Mónica Fernández-Aparicio

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.

Breeding approaches for crenate broomrape (Orobanche crenata Forsk.) management in pea (Pisum sativum L.).

BACKGROUND Pea cultivation is strongly hampered in Mediterranean and Middle East farming systems by the occurrence of Orobanche crenata Forsk. Strategies of control have been developed, but only marginal successes have been achieved. Most control methods are either unfeasible, uneconomical, hard to achieve or result in incomplete protection. The integration of several control measures is the most desirable strategy. RESULTS [corrected] Recent developments in control are presented and re-evaluated in light of recent developments in crop breeding and molecular genetics. These developments are placed within a framework that is compatible with current agronomic practices. CONCLUSION The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow. The application of knowledge gained from basic genomic research and genetic engineering will contribute to more rapid pea improvement for resistance against O. crenata and/or the herbicide.

The Parasitic Plant Genome Project: New Tools for Understanding the Biology of Orobanche and Striga

Abstract The Parasitic Plant Genome Project has sequenced transcripts from three parasitic species and a nonparasitic relative in the Orobanchaceae with the goal of understanding genetic changes associated with parasitism. The species studied span the trophic spectrum from free-living nonparasite to obligate holoparasite. Parasitic species used were Triphysaria versicolor, a photosynthetically competent species that opportunistically parasitizes roots of neighboring plants; Striga hermonthica, a hemiparasite that has an obligate need for a host; and Orobanche aegyptiaca, a holoparasite with absolute nutritional dependence on a host. Lindenbergia philippensis represents the closest nonparasite sister group to the parasitic Orobanchaceae and was included for comparative purposes. Tissues for transcriptome sequencing from each plant were gathered to identify expressed genes for key life stages from seed conditioning through anthesis. Two of the species studied, S. hermonthica and O. aegyptiaca, are economically important weeds and the data generated by this project are expected to aid in research and control of these species and their relatives. The sequences generated through this project will provide an abundant resource of molecular markers for understanding population dynamics, as well as provide insight into the biology of parasitism and advance progress toward understanding parasite virulence and host resistance mechanisms. In addition, the sequences provide important information on target sites for herbicide action or other novel control strategies such as trans-specific gene silencing. Nomenclature: Egyptian broomrape, Orobanche aegyptiaca (Pers.) (Syn. Phelipanche aegyptiaca) ORAAE; Lindenbergia philippensis (Cham. & Schltdl.) Benth. LINPH; yellowbeak owls-clover, Triphysaria versicolor (Fisch. & C.A. Mey) TRVEV; purple witchweed, Striga hermonthica, (Del.) Benth. STRHE.

The role of strigolactones in host specificity of Orobanche and Phelipanche seed germination

Strigolactones are apocarotenoids regulating shoot branching. They are also known to be exuded by plant roots at very low concentrations, stimulating hyphal branching of arbuscular mycorrhizal fungi and germination of root parasitic weed seeds. We show that strigolactones play a major role in host specificity of Orobanche and Phelipanche (the broomrapes) seed germination. This observation confirms that hostderived germination stimulants are an important component determining the host specificity of these parasitic plants. Weedy broomrape species were less specialized in germination requirements than the nonweedy species except for O. cumana and O. foetida var. broteri. Similar results were obtained with the root exudates. Some species, such as P. aegyptiaca and O. minor, showed a broad spectrum of host specificity in terms of seed germination, which was stimulated by exudates from the majority of species tested, whereas others, such as O. cumana, O. hederae and O. densiflora, were highly specific. Some species, such as O. minor, P. aegyptiaca and P. nana, were responsive to the three strigolactones studied, whereas others were induced by only one of them, or did not respond to them at all. The synthetic strigolactone analogue GR24, generally used as a standard for germination tests, was not effective on some Orobanche and Phelipanche species. Seeds of some species that did not respond to GR24 were induced to germinate in the presence of fabacyl acetate or strigol, confirming the role of strigolactones in host specificity.

Innovations in parasitic weeds management in legume crops. A review

Parasitic weeds decrease severely the production of major grain and forage legumes. The most economically damaging weeds for temperate legumes are broomrapes, in particular Orobanche crenata. Broomrape species such as Orobanche foetida, Orobanche minor, and Phelipanche aegyptiaca can also induce high local damage. Other parasitic weeds such as Striga gesnerioides and Alectra vogelii decrease yield of legume crops throughout semi-arid areas of sub-Saharan Africa. Dodders such as Cuscuta campestris can be damaging for some crops. Here, we review methods to control parasitic weeds. Preventing the movement of weed seeds into uninfested areas is a crucial component of control. Once a field is infested with parasitic weeds, controlling its seed production is very difficult. The only effective way to cope with parasitic weeds is to apply an integrated approach. Seedbank demise can be achieved by fumigation and solarization. However, this method is not economically feasible for low-value and low-input legume crops. A number of cultural practices including delayed sowing, hand weeding, no-tillage, nitrogen fertilization, intercropping, or rotations can contribute to seed bank demise. Other strategies such as suicidal germination, activation of systemic acquired resistance, biocontrol or target site herbicide resistance are promising solutions that are being explored but are not yet ready for direct application. The only methods currently available to farmers are the use of resistant varieties and chemical control, although both have their limitations. Chemical control with systemic herbicides such as glyphosate or imidazolinones at low rates is possible. Advances in modeling and the availability of new technologies allow the development of precision agriculture or site-specific farming. The most economical and environmentally friendly control option is the use of resistant crop varieties; however, breeding for resistance is a difficult task considering the scarce and complex nature of resistance in most crops. These strategies for parasitic weed management in legume crops will be presented and critically discussed.

Comparative Transcriptome Analyses Reveal Core Parasitism Genes and Suggest Gene Duplication and Repurposing as Sources of Structural Novelty

The origin of novel traits is recognized as an important process underlying many major evolutionary radiations. We studied the genetic basis for the evolution of haustoria, the novel feeding organs of parasitic flowering plants, using comparative transcriptome sequencing in three species of Orobanchaceae. Around 180 genes are upregulated during haustorial development following host attachment in at least two species, and these are enriched in proteases, cell wall modifying enzymes, and extracellular secretion proteins. Additionally, about 100 shared genes are upregulated in response to haustorium inducing factors prior to host attachment. Collectively, we refer to these newly identified genes as putative “parasitism genes.” Most of these parasitism genes are derived from gene duplications in a common ancestor of Orobanchaceae and Mimulus guttatus, a related nonparasitic plant. Additionally, the signature of relaxed purifying selection and/or adaptive evolution at specific sites was detected in many haustorial genes, and may play an important role in parasite evolution. Comparative analysis of gene expression patterns in parasitic and nonparasitic angiosperms suggests that parasitism genes are derived primarily from root and floral tissues, but with some genes co-opted from other tissues. Gene duplication, often taking place in a nonparasitic ancestor of Orobanchaceae, followed by regulatory neofunctionalization, was an important process in the origin of parasitic haustoria.

Identification of quantitative trait loci for specific mechanisms of resistance to Orobanche crenata Forsk. in pea (Pisum sativum L.)

Crenate broomrape (Orobanche crenata) is the major constraint for pea cultivation in the Mediterranean Basin and Middle East. Cultivation of resistant varieties would be the most efficient, economical and environmentally friendly way to control this parasite. However, little resistance is available within cultivated pea. Promising sources of resistance have been identified in wild peas but their use in breeding programs is hampered by the polygenic nature of the resistance. The identification of molecular markers linked to the resistance would allow tracking of the underlying genes, facilitating their introgression into pea cultivars. The main objective of this study was the identification of genomic regions associated with resistance to O. crenata. A RIL (Recombinant Inbred Lines) population derived from a cross between a resistant accession of the wild pea Pisum sativum ssp. syriacum, and a susceptible pea variety was screened for resistance to O. crenata under field conditions during two seasons. In addition, resistance reactions at different stages of the O. crenata infection cycle were assessed using a Petri dish method. The approach allowed the identification of four Quantitative Trait Loci (QTL) associated with field resistance, assessed as the number of emerged broomrape shoots per pea plant under field conditions. These identified QTLs explained individually from 10 to 17% of the phenotypic variation. In addition QTLs governing specific mechanisms of resistance, such as low induction of O. crenata seed germination, lower number of established tubercles per host root length unit, and slower development of tubercles were also identified. Identified QTLs explained individually from 8 to 37% of the variation observed depending on the trait. Host plant aerial biomass and root length were also assessed and mapped. Both traits were correlated with the level of O. crenata infection and three out of the four QTLs controlling resistance under field conditions co-localized with QTLs controlling plant aerial biomass or root length. The relationship observed among these traits and resistance is discussed.

Polyphenols, Including the New Peapolyphenols A−C, from Pea Root Exudates Stimulate Orobanche foetida Seed Germination

Three new polyphenols, named peapolyphenols A-C, together with an already well-known polyphenol and a chalcone (1-(2,4-dihydroxyphenyl)-3-hydroxy-3-(4-hydroxyphenyl)-1-propanone and 1-(2,4-dihydroxyphenyl)-3-(4-methoxyphenyl)propenone) were isolated from pea root exudates. They were found to strongly stimulate Orobanche and Phelipanche species seed germination. Interestingly, only peapolyphenol A, 1,3,3-substituted propanone, and 1,3-disubstituted propenone had specific stimulatory activity on O. foetida, excluding any other Orobanche or Phelipanche species tested. This species specificity is relevant, as O. foetida does not respond to the synthetic strigolactone analogue GR24, commonly used as a standard for germination assays. As characterized by spectroscopic methods, peapolyphenols A-C proved to be differently functionalized polyphenols with hydroxy and methoxy groups on both the aromatic rings and the propyl chain.

Differential expression proteomics to investigate responses and resistance to Orobanche crenata in Medicago truncatula

BackgroundParasitic angiosperm Orobanche crenata infection represents a major constraint for the cultivation of legumes worldwide. The level of protection achieved to date is either incomplete or ephemeral. Hence, an efficient control of the parasite requires a better understanding of its interaction and associated resistance mechanisms at molecular levels.ResultsIn order to study the plant response to this parasitic plant and the molecular basis of the resistance we have used a proteomic approach. The root proteome of two accessions of the model legume Medicago truncatula displaying differences in their resistance phenotype, in control as well as in inoculated plants, over two time points (21 and 25 days post infection), has been compared. We report quantitative as well as qualitative differences in the 2-DE maps between early- (SA 27774) and late-resistant (SA 4087) genotypes after Coomassie and silver-staining: 69 differential spots were observed between non-inoculated genotypes, and 42 and 25 spots for SA 4087 and SA 27774 non-inoculated and inoculated plants, respectively. In all, 49 differential spots were identified by peptide mass fingerprinting (PMF) following MALDI-TOF/TOF mass spectrometry. Many of the proteins showing significant differences between genotypes and after parasitic infection belong to the functional category of defense and stress-related proteins. A number of spots correspond to proteins with the same function, and might represent members of a multigenic family or post-transcriptional forms of the same protein.ConclusionThe results obtained suggest the existence of a generic defense mechanism operating during the early stages of infection and differing in both genotypes. The faster response to the infection observed in the SA 27774 genotype might be due to the action of proteins targeted against key elements needed for the parasites successful infection, such as protease inhibitors. Our data are discussed and compared with those previously obtained with pea [1] and transcriptomic analysis of other plant-pathogen and plant-parasitic plant systems.

Soyasapogenol B and trans-22-dehydrocam- pesterol from common vetch (Vicia sativa L.) root exudates stimulate broomrape seed germination

BACKGROUND Orobanche and Phelipanche species (the broomrapes) are root parasitic plants, some of which represent serious weed problems causing severe yield losses on important crops. Control strategies have largely focused on agronomic practices, resistant crop varieties and herbicides, albeit with marginal success. An alternative control method is the induction of suicidal seed germination with natural substances isolated from root exudates of host and non-host plants. RESULTS Soyasapogenol B [olean-12-ene-3,22,24-triol(3β,4β,22β)] and trans-22-dehydrocampesterol [(ergosta-5,22-dien-3-ol, (3β,22E,24S)] were isolated from Vicia sativa root exudates. They were identified by comparing their spectroscopic and optical properties with those reported in the literature. Soyasapogenol B was very specific, stimulating the germination of O. minor seeds only, whereas trans-22-dehydrocampesterol stimulated P. aegyptiaca, O. crenata, O. foetida and O. minor. CONCLUSION Soyasapogenol B and trans-22-deydrocampesterol were isolated for the first time from Vicia sativa root exudates, and their biological activity as stimulants of Orobanche or Phelipanche sp. seed germination was reported.