Applied Soil Ecology xxx (xxxx) xxxx

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Sinorhizobium spp inoculation alleviates the effect of Fusarium oxysporum on

Medicago truncatula plants by increasing antioxidant capacity and sucrose
accumulation
Marwa Batninia,b,c, Miguel Lopez-Gomezc, Francisco Palmac, Imen Haddoudia,b, Nadia Kallalaa,b,
⁎
Kais Zribia, Moncef Mrabeta, Haythem Mhadhbia,
a
Laboratory of Legumes, Center of Biotechnology of Borj Cedria (LL, CBBC), Hammam-Lif, Tunisia
b
University Tunis El Manar (UTM), Faculty of Sciences of Tunis (FST), 2092 Tunis, Tunisia
c
Department of Plant Physiology, Faculty of Sciences, University of Granada, de Fuentenueva s/n, 18071 Granada, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, the effect of the phyto-pathogenic fungus Fusarium oxysporum on Medicago truncatula-Sinorhizobium
Medicago truncatula symbiosis performance was investigated. Fusarium oxysporum (KLR13) was inoculated on two genotypes of M.
F. oxysporum truncatula, one tolerant (Jemalong A17) and one susceptible (TN1.11), in symbiosis with Sinorhizobium meliloti
Sinorhizobium (TII7), Sinorhizobium medicae (SII4) or fertilized with KNO3. Fungus infection reduced significantly biomass
Sucrose
production and photosynthetic related parameters of non-symbiotic M. truncatula A17 and TN1.11. Reduction
Antioxidant enzymes
was less pronounced in A17, where a lower accumulation of H2O2 and lipid peroxidation (MDA) in concomitance
Nitrogen fixation
to high activities of antioxidant enzymes, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) was
detected. In symbiosis, the fungus affected the nodule number and weight and inhibited the nitrogen fixing
capacity (NFC), as well as the fructose and glucose contents. Nevertheless, inoculation with S. meliloti and S.
medicae alleviated the effect of F. oxysporum on plant biomass production compared to the KNO3 fertilized plants.
Moreover, the Sinorhizobium inoculation decreased H2O2 and MDA over-production caused by the fungal in-
fection in leaves of both genotypes. This tolerance enhancing effect was more pronounced in S. medicae-TN1.11
symbiosis, where the relative maintenance of photosynthesis related parameters, growth capacity and NFC
performance was concomitant to the induction of SOD and CAT activities and an increase of the sucrose content
in leaves. For A17 genotype, both Sinorhizobium strains induced an increase of the proline and sucrose accu-
mulation. In conclusion, F. oxysporum effect on M. truncatula was mitigated when plants were in symbiosis with
Sinorhizobium species, mainly Sinorhizobium medicae that alleviated the oxidative stress and enhanced the ac-
cumulation of sucrose as energy source in TN1.11 genotype, which attenuates its susceptibility to F. oxysporum
infection.

1. Introduction
In nature, plants interact with several microorganisms which can be
either beneficial or detrimental for plant growth and development
(Ballhorn et al., 2014). Therefore distinguishing between a mutualistic
and threatening intruders and responding appropriately are of para-
mount importance to plants to survive (Häffner and Diederichsen,
2016). Legumes represent a suitable model to study how these inter-
actions overlap due to their unique capacity to establish symbiosis with
nitrogen fixing bacteria, rhizobia (Herridge et al., 2008) and to form a
host for a broad range of pathogens, i.e. fungi (Rispail and Rubiales,
2014). Several studies have demonstrated that this simultaneous
interactions could be harmful for the mutualistic changes and could
inhibit the symbiotic establishment (López-Gómez et al., 2012;
Chihaoui et al., 2014; Ballhorn et al., 2014; Marchetto and Power,
2018).The soil-borne pathogen Fusarium oxysporum is mentioned as soil
inhabitant that cause severe diseases and losses to legume production
annually (Eke et al., 2016). It is responsible for a major loss of seed
germination capacity and root rot in host plants that could be re-
sponsible of up to 100% yield loss in susceptible cultivars (Ramírez-
Suero et al., 2010; Belete et al., 2013). Infection of roots with hemi-
biotrophic fungus F. oxysporum occurs with conidia that were in the soil
living on dead tissues or on host plant's surface (Di Pierto et al., 2003).
Medicago truncatula, the model plant for legumes, is a host plant for

⁎
Corresponding author.
E-mail address: Haythem.mhadhbi@cbbc.rnrt.tn (H. Mhadhbi).

https://doi.org/10.1016/j.apsoil.2019.103458
Received 18 April 2019; Received in revised form 22 November 2019; Accepted 25 November 2019
0929-1393/ © 2019 Published by Elsevier B.V.

Please cite this article as: Marwa Batnini, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.103458
M. Batnini, et al.
broad range of pathogenic fungi that cause huge losses in legumes field
production, and it is a symbiotic partner to many beneficial micro-
organisms such as rhizobia and mycorrhiza (Rey et al., 2015). Thus, it
presents a fitting model to investigate how these interactions interfere.
Several studies investigated the effect of biotic and abiotic stress on M.
truncatula (Rispail et al., 2015; Djébali et al., 2009; López-Gómez et al.,
2012) and highlighted different mechanisms such as generation of re-
active oxygen species (ROS) which function as signaling molecules to
avoid the invasion of the hostile microbes by strengthening the cell wall
(Mittler, 2017). But ROS accumulation causes an oxidative stress which
needs to be scavenged by an antioxidant enzyme such as superoxide
dismutase, catalase and peroxidase (Able, 2003). In first steps of in-
teraction, rhizobia, more precisely the lipochito-oligosaccharides Nod
Factors, might be perceived as intruders by legumes, thus ROS pro-
duction is generated (Damiani et al., 2016). It was hypothesized that
Nod Factors may activate a first cascade of ROS involved in nodule
formation and development, and inhibit a second cascade involved in
plant defense reactions (Toth and Stacey, 2015). Therefore, it is ne-
cessary for the symbiotic establishment to maintain the Redox home-
ostasis under tight control to avoid oxidative stress and act as signaling
pathway.
Legume-rhizobia interaction and defense responses in plants are
highly demanding in terms of energy, ATP (Bolton, 2009; Liu et al.,
2018). Several studies stated that sugars are the initial carbon source in
nodules which, subsequently, will be oxidized by the bacteroid to
provide ATP for nitrogen fixation and it is the first substrates to supply
energy for defense responses in plants (Yamada and Osakabe, 2018;
Sasse et al., 2018; Singh et al., 2019). Sugars facilitate timely ROS
production at the beginning of infection, and enhance fortification of
cell walls (Garg et al., 2004; Morkunas et al., 2016; Li et al., 2017a, b).
Thus, they present a major factor that controls both the establishment
of symbiosis and abolishing pathogenic invasion (Moghaddam and Van
Den Ende, 2012).
Proline has been reported in studies on Bradyrhizobium japonicum
(Kohl et al., 1988) and S. meliloti (Jimenez-Zurdo et al., 1997) as an-
other source of energy to supply to the bacteroid to insure nitrogen
fixation. Proline plays a role in plant defense against pathogens
(Senthil-Kumar and Mysore, 2013), Fabro et al. (2007) showed that
proline content was increased in Arabidopsis thaliana when defending a
pathogen attack. It was reported that increment in proline content in
infected cells prevent or delay its death, and may decrease the disease
progression (Qamar and Senthil-Kumar, 2015).
Previous studies (Mrabet et al., 2011; Bahroun et al., 2018) reported
that S. meliloti in symbiosis with M. truncatula and Rahnella aquatilis or
Pseudomonas yamanorum in symbiosis with Faba bean (Vicia faba) have
an antagonistic capacity against Phoma medicaginis and F. solani by
reducing disease symptoms severity and enhancing biomass production.
However others (Chihaoui et al., 2014) reported that Phoma medicaginis
can affect the symbiotic performances of S. meliloti in symbiosis with M.
truncatula by colonizing the nodules and limiting their N2 fixation. The
present study was performed on two contrasting M. truncatula geno-
types face to F. oxysporum infection (unpublished data), Jemalong A17
(relatively resistant) and TN1.11 (susceptible), in symbiosis with two
efficient Sinorhizobium species S. meliloti and S. medicae infected or not
with F. oxysporum. Nitrogen fixing capacity and photosynthesis related
parameters were measured and in parallel redox status, proline content
and carbohydrates contents were determined. The hypothesis behind
this research is that Sinorhizobium species will protect M. truncatula
plants against the pathogen attack by adjusting biochemical changes to
enhance plant growth.
2. Materials and methods
2.1. Bacterial strains and growth conditions
Sinorhizobium meliloti (TII7) and Sinorhizobium medicae (SII4) strains
2
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(Zribi et al., 2005; Zribi et al., 2004) were used to inoculate Medicago
truncatula plants. Bacteria were grown in liquid yeast extract mannitol
medium (YEM) (Vincent, 1970) in an incubator at 150 rpm and 28 °C
until obtain a final concentration of 109 cfu mL−1.
2.2. Fungal material, inoculum preparation
F. oxysporum, KLR13, (Genbank reference # MK615110), from the
laboratory collection, isolated from Faba been and known for its viru-
lence against M. truncatula (Batnini et al., unpublished data), was used
for infection of Medicago truncatula plants. To prepare the spore sus-
pensions, six fungal agar-discs (6 mm in Ø) from 21 days-old culture
were sub-cultured in 100 mL liquid Potato Dextrose Broth medium
(PDB) at 27 °C with shaking at 160 rpm. The obtained suspensions were
filtered and adjusted to 106 conidia mL−1.
2.3. Plant material, inoculation and growth conditions
Two Medicago truncatula genotypes A17 and TN1.11 previously
characterized as respectively resistant and susceptible to Fusarium
oxysporum infection (Batnini et al., unpublished data) were used. Seeds
were scarified and germinated as previously described (Mhadhbi et al.,
2005). Similar sized germinated seedlings were selected and grown in
sterile-sand pots. Pots ware placed in a glass house at 25/22 °C day/
night temperatures, 60–70% relative humidity, and 16/8 h photo-
period. Plants were regularly watered with a free‑nitrogen nutritive
solution for plants in symbiosis as described by Vadez et al. (1996) and
modified by Mhadhbi et al. (2005). This solution contains: macro-
nutrients in mM: MgsO4 (1), KH2 PO4 (0.25), K2SO4 (0.7), and CaCl2
(1.65); micronutrients (μM): MnSO4·H2O (6.6), CuSO4·5H2O (1.56),
Na2MoO4·7H2O (0.12), CoSO4·7H2O (0.12), ZnSO4·7H2O (1.55) and
H3BO3 (4) and iron was added as Siquestrene (1.26 iron mg L−1). Ni-
trogen fertilized plants were watered with KNO3 enriched solution
(2 mM). 48 h old seedlings were inoculated with S. meliloti and S.
medicae (109 cfu mL−1). Three days later, seedlings were inoculated
with F. oxysporum (KLR13) following Hagland (Haglund, 1989) de-
scription with some modifications. Seedlings were removed from the
sand and washed with water. Roots were trimmed as described by Bani
et al. (2012) and dipped in the conidial suspension
(106 microconidia mL−1) for 7 min. Control plant were immersed in
sterile water. Then, seedlings were re-planted in the pots.
The experiment had a completely randomized block design. To
determine sugars, H2O2, MDA, antioxidant enzymes, and proline con-
tents, 4 biological replications were set up with six plants per replica-
tion. At the harvest time samples from each 6 plants (leaves and roots
separately) were homogenized and 3 technical repetitions were mea-
sured for each biological replication. For “in situ” analyzed parameters
(ARA, photosynthesis) and biomass analysis, 9 replications, with one
plant per replication were set up. These replications had the following
treatments: KNO3-fertilized treatment control, T (C), KNO3-fertilized
treatment infected with F. oxysporum, T (KLR13), Bacteria: S. meliloti,
TII7 (C), and S. medicae, SII4 (C), used separately and alone.
Combinations: inoculation with S. meliloti subsequently infected with F.
oxysporum, TII7 (KLR13), and inoculation with S. medicae subsequently
infected with F. oxysporum, SII4 (KLR13). This experiment was main-
tained for 6 weeks.
2.4. Plant biomass
After 6 weeks, plants were separated into shoots, roots, and nodules
which were rinsed with distilled water. Then samples of shoots and
roots were placed at 65 °C to dry for three days, until constant weights,
to determine the dry weights. Fresh samples were stored at −80 °C
until biochemical analyses.
M. Batnini, et al. Applied Soil Ecology xxx (xxxx) xxxx

2.5. Soluble carbohydrates content

Glucose, fructose and sucrose were extracted, separated and quan-

tified by ion chromatography with pulsed amperometric detection
(Dionex ICS-3000 chromatograph) according to Palma et al. (2014).

2.6. Lipid peroxidation and hydrogen peroxide assays

Lipid peroxidation was assayed using the thiobarbituric acid

(TBARS) method as described by Heath and Parcker (1968). 200 mg of
fresh material was grinded in 4 mL of a 1% trichloroacetic acid (TCA)
solution. The homogenate was centrifuged at 12,000g for 15 min, then
0.5 mL of the supernatant was added to tubes containing 3 mL of the
solution (0.5% thiobarbituric acid (TBA) in 20% TCA) which then were
incubated at 95 °C for 2 h. The reaction was stopped by placing the
tubes in an ice bath. Subsequently, tubes were centrifuged at 9000 ×g
for 10 min. The concentration of the malondialdehyde complex (MDA-)
TBA was determined by measuring the optical density of the super-
natant at 532 and 600 nm and was expressed using the molar extinction
coefficient of 155 mM−1 cm−1.
Hydrogen peroxide, in leaves and roots, was quantified using the KI
method, as described by Velikova et al. (2000). All extractions were
done in ice bath: 500 mg of root or leave samples were homogenized
with 0.1% (w/v) TCA (5 mL). The mixture was centrifuged for 15 min
at 12,000 ×g, then 0.5 mL (10 mM) phosphate buffer (pH 7) were
Fig. 1. Effect of F. oxysporum infection on: (a) shoot dry weight (SDW) and (b)
added to 0.5 mL of the supernatant and 1 mL 1 M KI. The content of root dry weight (RDW) of Medicago truncatula genotypes Jemalong A17 and
H2O2 was determined spectrophotometrically at 390 nm. TN1.11 inoculated, with Sinorhizobium meliloti (TII7) and Sinorhizobium medicae
(SII4). T (C): control plants nitrogen-fertilized, T (KLR13): plants nitrogen-fer-
2.7. Antioxidant enzymes assays tilized infected with F. oxysporum, TII7 (C): control plants inoculated with S.
meliloti, TII7 (KLR13): plants inoculated with TII7 and infected with F. oxy-
Levels of guaiacol peroxidase (POX), superoxide dismutase (SOD) sporum, SII4 (C): control plants inoculated with S. medicae, SII4 (KLR13): plants
and catalase (CAT) were measured spectrophotometrically. Extract inoculated with SII4 and infected with F. oxysporum. Bars with the same lower
preparation and enzymes' activities measure and calculation were case letter are not significantly different at P ≤ .05.
performed as described by Chihaoui et al. (2014). Samples of leaves and
roots were crushed to a fine powder in a mortar with liquid nitrogen. To using pure acetylene and ethylene as internal standards, as detailed by
extract soluble proteins, 500 mg of the powder were re-suspended, in Mhadhbi et al. (2009).
ice, in 2 mL of extraction buffer containing: 50 mM potassium phos-
phate buffer (pH 7.8)0.1 mM EDTA, 0.1 mM PMSF and 10 mM DTT.
2.10. Statistical analyses
The homogenate was then aliquoted in new tubes after centrifugation at
13000 ×g for 20 min. Protein concentration was measured using the
Variance analysis of data (three-way ANOVA) was performed using
(Bradford, 1976) method using a protein assay kit (Bio-Rad, Munched,
the SPSS 18 program, and means were separated according to the HSD
Germany) and BSA (bovine serum albumin) as a standard (Sigma, St.
Tukey test at P ≤ .05. Data shown for biomass, ARA, and photo-
Louis, USA). POX activity was determined at 470 nm during 1 min
synthetic related parameters are means of 9 replicates. For biochemical
(ε = 26.6 mM−1 cm−1) by its ability to produce 1 μmol min−1 of
analyses data are means of 4 replicates.
guaiacol oxidized in the presence of H2O2 according to the method
described by Anderson et al. (1995). SOD activity was assayed ac-
cording to the method of Yu (1999) in measuring the ability of the 3. Results
enzyme to inhibit the photochemical reduction of NBT (Sigma). CAT
activity is determined by following the decomposition of H2O2 at 3.1. Plant growth and biomass production
240 nm (ε = 36 M−1 cm−1) (Anderson et al., 1995).
Fungal infection reduced significantly biomass production in both
2.8. Proline content genotypes A17 and TN1.11 (Fig. 1a, b) in non-symbiotic conditions.
The three way ANOVA analysis (Table 1) showed a significant effect on
Proline was extracted using 0.5 g of sample (leaves) and 4 mL of shoot dry weight (SDW) and root dry weight (RDW) mainly for the
extraction medium (ethanol:chloroform:water) and the homogenate genotype and treatment factors and their interaction as well as inter-
was centrifuged at 4 °C and 3500 ×g for 10 min (Irigoyen et al., 1992). actions involving bacterial strain. SDW was 1.65 g plant−1,
The supernatant was separated into aqueous and chloroform phases by 0.38 g plant−1, 1.03 g plant−1 and 0.28 g plant−1 for control nitrogen-
the addition of chloroform (5 mL) and water (3 mL). Proline was de- fertilized plants, T (C), and infected plants, T (KLR13), of A17 and
termined from the aqueous phase and quantified by means of a col- TN1.11, respectively. In symbiosis implicating A17, Inoculation with S.
orimetric reaction with ninhydrin reagent. Standard curve was pre- meliloti, TII7 (KLR13), and S. medicae, SII4 (KLR13), antagonized the
pared with L-proline and results were expressed as mmol g FW−1. SDW reduction caused by F. oxysporum by 2.48 times and 4.12 times
compared to infected nitrogen fertilized plants T (KLR13), respectively.
2.9. Acetylene reduction assay (ARA) While in TN1.11, only S. medicae was able to alleviate F. oxysporum
effect on SDW by 1.90 times. The same enhancement effect of Si-
Nitrogenase (EC 1.7.9.92) was assayed by acetylene reduction ac- norhizobium spp inoculation was observed on RDW of both genotypes
tivity (ARA), in situ, by gas chromatography with a Porapak-T column (Fig. 1a, b).

3
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Table 1
Results of three-way analysis of the effect of strain (S), genotypes (G), and treatment (T) and their interaction (S ∗ T ∗ G) on shoots dry weight (SDW), roots dry
weight (RDW), antioxidant enzyme activities in leaves POX L, CAT L, SOD L, and in roots POX R, CAT R and SOD R, proline, glucose, fructose and sucrose content in
leaves of Medicago truncatula genotypes.
SDW RDW POX L POX R CAT L CAT R SOD L SOD R Proline Glucose Fructose Sucrose

⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎

S 0.073 3.722 0.942 65.549 57,384 655 28,516 20,740 272 2874 69,603 67,028⁎⁎⁎
G 211.138⁎⁎⁎ 175.822⁎⁎⁎ 3.300 2.845 32,159⁎⁎⁎ 13,846⁎⁎ 334 6800⁎ 381 3114 3816 3163
T 50.902⁎⁎⁎ 7.424⁎ 3.692⁎ 13.781⁎⁎⁎ 13,091⁎⁎⁎ 40,839⁎⁎⁎ 21,604⁎⁎⁎ 17,432⁎⁎⁎ 24,424⁎⁎⁎ 8736⁎⁎ 20,115⁎⁎⁎ 77,344⁎⁎⁎
S∗G 7.259⁎ 0.118 6.788⁎ 0.800 18,577⁎⁎⁎ 906 11,618⁎ 265 2711 1074 24,358⁎⁎⁎ 30,379⁎⁎⁎
S∗T 43.235⁎⁎⁎ 4.384⁎ 0.374 19.642⁎⁎⁎ 53,907⁎⁎⁎ 3210 16,871⁎⁎⁎ 39,390⁎⁎⁎ 58,272⁎⁎⁎ 6986⁎ 28,615⁎⁎⁎ 26,960⁎⁎⁎
G∗T 45.965⁎⁎⁎ 3.08 1.957 9.779⁎⁎ 29,256⁎⁎⁎ 11,221⁎⁎⁎ 903 9511⁎⁎ 702 4023⁎ 7054⁎ 1341
S∗G∗T 42.732⁎⁎⁎ 2.424 1.430 1.114 18,145⁎⁎⁎ 1809 14,354⁎⁎⁎ 3717⁎ 14,237⁎⁎⁎ 16,155⁎⁎⁎ 30,549⁎⁎⁎ 6215⁎

Numbers represent F-value.

⁎
P < .05.
⁎⁎
P < .01
⁎⁎⁎
P < .0001.

3.2. Photosynthesis related parameters 3.4. Hydrogen peroxide (H2O2) and lipid peroxidation (MDA) contents

In nitrogen fertilized infected plants, F. oxysporum decreased net
assimilation (AN) and stomatal conductance (gs) in TN1.11 while it
affected only gs in A17 (Fig. 2a, b) when compared to nitrogen fertilized
control plants. In combined treatments symbiosis-F. oxysporum both S.
meliloti and S. medicae didn't alleviate the negative effect of F. oxy-
sporum on gs in A17 plants. Moreover a decrease in intracellular CO2
concentration (Ci) was observed (Fig. 2c). However, within TN1.11 S.
medicae antagonized the effect of the fungal infection on AN and gs
compared to the nitrogen fertilized infected plants and increased AN by
2.08 times and gs by 1.25 times.
3.3. Changes in soluble carbohydrates content in leaves
The analyses of variance for carbohydrates content showed a high
and significant contribution of strain, Treatment and their interaction
along with strain-genotype interaction effect (Table 1). Within A17
nitrogen fertilized plants, F. oxysporum increased glucose content by
54.62% when compared to control plants. In treatments S. meliloti and
S. medicae, glucose increased by 54.61% and by 39.23% respectively
compared to nitrogen fertilized control plants, while in combined
treatments S. meliloti-F. oxysporum and S. medicae-F. oxysporum the
accumulation of glucose decreased by 67.71 and 16.36%, in leaves of
A17 when compared to F. oxysporum treatment (Fig. 3a). For TN1.11
plants, glucose content in leaves of plants infected with F. oxysporum
decreased significantly compared to nitrogen fertilized control plants.
The same behaviour was observed in leaves of plants inoculated with S.
meliloti and with S. medicae.
For fructose content, it increased by 40.69% in A17 nitrogen ferti-
lized infected plants compared to control plants (Fig. 3b). In symbiosis,
it increased by 78.15% in S. meliloti-A17 plants compared to nitrogen
fertilized control plants. Under combined treatments S. meliloti-F. oxy-
sporum and S. medicae-F. oxysporum, the accumulation of fructose de-
creased significantly by 71.95% and by 33.49% when comparing to F.
oxysporum treatment, respectively. For TN1.11, fructose accumulation
decreased significantly in nitrogen fertilized infected plants and in
symbiosis with both Sinorhizobium when compared to nitrogen control
plants (Fig. 3b).
Regarding sucrose content, it decreased by 44.29% in A17 nitrogen
fertilized infected plants compared to control plants (Fig. 3c). In com-
bined treatments, the accumulation of sucrose increased by 56.24%
under S. meliloti-F. oxysporum combination when compared to F. oxy-
sporum treatment. In TN1.11 infected plants sucrose accumulation in-
creased by 1.81 times compared to control plants. In combined treat-
ments it increased by 3.64 and 5.95 times in S. meliloti-F. oxysporum and
S. medicae-F. oxysporum when compared to F. oxysporum treatment,
respectively.
In leaves of nitrogen fertilized infected A17 plants, F. oxysporum
increased significantly H2O2 level (Fig. 4a) when compared to control
plants. In symbiosis implicating A17-S. medicae and infected with F.
oxysporum, H2O2 production decreased significantly when compared to
F. oxysporum treatment. For TN1.11 genotype, H2O2 production in-
creased significantly in leaves of nitrogen fertilized infected plants
when compared to control plants. In combined treatments, S. medicae-F.
oxysporum and S. meliloti-F. oxysporum, level of H2O2 decreased by
51.74 and 19.56%, respectively when compared to F. oxysporum
treatment (Fig. 4a). In roots of A17 plants in interaction with S. meliloti
and F. oxysporum, hydrogen peroxide production increased significantly
by 68.05 when compared to F. oxysporum treatment (Fig. 4b). For
TN1.11, production of H2O2 in roots in interaction with S. medicae and
F. oxysporum decreased by 13% when compared to F. oxysporum
treatment (Fig. 4b).
In nitrogen fertilized plants, MDA content in infected leaves of A17
increased by 32.80% when compared to control plants (Fig. 4c). In
combined treatment, S. meliloti-F. oxysporum, MDA production de-
creased by 21.25% when compared to F. oxysporum treatment. While an
accumulation of MDA was observed in the combined treatment S.
medicae-F. oxysporum. In TN1.11nitrogen fertilized plants, MDA content
in leaves infected with F. oxysporum increased by 52.51% when com-
pared to control plant (Fig. 4d). In combined treatments S. meliloti-F.
oxysporum and S. medicae-F. oxysporum, MDA content decreased by
21.32 and 82.87%, respectively, compared to F. oxysporum treatment.
While in roots interacting with S. medicae and F. oxysporum, MDA
content increased significantly by almost 1.5 times compared to F.
oxysporum treatment (Fig. 4d).
3.5. Antioxidant enzyme responses to fungal infection
The ANOVA for the antioxidant enzymes activities in leaves and
roots (superoxide dismutase (SOD), catalase (CAT), and peroxidase
(POX)) showed a significant effect mainly for the factors: strains (S) and
treatment (T) and their interaction (Table 1). When taking nitrogen
fertilized treatments, F. oxysporum caused a significant induction in the
activity of SOD in roots and leaves and POX and CAT in roots of A17
plants. While, within TN1.11 no significant enhancement in activities
was observed (Fig. 5). In symbiosis implicating TN1.11 and S. meliloti,
CAT activity in leaves increased by 14.94 times compared to nitrogen
fertilized infected plants (Fig. 5b). In the same line, S. medicae, en-
hanced SOD and CAT activities in leaves by 96.68% and by 9.07 times,
respectively (Fig. 5a, b) but no enhancement was observed in roots
(Fig. 5d, e, f). However for symbiosis implicating A17, SOD and POX
activity in leaves, as well as in roots, showed a significant decrease
when compared to nitrogen fertilized infected plants.

4
M. Batnini, et al.
Fig. 2. Effect of F. oxysporum infection on: (a) net assimilation of CO2 (AN), (b) stomatal conductance (Gs) and (c) intercellular CO2 concentrations (Ci) of M.
truncatula genotypes Jemalong A17 and TN1.11, inoculated with S. meliloti and S. medicae. Bars with the same lower case letter are not significantly different at
P ≤ .05.
3.6. Effect of fungal infection on proline content in leaves
Proline content showed a non-significant difference between in-
fected and control nitrogen fertilized plants in both genotypes (Fig. 6).
For combined treatments implicating S. meliloti-F. oxysporum and S.
medicae-F. oxysporum, plants of A17 showed an enhanced accumulation
of proline by 95% and by 58%, respectively, compared to infected ni-
trogen fertilized plants. While no significant changes were observed in
symbiosis implicating TN1.11 (Fig. 6).
3.7. Effect of fungal infection on nodulation and nitrogen fixation capacity
(ARA)
Generally, fungal infection affected the nodule number (NN) and
the nodule fresh weight (NFW) in M. truncatula genotypes inoculated
with both bacterial strains (Fig. 7a, b). In combined treatment im-
plicating A17, S. melliloti, and F. oxysporum NN was significantly re-
duced by 45% when compared to control S. meliloti plants. In TN1.11, F.
5
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oxysporum infection decreased significantly NN by almost 75% and
NFW by almost 68% in both symbiotic associations involving S. meliloti,
and S. medicae. Changes of NN and NFW were concomitant to changes
in nitrogen fixing capacity (NFC), as F. oxysporum decreased sig-
nificantly acetylene reduction activity (ARA) in both genotypes in-
oculated with both Sinorhizobium strains (Fig. 7c). In A17 genotype,
plants in the combined treatments S. meliloti-F. oxysporum and S. med-
icae-F. oxysporum marked a reduction in ARA by 71.75% and by 61%,
respectively. While For TN1.11 genotype, plants in the combined
treatment S. medicae-F. oxysporum, maintained relatively NFC (Fig. 7c).
4. Discussion
Plants recruit defense mechanisms when they are attacked by
harmful fungi, something that can affect their mutualistic relationship
with rhizobia. In our study, the effect of dual inoculation with F. oxy-
sporum (KLR13) and Sinorhizobium strains, S. meliloti (TII7) and S.
medicae (SII4), on two contrasting genotypes of M. truncatula (on their
M. Batnini, et al.
Fig. 3. (a) Glucose content, (b) fructose content, (c) sucrose content in leaves of M. truncatula genotypes A17 and TN1.11, inoculated or not with S. meliloti and S.
medicae and infected or not with F. oxysporum. Bars with the same lower case letter are not significantly different at P ≤ .05.
response to the infection with F. oxysporum) A17 (relatively resistant)
and TN1.11 (susceptible), (unpublished data), was investigated. Our
results (Fig. 1) showed that fungal infection caused loss in biomass
production, SDW and RDW (Fig. 1) in both genotypes, which is in
agreement with ample studies (Ramírez-Suero et al., 2010; Anderson
et al., 2013). In A17 this reduction was concomitant to a maintenance
of AN and Ci, and to a reduction in Gs (Fig. 2). The reduction in stomatal
conductance could be attributed to the accumulation of H2O2 observed
in leaves (Fig. 4a), though the increased activities of SOD, CAT and POX
(Fig. 5) as a scavenging system to decrease ROS levels (Pei et al., 2000;
Agarwal and Shaheen, 2007; García-Limones et al., 2009). The main-
tenance of photosynthesis was accompanied to a decline in sucrose
content but to an accumulation of hexoses, glucose and fructose, which
agrees with findings of Li et al. (2017a, b) who reported that accu-
mulation of sugars in sweet potato resistant cultivar increased under
infection with F. oxysporum Schlecht. f. sp. batatas. Since A17 is the
tolerant genotype, at least partially, this higher resistance could be
explained or caused by the accumulation of hexoses. The increase in
hexoses levels is generally believed as one of the important and basic
6
Applied Soil Ecology xxx (xxxx) xxxx
mechanisms to contribute to up-regulation of defences in the interac-
tion of pathogens with plants (Berger et al., 2007). Hexoses act as signal
molecules and activate the induction of several PR genes which encode
for proteins that represent a key response in plant defense against pa-
thogens. These proteins are part of systemic acquired resistance and
they can act as antimicrobial by attacking molecules of cell wall of the
pathogen (Herbers et al., 1996; Thibaud et al., 2004; Gómez-ariza et al.,
2007; Giardina, 2014). Combining all these data together, it seems that
the less susceptible A17 invested photosynthesis products, carbohy-
drates, in defending F. oxysporum at the expense of growth. Inoculation
with S. meliloti and S. medicae increased biomass production compared
to nitrogen fed plants. This could be explained by the fact that the
amount of nitrogen provided to the plant is not sufficient to allow full
development of the plant. In other hands, these results are consistent
with several studies on legumes species (López et al., 2008; Mrabet
et al., 2011) showing that rhizobial inoculation could enhance growth
parameters under abiotic or biotic stress. This increment was con-
comitant to an increment of AN, a decrease in hexoses and high pro-
duction of H2O2 in leaves and roots and MDA in leaves, and a decrease
M. Batnini, et al. Applied Soil Ecology xxx (xxxx) xxxx

Fig. 4. (a) Hydrogen peroxide in leaves (H2O2 L), (b) hydrogen peroxide in roots (H2O2 R), (c) lipid peroxidation in leaves (MDA L), (d) lipid peroxidation in roots
(MDA R) of M. truncatula genotypes A17 and TN1.11, inoculated or not with S. meliloti and S. medicae and infected or not with F. oxysporum. Bars with the same lower
case letter are not significantly different at P ≤ .05.

in antioxidant enzymes activities (Fig. 5). These data could suggest an
occurrence of oxidative stress in A17 plants inoculated with Sinorhizo-
bium spp and infected with F. oxysporum. Though the accumulation of
proline in leaves (Fig. 7) which agrees with findings in Fabro et al.
(2004) and Rizzi et al. (2015), who showed that in Arabidopsis thaliana,
proline biosynthesis can be activated by incompatible interaction with
Pseudomonas syringae pv. Tomato. This accumulation would trigger a
hypersensitive response and act as ROS scavenger to protect against
H2O2-induced cell death by limiting disease progression and delaying
cell death (Qamar and Senthil-Kumar, 2015). On the other hand, some
studies on Bradyrhizobium japonicum (King et al., 2000) and Sinorhizo-
bium meliloti (Jimenez-Zurdo et al., 1997) presented proline as an en-
ergy source for the bacteroid to enhance nitrogen fixation. The accu-
mulation of proline along with the accumulation of sucrose in leaves
(Fig. 3) which will be subsequently exported to nodules as energy
source to enhance NFC (Sassi-aydi et al., 2012; Staudinger et al., 2016;
Kafle et al., 2018), were associated with a decline in ARA activity of
both Sinorhizobium strains but no defectiveness in NFW and NN
(Fig. 7). These findings agree with many studies that demonstrated the
negative effect of salt stress and Phoma medicagenis infection on

7
M. Batnini, et al. Applied Soil Ecology xxx (xxxx) xxxx

Fig. 5. Effect of inoculation with F. oxysporum on antioxidant enzymes activities: (a) superoxide dismutase in leaves (SOD L), (b) catalase in leaves (CAT L), (c)
peroxidase in leaves (POX L), (d) superoxide dismutase in roots (SOD R), (e) catalase in roots (CAT R), (f) peroxidase in roots (POX R) of M. truncatula genotypes A17
and TN1.11, inoculated or not with S. meliloti and S. medicae and infected or not with F. oxysporum. Bars with the same lower case letter are not significantly different
at P ≤ .05.

Fig. 6. Proline accumulation in leaves of M. truncatula genotypes A17 and TN1.11, inoculated or not with S. meliloti and S. medicae and infected or not with F.
oxysporum. Bars with the same lower case letter are not significantly different at P ≤ .05.

8
M. Batnini, et al. Applied Soil Ecology xxx (xxxx) xxxx

Fig. 7. (a) Nodules number (NN), (b) nodules fresh weight (NFW) and (c) acetylene reduction assay (ARA) of M. truncatula genotypes A17 and TN1.11 inoculated
with S. meliloti and with S. medicae and infected or not with F. oxysporum. Bars with the same lower case letter are not significantly different at P ≤ .05.

nitrogenase activity in M. truncatula (Mayoral et al., 1989; Mhadhbi
et al., 2011; Chihaoui et al., 2014; López-Gómez et al., 2016; Marchetto
and Power, 2018). Although it remains speculative, this reduction in
NFC could be due to the fact that F. oxysporum may cause a dearth in
nutrients, nitrogen fixing substrates, supplied to the bacteroid. As it was
reported that it either clogs the conductor vessels (xylem and phloem)
and subsequently lead to a water deficit and disturbed translocation of
nutrients or penetrates the parenchymal cells and absorb nutrients, and
as a result a dearth in nutrients in infected plants (Bishop and Cooper,
1983; Pshibytko et al., 2006; Czymmek et al., 2007; Bani et al., 2018).
Moreover, the accumulation of sucrose and proline contents in leaves
and NFC decrement could be a consequence of the damage produced by
stressful condition, more than that of a protective or nutrition control
strategy (López et al., 2008). Curtis et al. (2004) showed that stressful
conditions can reduce the ability of some bacteroid to catabolize ac-
cumulated proline and use the energy derived from its oxidation, and
thus resulting in steeper decline of the N2 fixation.
Regarding the susceptible genotype TN1.11, when submitted to
infection with F. oxysporum, the reduction in biomass was concomitant
to a decline in all photosynthesis related parameters and in hexoses
accumulation which agrees with Jones et al. (2011) who reported that
susceptible rice cultivars showed a decrease in level of metabolites in-
volved in energy supply and metabolism, such as sugars, after an in-
fection with Magnaporthe grisea. This could be due to the fact that fungal

9
M. Batnini, et al.
infection could provoke sugar starvation and metabolism disorder
(Morkunas and Ratajczak, 2014). Also TN1.11 accumulated H2O2 and
MDA in leaves with a non-activation of antioxidant enzymes that sca-
venge the excess of H2O2 and suppress the toxic effect of MDA by
avoiding and limiting its formation. These findings agree with several
studies that reported the inability of the sensitive cultivar to develop an
antioxidant system and avoid the oxidative stress caused by the fungal
attack (Able, 2003; Djebali et al., 2007). Interestingly, inoculation of
TN1.11 with S. medicae enhanced SDW and RDW production compared
to nitrogen fertilized infected plants, in concomitance to maintaining
photosynthesis related parameters, fructose accumulation, decrement
in H2O2 production in leaves and roots and MDA in leaves and to a
development of the antioxidant system SOD, CAT and POX which will
alleviate the oxidative stress and maintain H2O2 to the lower level of
plant's defense response and allow the good reception of the symbiont
(Damiani et al., 2016). Taking all these data together, it seems that S.
medicae enhanced, partly, the defense status of TN1.11. This enhance-
ment could be attributed, at least partly, to the protective and priming
effects role attributed to rhizobia as reported in Arfaoui et al. (2007)
who has showed that inoculating cheakpea seedlings with Rhizobia
isolates induces expression of plant defense-related enzymes prior to
infection with the pathogen Fusarium oxysporum f. sp. ciceris. Moreover,
the accumulation of MDA in roots of plants inoculated with S. medicae
and infected with F. oxysporum could be attributed to the over-
estimation of MDA under stressful conditions as it can interfere with
carbohydrates determination as reported in Taulavuori et al. (2001).
Indeed we found, in this treatment, an accumulation of fructose and
sucrose which was concomitant to a reduction in NN and NFW but a
relative sustain of ARA activity. This positive effect of S. medicae on
fructose accumulation could enhance TN1.11 defense as it was reported
that fructose is involved in the abscisic acid (ABA)- and ethylene-sig-
naling pathway (Cho and Yoo, 2011; Li et al., 2011). Together with the
enhanced redox status which ultimately avoids reductions in both
photosynthesis and growth (Clemente-Moreno et al., 2019), it seems
that TN1.11 in partnership with S. medicae hindered, partially, F. oxy-
sporum growth and managed to translocate nutrients as sucrose to the
bacteroid which may explain the relative sustain of ARA activity
(Fig. 7c).
5. Conclusion
Infection of the relatively resistant A17 genotype of M. truncatula by
F. oxysporum leads to a maintenance of photosynthesis in concomitance
with development of an antioxidant system and accumulation of
hexoses to defend of the fungal growth. But under inoculation with
Sinorhizobium spp strains high production of H2O2 and a non-activation
of antioxidant enzymes in concomitance to a steeper decline in NFC
were observed. By the contrary, the susceptible genotype TN1.11 which
along with biomass reduction an oxidative stress occurred in nitrogen
fertilized infected plants. Notably, inoculation with S. medicae main-
tained higher growth in concomitance with (i) accumulation of fructose
and sucrose that are described as primary mechanisms to be involved in
both defense and successful symbiotic establishment, (ii) reduced oxi-
dative stress via the induction of antioxidant enzyme activities and (iii)
relative maintenance of NFC. This positive effect merits deeper atten-
tion by the implementation of molecular genetics and omics technolo-
gies to further our understanding on the overlap of mechanisms in-
volved in symbiosis and disease interactions, and will provide progress
in engineering resistant plants/symbioses with capability of making
mutualistic relations with beneficial microorganisms.
Acknowledgment
The author is grateful to the Centre of Biotechnology of Borj Cedria,
and the Department of Plant Physiology in the University of Granada to
carry out this study. This research was supported by the Ministry of
10
Applied Soil Ecology xxx (xxxx) xxxx
Higher Education and Scientific Research, Tunisia (Grant: LL-CBBC,
2015–2018) and by scholarship from University of Tunis El Manar,
Tunisia.
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