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Growth-defense tradeoffs and source-sink relationship during both faba bean and lentil interactions with Orobanche crenata Forsk
Crop Protection 127 (2020) 104924
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Growth-defense tradeoffs and source-sink relationship during both faba bean and lentil interactions with Orobanche crenata Forsk Mounia Ennami a, b, 1, Joseph Mbasani-mansi a, Fatima Zahra Briache a, Nada Oussible b, Fatima Gaboun a, Lamiae Ghaouti b, Loubna Belqadi b, Michel Edmond Ghanem c, Kamal Aberkani d, James Westwood e, Rachid Mentag a, * a
National Institute of Agricultural Research of Morocco (INRA), CRRA-Rabat, Biotechnology Unit, Rabat, Morocco Agronomic and Veterinary Institute Hassan II (IAV), Plant Biotechnology Department, Rabat, Morocco Crop Physiology Laboratory, International Center for Agricultural Research in the Dry Areas (ICARDA), Rabat, Morocco d University Mohammed First, Faculty of Science and Technology (FST), Al, Hoceima, Morocco e Virginia Tech, Department of Plant Pathology, Physiology and Weed Science, Blacksburg, VA, USA b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Vicia faba L. Lens culinaris medik. Orobanche crenata Susceptible and resistant cultivars Biomass partitioning Source-sink relationship
Orobanche crenata Forssk., a root holoparasitic weed, represents a major biotic threat of legumes in the Medi terranean region. O. crenata could reduce the yield up to 90%. In this study, the effect of O. crenata on biomass production and partitioning of some faba bean and lentil cultivars were investigated through pot assays. During the first stage of infection, reductions in host biomass were observed on susceptible and resistant faba bean cultivars (53.84 and 27.02%, respectively for Lobab and Misr 3) and on susceptible lentil cultivar (Zaaria). However, during the last stage (host plant maturity), combined biomasses of susceptible faba bean (35.69 g) and lentil (8 g) cultivars were similar to those of non-infested plants (32.45 and 6.62 g, respectively). Considering biomass partitioning over the various host parts, O. crenata parasitism on susceptible faba bean and lentil cul tivars greatly increased host root dry mater, but delayed and reduced host reproduction. The relative weight of parasite and host organs were also studied. Thus, a marked decrease of root relative weight was observed during the last developmental stages (up to 63%) accompanied simultaneously with the increase of relative parasitic weight (35.87%). Results suggested that legumes-O. crenata interactions were governed by growth-defense tradeoffs during the early stage of infection, whereas source-sink relationships explained the dry weight diver sion from host to parasite during the last phases of the interaction.
1. Introduction Orobanche crenata Forssk. is a chlorophyll-lacking root parasite. This parasitic weed is considered to be the most widespread parasitic plant species in the Mediterranean region, especially in North Africa and the Near East and Western Asia (Parker, 2009; Joel et al., 2011). O. crenata parasitizes several host species from the family Fabaceae, within which the main host crops are faba bean (Vicia faba L.), chickpea (Cicer arie tinum L.), pea (Pisum sativum L.), lentil (Lens culinaris Medik.), vetches (Vicia benghalensis L.), and many forage legumes (Goldwasser et al., 2000; Rubiales et al., 2009; Abu Irmailah and Haddad, 2011). In Morocco, O. crenata infestation was officially reported on faba bean in 1943 in the Fez region. Since then, the parasite has spread to
other areas (Zair, Pre-Rif, Chaouia, Sais, Fez, Meknes, Taounate, Sidi Kacem, Chefchaouen, Doukala, and Abda) (Labrada, 2007; FAO, 2014). The estimated average yield losses due to parasitism range from seven to 90%, depending on host plants, environmental factors, and soil infes tation level (Sauerborn, 1991; Torres et al., 2006; Ennami et al., 2017). In fact, a single O. crenata spike produces thousands of tiny seeds that can remain viable in the soil for more than 10 years and germinate in response to chemical signals exuded from host roots (Linke and Saxena, 1991; Zhou et al., 2004). Successful attachment to the host root requires development of the haustorium, a specialized structure that ensures connection between the host vascular tissue and the parasite. After attachment the parasite obtains from its host all water and mineral nutrients needed for growth into a tubercle, a bulbous structure from
* Corresponding author. National Institute of Agricultural Research (INRA), CRRA, Biotechnology Unit, Rabat, Morocco. E-mail address: [email protected] (R. Mentag). 1 Current address: National Institute of Agricultural Research (INRA), CRRA, Biotechnology Unit, Rabat, Morocco. https://doi.org/10.1016/j.cropro.2019.104924 Received 19 February 2018; Received in revised form 21 June 2019; Accepted 17 August 2019 Available online 18 August 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.
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which a shoot arises and emerges from the soil to flower and set seeds (Westwood, 2000; Rodríguez-Conde et al., 2004). Various control strategies; cultural, chemical, breeding, etc. have been employed to control Orobanche (Bayoumi et al., 2014; Kannan and Zwanenburg, 2014; Trabelsi et al., 2016). Unfortunately, none of them have proven to be both effective and economically feasible due to technical challenges, economic constraints, and the complexity of host-parasite interactions (Grenz et al., 2005; Rubiales et al., 2009). The development of effective Orobanche control strategies requires a better understanding of the host plant-Orobanche interaction acting at morphological and physiological levels in contrasting cultivars (sus ceptible and resistant). However, most research on the general features of host infection by Orobanche species has been conducted using sus ceptible host cultivars under field trials such as the interactions between �ndez-Aparicio et al., 2016; Trabelsi et al., faba bean and O. crenata (Ferna 2015, 2016; Ennami et al., 2017) and O. ramosa with tomato (Maur omicale et al., 2008). Analyses of these interactions showed highly significant differences among host cultivars in response to these para sitic weeds, especially in considering parameters of biomass production and grain yield. Leaf chlorophyll fluorescence can be used as a physiological indi cator of plant response to stress (Strasser et al., 2000; Strasser and Tsimilli-Michael, 2001). Many researchers used such photosynthetic parameters to evaluate plant responses under various stress conditions such as water deficit (Duraes et al., 2001), temperature (Yamada et al., 1996), salinity or nitrogen deficit (Duraes et al., 2001; Moradi and Ismail, 2007), and herbicide effects (Pavlovi�c et al., 2007; Bo�zi�c et al., 2010), etc. Some studies have assessed the effects of parasitic weeds on photosynthesis of host plants, including Cowpea (Vigna unguiculata L. Walp.)/Striga gesnerioides Willd.; Ambrosia trifida L./Cuscuta campestris Yunck.; and Solanum lycopersicum L./O. ramosa L. interactions (Hibberd et al., 1996; Mauromicale et al., 2008; Vrbnicanin et al., 2013). These authors concluded that parasitic weeds act as a competitor sink for host assimilates, causing damage principally via its negatives effect on the host photosynthetic machinery (photosystems I and photosystems II). Despite the high impact of O. crenata on legume species in Morocco, the host physiological changes in response to O. crenata have not been characterized. The main objective of this study was to investigate the effects of O. crenata on the agro-morphological and chlorophyll fluo rescence parameters of susceptible and resistant cultivars of faba bean and lentil under controlled conditions. These parameters were moni tored over the host growth cycle and also considered in light of the developmental progression of the parasite through different phenolog ical stages.
were developed and characterized at the Regional Center of Agriculture Research of Rabat, National Institute of Agriculture Research (INRA-Morocco). Orobanche crenata seeds were collected in 2014 from mature O. crenata plants growing in legume fields at the Marchouch experi mental station of National Institute of Agriculture Research (INRAMorocco) (Table 1). Before use, all seeds were stored dry in the dark at room temperature. 2.2. Experimental design Effects of O. crenata on agro-morphological and chlorophyll fluo rescence of faba bean and lentil plants were monitored in 6 L plastic pots following the modified pot testing method developed by Rubiales et al. (2005a). Pots were filled with soil–peat (3:1, v:v). For each legume species, the experiment was designed as a randomized split-plot with four replicates. Indeed, two separate experiments were set up, one for faba bean cultivars and the other one for lentil cultivars. The trials were conducted in greenhouse under natural day-light conditions at the Regional Center for Agricultural Research-INRA Rabat, Morocco (33� 580 1700 N; 6� 500 5900 W in north-western Morocco). Non-infested soil (autoclaved soil at 120 � C for 45 min) and artificially infested soil by O. crenata (autoclaved soil supplemented by 30 mg of O. crenata seeds per 1 kg of substrate) were considered as main plots. The subplots consisted on susceptible and resistant cultivars of faba bean or lentil. This led to 40 pots different “host cultivars/Orobanche treatment” as sociations. Five lentil seeds or one faba bean seed were sown in each pot. Indeed, seeds of each legume species were manually sown to a depth of 4 cm. 2.3. Kinetics of measurement of the different parameters Pots were sown on January 26th 2016. Kinetics of O. crenata development on both legume species were conducted through pot uprooting and root systems were gently washed under running tap water (Fern� andez-Aparicio et al., 2016). Uprooting of hosts (Five lentil plants or one faba bean plant) was performed 42 days after planting (DAP); means two weeks before O. crenata attack (Bud set “First individual flower buds visible outside of leaves but still closed”); at 56 DAP (Flowering “between Full flowering and end of flowering (first pods visible)”); at 70 DAP (Plant expansion “10% of pods have reached final length”); at 84 DAP (Plant expansion “more than 50% of pods have reached final length”), and at 98 DAP (Faba bean maturity). 2.4. Agro-morphological and O. crenata infestation measurements
2. Materials and methods
Ten agro-morphological parameters were measured for host plants: plant height; number of vegetative tillers; fresh and dry weight of vegetative tillers (g); seed number and dry weight (g) per plant; number of root-nodules (used as proxy to measure the impact of the parasite on the nodule formation on a 0 to 5 scale); fresh and dry weight of root (g); and root volume (Cubero et al., 1993; Rubiales et al., 2005b). Two in dexes of O. crenata Infestation Events (OIE) were measured: number (NOIE) and dry weight of emerged and underground (DwOIE) (Ennami et al., 2017; Briache et al., 2019; Mbasani-Mansi et al., 2019). Regarding, hosts and parasitic parts, dry weight was scored after oven
2.1. Plant material Two faba bean cultivars were used; cv. Misr 3 that was selected in Egypt and recently in Morocco for its resistance to O. crenata (Attia et al., 2013; Briache et al., 2019) and cv. Lobab, registered in the Moroccan catalogue since 1985, was recently confirmed as susceptible to O. crenata (Briache et al., 2019). For lentil, two cultivars, registered in Moroccan catalog were used. Zaaria (O. crenata-susceptible) and Bakria (O. crenata-moderately resistant) (Mbasani-Mansi et al., 2019). Lentil Table 1 Coordinates, Climate, and soil characteristics of experimental locations. Location Marchouch
a b
Coordinates Longitude Latitude Altitude
a
6.72 33.62a 398b
Soil type (FAO)
Soil texture
pH
Rainfall (mm)
Temperature (� C)
Luvisols (Chromic Luvisols)
silty clay
6
407
23.4 (minimum of 1 and a maximum of 45)
decimal degrees. Meters. 2
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drying at 80 � C for 48 h and assessed during the uprooting step at 42, 56, 70, 84, and 98 DAP. Infestation intensity was estimated by counting Orobanche stages developed on host root system, while phenological stages of OIEs were recorded using a modified scale (Briache et al., 2019) from 1 to 6 (S1: attachments on root; S2: tubercles; S3: small buds; S4: large buds; S5: underground stems; and S6: emerged spikes). �nde Additional parameters were calculated as suggested by Ferna z-Aparicio et al. (2016) to determine the biomass partitioning within the host-O. crenata relationship and also to evaluate the effect of O. crenata infestation on each legume species. These parameters were: total host biomass (vegetative tillers, seeds, and roots); host reproductive biomass (seed dry weight); O. crenata dry weight (emerged and underground OIEs); and combined biomass (vegetative tillers, seeds, and root dry weights of legume species and dry weight of emerged and underground OIEs). In addition, the relative weight of the various host parts was calculated as: root relative weight (proportions of root dry weight/ combined biomass); relative host reproductive weight (proportion of host seed dry weight/combined biomass) and relative O. crenata weight (proportion of O. crenata dry weight/combined biomass). All these pa rameters were calculated at the five uprooting stages (42, 56, 70, 84, and 98 DAP).
respectively. Considering the number of O. crenata infestation events, the resistant and susceptible cultivars differed significantly (P � 0.05), especially during the late stages of infection (70, 84, and 98 DAP). For both legume species, no Orobanche infestation event was recorded at the first time point (42 DAP). At the last time point (98 DAP), the NOIE on Misr 3 and Bakria were 8.5 and 5.75, respectively, against 14 and 15.5 recorded for the susceptible cultivars Lobab and Zaaria (Fig. 1). For both faba beans, the first O. crenata attachments were recorded at 56 DAP (Figs. 1 A, Fig. 2 A and F). This parasite developed rapidly on the sus ceptible host cultivar (Lobab) and O. crenata phenological stage 6 (emergent spikes) was reached 70 DAP (Fig. 1 A; Fig. 2 G), while it was recorded 98 DAP for the cultivar Misr 3 (Fig. 1 A; Fig. 2 D). For lentils, O. crenata developed rapidly on Zaaria compared to the Bakria cultivar. The first O. crenata attachment was reported at 56 DAP (Fig. 1 B). On the susceptible cultivar (Zaaria), O. crenata reached the final stage of development (Emergent spikes) at 70 DAP (Fig. 1 B). This stage was achieved only at 98 DAP on the more resistant Bakria cultivar (Fig. 1 B). 3.2. Effect of O. crenata on faba bean and lentil agro-morphological parameters The biomass of parasite-free host plants was higher than that of infested plants. The presence of O. crenata reduced the biomass of both faba bean cultivars, but this reduction was more important on Lobab than Misr 3 (Fig. 3). Compared to non-infested plants on susceptible cultivar, reduction rates induced by O. crenata were significant, up to 53.8, 31.7, 36.9, 62.1, and 59.3%, respectively, for 42, 56, 70, 84, and 98 DAP (Fig. 3 A). In contrast, on resistant cultivar, the only significant reduction rates were noted at 42, 56, and 84 DAP, respectively, for 27.0, 34.0, and 53.4% (Fig. 3 B). Regarding faba bean cultivars, additional differences were found between the dry weights of the non-infested system (Fig. 3 A”� Dry weight of non-infested system”) and combined biomass (Fig. 3 A”◆ Dry weight of infested system (combined biomass with O. crenata)”). In fact, compared to total dry weight produced on non-infested system, O. crenata reduced significantly the combined biomass of the infested Lobab cultivar at 42, 56, and 70 DAP. However, these reductions decreased at 84 DAP, and by 98 DAP the combined biomass of infested plants was equal to the total dry weight of the noninfested system (Fig. 3 A). For the resistant Misr 3 cultivar, reductions were observed at 56 and 84 DAP (Fig. 3 B). Considering O. crenata biomass (Fig. 3 B “〇 O. crenata dry weight”), this parameter remained low on Misr 3 cultivar and never exceed 5.41 g at 98 DAP (Fig. 3 B). In contrast, the biomass of O. crenata growing on the susceptible cultivar (Fig. 3 A “▴ O. crenata dry weight”) surpassed host dry matter at 84 and 98 DAP. For lentils, the data indicate almost the same pattern as observed on faba beans. Thus, for both lentil cultivars, higher biomass values were noted for non-infested plants compared to the infested plants (Fig. 4). Compared to non-infested plants (Fig. 4 A “� Dry weight of non-infested system (host biomass without O. crenata)”), significant biomass re ductions of 30.46 and 47.37% were observed on Zaaria dry weight (Fig. 4 A ⃞ “ Host dry weight of infested system (combined biomass without O. crenata)”), respectively for 56 and 70 DAP. However, re ductions were not significant after these times. On Bakria cultivar, no significant host biomass differences were recorded in parasite-free plants and during parasitism (Fig. 4 B). Regarding O. crenata biomass, this parameter was greater when grown on Zaaria plants, reaching 2.88 g at 98 DAP. Whereas on Bakria resistant cultivar, O. crenata biomass never exceed 0.84 g at the final time point (98 DAP). Further more, using Orobanche, the resistance is not only the capacity of the host plant to limit the development of the parasite but also the capacity of that plant to produce under such Orobanche parasitism pressure. For the susceptible faba bean cultivar, a marked effect of infestation by O. crenata on biomass partitioning over the various plant parts was recorded (Fig. 5 B). For non-infested plants, the biomasses of roots were 19, 33, 24, 14, and 12% of the total plant weight for 42, 56, 70, 84, and
2.5. Chlorophyll fluorescence measurements Fully expanded leaves were illuminated with a saturated light pulse after 30 min of darkness (Ghanem et al., 2008) using an Imaging-PAM 91090 Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Ger many) (Walz, 2009). The purpose of the darkness before measurements is to enable plastoquinone QA of PS II to become oxidized and allow for maximal photochemical quenching. They were illuminated during a 3 s (x 1 flash) of bright LED lamp: peak emission 470 nm; standard excita tion intensity: 0.5 μmol quanta m-2 s-1 PAR, maximal actinic intensity: 3000 μmol m-2 s-1 PAR, maximal saturation pulse intensity: 6000 μmol quanta μmol m-2 s-1. Measurements of chlorophyll fluorescence pa rameters were made during sunny and clear sky conditions (noon to 14:00). Imaging-PAM software records the values of all pixels within a single area of interest and calculates the average. Effective quantum yield of open photosystem II (PSII) was calculated according to (Genty et al., 1989) using the formula: (Fm’-F)/Fm’. This parameter was measured on all faba bean and lentil cultivars and on non-infested and infested soil by O. crenata. Two specific leaves per plant were chosen to monitor senescence. A young expanding leaf, identified as leaf number 4 (from the bottom), and leaf number 5. Each leaf was measured at three areas of interest in the intercostal regions close to the main vein also to secondary veins. Indeed, each data point represents an average of 120 repetitions of leaf samples � standard deviation; means of 20 plants x (2 leaves/plants) x (3 points/leaf) were chosen. Fluores cence measurements were performed at 35, 42, 49, 56, 63, 70, 77, 84, 91, and 98 days after planting of faba bean cultivars and at 42, 56, 70, 84, and 98 days after planting in the case of lentil cultivars. 2.6. Statistical analysis The treatment effects were analyzed using analysis of variance (ANOVA) in the SAS statistical analysis program (version 9.1; SAS, North Carolina, USA). ANOVA were conducted to assess whether there was a significant variation for each studied variable. Treatment means were compared using least significant difference (LSD) test at alpha ¼ 0.05. 3. Results 3.1. O. crenata development on faba bean and lentil cultivars Monitoring parasite developmental progression on faba bean and lentil cultivars confirmed the resistance level of both Misr 3 and Bakria, 3
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Fig. 1. Development of O. crenata phenological stages on (A): faba bean cultivars (Lob: Lobab and Misr: Misr 3) and (B): lentil cultivars (Bak: Bakria and Zaa: Zaaria), grown in pots assay for 14 weeks and harvested to observe parasitism at regular intervals (42, 56, 70, 84, 98 days after planting). Phenological stages are: (S1): Attachments; (S2): Tubercles; (S3): Small buds; (S4): Large buds; (S5): Underground stems; and (S6): Emergent spikes. Data are means of 8 replicates for each cultivar at each time point.
Fig. 2. Infestation levels of the two faba bean cultivars tested on non-infested and parasitized soil by O. crenata in a pot experiment over 14 weeks. Observations were carried out on infested resistant cultivar Misr 3 at 56 (A), 70 (B), 84 (C), 98 (D) and infested susceptible cultivar Lobab at 56 (F), 70 (G), 84 (H), 98 (I). Non-infested Misr 3 and Lobab were recorded at 98 DAP (E and J respectively).
98 DAP, respectively (Fig. 5 A). A relative increase in host biomass was observed on infested system, with dry weights of 27, 36, 28, 34, and 17% respectively for 42, 56, 70, 84, and 98 DAP (Fig. 5 B). Furthermore, the parasite attack affected seed production of the Lobab cultivar, as seed production on infested hosts was limited two weeks compared to the non-infested system. No differences were observed between noninfested and infested plants for host vegetative tillers dry matter of the Lobab cultivar. Over the course of the experiment biomass allocated to vegetative shoot tissues decreased gradually from 80.77 to 33.90% for the non-infested system (Fig. 5 A) and from 77.22 to 41.70% for the infested system (Fig. 5 B). Regarding the resistant Misr 3 cultivar, no significant differences were detected in dry matter partitioning over the
various plant parts between non-infested (Fig. 5 C) and infested systems (Fig. 5 D). Although both lentil cultivars responded to O. crenata infestation, the Zaaria (susceptible cultivar) was more negatively affected than the resistant Bakria cultivar (Fig. 6). Infestation by O. crenata resulted in a clear increase of biomass allocated to roots of Zaaria cultivar. Root tissue accounted for 29, 41, 44, 33, and 13% of total biomass at 42, 56, 70, 84, and 98 DAP (Fig. 6 B). By comparison, in the non-infested system, biomass allocated to roots were 54, 34, 17, 3, and 3% for 42, 56, 70, 84, and 98 DAP, respectively (Fig. 6 A). For this host, seed production as a fraction of the total dry weight decreased significantly in parasitized plants compared to non-infested hosts (Fig. 6 A and B). Regarding host 4
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Fig. 3. Relationship between total dry weight of host plants and O. crenata infection on faba bean (A) Lobab and (B) Misr 3 cultivars. 〇 Dry weight of non-infested system (host biomass without O. crenata); ◆ Dry weight of infested system (combined biomass with O. crenata); □ Host dry weight of infested system (combined biomass without O. crenata); and ▴ O. crenata dry weight. Data are means of 8 replicates of each cultivar for each time point. Bars indicate standard error.
Fig. 4. Relationship between total dry weight of lentil cultivars and O. crenata infection on (A) Zaaria and (B) Bakria. 〇 Dry weight of non-infested system (host biomass without O. crenata); ◆ Dry weight of infested system (combined host and with O. crenata biomass); □ Host dry weight of infested system (combined biomass without O. crenata); and ▴ O. crenata dry weight. Data are means of 8 replicates of each cultivar for each time point. Bars indicate standard error.
Fig. 5. Percentage of total biomass allocated to seeds, vegetative tillers and roots of the Lobab variety of faba bean when plants are (A) non-infested and (B) infested by O. crenata. Same for the Misr 3 cultivar on (C) non-infested and (D) O. crenata infested system.
vegetative dry matter of Zaaria cultivar, a negative effect of infestation was observed, as biomass increased gradually from 45.55 to 79.31% in the non-infested system (Fig. 6 A), but was reduced to 58.77, 55.55, and
65.87% of total biomass at 56, 70, and 84 DAP, respectively (Fig. 6 B). The resistant Bakria cultivar showed no O. crenata effect on dry matter partitioning over the various plant parts between non-infested (Fig. 6 C) 5
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Fig. 6. Percentage of total biomass allocated to vegetative tillers, roots, and seeds of lentil Zaaria cultivar when (A) non-infested and (B) infested by O. crenata. Likewise, the Bakria cultivar when (C) non-infested and (D) infested by O. crenata.
and infested systems (Fig. 6 D). In addition to dry matter partitioning over the various plant parts within the two cultivars, we also measured relative distribution of dry weight from source to different sinks (plant organs and parasite) in the absence and presence of O. crenata. Thus, in the presence of O. crenata and during the first two time point (42 and 56 DAP), the host root represents the main sink in both cultivars (Fig. 7). However, after 70 DAP, the relative weight of the parasite on Misr 3 reached maximum of only 21.5% at 84 DAP whereas the parasite relative weight on Lobab increased dramatically and reached 60% at the same time point. Within non-infested system, host reproductive relative weight of Lobab increased gradually from 9.24, 28.17, and 53.89%, respectively, for 70, 84, and 98 DAP (Data note shown). However, in the presence of O. crenata, host seed relative weight was, respectively, 0, 3.97, and 14.90% (Fig. 7 A). Regarding Misr 3 cultivar, host reproduction relative weight increased gradually on both systems (non-infested and infested) reaching respectively, on infested system, 9.17, 20.84, and 40.63% at 70, 84, and 98 DAP. O. crenata parasitism of the lentil Zaaria cultivar significantly increased relative root weight at 56 DAP, while seed relative weight was
significantly decreased compared to non-infested plants (Fig. 8 A). However, on the Bakria cultivar, host root relative weight showed only a downward trend during over time and host seed relative weight reached 16% by 84 DAP (Fig. 8 B). Relative parasite weight on Zaaria increased rapidly from 30% at 42 DAP to 3% at 98 DAP (Fig. 8 A). However, on the Bakria cultivar, the increase rate of relative parasitic weight was much lower (Fig. 8 B). 3.3. Effect of O. crenata on leaf chlorophyll fluorescence of faba bean and lentil No significant differences were recorded on the Effective PSII quantum yield between infested and non-infested cultivars of faba bean cultivars (Lobab and Misr 3) during the three first time points (35, 42, and 49 DAP) (Fig. 9 A). However, beyond these dates, infested Lobab cultivar showed a noticeable decrease, with 0.600 recorded by 91 DAP compared to 0.735 on the non-infested system. Responses of lentil parasitized by O. crenata were not observed on the Zaaria cultivar, except for day 70 where effective PSII quantum yield of the non-infested system was higher than infested system (Fig. 9 B). For the Bakria
Fig. 7. Relationship between relative weight and O. crenata infection on faba bean cultivars (A) Lobab and (B) Misr 3. ▴ Root relative weight; ■ O. crenata relative weight; and ◆ Host reproductive relative weight. Relative weights were calculated by dividing the given sink (host root, parasite, and host seed) by the total combined biomass. Data are means of four replicates for each treatment with SE indicated by vertical lines. 6
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Fig. 8. Relationship between relative weight and O. crenata infection on lentil cultivars (A) Zaaria and (B) Bakria. ▴ Root relative weight; ■ O. crenata relative weight; and ◆ Host reproductive relative weight. Relative weights were calculated by dividing the given sink (host root, parasite, and host seed) by the total combined biomass. Data are means of four replicates for each treatment replicates with SE indicated by vertical lines.
cultivar, no significant difference was noted between treatments except at 84 DAP, where Effective PSII quantum yield was higher for Bakria with O. crenata than Bakria without O. crenata.
points (35, 42, and 49 DAP) (Fig. 9 A and B). Our findings are in agreement with (Vrbnicanin et al., 2013), who studied the effect of Cuscuta campestris on morphological and chlorophyll fluorescence pa rameters on infested and non-infested Ambrosia trifida plants, reported that this chlorophyll fluorescence parameter (Effective PSII quantum yield (ϕPSII)) presented identical values for both infested and control plants during the first days of parasite attack. A more likely explanation for this biomass reduction could be attributed to growth-defense tradeoffs. In fact, in the presence of a parasite, host plants are con strained to assign a part of their resources, which were initially directed to growth, toward defense (Huot et al., 2014). Decreased growth with concomitant increases in defenses has been widely documented (Heidel et al., 2004; Zavala et al., 2004; Kempel et al., 2011; Meldau et al., 2012) and hormone crosstalk within the host was proposed as a major player in regulating this growth–defense balance (Huot et al., 2014). Metabolic costs involved in plant defense are considerable, as each defensive compound requires precursor molecules and energy (Gershenzon, 1994; Züst and Agrawal, 2017). Our results showed that the negative impact on growth (combined host biomass) was noticeable until 70 DAP on susceptible cultivar; while for the resistant Misr 3 cultivar this reduction was observed until 56 DAP. However, for this resistant cultivar and during 84 DAP; an unexpected noticeable decrease on host biomass was recorded (Fig. 3 B). This reduction in host growth may suggest the activation of defense mechanisms maintaining O. crenata infestation level at 5.4 NOIE per plant. By the end of the experiments, combined biomass (dry weight of host plant and parasite) of faba bean and lentil cultivars were similar to dry weight of host plant on non-infested systems (Figs. 3 A and Fig. 4 A). The difference in host biomass between infested and non-infested systems was accounted directly by O. crenata tissues. This indicates that the system has a maximum biomass and the difference in dry weight
4. Discussion Biomass production, partitioning, and sink strength of host plants infested by parasitic weeds have been usually studied under open-field conditions (Rubiales, 2010; McCormick et al., 2006; Fern� andez-Apar icio et al., 2016). However, due to technical constraints, host root sys tems are rarely considered in these field trials. For that purpose, we used controlled conditions (pots) to allow consideration of host roots during the study of Orobanche-host plant interactions. Furthermore, the pot assay allows the control of climatic variables, inoculum origin and density, and allows better monitoring of both emerged and underground Orobanche development (Rubiales et al., 2005a; Ennami et al., 2017). At the first time point (42 DAP), when no O. crenata considerable attachment events were observed, the presence of O. crenata seeds, at germination and fixation stages, reduced host biomass of faba bean cultivars Lobab and Misr 3 to 53.84 and 27.02% respectively compared to non-infested plants (Fig. 3). Similar host biomass reductions were observed, during beginning stages of infestation, in sorghum sensitive and tolerant cultivars infested by Striga hermonthica (45 and 25%) (Van Ast et al., 2000). This reduction could be due to a phytotoxic or path ological effect of this parasitic weed on host plant immediately after parasite infection of the host. Huot et al. (2014) suggested that negative impact on plant growth induced by pathogen could be attributed to growth–defense tradeoffs and/or a result of reduced photosynthesis. The latter hypothesis does not seem plausible in our case because no sig nificant differences were recorded on effective PSII quantum yield be tween infested and non-infested cultivars during the first three time
Fig. 9. Average chlorophyll fluorescence (Effective PSII quantum yield Y (II)) measured weekly (from 35 to 98 DAP) for (A): susceptible and resistant faba bean cultivars either infested or non-infested by O. crenata. ◆Lobab with O. crenata; ■ Lobab without O. crenata; ▴ Misr 3 with O. crenata; and � Misr 3 without O. crenata. (B): susceptible and resistant lentil cultivars either infested and non-infested. ◆ Zaaria with O. crenata; ■ Zaaria without O. crenata; ▴ Bakria with O. crenata; and 〇 Bakria without O. crenata. 7
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are a strong indication that breeding practices based on increasing yield of resistant cultivars should consider minimizing biomass losses at initial host development stages. Furthermore, most available researches have been performed in field experiments, neglected therefore characterizing of both emerged and underground parasites and also root relative weight, which reflect knowledge insufficiency of Orobanche-legumes interaction. For that, pots assays of this research contributed to identify and address these knowledge gaps. In addition, elucidating defense mechanisms of faba bean and lentil through physiological and enzy matic studies (key enzymes involved in such mechanisms “PAL, POX, and SOD) will clarify this growth–defense balance.
allocation can be attributed to a source-sink interactions. Comparable trends have been previously observed during O. cernua–tobacco, O. ramosa-faba bean, and O. aegyptiaca-tomato associations, and similar justifications were proposed (Hibberd et al., 1998, 1999). In contrast, significant differences have been found between host and parasite combined biomass and the biomass of non-infested system for O. crenata-faba bean, Striga hermonthica-sorgum, O. ramosa-tomato, O. minor-white clover, and Cuscuta-cowpea interactions (Barker et al., 1996; Hibberd et al., 1996; Gurney et al., 1999; Mauromicale et al., �ndez-Aparicio et al., 2016). According to Gurney et al. 2008; Ferna (1999), alteration of host metabolism by introduction of novel com pounds active at low concentrations or by influence on host growth regulators were suggested as mechanisms of host biomass reduction. Information on effects of O. crenata on biomass partitioning across the various host plant organs is still lacking. Other parasitic weeds have been shown to have a marked effect on biomass partitioning over the various plant compartments. Van Ast et al. (2000) indicated that biomass reduction on sorghum plants was accompanied by the increase of their root system after infection by Striga hermonthica. Another study showed that Cuscuta campestris, an obligate stem parasite, tends to in crease resource allocated to the aboveground parts (with more leaves than stems) of Mikania micrantha, and decrease the host root system (Shen et al., 2005). This is supported by our results in which O. crenata may have shifted susceptible cultivar resources through: i) increase of host root dry mater; ii) maintaining host vegetative parts; and iii) delay and reduction of host reproduction (Fig. 5). Across its influence, O. crenata seems to reinforce host root system, which is its unique point of attachment. Also, in order to maximize uptake of photosynthates from sensitive plants, this holoparasite tends to sustain host aboveground parts and redirect a part of host productivity to its own advantage. However, O. crenata fails to exercise these effects on faba bean and lentil resistant cultivars. We aimed to compare the strength of parasite and host sinks, so used relative distribution of dry weight based on ratio of sink dry matter to �ndez-Aparicio et al. (2016). combined biomass as suggested by Ferna Few studies have focused on the relative distribution of parasite and hosts dry weight compartments, and these have been limited to tradeoffs between reproductive and parasitic sinks only. Due to use of field trials, the relative weight of roots has been neglected. Our results show that during the two first time points (42 and 56 DAP) a dominance of root relative weight was observed in both susceptible and resistant faba bean and lentil cultivars (Fig. 7; Fig. 8). After these dates, parasitism didn’t influence root relative weight of the resistant cultivars, but the suscep tible cultivars showed a marked decrease in root relative weight that was reflected in an increase in relative parasitic weight. Similarly, Fern� an dez-Aparicio et al. (2016) using infection severity gradient on faba bean sensitive cultivar, reported that relative parasitic weight increased remarkably even at low infection severity. Delavault (2015) reported also that Orobanche sink strength derives from reduction of its osmotic potential, due to high accumulation levels of osmotically active com pounds such as cations, sugars, amino acids, and polyols.
Acknowledgements This research was supported by National Institute of Agricultural Research of Morocco and Ministry of Higher Education, Scientific Research and Professional Training of Morocco (MESRSFC) through funding of MEDILEG project within the European Union 7th Framework program for research, technological development and demonstration (ERA-Net Project, ARIMNet). And National Institute of Food and Agri culture grant no. 135997 to J.H.W. is acknowledged. References Abu Irmailah, B., Haddad, N., 2011. Indication of tolerance to broomrapes (orobanche crenata forsk.) in wild lentil collected from Jordan. Dirasat: Shari’a and Law Sciences 37. Attia, S.M.M.M., El-Hady, H.A., Saber, M.A., Omer, S.A., Khalil, M.S.A., A.A.M., Ashrei, R., A.M., Abd-Elrahman, M.A.M., Ibrahim, Z.,E., Ghareeb, T.S., ElMarsafawy, E.H., El-Harty, E.A.A., El-Emam, F.H., Shalaby, A.G., Helal, A.M., ElGarhy, E.M., Rabie, M., Abdeen, M., El-Noby, K.M.M., Yamani, A.E.-A.H.T., 2013. Misr 3, a new orobanche tolerant faba bean variety Egyptian. J. Plant Breed. 17, 143–152. Barker, E., Press, M., Scholes, J., Quick, W., 1996. Interactions between the parasitic angiosperm Orobanche aegyptiaca and its tomato host: growth and biomass allocation. New Phytol. 133, 637–642. Bayoumi, T., Ammar, S., El-Bramawy, M., MA, E., 2014. Effect of Some Broomrape Control Methods on Growth and Seed Yield Attributes of Faba Bean (Vicia Faba L.) Cultivars Agricultural Research Journal. Suez Canal University, 1. Briache, F.Z., Ennami, M., Mbasani-Mansi, J., Gaboun, F., Abdelwahd, R., Fatemi, Z.E.A., El-Rodeny, W., Amri, M., Triqui, Z.E.A., Mentag, R., 2019. Field and controlled conditions screenings of some faba bean (Vicia faba L.) genotypes for resistance to the parasitic plant Orobanche crenata Forsk. and investigation of involved resistance mechanisms. J. Plant Dis. Prot. 1–14. Bo�zi�c, D., Vrbni�canin, S., Elezovi�c, I., Sari�c, M., 2010. Chlorophyll Fluorescence in Vivo as the Indicator of Sunflower Susceptibility to Als-Inhibiting Herbicides, 15th EWRS Symposium. Cubero, J., Pieterse, A., Khalil, S., Sauerborn, J., 1993. Screening techniques and sources of resistance to parasitic angiosperms. Euphytica 73, 51–58. Delavault, P., 2015. Knowing the parasite: biology and genetics of orobanche. Helia 38, 15–29. Duraes, F., Gama, E., Magalhaes, P., Marriel, I., Casela, C., Oliveira, A., Luchiari Jr., A., Shanahan, J., 2001. The Usefulness of Chlorophyll Fluorescence in Screening for Disease Resistance, Water Stress Tolerance, Aluminium Toxicity Tolerance, and N Use Efficiency in Maize, Seventh Eastern and Southern Africa Regional Maize Conference 11th, pp. 356–360. Ennami, M., Briache, F.Z., Gaboun, F., Abdelwahd, R., Ghaouti, L., Belqadi, L., Westwood, J., Mentag, R., 2017. Host differentiation and variability of Orobanche crenata populations from legume species in Morocco as revealed by cross-infestation and molecular analysis. Pest Manag. Sci. https://doi.org/10.1002/ps.4536. FAO, 2014. The problem of orobanche spp in Africa and Near East. Food and agriculture organization of the united nations. http://www.fao.org/agriculture/crops/thematic -sitemap/theme/biodiversity/weeds/issues/oro/en/. Fern� andez-Aparicio, M., Flores, F., Rubiales, D., 2016. The effect of Orobanche crenata infection severity in faba bean, field pea, and grass pea productivity. Front. Plant Sci. 7. Genty, B., Briantais, J.-M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta Gen. Subj. 990, 87–92. Gershenzon, J., 1994. The Cost of Plant Chemical Defense against Herbivory: A Biochemeical Perspective, Insect-Plant Interactions. CRC Press, pp. 105–173. Ghanem, M.E., Albacete, A., Martínez-Andújar, C., Acosta, M., Romero-Aranda, R., Dodd, I.C., Lutts, S., P�erez-Alfocea, F., 2008. Hormonal changes during salinityinduced leaf senescence in tomato (Solanum lycopersicum L.). J. Exp. Bot. 59, 3039–3050. Goldwasser, Y., Plakhine, D., Kleifeld, Y., Zamski, E., Rubin, B., 2000. The differential susceptibility of vetch (vicia spp.) to orobanche aegyptiaca: anatomical studies. Ann. Bot. 85, 257–262.
5. Conclusion The prevalence of O. crenata on legume fields in the Mediterranean region is a serious problem. Unfortunately, despite of this high impact of O. crenata on legumes in Morocco, the topic of competitive relationship between this parasitic weeds and its legume plant crops remains poorly understood. In this study we investigated how Orobanche affects the growth of infested faba bean and lentil cultivars. As this way of research are important in order to provide intervention strategies on breeding programs. Our results show that parasite infections severely reduce biomass of hosts at the first stages of infection on both resistant and susceptible faba bean cultivars. It is likely that plant growth–defense tradeoffs have a direct negative impact in host resource allocation. Observed biomass differences between infested and non-infested system 8
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Crop Protection 127 (2020) 104924 Rubiales, Diego , A., P�erez-de-Luque, M.o., Fernandez-Aparico, J.C.S., Roman, Bel� en, Kharrat, Mohamed, Khalil, Shaban, Joel, Daniel M., Riches, Charlie, 2005. Screening Techniques and Sources of Resistance against Parasitic Weeds in Grain Legumes. Springer. Rubiales, D., Moreno, M., Sillero, J., 2005. Search for resistance to crenate broomrape (Orobanche crenata Forsk.) in pea germplasm. Genet. Resour. Crop Evol. 52, 853–861. Rubiales, A.,D.M., Castillejo, M., Fernandez-Aparicio, E., Prats, J., C Sillero, A.P.-d.-L., Rispail, Nicolas, Fondevilla, Sara, 2009. Breeding approaches for crenate broomrape (Orobanche crenata Forsk.) management in pea. Pest Manag. Sci. 65, 553–559. Rubiales, M.F.-A.D., 2010. Characterisation of resistance to crenate broomrape (Orobanche crenata Forsk.) in Lathyrus cicera L. Euphytica 173, 77–84. Sauerborn, J., 1991. Parasitic Flowering Plants: Ecology and Management. Verlag Josef Margraf. Shen, H., Ye, W., Hong, L., Cao, H., Wang, Z., 2005. Influence of the obligate parasite Cuscuta campestris on growth and biomass allocation of its host Mikania micrantha. J. Exp. Bot. 56, 1277–1284. Strasser, R., Tsimilli-Michael, M., 2001. Stress in plants, from daily rhythm to global changes, detected and quantified by the JIP-test. Chem. Nouv. 75, 3321–3326. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. Probing photosynthesis: mechanisms, regulation and adaptation 445–483. Torres, A.M., Avila, C.M., Satovic, Z., Rubiales, D., Sillero, J.C., Cubero, J.I., Rom� an, B., Moreno, M.T., 2006. Faba bean breeding for resistance against biotic stresses: towards application of marker technology. Euphytica 147, 67–80. Trabelsi, I., Abbes, Z., Amri, M., Kharrat, M., 2015. Performance of faba bean genotypes with Orobanche foetida Poir. and Orobanche crenata Forsk. infestation in Tunisia. Chil. J. Agric. Res. 75, 27–34. Trabelsi, I., Abbes, Z., Amri, M., Kharrat, M., 2016. Study of some resistance mechanisms to Orobanche spp. infestation in faba bean (Vicia faba L.) breeding lines in Tunisia. Plant Prod. Sci. 19, 562–573. Van Ast, A., Bastiaans, L., Kropff, M., 2000. A comparative study on Striga hermonthica interaction with a sensitive and a tolerant sorghum cultivar. Weed Res. 40, 479–493. Vrbnicanin, S., Saric-Krsmanovic, M., Bozic, D., 2013. The Effect of Field Dodder (Cuscuta Campestris Yunck.) on Morphological and Fluorescence Parameters of Giant Ragweed. Ambrosia trifida L. https://doi.org/10.2298/PIF1301057V Walz, H., 2009. Imaging-Pam M-Series Chlorophyll Fluorescence System. Instrument Description and Information for Users. Heinz Walz GmbH, p. 207. Westwood, J.H., 2000. Characterization of the Orobanche-Arabidopsis system for studying parasite-host interactions. Weed Sci. 48, 742–748. Yamada, M., Hidaka, T., Fukamachi, H., 1996. Heat tolerance in leaves of tropical fruit crops as measured by chlorophyll fluorescence. Sci. Hortic. 67, 39–48. Zavala, J.A., Patankar, A.G., Gase, K., Baldwin, I.T., 2004. Constitutive and inducible trypsin proteinase inhibitor production incurs large fitness costs in Nicotiana attenuata. Proc. Natl. Acad. Sci. U.S.A. 101, 1607–1612. Zhou, W., Yoneyama, K., Takeuchi, Y., Iso, S., Rungmekarat, S., Chae, S., Sato, D., Joel, D., 2004. In vitro infection of host roots by differentiated calli of the parasitic plant Orobanche. J. Exp. Bot. 55, 899–907. Züst, T., Agrawal, A.A., 2017. Trade-offs between plant growth and defense against insect herbivory: an emerging mechanistic synthesis. Annu. Rev. Plant Biol. 68, 513–534.
Grenz, J.H., Manschadi, A.M., Uygur, F.N., Sauerborn, J., 2005. Effects of environment and sowing date on the competition between faba bean (Vicia faba) and the parasitic weed Orobanche crenata. Field Crop. Res. 93, 300–313. Gurney, A., Press, M., Scholes, J., 1999. Infection time and density influence the response of sorghum to the parasitic angiosperm Striga hermonthica. New Phytol. 143, 573–580. Heidel, A.J., Clarke, J.D., Antonovics, J., Dong, X., 2004. Fitness costs of mutations affecting the systemic acquired resistance pathway in Arabidopsis thaliana. Genetics 168, 2197–2206. Hibberd, J., Quick, W., Press, M., Scholes, J., 1996. The influence of the parasitic angiosperm Striga gesnerioides on the growth and photosynthesis of its host, Vigna unguiculata. J. Exp. Bot. 47, 507–512. Hibberd, J., Quick, W., Press, M., Scholes, J., 1998. Can source–sink relations explain responses of tobacco to infection by the root holoparasitic angiosperm Orobanche cernua? Plant Cell Environ. 21, 333–340. Hibberd, J., Quick, W., Press, M., Scholes, J., Jeschke, W., 1999. Solute fluxes from tobacco to the parasitic angiosperm Orobanche cernua and the influence of infection on host carbon and nitrogen relations. Plant Cell Environ. 22, 937–947. Huot, B., Yao, J., Montgomery, B.L., He, S.Y., 2014. Growth–defense tradeoffs in plants: a balancing act to optimize fitness. Mol. Plant 7, 1267–1287. Joel, D.M., Chaudhuri, S.K., Plakhine, D., Ziadna, H., Steffens, J.C., 2011. Dehydrocostus lactone is exuded from sunflower roots and stimulates germination of the root parasite Orobanche cumana. Phytochemistry 72, 624–634. Kannan, C., Zwanenburg, B., 2014. A novel concept for the control of parasitic weeds by decomposing germination stimulants prior to action. Crop Protect. 61, 11–15. Kempel, A., Sch€ adler, M., Chrobock, T., Fischer, M., van Kleunen, M., 2011. Tradeoffs associated with constitutive and induced plant resistance against herbivory. Proc. Natl. Acad. Sci. 108, 5685–5689. Labrada, R., 2007. Progress on Farmers Training on Parasitic Weed Management Food and Agriculture Organization of the United Nations Rome. Linke, K., Saxena, M., 1991. Study on viability and longevity of Orobanche seed under laboratory conditions. Progress in Orobanche Research 110–114. Mauromicale, G., Monaco, A.L., Longo, A.M., 2008. Effect of branched broomrape (Orobanche ramosa) infection on the growth and photosynthesis of tomato. Weed Sci. 56, 574–581. Mbasani-Mansi, J., Briache, F.Z., Ennami, M., Gaboun, F., Benbrahim, N., Triqui, Z.E.A., Mentag, R., 2019. Resistance of Moroccan lentil genotypes to Orobanche crenata infestation. J. Crop Improv. 1–21. McCormick, A., Cramer, M., Watt, D., 2006. Sink strength regulates photosynthesis in sugarcane. New Phytol. 171, 759–770. Meldau, S., Ullman-Zeunert, L., Govind, G., Bartram, S., Baldwin, I.T., 2012. MAPKdependent JA and SA signalling in Nicotiana attenuata affects plant growth and fitness during competition with conspecifics. BMC Plant Biol. 12, 213. Moradi, F., Ismail, A.M., 2007. Responses of photosynthesis, chlorophyll fluorescence and ROS-scavenging systems to salt stress during seedling and reproductive stages in rice. Ann. Bot. 99, 1161–1173. Parker, C., 2009. Observations on the current status of Orobanche and Striga problems worldwide. Pest Manag. Sci. 65, 453–459. Pavlovi�c, D., Vrbni�canin, S., Bo�zi�c, D., Simon�ci�c, A., 2007. Abutilon theophrasti medic. Population responses to atrazine. J. Cent. Eur. Agric. 8, 435–442. Rodríguez Conde, M., Moreno, M., Cubero, J., Rubiales, D., 2004. Characterization of the Orobanche–Medicago truncatula association for studying early stages of the parasite–host interaction. Weed Res. 44, 218–223.
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Growth-defense tradeoffs and source-sink relationship during both faba bean and lentil interactions with Orobanche crenata Forsk Mounia Ennami a, b, 1, Joseph Mbasani-mansi a, Fatima Zahra Briache a, Nada Oussible b, Fatima Gaboun a, Lamiae Ghaouti b, Loubna Belqadi b, Michel Edmond Ghanem c, Kamal Aberkani d, James Westwood e, Rachid Mentag a, * a
National Institute of Agricultural Research of Morocco (INRA), CRRA-Rabat, Biotechnology Unit, Rabat, Morocco Agronomic and Veterinary Institute Hassan II (IAV), Plant Biotechnology Department, Rabat, Morocco Crop Physiology Laboratory, International Center for Agricultural Research in the Dry Areas (ICARDA), Rabat, Morocco d University Mohammed First, Faculty of Science and Technology (FST), Al, Hoceima, Morocco e Virginia Tech, Department of Plant Pathology, Physiology and Weed Science, Blacksburg, VA, USA b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Vicia faba L. Lens culinaris medik. Orobanche crenata Susceptible and resistant cultivars Biomass partitioning Source-sink relationship
Orobanche crenata Forssk., a root holoparasitic weed, represents a major biotic threat of legumes in the Medi terranean region. O. crenata could reduce the yield up to 90%. In this study, the effect of O. crenata on biomass production and partitioning of some faba bean and lentil cultivars were investigated through pot assays. During the first stage of infection, reductions in host biomass were observed on susceptible and resistant faba bean cultivars (53.84 and 27.02%, respectively for Lobab and Misr 3) and on susceptible lentil cultivar (Zaaria). However, during the last stage (host plant maturity), combined biomasses of susceptible faba bean (35.69 g) and lentil (8 g) cultivars were similar to those of non-infested plants (32.45 and 6.62 g, respectively). Considering biomass partitioning over the various host parts, O. crenata parasitism on susceptible faba bean and lentil cul tivars greatly increased host root dry mater, but delayed and reduced host reproduction. The relative weight of parasite and host organs were also studied. Thus, a marked decrease of root relative weight was observed during the last developmental stages (up to 63%) accompanied simultaneously with the increase of relative parasitic weight (35.87%). Results suggested that legumes-O. crenata interactions were governed by growth-defense tradeoffs during the early stage of infection, whereas source-sink relationships explained the dry weight diver sion from host to parasite during the last phases of the interaction.
1. Introduction Orobanche crenata Forssk. is a chlorophyll-lacking root parasite. This parasitic weed is considered to be the most widespread parasitic plant species in the Mediterranean region, especially in North Africa and the Near East and Western Asia (Parker, 2009; Joel et al., 2011). O. crenata parasitizes several host species from the family Fabaceae, within which the main host crops are faba bean (Vicia faba L.), chickpea (Cicer arie tinum L.), pea (Pisum sativum L.), lentil (Lens culinaris Medik.), vetches (Vicia benghalensis L.), and many forage legumes (Goldwasser et al., 2000; Rubiales et al., 2009; Abu Irmailah and Haddad, 2011). In Morocco, O. crenata infestation was officially reported on faba bean in 1943 in the Fez region. Since then, the parasite has spread to
other areas (Zair, Pre-Rif, Chaouia, Sais, Fez, Meknes, Taounate, Sidi Kacem, Chefchaouen, Doukala, and Abda) (Labrada, 2007; FAO, 2014). The estimated average yield losses due to parasitism range from seven to 90%, depending on host plants, environmental factors, and soil infes tation level (Sauerborn, 1991; Torres et al., 2006; Ennami et al., 2017). In fact, a single O. crenata spike produces thousands of tiny seeds that can remain viable in the soil for more than 10 years and germinate in response to chemical signals exuded from host roots (Linke and Saxena, 1991; Zhou et al., 2004). Successful attachment to the host root requires development of the haustorium, a specialized structure that ensures connection between the host vascular tissue and the parasite. After attachment the parasite obtains from its host all water and mineral nutrients needed for growth into a tubercle, a bulbous structure from
* Corresponding author. National Institute of Agricultural Research (INRA), CRRA, Biotechnology Unit, Rabat, Morocco. E-mail address: [email protected] (R. Mentag). 1 Current address: National Institute of Agricultural Research (INRA), CRRA, Biotechnology Unit, Rabat, Morocco. https://doi.org/10.1016/j.cropro.2019.104924 Received 19 February 2018; Received in revised form 21 June 2019; Accepted 17 August 2019 Available online 18 August 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.
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which a shoot arises and emerges from the soil to flower and set seeds (Westwood, 2000; Rodríguez-Conde et al., 2004). Various control strategies; cultural, chemical, breeding, etc. have been employed to control Orobanche (Bayoumi et al., 2014; Kannan and Zwanenburg, 2014; Trabelsi et al., 2016). Unfortunately, none of them have proven to be both effective and economically feasible due to technical challenges, economic constraints, and the complexity of host-parasite interactions (Grenz et al., 2005; Rubiales et al., 2009). The development of effective Orobanche control strategies requires a better understanding of the host plant-Orobanche interaction acting at morphological and physiological levels in contrasting cultivars (sus ceptible and resistant). However, most research on the general features of host infection by Orobanche species has been conducted using sus ceptible host cultivars under field trials such as the interactions between �ndez-Aparicio et al., 2016; Trabelsi et al., faba bean and O. crenata (Ferna 2015, 2016; Ennami et al., 2017) and O. ramosa with tomato (Maur omicale et al., 2008). Analyses of these interactions showed highly significant differences among host cultivars in response to these para sitic weeds, especially in considering parameters of biomass production and grain yield. Leaf chlorophyll fluorescence can be used as a physiological indi cator of plant response to stress (Strasser et al., 2000; Strasser and Tsimilli-Michael, 2001). Many researchers used such photosynthetic parameters to evaluate plant responses under various stress conditions such as water deficit (Duraes et al., 2001), temperature (Yamada et al., 1996), salinity or nitrogen deficit (Duraes et al., 2001; Moradi and Ismail, 2007), and herbicide effects (Pavlovi�c et al., 2007; Bo�zi�c et al., 2010), etc. Some studies have assessed the effects of parasitic weeds on photosynthesis of host plants, including Cowpea (Vigna unguiculata L. Walp.)/Striga gesnerioides Willd.; Ambrosia trifida L./Cuscuta campestris Yunck.; and Solanum lycopersicum L./O. ramosa L. interactions (Hibberd et al., 1996; Mauromicale et al., 2008; Vrbnicanin et al., 2013). These authors concluded that parasitic weeds act as a competitor sink for host assimilates, causing damage principally via its negatives effect on the host photosynthetic machinery (photosystems I and photosystems II). Despite the high impact of O. crenata on legume species in Morocco, the host physiological changes in response to O. crenata have not been characterized. The main objective of this study was to investigate the effects of O. crenata on the agro-morphological and chlorophyll fluo rescence parameters of susceptible and resistant cultivars of faba bean and lentil under controlled conditions. These parameters were moni tored over the host growth cycle and also considered in light of the developmental progression of the parasite through different phenolog ical stages.
were developed and characterized at the Regional Center of Agriculture Research of Rabat, National Institute of Agriculture Research (INRA-Morocco). Orobanche crenata seeds were collected in 2014 from mature O. crenata plants growing in legume fields at the Marchouch experi mental station of National Institute of Agriculture Research (INRAMorocco) (Table 1). Before use, all seeds were stored dry in the dark at room temperature. 2.2. Experimental design Effects of O. crenata on agro-morphological and chlorophyll fluo rescence of faba bean and lentil plants were monitored in 6 L plastic pots following the modified pot testing method developed by Rubiales et al. (2005a). Pots were filled with soil–peat (3:1, v:v). For each legume species, the experiment was designed as a randomized split-plot with four replicates. Indeed, two separate experiments were set up, one for faba bean cultivars and the other one for lentil cultivars. The trials were conducted in greenhouse under natural day-light conditions at the Regional Center for Agricultural Research-INRA Rabat, Morocco (33� 580 1700 N; 6� 500 5900 W in north-western Morocco). Non-infested soil (autoclaved soil at 120 � C for 45 min) and artificially infested soil by O. crenata (autoclaved soil supplemented by 30 mg of O. crenata seeds per 1 kg of substrate) were considered as main plots. The subplots consisted on susceptible and resistant cultivars of faba bean or lentil. This led to 40 pots different “host cultivars/Orobanche treatment” as sociations. Five lentil seeds or one faba bean seed were sown in each pot. Indeed, seeds of each legume species were manually sown to a depth of 4 cm. 2.3. Kinetics of measurement of the different parameters Pots were sown on January 26th 2016. Kinetics of O. crenata development on both legume species were conducted through pot uprooting and root systems were gently washed under running tap water (Fern� andez-Aparicio et al., 2016). Uprooting of hosts (Five lentil plants or one faba bean plant) was performed 42 days after planting (DAP); means two weeks before O. crenata attack (Bud set “First individual flower buds visible outside of leaves but still closed”); at 56 DAP (Flowering “between Full flowering and end of flowering (first pods visible)”); at 70 DAP (Plant expansion “10% of pods have reached final length”); at 84 DAP (Plant expansion “more than 50% of pods have reached final length”), and at 98 DAP (Faba bean maturity). 2.4. Agro-morphological and O. crenata infestation measurements
2. Materials and methods
Ten agro-morphological parameters were measured for host plants: plant height; number of vegetative tillers; fresh and dry weight of vegetative tillers (g); seed number and dry weight (g) per plant; number of root-nodules (used as proxy to measure the impact of the parasite on the nodule formation on a 0 to 5 scale); fresh and dry weight of root (g); and root volume (Cubero et al., 1993; Rubiales et al., 2005b). Two in dexes of O. crenata Infestation Events (OIE) were measured: number (NOIE) and dry weight of emerged and underground (DwOIE) (Ennami et al., 2017; Briache et al., 2019; Mbasani-Mansi et al., 2019). Regarding, hosts and parasitic parts, dry weight was scored after oven
2.1. Plant material Two faba bean cultivars were used; cv. Misr 3 that was selected in Egypt and recently in Morocco for its resistance to O. crenata (Attia et al., 2013; Briache et al., 2019) and cv. Lobab, registered in the Moroccan catalogue since 1985, was recently confirmed as susceptible to O. crenata (Briache et al., 2019). For lentil, two cultivars, registered in Moroccan catalog were used. Zaaria (O. crenata-susceptible) and Bakria (O. crenata-moderately resistant) (Mbasani-Mansi et al., 2019). Lentil Table 1 Coordinates, Climate, and soil characteristics of experimental locations. Location Marchouch
a b
Coordinates Longitude Latitude Altitude
a
6.72 33.62a 398b
Soil type (FAO)
Soil texture
pH
Rainfall (mm)
Temperature (� C)
Luvisols (Chromic Luvisols)
silty clay
6
407
23.4 (minimum of 1 and a maximum of 45)
decimal degrees. Meters. 2
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drying at 80 � C for 48 h and assessed during the uprooting step at 42, 56, 70, 84, and 98 DAP. Infestation intensity was estimated by counting Orobanche stages developed on host root system, while phenological stages of OIEs were recorded using a modified scale (Briache et al., 2019) from 1 to 6 (S1: attachments on root; S2: tubercles; S3: small buds; S4: large buds; S5: underground stems; and S6: emerged spikes). �nde Additional parameters were calculated as suggested by Ferna z-Aparicio et al. (2016) to determine the biomass partitioning within the host-O. crenata relationship and also to evaluate the effect of O. crenata infestation on each legume species. These parameters were: total host biomass (vegetative tillers, seeds, and roots); host reproductive biomass (seed dry weight); O. crenata dry weight (emerged and underground OIEs); and combined biomass (vegetative tillers, seeds, and root dry weights of legume species and dry weight of emerged and underground OIEs). In addition, the relative weight of the various host parts was calculated as: root relative weight (proportions of root dry weight/ combined biomass); relative host reproductive weight (proportion of host seed dry weight/combined biomass) and relative O. crenata weight (proportion of O. crenata dry weight/combined biomass). All these pa rameters were calculated at the five uprooting stages (42, 56, 70, 84, and 98 DAP).
respectively. Considering the number of O. crenata infestation events, the resistant and susceptible cultivars differed significantly (P � 0.05), especially during the late stages of infection (70, 84, and 98 DAP). For both legume species, no Orobanche infestation event was recorded at the first time point (42 DAP). At the last time point (98 DAP), the NOIE on Misr 3 and Bakria were 8.5 and 5.75, respectively, against 14 and 15.5 recorded for the susceptible cultivars Lobab and Zaaria (Fig. 1). For both faba beans, the first O. crenata attachments were recorded at 56 DAP (Figs. 1 A, Fig. 2 A and F). This parasite developed rapidly on the sus ceptible host cultivar (Lobab) and O. crenata phenological stage 6 (emergent spikes) was reached 70 DAP (Fig. 1 A; Fig. 2 G), while it was recorded 98 DAP for the cultivar Misr 3 (Fig. 1 A; Fig. 2 D). For lentils, O. crenata developed rapidly on Zaaria compared to the Bakria cultivar. The first O. crenata attachment was reported at 56 DAP (Fig. 1 B). On the susceptible cultivar (Zaaria), O. crenata reached the final stage of development (Emergent spikes) at 70 DAP (Fig. 1 B). This stage was achieved only at 98 DAP on the more resistant Bakria cultivar (Fig. 1 B). 3.2. Effect of O. crenata on faba bean and lentil agro-morphological parameters The biomass of parasite-free host plants was higher than that of infested plants. The presence of O. crenata reduced the biomass of both faba bean cultivars, but this reduction was more important on Lobab than Misr 3 (Fig. 3). Compared to non-infested plants on susceptible cultivar, reduction rates induced by O. crenata were significant, up to 53.8, 31.7, 36.9, 62.1, and 59.3%, respectively, for 42, 56, 70, 84, and 98 DAP (Fig. 3 A). In contrast, on resistant cultivar, the only significant reduction rates were noted at 42, 56, and 84 DAP, respectively, for 27.0, 34.0, and 53.4% (Fig. 3 B). Regarding faba bean cultivars, additional differences were found between the dry weights of the non-infested system (Fig. 3 A”� Dry weight of non-infested system”) and combined biomass (Fig. 3 A”◆ Dry weight of infested system (combined biomass with O. crenata)”). In fact, compared to total dry weight produced on non-infested system, O. crenata reduced significantly the combined biomass of the infested Lobab cultivar at 42, 56, and 70 DAP. However, these reductions decreased at 84 DAP, and by 98 DAP the combined biomass of infested plants was equal to the total dry weight of the noninfested system (Fig. 3 A). For the resistant Misr 3 cultivar, reductions were observed at 56 and 84 DAP (Fig. 3 B). Considering O. crenata biomass (Fig. 3 B “〇 O. crenata dry weight”), this parameter remained low on Misr 3 cultivar and never exceed 5.41 g at 98 DAP (Fig. 3 B). In contrast, the biomass of O. crenata growing on the susceptible cultivar (Fig. 3 A “▴ O. crenata dry weight”) surpassed host dry matter at 84 and 98 DAP. For lentils, the data indicate almost the same pattern as observed on faba beans. Thus, for both lentil cultivars, higher biomass values were noted for non-infested plants compared to the infested plants (Fig. 4). Compared to non-infested plants (Fig. 4 A “� Dry weight of non-infested system (host biomass without O. crenata)”), significant biomass re ductions of 30.46 and 47.37% were observed on Zaaria dry weight (Fig. 4 A ⃞ “ Host dry weight of infested system (combined biomass without O. crenata)”), respectively for 56 and 70 DAP. However, re ductions were not significant after these times. On Bakria cultivar, no significant host biomass differences were recorded in parasite-free plants and during parasitism (Fig. 4 B). Regarding O. crenata biomass, this parameter was greater when grown on Zaaria plants, reaching 2.88 g at 98 DAP. Whereas on Bakria resistant cultivar, O. crenata biomass never exceed 0.84 g at the final time point (98 DAP). Further more, using Orobanche, the resistance is not only the capacity of the host plant to limit the development of the parasite but also the capacity of that plant to produce under such Orobanche parasitism pressure. For the susceptible faba bean cultivar, a marked effect of infestation by O. crenata on biomass partitioning over the various plant parts was recorded (Fig. 5 B). For non-infested plants, the biomasses of roots were 19, 33, 24, 14, and 12% of the total plant weight for 42, 56, 70, 84, and
2.5. Chlorophyll fluorescence measurements Fully expanded leaves were illuminated with a saturated light pulse after 30 min of darkness (Ghanem et al., 2008) using an Imaging-PAM 91090 Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Ger many) (Walz, 2009). The purpose of the darkness before measurements is to enable plastoquinone QA of PS II to become oxidized and allow for maximal photochemical quenching. They were illuminated during a 3 s (x 1 flash) of bright LED lamp: peak emission 470 nm; standard excita tion intensity: 0.5 μmol quanta m-2 s-1 PAR, maximal actinic intensity: 3000 μmol m-2 s-1 PAR, maximal saturation pulse intensity: 6000 μmol quanta μmol m-2 s-1. Measurements of chlorophyll fluorescence pa rameters were made during sunny and clear sky conditions (noon to 14:00). Imaging-PAM software records the values of all pixels within a single area of interest and calculates the average. Effective quantum yield of open photosystem II (PSII) was calculated according to (Genty et al., 1989) using the formula: (Fm’-F)/Fm’. This parameter was measured on all faba bean and lentil cultivars and on non-infested and infested soil by O. crenata. Two specific leaves per plant were chosen to monitor senescence. A young expanding leaf, identified as leaf number 4 (from the bottom), and leaf number 5. Each leaf was measured at three areas of interest in the intercostal regions close to the main vein also to secondary veins. Indeed, each data point represents an average of 120 repetitions of leaf samples � standard deviation; means of 20 plants x (2 leaves/plants) x (3 points/leaf) were chosen. Fluores cence measurements were performed at 35, 42, 49, 56, 63, 70, 77, 84, 91, and 98 days after planting of faba bean cultivars and at 42, 56, 70, 84, and 98 days after planting in the case of lentil cultivars. 2.6. Statistical analysis The treatment effects were analyzed using analysis of variance (ANOVA) in the SAS statistical analysis program (version 9.1; SAS, North Carolina, USA). ANOVA were conducted to assess whether there was a significant variation for each studied variable. Treatment means were compared using least significant difference (LSD) test at alpha ¼ 0.05. 3. Results 3.1. O. crenata development on faba bean and lentil cultivars Monitoring parasite developmental progression on faba bean and lentil cultivars confirmed the resistance level of both Misr 3 and Bakria, 3
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Fig. 1. Development of O. crenata phenological stages on (A): faba bean cultivars (Lob: Lobab and Misr: Misr 3) and (B): lentil cultivars (Bak: Bakria and Zaa: Zaaria), grown in pots assay for 14 weeks and harvested to observe parasitism at regular intervals (42, 56, 70, 84, 98 days after planting). Phenological stages are: (S1): Attachments; (S2): Tubercles; (S3): Small buds; (S4): Large buds; (S5): Underground stems; and (S6): Emergent spikes. Data are means of 8 replicates for each cultivar at each time point.
Fig. 2. Infestation levels of the two faba bean cultivars tested on non-infested and parasitized soil by O. crenata in a pot experiment over 14 weeks. Observations were carried out on infested resistant cultivar Misr 3 at 56 (A), 70 (B), 84 (C), 98 (D) and infested susceptible cultivar Lobab at 56 (F), 70 (G), 84 (H), 98 (I). Non-infested Misr 3 and Lobab were recorded at 98 DAP (E and J respectively).
98 DAP, respectively (Fig. 5 A). A relative increase in host biomass was observed on infested system, with dry weights of 27, 36, 28, 34, and 17% respectively for 42, 56, 70, 84, and 98 DAP (Fig. 5 B). Furthermore, the parasite attack affected seed production of the Lobab cultivar, as seed production on infested hosts was limited two weeks compared to the non-infested system. No differences were observed between noninfested and infested plants for host vegetative tillers dry matter of the Lobab cultivar. Over the course of the experiment biomass allocated to vegetative shoot tissues decreased gradually from 80.77 to 33.90% for the non-infested system (Fig. 5 A) and from 77.22 to 41.70% for the infested system (Fig. 5 B). Regarding the resistant Misr 3 cultivar, no significant differences were detected in dry matter partitioning over the
various plant parts between non-infested (Fig. 5 C) and infested systems (Fig. 5 D). Although both lentil cultivars responded to O. crenata infestation, the Zaaria (susceptible cultivar) was more negatively affected than the resistant Bakria cultivar (Fig. 6). Infestation by O. crenata resulted in a clear increase of biomass allocated to roots of Zaaria cultivar. Root tissue accounted for 29, 41, 44, 33, and 13% of total biomass at 42, 56, 70, 84, and 98 DAP (Fig. 6 B). By comparison, in the non-infested system, biomass allocated to roots were 54, 34, 17, 3, and 3% for 42, 56, 70, 84, and 98 DAP, respectively (Fig. 6 A). For this host, seed production as a fraction of the total dry weight decreased significantly in parasitized plants compared to non-infested hosts (Fig. 6 A and B). Regarding host 4
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Fig. 3. Relationship between total dry weight of host plants and O. crenata infection on faba bean (A) Lobab and (B) Misr 3 cultivars. 〇 Dry weight of non-infested system (host biomass without O. crenata); ◆ Dry weight of infested system (combined biomass with O. crenata); □ Host dry weight of infested system (combined biomass without O. crenata); and ▴ O. crenata dry weight. Data are means of 8 replicates of each cultivar for each time point. Bars indicate standard error.
Fig. 4. Relationship between total dry weight of lentil cultivars and O. crenata infection on (A) Zaaria and (B) Bakria. 〇 Dry weight of non-infested system (host biomass without O. crenata); ◆ Dry weight of infested system (combined host and with O. crenata biomass); □ Host dry weight of infested system (combined biomass without O. crenata); and ▴ O. crenata dry weight. Data are means of 8 replicates of each cultivar for each time point. Bars indicate standard error.
Fig. 5. Percentage of total biomass allocated to seeds, vegetative tillers and roots of the Lobab variety of faba bean when plants are (A) non-infested and (B) infested by O. crenata. Same for the Misr 3 cultivar on (C) non-infested and (D) O. crenata infested system.
vegetative dry matter of Zaaria cultivar, a negative effect of infestation was observed, as biomass increased gradually from 45.55 to 79.31% in the non-infested system (Fig. 6 A), but was reduced to 58.77, 55.55, and
65.87% of total biomass at 56, 70, and 84 DAP, respectively (Fig. 6 B). The resistant Bakria cultivar showed no O. crenata effect on dry matter partitioning over the various plant parts between non-infested (Fig. 6 C) 5
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Fig. 6. Percentage of total biomass allocated to vegetative tillers, roots, and seeds of lentil Zaaria cultivar when (A) non-infested and (B) infested by O. crenata. Likewise, the Bakria cultivar when (C) non-infested and (D) infested by O. crenata.
and infested systems (Fig. 6 D). In addition to dry matter partitioning over the various plant parts within the two cultivars, we also measured relative distribution of dry weight from source to different sinks (plant organs and parasite) in the absence and presence of O. crenata. Thus, in the presence of O. crenata and during the first two time point (42 and 56 DAP), the host root represents the main sink in both cultivars (Fig. 7). However, after 70 DAP, the relative weight of the parasite on Misr 3 reached maximum of only 21.5% at 84 DAP whereas the parasite relative weight on Lobab increased dramatically and reached 60% at the same time point. Within non-infested system, host reproductive relative weight of Lobab increased gradually from 9.24, 28.17, and 53.89%, respectively, for 70, 84, and 98 DAP (Data note shown). However, in the presence of O. crenata, host seed relative weight was, respectively, 0, 3.97, and 14.90% (Fig. 7 A). Regarding Misr 3 cultivar, host reproduction relative weight increased gradually on both systems (non-infested and infested) reaching respectively, on infested system, 9.17, 20.84, and 40.63% at 70, 84, and 98 DAP. O. crenata parasitism of the lentil Zaaria cultivar significantly increased relative root weight at 56 DAP, while seed relative weight was
significantly decreased compared to non-infested plants (Fig. 8 A). However, on the Bakria cultivar, host root relative weight showed only a downward trend during over time and host seed relative weight reached 16% by 84 DAP (Fig. 8 B). Relative parasite weight on Zaaria increased rapidly from 30% at 42 DAP to 3% at 98 DAP (Fig. 8 A). However, on the Bakria cultivar, the increase rate of relative parasitic weight was much lower (Fig. 8 B). 3.3. Effect of O. crenata on leaf chlorophyll fluorescence of faba bean and lentil No significant differences were recorded on the Effective PSII quantum yield between infested and non-infested cultivars of faba bean cultivars (Lobab and Misr 3) during the three first time points (35, 42, and 49 DAP) (Fig. 9 A). However, beyond these dates, infested Lobab cultivar showed a noticeable decrease, with 0.600 recorded by 91 DAP compared to 0.735 on the non-infested system. Responses of lentil parasitized by O. crenata were not observed on the Zaaria cultivar, except for day 70 where effective PSII quantum yield of the non-infested system was higher than infested system (Fig. 9 B). For the Bakria
Fig. 7. Relationship between relative weight and O. crenata infection on faba bean cultivars (A) Lobab and (B) Misr 3. ▴ Root relative weight; ■ O. crenata relative weight; and ◆ Host reproductive relative weight. Relative weights were calculated by dividing the given sink (host root, parasite, and host seed) by the total combined biomass. Data are means of four replicates for each treatment with SE indicated by vertical lines. 6
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Fig. 8. Relationship between relative weight and O. crenata infection on lentil cultivars (A) Zaaria and (B) Bakria. ▴ Root relative weight; ■ O. crenata relative weight; and ◆ Host reproductive relative weight. Relative weights were calculated by dividing the given sink (host root, parasite, and host seed) by the total combined biomass. Data are means of four replicates for each treatment replicates with SE indicated by vertical lines.
cultivar, no significant difference was noted between treatments except at 84 DAP, where Effective PSII quantum yield was higher for Bakria with O. crenata than Bakria without O. crenata.
points (35, 42, and 49 DAP) (Fig. 9 A and B). Our findings are in agreement with (Vrbnicanin et al., 2013), who studied the effect of Cuscuta campestris on morphological and chlorophyll fluorescence pa rameters on infested and non-infested Ambrosia trifida plants, reported that this chlorophyll fluorescence parameter (Effective PSII quantum yield (ϕPSII)) presented identical values for both infested and control plants during the first days of parasite attack. A more likely explanation for this biomass reduction could be attributed to growth-defense tradeoffs. In fact, in the presence of a parasite, host plants are con strained to assign a part of their resources, which were initially directed to growth, toward defense (Huot et al., 2014). Decreased growth with concomitant increases in defenses has been widely documented (Heidel et al., 2004; Zavala et al., 2004; Kempel et al., 2011; Meldau et al., 2012) and hormone crosstalk within the host was proposed as a major player in regulating this growth–defense balance (Huot et al., 2014). Metabolic costs involved in plant defense are considerable, as each defensive compound requires precursor molecules and energy (Gershenzon, 1994; Züst and Agrawal, 2017). Our results showed that the negative impact on growth (combined host biomass) was noticeable until 70 DAP on susceptible cultivar; while for the resistant Misr 3 cultivar this reduction was observed until 56 DAP. However, for this resistant cultivar and during 84 DAP; an unexpected noticeable decrease on host biomass was recorded (Fig. 3 B). This reduction in host growth may suggest the activation of defense mechanisms maintaining O. crenata infestation level at 5.4 NOIE per plant. By the end of the experiments, combined biomass (dry weight of host plant and parasite) of faba bean and lentil cultivars were similar to dry weight of host plant on non-infested systems (Figs. 3 A and Fig. 4 A). The difference in host biomass between infested and non-infested systems was accounted directly by O. crenata tissues. This indicates that the system has a maximum biomass and the difference in dry weight
4. Discussion Biomass production, partitioning, and sink strength of host plants infested by parasitic weeds have been usually studied under open-field conditions (Rubiales, 2010; McCormick et al., 2006; Fern� andez-Apar icio et al., 2016). However, due to technical constraints, host root sys tems are rarely considered in these field trials. For that purpose, we used controlled conditions (pots) to allow consideration of host roots during the study of Orobanche-host plant interactions. Furthermore, the pot assay allows the control of climatic variables, inoculum origin and density, and allows better monitoring of both emerged and underground Orobanche development (Rubiales et al., 2005a; Ennami et al., 2017). At the first time point (42 DAP), when no O. crenata considerable attachment events were observed, the presence of O. crenata seeds, at germination and fixation stages, reduced host biomass of faba bean cultivars Lobab and Misr 3 to 53.84 and 27.02% respectively compared to non-infested plants (Fig. 3). Similar host biomass reductions were observed, during beginning stages of infestation, in sorghum sensitive and tolerant cultivars infested by Striga hermonthica (45 and 25%) (Van Ast et al., 2000). This reduction could be due to a phytotoxic or path ological effect of this parasitic weed on host plant immediately after parasite infection of the host. Huot et al. (2014) suggested that negative impact on plant growth induced by pathogen could be attributed to growth–defense tradeoffs and/or a result of reduced photosynthesis. The latter hypothesis does not seem plausible in our case because no sig nificant differences were recorded on effective PSII quantum yield be tween infested and non-infested cultivars during the first three time
Fig. 9. Average chlorophyll fluorescence (Effective PSII quantum yield Y (II)) measured weekly (from 35 to 98 DAP) for (A): susceptible and resistant faba bean cultivars either infested or non-infested by O. crenata. ◆Lobab with O. crenata; ■ Lobab without O. crenata; ▴ Misr 3 with O. crenata; and � Misr 3 without O. crenata. (B): susceptible and resistant lentil cultivars either infested and non-infested. ◆ Zaaria with O. crenata; ■ Zaaria without O. crenata; ▴ Bakria with O. crenata; and 〇 Bakria without O. crenata. 7
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are a strong indication that breeding practices based on increasing yield of resistant cultivars should consider minimizing biomass losses at initial host development stages. Furthermore, most available researches have been performed in field experiments, neglected therefore characterizing of both emerged and underground parasites and also root relative weight, which reflect knowledge insufficiency of Orobanche-legumes interaction. For that, pots assays of this research contributed to identify and address these knowledge gaps. In addition, elucidating defense mechanisms of faba bean and lentil through physiological and enzy matic studies (key enzymes involved in such mechanisms “PAL, POX, and SOD) will clarify this growth–defense balance.
allocation can be attributed to a source-sink interactions. Comparable trends have been previously observed during O. cernua–tobacco, O. ramosa-faba bean, and O. aegyptiaca-tomato associations, and similar justifications were proposed (Hibberd et al., 1998, 1999). In contrast, significant differences have been found between host and parasite combined biomass and the biomass of non-infested system for O. crenata-faba bean, Striga hermonthica-sorgum, O. ramosa-tomato, O. minor-white clover, and Cuscuta-cowpea interactions (Barker et al., 1996; Hibberd et al., 1996; Gurney et al., 1999; Mauromicale et al., �ndez-Aparicio et al., 2016). According to Gurney et al. 2008; Ferna (1999), alteration of host metabolism by introduction of novel com pounds active at low concentrations or by influence on host growth regulators were suggested as mechanisms of host biomass reduction. Information on effects of O. crenata on biomass partitioning across the various host plant organs is still lacking. Other parasitic weeds have been shown to have a marked effect on biomass partitioning over the various plant compartments. Van Ast et al. (2000) indicated that biomass reduction on sorghum plants was accompanied by the increase of their root system after infection by Striga hermonthica. Another study showed that Cuscuta campestris, an obligate stem parasite, tends to in crease resource allocated to the aboveground parts (with more leaves than stems) of Mikania micrantha, and decrease the host root system (Shen et al., 2005). This is supported by our results in which O. crenata may have shifted susceptible cultivar resources through: i) increase of host root dry mater; ii) maintaining host vegetative parts; and iii) delay and reduction of host reproduction (Fig. 5). Across its influence, O. crenata seems to reinforce host root system, which is its unique point of attachment. Also, in order to maximize uptake of photosynthates from sensitive plants, this holoparasite tends to sustain host aboveground parts and redirect a part of host productivity to its own advantage. However, O. crenata fails to exercise these effects on faba bean and lentil resistant cultivars. We aimed to compare the strength of parasite and host sinks, so used relative distribution of dry weight based on ratio of sink dry matter to �ndez-Aparicio et al. (2016). combined biomass as suggested by Ferna Few studies have focused on the relative distribution of parasite and hosts dry weight compartments, and these have been limited to tradeoffs between reproductive and parasitic sinks only. Due to use of field trials, the relative weight of roots has been neglected. Our results show that during the two first time points (42 and 56 DAP) a dominance of root relative weight was observed in both susceptible and resistant faba bean and lentil cultivars (Fig. 7; Fig. 8). After these dates, parasitism didn’t influence root relative weight of the resistant cultivars, but the suscep tible cultivars showed a marked decrease in root relative weight that was reflected in an increase in relative parasitic weight. Similarly, Fern� an dez-Aparicio et al. (2016) using infection severity gradient on faba bean sensitive cultivar, reported that relative parasitic weight increased remarkably even at low infection severity. Delavault (2015) reported also that Orobanche sink strength derives from reduction of its osmotic potential, due to high accumulation levels of osmotically active com pounds such as cations, sugars, amino acids, and polyols.
Acknowledgements This research was supported by National Institute of Agricultural Research of Morocco and Ministry of Higher Education, Scientific Research and Professional Training of Morocco (MESRSFC) through funding of MEDILEG project within the European Union 7th Framework program for research, technological development and demonstration (ERA-Net Project, ARIMNet). And National Institute of Food and Agri culture grant no. 135997 to J.H.W. is acknowledged. References Abu Irmailah, B., Haddad, N., 2011. Indication of tolerance to broomrapes (orobanche crenata forsk.) in wild lentil collected from Jordan. Dirasat: Shari’a and Law Sciences 37. Attia, S.M.M.M., El-Hady, H.A., Saber, M.A., Omer, S.A., Khalil, M.S.A., A.A.M., Ashrei, R., A.M., Abd-Elrahman, M.A.M., Ibrahim, Z.,E., Ghareeb, T.S., ElMarsafawy, E.H., El-Harty, E.A.A., El-Emam, F.H., Shalaby, A.G., Helal, A.M., ElGarhy, E.M., Rabie, M., Abdeen, M., El-Noby, K.M.M., Yamani, A.E.-A.H.T., 2013. Misr 3, a new orobanche tolerant faba bean variety Egyptian. J. Plant Breed. 17, 143–152. Barker, E., Press, M., Scholes, J., Quick, W., 1996. Interactions between the parasitic angiosperm Orobanche aegyptiaca and its tomato host: growth and biomass allocation. New Phytol. 133, 637–642. Bayoumi, T., Ammar, S., El-Bramawy, M., MA, E., 2014. Effect of Some Broomrape Control Methods on Growth and Seed Yield Attributes of Faba Bean (Vicia Faba L.) Cultivars Agricultural Research Journal. Suez Canal University, 1. Briache, F.Z., Ennami, M., Mbasani-Mansi, J., Gaboun, F., Abdelwahd, R., Fatemi, Z.E.A., El-Rodeny, W., Amri, M., Triqui, Z.E.A., Mentag, R., 2019. Field and controlled conditions screenings of some faba bean (Vicia faba L.) genotypes for resistance to the parasitic plant Orobanche crenata Forsk. and investigation of involved resistance mechanisms. J. Plant Dis. Prot. 1–14. Bo�zi�c, D., Vrbni�canin, S., Elezovi�c, I., Sari�c, M., 2010. Chlorophyll Fluorescence in Vivo as the Indicator of Sunflower Susceptibility to Als-Inhibiting Herbicides, 15th EWRS Symposium. Cubero, J., Pieterse, A., Khalil, S., Sauerborn, J., 1993. Screening techniques and sources of resistance to parasitic angiosperms. Euphytica 73, 51–58. Delavault, P., 2015. Knowing the parasite: biology and genetics of orobanche. Helia 38, 15–29. Duraes, F., Gama, E., Magalhaes, P., Marriel, I., Casela, C., Oliveira, A., Luchiari Jr., A., Shanahan, J., 2001. The Usefulness of Chlorophyll Fluorescence in Screening for Disease Resistance, Water Stress Tolerance, Aluminium Toxicity Tolerance, and N Use Efficiency in Maize, Seventh Eastern and Southern Africa Regional Maize Conference 11th, pp. 356–360. Ennami, M., Briache, F.Z., Gaboun, F., Abdelwahd, R., Ghaouti, L., Belqadi, L., Westwood, J., Mentag, R., 2017. Host differentiation and variability of Orobanche crenata populations from legume species in Morocco as revealed by cross-infestation and molecular analysis. Pest Manag. Sci. https://doi.org/10.1002/ps.4536. FAO, 2014. The problem of orobanche spp in Africa and Near East. Food and agriculture organization of the united nations. http://www.fao.org/agriculture/crops/thematic -sitemap/theme/biodiversity/weeds/issues/oro/en/. Fern� andez-Aparicio, M., Flores, F., Rubiales, D., 2016. The effect of Orobanche crenata infection severity in faba bean, field pea, and grass pea productivity. Front. Plant Sci. 7. Genty, B., Briantais, J.-M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta Gen. Subj. 990, 87–92. Gershenzon, J., 1994. The Cost of Plant Chemical Defense against Herbivory: A Biochemeical Perspective, Insect-Plant Interactions. CRC Press, pp. 105–173. Ghanem, M.E., Albacete, A., Martínez-Andújar, C., Acosta, M., Romero-Aranda, R., Dodd, I.C., Lutts, S., P�erez-Alfocea, F., 2008. Hormonal changes during salinityinduced leaf senescence in tomato (Solanum lycopersicum L.). J. Exp. Bot. 59, 3039–3050. Goldwasser, Y., Plakhine, D., Kleifeld, Y., Zamski, E., Rubin, B., 2000. The differential susceptibility of vetch (vicia spp.) to orobanche aegyptiaca: anatomical studies. Ann. Bot. 85, 257–262.
5. Conclusion The prevalence of O. crenata on legume fields in the Mediterranean region is a serious problem. Unfortunately, despite of this high impact of O. crenata on legumes in Morocco, the topic of competitive relationship between this parasitic weeds and its legume plant crops remains poorly understood. In this study we investigated how Orobanche affects the growth of infested faba bean and lentil cultivars. As this way of research are important in order to provide intervention strategies on breeding programs. Our results show that parasite infections severely reduce biomass of hosts at the first stages of infection on both resistant and susceptible faba bean cultivars. It is likely that plant growth–defense tradeoffs have a direct negative impact in host resource allocation. Observed biomass differences between infested and non-infested system 8
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