Phytochemistry 87 (2013) 6577

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Plant secondary metabolite proling evidences strain-dependent effect

in the AzospirillumOryza sativa association
Amel Chamam a, Herv Sanguin a,1, Floriant Bellvert a, Guillaume Meiffren a, Gilles Comte a,
Florence Wisniewski-Dy a, Cdric Bertrand b, Claire Prigent-Combaret a,
a
b

CNRS, UMR 5557, Ecologie Microbienne, Universit Lyon 1, Universit de Lyon, F-69622 Villeurbanne, France
Universit de Perpignan Via Domitia, Laboratoire de Chimie des Biomolcules et de lEnvironnement, EA 4215, F-66860, Perpignan, France

a r t i c l e

i n f o

Article history:
Received 3 August 2012
Received in revised form 8 November 2012
Available online 22 December 2012
Keywords:
Azospirillum
PGPR
Oryza sativa
Root colonization
Phenolic metabolites
Secondary metabolite proling

a b s t r a c t
Azospirillum is a plant growth-promoting rhizobacterium (PGPR) able to enhance growth and yield of
cereals such as rice, maize and wheat. The growth-promoting ability of some Azospirillum strains appears
to be highly specic to certain plant species and cultivars. In order to ascertain the specicity of the associative symbiosis between rice and Azospirillum, the physiological response of two rice cultivars, Nipponbare and Cigalon, inoculated with two rice-associated Azospirillum was analyzed at two levels: plant
growth response and plant secondary metabolic response. Each strain of Azospirillum (Azospirillum lipoferum 4B isolated from Cigalon and Azospirillum sp. B510 isolated from Nipponbare) preferentially
increased growth of the cultivar from which it was isolated. This specic effect is not related to a defect
in colonization of host cultivar as each strain colonizes effectively both rice cultivars, either at the rhizoplane (for 4B and B510) and inside the roots (for B510). The metabolic proling approach showed that, in
response to PGPR inoculation, proles of rice secondary metabolites were modied, with phenolic compounds such as avonoids and hydroxycinnamic derivatives being the main metabolites affected. Moreover, plant metabolic changes differed according to Azospirillum strain  cultivar combinations; indeed,
4B induced major secondary metabolic prole modications only on Cigalon roots, while B510, probably
due to its endophytic feature, induced metabolic variations on shoots and roots of both cultivars, triggering a systemic response. Plant secondary metabolite proling thereby evidences the specic interaction
between an Azospirillum strain and its original host cultivar.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Plant development and growth are strongly inuenced by biotic
and abiotic factors encountered by roots within soils. Certain soil
microbial populations can benet the plant by improving its
growth, development and health. This is typically the case of plant
growth-promoting rhizobacteria (PGPR), establishing associative
symbiotic interactions with their host plant (Barea et al., 1998;
Bloemberg and Lugtenberg, 2001; Raaijmakers et al., 2009;
Richardson et al., 2009). PGPR, whose growth are stimulated by
root exudates, are, in return, able to colonize plant root systems
and contribute to enhance plant growth through a variety of mechanisms including direct effects on nutrient uptake (e.g. N2-xation,
P-mobilization, iron-chelation), and on root growth through the

Corresponding author. Address: CNRS, UMR 5557, Ecologie Microbienne,

Universit Lyon 1, 43 bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Tel.: +33 4 72 44 58 89; fax: +33 4 72 43 12 23.
E-mail address: claire.prigent-combaret@univ-lyon1.fr (C. Prigent-Combaret).
1
Present address: CIRAD, UMR LSTM, F-34398 Montpellier, France.
0031-9422/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.11.009

production of phytohormones (auxins, giberrellins and cytokinins)

(Richardson et al., 2009; Shaharoona et al., 2007; Yang et al., 2009).
They can also protect plant against root and/or foliar pathogens
(Raaijmakers et al., 2009) through antibiotic production, competition for ecological niches, and induction of systemic resistance
(Antoun and Prvost, 2005; Lugtenberg and Kamilova, 2009;
Rezzonico et al., 2005).
PGPR belong to a wide range of genera like Acetobacter,
Azospirillum, Bacillus, Burkholderia, Herbaspirillum, Paenibacillus,
Phyllobacterium, Pseudomonas or even rhizobia (Desbrosses et al.,
2009; Kumar et al., 2011; Lugtenberg and Kamilova, 2009;
Richardson et al., 2009; Saharan and Nehra, 2011). They can stimulate the growth of several important crops and grasses both in
greenhouse and in eld trials under various soils and climatic conditions (Bashan et al., 2004; Shaharoona et al., 2007; Steenhoudt
and Vanderleyden, 2000; Veresoglou and Menexes, 2010). However, discrepancies were observed in the outcome of PGPR inoculations because plant growth promotion depends on many edaphic,
ecological and biotic factors that are quite difcult to manage altogether. Among PGPR, Azospirillum is considered as one of the most
important rhizobacterial genus for improvement of plant growth

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A. Chamam et al. / Phytochemistry 87 (2013) 6577

and crop yield worldwide, reaching commercialization in several

countries like Mexico (Bashan et al., 2004; Jacoud et al., 1998;
Okon and Labandera-Gonzalez, 1994).
Plant-benecial effects of Azospirillum strains result mostly in
morphological and physiological changes of the root system (Khalid
et al., 2004; Richardson et al., 2009). They are able to increase root
proliferation and elongation, which in turn leads to enhance
the capacity of plants to access to essential nutrients and water
(Richardson et al., 2009). Indeed, they stimulate the growth of their
host plant by producing phytohormones, especially indole-3-acetic
acid, and by deaminating 1-aminocyclopropane-1-carboxylate
(ACC), the precursor of ethylene, a gaseous plant hormone inhibiting root growth (Baldani et al., 1979; Prigent-Combaret et al.,
2008; Steenhoudt and Vanderleyden, 2000; Tarrand et al., 1978).
Azospirillum mostly colonized the surface of roots but some
strains, such as Azospirillum brasilense Sp245 and Azospirillum sp.
B510, can both colonize the root surface and be recovered from
the interior of surface-sterilized plant roots or above-ground parts
and are thereby dened as endophytes (Elbeltagy et al., 2001;
Rothballer et al., 2003; Kaneko et al., 2010; Yasuda et al., 2009).
It appears that the extent of internal colonization depends both
on the strain, and the host plant (Elmerich, 2007). Besides their
plant growth promotion effect, endophytes are able to increase
the resistance of the host plant against pathogens and parasites,
through induction of disease resistance mechanisms independent
of salicylic acid-signaling (Yasuda et al., 2009). Azospirillum endophytes have been proposed to be more efcient in promoting
growth and health when inoculated onto their corresponding host
plant, but no experiment has been designed to address this
question.
Despite growing efforts to study Azospirillum plant benecial
traits and plant root colonization patterns, the growth response
of crops to Azospirillum is not completely predictable. On one
side, differential varietal response has been reported for several
crops, notably for wheat, sorghum, corn and rice, in response to
Azospirillum inoculation (Charyulu et al., 1985; Sasaki et al.,
2010; Saubidet and Barneix, 1998; Vargas et al., 2012; Veresoglou and Menexes, 2010). On the other side, differences in yield
were reported after the inoculation of a single rice or wheat cultivar by several Azospirillum lipoferum or A. brasilense strains
(Garca de Salamone et al., 2010; Saubidet and Barneix, 1998).
These observations have so far not been strengthened by molecular studies.
In order to decipher the molecular mechanisms governing the
specicity of the associative symbiosis between Azospirillum
strains and plant, a multiple-step project was developed, of which
the initial objective is to ascertain the specicity of the physiological response of host cultivars to Azospirillum strains. Thus, the purpose of the present work was to compare the growth and
metabolic responses of two cultivars of Oryza sativa japonica, Cigalon and Nipponbare, to the inoculation with two Azospirillum
strains isolated from each cultivar, A. lipoferum 4B isolated from
the surface of Cigalon roots in Camargue (Thomas-Bauzon et al.,
1982) and Azospirillum sp. B510, a rice endophyte, isolated from
Nipponbare surface-sterilized above-ground parts (Elbeltagy
et al., 2001). More accurately, the rice root colonization pattern
of each strain on both cultivars was rst compared. Second, the
overall plant growth promotion effects and root architecture modications induced by each Azospirillum strain on both rice cultivars
was compared. Third, proling of plant secondary metabolites
from both root and shoot parts were compared between uninoculated and Azospirillum-inoculated plants. This study thus constitutes the rst comparative metabolomic analysis of the responses
of two rice cultivars to bacterial inoculation by Azospirillum differing in their root-colonization strategies and relationships with the
host plant.

2. Results and discussion

2.1. A. lipoferum 4B colonizes the outer surface of rice epidermal cells
whereas Azospirillum sp. B510 also colonizes the interior of roots
Root colonization of both rice cultivars was analyzed 7 days
after inoculation with A. lipoferum 4B or Azospirillum sp. B510.
Confocal laser scanning microscopic (CLSM) observations showed
that Enhanced Green Fluorescence Protein (EGFP)-labeled cells of
both strains preferentially colonized the rhizoplane of active root
zones, such as the apical root zone (Fig. 1AD), the root hair zone
(Fig. 1EH), and sites of lateral root emergence (Fig. 1IL), a colonization pattern already documented in Azospirillum spp. (Zhu et al.,
2002; Pothier et al., 2007; Couillerot et al., 2011). Non-uniform
bacterial colonization along the root can be explained by varying
root rhizodeposition patterns (Compant et al., 2010; Dennis et al.,
2010). Rhizodeposits include mucilages, volatiles, and soluble lysates and exudates that are released from damaged and intact cells,
respectively. The active root zones such as the root cap and the
meristematic root regions are major sites of root exudation towards which rhizobacteria are chemoattracted (Dennis et al.,
2010; Lugtenberg and Kamilova, 2009). Azospirillum cells were
found as single cells as well as small clumps on the root surface
(Fig. 1A, C and L). Both strains did not differ from each other in
their pattern of colonization of the surface of rice roots (namely,
the rhizoplane colonization) and moreover they displayed the
same colonization pattern when inoculated onto Cigalon (Fig. 1A,
B, E, F, I and J) and onto Nipponbare (Fig. 1C, D, G, H, K and L).
In order to check whether the studied Azospirillum strains were
able to colonize the rhizoplane only or both the rhizoplane and the
interior of roots, serial optical sections were made through Cigalon
and Nipponbare root samples, and especially at the point of lateral
root emergence (Fig. 2). Whatever the rice cultivar, 4B cells were
observed only in the rhizoplane (Fig. 2A); B510 cells were found
both at the surface and inside the root (Fig. 2B) without induction
of visible disease symptoms, conrming its endophytic colonization of rice. B510 cells were present 20 lm below the root surface,
a depth corresponding to the cortex (Marcel et al., 2010).
Thus, while B510 was able to colonize the interior of rice roots,
4B only colonized the rhizoplane, a feature observed on both rice
varieties. However, both strains display an equal weak ability to
degrade plant cell wall compounds in vitro (Elbeltagy et al., 2001;
Wisniewski-Dy et al., 2011). Thus, their differential ability to colonize internally the host plant might be related to a differential
regulation of the corresponding gene(s) or to the involvement of
other properties for endophytic colonization, such as twitching
motility (Bhm et al., 2007). According to microscopic observations, no signicant differences were found in the colonization
ability of the endophytic strain B510 onto its host cultivar, Nipponbare, versus its non-host cultivar, Cigalon. The same observation
was made with the non-endophytic strain 4B.

2.2. Azospirillum strains promote growth and modify root architecture

of their host rice cultivar
The effects of A. lipoferum 4B and Azospirillum sp. B510 on plant
growth and root development of Cigalon and Nipponbare were
compared after 7 days of contact, under gnotobiotic conditions.
In presence of 4B, shoot dry weight was signicantly enhanced
by 25% and 45% for respectively Nipponbare and Cigalon, compared to uninoculated plants (Table 1). Similarly, dry weight of
Nipponbare and Cigalon root systems signicantly increased by
25% and 34% when inoculated by 4B, respectively (Table 1). A
greater effect of 4B was observed on Cigalon (from which it was
isolated) compared to Nipponbare. While it had no signicant

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A. Chamam et al. / Phytochemistry 87 (2013) 6577

Cigalon
A. lipoferum 4B

Azospirillum sp. B510

Apical root zone

x
am

x
am

Azospirillum sp. B510

D
ez
x

cc
cc
rc

rc

apex x

bc

rh

H
rh

Root hair zone

Lateral root emergence

Nipponbare
A. lipoferum 4B

rs
x
rh

rs
x

x rs

rh
rs
x

K
lr

lr

L
cc

lr

Fig. 1. Confocal laser scanning microscope images of A. lipoferum 4B (A, C, E, G, I, K) and Azospirillum sp. B510 (B, D, F, H, J, L) on roots of rice Cigalon (A, B, E, F, I, J) and
Nipponbare (C, D, G, H, K, L) at 7 days after bacterial inoculation. Cells expressing EGFP are green and gray backgrounds correspond to the root image formed by the
transmitted light. Studied Azospirillum strains preferentially colonized the apical root zone (AD), the root hair zone (EH) and sites of lateral root emergence (IL). Images are
representative of the analysis of at least 10 images per condition. am, apical meristem; bc, border cells; cc, cell clumps; ez, elongation zone; lr, lateral root; lre, lateral root
emergence; rc, root cap; rh, root hairs.

effect on Cigalon, B510 had a signicant effect on shoots and roots

of Nipponbare, from which it was isolated, with respectively 29%
and 32% of dry weight increases (Table 1).
Extensive root architecture analyses were also conducted using
the WinRHIZO software to investigate the effect of Azospirillum
inoculation. Compared to uninoculated plants, root systems underwent major architectural changes when inoculated with A. lipoferum 4B whereas minor effects were observed with Azospirillum
sp. B510 (Table 2). Signicant enhancements were observed with
4B inoculation on the total root length, root surface and number
of roots of both cultivars, total root volume of Cigalon, and length
of the principal root of Nipponbare. A better promoting effect on
Cigalon was observed compared to Nipponbare, with an increase
of the total root length, root surface and number of roots, by 58%,
58%, and 80% respectively, for Cigalon, against 43%, 31%, and 57%
for Nipponbare. On the contrary, limited effects were observed
with B510 inoculation on the root system architecture of both cultivars, with an increase of the length of the principal root of Nipponbare while it decreased for Cigalon and an increase of the
root diameter of Cigalon (Table 2).
Thus, depending on the Azospirillum strain  rice cultivar combination, the type and number of root parameters signicantly affected by bacterial inoculation differed and the phytostimulatory
effects of A. lipoferum 4B and Azospirillum sp. B510 were higher

on their respective original rice cultivar. However, the endophytic

strain B510 appeared to have little effect on root morphology on
both rice cultivars compared to the non-endophytic strain in the
studied conditions. It has been often suggested that endophytes
are more likely to promote plant growth compared to bacteria
exclusively colonizing the rhizosphere because they achieve close
contact with the plant tissues and escape competition with the rhizosphere microbiome (Chanway et al., 2000; Conn et al., 1997;
Dbereiner, 1992). In gnotobiotic conditions, despite weak effects
on root morphology, B510 induced the highest increase of shoot
and root biomass on its host cultivar, Nipponbare, conrming eld
and greenhouse experiments performed by Isawa et al. (2010) in
Japan where B510 was shown to enhance the growth of leaves
and the production of seeds.
2.3. Azospirillum inoculation alters rice secondary metabolite proles
To determine rice metabolomic responses to Azospirillum inoculation, at the early stages of interaction, metabolite proling
was performed on 10-day-old rice plant methanolic extracts (from
shoots and roots) by Reversed-Phase Liquid Chromatography Mass
Spectrometry (RP-HPLCMS). Chromatographic analyses were
done at 280 nm, a wavelength allowing the detection of a wide
range of secondary metabolites and particularly phenolic

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A. Chamam et al. / Phytochemistry 87 (2013) 6577

lr

4B

20 m

4B
4B

lr

28 m

B510

lr

B510

B510

Fig. 2. Orthogonal optical sections of lateral roots of Nipponbare inoculated with

A. lipoferum 4B (A) and Azospirillum sp. B510 (B). Cells expressing EGFP are green
and red backgrounds correspond to the root image stained with propidium iodide.
The central views framed in blue show xy focal plans from the z-stacks. The red
and green dashed lines represent vertical optical cuts through the z-stacks, which
result in the side images framed in red and green, respectively. In these side views,
the blue dashed lines mark the position where the central view images are located
within the z-stacks. Images were all taken at the emergence of lateral roots. Strain
4B was only found at the surface of roots (A) whereas strain B510 was found both at
the surface and inside roots (B). Endophytic colonization of B510 at the emergence
of a lateral root is indicated by a white circle (B). lr, lateral root.

Table 1
Effect of Azospirillum inoculations on rice shoot and root dry weights.
Treatments

Shoot dry weight

(mg/plant)

Root dry weight

(mg/plant)

Cv. Cigalon
Control
A. lipoferum 4B
Azospirillum sp. B510

1.36 0.08 b
1.97 0.14 a
1.14 0.11 b

1.31 0.07 b
1.76 0.14 a
1.02 0.07 b

Cv. Nipponbare
Control
A. lipoferum 4B
Azospirillum sp. B510

4.20 0.77 b
5.23 0.21 a
5.43 0.31 a

2.12 0.38 b
2.66 0.11 a
2.80 0.19 a

Data shown are mean standard error, n = 4. For each data row and rice cultivar,
statistical differences between treatments are indicated with lowercase letters
(analysis of variance and Fishers least signicant difference test; P < 0.05). Cv.:
cultivar.

compounds. Chromatographic proles of shoot methanolic extracts from uninoculated plants were composed of 45 integrated
peaks for Cigalon and 39 for Nipponbare, 37 peaks being in common between the two cultivars. In the same way, chromatographic
proles of root methanolic extracts were composed of 74 peaks for
Cigalon and 70 for Nipponbare, with 63 common peaks.
After 7 days of contact with Azospirillum strains, secondary metabolic proles of rice shoot and root extracts underwent variations,
mostly in the relative intensity of peaks rather than in the appearance/disappearance of peaks. Chromatographic proles were further compared using Principal Component Analysis (PCA) and
Hierarchical Cluster Analysis (HCA) methods. First, using retention
times and peak areas at 280 nm, two data matrices (for shoot (data
not shown) and root extracts) were built in order to carry out HCA
(Fig. S1). At a bootstrap value of 100%, rice root extracts grouped in
two principal clusters (i.e. clusters Cig and Nip) corresponding to
each rice cultivar (Fig. S1). This analysis shows the disparity of
plant secondary metabolism between rice cultivars, a feature previously reported on 3000 rice accessions in the course of a study of
allelopathic potential of rice varieties (Kong, 2002).
Second, four matrices were built separately for each cultivar
and each plant extract. PCA performed on chromatographic proles
of root and shoot extracts showed signicant differences according
to biological treatments, with percentages of the rst axes higher
than 9.3% for each of the four PCA (Fig. 3). The data variability explained by the rst and second axes of the PCA represents 25.8%
and 46.1% of the total variability for Cigalon shoot and root extracts, respectively, and 15.2% and 34.1% for Nipponbare shoot
and root extracts, respectively (Fig. 3). Discriminant analysis of secondary metabolite proles from Cigalon root extracts showed signicant differences between all three treatments (uninoculated, A.
lipoferum 4B, Azospirillum sp. B510) (Fig. 3C) unlike proles from
Nipponbare root extracts (Fig. 3D). HCA on root chromatographic
data conrmed PCA results (Fig. S1). In a lesser extent, signicant
differences were observed in case of shoot extracts for both cultivars between the B510 treatment and the two others (Fig. 3A
and B). As previously observed with maize (Walker et al., 2011),
the associative symbiosis between Azospirillum PGPR and the host
plant had a signicant impact on plant secondary metabolism. Secondary metabolite proling is thus effective in revealing effects of
inoculation on plant physiology. These results suggest that host
plant recognition of Azospirillum might be strain specic, and triggers a specic physiological response by the host plant.
Once inoculated onto Nipponbare, A. lipoferum 4B induced the
highest plant growth promoting effects compared to Azospirillum
sp. B510, whereas B510 leads to the highest effect on secondary
metabolism. It is worth noting that major secondary metabolism
responses could arise on host plant, although morphological effects
on root architecture were hardly visible. Such a discrepancy between the inoculation effects on plant growth and plant secondary
metabolism has already been reported (Walker et al., 2011). This
suggests that these bacteria can have effects on the secondary
metabolism of plants without inuencing the physiological functions involved in primary metabolism and plant development. Azospirillum sp. B510 is the only studied strain able to induce
signicant modications of secondary metabolism on rice shoots,
suggesting systemic responses of rice cultivars to B510 inoculation.
As this strain has the ability to enter inside the cortex of rice roots
(as evidenced in this work) and to colonize the interior of shoots
(Elbeltagy et al., 2001), it may severely affect the cellular physiology of the host plant, triggering plant systemic responses and profound changes of secondary metabolism. Accordingly, several
studies have reported that even if endophytic PGPR promote plant
growth, they nevertheless induce stress and defense responses,
causing changes in plant metabolites that lead to a ne control
of bacterial populations inside plant tissues (De Matos Nogueira

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A. Chamam et al. / Phytochemistry 87 (2013) 6577

Table 2
Effect of Azospirillum inoculations on rice root architecture.
Treatments

Principal root length

(cm/plant)

Total root length (cm/

plant)

Root surface (cm2/

plant)

Root diameter (lm/

plant)

Root volume (mm3/

plant)

Number of roots per

plant

Cv. Cigalon
Control
A. lipoferum 4B
Azospirillum sp.
B510

7.03 0.42 a
8.25 0.33 a
5.18 0.34 b

23.29 3.34 b
36.85 3.02 a
22.28 4.41 b

2.55 0.32 b
4.04 0.35 a
2.93 0.63 b

366.78 17.79 b
358.80 5.72 b
428.83 31.77 a

22.73 2.75 b
35.88 3.59 a
31.15 7.58 ab

91.07 19.89 b
164.31 12.27 a
84.7 11.50 b

Cv. Nipponbare
Control
A. lipoferum 4B
Azospirillum sp.
B510

9.12 0.43 b
10.57 0.21 a
10.12 0.37 a

46.35 5.65 b
66.33 6.04 a
54.84 4.21 b

5.37 0.57 b
7.08 0.38 a
6.21 0.47 ab

376.29 12.08
348.93 16.33
365.15 9.10

50.25 4.74
61.06 2.93
56.34 4.61

169.50 21.31 b
266.31 23.66 a
205.64 11.66 b

Data shown are mean standard error, n = 4. For each root system parameter and rice cultivar studied, statistical differences between treatments are indicated with
lowercase letters (analysis of variance and Fishers least signicant difference test; P < 0.05). For Nipponbare, root diameter and total root volume parameters were not
signicantly different between treatments. Cv.: cultivar.

A. lipoferum 4B
Azospirillum sp. B510
Non-inoculated

Axis 2 (6.2%)

Axis 2 (6.8%)

A. lipoferum 4B
Azospirillum sp. B510
Non-inoculated

Axis 1 (19%)

Axis 1 (9.3%)

Non-inoculated

Axis 2 (12.9%)

Axis 2 (11.6%)

A. lipoferum 4B

Azospirillum sp. B510

A. lipoferum 4B
Non-inoculated

Azospirillum sp. B510

Axis 1 (34.5%)

Axis 1 (21.2%)

Fig. 3. Discriminant principal component analysis (PCA) performed on chromatographic data obtained for each methanolic extract of rice after 7 days of contact with or
without Azospirillum strains. Analyses were based on peak areas and retention times. Each point represents pooled extracts of the same treatment (nine plants). (A) Cigalon
shoot extracts; (B) Nipponbare shoot extracts; (C) Cigalon root extracts; (D) Nipponbare root extracts. The weight of each axis is indicated.

et al., 2001; Mich et al., 2006; Rosenblueth and Martinez-Romero,

2006). Moreover, rice inoculated with the endophyte Azospirillum
sp. B510 was shown to display signicant resistance against rice
blast disease caused by the fungus Magnaporthe oryzae and rice
blight disease caused by the bacterium Xanthomonas oryzae pv.
oryzae (Yasuda et al., 2009). Interestingly, pathogenesis-related
genes and salicylic acid accumulation were not induced, suggesting
that the priming effect of the endophyte Azospirillum sp. B510 may

involve a novel type of plant resistance mechanism, independent of

salicylic acid signaling (Yasuda et al., 2009).
2.4. Phenolic acids and avonoids are the most discriminant rice
secondary metabolites affected by Azospirillum
PCA and HCA data analyses enabled us to highlight rice metabolites differentially produced in presence of each Azospirillum

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A. Chamam et al. / Phytochemistry 87 (2013) 6577

strain. Most of those discriminant metabolites showed variations

in their relative intensity according to biological treatments rather
than appearance or disappearance in some conditions.
Based on RP-HPLC retention times, UV and mass spectra, and
literature data, the families or names of discriminant metabolites
were identied (Table 3). Most discriminant secondary metabolites are phenolic compounds belonging to three main polyphenol
classes, hydroxycinnamic acid derivatives (n = 10), avonoids
(n = 17), and alkylresorcinols (n = 4) (Table 3) (Fig. 4). Several discriminant phenolic compounds are detected only in one of the
two rice cultivars. Only a few discriminant compounds are found
in both rice cultivars, like tryptophan, avonoids with retention
times of 26.51 (n 9), 32.78 (n 14), 33.61 (n 15), and

46.11 min (n 26), 5-tridecylresorcinol and 5-pentadecylresorcinol. These results highlight that the associative symbiosis between
Azospirillum and rice has a signicant impact on plant phenolic
secondary metabolites. Variations in secondary metabolic proles,
including phenolic metabolites, have already been observed in
other PGPRplant interactions such as Rhizobiumrice, Azospirillummaize, AzospirillumPseudomonasGlomusmaize (Mishra
et al., 2006; Walker et al., 2011, 2012). Indeed, inoculation of rice
with Rhizobium strains induced accumulation of gallic, cinnamic,
ferulic and tannic acids (Mishra et al., 2006). In maize, Azospirillum
affected the biosynthetic pathways of benzoxazine derivatives,
such as benzoxazinones and benzoxazolinones (Walker et al.,
2011).

Table 3
Spectral data of rice discriminant compounds.
Compound
number

Retention times
(min)

Name or familya

UV (nm)b

Molecular
mass (Da)

ESI-MS positive (m/z)

Cultivarse

1
2
3
4
5
6
60
7
8
9

11.01
14.14
15.34
17.18
19.58
21.67
22.28
23.83
25.02
26.51

266, 290sh
222, 262sh
272, 278, 286sh
300sh, 318
302sh, 312
306sh, 324
300sh, 326
306sh, 330
275, 330sh
262sh, 272, 350

390
nd
204
nd
164
368
368
356
598
580

27.34
31.35

272, 350
300sh, 312

nd
nd

413 [M+Na]+, 391 [M+H]+

nd
205 [M+H]+, 188 [MNH3]+
nd
165 [M+H]+, 147 [MH2O+H]+
369 [M+H]+, 391 [M+Na]+
369 [M+H]+, 391 [M+Na]+
379 [M+Na]+, 735 [2M+Na]+
621 [M+Na]+, 581 [MH2O+H]+
581 [M+H]+, 563 [MH2O+H]+, 461
[M120+H]+
nd
nd

Nipd
Nipd
Cigd, Nipd
Nipd
Cigc
Cigc
Cigd
Nipd
Nipd
Cigc, Nipc

10
11
12

31.64

nd
nd
Tryptophan
Hydroxycinnamic acid
p-Coumaric acid
Feruloylquinic acid
Feruloylquinic acid
Feruloylhexose
Flavanone
Luteolin-6-C-arabinoside-8C-glucoside
Flavonoid
Hydroxycinnamic acid
derivative
Isoorientin-200 -glucoside

264sh, 272, 352

610

13

32.59

277, 310sh

14
15
16

32.78
33.61
35.09

17
18

35.18
35.67

19
20

36.41
36.53

Dihydroavone/
dihydroavonol
Flavonoid
Flavone
Apigenin-6-C-arabinoside-8C-glucoside
Hydroxycinnamic acid
6-C-Diglucose coumaroyl
isoscoparin
Apigenin derivative
Isoscoparin-200 -glucoside

21
22

37.36
37.89

Flavonoid
Flavonoid

23

39.32

24

42.02

25

44.15

26
27
28
29
30
31
32
33
34

46.11
50.42
58.11
60.91
61.11
68.31
68.75
69.38
69.73

Dihydroavone/
dihydroavonol
Dihydroavone/
dihydroavonol
Dihydroavone/
dihydroavonol
Flavone
Hydroxycinnamic acid
Hydroxycinnamic acid
nd
Hydroxycinnamic acid
5-Tridecylresorcinol
Alkylresorcinol#
Alkylresorcinol#
5-Pentadecylresorcinol#

Nipd
Cigd
Cigc

nd

611 [M+H]+, 449 [M162+H]+, 329

[M282+H]+
nd

272, 240
260 sh, 270, 350
274, 338

nd
520
564

nd
543 [M+Na]+, 503 [MH2O+H]+
565 [M+H]+, 587 [M+Na]+

Cigc, Nipc
Cigd, Nipd
Cigc

302sh, 314
272, 302sh, 324,
346sh
272, 302sh, 334
270, 350

448
756

Nipd
Cigc

272, 350
274sh, 280,
296sh
276, 296sh

nd
nd

471 [M+Na]+, 449 [M+H]+

757 [M+H]+, 595 [M162+H]+, 433 [M162
162+H]+
nd
625 [M+H]+, 647 [M+Na]+, 463 [M162+H]+ ,
343 [M282+H]+
nd
nd

Cigd
Cigd

800

801 [M+H]+, 823 [M+Na]+

Cigd

276, 296sh

nd

nd

Cigd

262, 292sh

nd

nd

Cigd

272, 284sh, 338

308sh, 330
302sh, 310
316
304sh, 314
276, 280
276, 280
276, 280
276, 280

688
nd
nd
318
nd
292
nd
nd
320

689
nd
nd
319
nd
293
nd
nd
321

nd
624

[M+H]+, 711 [M+Na]+, 527 [M162+H]+

[M+H]+, 341 [M+Na]+, 659 [2M+Na]

[M+H]+

[M+H]+

Cigd

Cigd
Cigc

Cigc, Nipc
Cigd
Cigd
Cigd
Cigd
Cigd, Nipd
Cigd
Nipd
Cigd, Nipd

nd, not determined; sh, spectral shoulder.

#
Proposal based on the UV spectrum.
a
Family or proposed name were given based on UV, MS data and literature data on rice or other plants.
b
Maximum spectral absorbance wavelengths (nm).
c
Identied in shoots.
d
Identied in roots.
e
Metabolites detected in Cigalon (Cig) and/or Nipponbare (Nip) cultivar. Literature data used are for compounds (5) Chung et al. (2001), Tian et al. (2004, 2005), Khanh
et al. (2007), and Jaiswal et al. (2010); (6 and 60 ) Jaiswal et al. (2010), Ferreres et al. (2008), Leiss et al. (2009), and Weisz et al. (2009); (9) Ferreres et al. (2008), Marin et al.
(2004), and Markham et al. (1998); (12) Kaneta and Sugiyama (1973), Ferreres et al. (2003, 2008), Marin et al. (2004), and Markham et al., 1998; (16) Qiu et al. (2009), Ferreres
et al. (2003, 2008), and Markham et al. (1998); (18) Besson et al. (1985); (20) Ferreres et al. (2008), Besson et al. (1985), and Markham et al. (1998); (3134) Suzuki et al.
(1996).

A. Chamam et al. / Phytochemistry 87 (2013) 6577

71

Fig. 4. Rice root secondary metabolites affected by inoculations with Azospirillum strains. Relative intensities of avonoids, hydroxycinnamic acid derivatives, unidentied
secondary metabolites and putative alkylresorcinols in Nipponbare roots (A, C, E, G) and Cigalon roots (B, D, F, H). White bars, uninoculated samples; gray bars, samples
inoculated with A. lipoferum 4B; black bars, samples inoculated with Azospirillum sp. B510. Compound numbers are indicated within brackets below retention times.
Statistical differences between treatments are indicated using letters ac (Fishers LSD test, P < 0.05).

72

A. Chamam et al. / Phytochemistry 87 (2013) 6577

Seventeen discriminant compounds (i.e. n 60 , 11, 13, 15, 19, 21

25, 2732, and 34) were responsible for the separation of Cigalon
root samples into two clusters (A, uninoculated plants and B, plants
inoculated with both strains) (Fig. S1). Those compounds were
hydroxycinnamic acid derivatives (ve out of 17), avonoids (eight
out of 17), putative alkylresorcinols (three out of 17) and another
unidentied metabolite (Table 3) (Fig. 4). Compared to Nipponbare, hydroxycinnamic acid derivatives and avonoids are the
most numerous and abundant discriminant compounds of Cigalon
roots. Following inoculation of Cigalon, the relative intensity of discriminant hydroxycinnamic acid derivatives was signicantly enhanced in plants inoculated with A. lipoferum 4B (i.e. n 11, 27
and 28) and Azospirillum sp. B510 (i.e. n 11, 28 and 30) compared
to uninoculated plants, except for the compound with retention
time of 22.28 min (n 60 ) (Fig. 4B), identied as a feruloylquinic
acid, an ester of quinic and ferulic acids (Jaiswal et al., 2010). This
phenolic compound is an intermediate in lignin biosynthesis
(Dauwe et al., 2007) and has been described as a factor that enhances plant resistance against the insect pest Frankliniella occidentalis (Leiss et al., 2009). Reduced relative intensity of feruloylquinic
acid in Azospirillum-inoculated rice cultivars might result from an
enhanced lignication of plant tissues in response to Azospirillum
strain and especially in response to the endophyte Azospirillum
sp. B510, which is able to trigger plant resistance (Yasuda et al.,
2009). Induction of lignication has been previously evidenced in
response to biocontrol PGPR (Ardebili et al., 2011). On the contrary,
variable effects on the relative intensities of discriminant avonoids were observed in presence of Azospirillum strains, some
being decreased (i.e. a dihydroavone/dihydroavonol (n 13), a
avone (n 15), an apigenin derivative (n 19) and a avonoid (n
21)) and others being increased (i.e. one avonoid (n 22), and
three dihydroavones/dihydroavonols (n 23, 24, 25)). Interestingly, for each compound, variations occurred in the same way
with both Azospirillum strains (Fig. 4D). Relative intensities of
alkylresorcinols were signicantly increased in presence of Azospirillum strains with the highest content being recovered in cigalon roots inoculated with B510 (Fig. 4F). Alkylresorcinols are
phenolic lipids with a hydrocarbon side chain. Among cereals, they
are found mainly in wheat, rye, triticale, and rice (Bouillant et al.,
1994; Suzuki et al., 1996; Ross et al., 2003). The antimicrobial
activity of alkylresorcinols has been widely reported (Mich
et al., 2003; Suzuki et al., 1996) and resorcinol has been described
as an allelochemical compound in rice (Kong, 2002). The amphiphilic characteristics of alkylresorcinols could promote the formation of a layer of phenolic lipids at the surface of roots acting as a
defensive boundary (Baerson et al., 2010). The ability of rhizobacteria to colonize plants despite the production of toxic compounds
suggests that they have developed strategies to cope with the
selective pressure exerted by plants.
Discriminant metabolites responsible for the separation of root
Nipponbare samples into two clusters correspond to compounds n
1, 2, 4, 7, 8, 10, 15, 17, 31, 33, and 34 (Fig. S1). Those compounds
are hydroxycinnamic acid derivatives (three out of 11), avonoids
(three out of 11), putative alkylresorcinols (three out of 11) and
other unidentied metabolites (two out of 11) (Table 3) (Fig. 4A,
C, E and G). In Nipponbare, the synthesis level of these metabolites
was signicantly modied only in presence of Azospirillum sp.
B510. Except the unidentied metabolite n 1, whose relative
intensity only increased with 4B (Fig. 4G), the relative intensities
of all discriminant hydroxycinnamic acid derivatives (i.e. compounds n 4, 7 and 17) were signicantly increased with B510 (Table 3) (Fig. 4A). Feruloylhexose (i.e. compound n 7) like
feruloylquinic acid is an intermediate in lignin biosynthesis
(Dauwe et al., 2007; Derikvand et al., 2008; Vanholme et al.,
2010), and might have, like other feruloyl oligosaccharides, antioxidant activity (Rao and Muralikrishna, 2006). This plant response

suggests that the endophytic B510 strain might induce a resistance

response in Nipponbare. By contrast, the relative intensity of the
three putative alkylresorcinol compounds decreased in Nipponbare roots, in presence of B510, a trend opposite to that observed
in Cigalon (Fig. 4E). For discriminant avonoids, two out of three
(n 10 and 15) showed a signicant decrease of their relative intensity when Nipponbare was inoculated with B510, while the last
one (n 8) showed a signicant increase (Fig. 4C).
Comparison between the two rice cultivars showed more dramatic changes of secondary metabolites in roots of Cigalon, with
a higher number of discriminant metabolites. This may be due to
differences between secondary metabolic contents of both uninoculated cultivars (data not shown). A. lipoferum 4B induced secondary metabolic variations only on its original host plant (Cigalon);
this can be correlated with a higher stimulation of plant growth
in the 4B  Cigalon combination. In presence of Azospirillum sp.
B510, variations in secondary metabolic proles were induced in
both cultivars, while a growth promotion was observed only on
its original host plant (Nipponbare). But as discussed above, bacteria can have effects on the secondary metabolism of plants without
inuencing plant development (Walker et al., 2011).
In addition to secondary metabolites, inoculation of both cultivars with strain 4B induced an accumulation of tryptophan in roots
(Fig. 5). Tryptophan is a pivotal precursor of secondary metabolites
in plants, such as glucosinolates and camalexin (Ishihara et al.,
2011; Kliebenstein, 2004). This amino acid is also the precursor
of auxins like indole acetic acid (IAA) in bacteria and in plants
(Bennett and Wallsgrove, 1994; Ishihara et al., 2011; Michiels
et al., 1989). Induction of tryptophan accumulation in inoculated
rice roots might lead to the induction of tryptophan-dependent
production of phytohormones. This result is consistent with the
observed phytostimulatory effects induced by A. lipoferum 4B on
both rice cultivars. Compared to uninoculated rice, tryptophan
accumulation in rice roots in response to 4B was slightly higher
in Cigalon (i.e. +148% increase) compared to Nipponbare (i.e.
+124% increase), which may explain the better PGPR effect of 4B
on Cigalon (Fig. 5).

2.5. Modications of rice shoot metabolic proles occur only in

response to the endophytic Azospirillum strain
Modications of secondary metabolic proles in rice shoots
were only signicant in plants inoculated with B510 (Fig. 6) but

Fig. 5. Modulation of the relative intensity of tryptophan in rice roots after

inoculation with Azospirillum strains. Relative intensity of tryptophan in Nipponbare roots (A) and Cigalon roots (B). White bars, uninoculated samples; gray bars,
samples inoculated with A. lipoferum 4B; black bars, samples inoculated with
Azospirillum sp. B510. Compound numbers are indicated within brackets below
retention times. Statistical differences between treatments are indicated using
letters a and b (Fishers LSD test, P < 0.05).

A. Chamam et al. / Phytochemistry 87 (2013) 6577

73

Fig. 6. Rice shoot secondary metabolites affected by inoculations with Azospirillum strains. Relative intensities of avonoids and hydroxycinnamic acid derivatives in Cigalon
shoots (A and C) and Nipponbare shoots (B). White bars, uninoculated samples; gray bars, samples inoculated with A. lipoferum 4B; black bars, samples inoculated with
Azospirillum sp. B510. Compound numbers are indicated within brackets below retention times. Statistical differences between treatments are indicated using letters a and b
(Fishers LSD test, P < 0.05).

their amplitude were less important in shoots than in roots. This

suggests the establishment of a systemic response in plants inoculated with the endophytic strain B510. The increase of several rice
secondary metabolites in shoots, in response to B510 inoculation,
might be part of a particular plant resistance mechanism induced
by this endophytic Azospirillum strain.
In shoots, discriminant secondary metabolites correspond to
avonoids in both cultivars (Fig. 6B and C) and to hydroxycinnamic
acid derivatives in Cigalon, notably p-coumaric acid and feruloylquinic (Fig. 6A) (Table 3). p-Coumaric acid is a hydroxy derivative
of cinnamic acid, and represents with ferulic acid one of the major
rice phenolic compounds derived from the phenylpropanoid pathway (Dixon et al., 2002; Khanh et al., 2007). This compound is involved in plant defense, and allelopathy in rice, and was found in
leaves, shoots, root exudates and rice rhizosphere (Khanh et al.,
2007). Feruloylquinic acid was also shown to confer plant resistance against pathogen fungus as Fusarium gramineum and Sclerotium rolfsii (Leiss et al., 2009; Sarma and Singh, 2003).
Discriminant avonoids identied in shoot parts were C-glycosilated avones, like luteolin-6-C-arabinoside-8-C-glucoside (n 9),
isoorientin-200 -glucoside (n 12), apigenin-6-C-arabinoside-8-Cglucoside (n 16), 6-C-diglucose coumaroyl isoscoparin (n 18),

and isoscoparin-200 -glucoside (n 20). C-glycosilated avones are

present in cereals such as rice and barley (Besson et al., 1985;
Brazier-Hicks et al., 2009; Ferreres et al., 2008; Markham et al.,
1998; Qiu et al., 2009). These avonoids were described to have
antioxidant and antimicrobial activity, but also to promote mycorrhizal symbioses (Ferreres et al., 2008; Markham et al., 1998; Qiu
et al., 2009; Brazier-Hicks et al., 2009). The increased production
of two of them (i.e. n 9 in Nipponbare and n 16 in Cigalon) in
shoots of B510-rice plants may be linked to the ability of this endophytic strain to trigger induced systemic resistance and/or disease
resistance mechanisms in rice (Compant et al., 2010; Yasuda et al.,
2009).
3. Conclusions
Both plant growth promotion assays and the metabolic proling
approach evidence that Azospirillum inoculation signicantly affects rice physiology and that plant responses differ according to
Azospirillum strain  cultivar combination. Higher stimulatory effects of the Azospirillum strains 4B and B510 were observed on
the cultivars from which they were isolated (i.e. Cigalon and
Nipponbare, respectively). Enhanced plant growth effects are

74

A. Chamam et al. / Phytochemistry 87 (2013) 6577

combined with major changes of root secondary metabolite proles, especially in the Cigalon4B interaction. Unlike strain 4B that
colonizes rice rhizoplane, the endophytic strain B510 induced signicant metabolism variations in the aerial parts of both cultivars
evidencing the activation of a plant systemic response. This study
demonstrates that plant secondary metabolite proling is relevant
to differentiate the physiological responses of rice cultivars to Azospirillum strains with distinct root-colonization strategies and ecological relationships with the host plant. This work constitutes the
rst step in our goal to elucidate the molecular bases of the specicity of Azospirillum-plant associative symbiosis (Drogue et al.,
2012). The forthcoming step is to decipher the plant and bacterial
functions and gene regulatory networks that control this specicity, by performing transcriptomic and functional analyses on both
partners from the four studied Azospirillum strain  O. sativa cultivar combinations.
4. Experimental
4.1. Plant material, bacterial strains and culture conditions
This study was performed on two rice (O. sativa L.) varieties
belonging to the Japonica group, i.e. cultivar Cigalon (Centre Franais du Riz, France), and cultivar Nipponbare (J.B. Morel, BGPI, Montpellier, France). A. lipoferum 4B, was isolated from the rhizosphere
of cultivar Cigalon in Camargue, France (Thomas-Bauzon et al.,
1982), and Azospirillum sp. B510, was isolated from surface-disinfected Nipponbare above-ground parts, in Japan (Elbeltagy et al.,
2001) and thus designated as endophyte. The Azospirillum strains
were grown at 28 C onto nutrient agar (Difco) supplemented with
0.0005% (w/v) bromothymol blue (i.e. NAB medium), or in Nfb
broth (Nelson and Knowles, 1978) supplemented with 1/40 (v/v)
Luria Bertani medium (Sambrook and Russell, 1989) containing
NaCl at only 5 g/L (i.e. Nfb). Antibiotics (Euromedex, Souffelweyersheim, France) were added at the following nal concentrations
(lg/mL): ampicillin (Ap), 100; gentamycin (Gm), 25.
4.2. Rice seed disinfection, pre-germination and inoculation by
Azospirillum strains
For all experiments, rice seeds from the two cultivars were surface sterilized as described by Pothier et al. (2007). Disinfected
seeds were germinated on sterile plant agar medium (8 g/L) (Sigma
Chemical Co., St. Louis, USA) for 3 days in the dark at 28 C. Seeds at
a same germination stage were selected for all the inoculation
experiments to limit intra-treatment variability. Bacterial inoculants were grown overnight in Nfb, at 28 C, and at 180 rpm. The
cells were collected by centrifugation at 7000 rpm during 20 min,
gently washed, and resuspended in 0.8% NaCl solution, and the suspensions adjusted to the nal required concentrations (see below).
4.3. Analysis of bacterial root colonization by confocal laser scanning
microscopy
Root colonization patterns of Azospirillum strains were assessed
on both rice cultivars under gnotobiotic systems. Both strains were
tagged with EGFP, so as to monitor bacterial cell colonization by
uorescence microscopy (Bloemberg et al., 2000). The Plac-egfp
plasmid pMP2444 was introduced by conjugation (as described
in Pothier et al., 2007) and tagged strains were selected on NAB
Gm Amp, at 28 C. Bacterial inoculants were obtained after overnight growth in liquid Nfb Gm25 as described above. Inoculation
of each strain on each rice cultivar was done by adding 100 ll of a
107 cells/mL suspension (i.e. leading to an inoculation level of
106 cells/plant) on seedlings. The same volume of 0.8% NaCl solu-

tion was added for uninoculated controls. Square plates

(120  120  17 mm; Greiner Bio-One Ltd., Stonehouse, UK) containing water agar plant (8 g/L) and four seedlings were used. Four
plants (i.e. two plants from two plates) were studied per treatment.
The plates were placed vertically for 7 days in a growth chamber at
28 C with 16 h of light (150 lE/m2/s) and at 22 C with 8 h of dark.
The whole experiment was repeated twice.
For confocal laser scanning microscopy (CLSM), samples 12 cm
in length were cut from different root zones (apical root, hair root,
and lateral root zones) and mounted in Aqua-Poly/Mount (Polysciences, Eppelheim, Germany). For observations of root internal
tissue colonization by Azospirillum, inoculated plants were incubated with 2 lM propidium iodide (PI) for 5 min and then were
gently washed three times in water. Root pieces (from the same
three peculiar zones) were mounted in Aqua Poly/mount. A 510
Meta microscope (Carl Zeiss, Le Pecq, France) equipped with argonkrypton and HeNe lasers was used for analysis of green uorescence emitted by bacteria (excitation at 488 nm and detection
at 510531 nm) and of red uorescence emitted by PI stained plant
cells (excitation at 488543 nm and detection at 563606 nm).
Green images were overlaid with the transmitted images (bright
eld mode) of the root pieces, or with the red images of the PI treated root pieces into a single image using LSM software release 3.5
(Carl Zeiss).
4.4. Plant growth promotion assay
To assess growth promotion of rice cultivars by Azospirillum
strains, 500 ll of a 2  109 cells/mL suspension were mixed with
50 mL of plant agar (8 g/L) (i.e. leading to a nal concentration of
2  107 cells/mL), which was introduced into 120  120  17 mm
square plates. Uninoculated plants were used as control; they were
obtained by adding 500 ll of NaCl 0.8% to 50 mL of plant agar. Five
disinfected seeds were then laid onto the plates, which were incubated vertically, for 7 days, in a growth chamber (using the same
conditions as described above). At 7 days, plants were collected,
and their root system architecture analyzed using WinRHIZO
2002c (Rgent Instrument Inc., Qubec City, Canada). Several
parameters (total root length, average root diameter, total root volume, root surface area and number of roots) were quantied. In
parallel, main root length, and root and shoot dry weights were
measured. Four plates containing ve plants were used per treatment. The whole experiment was done twice. Results are expressed as the means SD (n = 4).
4.5. Secondary metabolites proling of rice
To analyze the secondary metabolic response of rice to Azospirillum strains, and determine whether it changes according to the
rice cultivar and the strain inoculated, a proling of secondary
metabolites was done from plant shoot and root extracts.
4.5.1. Plant shoot and root extracts
From plants grown in the same way as in the plant-growth promoting assay, roots and shoots were separated. Roots from one
condition were randomly pooled by nine and three sets of nine
plant roots were analyzed per condition. The three shoot samples
were prepared in the same way. Plant samples were dipped into liquid nitrogen to avoid enzymatic reactions, and dried by lyophilization using a freeze-dryer (Alpha 1-4 LSC Christ, Osterode,
Germany). Dry weights were determined. One stainless steel ball
(5 mm diameter, VWR International S.A.S, France) was placed in
each tube containing dried roots and shoots. Eppendorfs were then
dipped into liquid nitrogen and crushed using a ball mill (Tissue
Lyser II, Qiagen, Courtaboeuf, France). Each plant sample was extracted with 1 mL of methanol (100%) for 10 mg of dry matter,

A. Chamam et al. / Phytochemistry 87 (2013) 6577

followed by sonication during 20 min. The extraction was performed twice and extracts were pooled. Solutions were then ltered through polytetrauoroethylene (PTFE) membranes
(CHROMAFIL Syringelters, MachereyNagel, France). Samples
were dried in a rotational vacuum concentrator system that includes a concentrator, a cold trap, a chemical trap and a rotor (CentriVap concentrator, Labconco, Kansas City, MO, USA), and
resuspended in methanol (100%) at a nal concentration of
10 mg dry extract/mL.
4.5.2. Chemical analysis of secondary metabolites
LCDAD: Rice root and shoot extracts (20 lL) were injected and
analyzed by reversed-phase liquid chromatography mass spectrometry (RP-HPLC), using an Agilent 1200 series HPLC (Agilent
Technologies, Santa Carla, CA, USA) containing an on-line degasser
(G132A), a quaternary pump module (G1311A), an auto sampler
(G1329A) and a diode array detector (DAD G1315B). The stationary
phase used was a column EC 4.6  250 mm Nucleodur Sphinx RP
5 lm (Macherey Nagel, Hoerdt, France) equipped with a guard column Gemini 3 lm C18, 30  4.6 mm (Phenomenex, Torrence, CA,
USA). The column was eluted at ow rate of 1 mL/min, with an
optimized gradient established using solvents A (formic acid 4
(v/v) in water) and B (formic acid 4 (v/v) in methanol (100%))
(Carloerba Reagents, Val de Reuil, France). Separation was carried
out by adding 5, 25, 40, 70, 95, 95, 5 and 5% solvent B at 0, 15,
30, 55, 65, 70, 73 and 83 min. Chromatograms were recorded and
processed using UVDAD detection at 254, 280 and 310 nm. At
280 nm, a wider spectrum of secondary metabolites, including
avonoids and phenylpropanoid acids was covered, while at 254
and 310 nm only avonoids and phenolic acids are detected,
respectively. The software ChemStation was used for data acquisition and peaks integration of chromatograms.
LCMS: Compounds separation and mass spectrometry analyses
of shoot and root rice extracts were carried out using an Agilent
1100 series HPLC system (Agilent Technologies, Santa Carla, CA,
USA), equipped with a degasser (G1322A), a binary pump module
(G1312), an automatic sampler (G1313A) and a diode array detector (DAD G1314A). Sample analysis and compound separation
were carried using the same conditions as described above (column, solvents, elution gradient). Data acquisition and peaks integration of chromatograms were done using the ChemStation
Agilent software.
For mass spectrometry analysis of extracts, an atmospheric
pressure ionization tted with an electrospray ionization source
(API-ES) in the positive-ion and negative-ion modes was used.
The ionization voltage was 3500 V, fragmentation was performed
at 70 and 200 and scan spectra from 60 to 1000 m/z.
4.6. Statistical analysis
The effects of treatments on plant biomass data, root architecture parameters, and contents of individual secondary metabolites
were analyzed using ANOVA followed by Fishers least signicant
difference (LSD) tests. Those analyses were conducted at P < 0.05,
using S-plus software 6.1 (TIBCO Software Inc., Palo Alto, CA).
Chemical analyses of rice root and shoot extracts were performed
on plants from three independent inoculation experiments. Similar
metabolite proling results were obtained from all experiments
(data not shown). The retention time of each peak in the chromatograms (at 280 nm) was aligned and its relative intensity recorded
in a matrix to perform discriminant principal component analysis
(PCA). PCA were performed using r software. Comparison of chromatographic proles of rice root extracts was made by linkage
hierarchical clustering analysis. For this purpose, data of 81 peak
areas for 30 samples were normalized and subjected to average
linkage hierarchical clustering with the Pearson correlation coef-

75

cient as a distance measure with the MeV 4.0 software (Saeed et al.,
2003). The reliability of sample and metabolite clusters was assessed with experiment bootstrapping (10,000 replications).
Acknowledgements
We are grateful to Benot Drogue for technical assistance and
Ren Bally for helpful discussion (UMR CNRS 5557, Ecologie Microbienne). We thank J.B. Morel (BGPI, Montpellier, France) for gift of
Nipponbare seeds. This work was supported by a fellowship from
the Centre National de la Recherche Scientique to A.C. and by
the ANR project AZORIZ (ANR-08-BLAN-0098). This work made
use of the technical platforms Centre Technologique des Microstructures and Serre at FR 41 (Universit Lyon 1) and Centre
dEtude des Substances Naturelles (UMR CNRS 5557, Ecologie
Microbienne).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.
2012.11.009.
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