South African Journal of Botany 108 (2017) 15–22

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South African Journal of Botany

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

Characterization of strigolactones produced by Orobanche foetida and

Orobanche crenata resistant faba bean (Vicia faba L.) genotypes and
effects of phosphorous, nitrogen, and potassium deficiencies on
strigolactone production
I. Trabelsi a,b,⁎, K. Yoneyama c, Z. Abbes a, M. Amri a,d, X. Xie c, T. Kisugi c, H.I. Kim c, M. Kharrat a, K. Yoneyama c
a
National Institute for Agricultural Research of Tunisia (INRAT), Rue Hédi Karray, 2080 Ariana, Tunisia
b
Faculty of Sciences of Bizerte, Carthage University, Jarzouna, 7021, Tunisia
c
Center for Bioscience Research and Education, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, Japan
d
Regional Field Crop Research Center of Beja (CRRGC), Route de Tunis, km 5, 9000 Béja, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, characterization of strigolactones (SLs) produced by some Tunisian faba bean genotypes
Received 22 January 2016 partially resistant to Orobanche foetida Poir. and O. crenata Forsk. was conducted by LC–MS/MS and the results
Received in revised form 4 July 2016 were compared with that of a susceptible genotype. Among the eight partially resistant genotypes grown hydro-
Accepted 9 September 2016
ponically, only the genotype G5 and the susceptible one G9 (Badï) exuded into growth media similar mixture of
Available online 29 September 2016
three SLs, orobanchol, orobanchyl acetate, and a novel SL at amounts detectable by LC–MS/MS. This implies that,
Edited by PN Hills for the resistant genotypes except for G5, impaired SL production confers resistance to Orobanche. The amounts of
orobanchol and orobanchyl acetate exuded by G5 and G9 were quantified by LC–MS/MS. Results showed that
Keywords: the susceptible genotype G9 produced larger amount of orobanchol than did the resistant genotype G5, and in
Faba bean both genotypes the amounts of orobanchol were significantly higher than that of orobanchyl acetate. Other
Resistance mechanisms, acting after induction of Orobanche seeds germination, could be implied in the resistance of geno-
Orobanche foetida type G5. Since nutrient availabilities have been shown to affect SL production, effects of N, P and K deficiencies on
Orobanche crenata SL production were studied for genotypes G5 and G9. Both N and P deficiencies enhanced SL exudation in both
Strigolactone
genotypes, while K deficiency did not affect it.
Nutrient deficiency
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
Tunisia

1. Introduction
Orobanche foetida Poir. and O. crenata Forsk. are root holoparasites
widely distributed in the Mediterranean area and inflicting severe
damages on faba bean (Vicia faba L.) production. Among different con-
trol strategies, breeding of Orobanche resistant cultivars appears to
be the most promising approach (Rubiales et al., 2006). However, resis-
tance against broomrape in legumes is scarce, of complex nature and of
low heritability, and these factors hamper the breeding of resistant cul-
tivars (Rubiales et al., 2014). Under these circumstances, understanding
mechanisms of naturally occurring resistance is expected to provide
possible targets for breeding.
Orobanche seeds remain dormant in the soil for many years until
they perceive germination stimulants released from host roots. Among
known germination stimulants, strigolactones (SLs) are the most active
stimulants inducing germination at as low as 10−12 M (Xie et al., 2010).
⁎ Corresponding author at: National Institute for Agricultural Research of Tunisia
(INRAT), Rue Hédi Karray, 2080 Ariana, Tunisia.
E-mail address: trabelsiimen11@yahoo.fr (I. Trabelsi).
More than 20 natural SLs have been characterized and novel ones are
being identified (Yoneyama et al., 2013a).
Since seeds of root parasitic weeds need chemical stimuli including
SLs for germination, reduced production and exudation of germination
stimulants is a good trait for Orobanche resistance (Yoder and Scholes,
2010). Low induction of Orobanche seed germination by host-root exu-
dates was reported in various legumes species including faba bean,
vetch, pea, chickpea, and grass pea (Rubiales et al., 2003; Sillero et al.,
2005; Pérez-de-Luque et al., 2005; Fernández-Aparicio et al., 2012),
sunflower (Labrousse et al., 2001, 2004) and tomato (Dor et al., 2011).
Fernández-Aparicio et al. (2014) reported that low SL production ap-
peared to confer resistance against Orobanche observed in some resis-
tant faba bean cultivars. However, impaired SL production may have
pleiotropic effects on plant growth and development (Kohlen et al.,
2011). In addition to their role as germination stimulants for root para-
sitic weeds, SLs function as a novel class of plant hormones regulating
shoot and root architectures (Gomez-Roldan et al., 2008; Umehara
et al., 2008; Kapulnik et al., 2011; Ruyter-Spira et al., 2011), photomor-
phogenesis (Shen et al., 2007), secondary growth (Agusti et al., 2011)
and leaf senescence (Snowden et al., 2005; Yamada et al., 2014). SLs

http://dx.doi.org/10.1016/j.sajb.2016.09.009
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
16 I. Trabelsi et al. / South African Journal of Botany 108 (2017) 15–22

have been shown to be essential for root colonization by arbuscular
mycorrhizal (AM) fungi (Akiyama et al., 2005; Besserer et al., 2006)
and enhance rhizobia root colonization in pea and alfalfa (Soto et al.,
2010; Foo and Davies, 2011) but may be inhibitory at higher concentra-
tions as reported for Medicago truncatula (De Cuyper et al., 2014).
The objective of the present study is to identify resistance mecha-
nisms of seven faba bean genotypes G1–G7 which have been recently
characterized as O. foetida and O. crenata resistant in the fields at
Beja and Ariana in Tunisia (Trabelsi et al., 2015). Najeh (G8) and Badï
(G9) were included as resistant and susceptible checks, respectively.
SLs produced by the faba bean genotypes were identified and quanti-
fied in order to examine if observed resistance is related to reduced
SL exudation. Moreover, because nutrient availabilities have been
shown to profoundly affect SL production (Yoneyama et al., 2007a,
2007b, 2012, 2013b; López-Ráez et al., 2008), effects of N, P, and K
deficiencies on SL productions in selected genotypes of faba bean were
also studied.
2. Materials and Methods
2.1. Plant material
Eight faba bean (Vicia faba L.) genotypes partially resistant to both
O. foetida and O. crenata including a released variety Najeh (G1, G2,
G3, G4, G5, G6, G7 and Najeh) and a susceptible check Badï (G9) were
used in the experiments (Table 1). Seeds of the nine genotypes were
harvested in 2011 from single selected plants grown separately under
insect-proof cages at Beja experimental station in the North-West of
Tunisia.
Seeds of O. foetida and O. crenata were collected from the parasites
on faba bean during the cropping season 2010–2011, respectively
from Beja and Ariana regions in Tunisia. O. minor seeds were collected
from the parasites on red clover plants in Utsunomiya, Japan.
2.3. Extraction of root exudates
The collected tap water growth media containing root exudates
(approx. 300 mL + washing) were extracted three times with 100 mL
of ethyl acetate. The ethyl acetate extracts were combined, washed
with 0.2 M K2HPO4 (pH 8.3), dried over anhydrous MgSO4 and concen-
trated in vacuo. The crude extracts were kept at 4 °C until use.
2.4. Identification of strigolactones by liquid chromatography–tandem
mass spectrometry (LC–MS/MS)
High performance liquid chromatography (HPLC) separation was
conducted with a U980 HPLC instrument (Jasco, Tokyo, Japan) fitted
with an ODS (C18) column (Mightysil RP-18, 2 × 250 nm, 5 μm: Kanto
Chemicals Co., Ltd., Tokyo, Japan). The crude extracts were dissolved
in 60% methanol and filtered through spin columns (Ultra-fee MC,
0.45 μm pore size; Millipore, Tokyo, Japan), and 3 μL was injected. The
mobile phase was 60% methanol in water and was changed to 100%
methanol 30 min after injection. The column was then washed with
100% methanol for 20 min. The flow rate was 0.2 mL min− 1 and the
column temperature was set to 40 °C.
Mass spectrometry was performed with a Quattro LC mass spec-
trometer (Micromass, Manchester, UK) equipped with an electrospray
source. The drying and nebulizing gas was nitrogen generated from
pressurized air in an N2G nitrogen generator (Parker-Hanifin Japan,
Tokyo, Japan). The nebulizer gas flow was set to approx. 100 Lh−1,
and the desolvation gas flow to 500 Lh−1. The interface temperature
was set to 400 °C and the source temperature to 150 °C. The capillary
and cone voltages were adjusted to orobanchol and the positive ioniza-
tion mode. MS/MS experiments were conducted using argon as the
collision gas and the collision energy was set to 16 eV. The collision
gas pressure was 0.15 Pa. Known strigolactones were detected by
using multiple reactions monitoring (MRM).

2.2. Hydroponic culture of faba bean 2.5. Quantification of strigolactones

Faba bean seeds were surface sterilized in sodium hypochlorite
(1.0%) containing 0.1% Tween-20 for 10 min and rinsed thoroughly
with sterile Milli-Q water. The seeds were soaked in Milli-Q water at
room temperature for 2 h and then sown in pots containing sterilized
sand and placed in a growth chamber for 7 d with a16h photoperiod
(120 μmol m−2.s−1) at 22 °C. Three healthy seedlings from each line
were selected and transferred to a stainless steel sieve lined with a
sheet of gauze moistened by placing it on the cup (9.5 cm in diameter,
17 cm deep, approx. 550 mL in volume) containing 350 mL of tap
water. The plants were grown for 20 d during which the tap water
growth media were replaced with fresh one every 2 d. Experiment was
repeated three times.
Among the nine faba bean genotypes studied, only G5 and the
susceptible check Badï (G9) were found to produce relatively large
amounts of SLs (orobanchol, orobanchyl acetate, and a novel SL) and
these genotypes were subjected to SL quantification. The plants were
grown hydroponically and growth media (tap water) were collected
as described before (Yoneyama et al., 2013a, 2013b). For quantification
of orobanchol and orobanchyl acetate, [6′-2H] orobanchol (400 pg) and
[6′-2H] orobanchyl acetate (200 pg) were added as internal standards to
the collected growth media prior to solvent extraction. Crude root ex-
tracts obtained as described before were analyzed by LC–MS/MS using
5-channel MRM. The transitions of m/z 369–272, 370–272, 411–254,
412–255, and 427–270 were monitored for orobanchol, orobanchol-d1,

Table 1
Pedigree, origin and main characteristics of faba bean genotypes used in the study.

Genotypes/Pedigree Origin/characteristics

G1: XAR-VF00.12–12–3-1-3-1
G2: XAR-VF00.13–8–3-1-1-1-1
G3: XAR-VF00.13–89–2-1-1-1-1
G4: XBJ92.10–27–1-1-1-1-1
G5: XBJ92–10–46-1-3-1-2-1-1-1-6-A
G6: XBJ90.04–6–2-1-1-4-C
G7: XBJ90.04–2–3-1-1-1-2 A
G8: Najeh
G9: Badï
Cross performed in Ariana (Tunisia) in 2000 between Tunisian breeding line resistant to O. foetida and large seeds population “Malti”.
Cross performed in Ariana (Tunisia) in 2000 between Tunisian breeding line resistant to O. foetida and faba bean small seeds breeding
lines selected by INRA Rennes (France)
Cross performed in Ariana (Tunisia) in 2000, between Tunisian breeding line resistant to O. foetida and faba bean small seeds breeding
lines selected by INRA Rennes (France)
Cross performed in Beja (Tunisia) in 1992 between faba bean breeding line selected for resistance to O. crenata by ICARDA and faba bean
small seeds selected by INRAT
Cross performed in Beja (Tunisia) in 1992 between faba bean breeding line selected for resistance to O. crenata by ICARDA and faba bean
small seeds selected by INRAT
Cross performed in Beja (Tunisia) in 1990 faba bean breeding line selected for resistance to O. crenata by ICARDA and faba bean small
seeds local population
Cross performed in Beja (Tunisia) in 1990 faba bean breeding line selected for resistance to O. crenata by ICARDA and faba bean small
seeds local population
Small seeded variety released in 2009/partial resistant variety to O. foetida and O. crenata
Small seeded variety released in 2004/susceptible to O. foetida and O. crenata
 I. Trabelsi et al. / South African Journal of Botany 108 (2017) 15–22
orobanchyl acetate, orobanchyl acetate-d1, and the novel SL, an isomer
of fabacyl acetate, respectively.
2.6. Germination tests with O. foetida, O. crenata, and O. minor seeds
The seeds of O. foetida, O. crenata and O. minor were surface-
sterilized in 70% ethanol for 3 min and in 1% sodium hypochlorite con-
taining 0.1% Tween-20 for 5 min. The seeds were rinsed thoroughly
with sterile Milli-Q water and air-dried. About 20 to 30 seeds were
sown on a 5 mm glass-fibre disc (Whatman GF/A, Tokyo, Japan). Ninety
discs were placed in a 9 cm petri dish lined with one layer of filter paper
wetted with 6 mL of sterile Milli-Q water. The petri dishes were sealed
with parafilm, wrapped with aluminium foil and incubated in the dark
at 22 °C for 8 d for O. foetida and O. crenata, and at 20 °C for 5 d for
O. minor. After conditioning, the glass-fibre discs were blotted to remove
excessive water. Each group of three discs was transferred to a separate
5 cm petri dish prepared as follows.
An aliquot of methanol solution (b 5 μL) of the crude sample was
added to the 5-cm Petri dish lined with a sheet of filter paper. The sol-
vent was allowed to evaporate before the discs carrying the conditioned
seeds were placed on the filter paper and treated with sterile Milli-Q
water (650 μL). The Petri dishes were sealed and placed in the dark
as for conditioning. After 8 d for O. foetida and O. crenata, and 5 d for
O. minor, germination rates were determined under binocular micro-
scope. Seeds treated with or without GR24 (10− 6 M) were always
included as positive and negative controls. All the experiments were
repeated three times and the data presented are mean ± s.e.
To assess the distribution of germination stimulation activity after
RP-HPLC separation, the eluate was collected every 30 s in 5-cm Petri
dishes lined with a sheet of filter paper. These dishes were then treated
as described for crude samples.
2.7. Effect of nutrient deficiency on strigolactone production
Both genotypes G5 and G9 were used in this experiment as resistant
and susceptible one to Orobanche, respectively. Three healthy 7-d-old
seedlings each prepared as described in the former section were trans-
ferred to small cups containing 350 mL of 1/2 Tadano and Tanaka (TT)
medium (Tadano and Tanaka, 1980) and incubated for 7 d. Before trans-
ferring the seedlings to the cups, cotyledons were removed so that the
seedlings became more sensitive to nutrient deficiency. After this accli-
mation period, the plants were subjected to N, P, or K deficiency in
which each nutrient was removed from the media and grown for 10 d
(Yoneyama et al., 2012) during which the growth media were replaced
with fresh ones every 2 d. After a 10 d-incubation in different nutrient
conditions, plants were harvested, separated into shoots and roots,
and length and fresh weight of shoots and roots, number of lateral
roots, and number of nodes were determined. SLs released to the
growth media over 48 h (d 9–d 10) were extracted with ethyl acetate
for the quantification as described in the former section.
In a separate experiment, the susceptible genotype Badï (G9) plants
were used to examine time-course characteristic of SL exudation under
nutrient deficiency. For this, three healthy 7-d-old seedlings each
without cotyledons, were transferred to small cups containing 350 mL
of 1/2 TT medium and incubated for 5 d as an acclimation. Then,
the seedlings were subjected to N, P, or K deficiency and grown for
another 13 d during which growth media containing root exudates
over 48 h were collected on 4, 6, 11, and 13 d after incubation in dif-
ferent nutrient conditions. Extraction of root exudates was conducted
as described in the former section. Three replications for each treatment
were conducted.
2.8. Statistical analysis
Statistical analyses were performed using the SPSS software
(Version 15.0 for Windows). Analyses of variance were conducted to
17
assess whether there was a significant variation for each studied vari-
able separately. Mean comparison was based on Tukey's multiple
ranges classification test at P = 0.05. Each experiment was replicated
three times (n = 3).
3. Results
3.1. Characterization of strigolactones produced by faba bean genotypes
grown hydroponically with tap water
Interaction between faba bean genotypes and both Orobanche spe-
cies, O. foetida and O. crenata, was studied in rhizotrons (unpublished
data). Results showed that, except for G5, Orobanche germination
rates were significantly lower with the partially resistant genotypes
tested than the susceptible genotype G9 (Badï) ranging from 4.8% to
10.3% and from 6.3% to 17.2% for O. foetida and O. crenata, respectively.
By contrast, G5 and G9 induced 40.8% and 59.7% germination of
O. foetida and 44.9% and 69.9% germination of O. crenata, respectively
Differences in Orobanche seed germination stimulations among the
genotypes may be attributable to quantitative or qualitative differences
in germination stimulants and/or germination inhibitors exuded by the
genotypes.
To characterize SLs, major germination stimulants, produced by faba
bean genotypes, we first used tap water as growth medium because in
general SL production is promoted when the plants are grown under
nutrient deficiency (Yoneyama et al., 2007a, 2007b; López-Ráez et al.,
2008). Therefore, faba bean plants were grown hydroponically in
tap water and root exudates were collected. The root exudates samples
were analyzed by LC–MS/MS for the presence of known SLs (Yoneyama
et al., 2007a). Among the nine genotypes studied, only G5 and the sus-
ceptible genotype G9 produced SLs at amounts detectable by LC–MS/MS
(Fig. 1). SLs were not detected from the root exudates of genotypes G1,
G2, G3, G4, G6, G7 and G8, over 28 d of experimental period (20 d of
hydroponic culture).
In the LC–MS/MS chromatograms of G5 and G9 root exudate
samples, distinct peaks in the transitions of m/z 369–272 (bottom),
411–254 (middle), and 427–270 (top) were detected as shown in
Fig. 1 in which MRM chromatograms of G9 root exudate sample col-
lected 20 d after transferring to the hydroponic culture (containing
strigolactones exuded over 48 h) are depicted. The peak eluted at
6.6 min in the channel monitoring the transition of m/z 427–270
(top) was a novel SL with molecular formula identical to that of fabacyl
acetate. The assignments of orobanchol (9.6 min) and orobanchyl ace-
tate (13.1 min) were confirmed by co-chromatography with natural
standards (data not shown). Similar data have been obtained for G5
root exudate sample.
The identification of SLs was further confirmed by germination assay
of fractions separated by reversed phase high performance liquid chro-
matography (RP-HPLC) for all the genotypes studied. For this, root exu-
dates samples collected 18 d after transferring to the hydroponics were
used. Germination stimulation activities were not observed from the
root exudates samples of G1, G2, G3, G4, G6, G7 and G8. The distribution
of germination stimulation activity on O. foetida, O. crenata, and O. minor
after RP-HPLC separation for G5 and G9 is presented in Fig. 2a, b. In the
root exudates samples of both G5 (Fig. 2a) and G9 (Fig. 2b), germination
stimulation activities on O. foetida, O. crenata, and O. minor were associ-
ated with the fractions whose retention times corresponded to those of
orobanchol (fraction 9.5) and orobanchyl acetate (fractions13 and
13.5). In addition to these known SLs, the activity observed in fraction
6.5 corresponded to the peak detected in the MRM channel monitoring
the transition of m/z 427–270 at a retention time of 6.6 min. Among
the three Orobanche species, O. minor appeared to be most sensitive to
germination stimulants exuded. The distribution of germination stimu-
lation activity is very similar for the different Orobanche species indi-
cating that these three SLs are major germination stimulants produced
by the faba bean genotypes examined in the present study.
18 I. Trabelsi et al. / South African Journal of Botany 108 (2017) 15–22
Fig. 1. 3-Channel-LC–MS/MS chromatograms of root exudates from the Orobanche–susceptible faba bean cultivar Badï (G9). The transitions monitored were m/z 427 N 270 (top), 411 N 254
(middle), and 369 N 272 (bottom) for novel strigolactone, orobanchyl acetate, and orobanchol, respectively.
3.2. Time-course characteristic of strigolactones exudation by faba bean
genotypes grown with tap water
The amounts of orobanchol and orobanchyl acetate exuded by
the resistant genotype G5 and susceptible genotype G9, grown hy-
droponically with tap water during 10 to 20 d after transplanting
Fig. 2. Distribution of germination stimulation activities after reversed phase HPLC
separation of root exudate extracts from two faba bean genotypes, G5 (a) and G9 (b).
Fraction indicates retention time (min) collected for assay. For example, fraction 2
contained samples eluted between 2.0 to 2.49 min. Black, gray and white bars indicate
Orobanche foetida, Orobanche crenata, and Orobanche minor germination rate (%),
respectively. Bars indicate means ± s.e.(n = 3).
to hydroponics, were quantified by LC–MS/MS (Fig. 3). For the novel
SL, only the peak areas in MRM chromatograms were compared.
Quantification was conducted in order to compare if there is a
quantitative difference in SL production between the resistant genotype
(G5) and the susceptible one (G9). During the experimental period,
the amounts of orobanchol and orobanchyl acetate exuded per plant
remained at similar levels for G9 and no significant differences were
recorded. By contrast, G5 showed significant variations in SL exuda-
tion over the experimental period. For both genotypes, amount of
orobanchol (2–3 ng per plant over 48 h) was about double that of
orobanchyl acetate (1–1.5 ng per plant over 48 h). Based on the peaks
areas of the novel SL, the amount exuded by G5 increased significantly
over 28 d while the susceptible G9 exuded the highest level of this SL
after 24 d. The susceptible genotype G9 exuded larger amounts of the
three SLs than did the resistant G5 (Fig. 3).
3.3. Effects of nutrient deficiency on strigolactone exudation by genotypes
G5 and G9
Significant effects of nutrient deficiency were recorded for both
genotypes G5 and G9 on some plant growth parameters (shoot fresh
weight, root fresh weight and lateral roots). Under different nutrient
deficiencies, the resistant genotype G5 plants showed a significant
decrease of the shoot fresh weight and the lateral root number. Under
P deficiency the susceptible genotype G9 plants showed significant in-
creases of root fresh weight and root length compared to the respective
control (Table 2).
Consequently, nutrient deficiency affected profoundly SL exudation
which was strongly promoted under N and P deficiency (Fig. 4). Under
the experimental conditions, amounts of orobanchol and orobanchyl
acetate exuded over 48 h were below LC–MS/MS detection limits
for the plants grown in the control (1/2 TT) medium and K deficient
conditions. For both genotypes G5 and G9, N and P deficiencies in-
creased SL production to similar levels (Fig. 4).
Time-course characteristics of SL exudation by the susceptible geno-
type G9 grown under different nutrient conditions are shown in Fig. 5.
In this case, the plants were acclimatized in 1/2 TT medium for 5 d
and thus the plants would be more sensitive to nutrient deficiency as
 I. Trabelsi et al. / South African Journal of Botany 108 (2017) 15–22 19

Fig. 4. Exudation of orobanchol and orobanchyl acetate from faba bean genotype G5
(a) and G9 (b) grown hydroponically under different nutrient conditions. Black and
white bars indicate amounts of orobanchol and orobanchyl acetate exuded per plant
over 48 h. Bars indicate means ± s.e.(n = 3).

P deficiency with incubation period. Under P deficiency, a reduction in

orobanchol exudation was observed after 13d incubation. Exudation
of orobanchyl acetate and the novel SL increased under N and P
deficiency with incubation period (Fig. 5). By contrast, exudation of
orobanchol in the plants grown in 1/2 TT medium and under K deficient
conditions decreased with incubation period and remained at very low
levels.

Fig. 3. Time-course characteristics of orobanchol (a), orobanchyl acetate (b) and
unidentified strigolactone (c) exuded by faba bean genotypes G5 and G9 per plant over
48 h. Bars indicate means ± s.e.(n = 3).
compared to the former experiment (Fig. 4) in which the plants had
been acclimatized for 7 d. Indeed, amounts of SLs exuded were larger
than those in the former experiment. Plant growth was more and se-
verely affected under nutrient deficiency compared to the control
with significant reductions in shoot length, shoot fresh weight, root
fresh weight and number of nodes especially under N deficiency
(Table 3). Higher amounts of SLs were exuded from the plants grown
under N deficiency than those grown under P deficiency (Fig. 5).
Exudation of orobanchol was relatively high even after 4 d incubation
in different nutrient conditions with significant increase under N and
4. Discussion
In Tunisia O. foetida and O. crenata cause serious yield losses mainly
on faba bean (Abbes and Kharrat, 2006; Abbes et al., 2007a, 2007b,
2009). One of the most promising approaches to mitigate these dam-
ages is breeding for genetic resistant (Abbes et al., 2007a; Kharrat
et al., 2010; Maalouf et al., 2011). Reduced production of Orobanche
seed germination stimulants “strigolactones” is considered among the
best possible resistance mechanisms to Orobanche. Faba bean cultivars
that exude stimulants in low levels are expected to be resistant resulting
thus in low Orobanche infection level because they will induce germina-
tion of only small portion of Orobanche seeds located very close to the
host roots (Fernández-Aparicio et al., 2012).
Significant differences of resistance to both O. foetida and O. crenata
were recorded between the genotypes studied in the present study

Table 2
Growth parameters of faba bean genotypes G5 and G9 grown under different nutrient conditions.

Genotype G5 G9

Nutrient normal –N –P –K normal –N –P –K

Shoot f. wt 1.05 ± 0.07b⁎ 0.65 ± 0.06a 0.66 ± 0.02a 0.77 ± 0.02a 0.96 ± 0.15a 0.64 ± 0.05a 0.85 ± 0.07a 0.68 ± 0.04a
Shoot length 82.7 ± 4.2a 67.3 ± 4.3a 66.0 ± 3.1a 69.0 ± 3.0a 78.0 ± 4.2a 64.3 ± 6.2a 68.7 ± 5.5a 66.0 ± 1.5a
Root f. wt 0.85 ± 0.03a 0.91 ± 0.04a 0.71 ± 0.07a 0.81 ± 0.04a 0.69 ± 0.05ab 0.67 ± 0.01a 0.87 ± 0.03b 0.60 ± 0.06a
Root length 204 ± 17a 197 ± 6a 196 ± 21a 192 ± 7a 165 ± 6a 168 ± 10a 192 ± 7a 172 ± 10a
Lateral roots 30.3 ± 1.9b 28.3 ± 1.5ab 28.0 ± 0.6ab 24.0 ± 1.0a 41.7 ± 2.9a 33.3 ± 0.9a 36.0 ± 1.5a 31.3 ± 3.8a
Nodes 4.7 ± 0.3a 4.0 ± 0a 4.0 ± 0a 4.0 ± 0a 4.0 ± 0a 3.7 ± 0.3a 4.0 ± 0a 4.0 ± 0a

The fresh weights of shoots and roots (g), shoot and root lengths (mm), and the numbers of lateral roots and nodes were determined on 10 days after incubation under different nutrient
conditions. Normal means 1/2 Tadano-Tanaka medium, and – N, − P, and – K indicate deficiency of each nutrient element.
⁎ Values within a row with different letters indicate significant differences (ANOVA, P b 0.05) for each genotype. Values are means ± s.e. (n = 3).
20 I. Trabelsi et al. / South African Journal of Botany 108 (2017) 15–22

genotypes studied is due to the reduced production of germination

stimulants and not to the production of germination inhibitors. Similar
results were reported by Fernández-Aparicio et al. (2014) where they
indicated that the low O. crenata, O. foetida, and P. aegyptiaca seed ger-
mination induced by both faba bean genotypes Navio and Quijote is
due to a low germination stimulant production. Navio and Quijote
were derived by individual plant selection from Tunisian genotypes
G8 (Najeh) and G6, respectively, suggesting that the low germination
stimulants production was inherited from relative parental lines G8
and G6. Orobanchol, orobanchyl acetate, and the novel SL seem to be
the major germination stimulants for O. foetida and O. crenata produced
by both susceptible genotype G9 (Badï) and the resistant genotype G5.
Yoneyama et al. (2012) detected 5-deoxystrigol by LC–MS/MS (pre-
sumably ent-2′-epi-5-deoxystrigol or 4-deoxyorobanchol) in addition
to orobanchyl acetate and orobanchol in the root exudates of 12
Fabaceae species including faba bean. However, the RP-HPLC fractions
corresponding to the retention time of this SL were inactive in the ger-
mination tests at the dilution used in this study indicating that it was a
minor germination stimulant of faba bean since only major stimulants
could be detected by germination assay. The root exudates of the sus-
ceptible faba bean genotype G9 (Badï) and the resistant genotype G5 in-
duced high germination of O. foetida, O. crenata, and especially O. minor
seeds. This is because that O. minor which has a wide host range (Parker
and Riches, 1993) and is more sensitive to orobanchol and orobanchyl
acetate as reported by Kim et al. (2010). Nevertheless, no Orobanche
seed germination were observed for genotypes G1, G2, G3, G4, G6, G7
and G8 after RP-HPLC separation of root exudates samples.
The resistant genotype G5 was found to produce SLs at levels similar
to that produced by susceptible genotype G9. Similar results were ob-
served in previous studies with some resistant genotypes which in-
duced relatively high parasite seed germinations compared to
susceptible genotypes (Khalaf and El-Bastawesy, 1989; Goldwasser
et al., 1997; Labrousse et al., 2001; Yoneyama et al., 2010). Those results
confirm that resistance to Orobanche is related to several mechanisms
acting at different levels.
The susceptible genotype G9 exuded relatively large amounts of
both orobanchol and orobanchyl acetate (Fig. 3). SL quantification
Fig. 5. Time-course characteristics of strigolactone exudation by faba bean genotype G9 from root exudates of genotypes G9 and G5 showed that the amounts
(Badï). Black cube circle, triangle, and lozenge indicate data from plants grown under N, of orobanchol exuded were always larger than those of orobanchyl ace-
P, K deficiency and in control media, respectively. Bars indicate means ± s.e.(n = 3).
tate suggesting that orobanchol may play an important role in the root
parasitic seed germination stimulation in faba bean.
under field conditions with low Orobanche attachment number com- No qualitative or quantitative differences were observed between
pared to the susceptible genotype G9 (Badï) (Trabelsi et al., 2015). the susceptible (G9) and the resistant (G5) genotypes in SL production.
Such behavior/resistance was confirmed by low seed germination and G5 produced three SLs, the novel SL, orobanchol and orobanchyl acetate,
delayed tubercle development for these genotypes in pots and rhizotron at levels similar to that produced by G9. This implies that the genotype
experiments (unpublished data). No SLs were detected from root exu- G5 may have other resistance mechanisms acting after Orobanche seed
dates of genotypes G1, G2, G3, G4, G6, G7 and G8, by LC–MS/MS in the germination such as mechanical barriers interrupting tubercle develop-
present study. These results confirm that, except for G5, the major resis- ment, as reported in a previous study for O. crenata- resistant Vicia faba
tance mechanism of the resistant genotype G8 (Najeh) and the other “Giza 402” and “Baraca” (Pérez-de-Luque et al., 2007).
In monocotyledonous plants, sorghum and maize, not only quantita-
tive differences but also qualitative differences in SL exudation are
Table 3
Growth parameters of faba bean genotype G9 (Badï) grown under different nutrient involved in their Striga resistance. In both plant species, resistant
conditions. cultivars produce and exude smaller amounts of 5-deoxystrigol, the
most stable SL among those produced by these plant species, than do
Nutrient Growth Normal –N –P –K
parameters
susceptible cultivars. Major SLs exuded from resistant cultivars are less
stable hydroxy-SLs, sorgomol and strigol (Yoneyama et al., 2010). In
Shoot f. wt 2.10 ± 0.20a⁎ 1.00 ± 0.1b 1.80 ± 0.07a 1.70 ± 0.15a
the case of faba bean genotypes studied, such a qualitative difference
Shoot length 153 ± 10a 108 ± 9b 148 ± 6a 130 ± 7ab
Root f. wt 1.50 ± 0.15ab 1.20 ± 0.09b 1.80 ± 0.07a 1.30 ± 0.05ab of SL production appears not to be involved in their Orobanche resis-
Root length 154 ± 5a 171 ± 0.00a 170 ± 7a 175 ± 11a tance because SL exudation profile of resistant genotype G5 was almost
Lateral roots 29.30 ± 3.38a 26.50 ± 2.10a 33.10 ± 1.15a 29.70 ± 3.25a identical to that of susceptible genotype G9.
Nodes 5.00 ± 0.00a 3.80 ± 0.20b 5.00 ± 0.00a 4.30 ± 0.26b
Nutrient availability has been demonstrated to regulate SL pro-
The fresh weights of shoots and roots (g), shoot and root lengths (mm), and the numbers duction and/or exudation in plants (Yoneyama et al., 2007a, 2007b;
of lateral roots and nodes were determined on 13 days after incubation under different López-Ráez et al., 2008; Umehara et al., 2008; Chiou and Lin, 2011).
nutrient conditions. Normal means 1/2 Tadano-Tanaka medium, and – N, − P, and – K
indicate deficiency of each nutrient element.
Nitrogen (N) and Phosphorus (P) are major nutrients limiting plant
⁎ Values within a row with different letters indicate significant differences (ANOVA, growth (Epstein and Bloom, 2005). Plants respond differently to the
P b 0.05). Values are means ± s.e. (n = 3). deficiency or the presence of nutrients resulting in various levels of SL
 I. Trabelsi et al. / South African Journal of Botany 108 (2017) 15–22
production. Nitrogen fertilization has been reported to inhibit Orobanche
seed germination (Abu Irmaileh, 1994; Pérez-de-Luque et al., 2010) and
was largely used as an Orobanche control method (Jain and Foy, 1992).
In the present study, we examined the effect of N, P, and K deficiency
on SL exudation from faba bean plants. Results showed that N and P de-
ficiencies enhanced SL production in faba bean genotypes G5 and G9 but
not K deficiency. Similar results were reported for rice (Umehara et al.,
2010; Jamil et al., 2011), sorghum (Yoneyama et al., 2007a), Chinese
milk vetch, marigold, lettuce, and wheat (Yoneyama et al., 2012). In
other plant species such as red clover (Yoneyama et al., 2007b), tomato
(López-Ráez et al., 2008; Yoneyama et al., 2012), and alfalfa (Yoneyama
et al., 2012), only P deficiency enhanced SL production.
Time-course characteristics of SL exudations by G9 under different
nutrient conditions indicate that exudation of orobanchol responds
most rapidly to nutrient deficiency than orobanchyl acetate and the
novel SL (Fig. 5). It seems reasonable that exudation of orobanchyl
acetate increased later since this SL may be derived from orobanchol
(Xie et al., 2010).
5. Conclusion
In the present study, most of the faba bean genotypes selected in the
field for their moderate resistance to O. foetida and O. crenata were
found to be low SL germination stimulants producers. Orobanchol,
orobanchyl acetate, and a novel SL are three major SLs produced
by faba beans. The similar production levels of these SLs observed
for both susceptible genotype G9 (Badï) and resistant genotype G5 sug-
gest that for the latter genotype other resistance mechanisms are in-
volved and that could be an inhibition of invading processes after seed
germination. Further studies are needed to clarify details related to
the resistance mechanism to Orobanche in genotype G5.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We wish to thank Ms. Yuki Ishii and Erina Sasase for assistance in
experiments, Professor Kohki Akiyama (Osaka Prefecture University)
and Professor Tadao Asami (The University of Tokyo) for their kinds
gifts of deuterated strigolactone standards and GR24, the Tunisian
Ministry of Agriculture (313/5793 in 2011 and 313/4926 in 2012) and
the Ministry of Higher Education and Scientific Research, for their finan-
cial support.
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