Analyses de spectroscopie infrarouge à transformée de Fourier (FTIR)

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INTRODUCTION

Flax (Linum usitatissimum) is a plant with an important economic interest in the French Normandy region. Flax fibers are traditionally used in the textile industry. Nowadays, the use of plant fibers in composite materials is increasing, firstly due to their physicochemical properties that reduce the size and weight of the materials while improving its mechanical strength, and secondly for economic and sustainable reasons. One of the main problem in using natural fibers is their physic and chemical variability. In flax, the bast fibers formed fibrous bundles, which provide strength to the stem. Fibers development is in direct relationship with plant growth and development (Morvan et al., 2003; Crônier et al., 2005). Thereby, biotic and/or abiotic stresses will have a direct impact on the fibers quality (Chemikosova et al., 2006).
Fusarium oxysporum forma specialis lini (Fol) is one of the main pathogens in flax crop, causing the Fusarium wilt, which provokes the lowering of crop yield and fibers quality. This telluric fungus penetrates predominantly by the roots at the intercellular junctions of cortical cells or through wounds (Perez-Nadales and Di Pietro, 2011). After penetrating the roots, the mycelium grows inter- and intracellularly through the cortex until it reaches the xylem vessels. Then, the fungus expends within the xylem by producing conidia that germinate to develop new mycelium (Bishop and Cooper, 1983; Perez-Nadales et al., 2014). F. oxysporum can survive in soil for extended period of time by producing chlamydospores (thick-walled survival structures) or by growing saprophytically on organic compounds until a new cycle of infection (Agrios, 2005).
The cell wall is the first physical barrier to pathogen infection. It is a dynamic structure composed by cellulose and hemicelluloses polysaccharides embedded in a pectin matrix, enzymes and non-enzymatic glycoproteins such as extensin and arabinogalactan-proteins (AGPs). The cell wall composition and rearrangement are strictly regulated in the different types of cells during their growth, development and plant response to abiotic and/or biotic stress factors (Tenhaken, 2015; Bellincampi et al., 2014). In land Plant, cellulose is composed of 36 β-(1,4)-glucan chains connected with hydrogen bonds and Van der Walls forces forming microfibrils with amorphous and crystalline zones. Linked to cellulose microfibrils, hemicelluloses are subdivided into: xyloglucan (XyG) (β-1,4-glucose chains substituted with xylose, xylose-galactose or xylose-galactose-fucose), xylan (chains of β-1,4-xylose that can be substituted with arabinose and/or glucuronic acid) and glucomannan (chains of glucose an mannose linked in β-(1,4)) (Cosgrove, 2005). Pectins are a complex and heterogeneous group of polysaccharides constituted of four different structural motifs: (i) homogalacturonan (HG), constituted of a linear chain of D-galacturonic acid secreted in the cell wall with a high degree of methylesterification (DM) which is regulated by the action of pectin methylestarases (PMEs), (ii) xylogalacturonan, chains of HG substituted with xylose, (iii) rhamnogalacturonan
I (RG-I), chains composed of the repeating α-1,4-galacturonic acid – α-1,2-rhamnose disaccharide substituted on the rhamnosyl residues with lateral chains of arabinan, galactan and/or arabinogalactan, and (iv) rhamnogalacturonan II (RG-II), constituted of a backbone of
α-1,4-galacturonic acid and four lateral chains containing a high diversity of monosaccharides (Mohnen, 2008). Structural proteins are also present in the cell wall. These proteins include the hydroxyproline-rich glycoproteins (HRGPs) that are composed of arabinogalactan-proteins (AGPs) and extensins. These glycoproteins consist of a protein backbone rich in serine-hydroxyproline units, separated by regions rich in tyrosine, lysine and histidine (Kieliszewski and Lamport, 1994). Upon infection of flax with Fol, numerous genes are up-regulated including cell wall-related genes coding HRGP, cell wall remodeling proteins such as expansins, xylosidases, galactosidases, xyloglucan endo-transglucosylases, pectin lyase, PMEs, polygalacturonases, their inhibitors (PMEI, polygalacturonase inhibitor, xyloglucan endoglucanase inhibitor) and lignin-related genes like phenylalanine ammonia lyases, peroxidases, laccases… (Galindo-Gonzales and Deyholos, 2016; Wojtasik et al., 2016). Interestingly, while overexpression of defense-related genes was observed in resistant flax cultivars, cell wall organization and biogenesis-related genes were down-regulated in susceptible ones (Dmitriev et al., 2017).
Numerous studies have reported the effect of beneficial microorganisms such as Plant Growth Promoting Rhizobacteria (PGPR) on plant growth and protection against pathogens. Several bacteria have been identified as PGPR, however, the two predominant genera are Pseudomonas spp and Bacillus spp (Beneduzi et al., 2012). Beside their ubiquities, members of the genus Bacillus offer advantages over other microorganisms as they are tolerant to fluctuating pH, temperature and osmotic conditions (Nicholson et al., 2000) and can colonize and establish robust interactions with roots by forming biofilms (Allard-Massicotte et al., 2016). In addition to their abilities to increase nutrient uptake and promote plant growth, these bacteria have been shown to produce antifungal compounds and induce plant resistance against pathogens (Vanittanakom et al., 1986, Duitman et al., 1999, Ongena et al., 2007, Beneduzi et al., 2012). The efficiency of Bacillus spp to limit Fusarium wilt has been demonstrated on different plant species such as tomato (Ajilogba et al., 2013), wheat (Grosu et al., 2015, Zalila-Kolsi et al., 2016), maize (Cavaglieri et al., 2005) or pepper (Yu et al., 2011).
To date, the best known ways to limit Fusarium wilt on flax are the selection of tolerant varieties and crop rotations (a flax culture every 7 years). However, the continuous generation of new pathogenic strains requires the development of alternative methods. Wojtasik and colleagues (2013) have proposed to use transgenic varieties, overproducing a β-(1,3)-glucanase. They reported in these overexpressing lines, an accumulation of pectins and phenolic compounds in the fibers, thus improving anti-oxidant properties without altering their mechanical properties.
In this study, we propose to use the Bacillus subtilis ATCC 6633 strain to limit Fusarium wilt on flax. We investigated first by cell imaging the impact of Fusarium oxysporum f. sp. lini (Fol) infection on the cell wall remodeling in two flax varieties (Aramis and Mélina) after different time points of infection and second by biochemical approaches, the impact of a pre-inoculation or not with B. subtilis then with Fol or not on disease development, stem composition and properties.

MATERIALS AND METHODS

Plant material. The two flax varieties (Aramis and Mélina) were provided by the agricultural cooperative Terre de Lin (76740 Saint-Pierre-Le-Viger, France). Seeds were sterilized with 70% (v/v) ethanol for 15 min, then 2.6 % (v/v) bleach for 15 min before being washed 10 times with sterilized water. Seeds were soaked during 10 min before sowing in pellets of compressed peat rehydrated in home-made Hoagland medium (Hoagland and Amon, 1950). Plants were grown in greenhouse with a photoperiod of 16 h at 24 °C days and 18 °C night with 60 % of humidity, and watered with Hoagland medium twice a week. At the cotyledon stage, plants were sprayed with 0.5 mg.mL-1 zinc sulphate. After 3 weeks, peat pellets were transferred into 2 L pots (4 pellets per pot) containing sterilized soil and watered via a drip system.
Bacterial strain. Bacillus subtilis ATCC 6633 was provided by the laboratory of Microbiologie, Signaux et Microenvironnement (LMSM, Normandie Univ, UNIROUEN, France). Bacteria were grown in 100 mL Luria-Bertani (LB) medium on a rotary shaker (150 rpm) at 37 °C. The inoculum
Fungal strain. F. oxysporum f sp. lini (Fol) 04-649 was isolated from the soil of a contaminated flax field (76550 Ambrumesnil, France) and provided by Terre de Lin. The mycelium was grown in Petri dishes on PDB medium (Difco™ Potato Dextrose Broth), supplemented with 1 % (w/v) of Agar (Sigma-Aldrich), at 25 °C with 12 h of light. To enhance the sporulation, part of the mycelium was harvested and incubated in Armstrong’s liquid medium (Armstrong and Armstrong, 1948) on a rotary shaker (120 rpm) at 25 °C with 12 h light for 5 days. The inoculum was then filtered through a Falcon™ Cell Strainer (40 µm pore), centrifuged (4000 g, 7 min, 4 °C) and rinsed 3 times with deionized water before being diluted to a final concentration at 106 conidia.mL-1 (Visser et al., 2004). Inoculation and harvests. Plants were inoculated with the bacteria, two weeks after sowing, by dipping 50 pellets in 1 L of physiological water containing 106 CFU.mL-1 for 1 h. A week later, plants were inoculated with the fungus by dipping 50 pellets in 1 L of deionized water containing 106 conidia.mL-1 for 1 h. Harvests were carried out 2 and 63 days post inoculation (dpi) with Fol for immunofluorescence studies and 8, 21, 42 and 63 dpi for biochemical analyses (Figure 1).

Table des matières

Chapitre I : Introduction générale
Partie 1 : Le lin, Linum usitatissimum
I.1.1 Historique
I.1.2 Anatomie et croissance du lin
I.1.2.1 Généralités
I.1.2.2 Les paramètres pédo-climatiques
I.1.2.3 Le développement de la plante
I.1.2.4 De la récolte à l’obtention des fibres
I.1.2.5 Les tiges de lin
Partie 2 : Les fibres et leurs propriétés
I.2.1 Les fibres ligno-cellulosiques du lin
I.2.1.1 Fibrogenèse des fibres de lin
I.2.1.2 La paroi cellulaire des fibres ligno-cellulosiques
I.2.1.2.1 La cellulose
I.2.1.2.2 Les hémicelluloses
I.2.1.2.3 Les pectines
I.2.1.2.4 Les lignines
I.2.1.2.5 Les protéines pariétales
I.2.2 Caractéristiques des fibres
I.2.2.1 Localisations des fibres
I.2.2.2 Propriétés
Partie 3 : Les défenses des plantes
I.3.1 La résistance active
I.3.1.1 La détection du pathogène
I.3.1.2 Transduction du signal et réponse immunitaire
I.3.1.3 Les médiateurs moléculaires
I.3.1.3.1 La voie des phénylpropanoïdes
I.3.1.3.2 La voie des octadecanoïdes
I.3.1.4 Implication de la paroi cellulaire dans la défense
I.3.1.5 Les protéines de défense
I.3.1.5.1 Les protéines PR
I.3.1.5.2 Les protéines R
I.3.2 Les pathogènes du lin
I.3.2.1 Les champignons du genre Fusarium
I.3.2.2 Cycle de développement de Fusarium oxysporum
Partie 4 : Les biocontrôles
I.4.1 Les stimulateurs des défenses des plantes
I.4.2 Les bactéries bénéfiques
I.4.2.1 Effets des PGPR sur le développement des plantes
I.4.2.2 Le pouvoir compétiteur des PGPR
Objectifs de la thèse
Chapitre II : Matériels et méthodes
II.1 Matériels
II.1.1 Les variétés de Linum usitatissimum L.
II.1.2 Fusarium oxysporum f. sp. lini
II.1.3 Les bactéries bénéfiques
II.1.4 La pregnénolone sulfate
II.2 Analyses phénotypiques
II.2.1 Notation des symptômes
II.2.2 Effets des traitements sur le développement des plantules de lin
II.3 Analyses microscopiques
II.3.1 Détection des dépôts de callose
II.3.2 Immuno-détection des composés pariétaux
II.3.2.1 Enrésinement
II.3.2.2 Préparation des coupes
II.3.2.3 Marquages immuno-histochimiques
II.3.2.4 Marquage de F. oxysporum
II.4 Analyses biochimiques et physico-chimiques
II.4.1 Analyses thermogravimétriques
II.4.2 Analyses de spectroscopie infrarouge à transformée de Fourier (FTIR)
II.5 Analyses de biologie moléculaire
II.5.1 Oligonucléotides utilisés en qPCR
II.5.2 Extraction et dosage des ARN
II.5.3 Rétrotranscription des ARN
II.5.4 Réaction de polymérisation en chaîne (PCR)
II.5.5 PCR quantitative en temps réel (qRT-PCR)
II.6 Analyses statistiques
Chapitre III : Résultats..
Partie 1 : Expérimentations in vitro
III.1.1 Effets des bactéries sur les plantes
III.1.1.1 Effet des différentes souches sur la croissance
III.1.1.2 Effet sur l’architecture racinaire
III.1.1.3 Production des dépôts de callose
III.1.2 Tests de pathogénicité
III.1.3 Effet de B. subtilis et d’une molécule élicitrice sur les mécanismes de défense
contre F. oxysporum (Article)
Partie 2 : Expérimentations en serre
II.2 Effet de B. subtilis sur le développement de la fusariose, impact sur les propriétés plantes (Article)
Chapitre IV : Discussion
Chapitre V : Conclusions Et Perspectives
Références bibliographiques