The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

Memoria para optar al título profesional de Ingeniero Agrónomo / La sarna del manzano causada por Venturia inaequalis es la enfermedad más importante del manzano en Chile y a nivel mundial, y su control está basado principalmente en el uso de fungicidas. En los últimos años se ha observado en Chile una aparente disminución del nivel de control de la enfermedad con los fungicidas comúnmente utilizados con este propósito y se especula sobre una posible pérdida de sensibilidad de poblaciones del patógeno. Durante las temporadas 2006-2009, se obtuvo una colección de 128 cepas del patógeno que provino de hojas de manzanos infectados con sarna recolectadas entre las regiones VII y IX. Se realizaron pruebas de sensibilidad a los fungicidas mancozeb, difenoconazole, fenarimol y pyrimethanil en aislados monoconidiales de cepas silvestres del patógeno con el objetivo de elaborar curvas basales de sensibilidad y compararlas con la sensibilidad de aislados monoconidiales provenientes de seis huertos comerciales sin evidencias de resistencia práctica. La sensibilidad en las pruebas in vitro se midió como la inhibición de crecimiento miceliar (difenoconazole, fenarimol, y pyrimethanil) ó la inhibición de la germinación de conidias (mancozeb). A partir de los resultados obtenidos se propusieron dosis discrimininatorias de 0,04; 1,0; 0,6; y 0,2 μg mL-1 para difenoconazole, fenarimol, mancozeb y pyrimethanil, respectivamente. La población de huertos comerciales estudiada presentó una menor sensibilidad a los fungicidas difenoconazole, fenarimol y mancozeb con valores de factor de resistencia 4,7; 5,8 y 2,1, respectivamente. La mayor diferencia de sensibilidad entre la población silvestre y la de huerto se observó en fenarimol, fungicida para el cual la población de aislados de huerto presentó el mayor cambio hacia una menor sensibilidad. Por el contrario, para pyrimethanil, la población de huerto no evidenció pérdida significativa de sensibilidad en relación a la población basal. / Apple scab caused by Venturia Inaequalis is the most important disease of apples worldwide, and its control is primarily based on fungicides. In recent years an apparent disease control failure has been observed in Chile. A potential loss of fungicide sensitivity from pathogen populations is speculated. A collection of 128 strains were isolated from diseased leaves collected between VII and IX regions, during 2006 to 2009. Sensitivity tests to the fungicides mancozeb, difenoconazole, fenarimol, and pyrimethanil were conducted on wild isolates to build local baseline sensitivity distributions and to compare with the sensitivity of strains from six different commercial orchards without evidence of practical resistance. In vitro tests were measured as inhibition of mycelial growth or conidial germination (mancozeb). Discriminatory doses of 0.04; 1.0; 0.6; and 0.2 μg mL-1 were proposed according to the in vitro monitoring of sensitivity to difenoconazole, fenarimol, mancozeb and pyrimethanil, respectively. Departure from the baseline distribution towards less sensitivity was found for difenoconazole, fenarimol and mancozeb with resistance factors of 4.7; 5.8 and 2.1 respectively. The highest difference between baseline and orchard population was observed for fenarimol which showed the higher shift towards less sensitivity. In contrast, pyrimethanil did not show a not significant shift.

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Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia. / Ph. D.

La plasticité phénotypique, i.e. la capacité d’un génotype à exprimer plusieurs phénotypes en fonction des conditions environnementales, représente une source de variabilité phénotypique, et tient donc un rôle majeur dans l’évolution. Dans un environnement variable, les stratégies généralistes, capables de maintenir une valeur d’aptitude phénotypique (fitness) stable sur une plus large gamme de conditions environnementales grâce à une plus grande plasticité de traits sous-jacents, devraient être favorisées. L’analyse de normes de réaction, au laboratoire et en conditions naturelles, de traits physiologiques et de traits liés à la fitness en fonction de la température, révèle chez le parasitoïde Venturia canescens, que (i) la variabilité thermique de l’habitat détermine la forme des normes de réaction selon le compromis entre stratégies généraliste et spécialiste, (ii) les différences entre individus généralistes et spécialistes se maintiennent face à des fluctuations de la température mimant les conditions naturelles et (iii) les individus subissant une plus grande variabilité thermique dans leur habitat possèdent une capacité d’acclimatation au froid plus importante. Ces résultats montrent l’existence de deux stratégies chez V. canescens et donc une différenciation de leur niche thermique, autorisant leur coexistence en environnement hétérogène / Phenotypic plasticity, i.e. the ability of a genotype to express several phenotypes depending on environmental conditions, represents an important source of phenotypic variability and so plays a major role in evolution. In a variable environment, generalist strategies, able to maintain a stable value of fitness over a wide range of environmental conditions, thanks to a greater plasticity of underlying traits, should be favored. The analysis of reaction norms of physiological traits and traits related to fitness as a function of temperature reveals in the parasitoid Venturia canescens that (i) thermal variability of the habitat determines the shape of reaction norms, according to the trade-off between generalist and specialist strategies, (ii) differences between generalist and specialist individuals are maintained face to rapid fluctuations in temperature that mimic natural conditions and (iii) individuals experiencing greater variability in their thermal habitat have a greater capacity for cold acclimation. These results indicate the existence of two different strategies in V. canescens and therefore a thermal niche differentiation, allowing their coexistence in a heterogeneous environment

Depuis la domestication du pommier, l’homme a progressivement sélectionné des variétés plus performantes, notamment pour la qualité du fruit, la productivité ou la résistance aux pathogènes. Les bases génétiques de ces caractères ont été explorées par cartographie en descendances F1 ne permettant d’explorer qu’une infime partie de la diversité génétique disponible.L’objectif de la thèse portait sur l’analyse des bases génétiques de caractères de qualité du fruit et de résistance du pommier à la tavelure et au feu bactérien dans des collections représentant une diversité plus large. Le génotypage de core collections de variétés anciennes s’est fait à l’aide de deux puces 8k et 480kSNPs ou grâce à du ré-séquençage de gènes. Des traces de différenciation génétique entre pommes à cidre et à couteau ont été identifiées et partiellement reliées à la voie des polyphénols. Après analyse de l’étendue du déséquilibre de liaison à large et fine échelle, une approche de génétique d’association a permis l’identification de régions génomiques associées à la variation de plusieurs caractères de qualité du fruit, dont le haut du groupe de liaison 16 rassemblant l’acidité (locus Ma), la fermeté, la jutosité et l’amertume (gène LAR). Pour la résistance au feu bactérien, une région contenant un homologue du gèneNPR1 (activateur de défenses) a été identifiée.Cette thèse a ainsi permis de préciser la localisation potentielle de QTLs identifiés préalablement par cartographie génétique et d’identifier de nouvelles ressources utiles dans de futurs programmes de sélection assistée par marqueurs. / Since apple domestication, humans have progressively selected improved varieties, especially for traits linked with fruit quality, productivity or resistance to pathogens. The genetic bases underlying these traits have been explored thanks to genetic mapping in F1 segregating populations that only allows the study of a small part of the available genetic diversity. The aim of this work was to analyze the genetic bases of fruit quality and disease resistance against apple scab and fire blight, in collections of old apple varieties representing a much larger diversity. Genotyping of core collections was performed either with arrays of 8k and 480k SNPs or by resequencing of chosen genes. Signs of genetic differentiation were identified between cider and dessert apples and were partially linked to the polyphenols pathway. After studying linkage disequilibrium, both on a large and a small scale, an association genetics approach allowed the identification of genomic regions associated with the variation of several fruit quality traits. Especially, the top of linkage group 16 was found to be linked with acidity (locus Ma), firmness, juiciness and bitterness (LAR gene). Concerning the resistance of apple to fire blight, a region containing a homolog of the NPR1 gene (defense activator) was identified. This thesis allowed the refining of the putative localization of previously identified QTLs and the identification of new genetic resources that could be useful in future selection programs using marker assisted selection.

Apple scab is the most important disease of apples in most of the world. The disease, caused by Venturia inaequalis, is controlled by numerous fungicide applications, regardless of the presence of inoculum in the orchard. Better timing of fungicide applications could be achieved if the airborne ascospore concentration (AAC) was considered in decision making. AAC can be measured in real time using spore traps. In this project, the relationship between AAC and lesions development was studied under controlled and natural conditions for five cultivars: Empire, McIntosh, Jonagold, Royal Gala, and Spartan. Potted trees were exposed to different airborne ascospore inoculum and the corresponding AAC were measured using spore traps. The spatial distribution of ascospores was studied in a commercial apple orchard plot. The potential ascospore dose (PAD) and the AAC were measured in 40 quadrats in the spring of 1999. (Abstract shortened by UMI.)