Variability of interactions between barrel medic (Medicago truncatula) genotypes and Orobanche species

A number of broomrapes ( Orobanche spp.) are major threats for grain legumes in the Mediterranean area. Previous studies have shown very high levels of resistance to Orobanche crenata in Medicago truncatula with little variation among accessions hampering, therefore, its use for genetic analysis. In order to identify alternative systems for ulterior genetic and genomic analysis, we studied early stages of the interaction between M. truncatula accessions and a range of Orobanche species. We found significant differences in the induction of germination on Orobanche aegyptiaca , Orobanche foetida var. broteri , Orobanche minor , Orobanche nana and Orobanche ramosa seeds. Accessions also varied in the number of O. aegyptiaca , O. crenata , O. foetida var. broteri , O. nana and O. ramosa attachments supported. Applications of the synthetic germination stimulant GR24 increased the germination level of Orobanche cumana and O. minor . No attachments were observed for any of these species because of physical barriers typical of host resistance. On the contrary, increase of O. nana germination by GR24 led to an increase in number of attachments albeit the normal development of the nodules was stopped in a later stage. The genetic variation observed for induction of germination and subsequent attachment will be useful for isolating and characterising genes involved in early stages of Orobanche –host plant interaction and for the study of the biosynthetic pathways of production of germination stimulants.     

IAA production during germination of Orobanche spp. seeds

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.     

Sunflower (Helianthus annuus L.) response to broomrape (Orobanche cernua Loefl.) parasitism: induced synthesis and excretion of 7-hydroxylated simple coumarins

The interaction of the parasitic plant Orobanche cernua with resistant and susceptible cultivars of Helianthus annuus L. was investigated. Using different bioassays to evaluate the early stages of the parasite life cycle (germination, attachment, penetration, and establishment), differences were observed between O. cernua-resistant and O. cernua-susceptible sunflower varieties. Germination of O. cernua seeds in the presence of resistant sunflower roots was approximately half that of germination in the presence of susceptible roots, and germinated seeds displayed enhanced browning symptoms. Parasite radicles or host-tissue around the contact point turned brown after O. cernua attachment to sunflower roots, especially in the resistant varieties. These observations suggested the possible accumulation of toxic compounds as a defence strategy in the resistant sunflower varieties. Sunflower 7-hydroxylated simple coumarins may play a defensive role against O. cernua parasitism by preventing successful germination, penetration and/or connection to the host vascular system. This hypothesis is supported by the following data: (i) coumarins inhibited the in vitro germination of O. cernua seeds induced by the strigol analogue GR(24) and caused a browning reaction in germinated seeds and (ii) resistant sunflowers accumulated higher levels of coumarins in roots and excreted greater amounts than susceptible varieties in response to O. cernua infection.     

Potential role of winter rape weeds in the extension of broomrape in Poitou-Charentes

In the Poitou-Charentes district, among the 82 species of winter rape weeds identified, 22 displayed a strong affinity for this crop (Brassica napus L.). In fields, 50% of these weeds were parasitized by Orobanche ramosa, playing the role of host plants. Greenhouse co-cultures (weed/Orobanche ramosa) showed that weeds non-parasitized in fields could be attacked by broomrape, developing a more or less complete cycle. In vitro co-cultures (weed/Orobanche ramosa) revealed that root exudates of non-parasitized weeds, in fields or in greenhouse co-cultures, could induce Orobanche ramosa seed germination, but not attachment. These weeds could play the role of false hosts.     

Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination

Six sunflower sesquiterpene lactone models which share structural features of the lactone rings of strigol and its synthetic analogues, the GR family, with different conformational flexibilities were tested as Orobanche cumana germination stimulants. Among them, parthenolide and 3,5-dihydroxydehydrocostus-lactone significantly increased O. cumana germination, presenting higher activity than GR-24, used as a standard in the germination bioassay. The effect of these two compounds is species-specific, showing no germination stimulant activity on other Orobanche spp. tested (O. crenata, O. ramosa and O. aegyptiaca). Data presented are discussed in terms of a structure-activity relationship.     

Conditioning duration and agents involved in broomrape seeds responding to germination stimulants

Weedy broomrape species, such as sunflower broomrape (Orobanche cumana Wallr.) and Egyptian broomrape [Phelipanche aegyptiaca Pers. (syn. O. aegyptiaca)], require a period of pre-conditioning before they can respond to germination stimulants. Thus, the sensitivity of weedy broomrape seeds to germination stimulants could be an important factor for broomrape control. In this study, the influence of conditioning agents, conditioning period (0–21 days) and germination stimulants on the germination of sunflower broomrape and Egyptian broomrape seeds was analyzed. Without conditioning, the sunflower and Egyptian broomrape seeds exhibited negligible germination responses to the stimulants. The germination rate of the broomrape seeds increased rapidly with conditioning period and reached a maximum under a conditioning period of 4–10 days; further prolonged conditioning resulted in a decrease in the germination rate. Gibberellic acid (GA₃) could not only break the dormancy of the sunflower and Egyptian broomrape seeds but also maintained the high sensitivity of these seeds even after 21 days of conditioning. Furthermore, 100 µM of GA₃ induced the germination of the Egyptian broomrape seeds. The stimulants that induced Egyptian broomrape germination were ranked in decreasing order as GR24 (76.8?%), strigol (76.1?%), tobacco root exudates (49.5?%), dehydrocostus lactones (DCL, 39.2?%), and maize root exudates (18?%). In contrast, GA₃ did not directly induce sunflower broomrape seed germination, which responded to strigol (62.8?%)?>?maize root exudates (58.2?%)?>?GR24 (57.9?%)?>?tobacco root exudates (41.6?%)?>?DCL (41.3?%). These results indicate specialized recognition of germination stimulants by sunflower and Egyptian broomrape. This study may contribute to a better understanding of parasitic weed germination and may lead to improved control strategies.     

Role of cAMP in Gibberellin Promotion of Seed Germination in <Emphasis Type="Italic">Orobanche minor</Emphasis> Smith

Adenosine 3′,5′-cyclic monophosphate (cAMP) is known as a key second messenger in many living organisms, regulating a wide range of cellular responses. In higher plants the function of cAMP is poorly understood. In this study, we examined the role of cAMP in seed germination of the root parasitic plant Orobanche minor whose seeds require preincubation in warm moist environments for several days, termed conditioning, prior to exposure to germination stimulants released from roots of host plants. Accumulation of endogenous cAMP was observed in the conditioned O. minor seeds. When the seeds were exposed to light or supraoptimal temperature during the conditioning period, cAMP did not accumulate and the seeds showed low germination rates after stimulation with strigol, a germination stimulant. Addition of membrane-permeable cAMP to the medium restored the germination rates of the seeds treated with light or supraoptimal temperature during the conditioning period, suggesting that cAMP functions during the conditioning period. The endogenous cAMP levels of the seeds conditioned in the light or at a supraoptimal temperature were elevated by treatment of the seeds with gibberellin (GA) during the conditioning period. Uniconazole, a potent inhibitor of GA biosynthesis, blocked elevation of the cAMP level. Furthermore, a correlation between the endogenous cAMP level and GA level was observed during the conditioning period. These results suggest that GAs elevate the cAMP level, which is required for the germination of O. minor seeds.     

Isolation of mannose 6-phosphate reductase cDNA,changes in enzyme activity and mannitol content in broomrape (Orobanche ramosa) parasitic on tomato roots

We are interested in developing a control strategy efficient at the early stages of subterranean development of Orobanche in the inhibition of mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), the key enzyme of mannitol production in the parasite. We examined M6PR gene expression during pre-conditioning, germination, procaulome growth, underground shoot development and emergence of Orobanche ramosa L. attached to tomato roots, the enzyme activity at each of the above stages and the level of stored mannitol in the parasite. A 1120-pb length cDNA isolated by 3' and 5'RACE was identified as a M6PR sequence by cDNA expression in E. coli and M6PR activity measurement. Only one M6PR gene was detected in O. ramosa following southern blot analysis. M6PR expression, analysed by RT-PCR, was constant from the pre-conditioned seed to the emergence of broomrape, i.e. M6PR expression is constitutive in Orobanche . M6PR activity was also detected in pre-conditioned seeds and attachment to tomato roots resulted in a two-fold increase in enzyme activity during tubercle enlargement and crown root formation. Hexose and mannitol accumulation was strongly enhanced in the attached parasite, with accumulation primarily in the shoot. These results support the prospect of utilizing M6PR inhibitors as early applied herbicides to control this parasite in the early stages of its development.     

Synthesis and seed germination stimulating activity of some imino analogs of strigolactones

Strigolactones are germination stimulants for seeds of the root parasitic weeds, Striga and Orobanche spp. The imino analog of GR24 showed moderate germination stimulating activity against the seeds of S. hermonthica. The seed germination stimulating activity of some phenyliminoacetates and phenyliminoacetonitriles was also examined. The degree of activity of the phenyliminoacetate was less than that of the phenylacrylates. On the other hand, the degree of activity of the phenyliminoacetonitrile was comparable to that of the phenylacrylonitriles. Among the tested compounds, the 3-pyridyliminoacetonitrile showed higher activity against the seeds of O. crenata than GR24. These findings demonstrate that it is not always essential to have the Michael acceptor of the C-D ring junction moiety which has been proposed to react with nucleophilic species presented at the target site to enhance the activity.     

Thermoinhibition uncovers a role for strigolactones in Arabidopsis seed germination

Strigolactones are host factors that stimulate seed germination of parasitic plant species such as Striga and Orobanche. This hormone is also important in shoot branching architecture and photomorphogenic development. Strigolactone biosynthetic and signaling mutants in model systems, unlike parasitic plants, only show seed germination phenotypes under limited growth condition. To understand the roles of strigolactones in seed germination, it is necessary to develop a tractable experimental system using model plants such as Arabidopsis. Here, we report that thermoinhibition, which involves exposing seeds to high temperatures, uncovers a clear role for strigolactones in promoting Arabidopsis seed germination. Both strigolactone biosynthetic and signaling mutants showed increased sensitivity to seed thermoinhibition. The synthetic strigolactone GR24 rescued germination of thermoinbibited biosynthetic mutant seeds but not a signaling mutant. Hormone analysis revealed that strigolactones alleviate thermoinhibition by modulating levels of the two plant hormones, GA and ABA. We also showed that GR24 was able to counteract secondary dormancy in Arabidopsis ecotype Columbia (Col) and Cape Verde island (Cvi). Systematic hormone analysis of germinating Striga helmonthica seeds suggested a common mechanism between the parasitic and non-parasitic seeds with respect to how hormones regulate germination. Thus, our simple assay system using Arabidopsis thermoinhibition allows comparisons to determine similarities and differences between parasitic plants and model experimental systems for the use of strigolactones.     

Fluridone and norflurazon, carotenoid-biosynthesis inhibitors, promote seed conditioning and germination of the holoparasite Orobanche minor

Fluridone and norflurazon, two carotenoid-biosynthesis inhibitors, shortened the conditioning period required by seeds of Orobanche minor in order to respond to the germination stimulant strigol. Neither fluridone nor norflurazon alone induced seed germination of O. minor , they promoted strigol-induced germination. In addition, these compounds restored the conditioning and germination of seeds at a supraoptimal temperature (30°C) as well as in the light. Gibberellic acid (GA₃) showed similar promotive and protective effects on the conditioning and germination of O. minor seeds. Although fluridone and norflurazon are known to prevent abscisic acid (ABA)-biosynthesis, and stresses such as supraoptimal temperatures have been reported to induce ABA accumulation in plants, the amount of ABA in the seeds or that released from the seeds into the conditioning media was not affected by the fluridone treatment and by exposure to the supraoptimal temperature. These results indicate that the promotive and protective effects of fluridone and norflurazon on the conditioning and germination of O. minor seeds would be attributed to other perturbations rather than the inhibition of ABA-biosynthesis.     

A Model of the Effects of a Wide Range of Constant and Alternating Temperatures on Seed Germination of FourOrobanche Species

Seeds of the obligate parasitic plants, Orobanche spp., wereconditioned in water or GA₃for 2 or 12 weeks and then stimulatedto germinate by the synthetic stimulant GR24. Temperature treatmentsduring the germination tests comprised 169 different constantand alternating temperature regimes on a two-dimensional gradientplate. Optimum temperatures for germination of seeds of O. aegyptiacaand O. crenata were 18–21 °C and 18 °C, respectively.However, longer conditioning periods slightly lowered the optimain both species, and the maximum germination percentage wasalso reduced due to an induction of secondary dormancy. At agiven mean temperature, more seeds germinated at constant thanat alternating temperatures. Results were analysed in termsof characteristics of alternating temperatures that appearedto control germination, i.e. mean temperature, maximum temperature,amplitude (difference between daily maximum and minimum temperatures)and thermoperiod (the time spent at the maximum temperatureeach day). Final germination was modelled on the basis of therebeing two prerequisites for germination: a minimum mean temperaturewhich must be exceeded and a maximum temperature above whichthe seed will not germinate. These two requirements were assumedto be independent and to be normally distributed in the seedpopulation so that final germination could be described by amultiplicative probability model. Because of the response tomaximum temperature, inhibitory effects were more evident atalternating temperatures. Amplitude and thermoperiod influencedthis effect of maximum temperature. The implications of thedetrimental effect of alternating temperatures for germinationofOrobanche spp. in the field are discussed. Copyright 1999Annals of Botany Company Orobanche aegyptiaca, O. crenata, O. cernua, O. minor, broomrape, seed germination, temperature, germination model, secondary dormancy.     

外源信号物质对肉苁蓉种子萌发与吸器形成的影响

研究氟草敏（norflurazon）、氟啶酮（fluridone）、GR24、赤霉素（GA3）4种外源信号物质对肉苁蓉种子萌发的影响,以及2,6-二甲氧基-对-苯醌（2,6-DMBQ）、5,8-二羟基萘醌（5,8-DHNQ）、阿魏酸（ferulic acid）3种外源信号物质对萌发的肉苁蓉种子吸器形成的影响,结果表明：氟草敏、氟啶酮、GR24对肉苁蓉种子的萌发均具有明显的促进作用,其中氟草敏作用最为显著,处理168h后肉苁蓉种子开始萌发,萌发率最高达65%;2,6-DMBQ对肉苁蓉种子吸器形成具有显著的促进作用,处理48h后肉苁蓉种子开始形成吸器,吸器形成率最高达50%。     

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.     

Fate of the soil seed bank of giant ragweed and its significance in preventing and controlling its invasion in grasslands

Giant ragweed (Ambrosia trifida, L. henceforth referred to as GR), an annual non‐native invasive weed, may cause health problems and can reduce agricultural productivity. Chemical control of GR in grasslands may have irreversible side effects on herbs and livestock. In an attempt to propose a solution to the harmful effects of GR on grasslands, this study explores the fate of its soil seed bank (SSB) and considers the physical control of its SSB reduction. By studying GR distributed in grasslands of the Yili Valley, Xinjiang, China, we measured the spatial and temporal changes in seed density, seed germination, dormancy, and death. We analyzed seed germination, dormancy, and death following different storage periods. The study analyzed population characteristics over time, including seed fate, and examined physical control methods for reducing the SSB density. The SSB of GR occurs in the upper 0–15 cm of soil in grasslands. Seed density in the SSB decreased by 68.1% to 82.01% from the reproductive growth period to the senescence period. More than 98.7% of the seeds were rotten, eaten, germinated, dispersed, or died within one year after being produced. The seed germination rate of the SSB decreased with the number of years after invasion. When stored for 0.5 or 3.5 years, seed germination rates fell by 40%, during which time seed death rate increased by almost 40%. When GR was completely eradicated for two consecutive years, the SSB and population densities decreased by >99%. The vast majority of GR seeds germinated or died within one year; the germination rate decreased significantly if the seeds were stored dry at room temperature for a long time. Newly produced seeds are the main source of seeds in the SSB. Therefore, thoroughly eradicating GR plants for several years before the seeds can mature provides an effective control method in grasslands.     

Responsiveness of Orobanche ramosa L. Seeds to GR 24 as Related to Temperature, Oxygen Availability and Water Potential During Preconditioning and Subsequent Germination

Broomrape (Orobanche ramosa L.) is a root holoparasite responsible for important yield losses in numerous crops, particularly in the Mediterranean area. In this paper, the effects of temperature, oxygen concentration and water potential of the medium on broomrape seed germination were investigated. Seeds became able to germinate in the presence of a strigol analogue (GR 24) only after a preincubation period for at least 3 days at 20 °C. Their responsiveness to GR 24 increased with increasing duration of their preconditioning at 20 °C, and was optimal after 2–3 weeks. The preconditioning treatment was effective at temperatures ranging from 10 to 30 °C. At the optimal temperature (20 °C), it required at least 1% oxygen in the atmosphere and remained effective at a water potential of the medium of –2 MPa. A too prolonged preincubation of seeds at sub- or supraoptimal temperatures (5 and 30 °C) resulted in induction of a secondary dormancy. Seeds preconditioned for 14 days at 20 °C germinated in the presence of 1 mg L^–1 GR 24 at temperatures ranging from 10 to 25 °C, and the thermal optimum was the same (20 °C) than that of preconditioning. At 20 °C, seeds were able to germinate in the presence of GR 24 under atmospheres containing at least 3% oxygen and at a water potential of the medium as low as –3 MPa. The differences observed in the effects of environmental factors on preconditioning efficiency and germination of preconditioned seeds suggest that both processes involve different mechanisms. The results obtained might also help to better understand the regulation of O. ramosa spread in temperate areas.     

Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation

* Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). * Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal branching assay with Gigaspora spp; and by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. * The root exudates of tomato cv. MoneyMaker induced O. ramosa seed germination and hyphal branching in AM fungi. Phosphate starvation markedly increased, and fluridone strongly decreased, this activity. Exudates of notabilis induced approx. 40% less germination than the wild-type. The LC-MS/MS analysis confirmed that the biological activity and changes therein were due to the presence of several strigolactones; orobanchol, solanacol and two or three didehydro-orobanchol isomers. * These results show that the AM branching factors and parasitic plant germination stimulants in tomato root exudate are strigolactones and that they are biosynthetically derived from carotenoids. The dual activity of these signalling compounds in attracting beneficial AM fungi and detrimental parasitic plants is further strengthened by environmental conditions such as phosphate availability.     

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.