The growth and development of broomrapes (Orobanche spp.) fully depends on the nutritional connection established between the parasitic plant and the root of the corresponding host plant. In thepresent study, narbon bean plants infected with Orobanche crenata were watered with different concentrations of sucrose (0.014, 0.044, 0.088 and 0.146 M) in order to evaluate its effect on the earlygrowth stages of the parasite. The germination of O. crenata seeds decreased with increasing sucrose concentrations and the number of infection attachments of crenata broomrapes decreased significantlywhen sucrose was present. A parallel experiment was conducted with identical sorbitol concentrations in order to determinate the role of the osmotic potential in the inhibition of the parasite growth. Ourresults showed that while low sucrose concentrations significantly reduced seed germination in O. crenata, similar concentrations of sorbitol have no significant effect thus indicating that the effect ofsucrose is not simply osmotic. Sucrose phytotoxicity was also studied by considering the dry weight of the host plants

González-Verdejo, CI

Guzmán, JR

Nadal, S

Román, B

Suso, MJ

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 8 (13), pp. 3027-3030, 6 July 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals    Full Length Research Paper  Sucrose effect on broomrape (Orobanche crenata) development on narbon bean (Vicia narbonensis L.)  S. Nadal1*, C. I. González-Verdejo1, J. R. Guzmán2, M. J. Suso3 and B. Román1  1Centro "Alameda del Obispo", IFAPA (Junta de Andalucía), Área de Mejora y Biotecnología, Apdo. 3092, 14080 Córdoba, Spain. 2Dpto. de Ingeniería Agroforestal. ETSIAM, Universidad de Córdoba, Córdoba, Spain. 3IAS-CSIC, Apdo. 4240, 14080, Córdoba, Spain.  Accepted 1 March, 2007  The growth and development of broomrapes (Orobanche spp.) fully depends on the nutritional connection established between the parasitic plant and the root of the corresponding host plant. In the present study, narbon bean plants infected with Orobanche crenata were watered with different concentrations of sucrose (0.014, 0.044, 0.088 and 0.146 M) in order to evaluate its effect on the early growth stages of the parasite. The germination of O. crenata seeds decreased with increasing sucrose concentrations and the number of infection attachments of crenata broomrapes decreased significantly when sucrose was present.  A parallel experiment was conducted with identical sorbitol concentrations in order to determinate the role of the osmotic potential in the inhibition of the parasite growth. Our results showed that while low sucrose concentrations significantly reduced seed germination in O. crenata, similar concentrations of sorbitol have no significant effect thus indicating that the effect of sucrose is not simply osmotic. Sucrose phytotoxicity was also studied by considering the dry weight of the host plants.   Key words: Sucrose, Orobanche crenata, Vicia narbonensis and broomrape control.   INTRODUCTION  Narbon bean (Vicia narbonensis L.) is an ancient grain and forage legume used for livestock feed in the Mediterranean region. It has a tap root system, erect habit, strong stems and it grows on neutral/alkaline soils with medium and low rainfall conditions, seed yielding up 1 t/ha under extremely arid conditions (104 mm) and up 2.5 t/ha on 400 mm rainfall (Berger et al., 2002). Although this species has a low incidence of disease and insect attack in the field, it is ddescribed as clearly susceptible to broomrape (Orobanche crenata Forsk.) (Linke et al., 1993; Nadal and Moreno, 2007). This parasitic plant attacks its seedlings to the root system of the host plant, establishes a connection with the vascular system of the host via a specialized organ (haustorium) and deprives the host of water, mineral nutrients and metabolites (Parker, 1991). In infested soils broomrape represents the major constraint for narbon bean crop and where this holoparasitic  plant  is  endemic,  it  is  necessary  to   use    *Corresponding author. E-mail: salvador.nadal.ext@junta-deandalucia.es.                                                        some control strategy. The understanding of metabolic and developmental aspects of root parasite is of great importance because each developmental stage is crucial for the growth and dispersal of these root parasites being each step a potential target for their control. Considering this, the finding of compounds that could interfer with the parasite in these initial phases could help in controlling it. In this sense, different control means such as ammonium-containing nutrients or essential amino acids have been already proposed to control Orobanche (Nandula et al., 1996; Vurro et al., 2006). Since sucrose is the main carbohydrate transferred from the host to the parasite (Aber 1984), this molecule can induce or repress a huge variety of genes which act as regulators of different processes such as germination, early seedling developments or tuberization (Rolland et al., 2002; Gibson, 2004). González-Verdejo et al. (2006) proposed the use of sucrose as a control method of Oro-banche ramosa on tomato plants, but no studies are alre-ady available regarding its effects on O. crenata- legu-mes interaction. The objective of the present study was to 3028         Afr. J. Biotechnol.    Table 1. Effect of different sucrose and sorbitol concentrations on the germination of O. crenata seeds and effect of sucrose on nodes per root length of O. crenata seeds. Values within a column with not letters in common differ significantly at p < 0.05.  % Germination Nodes/root lenght Sucrose concentration (M) Sucrose Sorbitol Sucrose 0 27.8 ab 32.2 0.20 a 0.014 31.1 a 27.1 0.07 b 0.044 13.6 c 22.1 0.03 b 0.088 16.5 c 23.6 0.03 b 0.146 21.1 bc 25.7 0.02 b CV 18.15 41.5 50.7   determinate the effects of different sucrose concentra-tions on the germination and nodulation of crenate broo-mrape on narbon bean growing under in vitro conditions, trying to find a differential situation where the target weed is sucrose inhibited but the crop is not.   MATERIALS AND METHODS  Seeds disinfection  Narbon bean seeds (sel 556 - 2376, broomrape susceptible entry from ICARDA) were surface-disinfected by immersing them first in 70% ethanol for 2 min, then in 3.5% sodium hypochlorite containing 0.1% Tween 20 for 10 min, and finally rinsing three times with sterile distilled water. O. crenata seeds were collected from plants parasitizing narbon bean plants in Córdoba (37º 51´ 42´´N and 4º 48´ W, Spain). Orobanche seeds were surface-sterilized by treating them for 2 h with a solution of 0.5% formaldehyde and 0.1 Tween 20, followed by 20 min incubation at 50ºC. Subsequently, seeds were rinsed three times with sterile distilled water as proposed by González-Verdejo et al. (2005).    Sucrose assays  Assays were performed following the methodology described by Pérez de Luque et al. (2004) with slight modifications. Narbon bean (V. narbonensis) seedlings, previously pre-germinated in vermicu-lite, and approximately 6 mg of O. crenata seeds were simultan-eously sown in 12 x 12 cm squared Petri dishes containing perlite and a sheet of glass-fibre filter (Whatman International, Kent, UK). Afterwards, the plates were wrapped in aluminium foil in order to maintain Orobanche seeds in the dark, watered and vertically stored under growth chamber conditions (21ºC, 16 h light). After 10 days of Orobanche conditioning period, sucrose (Merk 141621) at different concentrations (0.014, 0.044, 0.088 and 0.146 M) was added to the watering water. Plates without any sucrose treatments were used as negative controls Four independent plates were set per each of the four treatments and per sugar assays. Percentages of seed germination were determined using a stereoscopic microscope. In order to refer the germination data to root length (germination rate per root cm.) the host root length was estimated by the intercept method of Tennant (1975). The final number of attachments and dry weight per plant were determined per plate at the end of the experiment. In a separated experiment, the same protocol was used, but in a broomrape free environment. The data of both experiments were analyzed separately by analysis of variance according to rando-mized blocks design, and the means were compared using the Tukey comparisons test at 0.05 probability level.  RESULTS AND DISCUSSION  O. crenata seed germination in presence of sucrose  The mean percentage values of O. crenata germination for the tested sucrose concentrations (0.014, 0.044, 0.088, 0.146 M) were 31.1, 13.6, 16.5 and 21.1% respec-tively (Table 1). The germination rate became significant-ly lower with the 0.044 M sucrose concentration, thus, indicating that only the lowest sucrose concentration in the watering solution could not reduce significantly the number of germinating seeds. In the case of O. ramosa germinating seeds in tomato roots, González et al. (2006) studying similar sucrose concentrations reported that low and intermediate concentrations (0.044 and 0.088 M) did not reduce the number of germinating seeds and only the highest concentration of sucrose (0.18 M) caused a moderate reduction on germination. Considering that in non-parasitic plants like Arabidop-sis, 90 - 100% of the seeds germinated in the presence of high glucose or sucrose (Laby et al., 2000) and in the case of O. ramosa only high concentrations alter signifi-cantly the germination of the parasitic seeds in the pre-sence of tomato roots, O. crenata seems to detect earlier the sucrose presence thus preventing seed germination at lower concentrations. In this sense, differential sensiti-vities to nitrogen containing nutrients have been obser-ved regarding germination between two different Oroban-che species: Orobanche aegyptiaca and O. ramosa (Nandula et al., 1996). The observation that O. crenata is more sensitive than O. ramosa to sucrose application indicates that there are inherent differences between these closely related species in response to this carbohy-drate.    O. crenata tubercles formation in presence of sucrose  Sucrose reduced significantly (p < 0.05) the number of nodes per root plant at all studied concentrations when compared to the control treatment (Table 1). The mean number of nodes per root length cm was 0.07, 0.03, 0.03 and  0.02  at  0.014,  0.044,  0.088  and  0.146 M sucrose Nadal et al.        3029    Table 2. Effects of different sucrose concentrations on dry weight of narbon bean plant under broomrape conditions and free broomrape conditions. Values within a column with no letters in common differ significantly at p < 0.05.  Sucrose concentration (M) Orobanche non inoculated  Orobanche inoculated 0 0.51 a 0.49 0.014   0.27 ab 0.45 0.044   0.35 ab 0.36 0.088   0.41 ab 0.40 0.146    0.16 b* 0.38 CV 43.2 28.4                                *Dead plants.    concentrations, being that of the control plate of 0.20 nodes per root cm. Sucrose exposure at the lowest concentration caused an effect statistically comparable to that caused at the highest one indicating that even the lowest sucrose concentration was enough to prevent tu-bercles formation in vitro when compared to the concen-trations needed to inhibit the germination stage. In this sense, all the treatments with sucrose avoided Oroban-che attachments at the time that prevented a successful seedling establishment and tubercle formation.  The avoidance of tubercles formation in the presence of sucrose can be explained by the fact that in Oroban-che, the exogenous supply of sucrose might mimic the later stages of infection when the parasite is connected to the host vascular system. With the exogenous application of sucrose the parasitic seedling receives a carbohydrate as it does when penetrating the host root, thus preventing the activation of genes involved in attachment. Avoidance of successful attachment of Orobanche to the host plant is a valuable mean for its control. In this sense, reports of inhibitory effects of nitrogen on the growth of Orobanche has been common in the literature for many years (Sauerborn, 1991; Parker and Riches, 1993) and field experiments were able to reduce the number of emerging Orobanche shoots with the applica-tion of ammonium sulphate and to increase the yield of faba bean significantly (Kukula and Masri, 1984). More-over, the exogenous application of appropriate amino acids to a root zone might result in control of O. ramosa as reported by Vurro et al. (2006). Just recently Gonza-lez-Verdejo et al. (2006) has also proposed sucrose as a possible control strategy for O. ramosa control.    Effect of osmotic potential   Since an appropriate osmotic potential plays a key role for seed germination, to determine if sucrose itself or changes in the osmotic potential after the treatments are responsible for the changes detected in the germination rate of O. ramosa, identical sucrose concentrations were also tested with sorbitol. In this study, while low sucrose concentrations significantly reduced seed germination in O. crenata, similar concentrations of sorbitol have no significant effect (Table 1) thus indicating that the effect of sucrose is not simply osmotic. This evidence has been also found by other authors when determining the effect of glucose against sorbitol and manitol in Arabidopsis thaliana (Price et al., 2003; Dekkers et al., 2004). In the case of Orobanche, Nandula et al. (1996) when studying the effect of nitrogen-containing nutrients also considered the osmotic potential as a mechanism for inhibition of germination in different species of Orobanche and conc-luded that although osmotic stress reduces germination in three different Orobanche species, O. crenata germi-nation percentage seemed not to be affected by the osmotic potential.   Sucrose effect on the dry weight of narbon plants   According to the host plant, the effect of sucrose was tested by determining the dry weight of no inoculated and inoculated narbon bean plants. The mean values of the control (no sucrose added) and the tested concentrations (0.014, 0.044, 0.088 and 0.146 M) in both situations (inoculated and non-inoculated plants), were those showed in Table 2. Although in the case of no inoculated plates, significant differences were found among treat-ments with decreasing values of host dry weight with increasing sucrose concentrations, no significant differen-ces were found for the same treatments in the inoculated plants. In this sense, even dead plants were observed in the 0.146 M treatment under broomrape-free conditions, evidencing a poorer growth on very high concentrations compared with moderate concentrations of sucrose due to osmotic stress. In contrast, we did not observe statis-tical differences among host plants under inoculated con-ditions, since broomrape parasite acts as a strong carbo-hydrate sink that reduced the damage provoked by suc-rose. Although the feasibility, effectiveness and application sucrose as a control strategy need to be determined, this work can be considered as an opening step in the study of sucrose as a regulator of broomrape germination. In the  future,  the  determination of the best timing and con- 3030         Afr. J. Biotechnol.    centration of sucrose as well as the evaluation of its application considering its stability and movement into the soil, in order to consider the potential of its direct applica-tion and to evaluate their influence in the environment, will be highly desirable. Moreover, although no phytoto-xicity is evident in inoculated plants, considering the fut-ure use of sucrose as a field control mean, the determi-nation of the minimum concentrations needed to control Orobanche will be of great importance since the carbohy-drate seems to clearly interfer with the normal root development of non infested host plants and phytotoxicity could compensate the benefit of the control provided. Finally, differences in sucrose sensitivity between O. crenata and O. ramosa suggested in this study and that reported by González-Verdejo et al. (2006) caution agai-nst generalizing about the efficacy of Orobanche control by sucrose application.    ACKNOWLEDGEMENTS  This research was funded by the Project INIA RTA2006-00031-00-00 of Spanish Government. The authors thank ICARDA Germplasm Bank for seeds, and Gema Gon-zález and María Dolores Madrid for technical assistance.   REFERENCES  Aber M (1984). Cyto-Physiological aspects of Orobanche crenata Forsk. parasitizing Vicia faba L. Third International Symposium on Parasitic Weeds, Aleppo. pp. 200-209. Berger JD, Robertson LD, Cocks PS (2002). Genotype x environment interaction for yield and other plant attributes among undomesticated Mediterranean Vicia species. Euphytica 126: 421-435. Dekkers BJ, Schuurmans JA, Smeekens SC (2004). Glucose delays seed germination in Arabidopsis thaliana. Planta 218: 579-588. Gibson SI (2004). Sugar and phytohormone response pathways: navigating a signalling network. J. Exp. Bot. 55: 253-264. 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Sucrose effect on broomrape (Orobanche crenata) development on narbon bean (Vicia narbonensis L.)

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Sucrose effect on broomrape (Orobanche crenata) development on narbon bean (Vicia narbonensis L.)

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