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International audience; Intragenesis is an important alternative to transgenesis to produce modified plants 27 containing native DNA only. A key point to develop such a strategy is the availability of 28 regulatory sequences controlling the expression of the gene of interest. With the aim of 29 finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora 30 (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes 31 encode oxidative enzymes involved in many physiological processes and have been 32 previously shown to be up-regulated during the Ea infection process. We found ten PPO and 33 two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 34 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-35 constitutive regulatory sequences respectively. Expression levels of reporter genes fused to 36 these promoters and transiently or stably expressed in apple were quantified after various 37 treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and 38 strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System 39 dependent manner. Altogether our results indicate that pKFDV02 did not keep its promises as 40 a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned 41 from apple, can be a useful component of intragenic strategies to create fire blight resistant 42 apple genotypes.

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.

International audience; Under natural growth conditions, plants experience various and repetitive biotic and abiotic stresses. Salicylic acid (SA) is a key phytohormone involved in the response to biotic challenges. Application of synthetic SA analogues can efficiently prime defense responses, and leads to improved pathogen resistance. Because SA analogues can result in long-term priming and memory, we identified genes for which expression was affected by the SA analogue and explored the role of DNA methylation in this memorization process. We show that treatments with an SA analogue can lead to long-term transcriptional memory of particular genes in Arabidopsis. We found that subsequent challenging of such plants with a bacterial elicitor reverted this transcriptional memory, bringing their expression back to the original pre-treatment level. We also made very similar observations in apple (Malus domestica), suggesting that this expression pattern is highly conserved in plants. Finally, we found a potential role for DNA methylation in the observed transcriptional memory behavior. We show that plants defective in DNA methylation pathways displayed a different memory behavior. Our work improves our understanding of the role of transcriptional memory in priming, and has important implication concerning the application of SA analogues in agricultural settings.

International audience; Apple fruit growth is the result of several factors: inherent demand (relative sink strength) of the fruit (defined by the demands for cell division and expansion growth, etc.), carbon assimilation by the source leaves (source strength), and the resulting allocation to the organ in question. It is thus a complex process involving source-sink interactions. In the present study, we designed an experimental system in which parts of fruit-bearing branches of two apple cultivars ("Fuji" and "Ariane") were isolated from the rest of the tree by girdling and then subjected to specific pruning and fruit removal treatments to create a wide range of global (branch-level) source-sink ratios. We monitored not only fruit kinetics but also photosynthesis as a response to light in leaves of the three different shoot types (i.e., the rosette, the bourse, and the vegetative shoots) to 1) study the impact of source-sink distance on carbon partitioning between fruits within the same branch and 2) to investigate the impact of source/sink ratio on fruit growth and leaf photosynthetic activity. Our results indicate 1) no significant differences among lateral fruits belonging to different ranks, and this independent of source availability; 2) that a modification of the source/sink ratio seems to be compensated by an alteration of the photosynthetic rate of leaves, with stronger and weaker values obtained for lower and higher ratios, respectively. Moreover, our results seem to suggest that two growing sinks together will upregulate photosynthesis rate more strongly than one growing sink does on its own, and this with the same leaf area per fruit. These results are discussed, and some hypotheses are put forward to explain them.

International audience; Apple orchard production is facing new environmental and societal challenges, resulting, in particular, in strong pressure to reduce pesticide use. Cider-apple production, for which the perfect visual aspect of fruits is not a marketability imperative, offers good opportunities to study production systems that are developing new agronomic strategies, which could be subsequently extended to all apple-production types. Agroecological infrastructures play an important role in providing shelter, food resources, or reproduction habitats to many arthropods. Consequently, setting-up agroecological infrastructures in the vicinity of or within orchards could increase natural enemy presence and thus improve the biological control of pests. In this study, we focused on Dysaphis plantaginea (Passerini), one of the major pests in apple orchards in Europe, which causes important economic production losses. During two years (2014 and 2015), we monitored the population dynamics of D. plantaginea, its natural enemies, and mutualistic ants in commercial production cider-apple orchards. The influences of the cider-apple cultivar, insecticide use, and distance to agroecological infrastructures (hedgerows and flower strips) were assessed. Our results suggest that flower strips favor an increase in natural enemy abundance in the vicinity of the orchards and could thus play an important role in the production system by improving the biological control of D. plantaginea.

Deciphering the genetic control of flowering and ripening periods in apple is essential for breeding cultivars adapted to their growing environments. We implemented a large Genome-Wide Association Study (GWAS) at the European level using an association panel of 1,168 different apple genotypes distributed over six locations and phenotyped for these phenological traits. The panel was genotyped at a high-density of SNPs using the Axiom®Apple 480 K SNP array. We ran GWAS with a multi-locus mixed model (MLMM), which handles the putatively confounding effect of significant SNPs elsewhere on the genome. Genomic regions were further investigated to reveal candidate genes responsible for the phenotypic variation. At the whole population level, GWAS retained two SNPs as cofactors on chromosome 9 for flowering period, and six for ripening period (four on chromosome 3, one on chromosome 10 and one on chromosome 16) which, together accounted for 8.9 and 17.2% of the phenotypic variance, respectively. For both traits, SNPs in weak linkage disequilibrium were detected nearby, thus suggesting the existence of allelic heterogeneity. The geographic origins and relationships of apple cultivars accounted for large parts of the phenotypic variation. Variation in genotypic frequency of the SNPs associated with the two traits was connected to the geographic origin of the genotypes (grouped as North+East, West and South Europe), and indicated differential selection in different growing environments. Genes encoding transcription factors containing either NAC or MADS domains were identified as major candidates within the small confidence intervals computed for the associated genomic regions. A strong microsynteny between apple and peach was revealed in all the four confidence interval regions. This study shows how association genetics can unravel the genetic control of important horticultural traits in apple, as well as reduce the confidence intervals of the associated regions identified by linkage mapping approaches. Our findings can be used for the improvement of apple through marker-assisted breeding strategies that take advantage of the accumulating additive effects of the identified SNPs. JU has been partially supported by an Early Stage Research Fellowship of the Institute of Advanced Studies (University of Bologna).

International audience; The impact of a cultivar mixture within the rows including the cvs Melrouge (low susceptibility to scab) and Pitchounette (Rvi6 resistant) was assessed during 2008e2009 in an organic apple orchard located in Rhône Valley (Drôme, France), on three important pests and diseases: scab, powdery mildew and rosy apple aphid. In this area, the resistance of Rvi6 gene was effective since the orchard plantation in 2004. The impact of the cultivar mixture was assessed per se, but also associated with sanitation practices to decrease the primary inoculum of scab and mildew, and compared to disease development on pure stands of Melrouge. The cultivar mixture showed a good efficiency to decrease the scab incidence on leaves and fruits; a significant effect on scab severity was observed in 2009 for the cultivar mixture plus sanitation practices. Our data confirmed the different susceptibility levels of the two cultivars for powdery mildew. Against powdery mildew, a favourable effect of cultivar mixture, at the condition it is associated with sanitation, was observed in case of a moderate level of the disease (2008). For rosy apple aphid, a favourable effect of cultivar mixture was observed on Melrouge in 2008 when Pitchounette has a good resistance level. In the context of organic production, the impact of the cultivar mixture seemed favourable for the control of the three pests and diseases.

International audience; Adequate protection of apple trees during the primary contamination period is a cornerstone for the management of apple scab. Correct timing of spring treatments is fundamental and thus, much effort has been devoted to forecasting ascospore release by Venturia inaequalis. Most models rely on degree-day accumulation starting from a biofix date established yearly on the basis of biological observations. Here, we explored the potential interest of using a single calendar date as a biofix and new types of time scales, with the help of numerical optimization with field-collected data. Using data acquired between 1996 and 2008, we assessed the daily rate of development for V. inaequalis primary inoculum by fitting generic time scale functions, a method which requires the smallest number of assumptions about the effect of temperature on the biological phenomenon. An optimal calendar biofix was established for Provence and the use of non-linear functions relating pseudothecial development rate to temperature for accumulating thermal time was compared with the usual linear response in standard degree-day models. A model was then constructed using 4 additional years of data for validation. The predictive value of the model was further improved by adjusting the time scale with “accelerating rules” to take into account the positive influence of rainy days on pseudothecial maturation prior to ascospore release. However, halting rules inserted in the time scale to account for dry days during the ascospore release period strongly reduced the predictive value of the model in the conditions of Southern France, suggesting the possible occurrence of strains adapted to dry conditions.

La pomme (Malus Domestica Borkh.), fruit largement répandu dans les pays tempérés est beaucoup consommée. Elle représente une source importante en composés phénoliques. Cette étude s’est intéressée aux polyphénols des tissus du parenchyme. La problématique s’oriente sur les effets de la texture sur la diffusion de ces molécules. L’originalité de l’approche repose sur l’association de la texture, de la pression osmotique et la diffusion des polyphénols. Les méthodes de caractérisations physiques et biochimiques ont permis de mesurer les changements à l´échelle macroscopique et les modifications chimiques qui s’opèrent dans les matrices végétales. Les résultats de l’étude du transfert de matière ont permis de mettre en évidence les différents facteurs pouvant influer sur les valeurs des coefficients de diffusion. La texture, l’épaisseur, la variété du fruit et la pression du milieu diffusant, constituent des facteurs pouvant influencer le transfert de matière. L’étude de l’évolution de composant de la paroi a montré des changements qui s’opèrent au cours de la diffusion. Des analyses microscopiques ont relevé les modifications à l’échelle cellulaire de la diffusion de procyanidines, polyphénols majoritaires et des interactions avec les composants pariétaux., Apple (Malus domestica Borkh. ) fruit widespread in temperate countries, is much consumed.It represents an important source of phenolic compounds. This study was interestedin polyphenol content of apple tissue parenchyma. The problem concerns effects of texturedegradation on the diffusion of polyphenols molecules. The originality of the approach isbased on the combination of texture, osmotic pressure and polyphenol leaching. Physicaland biochemical methods were used to measure changes at macroscopic scale and chemicalchanges occurring in the parenchymateous tissue . The study of mass transfer highlightedvarious factors that may affect apparent coefficient diffusion. The result showed that thedisintegration of texture , thickness, apple variety and osmotic pressure of leaching mediacan influence mass transfer yield. The study of the Cell walls components showed changesthat occur during leaching process. Light microscopic analysis revealed changes at cellularscale, procyanidins the major polyphenols, leaching phenomena and also interactionswith cell walls matrix.

It is generally postulated that at the tree scale a drought-related decrease in hydraulic conductance is balanced by a decrease of leaf area. We hypothesized that, at the individual leaf scale, drought affects the allometry between leaf area or mass and hydraulics, leading to a non-linear relationships between these traits. The study was conducted on well-watered and on water-stressed shoots of several apple genotypes covering an extended range of leaf area. Working on dried leaves, we measured leaf lamina area and mass and analyzed their relationships with the maximal xylem hydraulic conductance of the water pathway through the parent shoot and the petiole connected to the leaf lamina. Drought decreased leaf area and mass in absolute values. It also changes the allometric relationships between these two variables: for a same decrease of leaf dry mass the water-stressed shoot had a lower decrease of leaf dry area than the well-watered shoot. Our study also showed that drought affected the stem-topetiole hydraulics with a higher hydraulic efficiency in the well-watered shoot compared to the waterstressed shoot. We discuss that, compared to the well-watered condition, drought not only decreased leaf size, but also reduced xylem efficiency through the stem-to-petiole pathway with regard to the leaf area and mass supplied.

National audience; La tavelure est certainement la maladie la plus redoutée dans les vergers de pommiers biologiques. Pour limiter les contaminations primaires au printemps, il est possible de réduire l'inoculum primaire en éliminant les feuilles mortes tombées au sol à l'automne, par broyage ou par ramassage. Ces deux méthodes ont déjà prouvé leur efficacité mais elles n'ont jamais été comparées. Un essai a été mis en place dans un verger commercial biologique de la Drôme afin de comparer ces deux méthodes de prophylaxie : balayage ou broyage des feuilles de l'inter-rang, complétés par l'enfouissement des feuilles sur le rang. Le retrait des feuilles par balayage a été plus efficace que la méthode de broyage et a permis de limiter plus fortement le développement des épidémies de tavelure au printemps suivant.

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International audience; The question as to why parasites remain generalist or become specialist is a key unresolved question in evolutionary biology. Ampelomyces spp., intracellular mycoparasites of powdery mildew fungi, which are themselves plant pathogens, are a useful model for studies of this issue. Ampelomyces is used for the biological control of mildew. Differences in mycohost phenology promote temporal isolation between sympatric Ampelomyces mycoparasites. Apple powdery mildew (APM) causes spring epidemics, whereas other powdery mildew species on plants other than apple cause epidemics later in the season. This has resulted in genetic differentiation between APM and non-APM strains. It is unclear whether there is genetic differentiation between non-APM Ampelomyces lineages due to their specialization on different mycohosts. We used microsatellites to address this question and found no significant differentiation between non-APM Ampelomyces strains from different mycohosts or host plants, but strong differentiation between APM and non-APM strains. A geographical structure was revealed in both groups, with differences between European countries, demonstrating restricted dispersal at the continent scale and a high resolution for our markers. We found footprints of recombination in both groups, possibly more frequent in the APM cluster. Overall, Ampelomyces thus appears to be one of the rare genuine generalist pathogenic fungi able to parasitize multiple hosts in natural populations. It is therefore an excellent model for studying the evolution of pathogens towards a generalist rather than host-specific strategy, particularly in light of the tritrophic interaction between Ampelomyces mycoparasites, their powdery mildew fungal hosts and the mildew host plants.

A QTL analysis reveals two major genomic regions involved in apple resistance to three races of [i]Venturia inaequalis[/i]. 10.Plant and Animal Genome (PAG)

Application of near infrared spectroscopy to the non-destructive assessment of the development stages of apples. 6. International Symposium on Fruit, Nut, and Vegetable Production Engineering

Session 4 : Breeding Aims and Objectives; Use of apple genetic resources in apple breeding programmes at Inra: present status and perspectives. European Malus Germplasm

La recherche de variétés de nouvelles pommes

Une nouvelle pomme : Baujade

Impératifs pour la conservation :[br/] - le froid ;[br/] - l'atmosphère de conservation;[br/] - empêcher la déshydratation;[br/] - Conserver les qualités organoleptiques[br/] Actions sur les maladies de conservation :[br/] - traitements spécifiques;[br/] - effets des conditions de conservation sur les maladies potentielles;[br/] - évolution possible.

Rubrique La rugosité des pommes - II; En 1977, l'Inra a proposé un mutant de "Golden Delicious" : "Lysgolden-Goldenir" à l'inscription au catalogue officiel des espèces et variétés cultivées en France. Il s'agit d'un mutant obtenu par traitement mutagène aux rayons Gamma du Cobalt 60, améliorant la présentation du fruit sans modifier sensiblement les qualités organoleptiques.

In vitro androgenesis in cultivated apple ([i]Malus x domestica Borkh[/i]). Impact of Biotechnology in Agriculture

The fireblight susceptibility of 75 named cv. of apple and of two Malus species has been evaluated through artificial inoculation, during 3 or 4 consecutive years for shoots inoculation and 2 consecutive years for flowers inoculation. The result of shoot inoculations are assessed in 5 classes of a varietals index susceptibilty (V.I.S.).

Rubrique Production recherche; Les producteurs, sous l'impulsion des ingénieurs et techniciens de développement, attachent de plus en plus d'importance à la qualité des fruits : qualité de présentation, mais aussi qualité organoleptique. - Application du test - Composition de la solution-test - Observations - Remarque importantes

L'expérimentation conduite en 1985 sur deux variétés de pommes "Golden Delicious" et "Granny Smith" a permis de compléter les travaux publiés sur l'évolution de la teneur en amidon des fruits au cours des cueillettes échelonnées, par l'étude de sa localisation et de sa régression sur les fruits entreposés au laboratoire ou au froid en atmosphère normale (+ 2°C).

Les producteurs, sous l'impulsion des ingénieurs et techniciens de développement, attachent de plus en plus d'importance à la qualité des fruits : qualité de présentation, mais aussi qualité organoleptique.[br/] - Application du test[br/] - Composition de la solution-test[br/] - Observations[br/] - Remarque importantes

National audience

L'étude des stades de sensibilité à la rugosité a été effectuée sur des pommes de la variété Golden Delicious par pulvérisation d'un fongicide cuprique aux divers stades de l'évolution du fruit ; les témoins n'ont reçu aucun traitement. La période d'induction de la rugosité s'étend depuis le stade D (bouton vert) jusqu'au-delà du stade J (fruit gros comme une noix). Le maximum de sensibilité se situe aux stades H et I.

L'étude au verger de l'action des facteurs induisant la rugosité nécessite la définition d'une méthode d'évaluation de ce phénomène. La pomme est divisée en 4 zones pour chacune desquelles les notes 0,1 et 2 sont données, suivant l'intensité de la rugosité. L'analyse statistique des résultats montre que seule la zone 3 ("joues" côté oculaire) peut être retenue. Un exemple d'application de la méthode est donné : action de 3 fongicides.

Histologie[br/] - Proposition d'une échelle de notation[br/] - Action des facteurs climatiques[br/] - Action des produits de traitement[br/] - Facteurs trophiques[br/] - Possibilités d'obtention de clones moins sensibles à cet accident ou totalement résistants[br/]

I- Introduction[br/] II- Comparaison de la qualité gustative de "Golden delicious" en différents vergers[br/] III- Influence de l'éclaircissage sur la qualité des fruits chez la variété Golden delicious[br/]

I- Matériel et méthodes[br/] II- Evolution de certaines caractéristiques de fruits pendant la période de récolte[br/] . poids moyen du fruit[br/] . pertes relatives en poids[br/] . couleur de l'épiderme du fruit[br/] . couleur des pépins[br/] . fermeté de la chair[br/] . teneur en amidon[br/] . évolution des caractères chimiques[br/] III- Evolution de la qualité gustative en fonction de la date de récolte[br/] . méthode et organisation des dégustations[br/] . observations générales et résultats[br/] . date critique de cueillette et différents critères[br/] IV- Qualité gustative et analyses chimiques

Introduction[br/] Histologie[br/] Facteurs climatiques[br/] Produits de traitements[br/] Facteurs intrinsèques et trophiques[br/] Différences entre fruits atteints de russetting et indemnes[br/] Conclusion-discussion[br/] Discussions (après conférence)