Petunia growth, mycorrhizal colonization and T. basicola development 

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Pathogen recognition: general elicitors

The signalling molecules produced by pathogens which plants are able to recognize and respond to are known as “elicitors”. Elicitors rapidly activate a range of plant defence responses that can be either sufficient to stop pathogen spread (incompatible interaction) or insufficient leading to disease (compatible interaction) (Nümberger et al., 2004). Identification of elicitors has unveiled similarities in the molecular basis of immunity in plants with that known for insects and animals (Paré et al., 2005). The first characterized microbial elicitors were predominantly oligosaccharides but later many other compounds were identified such as flagellin or cold-shock protein produced by bacteria, and necrosis-inducing proteins, transglutaminase, elicitins or β-glucans produced by fungi. Altogether, they were called PAMPs for “pathogen associated molecular patterns” (see Nümberger et al., 2004). However, not only pathogenic microorganisms possess these patterns and therefore a broader term was introduced, MAMPs, that substitutes the word pathogen by microbe (Ausubel, 2005). In addition to these exogenous elicitors produced by microbes, plant endogeneous elicitors of defence responses that are generated as a result of physical and/or chemical cleavage of the plant cell wall have also been identified since a long while (Hahn et al., 1981).

Defence responses in shoots and roots

In leaves, PAMP recognition via pattern recognition receptors (PRRs) activates a basal resistance, called PAMP-triggered immunity (PTI), which is translated by different plant responses such as oxidative burst and/or nitric oxide production, the biosynthesis of particular phytohormones like salicylic acid, jasmonate or ethylene, as well as a complex cascade of calcium dependent and 13 mitogen-activated protein kinases that leads to the activation of transcription factors and in turn of defence response genes (Nümberger et al., 2004). Faced with this plant immunity, pathogens have co-evolved a strategy in which they secrete small effector molecules into the host cell to suppress PTI and establish a compatible interaction. In turn, plants have developed another recognition system, based on ‘R’ proteins, to detect these pathogen effectors and induce a secondary immune response known as effector-triggered immunity (ETI) (Pieterse et al., 2009). ETI activates a signalling pathway which leads to programmed hypersensitive cell death in order to restrict pathogen invasion and therefore prevent intact tissues from further damage (De Wit, 1998). Although, there have been major advances in the understanding of host shoot-pathogen interactions, relatively little is known about PAMP-mediated responses in roots (Millet et al., 2010). Root pathogens play an important economical role; monetary losses annually in the US due to soil-borne pathogens of vegetables, fruits or field crops have been estimated at 4 billion US $ (Lumbsden et al., 1995).
Pathogens do not necessarily discriminate between different plant organs, and shoots as well as roots can be targets of the same pathogenic strain. It has been suggested that root pathogens induce no or only weak responses in order to reduce plant fitness costs. Studies focussing on A. thaliana to compare leaf and root responses to different PAMPS or MAMPs have pointed to the presence of orchestrated and tissue-specific plant, as well as potential pathogen-encoded, mechanisms to block elicited signalling pathways in roots (Millet et al., 2010). However, further studies are needed to better understand plant defence in roots against biotrophic or necrotrophic pathogens and how MAMP and/or effector signalling pathways are involved in compatible interactions with beneficial microbes.

Phytohormones and induced resistance in plants

Phytohormones are plant chemical messengers that play an important role in growth and development processes and all are known to be also involved in plant responses against biotic stresses (Bari and Jones, 2009). Those considered to play major roles in defence responses include salicylic acid (SA), ethylene (ET) and jasmonic acid (JA). Attack by diverse pathogens results in changes in the level of these phytohormones and thereafter in the expression of defence related genes (Adie et al., 2007; Robert–Seilaniantz et al., 2007). The types of phytohormones implicated in signalling pathways leading to defence gene regulation appear however to depend on whether the pathogen concerned is biotrophic or necrotrophic (Bari and Jones, 2009) (Fig. 2).

Exchange of nutrients

Arbuscules represent a checkpoint between the two mycorrhizal symbionts where a high transporting activity occurs not only from plant to fungus, but also in the direction fungus to plant, via the symbiotic interface made up of the plant periarbuscular membrane and fungal plasma membrane separated by an apoplastic zone (Hause and Fester, 2005).
In AM plants, there is a net increase in photosynthesis which results in a photoassimilate increase in AM roots, estimated to be up to 20% (Bago et al., 2000). Carbohydrates from “source leaves” are transferred as sucrose via the phloem to the “root sink” and converted into glucose plus fructose (Blee and Anderson, 1998). Glucose seems to be transferred to the fungal symbiont (Solaiman and Saito, 1997; Boldt et al., 2011). However, a monosaccharide transporter recently isolated from G. intraradices did not only transport glucose, but also xylose indicating plant cell wall sugars as alternative carbon source for AM fungi (Helber et al., 2011). Localisation of its expression, moreover, suggested that the transfer of carbohydrates does not solely occur at the arbuscules but also at other intraradical hyphae.
In mycorrhizal plants, the pathway of direct uptake of inorganic phosphate (Pi) from the soil at the root surface is suppressed and replaced by the mycorrhizal pathway that involve import of Pi into fungal hyphae via Pi transporters, translocation of Pi to the arbuscule interface, and release to root cells where plant Pi transporters transfer the Pi into cortical cells (Bucher, 2007; Smith et al., 2011). Many plant Pi transporters have been characterized and classified into high or low affinity transporters, of which some are AM specific.
Although improved nutrient assimilation by AM associations concerns mainly Pi, the fungal partner can also provide the host plant with N (Hawkins et al., 2000). The current model predicts that nitrate and ammonium are taken up by the extraradical mycelium, arginine is transported in the fungal hyphae and ammonium is finally transferred towards the plant (Govindarajulu et al., 2005; Chalot et al., 2006; Guether et al., 2009). Fungal transport capacities for N and P are in a similar range (Smith and Read, 2008), but the plant needs ten times more N than P, so that the fungal-mediated transfer of N is probably of less importance for mycorrhizal effects on plant growth.

Bioprotection against environmental stress

In addition to influencing plant nutrition, AM fungi improve the performance of their hosts on polluted soils (Aloui et al., 2009; Rivera-Becerril et al., 2002; Gonzalez-Chavez et al., 2002), under drought stress (Augé, 2001) or at high salt concentrations (Ruiz–Lozano et al., 1996). Consequently, AM contributions have been investigated in different fields like landscape regeneration, alleviation of desertification or bioremediation of contaminated soils (Jeffries et al., 2003).
The mechanisms contributing to such tolerance against abiotic stresses in AM plants are not fully understood (Schützenduebel and Polle, 2002). Pathways of heavy metal chelation do not appear to operate in such AM-enhanced tolerance (Rivera-Becerril et al., 2005) and recent investigations have indicated the implication of anti-oxidative activities through, in particular, reactive oxygen species (ROS) accumulation (Aloui et al., 2009). In fact, several observations have shown that AM induced tolerance against different abiotic stresses (heavy metals, salt or drought) may be ROS-dependent (Ruiz-Lozano et al., 1996, 2001; Bowler and Fluhr, 2000; Huang et al., 2010). In parallel to enhanced plant anti-oxidant activities, it was shown that on the fungal side accumulation of six glutathione-S-transferases was up-regulated in extraradical hyphae of G. intraradices growing in a heavy metal contaminated soil (Waschke et al., 2006).

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Petunia genus: origin and interest

The genus Petunia (first established by Jessieu in 1803) assembles commercially important flowering plants originating from South America. The name petunia derives from “Petum” meaning “tobacco” in the language Tupi-Guarani. The geographic distribution of the genus includes temperate and subtropical regions of Argentina, Uruguay, Paraguay, Bolivia, and Brazil, with a centre of high diversity in southern Brazil.
Petunia is an ornamental crop of high economic interest. Many advantages make the culture of petunias favourable for gardeners, such as their easy growth, their versatility and a huge range of colours and flower shapes. One very important quality is their relatively high tolerance to drought, probably related to their origin. For all these reasons, petunias belong to the most sold bedding plants worldwide. For greenhouse growers, petunia is listed as the top genus grown per number of plants sold (Tambascio, 2007).
Plant geneticists’ interest in petunia began in the late fifties of the last century. Predicting flower colours on the basis of Mendel’s laws enabled the definition of over thirty genes involved in flavonoid biosynthesis (Gerats and Vandenbussche, 2005). Moreover, the finding in petunia of reversible co-suppression of homologous genes (Napoli et al., 1990) had an unexpected outcome in 1998 with the revolutionary discovery of RNA interference (RNAi) (Fire, 1999).

Petunia hybrida Mitchell: advantages and qualities

Petunia hybrida, derived from crosses between Petunia axillaris (large white flower) and Petunia integrifolia (purple flower), is the most widely cultivated of the 30 extant petunia species. P. hybrida Mitchell variety is an inbred colchidiploid (2 n =14) and has a relatively large genome (1200-1500 Mbp) (Mishiba et al., 2000; Bossolini et al., 2011). It is characterised by white flowers that produce a strong fragrance in the evening and at night (Verdonk et al., 2005). The hybrid has been considered as a genetic model plant since the early 1980s (Gerats and Vandenbussche, 2005). In particular, the high mutation rate in the P. hybrida line W138 has turned out to be very useful for mutant screens especially after the molecular basis for the mutations was shown to be the non-autonomous transposable element dTph1 (Gerats et al., 1990). Because transformation of petunia is also applicable (Conner et al., 2009), forward and reverse genetic approaches are nowadays possible (Wegmüller et al., 2008). Together with a large EST collection, commercially available microarrays (Breuillin et al., 2010) and the currently on-going genomic sequencing (Franken and Drüge, personal communication), petunia has become an interesting model for studies on the genetics and the molecular physiology of plants (Bossilini et al., 2011).
The fact that reverse genetics can be used as a strategy with petunia has led to the isolation of a petunia mutant, pam1 (penetration and arbuscule morphogenesis1), which is affected in the development of AM. The corresponding gene has been characterized as a VAPYRIN homologue with 11 ankyrin repeats which could be involved in the transport via the tonoplast of a component with an essential function during intracellular colonization by AM fungi (Sekhara Reddy et al., 2007; Feddermann et al., 2010). In contrast to previously described tomato and maize mutants which are affected at early stages of root colonization or have reduced level of mycorrhization (Barker et al., 1998; David-Schwartz et al., 2001, 2003; Paskowski et al., 2006), the pam1 mutant is defect in intracellular accommodation, arbuscular development and morphogenesis of the fungal endosymbiont (Sekhara Reddy et al., 2007) and can contribute to our understanding of the AM-specific SYM pathway at later stages of the symbiosis.

Petunia in ornamental crop production

Ornamental crops like petunia are mainly produced as potted plants in artificial substrates, and their marketability is greatly influenced by conditions used during their production, such as substrate quality, drainage, irrigation, water quality and fertilization (Chavez et al., 2008). Soilless culture substrates associated with rich fertilizer regimes are increasingly applied to meet present-day consumer demands for ornamental and nursery plants (Gruda, 2009). Whilst these offer significant advantages for high crop yield and product quality through complete control over water and nutrient supplies (Grillas et al., 2001), the use of substrates with poor or noion exchange capacity can lead to mineral nutrient losses or to short-term unintentional exposure of plants to high ion concentrations. This in turn results in short periods of salt stress which reduce vigour and yield and are detrimental, if not lethal, especially for young plants (Rosendahl and Rosendahl, 1990). Moreover, ornamental petunia production is confronted with attack by root pathogens like Fusarium oxysporum, Rhizoctonia solani or Thielaviopsis basicola (Dreistadt, 2001) which cause high losses in greenhouses.

Table of contents :

I- General Introduction 
1.1- Introduction
1.2- Plant/pathogen interactions
1.2.1- Plant defence
1.2.2- Pathogen recognition: general elicitors
1.2.3- Defence responses in shoots and roots
1.2.4- Phytohormones and induced resistance in plants
1.3– Arbuscular mycorrhiza
1.3.1– AM development
1.3.1.1- Presymbiosis
1.3.1.2- Symbiotic phase
1.3.2– Arbuscular mycorrhiza functions 23 1.3.2.1- Exchange of nutrients
1.3.2.2- Bioprotection against environmental stress
1.4– Mycorrhiza–induced resistance (MIR)
1.5– AM in the Solanaceae
1.6- Petunia hybrida Mitchell: a model plant
1.6.1- Petunia genus: origin and interest
1.6.2- Petunia hybrida Mitchell: advantages and qualities
1.6.3- Petunia in ornamental crop production
1.7- Thesis objectives
2- Materials and Methods 
2.1- Biological materials
2.2- Petunia propagation
2.3- Petunia mycorrhization
2.4- Determination of shoot biomass, water content and phosphorus concentration
2.5- Salt stress treatment
2.6- Fungal pathogen inoculation
2.7- Disease severity (DS) estimation
2.8- RNA extraction from petunia roots and first-strand cDNA synthesis
2.9- Reverse transcriptase (RT)-PCR
2.10- Real-time RT-PCR
2.11- Relative gene expression (R)
2.12- Statistical analysis
3- Results 
3.1- Chapter I
I.1- Petunia mycorrhization studies
I.2- Results
I.2.1- Mycorrhiza development, plant growth and phosphate nutrition
I.2.2- Salt stress
I.3- Discussion
3.2- Chapter II
II.1- Introduction
II.1.1- Pythium aphanidermatum (Edson) Fitzp.
II.1.2- Fusarium oxysporum Schlecht
II.1.3- Rhizoctonia solani Kühn
II.1.4- Thielaviopsis basicola (Berk. and Broome) Ferraris (syn. Chalara elegans)
II.2- Results
II.2.1- Pathogen selection
II.2.1.1- Pathogenicity tests in vitro
II.2.1.2- Pathogenicity tests in vivo
II.2.2- Time course infection with T. basicola
II.2.2.1- Root necrosis and leaf symptoms
II.2.2.2- Molecular detection of T. basicola
II.3- Discussion
3.3- Chapter III
III.1- Introduction
III.2- Results
III.2.1- Comparison of the effect of three AM fungi in the petunia/T. basicola pathosystem
III.2.2- Effect of G. mosseae on cuttings in the petunia/T. basicola pathosystems
III.2.3- Optimization of G. mosseae-induced bioprotection against T. basicola
III.3- Discussion
3.4- Chapter IV
IV.1- Introduction
IV.2- AM-related plant genes
IV.3- SA- and JA- regulated plant defense genes
IV.4- Plant defense genes with other functions
IV.5- Results
IV.5.2- Expression of SAR or ISR-related defense genes
IV.5.3- Expression of defense genes with different functions
IV- Discussion
3.4- Chapter V
V.1- Introduction
V.2- Results
V.2.1- Petunia growth, mycorrhizal colonization and T. basicola development
V.2.2- Petunia gene expression
V.3- Discussion
Concluding remarks
References

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