Constitutive components of BR resistance: plant cuticle, a multi-component barrier 

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Differentiation of Monilinia species

By observation with naked eye, it is possible to identify the differences between the three agents of monilioses in fruit in orchard conditions (Mercier, 2009). M. fructigena has colour ranging from white to light beige, large (1.5 mm on average) conidiospores tufts, and disposition in concentric circles in the fruit. M. fructicola has brown-coloured, medium size (1 mm on average) conidiospores tufts and 10% black spots. M. laxa can be distinguished by greenish-grey conidiospores tufts less than 0,5 mm on average that cover the whole infected surface. However the differentiation in fruit between M. laxa and M. fructicula may sometimes be difficult and the use of molecular techniques is required (Figure 2).
Studies to identify the Monilinia species reported that, in culture medium with potato dextrose and agar (PDA) at 22oC, M. laxa is characterized by concentric rings of mycelium with lobbed margins, while in M. fructigena it is possible to observe fragmented radial colonies. Differences in colony growth rates between the three species were observed (20 – 25ºC). The highest growth rate on PDA was found for M. fructicola, followed by M. fructigena and M. laxa respectively. However, M. laxa showed the biggest lesion growth rate on peach fruit (Hu et al, 2011). In culture medium it is possible to analyse characters as conidial size and germ tube morphology. These methods have been used since 1920 and their simplicity makes them useful still (Ioos & Frey, 2000). Differences in conidia size among the species are reported. On average the conidia size of M. laxa is smaller compared to M. fructigena, 13×9 µm and 22×12 µm, respectively. M. fructigena produces one or two germ tubes per conidium, and M. laxa and M. fructicola isolates consistently produce only one germ tube per conidium (Hu et al, 2011).
Several molecular biology techniques (mostly based in the Polymerase Chain Reaction, PCR) have been used to develop reliable and sensitive methods to identify and detect Monilinia species. Fulton and Brown (Fulton & Brown, 1997), proposed the study of the small sub unit of ribosomal DNA (rDNA) to differentiate Monilinia isolates from the three major species. Many PCR protocols for Monilinia spp. identification, based on the comparison of internal transcribed spacers, sequence between the 18S small and the 28S rDNA subunits of Monilinia genes, have been proposed (Ioos & Frey, 2000), (Boehm et al, 2001), (van Brouwershaven et al, 2010). Ma et al (Ma et al, 2003) and Hu et al (Hu et al, 2011) reported a detection and identification method of Monilinia fungi based on species-specific microsatellites (Hu et al, 2011), (Ma et al, 2003). Identification methods based on amplified fragment length polymorphism (AFLP) are also reported (Gril et al, 2008), (Gril et al, 2010). In addition, molecular techniques have been developed for species identification on quiescent fruit infections of stone fruit (Côté et al, 2004), and for the early detection of infections in cherry fruit (Forster & Adaskaveg, 2000). In Banks et al (Banks et al, 1997), monoclonal antibodies are reported to be useful for identification and detection of Monilinia spp. in pome and stone fruit (Banks et al, 1997). Some of these approaches have set the basis for several studies about morphological and molecular diversity of Monilinia spp., describing the geographical distribution and host range of the three main species of Monilinia that caused BR of stone and pome fruits, (Gell et al, 2007), (Petroczy & Palkovics, 2006), (De Cal et al, 2009).

Host range and distribution of Monilinia spp.

M. fructigena is an economically important BR-agent that has been associated with European BR of pome fruits (Holst-Jensen et al, 1997a), (Byrde & Willetts, 1977). However, its occurrence in stone fruits has also been well documented in Europe (Larena et al, 2005), (Villarino et al, 2013), Brazil (Lichtemberg et al, 2014) and China (Zhu et al, 2011).
M. laxa has been historically associated with European blossom blight and BR of stone (Byrde & Willetts, 1977), (Villarino et al, 2013) and pome fruit (Muñoz et al, 2008), (Lesik, 2013). However in the last two decades it has been also reported in different regions of the world, including Brazil (Lichtemberg et al, 2014), (Souza et al, 2008), United States (Snyder & Jones, 1999), (Villani & Cox, 2010),(Cox et al, 2011), China (Zhu et al, 2011) and Iran (Nasrollanejad & Ghasemnezhad, 2009).
M. fructicola (G.Wint) is the most widely distributed species, occurring in Asia, North and South America, New Zealand and Australia (Fan et al, 2010), (Latorre et al, 2014). In Europe, it was a quarantine pathogen until early 2014, when it was removed from the European quarantine pest list due to its current spread in the following countries: France (Lichou J., 2002), Hungary (Petroczy & Palkovics, 2006), Switzerland (Bosshard et al, 2006), (Hilber-Bodmer et al, 2010) Germany (Grabke et al, 2011), Czech Republic (Duchoslavova et al, 2007), Slovenia (Munda & Marn, 2010), Italy (Pellegrino et al, 2009), (Martini et al, 2013) Austria (subsequently erradicated) (Jänsch et al, 2012), Poland (Poniatowska et al, 2013), Slovakia (Ondejková et al, 2010), Serbia and Spain (De Cal et al, 2009).
The low genetic diversity found in Spanish and French populations of M. fructicola, compared with American or New Zealand diversity, indicates few and recent introduction events of the pathogen to Europe (Villarino et al, 2012). In addition to its wide distribution, M. fructicola has been reported to infect other hosts such as Cornelian cherry (Beckerman & Creswell, 2014) and others that do not belong to Rosaceae family, for example grapes (Sholberg et al, 2003) and dragon fruit (Abd Ghani et al, 2011).
These three species share high levels of DNA similarities. M. fructicola and M. fructigena exhibited 97,5% sequences identity while M. laxa and M. fructigena displayed more than 99,1% for the Cyt b gene (Hily et al, 2011). In this way, we may expect that part of the knowledge acquired from one species may be extrapolated to the other members of Monilinia genus.
A fourth species, M. polystroma (also called ‘Asiatic Brown Rot’) is native of Japan, where it had been formerly confounded with M. fructigena. It was described as a new species after finding significant biological and morphological characteristics with respect to European isolates of M.

Penetration sites in relation to fruit growth

As stated before, in this review we only discuss aspects of fruit infection. Different biologic mechanisms may be involved in pathogenesis of fruit and flowers by Monilinia spp., suggested by an absence of correlation between blossom bight occurrence and fruit rot impact, after artificial inoculation of M. fructicola, in Brazilian cultivars and selections of peach (Wagner Júnior et al, 2005). In fruit, Monilinia spp. has often been considered as an opportunistic fungi that may enter in the tissue only via naturally occurring entry points. Therefore, many studies have focused on these entrances or employed infection tests injuring the fruit first. Although in most of the cases the fungus penetrates using ‘open doors’, (Figure 3 F), most of the species may also be able to penetrate fruit through intact surface, after the establishment of latent or quiescent infections. For example, the penetration of M. fructicola in immature apricot fruit was reported to occur through wounds, stomata, (Figure 3 B and C), intact cuticle or via trichoma bases, (Figure 3 A) (Wad & Cruickshank, 1992b). The same way in peach, hyphae infect fruits by either degrading the cuticle and epidermal tissue (Bostock et al, 1999) or directly entering through pre-existing skin microcracks (Figure 3 D and E). Fungus incidence is greater if the fruit has small cracks or wounds (De Cal et al, 2013). It has been reported that M. fructigena infects fruit via wounds only, in contrasts to M. laxa that may infect both healthy and wounded fruit (Xu et al, 2007). Indeed, infection may depend on which site is most frequently encountered by fungal germ tubes. Penetration site may also depend on the developmental stage of the fruit. For example, stomata are the preferred sites in the case of unripe peaches only. Curtis (Curtis, 1928) found that apricots were penetrated through cuticle and stomata, plums via stomata, and nectarines through the cuticle. Sharma and Kaul (Sharma & Kaul, 1990) described the penetration of apple under laboratory conditions by M. fructigena through lenticels.

Fruit susceptibility evolves along fruit development

The stages of development of fruit are very important to understand the occurrence of BR, since the dramatic changes in fruit physiology and biochemical composition are in sync with changes in the susceptibility to BR infection (De Cal et al, 2013), (Wad & Cruickshank, 1992a),(Biggs & Northover, 1988).
The first stage starts after ovule fertilization, petal fall and ends when stone starts lignifying. In this stage the fruit is photosynthetically active, displays intense transpiration activity, and shows the highest nutrient content (Thomidis et al, 2007), resulting in a high susceptibility to BR, probably due, in part, to the fact that stomata are active, and offer an entrance opportunity to the pathogen (Curtis, 1928).
The second stage, also known as “pit hardening”, is the stage most resistant to infection by Monilinia spp. (De Cal et al, 2013), (Mari et al, 2003). This stage is characterized by intense metabolite activity of secondary compounds, like catechin, epicatechin and phenolic compounds, associated with the lignification of the endocarp, occurring in this stage. In order to find genes whose expression is involved in the synthesis of compounds conferring pathogen resistance, Guidarelli et al (Guidarelli et al, 2014), compared gene expression profiles obtained by microarray analysis of susceptible phase (stage S1) and resistant phase (S2) RNA samples from peel fruit, finding dramatic changes in the expression of phenylpropanoid and jasmonate-related genes, and thus supporting a potential role of these compounds in BR resistance along fruit development. At the third stage, the highest cell expansion is observed and colour changes from greenish to yellow to red. This stage ends with physiological maturity. Stone fruits become increasingly susceptible to pathogens as they mature and ripen, enabling quiescent infections to become active and new infections to begin. Associated with this increased susceptibility, structural changes in the fruit surface take place, such as thinning and fracturing of the cuticle, changes in fruit surface chemistry (e.g. production of sugars, decline of phenolic compounds and organic acids, etc.), structure and integrity of fruit mesocarp (Bostock et al, 1999).
Notably, various works in different Prunus species have observed a shift in the latent infection rate across the diverse stages of fruit development (Gell et al, 2008), (Northover & Cerkauskas, 1994),(Keske et al, 2011). However, the results vary among studies, probably due to differences in methodology and cultivars used in those studies. For instance, Lou and Michailides (Luo et al, 2001) observed that pit hardening of prunes presented the lowest rates of latent infections, differing from other works reporting a minimum rate of latent infections at the embryo growth stage (Gell et al, 2008), (Keske et al, 2011).

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Infection by direct penetration of the cuticle

After conidial germination, Monilinia species are able to develop appressoria to establish a latent infection and ease the penetration of the intact cuticle when fruit maturity conditions allow colonization (Fourie & Holz, 2003). This structure allows adhesion of the pathogen to the surface of the host during infection (Lee & Bostock, 2006). Direct penetration of Monilinia spp. is enhanced by its production of cutinases (Bostock et al, 1999), whose redox-mediated over-expression results in an increased fungal virulence of M. fructigena in stone fruit (Lee et al, 2010). More details about the infection process are given in chapter 4.

Infection through the trichomes basis

A dense layer of trichomes covers the surface of the peach fruit. The infection can occur in both pubescent and not-pubescent peach fruit. The role of trichomes in the infection remains controversial. Indeed, trichomes may protect the fruit in two ways: 1) Directly: exudates from trichome gland may act as fungicide and 2) Indirectly: the high density of trichomes could prevent the formation of “water film” important to spore germination. In contrast, trichome basis fracture can result in epidermis crack, resulting in points for fungal entrance (Silva et al, 2005), (Fernandez et al, 2011). Smith (Smith, 1936) showed that removing pubescence by means of brushing reduced the time of infection development, suggesting that the spores could reach fruit surface more directly. Other studies (Wad & Cruickshank, 1992b) affirmed that M. fructicola is able to penetrate apricots at hair bases. Similar results were found on mature peaches (Curtis, 1928), (Hall, 1971). Finally, is not yet clear whether nectarines are more resistant or susceptible to BR compared to peaches. Large variations of trichomes density and length and, more generally, of fruit surface, between varieties make comparisons between studies and drawing general conclusions a very hard task.

Infection through skin cracks and wounds

Cuticular crack is defined as the physical failure of the fruit skin, caused by forces of growth as turgor pressure within the fruit cells or hydration of fruit fresh acting on the skin (Milad & Shackel, 1992). Cuticular cracks on nectarine fruit occur during the final fruit growth stage (Gibert et al, 2007), (Gibert et al, 2009),(Gibert et al, 2010). Micro-cracks and cracks can develop on the surface of fruit when the growth speed of the internal cells is more rapid than epidermal cell growth. In this case, a time lag between fruit growth and cutin deposit can occur and provoke zones of weakness that may evolve into microcracks. Several factors contribute to fruit cracking, often in interactions, such as unbalanced water flux into and out of the fruit, maximal elastic limit of the cuticle, cuticle strain, and absence of cuticular membrane deposition. Observations of the fruit skin have shown that the cracks are frequently initiated around the lenticels (Brown & Considine, 1982), (Figure 3D, 3E and Figure 5).
Larger fruits can present high cuticular crack densities, which may represent more than 10% of the fruit surface area (Gibert et al, 2007).
One of the first studies on M. laxa penetration in micro-cracks (Nguyen-The et al, 1989), observed a significant number of cracks and micro-cracks organized radially around lenticels and noticed that germinating conidia of M. laxa tended to accumulate in the micro-cracks in an anarchic pattern and without apparent direct attraction by micro-cracks, despite the fact that the germ tubs grew inside of them. However Borve et al (Borve et al, 2000), demonstrated a clear link between cracking and BR in cherries, by finding significant correlations between the cultivar-specific amount of micro-cracks and the resulting incidence of BR.
Skin wounding deprives the fruit of its main barrier to biotic stress agents, as demonstrated in several reports (Xu et al, 2007), (Hong et al, 1998), where BR infection rates obtained after infecting wounded regions of the fruit were significantly higher than infecting intact fruit regions. Effect of presence of skin barrier in BR resistance was investigated on apricot, peach and plum fruit, to find resistant genotypes (Pascal et al, 1998). Injured-fruit infection developed on all fruit with quite similar speed in all species. On the contrary, when uninjured fruit were infected, large variability was observed between genotypes of a same species and between species. These observations suggest that few resistant factors may be expressed at the flesh level and that resistance factors were no more efficient when the fruit was injured. However, Ogundiwin et al (Ogundiwin et al, 2008), explored larger genetic diversity by evaluating 81 peach genotypes by infection on wounded and unwounded fruit. The authors observed variability in both cases and suggested that BR resistance is associated with the pericarp or the mesocarp or both, depending on the genotype (Ogundiwin et al, 2008). Nonetheless, more recently the same group further explored the variability of infection reaction after wounding of a canning peach progeny (Martinez-Garcia et al, 2013), concluding that wounding the fruit generally abrogated any resistance to brown rot. Resistance factors at the level of the flesh (wounded fruit) may not provide total resistance to infection but may slightly act on the speed of lesion propagation. To further explore these potential factors of resistance, large trials considering a high replicate number on highly contrasted germplasm panels may be needed.
In conclusion, it is evident that stomata, lenticels, pores, cracks and microcracks offer preferential entry sites for Monilinia and make fungi colonization easier. Number of stomata, lenticels and pores may be under genetic control, but structure may be influenced by environment conditions. As for cracks and microcracks, genetic determinism has not been investigated, but studies have demonstrated the effect of cultural practices (e.g. irrigation and thinning) on their density (Gibert et al, 2007).

Table of contents :

Chapitre 1: Synthèse bibliographique sur la pourriture brune chez les Prunus 
1 Introduction
2 Monilinia spp. fungi cause brown rot
2.1 Taxonomy
2.2 Differentiation of Monilinia species
2.3 Host range and distribution of Monilinia spp
3 Penetration sites in relation to fruit growth
3.1 Fruit susceptibility evolves along fruit development
3.2 Infection by direct penetration of the cuticle
3.3 Infection through the trichomes basis
3.4 Infection through stomata
3.5 Infection through skin cracks and wounds
4 Infection development
4.1 Adhesion to the cuticle and germination
4.2 Latent infection
4.3 Appressorium formation and hypha penetration
4.4 Appressoria melanization increase pathogenicity
4.5 pH lowering regulates the expression of pathogenicity genes.
4.6 Biochemical arsenal of Monilinia spp.
4.7 Post-penetration
5 Host factors for BR resistance/susceptibility in fruit
5.1 Constitutive components of BR resistance: plant cuticle, a multi-component barrier
5.2 Phenolic acids and their redox-mediated role in fungal inhibition
5.3 Active mechanisms in response to pathogen attack: defence proteins
5.4 ROS, oxidative stress and programmed cell death
6 Breeding for brown rot (BR) resistance
6.1 Genetic resources, breeding programs and phenotyping strategies
6.2 Field-borne inoculum assessment
6.3 Artificial infection assessment
6.4 QTL of resistance
7 Conclusion
Chapit􀆌e 2 : Etude de la p􀆌o􀄏a􀄏ilité d’i􀅶fe􀄐tio􀅶 au 􀄐ou􀆌s du développe􀅵e􀅶t du f􀆌uit, e􀅶 lie􀅶 ave􀄐 les caractéristiques structurales et biochimiques du fruit 
1 Introduction
2 Material and Methods
2.1 Plant material
2.2 Fruit sampling
2.3 Extraction
2.4 Analysis of wax compounds
2.5 HPLC Analysis
2.6 Cuticular conductance
2.7 Monilinia susceptibility
2.8 Statistical analysis
3 Results
3.1 Evolution of fruit characteristics during development
3.2 Fruit growth
3.3 Fruit surface conductance
3.4 Fruit susceptibility to M. laxa
3.5 Identification and characterization of fruit cuticular compounds
3.6 Cuticular wax composition
3.7 Identification of secondary compounds
3.8 Developmental variations of wax and surface compounds
3.9 Relationships between wax and surface compounds and fruit characteristics
3.10 Do wax and surface compounds correlate with Monilia laxa infection probability?
4 Discussion
4.1 New compounds detected in fruit surface
4.2 Variations with cultivars and between years
4.3 Evolution of wax and surface compounds during development
4.4 Role of wax and surface compounds in preventing fruit gas exchanges
4.5 Potential effect of wax and surface compounds on brown rot infection
5 Conclusion
Chapitre 3: Exploration des caractéristiques physiques du fruit immature en relation avec la sensibilité à M. laxa 
1 Introduction
2 Materials and methods
2.1 Vegetal material
2.2 Fruit sampling
2.4 Infection tests
2.5 Fruit surface conductance assessment
2.6 Stomata number estimation
2.7 Genetic linkage map construction and QTL analysis
2.8 Projection of QTL on a physical map
2.9 Statistical analysis
3 Results
3.1 Physical characteristics of young fruit of the population
3.2 Infection probability
3.3 Links between fruits characteristics and fungal susceptibility
3.4 QTL location
4 Discussion
4.1 Marker density and population size – factors controlling QTL detection
4.2 Counting stomata number on a mapping population
4.3 Exploring the link between stomata number and conductance
4.4 Identification of loci governing surface conductance
4.6 Identification of loci governing infection probability and progression
4.7 Hypotheses to explain the variations in susceptibility of young peach to M. laxa
5 Conclusion
Chapter 4: Etude de la variabilité génétique et cartographie QTL de la résistance à la pourriture brune (Monilinia laxa) dans une descendance interspécifique issue d’un croisement entre Prunus persica et P. davidiana 
1 Introduction
2 Materials and methods
2.1 Plant material
2.2 Monilinia laxa strain
2.3 Infection tests
2.4 Phenotyping
2.5 Measurements acquired in the laboratory test
2.6 Fruit surface conductance assessment
2.7 Genetic linkage map construction and QTL analysis
3.1 Screening for brown rot resistance with infection tests
3.2 High variability of scoring between years
3.3 Natural infection of stems in spring
3.4 Natural infection of fruit in the orchard in 2015
3.5 Comparison between infection methodologies
3.6 Survey of infection progression through lab tests
3.7 Fruit surface conductance
3.8 QTL location
4 Discussion
4.1 The BC2 progeny: an interspecific back cross as a potential source of resistance to M. laxa
4.2 Infection tests for scoring brown rot resistance: with or without wounding fruit?
4.3 Infection tests for scoring brown rot resistance: drop or spray, orchard or lab, natural or artificial infections?
4.4 Trying to explain the high instability between years
4.5 The QTL for brow rot resistance: where are we now?
5 Conclusion
Chapter 5: Etude de la variabilité des composés d’épiderme des f􀆌uits d’u􀅶e des􀄐e􀅶da􀅶􀄐e i􀅶te􀆌spé􀄐ifi􀆋ue issue d’un croisement entre Prunus persica et P. davidiana et liens avec la susceptibilité à la pourriture brune 
1 Materials and Methods
1.1 Fruit sampling
1.2 Epidermis preparation
1.3 Extraction
1.4 HPLC analyses
2 Results and discussion
2.1 Epidermis compounds showed great variations within the BC2 progeny
2.2 Some compounds were highly correlated between each other
2.3 Peaches and nectarines displayed huge differences
2.4 A hundred QTL were detected for epidermis compounds
2.5 Some compounds exhibited significant relationships with infection traits
2.6 Colocation of QTL for epidermis compounds and M. laxa susceptibility were observed
3 Conclusions
CONCLUSION GENERALE ET PERSPECTIVES 

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