Orobanche and Phelipanche spp. - American Chemical Society

Article pubs.acs.org/JAFC

Low Strigolactone Root Exudation: A Novel Mechanism of Broomrape (Orobanche and Phelipanche spp.) Resistance Available for Faba Bean Breeding Mónica Fernández-Aparicio,*,†,‡,§ Takaya Kisugi,§ Xiaonan Xie,§ Diego Rubiales,† and Koichi Yoneyama§ †

Institute for Sustainable Agriculture, IAS-CSIC, Apdo. 4084, E-14080, Córdoba, Spain Dep. Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, Virginia 24061, United States § Weed Science Center, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan ‡

ABSTRACT: Faba bean yield is severely constrained in the Mediterranean region and Middle East by the parasitic weeds Orobanche crenata, O. foetida, and Phelipanche aegyptiaca. Seed germination of these weeds is triggered upon recognition of host root exudates. Only recently faba bean accessions have been identified with resistance based in low induction of parasitic seed germination, but the underlying mechanism was not identified. Strigolactones are a group of terpenoid lactones involved in the host recognition by parasitic plants. Our LC-MS/MS analysis of root exudates of the susceptible accession Prothabon detected orobanchol, orobanchyl acetate, and a novel germination stimulant. A time course analysis indicated that their concentration increased with plant age. However, low or undetectable amounts of these germination stimulants were detected in root exudates of the resistant lines Quijote and Navio at all plant ages. A time course analysis of seed germination induced by root exudates of each faba bean accession indicated important differences in the ability to stimulate parasitic germination. Results presented here show that resistance to parasitic weeds based on low strigolactone exudation does exist within faba bean germplasm. Therefore, selection for this trait is feasible in a breeding program. The remarkable fact that low induction of germination is similarly operative against O. crenata, O. foetida, and P. aegyptiaca reinforces the value of this resistance. KEYWORDS: parasitic plant, broomrape, Vicia faba, genetic resistance, seed germination

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germination is available in some legumes,22−27 in sunflower,28−30 and in tomato.31 Similarly, low induction of Striga seed germination has been identified in sorghum32 and in maize.33 Faba bean (Vicia faba) is an important legume crop in northern Europe, the Mediterranean area, China, and Australia34,35 that is heavily parasitized by three species of broomrapes, Orobanche crenata, O. foetida, and Phelipanche aegyptiaca.36 Only moderate levels of incomplete resistance are available in faba bean cultivars and germplasm, 37,38 based on resistance mechanisms hampering broomrape tubercle development, but no seed germination.39 Only recently faba bean lines have been identified that induce low induction of broomrape seed germination.36 The objective of this study is to elucidate if the absence of strigolactone exudation to the rhizosphere is the responsible mechanism that allows these faba bean cultivars to escape from the broomrape infestation.

INTRODUCTION Strigolactones are a group of terpenoid lactones derived from carotenoids. Their structure consists of a tricyclic lactone connected to a second lactone via an enol ether bridge.1 Strigolactones are plant hormones regulating shoot branching, leaf senescence, hypocotyl/mesocotyl elongation, root architecture, and germination.2−9 They inhibit shoot branching,3,4 and their synthesis is increased under nutrient starvation.10,11 Therefore, strigolactones contribute to plant adaptation in poor soils by reducing branching and consequently the nutrient needs.12 As an indirect method of ecological adaptation, plants exude strigolactones to the rhizosphere, promoting arbuscular mycorrhizal fungi symbiosis13−15 which alleviates the plant from nutrient deficiency by increasing the supply of inorganic nutrients, mainly nitrogen and phosphorus.14,16 Collaterally, root parasitic weeds such as Striga and Orobanche have evolved the ability to eavesdrop the plant-to-symbiont strigolactones mediated communication.17,18 Seed germination of these weeds is triggered upon recognition of host-derived strigolactones, thus coordinating parasitic germination with the perception of their hosts.1 Due to their complete dependence from host derived nutrient and water supply, the inhibition of the early stages of the parasitic life cycle, such as of parasitic seed germination, is the obvious target for parasitic weed control.19 Breeding for parasitic weed resistance is the most economic and environmentally friendly method for parasitic weed control. However, few sources of resistance based on low induction of parasitic seed germination have been identified until now.20,21 Germplasm with lower induction levels of broomrape seed © 2014 American Chemical Society

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MATERIALS AND METHODS

Plant Material. Faba bean lines used in this work were Prothabon as susceptible line (positive control) and Navio and Quijote as resistant lines. Faba bean breeding lines Navio and Quijote were developed after selection under field conditions at Córdoba, Spain, for resistance to Orobanche crenata and agronomic performance within germplasm earlier selected in Tunisia for resistance to O. foetida.36 Received: Revised: Accepted: Published: 7063

February 21, 2014 June 27, 2014 June 28, 2014 June 28, 2014 dx.doi.org/10.1021/jf5027235 | J. Agric. Food Chem. 2014, 62, 7063−7071

Journal of Agricultural and Food Chemistry

Article

Figure 1. Structures of strigolactones used in this work: orobanchol (1), orobanchyl acetate (2), 5-deoxystrigol (3), 7α-hydroxyorobanchyl acetate (4), 7β-hydroxyorobanchyl acetate (5), fabacyl acetate (6), strigol (7), strigyl acetate (8), sorgomol (9), ent-2′-epi-orobanchol (10), and ent-2′-epiorobanchyl acetate (11). The parasitic plant species used were O. crenata (population collected on faba bean in Spain), O. foetida (collected on faba bean in Tunisia), and P. aegyptiaca (collected on faba bean in Israel). Parasitic seeds were collected from dry inflorescences using a 0.6 mm mesh-size sieve (Filtra, Barcelona, Spain) and stored dry in the dark at 4 °C until use. Faba Bean Root Exudate Collection. Faba bean seeds were surface sterilized with 4% sodium hypochlorite containing 0.02% Tween 20. Then, they were rinsed three times with sterile distilled water and placed on moistened filter paper inside Petri dishes to allow imbibition and germination. Four days after imbibition, germinated faba been seeds (radicle from 0.5 to 3 cm long) were transferred to pots filled with sterile Perlite in a growth chamber (22 °C, 12/12 h d/night regime and 200 μmol/m2/s irradiance). Plants were always watered with tap water and therefore suffering from nutrient starvation. Seven days after imbibition faba bean plant epicotyls emerged from Perlite, and 12 d after imbibition the first two leaves were expanded. Two, three, and four weeks after imbibition, faba bean plants were removed from the perlite and the roots were carefully washed with sterile distilled water. Faba bean plants were individually placed in tubes by immersing the roots for 24 h in sterile distilled water, allowing them to release the root exudates. The root solutions containing the faba bean root exudate were collected, and the total faba bean root contained in each tube was weighed. As exudation might vary accordingly with the amount of faba bean roots in each tube, which varied with faba bean plants and harvesting times, root solution per each individual plant was adjusted with sterile distilled water to achieve equivalent concentration of faba bean fresh root weight/mL of root solution across treatments. Three final concentrations were tested for all treatments in broomrape germination: 0.02 g of faba bean root (fresh weight)/mL of hydroponic media (root solution) and, in order to be sure that broomrape seeds could recognize any germination factor present in the root exudates from the resistant cultivars, we used two additional highly concentrated root exudate solutions: 0.04 and 0.1 g/

mL. Those concentrations are higher than those normally used for faba beanbroomrape seed germination bioassays.36 Parasitic Seed Surface Sterilization and Conditioning. Orobanche and Phelipanche seeds were surface sterilized with 0.2% (w/v) formaldehyde containing 0.02% (v/v) Tween 20, rinsed with sterile distilled water, and dried for 60 min in a laminar airflow cabinet. Approximately 100 seeds of each broomrape species were placed separately on glass fiber filter paper (GFFP) discs of 1.5 cm diameter moistened with 120 μL of sterile distilled water and conditioned in a 10 cm sterile Petri dish in the dark at 20 °C for 10 d. Root Exudate Bioassay on Broomrape Germination. GFFP discs containing 10 d-conditioned seeds of each species as described above were transferred onto a sterile sheet of paper to remove the excess of water used during seed conditioning and dry discs transferred to new 10 cm sterile Petri dishes. Differences in the ability displayed by each faba bean accession to induce broomrape germination was studied by applying triplicated aliquots of 100 μL of root exudate collected from each faba bean plant (four plants per faba bean line) at each harvesting time. Sterile distilled water was used as negative control. GR24 (10−6 M) was used as a second positive control in addition to the susceptible cultivar Prothabon. Distilled water and GR24 were applied on three additional discs of each Orobanche and Phelipanche species. Seeds were stored in the dark at 20 °C for 7 d to allow germination. The germination was scored for each disc by determining the number of germinated seeds on 100 seeds using a stereoscopic microscope. Seeds were considered germinated when radicle was visible through the seed coat. Strigolactone Analysis and Quantitation. Root Exudate Collection and Purification Using Ethyl Acetate. Root exudates were collected as described previously.40 Faba bean lines used were Prothabon as susceptible line and Navio and Quijote as resistant lines. Faba bean seeds were surface-sterilized in 70% ethanol and then with 1% NaClO. After being thoroughly rinsed with sterile distilled water, seeds were imbibed in water to allow germination for 2 d. Five seedlings were 7064

dx.doi.org/10.1021/jf5027235 | J. Agric. Food Chem. 2014, 62, 7063−7071


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Figure 2. Effect of plant age, accession, and concentration of root exudates on germination of seeds of Orobanche crenata. Vertical bars represent standard error for n = 4. Analysis of variance was applied to transformed replicate data. For each plant age, bars with different letters are significantly different according to the Tukey test (p = 0.05). Negative control (distilled water) = 0%. Positive control (GR24, 10−6 M) = 61%. to 500 L/h. The interface temperature was set to 400 °C and the source temperature to 150 °C. The capillary and cone voltages were adjusted to orobanchol (1) and to the positive ionization 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. The mass spectrometer was operated in positive ESI mode. Sample injection volume was 5 μL. Multiple reaction monitoring (MRM) was used for identification of strigolactones in root exudates and extracts by comparing retention times with 10 synthetic and natural strigolactone standards at 1 μM solution: orobanchol (1), orobanchyl acetate (2), 5deoxystrigol (3), 7α-hydroxyorobanchyl acetate (4), 7β-hydroxyorobanhyl acetate (5), fabacyl acetate (6), strigol (7), strigyl acetate (8), sorgomol (9), ent-2′-epi-orobanchol (10), and ent-2′-epi-orobanchyl acetate (11) (Figure 1).40,41 Strigolactone Assays on Broomrape Germination. The effects of three major strigolactones in legume crops were tested on germination of broomrape species. The strigolactones detected in root exudates of faba bean lines Prothabon, Quijote, and Navio (i.e., orobanchol (1) and orobanchyl acetate (2)) were used in germination assays of seeds of O. crenata, O. foetida, and P. aegyptiaca. Although not detected in faba bean in the present work, 5-deoxystrigol (3) was also studied as it is known as a major strigolactonein Fabaceae.40 These three strigolactones were tested at 10−6, 10−7, and 10−8 M on GFFP discs containing 10-d conditioned seeds of each parasitic species. GFFP discs containing the parasitic seeds conditioned as described above were transferred into a sterile sheet of paper to remove the excess of water and transferred to new 10 cm sterile Petri dishes. Each strigolactone was dissolved in acetone and diluted with sterile Milli-Q water to a final concentration of 0.7% of acetone. Aliquots of 100 μL of orobanchol (1), orobanchyl acetate (2), or 5-deoxystrigol (3) at each concentration were applied to triplicated GFFP discs containing parasitic seeds. This was done for each broomrape species separately. Petri dishes were sealed with Parafilm and incubated in the dark at 20 °C for 7 d. Milli-Q water containing 0.7% acetone was used as negative control. The germination was scored as described above. Statistical Analysis. Experiments were performed using a completely randomized design. Percentage data were approximated to normal frequency distribution by means of angular transformation (180/Π × arcsine (sqrt[%/100]) and subjected to analysis of variance (ANOVA) using SPSS software for Windows, version 21.0 (SPSS Inc., Chicago, Illinois, USA). The significance of mean differences among treatments was evaluated by Tukey HSD test. Null hypothesis was rejected at the level of 0.05.

transferred to a plastic cup (9 cm in diameter at top, 7.5 cm in diameter at the bottom, 8 cm deep, with 7 mm diameter holes on the bottom) placed in a larger plastic cup (8 cm in diameter at top, bottom 5.7 cm in diameter at the bottom, 13.5 cm deep, approximately 500 mL in volume) containing 400 mL of tap water as the culture medium. The culture medium (tap water) was frequently refreshed to allow aeration. The plants were grown in a growth chamber with a 14/10 h photoperiod at 23 °C. At 1, 2, 4, 6, 8, and 10 weeks after seed imbibition, the culture medium (tap water) was refreshed, allowing faba bean plants to release their root exudates in fresh tap water for 24 h before collection. One plastic cup was used for each faba bean line and stage of development. The tap water root exudates were extracted twice with ethyl acetate. Ethyl acetate solutions were combined, washed with 0.2 M K2HPO4 (pH 8.3), dried over anhydrous MgSO4, and concentrated in vacuo. Root Exudate Collection and Purification Using C18 SEPAK Cartridge. Faba bean seeds from cultivars Prothabon, Navio, and Quijote were surface sterilized and placed on sterile moistened filter paper inside Petri dishes to promote germination. Four-days-old faba been seedlings were individually transferred from the Petri dish to pots filled with sterile perlite in a growth chamber with a 14/10 h photoperiod at 23 °C. Plants were watered with tap water. After 2 weeks of cultivation in the pots, faba bean plants were removed from the Perlite and roots carefully washed with tap water. Faba bean plants were individually placed in 50 mL flasks containing sterile distilled water. Faba bean plants released in the tap water their root exudates during 24 h. Individually collected root exudate of four replicated plants per cultivar was individually loaded and purified in pre-equilibrated C18 SEPAK cartridges according with previous protocols.36 Strigolactone Analysis and Quantitation Using Liquid Chromatography−Tandem Mass Spectrometry. Identification of strigolactones was performed using a tandem mass spectrometer (Quattro LC Mass Spectrometer, Micromass, Manchester, U.K.) equipped with an electrospray ionization (ESI) source and coupled to a UPLC system (HITACHI Lachrom Ultra HPLC system, Hitachi, Japan). Chromatographic separation was achieved on a 2.1 mm × 50 mm, 2.0 μm, LColumn2 ODS column (CERI, Japan) by applying a methanol−water (MeOH-H2O) gradient as follows: 0−3 min, 30−45% methanol; 3−8 min, 45−50% methanol; 8−12 min, 50−70% methanol; 12−15 min, 70−100% methanol, maintained for 3 min, then returning to 30% methanol over 1 min. Finally, the column was re-equilibrated under these conditions for 3 min, prior to the next run. The column was operated at 40 °C with a flow rate of 0.2 mL/min. 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 approximately 100 L/h and the desolvation gas flow 7065



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Figure 3. Effect of plant age, accession, and concentration of root exudates on germination of seeds of Orobanche foetida. Vertical bars represent standard error for n = 4. Analysis of variance was applied to transformed replicate data. For each plant age, bars with different letters are significantly different according to the Tukey test (p = 0.05). Negative control (distilled water) = 0%. Positive control (GR24, 10−6 M) = 0%.

Figure 4. Effect of plant age, accession and concentration of root exudates on germination of seeds of Phelipanche aegyptiaca. Vertical bars represent standard error for n = 4. Analysis of variance was applied to transformed replicate data. For each plant age, bars with different letters are significantly different according to the Tukey test (p = 0.05). Negative control (distilled water) = 0%. Positive control (GR24, 10−6 M) = 91%.

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RESULTS AND DISCUSSION Time Course Analysis on Stimulatory Effect of Faba Bean Root Exudates on Orobanche and Phelipanche Seed Germination. The effects of faba bean cultivar (resistant cultivars: Quijote and Navio; and susceptible cultivar: Prothabon), plant age (2, 3 and 4 weeks old), and root exudate concentration (0.02, 0.04, and 0.1 g of faba bean root/mL of root solution) on germination of seeds of O. crenata, O. foetida, and P. aegyptiaca are shown in Figures 2, 3 and 4, respectively. In all cases, null germination was observed when broomrape seeds were treated with negative control (distilled water). Significant effect in broomrape germination was observed for cultivar, (ANOVA, p < 0.001). Root exudates of the highly resistant cultivar Quijote induced the lowest levels of parasitic germination on all the broomrape species tested at all harvesting times (11.3% ± 2.1) followed by the moderately resistant cultivar Navio (35% ± 2.5). The susceptible cultivar Prothabon used as a

positive control showed the highest stimulatory activity on germination of broomrape species (48% ± 3.1). Significant effects in broomrape germination were observed both for plant age and the interaction plant age × cultivar (ANOVA, p < 0.001, p < 0.001 respectively). The effect of plant age was different in the different faba bean cultivars tested. The induction potential of parasitic germination by Quijote root exudate was not significantly different across life stages; however, root exudates collected from older Navio and Prothabon plants induced higher germination levels on O. crenata and O. foetida seeds. Significant effect in broomrape germination was observed for broomrape species (ANOVA, p < 0.001). P. aegyptiaca was the parasitic species more responsive when exposed to faba bean root exudates. The germination average induced to P. aegyptiaca seeds by all faba bean cultivars at all harvesting times and root exudate concentrations was 59.4% ± 2.8, being significantly higher than 7066



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Figure 5. Liquid chromatography tandem mass spectrometry analysis of (A, B, C) faba bean susceptible line (Prothabon), (D, E, F) faba bean moderately resistant line (Navio), and (G, H, I) faba bean resistant line (Quijote). Root exudates were analyzed on 2-week-old faba bean plants (green line), 4-week-old faba bean plants (purple line), and 8-week-old faba bean plants (black line). (J) Representative UPLC chromatogram from a 4-weekold Prothabon root exudate sample.

the average induced to O. crenata (15.4% ± 2.0) and to O. foetida (22.2% ± 2.1). For each broomrape species tested, significant effects in germination were observed for root exudate concentration

(ANOVA, p < 0.001). Germination of broomrape species was not significantly affected by the interaction concentration × cultivar. Thus, we rule out the hypothesis that the resistance to broomrape in cultivars Quijote and Navio based on low levels of 7067



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Table 1. Quantitation (According to the Peak Area) of the Strigolactones Orobanchol (1) and Orobanchyl Acetate (2) in the Root Exudates of Resistant Cultivars Quijote and Navio and Susceptible Cultivar Prothabona amount of strigolactones and unknown germination stimulant (peak area) 2-week-old orobanchol (1) orobanchyl acetate (2) unknown a

4-week-old

8-week-old

Quijote

Navio

Prothabon

Quijote

Navio

Prothabon

Quijote

Navio

Prothabon

26.0 12.6 0.0

32.2 39.0 0.0

0.0 0.0 0.0

65.8 84.2 170.6

0.0 0.0 0.0

407.6 2467.0 1264.6

97.2 221.8 566.6

0.0 0.0 0.0

1200.6 5280.8 2352.8

Quantitation was also performed on the unknown germination stimulant.

orobanchyl acetate (2), using comparisons with 11 strigolactone standards (Figure 1). In addition, a peak at retention time 6.25 was collected and proved for germination stimulatory activity. This peak corresponds to an unknown germination stimulant and is currently under characterization. The susceptible cultivar Prothabon was the cultivar that exuded relatively larger amounts of strigolactones followed by Navio. The highly resistant cultivar Quijote was the cultivar whose root exudates contained the lowest amount of strigolactones. There were also differences between cultivars in the timing of strigolactone detection along the faba bean development. Table 1 shows the pattern of strigolactone exudation at 2, 4, and 8 weeks. At early growth stages (1 and 2 weeks old), none or negligible amounts of strigolactones were detected in root exudates of any of the faba bean lines tested. These contents remained low as time increased in Navio but increased markedly in Prothabon. Strigolactones levels in Quijote were under detection limits in young plants. As Quijote plants grew, orobanchol (1) and orobanchyl acetate (2) were detected in their root exudates but their levels were very low across all life stages studied. Quijote plants exuded 83.8% less orobanchol (1), 96.6% less orobanchyl acetate (2), and 86.6% less of the unknown germination stimulant than Prothabon plants when compared at 4 weeks old and 91.9% less orobanchol (1), 95.8% less orobanchyl acetate (2), and 75.9% less unknown germination stimulant than Prothabon plants when compared at 8 weeks old. In order to further confirm low exudation of strigolactones in Quijote plants, we analyzed by LC-MS/MS the root exudates individually collected from four replicated plants of Quijote, Navio, and Prothabon growing in 50 mL tubes. Previous to root exudate purification and LC-MS/MS analysis, aliquots of root exudate (at concentration 0.02 g/mL) of each plant and cultivar were tested on P. aegyptiaca and O. crenata germination. Differences in activity of the root exudates between susceptible control and resistant cultivars were confirmed, Prothabon root exudates being highly active on broomrape germination and Navio showing low activity, and very low or negligible levels of broomrape germination were induced by Quijote (data not shown). Our LC-MS/MS analysis showed that strigolactone levels, if any, were under detection limits for Quijote samples, while a peak corresponding to orobanchyl acetate (2) was detected in all Navio samples. In all Prothabon samples the peak corresponding to the unknown germination stimulant was always observed. This peak was collected and probed actively for germination stimulatory activity. This novel germination stimulant is currently under characterization. Differential Effect of Pure Strigolactones on Orobanche and Phelipanche Seed Germination. Figure 6 shows the effects of strigolactone (orobanchol (1), orobanchyl acetate (2), and 5-deoxystrigol (3)) and strigolactone concentration (10−6 M, 10−7 M, and 10−8 M) on germination of seeds of

broomrape seed germination is due to presence of germination inhibitors at the concentrations tested in this work. Root exudates of the resistant faba bean cultivar Quijote induced negligible levels of seed germination of O. crenata at all concentrations and harvesting times tested. They showed also a negligible activity on germination of O. foetida at 0.02 g/mL (1.0% ± 0.3) and at 0.04 g/mL (2.0% ± 1.2) at all harvesting times tested. Only at the highest root exudate concentration (0.1 g/mL), Quijote root exudates stimulated some O. foetida germination, that although low was significant at 3 weeks (27.7% ± 6.7) but not at 2 and 4 weeks of plant cultivation. P. aegyptiaca germination was induced by 2- and 3-week-old Quijote plants (39.1% ± 6.9 and 38.1% ± 8.8), but this activity significantly decreased at 4-week-old to 7.4% ± 4.4. Quijote root exudate concentration had significant effect on P. aegyptiaca germination only when seeds were exposed to root exudates collected from 3-week-old Quijote plants. As described before, root exudates of Navio induced higher germination than Quijote although still much lower than Prothabon. Root exudates of Navio plants showed activity on the three tested species, but this was higher on P. aegyptiaca seeds (64.1% ± 2.7) than on O. crenata (18.3% ± 3.1) and O. foetida (26.7% ± 3.1) seeds. Prothabon root exudates induced germination of the three parasitic species tested. The average germination at all harvesting times and root exudate concentration for P. aegyptiaca, O. crenata, and O. foetida was 81.9% ± 1.5; 25.9% ± 3.7; and 32.3% ± 3.8, respectively. The Prothabon stimulation of broomrape seeds increased with harvesting time reaching maximum levels of germination for each broomrape species when Prothabon plants were 4 weeks old. LC-MS/MS Analysis for Strigolactone Identification and Quantitation in Faba Bean Root Exudate. Root exudates collected from each faba bean cultivar were screened for the presence of strigolactones by LC-MS/MS. This analysis was repeated across faba bean development, 6 times at 1, 2, 4, 6, 8, and 10 weeks of cultivation. Strigolactone profiles of root exudates from 1- and 2-week-old plants were similar for each faba bean cultivar. Strigolactone profiles for 6-, 8-, and 10-week-old plants were similar for each faba bean cultivar. LC-MS/MS analysis indicated that the same strigolactones were present in root exudates of all faba bean cultivars studied (Figure 5, Table 1). However, there were differences between cultivars in the pattern of strigolactone exudation. We observed a link between the low amounts of strigolactones exuded by Quijote and Navio, the low level of broomrape germination observed in vitro in this work and in previous reports,36 and the high level of broomrape resistance observed in the field in these resistant cultivars.36 For all faba bean cultivars the LC-MS/MS chromatograms showed two peaks at retention times 9.10 and 12.84 min, and they were identified as orobanchol (1) and 7068



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0.001). The stimulatory effects of strigolactones on O. crenata seed germination increased at increased concentrations, being maximum (77.7% ± 2.2) at 10−6 M. This decrease was more marked for 5-deoxystrigol (3), which induced 6.3% ± 1.3% of O. crenata germination at 10−8 M, and moderate for orobanchyl acetate (2) (21.3% ± 2.2). On the contrary orobanchol (1) and orobanchyl acetate (2) effects on O. foetida germination increased as the strigolactone concentration decreases, showing a maximum germination rate of 74.5% ± 1.8 at 10−8 M. Remarkably, 5-deoxystrigol (3) was not effective inducing the germination of O. foetida seeds at the concentrations tested. High germination rates were observed on P. aegyptiaca seeds with all strigolactones and strigolactone concentrations. Several measures are available for broomrape management; however, they are not fully applicable in a low input crop such as faba bean.37,42 Therefore, development of resistant cultivars is the most desirable strategy.42 Resistance in faba bean against broomrape has been based on mechanisms hampering the parasitic penetration and development in the host.39 Only recently, resistance due to reduced parasitic seed germination has been discovered in faba bean against O. crenata, O. foetida, and P. aegyptiaca.36,43 In the later study we have shown that the reduced susceptibility observed in the field by the cultivar Quijote was correlated with reduced stimulatory activity of broomrape in the in vitro germination test, but the underlying mechanisms were not determined. Our LC-MS/MS analysis of root exudates from three faba bean cultivars has detected orobanchol (1) and orobanchyl acetate (2) both in susceptible and resistant cultivars. We have not detected 5-deoxystrigol (3), in spite that this strigolactone is common in the Fabaceae, as well as orobanchol (1) and orobanchyl acetate (2).40 In addition to these known strigolactones, a third unknown germination stimulant was found. A time course quantitation of strigolactone according to the peak area in LC-MS/MS44 showed that the pattern of strigolactone exudation differs within faba bean cultivars and directly related with low levels of broomrape germination observed in vitro. As the roots of the susceptible cultivar Prothabon developed, strigolactone exudation increased greatly, this fact being in agreement with previous findings that production and exudation of strigolactones vary significantly with plant growth conditions and growth stages.1 On the contrary, the resistant faba bean lines Quijote and Navio exuded low or undetected levels of strigolactones at all plant ages studied. Reduced broomrape infestation in the resistant lines might depend on a combination of low concentration of strigolactones in root exudate and their exudation pattern along the life stages that could avoid the correct timed encounter between parasites and host. According with the results of strigolactone quantitation by LCMS/MS, the germination tests carried out on seeds of three broomrape species showed that parasitic germination increased with the age of Prothabon plants from which root exudates were collected. Small differences between LC-MS/MS and the germination test were observed for the resistant varieties. As described before, LC-MS/MS analysis detected low or undetected amount of strigolactones in root exudates of Quijote and Navio at all life stages but their relative quantitation across the life cycle showed a small increase with age in Quijote but a small decrease in Navio plants. However, broomrape seed germination increased as Navio plants grew older and in Quijote plants the broomrape germination decreased when exposed to 4-

Figure 6. Effects of orobanchol (1), orobanchyl acetate (2), and 5deoxystrigol (3) at decreasing concentrations on seed germination of O. crenata (A), O. foetida (B), and P. aegyptiaca (C). Vertical bars represent standard error for n = 3. The same letter per broomrape species indicates that differences are not statistically significant between SL and SL concentrations (Tukey test, p < 0.05). Negative controls (distilled water) = 0%, for O. crenata, O. foetida, and P. aegyptiaca.

O. crenata, O. foetida, and P. aegyptiaca. In all cases, null germination was observed when broomrape seeds were treated with distilled water (negative control). We observed significant differences in parasitic seed germination depending on the strigolactone tested (p < 0.001), the concentration applied (p < 0.001), the parasitic species tested (p < 0.001), and their interactions (p < 0.001). Seeds belonging to O. crenata and O. foetida were more sensitive to orobanchol (1) and orobanchyl acetate (2) than to 5deoxystrigol (3). The effect of strigolactone and strigolactone concentration was highly significant (ANOVA, p < 0.001 and p < 0.001 respectively for O. crenata seeds and p < 0.001 and p = 0.007 respectively for O. foetida seeds). Germination of P. aegyptiaca seeds was very sensitive to all strigolactones and strigolactone concentrations tested. The effect of strigolactone and strigolactone concentration was not significant (ANOVA, p = 0.543 and p = 0.077 respectively) showing a germination rate close to 90% in all cases. The interaction between broomrape species × strigolactone × strigolactone concentration was highly significant (ANOVA, p < 7069



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would be a great advantage as the resistances available before were of complex inheritance and, therefore, more difficult to breed for.37 The use of molecular markers tightly linked to specific resistance mechanisms will allow their tracking in segregating populations, facilitating their pyramiding with other genes. The remarkable fact that low induction of germination is similarly operative against O. crenata, O. foetida, and P. aegyptiaca reinforces the value of this resistance.

week-old plants. It is important to remark that Quijote root exudates induced low germination of P. aegyptiaca, a broomrape species highly sensitive to strigolactones. Also, they induced germination of O. crenata and O. foetida seeds only observed when applied at the highest concentrations, which was much higher than those commonly used in germination tests until now. Several factors may determine the observation of low but still significant broomrape germination when low or negligible levels of strigolactones were detected by LC-MS/MS. First, plant growth and root exudate collection for germination bioassays and strigolactone quantitation were carried out in independent laboratories. Activity of root exudates may change from experiment to experiment as production and exudation of strigolactones vary significantly with different environmental conditions.1These variations may be more remarkable when the production of germination stimulants is low as is the case for Quijote and Navio cultivars. Second, germination tests when carried out on seeds of the highly strigolactone-sensitive broomrape species such as P. aegyptiaca and O. minor are considered more sensitive for strigolactone detection than LCMS/MS analysis. Third, LC-MS/MS analysis only detects major strigolactones from the myriad of compounds exuded by plant roots. Non-strigolactone-like molecules of unknown nature present in host root exudate can induce germination of the broomrape species tested in this work.45,46 And last, faba bean samples analyzed by LC-MS/MS were obtained by ethyl acetate extraction washed with 0.2 M K2HPO4 to remove acidic substances. This procedure was done in order to facilitate direct comparisons between crude samples from different plants and cultivars by removing interference of acidic substances with electrospray ionization of strigolactones. This procedure may lead to removal of additional substances other than strigolactones in the faba bean root exudates with potential for broomrape germination.45,46 We have studied the effect of three major strigolactones typically found in legumes (orobanchol (1), orobanchyl acetate (2), and 5-deoxystrigol (3)), on three broomrape species that parasitize legumes but differ in their host ranges. P. aegyptiaca seeds highly germinated when exposed to all strigolactones, at all concentrations tested, confirming the sensitive nature of this broomrape species to strigolactones that correlates with the broad host range of this species.47 Intriguing results were found for the more host-specific species O. crenata and O. foetida. First, in both broomrape species, orobanchol (1) induced much higher germination than orobanchyl acetate (2). Orobanchyl acetate (2) with less activity on O. foetida and O. crenata was found to be more abundant in root exudates of faba bean. This fact could point to a germination tuning between parasitic and host species through strigolactone concentration. In addition, 5-deoxystrigol (3) was not found in the faba bean root exudate tested in this work and was not active in inducing germination of O. fetida, a broomrape species with a very narrow host range for few legumes.47 However, 5-deoxystrigol (3) was active inducing germination of O. crenata, a species that, although still being rather host-specific, expands its host range to other host families with different strigolactone exudation profiles, confirming previous observations.48 Results presented here show that low strigolactone exudation does exist within faba bean germplasm, contributing to resistance to broomrape. Therefore, selection for this trait is feasible in a breeding program. Inheritance of this trait should be studied, and it seems feasible that it might be simple inheritance, as was the case for low induction of Striga germination in sorghum.49,50 This

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AUTHOR INFORMATION

Corresponding Author

*Tel.: + 34 957499215. Fax: + 34 957499252. E-mail: monica. [email protected]. Funding

This work was supported by the Program for Promotion of Basic and Applied Researches for Innovation in Bio-oriented Industry to K.Y., T.K., and X.X. and to projects FP7-LEGATO and AGL2011-22524, cofinanced by FEDER funds to D.R. M.F.-A. has received the support of the European Union, in the framework of the International Outgoing European Marie Curie Postdoctoral Fellowship Programme (PIOF-GA-2009252538), and from the Japan Society for the Promotion of Science Postdoctoral Fellowship ID noP 13390. Notes

The authors declare no competing financial interest.

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REFERENCES

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Orobanche and Phelipanche spp. - American Chemical Society

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