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.     

An improved axenic system for studying pre-infection development of the parasitic plant Orobanche ramosa

BACKGROUND AND AIMS: Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control early stages of host infection in Orobanche are poorly understood, partly due to the lack of experimentally tractable in vitro systems that allow the efficient application of molecular tools. Here an improved axenic system for the analysis of pre-infection stages in O. ramosa in the absence of the host plant is described. METHODS: An optimized protocol for seed disinfection, based on formaldehyde, was developed. Orobanche ramosa seeds were conditioned in Petri dishes with filter paper, stimulated by addition of the synthetic strigol analogue GR24, and the percentage of germination as well as attachment-organ formation was determined. KEY RESULTS: Treatment of O. ramosa seeds with tobacco-root exudate or with GR24 resulted in highly reproducible germination rates around 70 %. A conditioning period of 8 d was both necessary and sufficient to allow optimal germination in response to GR24. Conditioned seeds that were dehydrated for several months remained fully responsive to GR24 without the need of a new conditioning period. Treatments as short as 5 min with GR24 were sufficient to fully and irreversibly induce the seed germination response. Approximately half of the germinated seeds initiated attachment-organ development. Similar rates of attachment organ induction were also detected in the rare cases of seeds that had germinated spontaneously on water. CONCLUSIONS: The results suggest that the conditioning period produces persistent changes in the seeds required for responsiveness to external stimulants. The rapid action of GR24 suggests that it may act via a receptor-mediated signalling mechanism. While germination in O. ramosa is induced by exogenous stimuli, attachment organ differentiation appears to be triggered by unknown endogenous signals. The new in vitro culture system will have useful applications for the molecular analysis of early stages of parasitic development in Orobanche.     

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.     

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.     

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.     

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.     

Recognition of root exudates by seeds of broomrape (Orobanche and Phelipanche) species

Background and Aims

The long co-existence of broomrapes and their hosts within the same environment has culminated in a strong adaptation and effective parasitism. As a first step of specialization in the parasitic process, seed receptors of parasitic plant species vary in their ability to recognize compounds released by their hosts. This work aims to investigate potential patterns for the reception requirements needed to activate germination within Orobanche and Phelipanche species.

Methods

Induction of the germination of seeds of nine Orobanche and Pheliphanche species by root exudates of 41 plant species was studied and subjected to biplot multivariate analysis.

Key Results

A high level of specialization in root exudate recognition was found in Orobanche densiflora, O. gracilis and O. hederae, which germinated almost exclusively in contact with root exudates from the plants they infect in nature. At the opposite extreme, Phelipanche aegyptiaca, P. ramosa and O. minor were highly generalist, germinating when in contact with the root exudates of most plant species. Orobanche crenata, O. cumana and O. foetida showed intermediate behaviour.

Conclusions

A universal germination stimulant for all broomrape species has not being identified to date. The synthetic stimulant GR24 is active against most of the weedy broomrape species, but fails with the non-weedy species tested in this study and with the very recent weedy species O. foetida. In addition, germination behaviour of broomrape species depends on the crop plant tested. Weedy broomrapes with a broad host spectrum respond better to the different exudates released by a wide range of crops and wild species than do non-weedy broomrapes, which have a narrow host spectrum and are more restricted to their host range. Root exudates of many plant species were active in stimulating germination of seeds of Orobanche and Phelipanche species for which they are not described as hosts, representing interesting examples of potential trap crops.Key words: Xenognosis, broomrape, root exudate, germination, Orobanche, Phelipanche     

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.     

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.     

A role for IAA in the infection of Arabidopsis thaliana by Orobanche aegyptiaca

BACKGROUND: Vascular continuity is established between a host plant and the root parasite broomrape. It is generally accepted that the direction of vascular continuity results from polar flow of auxin. Our hypothesis was that chemical disruptions of auxin transport and activity could influence the infection of the host by the parasite. METHODS: A sterile system for the routine infection of Arabidopsis thaliana seedlings in Nunc cell culture plates by germinated seeds of Orobanche aegyptiaca was developed. This method permitted a quantitative assay of the rate of host infection. The three-dimensional structure of the vascular contacts was followed in cleared tissue. IAA (indole acetic acid) or substances that influence its activity and transport were applied locally to the host root. RESULTS: The orientation of the xylem contacts showed that broomrape grafts itself upon the host by acting hormonally as a root rather than a shoot. Local applications of IAA, PCIB (p-chlorophenoxyisobutyric acid) or NPA (naphthylphthalamic acid) all resulted in drastic reductions of Orobanche infection CONCLUSIONS: Broomrape manipulates the host by acting as a sink for auxin. Disruption of auxin action or auxin flow at the contact site could be a novel basis for controlling infection by Orobanche.     

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.     

Exogenous amino acids inhibit seed germination and tubercle formation by Orobanche ramosa (Broomrape): Potential application for management of parasitic weeds

Parasitic plants are among the most problematic weeds that are responsible for major losses to many crops. Early growth stages, such as seed germination stimulated by host root exudates and tubercle development, are key phases for the development of these parasites. Inhibition of these early phases by naturally occurring compounds could be a general strategic option for management of parasitic plants. In this study, we report that certain natural amino acids cause severe physiological disorders of germinating broomrape seeds. In particular, methionine was able to inhibit almost totally the germination of seeds of Orobanche ramosa when applied at a concentration of 2 mM. Applied to tomato roots, methionine strongly reduced the number of developing tubercles of the parasite. These findings suggest that: (1) appropriate amino acids applied exogenously to a root zone might result in control of parasitic plants such as Orobanche sp.; (2) amino acid excreting microbes introduced into the crop rhizosphere might control root parasitism; and (3) amino acid producing broomrape pathogens might be selected in order to enhance their virulence.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Stimulation of Orobanche ramosa seed germination by fusicoccin derivatives: a structure-activity relationship study

A structure-activity relationship study was conducted assaying 25 natural analogues and derivatives of fusicoccin (FC), and cotylenol, the aglycone of cotylenins, for their ability to stimulate the seed germination of the parasitic species Orobanche ramosa. Some of the compounds tested proved to be highly active, being 8,9-isopropylidene of the corresponding FC aglycone and the dideacetyl derivative the most active FC derivatives. In both groups of glucosides and aglycones (including cotylenol), the most important structural feature to impart activity appears to be the presence of the primary hydroxy group at C-19. Furthermore, the functionalities and the conformation of the carbotricyclic ring proved to play a significant role. The dideacetyl derivative of FC, being easily and rapidly obtainable in high yield starting by FC, could be of interest for its practical application as a stimulant of Orobanche ramosa seed germination, inducing the "suicidal germination", an interesting approach for parasitic plant management.     

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.     

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.     

Reduced Germination of Orobanche cumana Seeds in the Presence of Arbuscular Mycorrhizal Fungi or Their Exudates

Broomrapes (Orobanche and Phelipanche spp) are parasitic plants responsible for important crop losses, and efficient procedures to control these pests are scarce. Biological control is one of the possible strategies to tackle these pests. Arbuscular Mycorrhizal (AM) fungi are widespread soil microorganisms that live symbiotically with the roots of most plant species, and they have already been tested on sorghum for their ability to reduce infestation by witchweeds, another kind of parasitic plants. In this work AM fungi were evaluated as potential biocontrol agents against Orobanche cumana, a broomrape species that specifically attacks sunflower. When inoculated simultaneously with O. cumana seeds, AM fungi could offer a moderate level of protection against the broomrape. Interestingly, this protection did not only rely on a reduced production of parasitic seed germination stimulants, as was proposed in previous studies. Rather, mycorrhizal root exudates had a negative impact on the germination of O. cumana induced by germination stimulants. A similar effect could be obtained with AM spore exudates, establishing the fungal origin of at least part of the active compounds. Together, our results demonstrate that AM fungi themselves can lead to a reduced rate of parasitic seed germination, in addition to possible effects mediated by the mycorrhizal plant. Combined with the other benefits of AM symbiosis, these effects make AM fungi an attractive option for biological control of O. cumana.     

小花草玉梅种子萌发过程中常见内源激素含量的变化及萌发机制

植物内源激素在调节种子休眠和萌发过程中具有极其重要的作用。本研究运用反式高效液相色谱(RP-HPLC)与紫外检测器联用的方法对小花草玉梅干种子、吸胀种子和露白种子中的赤霉素GAs、脱落酸ABA、玉米素ZT和生长素IAA含量进行了检测,旨在研究植物内源激素水平在种子萌发过程中的变化是否直接关系着小花草玉梅种子在光照或黑暗条件下的萌发能力。结果显示,种子吸胀过程中,光照促进了ZT同时抑制了ABA的积累,并且ZT对ABA萌发抑制作用的解除也受光的促进,露白种子中的ABA/ZT,ABA/GAs和(ABA+IAA)/(GAs+ZT)水平在黑暗条件下高于光照条件,上述均是导致光照条件下种子萌发率较高的重要原因;相对于干种子,IAA含量在种子吸胀初期急速下降,(ABA+IAA)/(GAs+ZT)在种子萌发过程中有所降低,而ABA/GAs却表现出明显的上升趋势;各激素水平所受光照的影响均在种子开始露白时显著减弱,另外,吸胀第9天是小花草玉梅种子萌发过程中激素变化的一个关键的时间节点。总之,种子萌发并非直接关系着GA含量的升高和ABA含量的降低。因光照直接促进了小花草玉梅种子的萌发,本研究认为高寒草甸充...