Biological and Transcriptomic Characterization of Pre-Haustorial Resistance to Sunflower Broomrape (Orobanche cumana W.) in Sunflowers (Helianthus annuus).

Infestations with sunflower broomrape (Orobanche cumana Wallr.), an obligatory root parasite, constitute a major limitation to sunflower production in many regions around the world. Breeding for resistance is the most effective approach to reduce sunflower broomrape infestation, yet resistance mechanisms are often broken by new races of the pathogen. Elucidating the mechanisms controlling resistance to broomrape at the molecular level is, thus, a desirable way to obtain long-lasting resistance. In this study, we investigated broomrape resistance in a confectionery sunflower cultivar with a robust and long-lasting resistance to sunflower broomrape. Visual screening and histological examination of sunflower roots revealed that penetration of the broomrape haustorium into the sunflower roots was blocked at the cortex, indicating a pre-haustorial mechanism of resistance. A comparative RNA sequencing between broomrape-resistant and -susceptible accessions allowed the identification of genes that were significantly differentially expressed upon broomrape infestation. Among these genes were ß-1,3-endoglucanase, ß-glucanase, and ethylene-responsive transcription factor 4 (ERF4). These genes were previously reported to be pathogenesis-related in other plant species. This transcriptomic investigation, together with the histological examinations, led us to conclude that the resistance mechanism involves the identification of the broomrape and the consequent formation of a physical barrier that prevents the establishment of the broomrape into the sunflower roots.

The Influence of the Plant Growth Regulator Maleic Hydrazide on Egyptian Broomrape Early Developmental Stages and Its Control Efficacy in Tomato under Greenhouse and Field Conditions.

Broomrapes (Phelipanche spp. and Orobanche spp.) are holoparasitic plants that cause tremendous losses of agricultural crops worldwide. Broomrape control is extremely difficult and only amino acid biosynthesis-inhibiting herbicides present an acceptable control level. It is expected that broomrape resistance to these herbicides is not long in coming. Our objective was to develop a broomrape control system in tomato (Solanum lycopersicum L.) based on the plant growth regulator maleic hydrazide (MH). Petri-dish and polyethylene-bag system experiments revealed that MH has a slight inhibitory effect on Phelipanche aegyptiaca seed germination but is a potent inhibitor of the first stages of parasitism, namely attachment and the tubercle stage. MH phytotoxicity toward tomato and its P. aegyptiaca-control efficacy were tested in greenhouse experiments. MH was applied at 25, 50, 75, 150, 300, and 600 g a.i. ha-1 to tomato foliage grown in P. aegyptiaca-infested soil at 200 growing degree days (GDD) and again at 400 GDD. The treatments had no influence on tomato foliage or root dry weight. The total number of P. aegyptiaca attachments counted on the roots of the treated plants was significantly lower at 75 g a.i. ha-1 and also at higher MH rates. Phelipanche aegyptiaca biomass was close to zero at rates of 150, 300, and 600 g a.i. ha-1 MH. Field experiments were conducted to optimize the rate, timing and number of MH applications. Two application sequences gave superior results, both with five split applications applied at 100, 200, 400, 700, and 1000 GDD: (a) constant rate of 400 g a.i. ha-1; (b) first two applications at 270 g a.i. ha-1 and the next three applications at 540 g a.i. ha-1. Based on the results of this study, MH was registered for use in Israel in 2013 with the specified protocol and today, it is widely used by most Israeli tomato growers.

Non-chemical Control of Root Parasitic Weeds with Biochar.

This study tested whether soil-applied biochar can impact the seed germination and attachment of root parasitic weeds. Three hypotheses were evaluated: (i) biochar adsorbs host-exuded signaling molecules; (ii) biochar activates plants' innate system-wide defenses against invasion by the parasite; and (iii) biochar has a systemic influence on the amount of seed germination stimulant produced or released by the host plant. Three types of experiments were performed: (I) pot trials with tomato (Solanum lycopersicum) infested with Phelipanche aegyptiaca PERS. (Egyptian broomrape) and three different types of biochar at concentrations ranging from 0 to 1.5% weight, wherein tomato plant biomass, P. aegyptiaca biomass, and number of P. aegyptiaca-tomato root attachments were quantified; (II) split-root biochar/no-biochar experiments under hydroponic growing conditions performed in polyethylene bags with tomato plant rootings, wherein P. aegyptiaca seed germination percentage and radicle attachment numbers were quantified; and (III) germination trials, wherein the effect of biochar adsorption of GR-24 (artificial germination stimulant) on P. aegyptiaca seed germination was quantified. Addition of biochar to the pot soil (Experiment I) resulted in lower levels of P. aegyptiaca infection in the tomato plants, mainly through a decrease in the number of P. aegyptiaca attachments. This led to improved tomato plant growth. In Experiment II, P. aegyptiaca seed germination percentage decreased in the biochar-treated root zone as compared with the no-biochar control root zone; P. aegyptiaca radicle attachment numbers decreased accordingly. This experiment showed that biochar did not induce a systemic change in the activity of the stimulant molecules exuded by the tomato roots, toxicity to the radicles, or a change in the ability of the radicles to penetrate the tomato roots. The major cause for the decrease in germination percentage was physical adsorption of the stimulant molecule by the biochar (Experiment III). Adding biochar to soil to reduce infections by root parasitic weeds is an innovative means of control with the potential to become an important strategy both for non-chemical treatment of this family of pests, and for enhancing the economic feasibility of the pyrolysis/biochar platform. This platform is often viewed as one of a handful of credible strategies for helping to mitigate climate change.

Secondary Effects of Glyphosate Action in Phelipanche aegyptiaca: Inhibition of Solute Transport from the Host Plant to the Parasite.

It is currently held that glyphosate efficiently controls the obligate holoparasite Phelipanche aegyptiaca (Egyptian broomrape) by inhibiting its endogenous shikimate pathway, thereby causing a deficiency in aromatic amino acids (AAA). While there is no argument regarding the shikimate pathway being the primary site of the herbicide's action, the fact that the parasite receives a constant supply of nutrients, including proteins and amino acids, from the host does not fit with an AAA deficiency. This apparent contradiction implies that glyphosate mechanism of action in P. aegyptiaca is probably more complex and does not end with the inhibition of the AAA biosynthetic pathway alone. A possible explanation would lie in a limitation of the translocation of solutes from the host as a secondary effect. We examined the following hypotheses: (a) glyphosate does not affects P. aegyptiaca during its independent phase and (b) glyphosate has a secondary effect on the ability of P. aegyptiaca to attract nutrients, limiting the translocation to the parasite. By using a glyphosate-resistant host plant expressing the "phloem-mobile" green fluorescent protein (GFP), it was shown that glyphosate interacts specifically with P. aegyptiaca, initiating a deceleration of GFP translocation to the parasite within 24 h of treatment. Additionally, changes in the entire sugars profile (together with that of other metabolites) of P. aegyptiaca were induced by glyphosate. In addition, glyphosate did not impair germination or seedling development of P. aegyptiaca but begun to exert its action only after the parasite has established a connection to the host vascular system and became exposed to the herbicide. Our findings thus indicate that glyphosate does indeed have a secondary effect in P. aegyptiaca, probably as a consequence of its primary target inhibition-via inhibition of the translocation of phloem-mobile solutes to the parasite, as was simulated by the mobile GFP. The observed disruption in the metabolism of major sugars that are abundant in P. aegyptiaca within 48 h after glyphosate treatment provides a possible explanation for this inhibition of translocation and might reflect a critical secondary effect of the herbicide's primary action that results in loss of the parasite's superior sink for solutes.

Maternal tissue is involved in stimulant reception by seeds of the parasitic plant Orobanche.

BACKGROUND AND AIMS: A fundamental element in the evolution of obligate root-parasitic angiosperms is their ability to germinate only in response to chemical stimulation by roots, to ensure contact with a nearby nourishing host. The aim of this study was to explore inheritance of the unique germination control in this group of plants. METHODS: Analysis was made of the segregation of spontaneous (non-induced) germination that appeared in hybrid progenies derived from crosses between Orobanche cernua and O. cumana, which, like all other Orobanche species, are totally dependent on chemical stimulation for the onset of germination, and show negligible spontaneous germination in their natural seed populations. KEY RESULTS AND CONCLUSIONS: F(1) and F(2) seeds did not germinate in the absence of chemical stimulation, but significant spontaneous germination was found in some F(3) seed families. This indicates that the prevention of non-induced germination in Orobanche seeds, i.e. dependence on an external chemical stimulation for seed germination, is genetically controlled, that this genetic control is expressed in a seed tissue with maternal origin (presumably the perisperm that originates from the nucellus) and that genetic variation for this trait exists in Orobanche species. Similar segregation results were obtained in reciprocal crosses, suggesting that stimulated germination is controlled by nuclear genes.

Seed ultrastructure and water absorption pathway of the root-parasitic plant Phelipanche aegyptiaca (Orobanchaceae).

BACKGROUND AND AIMS: Obligate root parasitic plants of the Orobanchaceae do not germinate unless they chemically detect a host plant nearby. Members of this family, like Orobanche, Phelipanche and Striga, are noxious weeds that cause heavy damage to agriculture. In spite of their economic impact, only a few light microscopical studies of their minute seeds have been published, and there is no knowledge of their ultrastructure and of the role each tissue plays during the steps preceding germination. This paper describes the ultrastructure of Phelipanche seeds and contributes to our understanding of seed tissue function. METHODS: Seeds of P. aegyptiaca were examined under light, scanning electron, transmission electron and fluorescence microscopy following various fixations and staining protocols. The results were interpreted with physiological data regarding mode of water absorption and germination stimulation. KEY RESULTS AND CONCLUSIONS: The endothelium, which is the inner layer of the testa, rapidly absorbs water. Its interconnected cells are filled with mucilage and contain labyrinthine walls, facilitating water accumulation for germination that starts after receiving germination stimuli. Swelling of the endothelium leads to opening of the micropyle. The perisperm cells underneath this opening mediate between the rhizosphere and the embryo and are likely to be the location for the receptors of germination stimuli. The other perisperm cells are loaded with lipids and protein bodies, as are the endosperm and parts of the embryo. In the endosperm, the oil bodies fuse with each other while they are intact in the embryo and perisperm. Plasmodesmata connect the perisperm cells to each other, and the cells near the micropyle tightly surround the emerging seedling. These perisperm cells, and also the proximal embryo cells, have dense cytoplasmic contents, and they seem to represent the two seed components that are actively involved in transfer of reserve nutrients to the developing seedling during germination.

Dehydrocostus lactone is exuded from sunflower roots and stimulates germination of the root parasite Orobanche cumana.

The germination of the obligate root parasites of the Orobanchaceae depends on the perception of chemical stimuli from host roots. Several compounds, collectively termed strigolactones, stimulate the germination of the various Orobanche species, but do not significantly elicit germination of Orobanche cumana, a specific parasite of sunflower. Phosphate starvation markedly decreased the stimulatory activity of sunflower root exudates toward O. cumana, and fluridone - an inhibitor of the carotenoid biosynthesis pathway - did not inhibit the production of the germination stimulant in both shoots and roots of young sunflower plants, indicating that the stimulant is not a strigolactone. We identified the natural germination stimulant from sunflower root exudates by bioassay-driven purification. Its chemical structure was elucidated as the guaianolide sesquiterpene lactone dehydrocostus lactone (DCL). Low DCL concentrations effectively stimulate the germination of O. cumana seeds but not of Phelipanche aegyptiaca (syn. Orobanche aegyptiaca). DCL and other sesquiterpene lactones were found in various plant organs, but were previously not known to be exuded to the rhizosphere where they can interact with other organisms.

Is seed conditioning essential for Orobanche germination?

BACKGROUND: Parasitic Orobanchaceae germinate only after receiving a chemical stimulus from roots of potential host plants. A preparatory phase of several days that follows seed imbibition, termed conditioning, is known to be required; thereafter the seeds can respond to germination stimulants. The aim of this study was to examine whether conditioning is essential for stimulant receptivity. RESULTS: Non-conditioned seeds of both Orobanche cumana Wallr. and O. aegyptiaca Pers. [syn. Phelipanche aegyptiaca (Pers.) Pomel] were able to germinate in response to chemical stimulation by GR24 even without prior conditioning. Stimulated seeds reached maximal germination rates about 2 weeks after the onset of imbibition, no matter whether the seeds had or had not been conditioned before stimulation. Whereas the lag time between stimulation and germination response of non-conditioned seeds was longer than for conditioned seeds, the total time between imbibition and germination was shorter for the non-conditioned seeds. Unlike the above two species, O. crenata Forsk. was found to require conditioning prior to stimulation. CONCLUSIONS: Seeds of O. cumana and O. aegyptiaca are already receptive before conditioning. Thus, conditioning is not involved in stimulant receptivity. A hypothesis is put forward, suggesting that conditioning includes (a) a parasite-specific early phase that allows the imbibed seeds to overcome the stress caused by failing to receive an immediate germination stimulus, and (b) a non-specific later phase that is identical to the pregermination phase between seed imbibition and actual germination that is typical for all higher plants.

Changes in the activity of the alternative oxidase in Orobanche seeds during conditioning and their possible physiological function.

The appearance of the activity of the cyanide insensitive, alternative oxidase (AOX), pathway of oxygen uptake was followed in seeds of Orobanche aegyptiaca during conditioning. The pathway becomes operative during conditioning, up to day three as determined by inhibition of oxygen uptake of the seeds by propyl gallate. At the same time an increasing percentage of oxygen uptake is insensitive to cyanide and an increased oxygen uptake, responsive to propyl gallate, is induced by brief salicylic acid treatment of seeds. By day six of conditioning, these responses decrease and the AOX pathway could not be detected in germinating seeds, after treatment with a germination stimulant. These results were confirmed by following the reaction of extracts of fractions enriched with mitochondria from the conditioned seeds, using a specific antibody against AOX. Treatment of the seeds with inhibitors of AOX during conditioning significantly inhibited their subsequent germination. Addition of hydrogen peroxide after 4 and 7 days of conditioning resulted in reduced germination. In addition treatment of seed with propyl or octyl gallate during conditioning reduced the infection of tomato plants by Orobanche seeds and the development of tubercles of the parasite on the host roots. These results together indicate that the operation of AOX during conditioning has a significant function on the subsequent germination behaviour and pathogenicity of the root parasite. Some potential practical applications of these findings are discussed.