RESUMEN
Phelipanche ramosa is a root parasitic plant fully dependent on host plants for nutrition and development. Upon germination, the parasitic seedling develops inside the infected roots a specific organ, the haustorium, thanks to the cell wall-degrading enzymes of haustorial intrusive cells, and induces modifications in the host's cell walls. The model plant Arabidopsis thaliana is susceptible to P. ramosa; thus, mutants in cell wall metabolism, particularly those involved in pectin remodeling, like Atpme3-1, are of interest in studying the involvement of cell wall-degrading enzymes in the establishment of plant-plant interactions. Host-parasite co-cultures in mini-rhizotron systems revealed that parasite attachments are twice as numerous and tubercle growth is quicker on Atpme3-1 roots than on WT roots. Compared to WT, the increased susceptibility in AtPME3-1 is associated with reduced PME activity in the roots and a lower degree of pectin methylesterification at the host-parasite interface, as detected immunohistochemically in infected roots. In addition, both WT and Atpme3-1 roots responded to infestation by modulating the expression of PAE- and PME-encoding genes, as well as related global enzyme activities in the roots before and after parasite attachment. However, these modulations differed between WT and Atpme3-1, which may contribute to different pectin remodeling in the roots and contrasting susceptibility to P. ramosa. With this integrative study, we aim to define a model of cell wall response to this specific biotic stress and indicate, for the first time, the role of PME3 in this parasitic plant-plant interaction.
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Interactions among plants have been long recognized as a major force driving plant community dynamics and crop yield. Surprisingly, our knowledge of the ecological genetics associated with variation of plant-plant interactions remains limited. In this opinion article by scientists from complementary disciplines, the international PLANTCOM network identified four timely questions to foster a better understanding of the mechanisms mediating plant assemblages. We propose that by identifying the key relationships among phenotypic traits involved in plant-plant interactions and the underlying adaptive genetic and molecular pathways, while considering environmental fluctuations at diverse spatial and time scales, we can improve predictions of genotype-by-genotype-by-environment interactions and modeling of productive and stable plant assemblages in wild habitats and crop fields.
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Ecosistema , Plantas , Genotipo , Fenotipo , Plantas/genéticaRESUMEN
Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species.
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Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.
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Compuestos Heterocíclicos con 3 Anillos/metabolismo , Hidrolasas/genética , Isotiocianatos/metabolismo , Lactonas/metabolismo , Orobanchaceae/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Europa (Continente) , Hidrolasas/química , Hidrolasas/metabolismo , Orobanchaceae/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Malezas/genética , Malezas/metabolismo , Alineación de SecuenciaRESUMEN
In angiosperms, the α/ß hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (-)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (-)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.
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Bryopsida/genética , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Lactonas/metabolismo , Orobanchaceae/fisiología , Proteínas de Plantas/genética , Bryopsida/metabolismo , Bryopsida/parasitología , Proteínas de Plantas/metabolismoRESUMEN
Strigolactones are a class of plant hormones involved in shoot branching, growth of symbiotic arbuscular mycorrhizal fungi, and germination of parasitic plant seeds. Assaying new molecules or compound exhibiting strigolactone-like activities is therefore important but unfortunately time-consuming and hard to implement because of the extremely low concentrations at which they are active. Seeds of parasite plants are natural integrator of these hormones since they can perceive molecule concentrations in the picomolar to nanomolar range stimulating their germination. Here we describe a simple and inexpensive method to evaluate the activity of these molecules by scoring the germination of parasitic plant seeds upon treatment with these molecules. Up to four molecules can be assayed from a single 96-well plate by this method. A comparison of SL-like bioactivities between molecules is done by determining the EC50 and the maximum percentage of germination.
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Bioensayo , Germinación/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Lactonas/metabolismo , Orobanche/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/farmacología , Semillas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Orobanche/embriología , Semillas/embriologíaRESUMEN
Parasitic plants rely on neighboring host plants to complete their life cycle, forming vascular connections through which they withdraw needed nutritive resources. In natural ecosystems, parasitic plants form one component of the plant community and parasitism contributes to overall community balance. In contrast, when parasitic plants become established in low biodiversified agroecosystems, their persistence causes tremendous yield losses rendering agricultural lands uncultivable. The control of parasitic weeds is challenging because there are few sources of crop resistance and it is difficult to apply controlling methods selective enough to kill the weeds without damaging the crop to which they are physically and biochemically attached. The management of parasitic weeds is also hindered by their high fecundity, dispersal efficiency, persistent seedbank, and rapid responses to changes in agricultural practices, which allow them to adapt to new hosts and manifest increased aggressiveness against new resistant cultivars. New understanding of the physiological and molecular mechanisms behind the processes of germination and haustorium development, and behind the crop resistant response, in addition to the discovery of new targets for herbicides and bioherbicides will guide researchers on the design of modern agricultural strategies for more effective, durable, and health compatible parasitic weed control.
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Seeds of the parasitic weed Phelipanche ramosa are well adapted to their hosts because they germinate and form haustorial structures to connect to roots in response to diverse host-derived molecular signals. P. ramosa presents different genetic groups that are preferentially adapted to certain hosts. Since there are indications that microbes play a role in the interaction especially in the early stages of the interaction, we studied the microbial diversity harbored by the parasitic seeds with respect to their host and genetic group. Twenty-six seed lots from seven cropping plots of three different hosts-oilseed rape, tobacco, and hemp-in the west of France were characterized for their bacterial and fungal communities using 16S rRNA gene and ITS (Internal transcribed spacer) sequences, respectively. First seeds were characterized genetically using twenty microsatellite markers and phenotyped for their sensibility to various germination stimulants including strigolactones and isothiocyanates. This led to the distinction of three P. ramosa groups that corresponded to their host of origin. The observed seed diversity was correlated to the host specialization and germination stimulant sensitivity within P. ramosa species. Microbial communities were both clustered by host and plot of origin. The seed core microbiota was composed of seventeen species that were also retrieved from soil and was in lower abundances for bacteria and similar abundances for fungi compared to seeds. The host-related core microbiota of parasitic seeds was limited and presumably well adapted to the interaction with its hosts. Two microbial candidates of Sphingobacterium species and Leptosphaeria maculans were especially identified in seeds from oilseed rape plots, suggesting their involvement in host recognition and specialization as well as seed fitness for P. ramosa by improving the production of isothiocyanates from glucosinolates in the rhizosphere of oilseed rape.
RESUMEN
Karrikins stimulate Arabidopsis thaliana germination, whereas parasitic weeds of the Orobanchaceae family have evolved to respond to host-exuded compounds such as strigolactones, dehydrocostus lactone, and 2-phenylethyl isothiocyanate. In Phelipanche ramosa, strigolactone-induced germination was shown to require one of the CYP707A proteins involved in abscisic acid catabolism. Here, germination and gene expression were analysed to investigate the role of CYP707As in germination of both parasitic plants and Arabidopsis upon perception of germination stimulants, after using pharmacological inhibitors and Arabidopsis mutants disrupting germination signals. CYP707A genes were up-regulated upon treatment with effective germination stimulants in both parasitic plants and Arabidopsis. Obligate parasitic plants exhibited both intensified up-regulation of CYP707A genes and increased sensitivity to the CYP707A inhibitor abscinazole-E2B, whereas Arabidopsis cyp707a mutants still positively responded to germination stimulation. In Arabidopsis, CYP707A regulation required the canonical karrikin signalling pathway KAI2/MAX2/SMAX1 and the transcription factor WRKY33. Finally, CYP707As and WRKY33 also modulated Arabidopsis root architecture in response to the synthetic strigolactone rac-GR24, and wrky33-1 exhibited a shoot hyperbranched phenotype. This study suggests that the lack of host-independent germination in obligate parasites is associated with an exacerbated CYP707A induction and that CYP707As and WRKY33 are new players involved in a variety of strigolactone/karrikin responses.
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Arabidopsis/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Germinación , Orobanchaceae/enzimología , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Furanos/metabolismo , Hidrolasas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Piranos/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Obligate root-parasitic plants belonging to the Orobanchaceae family are deadly pests for major crops all over the world. Because these heterotrophic plants severely damage their hosts even before emerging from the soil, there is an unequivocal need to design early and efficient methods for their control. The germination process of these species has probably undergone numerous selective pressure events in the course of evolution, in that the perception of host-derived molecules is a necessary condition for seeds to germinate. Although most of these molecules belong to the strigolactones, structurally different molecules have been identified. Since strigolactones are also classified as novel plant hormones that regulate several physiological processes other than germination, the use of autotrophic model plant species has allowed the identification of many actors involved in the strigolactone biosynthesis, perception, and signal transduction pathways. Nevertheless, many questions remain to be answered regarding the germination process of parasitic plants. For instance, how did parasitic plants evolve to germinate in response to a wide variety of molecules, while autotrophic plants do not? What particular features are associated with their lack of spontaneous germination? In this review, we attempt to illustrate to what extent conclusions from research into strigolactones could be applied to better understand the biology of parasitic plants.
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Germinación , Lactonas/metabolismo , Orobanchaceae/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Semillas/crecimiento & desarrollo , Orobanchaceae/crecimiento & desarrollo , Malezas/crecimiento & desarrollo , Malezas/metabolismo , Transducción de SeñalRESUMEN
The heterotrophic lifestyle of parasitic plants relies on the development of the haustorium, a specific infectious organ required for attachment to host roots. While haustorium development is initiated upon chemodetection of host-derived molecules in hemiparasitic plants, the induction of haustorium formation remains largely unknown in holoparasitic species such as Phelipanche ramosa. This work demonstrates that the root exudates of the host plant Brassica napus contain allelochemicals displaying haustorium-inducing activity on P. ramosa germinating seeds, which increases the parasite aggressiveness. A de novo assembled transcriptome and microarray approach with P. ramosa during early haustorium formation upon treatment with B. napus root exudates allowed the identification of differentially expressed genes involved in hormone signaling. Bioassays using exogenous cytokinins and the specific cytokinin receptor inhibitor PI-55 showed that cytokinins induced haustorium formation and increased parasite aggressiveness. Root exudates triggered the expression of cytokinin-responsive genes during early haustorium development in germinated seeds, and bio-guided UPLC-ESI(+)-/MS/MS analysis showed that these exudates contain a cytokinin with dihydrozeatin characteristics. These results suggest that cytokinins constitutively exudated from host roots play a major role in haustorium formation and aggressiveness in P. ramosa.
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Brassica napus/parasitología , Citocininas/metabolismo , Orobanche/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Orobanche/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiologíaRESUMEN
Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (Phelipanche aegyptiaca, Orobanche cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa, and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.
RESUMEN
BACKGROUND: Some radiation-mutagenised chickpea mutants potentially resistant to the broomrape, Orobanche foetida Poir., were selected through field trials. The objectives of this work were to confirm resistance under artificial infestation, in pots and mini-rhizotron systems, and to determine the developmental stages of broomrape affected by resistance and the relevant resistance mechanisms induced by radiation mutagenesis. RESULTS: Among 30 mutants tested for resistance to O. foetida, five shared strong resistance in both pot experiments and mini-rhizotron systems. Resistance was not complete, but the few individuals that escaped resistance displayed high disorders of shoot development. Results demonstrated a 2-3-fold decrease in stimulatory activity of root exudates towards broomrape seed germination in resistant mutants in comparison with non-irradiated control plants and susceptible mutants. Resistance was associated with an induction of broomrape necrosis early during infection. When infested, most of the resistant mutants shared enhanced levels of soluble phenolic contents, phenylalanine ammonia lyase activity, guaiacol peroxidase activity and polyphenol oxidase activity, in addition to glutathione and notably ascorbate peroxidase gene expression in roots. CONCLUSION: Results confirmed enhanced resistance in chickpea radiation-mutagenised mutants, and demonstrated that resistance is based on alteration of root exudation, presumed cell-wall reinforcement and change in root oxidative status in response to infection. © 2016 Society of Chemical Industry.
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Cicer/genética , Orobanche/fisiología , Malezas/fisiología , Biomarcadores , Catecol Oxidasa/metabolismo , Cicer/fisiología , Cicer/efectos de la radiación , Germinación , Mutagénesis/efectos de la radiación , Peroxidasa/metabolismo , Fenotipo , Exudados de Plantas/farmacología , Raíces de Plantas/fisiología , Semillas/crecimiento & desarrolloRESUMEN
The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in concordance with the PrSUT3 mRNA accumulation in multiple sink areas including apical meristem, scale-leaf primordia, immature vasculature and even storage parenchyma. However, the PrSUT3 transcripts did not accumulate in the mature phloem cells. The transient expression experiments in Arabidopsis protoplasts suggested a tonoplast localization of PrSUT3, for which nevertheless the involvement in intracellular sucrose transport needs clarification.
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Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1.
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Sistema Enzimático del Citocromo P-450/metabolismo , Lactonas/farmacología , Orobanche/fisiología , Semillas/fisiología , Ácido Abscísico/metabolismo , Azacitidina/farmacología , Secuencia de Bases , Sistema Enzimático del Citocromo P-450/genética , Metilación de ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Germinación/efectos de los fármacos , Hidroxiurea/farmacología , Datos de Secuencia Molecular , Orobanche/efectos de los fármacos , Latencia en las Plantas/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/parasitología , Semillas/efectos de los fármacos , Análisis de Secuencia de ADNRESUMEN
An AccQâ¢Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQâ¢Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.
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Amoníaco/análisis , Brassica napus/metabolismo , Glutamina/análisis , Nitrógeno/análisis , Orobanche/metabolismo , Raíces de Plantas/metabolismo , Amoníaco/metabolismo , Brassica napus/química , Brassica napus/parasitología , Cromatografía Líquida de Alta Presión/métodos , Glutamina/metabolismo , Límite de Detección , Nitrógeno/metabolismo , Isótopos de Nitrógeno , Orobanche/química , Raíces de Plantas/química , Raíces de Plantas/parasitología , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
BACKGROUND: Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope. RESULTS: We developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann's protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose-response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves. CONCLUSION: The developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination.
RESUMEN
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.