Packer 1968), with some modifications (Dhindsa et al.
1981; Zhang and Kirkham 1994).
Free proline was estimated following the method of
Bates et al. (Bates et al. 1973).
Measurement of the relative water content
RWC was measured following the method described by
Gonzalez and Gonzalez-Vilar (Gonzalez L 2003).
Drought stress experiment
Measurement of the antioxidant enzyme activity
Ninety At. lancea plantlets were chosen for drought
experiments, including 45 E+ and 45 E. All of the chosen plantlets were approximately the same size. A
PEG6000 solution was used to simulate drought conditions (Khan et al. 2011). The treatment was classified into
six groups as follows. Group CKE+ was watered regularly
(2400 ml water for 12 days) and previously inoculated
with Ac. strictum. Group MDE+ was watered with a 10%
PEG6000 solution (w/v, 2400 ml solution for 12 days)
and inoculated with Ac. strictum. Group SDE+ was
watered with a 30% PEG6000 solution (w/v, 2400 ml
solution for 12 days) and inoculated with Ac. strictum.
Groups CKE, MDE and SDE corresponded with
CKE+, MDE+ and SDE+, but were free from endophytes.
Drought treatment continued for 12 days. The levels of
soluble sugars, soluble proteins, malondialdehyde (MDA)
and proline; the activities of three antioxidant enzymes,
catalase (CAT), peroxidase (POD), and superoxide
dismutase (SOD); and relative water content (RWC) were
all measured at 0, 3, 6 and 9 days after drought treatment. At the ninth day during drought treatment, the
amount of abscisic acid (ABA) was measured in leaves
and roots. Finally, on the twelfth day during drought
treatment, we collected all of the remaining plantlets and
For the estimation of CAT, leaves were homogenized in a
medium composed of 50 mmol l1 potassium phosphate
buffer (pH 70), 01 mmol l1 EDTA and 1 mmol l1
dithiothreitol. CAT activity was measured using the
method of Beers and Sizer (Beers and Sizer 1952). The
assay solution (3 ml) contained 50 mmol l1 phosphate
buffer (pH 70), 59 mmol l1 H2O2 and 01 ml enzyme
extract. The decrease in absorbance of the reaction solution at 240 nm was recorded every 20 s. An absorbance
change of 001 min1 was defined as 1 U of CAT
activity.
For the estimation of POD, leaves were homogenized
in a medium composed of 50 mmol l1 potassium phosphate buffer (pH 70), 01 mmol l1 EDTA and
1 mmol l1 dithiothreitol. POD activity was measured
using the method of Chance and Maehly (Chance and
Maehly 1955), with some modifications. The activity
assay solution (3 ml) contained 50 mmol l1 phosphate
buffer (pH 70), 20 mmol l1 guaiacol, 40 mmol l1
H2O2 and 01 ml enzyme extract. The reaction was initiated by the addition of enzyme extract. The increase in
absorbance of the reaction solution at 470 nm was
recorded every 20 s. One unit of POD activity was
defined as an absorbance change of 001 min1.
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T. Yang et al.
For the estimation of SOD activity, leaves were homogenized in a medium composed of 50 mmol l1 potassium
phosphate buffer (pH 70), 01 mmol l1 EDTA and
1 mmol l1 dithiothreitol, as described by Dixit et al.
(Dixit et al. 2001). SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of
nitroblue tetrazolium following the method of Giannopolitis and Reis (Giannopolitis and Ries 1977). One unit of
SOD activity was defined as the amount of enzyme causing 50% inhibition of the photochemical reduction of
nitroblue tetrazolium.
All of the enzyme activities are expressed on a fresh
weight basis and were measured using a common tissue
extract.
Measurement of ABA
The roots and leaves of plantlets were collected at the
ninth day during drought treatment. Per replicate, 1 g of
root tissue and 05 g of leaf tissue were transferred to a
precooled mortar. The plant tissue was grounded to powder via liquid nitrogen. The powder was placed in a 50ml centrifuge tube, and precooled 80% methanol was
used to wash the mortar and then transferred to the centrifuge tube, making the final volume 15 ml. The samples
were digested at 4°C for 12 h and suction-filtered. The
filtrate was amended with 10 ml of methanol at 4°C for
2 h to digest the filter residue. The samples were suctionfiltered and the filtrate was merged. The filtrate was then
decompression-concentrated to aqueous phase and acidized to pH 28–30 using hydrochloric acid. ABA was
extracted three times with ethyl acetate and was then
decompression-concentrated to dryness. Finally, 1 ml of
methanol was added to each sample to dissolve the ABA
and filtered with a 045-lm microfilter. The extract was
preserved at 4°C.
High-performance liquid chromatography (HPLC) (Agilent Technologies 1200 series, Santa Clara, CA) was used
to determine levels of ABA in the different samples. The
HPLC conditions were given as follows. The mobile phase
was acetonitrile (purified): acetic acid (18% v/v) = 1 : 1.
The column (46*250 5 lm; Hanbon Sci. & Tech,
Huai’an, Jiangsu, China) temperature was 25°C. The flow
speed was 05 ml min1, and the ultraviolet absorption
wavelength was 262 nm. An ABA standard was purchased
from Sigma Corporation (Shanghai, China).
Statistical analysis
Data were compiled using SigmaPlot 120 (Systat software
Inc., San Jose, CA). Values are represented as the
mean standard deviation (SD) of three or six replicates
for each treatment. All data were analysed using one-way
1438
and two-way analyses of variance (ANOVA) by SPSS 20.0
(SPSS, Chicago, IL). Duncan’s multiple range test was used
to measure the difference among the means (P = 005).
Results
Colonization of Acremonium strictum and survival after
acclimatization
After acclimatization, Ac. strictum was re-isolated from
the E+ groups. Due to inoculation via the meristem culture system, the rate of infection by the endophyte was
100%. In addition to colonization on leaves, AL16 was
detected within and between root cortical cells (Fig. 1).
In particular, some root cortical cells filled with red stain
seemed to indicate cell necrosis (Fig. 1f).
Inoculation with Ac. strictum significantly enhanced
the survival rate during acclimatization (Figure S3) with
8028 625% in the E+ group and only 625 722%
in the E group.
Plantlet physiological responses
Leaf soluble sugars and soluble proteins were measured at
0, 3, 6 and 9 days of drought treatment. The variations
of the soluble sugars and proteins were complex. For soluble sugars (Fig. 2a), (i) endophyte inoculation decreased
soluble sugars in the regularly watered groups
(CKE+ < CKE). (ii) Drought treatment decreased soluble sugars in the endophyte-free groups, especially for
severe drought (SDE < CKE). (iii) With mild drought
treatment, the presence of endophytes significantly
enhanced soluble sugars (MDE+ > MDE/CKE+). For
soluble proteins (Fig. 2b), (i) initial endophyte inoculation decreased soluble proteins in the regularly watered
groups (CKE+ < CKE; 0–6th day), while after the 6th
day of drought treatment, soluble proteins increased in
the endophyte-inoculated groups (CKE+ > CKE; 6th–
9th day). (ii) Soluble proteins increased after 6 days of
drought treatment (e.g. SDE > MDE > CKE; 6th–
9th day). (iii) With mild drought treatment, the presence
of endophytes significantly enhanced soluble proteins
(MDE+ > SDE+/CKE+).
For malondialdehyde (MDA) in leaves (Fig. 3a), (i)
endophyte inoculation slightly increased MDA in the regularly watered groups (CKE+ > CKE). (ii) In both
endophyte-inoculated groups and endophyte-free groups,
drought treatment increased MDA (SD > MD > CK; E+
or E). (iii) Initial endophyte inoculation with mild
drought treatment increased MDA (MDE+ > MDE; 0th
day), but later endophyte inoculation significantly
deceased MDA (MDE+ < MDE; 3th–9th day). In the
MDE+ group, endophytes modulated the increase in
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
T. Yang et al.
Drought degree constrains FEs’ benefits on plants
(a)
(b)
(c)
(d)
(e)
(f)
Figure 1 The establishment of symbiosis between Acremonium strictum and Atractylodes lancea plantlets. (a) Leaf tissue of endophyte-free
groups only contains plant cells and tissues. (b and c) Leaf tissue of endophyte-inoculated groups. The arrows point to AL16 endophytic fungi.
(d) Root cortical cells of endophyte-free groups. (e) Root cortical cells of endophyte-inoculated groups. The arrow points to AL16 endiphytic fungi.
(f) Root cortical cells of endophyte-inoculated groups. The arrow points to root cortical cells that are necrotic due to AL16 infection. Scale bar in
d–f = 10 lm.
MDA. However, with severe drought treatment, endophytes did not significantly affect the MDA level. For
proline (Fig. 3b), (i) endophyte inoculation did not affect
proline in the regularly watered groups. (ii) Drought
treatment increased proline (SD > MD > CK; E+ or E).
(iii) Endophyte presence significantly enhanced proline
with mild drought treatment (MDE+ > MDE), but not
with severe drought treatment or regular watering
(SDE+ SDE; CKE+ CKE).
A variation in the relative water content (RWC) of the
leaves was also observed (Fig. 4): (i) virtually no effects
of endophyte inoculation on RWC were observed in the
regularly watered groups. (ii) Drought treatment
decreased RWC significantly (SDE < MDE < CKE).
(iii) With mild drought treatment, the presence of endophytes did not significantly affect RWC.
Catalase (CAT), peroxidase (POD) and superoxide
dismutase (SOD) were chosen as representative antioxidants to estimate the plantlets’ responses to drought and
endophytes. For CAT (Fig. 5a), (i) endophyte inoculation
slightly increased CAT in the regularly watered groups
(CKE+ > CKE). CAT activity increased with time and
reached its peak at the 6th day, afterwards remaining stable. (ii) Drought treatment increased CAT, but the CAT
activity in the MDE group is similar to that in the
SDE group. (iii) With mild drought treatment, endophyte presence significantly increased CAT. For POD
(Fig. 5b), (i) endophyte inoculation did not affect POD
in the regularly watered groups. (ii) Drought treatment
increased POD, which reached its peak at the 3rd day,
but the POD activity in the MDE group is similar to
that in the SDE group. (iii) Endophyte presence significantly increased POD with mild drought treatment
(MDE+ > MDE), but not with severe drought treatment or regular watering (SDE+ SDE; CKE+
CKE). For SOD (Fig. 5c), (i) endophyte inoculation
did not affect SOD in the regularly watered groups,
except for the 6th day. SOD activity increased with time
and reached its peak at the 6th day. (ii) Drought treatment increased SOD (SD > MD > CK; E+ or E). (iii)
With mild drought treatment, endophyte presence significantly increased SOD (MDE+ > MDE). On the 6th
day, the maximum SOD value was reached in the MDE+
group, while the SOD value of the SDE+ group was significantly lower at that time.
Abscisic acid (ABA) is the most important phytohormone in drought response, and it triggers the closing of
stomata, thus reducing water loss. On the 9th day postdrought treatment, roots and leaves of plantlets of At. lancea were collected and ABA was measured (Table 1). (i)
Endophyte inoculation significantly increased ABA in the
regularly watered groups (CKE+ > CKE). (iii) Drought
treatment also significantly increased ABA (SD > MD >
CK; E+ or E). (iii) With mild drought treatment, endo-
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
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Drought degree constrains FEs’ benefits on plants
T. Yang et al.
Soluble sugar content (µg g–1 Fw)
(a) 12 000
d
11 500
11 000
d
a
10 500
d
c
10 000
c
9500
c
b
b
b
9000
a
a
8500
8000
a
0 day
3 day
6 day
9 day
Time (day for drought treatment)
Soluble protein content (µg g–1 Fw)
(b) 5400
5200
d
e
d
5000
d
4800
c
bc
abc
ab
c
b
4600
4400
c
3800
a
b
a
4200
4000
a
b
a
0 day
3 day
6 day
9 day
Time (day for drought treatment)
phyte inoculation significantly increased ABA in both
roots and leaves. However, when the host plant suffered
severe drought, endophytes did not affect ABA in roots. In
summary, endophytes seem to be able to enhance ABA
content, especially in leaves (Table 1).
Basic plantlet growth traits
After 12 days of drought treatment, all of the remaining
plantlets were collected and the basic growth traits were
measured, including the root branch number, tiller number, total tiller length, root fresh weight, shoot fresh
weight and root:shoot ratio (Table 2). (i) In the regularly
watered groups, E+ had more root branches, but its root
fresh weight was lower. E+ and E had similar tiller
numbers, but compared with E+, the total tiller length of
E was greater. The root:shoot ratios of E+ and E were
1440
Figure 2 Levels of soluble sugars and
proteins in fresh leaves. (a) Soluble sugar
content. (b) Soluble protein content. E+
treatment groups were inoculated with the
endophyte Acremonium strictum; E
indicates endophyte-free. CK indicates control
groups, which were watered regularly. MD
indicates mild drought treatment, watered
with a 10% PEG6000 solution. SD indicates
severe drought treatment, watered with a
30% PEG6000 solution. Values are the
means SD, n = 3. The test method was the
Duncan test. Values in the same column
followed by different letters differ significantly
at P ≤ 005. (
) CKE+; (
) CKE; (
)
MDE+; (
) MDE; (
) SDE+; (
) SDE.
approximately equal. (ii) With mild drought treatment,
E+ had more root branches and its root fresh weight was
significantly higher than E. E+ also had higher tiller
numbers and greater total tiller length. However, the
shoot fresh weights of E+ and E were similar. The root:
shoot ratio of E+ was higher. With mild drought treatment, endophyte inoculation significantly stimulated the
growth of the plantlets’ roots. (iii) When plantlets experienced severe drought, endophyte presence failed to significantly promote root growth. The shoot and root fresh
weights of E+ and E were similar.
Interactions between the effects of drought and
endophytes
Physiological traits and basic growth traits of At. lancea
plants were measured and analysed. For some traits, the
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
T. Yang et al.
Drought degree constrains FEs’ benefits on plants
(a) 550
MDA content (nmol g–1 Fw)
500
e
e
d
450
d
d
400
c
c
350
c
300
b
b
b
b
a
a
a
250
a
200
3 day
0 day
6 day
9 day
Time (days for drought treatment)
(b) 2000
1800
Figure 3 Levels of MDA and proline in fresh
leaves. (a) MDA content. (b) Proline content.
E+ treatment groups were inoculated with
the endophyte Acremonium strictum; E
indicates endophyte-free. CK indicates control
groups which were watered regularly. MD
indicates mild drought treatment, watered
with a 10% PEG6000 solution. SD indicates
severe drought treatment, watered with a
30% PEG6000 solution. Values are the
means SD, n = 3. The test method was the
Duncan test. Values in the same column
followed by different letters differ significantly
at P ≤ 005. (
) CKE+; (
) CKE; (
)
MDE+; (
) MDE; (
) SDE+; (
) SDE.
Proline content (µg g–1 Fw)
d
e
d
1600
d
1400
c
1200
c
c
1000
b
b
b
800
600
400
200
b
a
0 day
effects of endophyte inoculation were more significant,
whereas drought treatment had more significant effects
on other traits. Interestingly, the drought degree seemed
to affect the variations of plant traits with or without
FEs. With mild drought treatment, the beneficial effects
of endophytes (promoting growth and conferring
drought resistance) were more pronounced (Fig. 6). To
systematically estimate the effects of drought, endophytes
and their interactions on host plantlets (ABA levels and
basic growth traits), two-way ANOVA was used (Table 3).
The plant hormone ABA, tiller number and tiller length
were all significantly affected by drought, endophytes and
their interactions. Although neither endophytes or
drought significantly affected root:shoot ratio individually, their interaction affected it significantly. Additionally, root branching was significantly affected by both
3 day
6 day
9 day
Time (days for drought treatment)
endophytes and drought individually but not by their
interaction.
Discussion
Effects of drought on plantlets
Increasing drought affects different aspects of plants to
varying degrees. With respect to plant ecology, drought
affects phenology, species’ distribution and net primary
production (Cleland et al. 2007; Thuiller et al. 2008;
Zhao and Running 2010). In individual plants, it mainly
changes physiology, growth, reproduction and survival
from root to leaf (Schroeder et al. 2001; Pennisi 2008).
In our drought experiment, drought treatment
decreased soluble sugars but increased soluble proteins,
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
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Drought degree constrains FEs’ benefits on plants
T. Yang et al.
95
d
90
a
c
b
c
RWC (%)
85
b
ab
80
75
b
a
70
65
a
0 day
3 day
a
9 day
6 day
Time (days for drought treatment)
especially at later stages of drought treatment. With regular watering treatments, At. lancea plantlets suck up water
from the ground and it leaks out of their stomata. The
stomata remain open to let CO2 in for photosynthesis,
but water loss from evapotranspiration is severe, especially under severe drought. Drought compelled plantlets
to close their stomata, thus decreasing photosynthesis.
Hence, the soluble sugars in leaves, which is the representative of plant nutrient status, decreased. Soluble proteins
increased at later stages of drought due to their roles in
permeation and antioxidation (e.g. antioxidant enzymes,
phytohormone receptors). Proline, a protective osmolyte,
was biosynthesized and accumulated in At. lancea plantlets when drought stress appeared. Proline alleviates the
effects of drought and helps maintain plantlet water
potential, which facilitates the extraction of water from
soil (Hanson et al. 1979). Another harmful product of
drought is reactive oxygen species (ROS) from misdirected photosystem electrons. Excess ROS damage the
plasmalemma and enhance MDA levels (Sgherri et al.
2000). With drought treatment, MDA levels increased, as
did those of three antioxidants (CAT, POD and SOD).
Although At. lancea was able to produce some antioxidant enzymes, this ability was not sufficient to cope with
severe drought. Drought treatment clearly caused some
harm to At. lancea (e.g. reduced RWC and increased
MDA), but the ABA content increased during drought
treatment. ABA can trigger the closing of stomata, thus
reducing water loss (Schroeder et al. 2001). Recently, several proteins have been reported to function as ABA
receptors, and ABA can regulate the expression of many
genes in plants involved in stress resistance, growth and
development (Fujii et al. 2009). It is hypothesized that
1442
Figure 4 RWC levels of fresh leaves. E+
treatment groups were inoculated with the
endophyte Acremonium strictum; E
indicates endophyte-free. CK indicates control
groups which were watered regularly. MD
indicates mild drought treatment, watered
with a 10% PEG6000 solution. SD indicates
severe drought treatment, watered with a
30% PEG6000 solution. Values are the
means SD, n = 3. The test method was the
Duncan test. Values in the same column
followed by different letters differ significantly
at P ≤ 005. (
) CKE+; (
) CKE; (
)
MDE+; (
) MDE; (
) SDE+; (
) SDE.
ABA is the core of an antidrought metabolic signalling
network in At. lancea affecting soluble proteins, soluble
sugars, proline, MDA, antioxidants and several growth
traits.
The effects of FEs on plantlets
As a ubiquitous biofactor for plants, FEs have been studied widely. Their relationships with plants involve a balance of antagonism instead of neutral interactions
(Schulz and Boyle 2005). FEs affect many aspects of
plants (Rodriguez et al. 2009). With our experimental
plant At. lancea, previous researches have shown that FEs
can effectively colonize At. lancea, modify its physiological and growth traits and induce defensive host responses
(Wang et al. 2011, 2012; Ren and Dai 2012, 2013; Yang
and Dai 2013; Yang et al. 2013b).
Comparison between CKE+ and CKE provides a suitable opportunity to analyse the singular effects of FEs on
At. lancea. FEs decreased soluble sugars in regularly
watered groups. FEs’ nutrients come solely from the host
plant, so soluble proteins and sugars levels were lower in
endophyte-inoculation groups. FE nutrient consumption
has been regarded as disadvantageous for this symbiosis
in general. Moreover, host defensive responses triggered
by FEs also consume nutrients. However, in normal conditions, this cost is strictly maintained within reasonable
limits (Schulz and Boyle 2005), and benefits can be seen,
for example enhanced survival rate during acclimatization
(Figure S3). However, soluble proteins increased in later
stages of the experiment. This fluctuation of protein content indicated that some soluble proteins in leaves are
highly relevant to plant defences. Additionally, without
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
T. Yang et al.
Drought degree constrains FEs’ benefits on plants
(a)
450
400
d
CAT activity (U g–1 Fw)
d
e
350
c
300
c
d
250
b
c
b
b
b
200
a
150
100
a
a
a
0 day
3 day
6 day
9 day
Time (days for drought treatment)
(b) 1000
d
POD activity (U g–1 Fw)
800
c
c
b
d
600
b
c
bc
b
400
200
0
a
0 day
a
3 day
a
a
6 day
9 day
Time (days for drought treatment)
(c)
400
e
380
d
360
SOD activity (U g–1 Fw)
Figure 5 Activities of three antioxidant
enzymes in fresh leaves. (a) CAT activity. (b)
POD activity. (c) SOD activity. E+ treatment
groups were inoculated with the endophyte
Acremonium strictum; E indicates
endophyte-free. CK indicates control groups
which were watered regularly. MD indicates
mild drought treatment, watered with a 10%
PEG6000 solution. SD indicates severe
drought treatment, watered with a 30%
PEG6000 solution. Values are the
means SD, n = 3. The test method was the
Duncan test. Values in the same column
followed by different letters differ significantly
at P ≤ 005. (
) CKE+; (
) CKE; (
)
) MDE; (
) SDE+; (
) SDE.
MDE+; (
d
340
c
320
c
c
300
b
280
b
b
260
a
240
220
a
a
a
200
180
0 day
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
3 day
6 day
9 day
Time (days for drought treatment)
1443
Drought degree constrains FEs’ benefits on plants
T. Yang et al.
Table 1 Abscisic acid levels in leaves and roots of Atractylodes lancea plantlets
Drought treatment
(concentration of PEG6000)
0%
10%
30%
Endophyte treatment
ABA content in roots
(ng/g FW)
Endophyte-inoculated
Endophyte-free
Endophyte-inoculated
Endophyte-free
Endophyte-inoculated
Endophyte-free
239445
197068
340547
292815
719091
713781
ABA content in leaves
(ng/g FW)
3175b
2116a
159d
3558c
44395e
3746e
59128
19662
89195
80829
13166
96517
1103b
3307a
1224d
974c
4019f
3379e
FW, fresh weight.
Values are the means SD, n = 6. The test method was the Duncan’s test. Values in the same column followed by different letters differ significantly at P ≤ 005.
Table 2 Basic plant growth traits after 12 days of drought treatment
Drought treatment
(concentration of
PEG6000)
0%
10%
30%
Endophyte treatment
Root branching
number
Endophyte-inoculated
Endophyte-free
Endophyte-inoculated
Endophyte-free
Endophyte-inoculated
Endophyte-free
323
26
287
19
287
223
34d
7bc
74cd
18a
23cd
14ab
Tiller number
107
12
107
86
67
73
14c
15c
11c
14b
1a
23ab
Total tiller
length (mm)
6017
7817
5867
5217
3717
440
537c
994d
619c
795bc
534a
1351ab
Root fresh
weigh (g)
155
208
253
137
124
136
009a
066b
065b
004a
003a
001a
Shoot fresh
weight (g)
074
095
071
077
057
069
017a
075a
011a
005a
002a
001a
Root:shoot
ratio
2097
2191
3579
1768
2186
1971
FW, fresh weight.
Values are the means SD, n = 6. The test method was the Duncan test. Values in the same column followed by different letters differ significantly at P ≤ 005.
SDE+ MDE+ CKE+ CKE– MDE– SDE–
MDE+
MDE–
E+
E–
CK
MD
SD
MDE+
drought treatment, FEs did not affect either RWC or proline content, which are more relevant to water stress
rather than FEs. However, FE inoculation increased MDA
1444
MDE–
Figure 6 Atractylodes lancea Plantlets in
different treatment groups after 12 days of
drought treatment. E+ treatment groups
were inoculated with the endophyte
Acremonium strictum; E indicates
endophyte-free. CK indicates the control
groups which were watered regularly. MD
indicates mild drought treatment, watered
with a 10% PEG6000 solution. SD indicates
severe drought treatment, watered with a
30% PEG6000 solution.
slightly in the regularly watered groups, which implies
that FEs caused damage to the plasmalemma. Microscopic morphological examination of At. lancea roots
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
1619
1355
0157
0213
0273
0856
0156
1551
3523
0695
0229
0042
revealed some root cortical cells filled with red stain,
indicating cell necrosis (Fig. 1f). Similar findings were
reported on a mutualistic symbiosis involving the famous
root FE Priformospora indica and barely (Deshmukh et al.
2006). Previously, the FEs of At. lancea (Gilmaniella sp.)
were found to enhance the activities of defence-related
enzymes including CAT, POD and SOD (Wang et al.
2012). However, the ability of our experimental FE
(Ac. strictum) to enhance defence-related enzyme activities was not as strong as that of Gilmaniella sp. The variation in CAT was most significant, similar to Wang
et al.’s research on Ac. strictum (Wang et al. 2013). FEs
from different taxa form different interactions with
At. lancea, which lead to selective stimulation of antioxidant enzymes. Interestingly, ABA was also significantly
enhanced by FEs. This implies that ABA is a core phytohormone relevant not only to drought stress but also to
biostress. Recent research on At. lancea and its endophytic bacteria has also indicated the importance of ABA
for mediating volatile oil biosynthesis in At. lancea
(Wang et al. 2014).
1769
9724
1608
<001
<001
<001
1758
6508
6607
0194
<001
<001
1
2
2
1356
1243
2384
<001
<001
<001
4898
1210
6058
<001
<001
<001
2378
4258
0529
<001
0023
0594
3938
6662
6477
<001
<001
<001
Interaction of drought and FEs and their dual effects on
plants
Fw, fresh weight.
Significant P-values are in bold. All of the data used here are the same as that in Tables 1 and 2.
P
F
F
F
P
F
F
P
F
df
F
P
F
P
Root branching
no.
Root ABA
Leaf ABA
Growth traits
Tiller no.
P
Root Fw
Total tiller
length
P
Shoot Fw
P
Root:Shoot ratio
Drought degree constrains FEs’ benefits on plants
Endophyte (E)
Drought (D)
E9D
Table 3 Two-way
ANOVA
results for ABA and basic growth traits of endophyte-inoculated or endophyte-free Atractylodes lancea plantlets grown under different drought treatments.
T. Yang et al.
When facing increasing drought stress, researchers used
to focus with great enthusiasm on discovering particular
FEs to solve this global problem (Sherameti et al. 2008;
Bae et al. 2009; Khan et al. 2013; Hubbard et al. 2014).
Although it is plausible (e.g. numerous studies have indicated that plant growth-promoting bacteria and fungi
positively affect plants subjected to drought stress (Compant et al. 2010)), drought can lead to changes in plant–
FE associations and FEs themselves (Zhang et al. 2011;
Giauque and Hawkes 2013). For example, Rudgers and
Swafford (2009) found Elymus virginicus benefited more
from FEs under daily watering than under drought. Similarly, in our experiment, the individual effects of drought
and Ac. strictum on certain plant physiological and
growth traits were obvious, but their interaction was
more significant.
Among all of the treatment groups, the soluble sugars
and proteins of the MDE+ group were highest in the later
period of drought. Although FEs consumed soluble sugars, they also effectively enhanced soluble sugars under
mild drought stress, which implies a certain degree of
potential for FE application. However, once environmental stress exceeds certain ranges, this benefit of FEs fades
away. The changes in soluble protein in the MDE+ group
in Fig. 2b indicated that the benefits of FEs decrease
slowly with prolonged drought time. This may show a
timing aspect to the effect of drought degree on the symbiosis of FEs and plants. It is supposed that as drought
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
1445
Drought degree constrains FEs’ benefits on plants
T. Yang et al.
continues, soluble sugars and proteins will all decrease.
Drought stress significantly increased the plantlets’ ROS,
which enhanced the MDA content, and even damaged
DNA. At day 0 of drought treatment, the MDA content
of the MDE+ group was higher than that of the MDE
group. Later, Ac. strictum reduced the rate at which
MDA increased (MDE+ < MDE at 3th–9th day). However, with severe drought treatment or regular watering,
this effect of the FE on MDA was not significant. For
proline and the three antioxidant enzymes, the FE provided significant beneficial effects only in the MDE+
group. Enhancement of proline under drought stress was
also found with Exophiala sp. LHL08, Pseudomonas fluorescens and Bacillus subtilis (Khan et al. 2011; Saravanakumar et al. 2011). Additionally, several studies have
demonstrated that FEs may produce mannitol, other carbohydrates and small molecules (proline) with antioxidant capacity (White and Torres 2010). FEs may provide
an arsenal of antioxidants for plants to cope with abiotic
stresses. Although RWC is a vital and intuitive trait for
plant drought resistance, Ac. strictum failed to enhance
RWC in host leaves. A possible reason is that the leaves
are small and we only used live and healthy leaves in our
experiment. Leaves wilted due to drought were excluded.
Perhaps for Ac. strictum and At. lancea, the FE prefers to
strengthen host tolerance to drought (e.g. antioxidants)
rather than sustain RWC like a sponge.
From Table 3, significant interactions of FE presence
and drought were found for ABA and the tiller numbers,
total tiller length, root fresh weight, shoot fresh weight
and root:shoot ratio. It was surprising to find that the
effects of Ac. strictum on At. lancea growth traits were
distinct under different degrees of drought. In the regularly watered groups, the FE had passive effects on the
root growth of At. lancea. In the mild drought groups,
the FE had positive effects on root growth. In the severe
drought groups, root and shoot fresh weight showed no
difference between E+ and E. However, the interaction
between 10% PEG6000 and the FE significantly stimulated root fresh weight and root:shoot ratio, even more
so than any other group, which implies that the correct
abiotic stress together with FE presence benefits plant
more in certain respects.
In our experiment, FE AL16, identified as Ac. strictum,
primarily benefits from its symbiosis with At. lancea, and
in return, it helps the plant cope with drought stress
when it is mild and not over a certain threshold (e.g.
only with 10% PEG6000 treatment in our research).
Drought can also affect the FE–plant association. For
example, when drought stress exceeds a certain threshold
(severe drought treatment), the beneficial effects of the
FE fade away. In summary, plant trait plasticity was conferred by the dual effects of drought stress and FEs, and
1446
these factors are interactive. Because the beneficial effects
of FEs are highly context dependent (Rodriguez et al.
2009), the role of FEs in a changing background should
be re-assessed (Emery and Rudgers 2013). Our next aim
is to unravel when these ‘beneficial’ FEs actually benefit
plants (Pineda et al. 2013).
Acknowledgements
We are grateful to the National Natural Science Foundation of China (NSFC, Nos. 31070443), a Project Funded
by the Priority Academic Program Development of Jiangsu Higher Education Institutions and Integration of
Production and Research Project of Nanjing Science and
Technology Commission (Nos. 201306019) for their
financial support. We also express our grateful appreciation to the reviewers and editorial staff for their time and
attention.
Conflict of Interest
The authors declare no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 The phylogenetic tree of rDNA ITS
sequences of the endophyte AL16 Acremonium strictum
Journal of Applied Microbiology 117, 1435--1449 © 2014 The Society for Applied Microbiology
T. Yang et al.
and related anamorphs and teleomorphs in the Moniliales. Bootstrap values are indicated above branch nodes.
Thielavia terrestris is used as an outgroup.
Figure S2 Morphology of endophyte AL16. (a) The
obverse side of the plate cultured for 7 days in the dark.
(b) Fungal auto-fluorescence clearly shows the typically
slimy head containing conidia above the metulae. (c) The
unique spore structure of endophyte AL16 after repeated
freeze-thaw for 65 days, the left end of this structure
Drought degree constrains FEs’ benefits on plants
including typically slimy head. Scale bar is 5 lm. (d) The
unique spore structure of endophyte AL16 after repeated
freeze-thaw for 65 days, the middle enlargement part of
this structure full of conidia. Scale bar is 5 lm.
Figure S3 The survival rate of plantlets after acclimatization. E+ group is 80.28 6.25%, while E group is
62.5 7.22% (Total plantlets number is 160). “*” means
significant difference at P = 0.05.
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