Characterization of a novel tomato mutant resistant to the weedy parasites Orobanche and Phelipanche spp.
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- Dor, E., Alperin, B., Wininger, S. et al. Euphytica (2010) 171: 371. doi:10.1007/s10681-009-0041-2
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Orobanche and Phelipanche, commonly known as broomrape, are dicotyledonous holoparasitic flowering plants that cause heavy economic losses in a wide variety of plant species. Breeding for Orobanche resistance is still one of the most effective management strategies for this weed. However, previous efforts to find broomrape-resistant tomato (Solanum lycopersicon) genotypes have been unsuccessful. Here, we report on the isolation and characterization of a fast-neutron-mutagenized M-82 tomato mutant, Sl-ORT1. The Sl-ORT1 mutant showed resistance to Phelipanche aegyptiaca as compared to cultivar M-82; segregation analysis suggested a single recessive ort1 allele. Sl-ORT1 broomrape resistance was reflected in a lower number of broomrapes per plant, reduced P. aegyptiaca fresh weight per plant, and the absence of broomrape’s negative effect on plant host growth and yield. Sl-ORT1 was shown to be resistant to high concentrations of P. aegyptiaca seeds, and to another three broomrape species: Phelipanche ramosa, Orobanche cernua, and Orobanche crenata. Grafting experiments demonstrated that roots, rather than shoots, are necessary for Sl-ORT1 broomrape resistance. In addition, Sl-ORT1 was shown to be resistant to broomrape under field conditions. Since yield parameters are slightly affected by the mutation, this resistance gene should be introduced into tomato varieties with different genetic backgrounds; this newly identified Orobanche-resistant mutant may be further utilized in breeding programs for Orobanche resistance.
Orobanche and Phelipanche, commonly known as broomrapes, are dicotyledonous holoparasitic flowering plants that cause heavy economic losses in legumes, crucifers, tomato, sunflower, tobacco and many other crops worldwide (Joel et al. 2007). In many Mediterranean countries, Phelipancheaegyptiaca (syn. Orobanche aegyptiaca) and Phelipanche ramosa (syn. Orobanche ramosa) are the most damaging broomrape species in tomato (Solanum lycopersicon) fields (Hershenhorn et al. 2009). Consequently, different control strategies have been developed and tested for broomrape management (see review by Joel et al. 2007; Rispail et al. 2007), but with no definitive or reliable solution for extensive tomato cropping systems.
Breeding for broomrape resistance is still one of the most effective and environmentally friendly management strategies for coping with this weed (Rubiales et al. 2003a). Successful breeding efforts have been made for many crops, such as chickpea (Rubiales et al. 2003a), faba bean (Nassib et al. 1982; Román et al. 2002; Torres et al. 2006), pea (Valderrama et al. 2004), sunflower (Pérez-Vich et al. 2004) and others. One common element in breeding for broomrape resistance is the on-going struggle for genetic broomrape-resistance traits in many crops, such as tomato. Furthermore, the parasite’s ability to overcome newly developed resistant varieties in a short period of time makes this aim even more elusive (Fernández-Martínez et al. 2000).
Previous efforts to find broomrape-resistant tomato genotypes have been unsuccessful (Abu-Gharbieh et al. 1978; Avdeyev et al. 2003; Dalela and Mathur 1971; Foy et al. 1988; Qasem and Kasrawi 1995). A tomato line (PZU-11) that was reported to be resistant to Phelipanche in the northern region of Eastern Europe (Avdeyev and Scherbinin 1977) failed to show similar resistance, in the field, in other locations (Foy et al. 1987; reviewed by Hershenhorn et al. 2009). These results, coming from the enormous effort that has been invested in searching for a broomrape resistance trait (from wild and primitive tomato genetic resources—see Hershenhorn et al. 2009), let us to the conclusion that the natural genetic variability supporting broomrape resistance is low. Under these circumstances, mutagenesis may provide a way of expanding genetic variation, which could be utilized in resistance-breeding programs (Van-Harten 1998). Chemical mutagenesis of tomato seeds using ethane methane sulfonate (EMS) was conducted in Bulgaria producing tomato lines with partial resistance to P. ramosa (Kostov et al. 2007).
Here we report on the isolation and characterization of a fast-neutron-mutagenized M-82 tomato mutant, Sl-ORT1, which shows considerable resistance to broomrape under both greenhouse and field growth conditions. This newly identified broomrape-resistant mutant may be further utilized in breeding programs for broomrape resistance.
Materials and methods
Seeds from tomato (Solanum lycopersicon) cultivar M-82 were obtained from Tarsis Agricultural Chemicals Ltd., Israel. For generation of mutants, seeds of cultivar M-82 were irradiated by fast neutrons at a dose of 60 Gy. M2 plants were screened for resistance against Phelipanche under field conditions at Kibbutz Usha, Israel, in the year 2000, and were allowed to self-pollinate. M3 progeny of plants with viable seeds were rescreened from each putative M2 mutant for inheritance of the mutant phenotype under nethouse conditions. Resistant plants were reevaluated for three additional consecutive generations.
Broomrape seeds were collected from Phelipanche aegyptiaca, Phelipanche ramosa, Orobanche cernua and Orobanche crenata inflorescences parasitizing tomato grown in Kibbutz Bet Ha’shita, potato grown in the Golan Heights, tomato grown in Bet Dagan, and chickpea grown in Kibbutz Kfar H’horesh, respectively. Broomrape seeds were stored and held in the dark at 4°C until use.
All experiments were conducted with 2- or 4-l pots (Tefen Nachsholim, Israel) using medium-heavy clay-loam soil containing on a dry weight basis 55% clay, 23% silt, 20% sand, 2% organic matter, pH 7.1. Fertilizer at a concentration of 0.6% (w/v) (Osmocote, ScottsMiracle-Gro, Marysville, OH) and broomrape seeds at a concentration of 20 ppm (20 mg seeds kg−1 soil), unless otherwise stated, were added to the soil. The above-mentioned components were mixed to homogeneity in a cement mixer, for about 10 min. Control pots did not contain broomrape seeds. The pots were placed in a nethouse and drip-irrigated.
All pot experiments were carried out with five replicates, unless otherwise indicated, and were repeated twice. Tomato seeds of cultivar M-82 and line Sl-ORT1 were germinated in Styrofoam growing trays and were transferred to pots 4 weeks after sowing.
Selection for Phelipanche-resistant mutants
Evaluation of Sl-ORT1 resistance
Effect of P. aegyptiaca on Sl-ORT1 growth in the greenhouse
M-82 and Sl-ORT1 plants were grown in pots artificially inoculated with P. aegyptiaca seeds. Every week, starting at 4 weeks after planting (WAP), the soil from five pots was gently removed under tap water and the root system was checked for unusual development, dents, necrotic areas and any marks indicating unsuccessful broomrape attachments. The effect of broomrape infection on growth and development of the two tomato lines, as expressed by tomato root and foliage weight, was recorded at each sampling date. Broomrape infections were counted as number of visible inflorescences per tomato plant. Total broomrape fresh weight, above and below ground, was recorded to calculate broomrape biomass supported by each tomato phenotype. The root and foliage weights of Sl-ORT1 and M-82 plants were determined with and without broomrape infection.
To ascertain the heredity of the resistance trait in the Sl-ORT1 line and to characterize these genes (i.e. whether recessive or dominant), the mutant line was crossed with M-82. The F1 generation was selfed and the F2 population was screened for Phelipanche resistance. The segregation ratios were calculated in two F2 populations, F2(2) and F2(6), in each population 85 individual F2 plants, to reflect the genetics and inheritance of the resistance trait. The broomrape shoots emerging from the soil were counted twice a week for each pot, starting with the first broomrape shoot protruding aboveground. At the termination of the experiment, the soil and roots were washed gently under tap water and the root system of each plant was evaluated for extent of underground stages of broomrape parasitism, i.e. tubercles, spiders (rooted tubercles), and underground inflorescences.
Effect of P. aegyptiaca seed concentration on P. aegyptiaca infectivity of line Sl-ORT1
A pot experiment was conducted in the greenhouse to evaluate the effect of Phelipanche seed concentration in the soil on Phelipanche parasitism on M-82 and Sl-ORT1 plants. Three broomrape seed concentrations, 20, 50 and 100 mg seeds kg−1 soil, were used in pots with one tomato plant per pot. The infection and growth conditions were as described above.
Sl-ORT1 resistance against different broomrape species
The spectrum of Sl-ORT1 resistance toward P. aegyptiaca, P. ramosa, O. cernua and O. crenata, four broomrape species present in the agricultural fields of Israel, was tested in pots under greenhouse conditions. The experiment was carried out in a nethouse under normal days with 14 h light for a period of 90 days. At the conclusion of the experiment, the soil in the pots was gently rinsed under tap water revealing the root system of each tomato plant. The roots were checked for broomrape parasitism. The number of broomrape tubercles (small rounded attachments on the host roots), spiders (parasites with root-like papillae), and inflorescences (elongated parasite shoots with a defined inflorescence stem) were counted per plant to evaluate the resistance of the mutant Sl-ORT1 against different broomrape species.
Evaluation of Sl-ORT1 resistance in the field
The ability of Sl-ORT1 plants to resist natural acquisition of infection under agronomical conditions and their agronomical productivity traits were evaluated. Plants were tested in a broomrape-infested and non-infested field for evaluation of crop productivity.
A field experiment was carried out in broomrape-infested field in Mavo Hama (Israel) in 2004, in a clay-loam soil with pH 7.2 and 1% organic matter, in which 160 Sl-ORT1 and 160 M-82 plants were planted on June 1 in a commercial tomato field naturally infested with P. aegyptiaca in a completely randomized design. The plants were planted in one row per bed and with 1 m distance between plants. Visible aboveground inflorescences (flowering shoots) were counted for each tomato plant from 40 to 100 days after planting to evaluate resistance.
For the non-broomrape-infested field, experiment was carried out in Ein H’mifratz (Israel) in 2006, in a clay-loam soil with 7.6 and 1.6% organic matter. On March 7, 160 Sl-ORT1 and 160 M-82 plants were planted in a commercial tomato field with no known history of P. aegyptiaca infestation. The experiment was design in completely randomized blocks with three replications per treatment. Each plot consisted of 10 m long bad with a width of 1.93 m. Plots were harvested three months after planting. Total and marketable yield was determined; the number of fruits of 25 plants from each plot was determined and weighted and 5 fruits from each of the 25 plants were taken for fruit diameter, length and brix measurements.
To ascertain which part of the tomato plant confers resistance to broomrape—the root system or the foliage, four types of grafting combinations were tested: (a) M-82 scion on M-82 rootstock (susceptible on susceptible), (b) M-82 scion on Sl-ORT1 rootstock (susceptible on resistant), (c) Sl-ORT1 scion on M-82 rootstock (resistant on susceptible), and (d) Sl-ORT1 scion on Sl-ORT1 rootstock (resistant on resistant). Non-grafted sensitive and resistant plants were used as controls. The experiment was carried out in a nethouse under 14 h of daylight for 60 days. The number of broomrape inflorescences emerging aboveground was counted twice a week, beginning at the first emergence of an aboveground inflorescence. Seven replicates (plants) were used for each grafting treatment and controls.
All experimental results were subjected to ANOVA by means of JMP Software, version 5.0 (SAS Institute Inc., Cary, NC). The data on resistance of Sl-ORT1 and M-82 to various broomrape species and on resistance of grafted plants were compared by least-significant differences (LSD) on the basis of the Tukey-Kramer Honestly Significant Difference test (P < 0.001). Data of all other experiments were separated by standard error of the differences (SED).
Botanical description of Sl-ORT1
The shoot morphology of Sl-ORT1 in comparison to M-82 plants is characterized in detail in Koltai et al. (2009). Briefly, a higher number of unsuppressed side branches emerged in Sl-ORT1 plants relative to M-82, from the first to seventh (±2) metamers, whereas higher Sl-ORT1 metamers and inflorescence were similar to those of M-82 (Fig. 1).
Effect of the resistance on P. aegyptiaca growth
Effect of Phelipanche parasitism on plant host growth and development
Necrotic areas were not observed in Sl-ORT1 roots grown in P. aegyptiaca-infested soil. Hence, a hypersensitive response can be excluded as a basis for Sl-ORT1 broomrape resistance.
Sl-ORT1 resistance against different levels of broomrape infestation
Sl-ORT1 resistance against different broomrape species
Determination of shoot or root requirement for broomrape-resistance phenotype
Sl-ORT1 performance under field conditions
Yield parameters of M-82 and Sl-ORT1 under field conditions. Experiment was carried out in Ein HaMifratz (2006) under non-infested field conditions. Values are mean of five replicates ± SE
Total yield (T/ha)
Marketable yield (%)
Fruit number (per plant)
Fruit diameter (mm)
Fruit length (mm)
65.0 ± 2.0
70 ± 2.4
39.5 ± 2.0
46.8 ± 1.8
34.0 ± 2.2
50 ± 2.9
38.0 ± 1.3
42.1 ± 1.4
In the present work, we describe a new broomrape-resistant mutant Sl-ORT1, which shows considerable resistance to broomrape under greenhouse conditions, as well as under field conditions. In the case of broomrape infection of Sl-ORT1, fewer plants were infected, and fewer broomrapes were present per plant. Moreover, this resistance was wide-ranging: Sl-ORT1 was resistant to the four different broomrape species tested. This trait may form the basis for introduction of broomrape-resistant lines in breeding programs. We therefore characterized Sl-ORT1 broomrape resistance in detail.
Previous efforts to understand broomrape resistance in different crops have revealed different and independent, or sometimes coordinated, induction of several defense mechanisms that are responsible for the resistance phenomenon. For example, low stimulation of broomrape seed germination (Labrousse et al. 2001; Rubiales et al. 2009), phytoalexin induction (Serghini et al. 2001), high levels of peroxidase activity (Goldwasser et al. 1999; Pérez-de-Luque et al. 2006a), lignification of host endodermis and xylem vessels (Dörr et al. 1994), cell wall deposition (Pérez-de-Luque et al. 2006a), development of an encapsulation layer in the cortical parenchyma (Pérez-de-Luque et al. 2005a, b), induction of pathogenesis-related (PR) proteins (Joel and Portnoy 1998), and sealing of host xylem vessels by mucilage deposition (Pérez-de-Luque et al. 2006b) have all been demonstrated. Callose deposition and reactive oxygen species (ROS) production and accumulation are also typical responses to biotic and abiotic stresses and are involved in the interaction between plants and their parasites (Pérez-de-Luque et al. 2005a, 2006b). Recently, Lejeune et al. (2006) demonstrated the involvement of a Solanum lycopersicon wall-associated kinase (LeWAK) in triggering the activation of defense reactions during the interaction of P. ramosa and tomato roots.
Detailed analysis of line Sl-ORT1 showed not only reduced, but also delayed infection. This combination of reduced and delayed infection may have a large effect on plant yield: even when infection does occur, it may develop only following anthesis, the stage determining yield potential and productivity.
As a prelude to its integration into breeding programs, we characterized the inheritance of the resistance trait. Segregation of resistance indicated that it is governed by a single recessive gene. Since yield parameters were found to be slightly affected by the mutation, this resistance gene should be introduced into tomato varieties with different genetic backgrounds.
To determine the requirement of root or shoot for the resistance response, we compared different grafting combinations between Sl-ORT1 and M-82 and non-grafted plants. The results of these experiments suggested that roots, rather than shoots, are necessary for acquisition of the resistance phenotype.
To further elucidate the mechanism of resistance in Sl-ORT1, we examined the phenotype of early stages of the Phelipanche-root interaction in the soil. Close visual inspection and binocular microscope examination of Sl-ORT1 roots grown in P. aegyptiaca-infested soil revealed no signs of a host hypersensitive response or of morphological deformation in the host or the parasite. This indicated that successful attachment and penetration of the parasite results in successful parasitism. Thus, the reduced Sl-ORT1 infection may result from deficiencies in the host-parasite interaction, before any contact is formed between the parasite germ tube and the host roots. The consistently low level of infection in Sl-ORT1 may be a result of a lack or reduced amount of stimulant secretion by the host, leading to reduced broomrape seed germination, and hence reduced infection level. The low level of Sl-ORT1 infection that still occurs may be due to a low level of spontaneous germination of broomrape seeds. Other possibilities may include root inhibitors that interact with broomrape germination or its stimulant, abolishing broomrape seed germination. Further experiments designed to elucidate this point are ongoing in our laboratories.
Several studies have attempted to identify low germination stimulant-producing lines in different crops, such as sunflower resistant to O. cumana (Jorrín et al. 1999; Labrousse et al. 2001), tomato with partial resistance to P. aegyptiaca (El-Halmouch et al. 2006), in some accessions of a range of legumes resistant to O. crenata, including vetches, peas, chickpea, grass peas (Rubiales et al. 2003b, c; Sillero et al. 2005; Pérezde-Luque et al. 2005a, b), sorghum and maize resistant to Striga hermonthica (Mohamed et al. 2001; Gurney et al. 2003), However, not a single outstanding broomrape-resistant variety is currently available. Sl-ORT1 offers an opportunity for resistance-breeding programs in tomato. Understanding the molecular biology of the resistance mechanisms will help in developing resistant varieties in other crops as well (Rubiales et al. 2009).
This project was partially supported by a grant from The Chief Scientist, Ministry of Agriculture in Israel to JH and YK, and by Zeraim Gedera Ltd., Israel