Molecular Identification of Broomrape Species from a Single Seed by High Resolution Melting Analysis

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 (P. aegyptiaca, O. 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 fulfils 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

The dynamics of faba bean (Vicia faba L.) parasitism by Orobanche foetida

The dynamics of Orobanche foetida parasitizing faba bean are examined using Petri dish experiments. Rates of broomrape seed germination and seedling attachment to the host roots were quantified on three resistant genotypes (the Egyptian line Giza 429, the Spanish cultivar Baraca, and the Tunisian cultivar Najeh [XBJ90.03-16-1-1-1]) and the susceptible cv. Bachaar. The percentage of O. foetida seed germination (11 to 38%) was lower near the roots of resistant host plants than it was near the roots of ‘Bachaar’ (67%). O. foetida parasitism was followed using three parametric logistic functions. In this way some major parameters of the infection process were quantified: the maximal number (Nmax) and the maximal rate (Rmax) of broomrape attachments to the host roots, the median time required for attachment (T50), the maximal percentage of established tubercles reaching the final growth stage at 70 days after inoculation (DAI) (%max), and the maximal rate of established tubercle growth (R’max). Broomrape attachment was lower and slower in resistant plants, as indicated by low Nmax and Rmax values combined with high T50 values. Furthermore the precocity of the resistant genotypes was correlated with low attachment. The parameters %max and R’max did not discriminate the susceptible cultivar Bachaar from Giza 429 or Baraca. On the other hand, the %max and the R’max were lower in the ‘Najeh’ plants. The findings indicated that both low attachment and limited growth of established tubercles contributed to resistance in the Najeh cultivar

Wild Helianthus species: A reservoir of resistance genes for sustainable pyramidal resistance to broomrape in sunflower

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.This study was performed in the frame of a 3-year project (ResODiv), funded by “Promosol” (the association of French Sunflower and Rapeseed Breeders for promoting these crops).Peer reviewe

Aspects moléculaires de la réponse de génotypes sensible et résistant de tournesol à l'Orobanche

NANTES-BU Sciences (441092104) / SudocSudocFranceF

Purification et clonage d'une rhamnogalacturonase tolérante à un substrat acétylé

Les rhamnogalacturonases (RGases) connues jusqu à présent sont gênées par la présence de substituants acétyles sur leur substrat, les rhamnogalacturonanes (RG). Une nouvelle RGase ayant la particularité de pouvoir dégrader des RG fortement acétylés a été détectée dans le mélange enzymatique commercial Driselase®, issu du basidiomycète Irpex lacteus. Afin de suivre la purification de cette enzyme, puis de la caractériser, des RG ayant un degré d acétylation de 45 ont été préparés par dégradation enzymatique et fractionnements chromatographiques de pectines de betterave. La RGase a ensuite été purifiée à partir de Driselase® en mettant en place et en optimisant différentes techniques de fractionnement permettant d éliminer les protéines contaminantes présentes dans le mélange. La RGase purifiée a une masse molaire de 55 kDa et ses optima de pH et température sont compris respectivement entre pH 4,5-5 et 40-50C. Les produits de dégradation de l enzyme ont été analysés par spectrométrie de masse afin de déterminer son mode d action. Il a alors été constaté que la RGase tolère la présence d acétyle sur l acide galacturonique réducteur des oligomères produits. Parallèlement un ADNc pleine longueur de 1460 pb codant pour une RGase a été isolé à partir de cultures d Irpex lacteus grâce à des stratégies RT et RACE PCR. Cet ADNc a été cloné chez le système d expression hétérologue Pichia pastoris afin de produire une RGase recombinante. Plusieurs clones présentant un haut niveau d expression ont été isolés. Des mesures d activité enzymatique pratiquées sur les milieux de culture de ces transformants ont montré qu une RGase recombinante a pu être produite avec succèsUp to now, known rhamnogalacturonases (RGases) are hampered by the presence of acetyl substituants on their substrate, the rhamnogalacturonan (RG). A new RGase which is able to degrade highly acetylated RG, was detected in the commercial enzymatic mixture Driselase® derived from the basidiomycete Irpex lacteus. To monitor the purification of this enzyme and to further characterize it, acetylated RG with a degree of acetylation of 45 has been prepared using enzymatic degradation and chromatographic fractionations of sugar beet pectin. The RGase was then purified from the Driselase® mixture performing different techniques of fractionation allowing to remove contaminating proteins present in the mixture. The purified RGase has a molecular mass of 55 kDa and its optimal pH and temperature are between pH 4.5-5 and 40-50C, respectively. The degradation products of the enzyme have been analyzed by mass spectrometry to investigate its mode of action. It has been shown that the RGase is tolerant to the presence of an acetyle on the reducing galacturonic acid of the product oligomers. In parallel, a 1460-bp full-length cDNA encoding a RGase was isolated from Irpex lacteus cultures using RT and RACE PCR strategies. The cDNA was cloned in the heterologous expression system, Pichia pastoris in order to produce a recombinant RGase. Several clones exhibiting high expression level have been selected. Enzymatic activities measurements realized on culture media of these transformants showed that a recombinant RGase was successfully producedNANTES-BU Sciences (441092104) / SudocSudocFranceF

Management of Infection by Parasitic Weeds: A Review

© 2020 by the authors.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.Financial support is acknowledged to M.F.-A. from the Spanish Ministry of Science, Innovation and Universities (AGL2017-87693-R and RYC-2015-18961), to P.D. from the French National Research Agency (ANR-16-CE20-0004), and to M.P.T. from the NSF (IOS-1213059 and IOS-1238057) and Kirkhouse Trust SCIO.Peer reviewe

Aspects moléculaires de la réponse d'Arabidopsis thaliana à l'infestation par l'holoparasite Orobanche ramosa

Li'nfestation de racines d'A. thaliana par l'holoparasite O. ramosa constitue un modèle intéressant pour l'étude moléculaire de la réponse de l'hôte à l'attaque par une plante parasite. Un système de co-culture in vitro a été développé et nous a permis d'analyser, grâce à des méthodes d'amplification par PCR, l'expression de quelques gènes appartenant aux voies de l'éthylène, des isoprénoïdes, des phénylpropanoïdes, du jasmonate, du stress oxydatif et des protéines PR. Une autre approche, non-ciblée, basée sur une stratégie de banque soustractive par PCR a été utilisée pour identifier des gènes induits deux heures après l'infestation d'A. thaliana. Ils sont impliqués dans la transduction du signal, l'inhibition de pectine methylesterases, la détoxification d'espèces réactives d'oxygène, la voie dépendante du jasmonate et le renforcement pariétal. A. thaliana répond à l'attaque d'O. ramosa en induisant les voies de l'éthylène et du jasmonate, mais pas celle de l'acide salicylique.The infection of A. thaliana roots with the holoparasite O. ramosa represents a useful model for a molecular study of the host plant response to a parasitic plant attack. An in vitro co-culture system was developed, and allowed us to investigate by PCR amplification methods the expression of some host genes involved in plant/pathogen interactions: ethylene, isoprenoid, phenylpropanoid, and jasmonate pathways, oxidative stress responses and PR proteins. A non-targeted study based on a suppression subtractive hybridization strategy was also used to identity genes that were induced two hours after A. thaliana infestation. These genes are also involved in pathways like signal transduction, pectin methylesterase inhibition, detoxification of reactive oxygen species, jasmonate-dependent pathway and cell wall reinforcement. When A. thaliana is infested by O. ramosa, jasmonate- and ethylene-dependent pathways were induced whereas no salicylic acid dependent defence has been detected.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Genetic differentiation and host preference reveal non-exclusive host races in the generalist parasitic weed Phelipanche ramosa

International audienceWe developed 20 microsatellite markers to genotype over 100 populations of the parasitic weed Phelipanche ramosa, which covers a wide host crop and geographic range. A representative core collection of 15 populations was also used in cross-infestation assays to study host preference during germination, attachment and shoot formation. We observed low genetic differentiation within most of the populations, but high genetic differentiation between populations partitioned into 3 genetic groups with different host preferences and geographic distributions. Genetic group 1 is detected exclusively in western France and on various host crops, notably winter oilseed rape (WOSR) and not hemp. Cross-infection assays confirmed its incompatibility with hemp and showed its preference for WOSR and tobacco in terms of germination and attachment success. The group 2 populations share a large geographic distribution in France and Europe, low germination success with WOSR and high germination success, attachment success and shoot formation with hemp, tobacco or tomato. The subclades 2a and 2b include most of the French populations in hemp crops in eastern France and in tobacco fields in several European countries respectively. The genetic analyses revealed the potential of the three groups to increase their geographic range in the future. Intermediate genetic groups showed higher intrapopulation diversity and represent potential stocks for new host race emergence. Those findings argue in favour of the existence of host races in P. ramosa and should be considered for appropriate management strategies, notably in breeding programmes for resistance against this parasitic weed

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