Genetic and genomic based approaches towards a better sustainability of sunflower Helianthus annuus resistance to downy mildew Plasmopara halstedii

Le mildiou du tournesol, causé by P. halstedii, est une des maladies les plus dévastatrice de la culture de tournesol (Helianthus annuus). Les gènes Pl qui confèrent une résistance totale et qualitative, ont été largement utilisés chez les cultivars. Probablement de type race-spécifique, les gènes Pl ont été progressivement dépassés du à la pression de sélection imposée sur l'agent pathogène. Ainsi depuis 10 ans, on assiste à une augmentation du nombre de races de P. halstedii dans les champs. La résistance quantitative a été détectée par Tourvieille et al. (2008) et avancée comme une solution pour contrer l'évolution rapide de l'agent pathogène. Deux QTLs ont été détectés sur LG10 et LG8 dans une population de RILs issus d'un croissement XRQ*PSC8 par Vear et al. (2008). En examinant les différents aspects de l'interaction plante-agent pathogène pour améliorer la durabilité de la résistance du tournesol contre le mildiou, les objectives de cette thèse sont (i) côté agent pathogène, améliorer la caractérisation génétique et génomique de P. halstedii (ii) côté plante, affiner la cartographie du QTL majeur localisé sur LG10 (QRM1) et (iii) comparer, par analyse transcriptomique, les voies génétiques régulés par Pl5 et QRM1-R. Le premier séquençage haut-débit a été effectué sur des échantillons de tournesol infecté et ainsi a permis d'augmenter les ressources génomiques disponibles de P. halstedii et de mettre en évidence les premiers effecteurs putatifs RXLR et CRN chez P. halstedii. Du côté de la plante, une cartographie fine de QRM1 a été effectué: une nouvelle banque BAC a été construite et " screenée " pour mettre en place une carte physique préliminaire de la région de QRM1 ; la carte génétique du LG10 a été construite et enrichie avec des marqueurs issus des BACs ; et une nouvelle méthode de phénotypage pour simuler la résistance quantitative dans un environnement contrôlé a été validé et utilisé pour phénotyper les nouvelles RILs obtenues. La région QRM1 a été réduite à 1.5 cM. En utilisant les puces Affymetrix de tournesol, l'analyse transcriptomique a révélé plusieurs voies génétiques régulées par l'interaction incompatible (Pl5*race 710) ou l'interaction quantitative dépendante de QRM1-R.Downy mildew, caused by P. halstedii, is one of the major diseases for sunflower (Helianthus annuus). Pl genes conferring full, qualitative resistance were discovered and extensively spread in cultivars. They appeared as race-specific and were progressively overcome due to the selection pressure on the pathogen. Thus since 10 years we are witnessing an increase of P. halstedii races in the fields. Quantitative resistance was found by Tourvieille et al. (2008) and proposed as a solution to counter the rapid evolution of the pathogen. Two QTLs were detected at LG10 and LG8 in a RIL population from XRQ*PSC8 by Vear et al. (2008). With the aim to improve the sustainability of sunflower resistance in gathering knowledge on several aspects of the plant*pathogen interaction, the objectives of this thesis are (i) on the pathogen side, to better characterize P. halstedii at the genetic and genomic level, (ii) on the plant side, to obtain a fine mapping of the main QTL located on LG10 (QRM1) and (iii) to compare the Pl5 gene and QRM1-R dependent genetic pathways by transcriptomic analysis. The first high-throughput sequencing was performed on infected sunflower which increased the available genomic resources of P. halstedii and highlighted first putative effectors RXLR and CRN of P. halstedii. On the plant side, we performed a fine mapping of QRM1: a new BAC library was built and screened to set up a preliminary physical map of the QRM1 region; genetic map of LG10 was enriched with BAC derived markers and a phenotyping method to mimic the quantitative resistance in controlled environment was validated and used to phenotype newly constructed RILs. The QRM1 region was restricted to 1.5 cM. Using Sunflower Affymetrix chip, transcriptomic analysis revealed different genetic pathways depending on incompatible interaction (Pl5 gene * race 710) or quantitative interaction managed by QRM1-R

Impact of <i>Beauveria bassiana</i> and <i>Metarhizium anisopliae</i> on the Metabolic Interactions between Cucumber (<i>Cucumis sativus</i> L.) and Cucumber Mosaic Virus (CMV)

In natural systems, plant–endophyte interactions are important for reducing abiotic and biotic stresses in plants by producing a variety of metabolites that protect plants from pathogens and herbivores. Biocontrol strategies are increasingly being used as a viable alternative to chemical pesticides. Entomopathogenic fungi (EPF) are one of them, and they have been touted as a successful method for biological pest control in plants. Because EPF strains are sensitive to environmental conditions when sprayed, the recently discovered endophytic behavior of several EPF strains has improved their management. Cucumber mosaic virus (CMV) is one of the most common and serious plant viruses worldwide, infecting over 1200 plant species and being spread by more than 80 aphid species. CMV control is directed towards the use of chemical insecticides to eradicate its insect vectors. Endophytic EPF is currently being studied to control plant virus infection, and antagonistic effects have been reported. Metabolomics is an emerging research field for plant metabolite profiling and is employed to study plant–endophyte interactions. In the present research, metabolomics approaches were conducted to gain information into mechanisms involved in defense against CMV in endophytes Beauveria bassiana and Metarhizium anisopliae (EPF)-treated diseased cucumber plants. In addition, CMV-induced metabolic changes in cucumber plants were investigated. Our analysis indicated large differences in cucumber metabolites due to endophytes application. In total, six hundred and thirty-one metabolites were differentially expressed in endophyte-treated CMV diseased cucumber plants. Regulation of different kinds of amino acids, organic acids, and phenylpropanoids metabolites could provide insight about plant defense mechanism against CMV pathogen. Important metabolites were found to be regulated in diseased cucumber plants due to fungal endophytes treatment that could possibly confer tolerance to CMV disease

Screening for antibacterial and antibiofilm activities in Astragalus angulosus.

AimIn a search for finding novel therapeutic agents, extracts from an endemic Lebanese plant, Astragalus angulosus, were evaluated for their potential in-vitro antibacterial and antibiofilm activities against three Gram-positive bacterial strains; Staphylococcus epidermidis (CIP444), Staphylococcus aureus (ATCC25923), and Enterococcus faecalis (ATCC29212); in addition to two Gram-negative strains, Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853).Materials and methodsThe plant was collected in April of 2013 and divided into several different portions, then its extracts were obtained by maceration using two different solvents. Extract analysis followed directly where microtiter broth dilution method was employed to assess antibacterial activity, while antibiofilm potential was tested using colorimetric method.ResultsWhole plant ethanolic extract showed the highest bacteriostatic effect at a concentration of 12.78 mg/ml and also was the most versatile exerting its effect against 3 different strains. Other extracts also exhibited an effect but at higher concentrations and each against a single strain. Regarding antibiofilm activity, the majority of the extracts were able to eradicate &gt;50% of S. epidermidis preformed biofilm, where the highest activity was obtained with flower fraction extracted in water, achieving 67.7% biofilm eradication at 0.2 mg/ml.ConclusionsThis plant possesses a promising potential in regard to eradicating bacteria and their biofilms and it is the first contributing step of establishing a library for the endemic Lebanese plants in this domain

Screening for antibacterial and antibiofilm activities in Astragalus angulosus

Aim: in a search for finding novel therapeutic agents, extracts from an endemic Lebanese plant, Astragalus angulosus, were evaluated for their potential in-vitro antibacterial and antibiofilm activities against three Gram positive bacterial strains; Staphylococcus epidermidis (CIP444), Staphylococcus aureus (ATCC25923) and Enterococcus faecalis (ATCC29212);.in addition to two Gram negative strains, Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853). Methods: the plant was collected in April of 2013 and it was divided into several different portions, then its extracts were obtained by maceration using two different solvents. Extract analysis followed directly, where microtiter broth dilution method was employed to assess antibacterial activity, while antibiofilm potential was tested using colorimetric method. Results: whole plant ethanolic extract showed the highest bacteriostatic effect at a concentration of 12.78 mg ml-1 and also was the most versatile exerting its effect against 3 different strains. Other extracts also exhibited an effect but at higher concentrations and each against a single strain. Regarding antibiofilm activity, the majority of the extracts were able to eradicate &gt;50% of Staphylococcus epidermidis preformed biofilm, where the highest activity was obtained with flower fraction extracted in water, achieving 67.7% biofilm eradication at 0.2 mg ml-1. Conclusions: This plant possesses a promising potential in regard to eradicating bacteria and their biofilms, and it is the first contributing step of establishing a library for the endemic Lebanese plants in this domain. [J Complement Med Res 2017; 6(1.000): 50-57

Identification of beneficial Lebanese Trichoderma spp. wheat endophytes

International audienceWheat is one of the most important crops in the world. Its production can be influenced by a diversity of beneficial and pathogenic rhizospheric microbes, including fungi. Amongst them, beneficial Trichoderma spp. can be used as alternatives to chemical fertilizers, as they are cheaper and harmless to the environment. Our study aimed to isolate, identify, and characterize Trichoderma spp. from Lebanon associated to wheat. Two Trichoderma strains belonging to T. afroharzianum, and T. guizhouense species, were isolated and found to be endophytes enhancing root growth and producing 2 IAA. Inoculation also improved seedling development, and boosted general production. These Trichoderma spp. have thus the capacity to be used as organic fertilizers for wheat

Consensus mapping of major resistance genes and independent QTL for quantitative resistance to sunflower downy mildew

International audienceMajor gene resistance to sunflower downy mildew (Plasmopara halstedii) races 304 and 314 was found to segregate independently from the resistance to races 334, 307 and 304 determined by the gene Pl2, already positioned on Linkage Group (LG) 8 of sunflower molecular maps. Using a consensus SSR-SNP map constructed from the INEDI RIL population and a new RIL population FU × PAZ2, the positions of Pl2 and Pl5 were confirmed and the new gene, denoted Pl21, was mapped on LG13, at 8 cM from Pl5. The two RIL populations were observed for their quantitative resistance to downy mildew in the field and both indicated the existence of a QTL on LG8 at 20-40 cM from the major resistance gene cluster. In addition, for the INEDI population, a strong QTL on LG10, reported previously, was confirmed and a third QTL was mapped on LG7. A growth chamber test methodology, significantly correlated with field results, also revealed the major QTL on LG10, explaining 65 % of variability. This QTL mapped in the same area as a gene involved in stomatal opening and root growth, which may be suggested as a possible candidate to explain the control of this character. These results indicate that it should be possible to combine major genes and other resistance mechanisms, a strategy that could help to improve durability of sunflower resistance to downy mildew

DataSheet_1_Identification of beneficial Lebanese Trichoderma spp. wheat endophytes.pdf

Wheat is one of the most important crops in the world. Its production can be influenced by a diversity of beneficial and pathogenic rhizospheric microbes, including fungi. Amongst them, beneficial Trichoderma spp. can be used as alternatives to chemical fertilizers, as they are cheap and harmless to the environment. Our study aimed to isolate, identify, and characterize Trichoderma spp. from Lebanon associated with wheat. Two Trichoderma strains belonging to T. afroharzianum, and T. guizhouense species, were isolated and found to be endophytes, enhancing root growth and producing Indole-3-acetic acid. Inoculation also improved seedling development, and increased plant growth and yield. Furthermore, the two strains inhibit Fusarium growth in vitro. These Trichoderma spp. have thus the capacity to be used as organic fertilizers for wheat.</p

Transcriptomic analysis of the interaction between <it>Helianthus annuus </it>and its obligate parasite <it>Plasmopara halstedii </it>shows single nucleotide polymorphisms in CRN sequences

<p>Abstract</p> <p>Background</p> <p>Downy mildew in sunflowers (<it>Helianthus annuus </it>L.) is caused by the oomycete <it>Plasmopara halstedii </it>(Farl.) Berlese et de Toni. Despite efforts by the international community to breed mildew-resistant varieties, downy mildew remains a major threat to the sunflower crop. Very few genomic, genetic and molecular resources are currently available to study this pathogen. Using a 454 sequencing method, expressed sequence tags (EST) during the interaction between <it>H. annuus </it>and <it>P. halstedii </it>have been generated and a search was performed for sites in putative effectors to show polymorphisms between the different races of <it>P. halstedii</it>.</p> <p>Results</p> <p>A 454 pyrosequencing run of two infected sunflower samples (inbred lines XRQ and PSC8 infected with race 710 of <it>P. halstedii</it>, which exhibit incompatible and compatible interactions, respectively) generated 113,720 and 172,107 useable reads. From these reads, 44,948 contigs and singletons have been produced. A bioinformatic portal, HP, was specifically created for in-depth analysis of these clusters. Using <it>in silico </it>filtering, 405 clusters were defined as being specific to oomycetes, and 172 were defined as non-specific oomycete clusters. A subset of these two categories was checked using PCR amplification, and 86% of the tested clusters were validated. Twenty putative RXLR and CRN effectors were detected using PSI-BLAST. Using corresponding sequences from four races (100, 304, 703 and 710), 22 SNPs were detected, providing new information on pathogen polymorphisms.</p> <p>Conclusions</p> <p>This study identified a large number of genes that are expressed during <it>H. annuus/P. halstedii </it>compatible or incompatible interactions. It also reveals, for the first time, that an infection mechanism exists in <it>P. halstedii </it>similar to that in other oomycetes associated with the presence of putative RXLR and CRN effectors. SNPs discovered in CRN effector sequences were used to determine the genetic distances between the four races of <it>P. halstedii</it>. This work therefore provides valuable tools for further discoveries regarding the <it>H. annuus/P. halstedii </it>pathosystem.</p