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.

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.

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.

Made available in DSpace on 2014-06-11T19:26:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-17Bitstream added on 2014-06-13T19:33:27Z : No. of bitstreams: 1 souza_mc_me_jabo.pdf: 333310 bytes, checksum: 37cfc8757cedd14ed24ec276ff06a4ea (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho teve por objetivos: (i) selecionar genótipos de tangerinas resistentes a infecção por Alternaria alternata, e estabelecer relações entre níveis de severidade da doença entre folhas e frutos, (ii) avaliar a contribuição de metabólitos na resistência de folhas maduras de tangerinas a A. alternata, (iii) selecionar genótipos de tangerina resistentes a infecção por Elsinoe fawcettii. Para tais experimentos foram utilizados 22 genótipos de tangerina pertencentes ao banco de germoplasma da Estação experimental de Citricultura de Bebedouro, Bebedouro-SP. (i) Dentre os materiais genéticos resistentes, foram constatados quatro genótipos de clementinas (Citrus clementina), seis mandarinas, sendo duas pertencentes a C. reticulata, duas a C. tangerina, uma a C. deliciosa e uma a C. nobilis; um tangelo (C. tangerina x C. paradisi); dois híbridos de mandarinas, sendo um resultante do cruzamento entre C. nobilis x C. deliciosa, e o outro de C. clementina x C. reticulata; um híbrido de tangor (C. clementina x C. sinensis) e dois híbridos de satsumas (C. unshiu x C. deliciosa.). Também foi constatado indícios de relação entre severidade de doença em folhas jovens destacadas e incidência natural de doença em frutos. (ii) Ao final dos experimentos, não foi observado influência desses metabólitos na defesa das folhas maduras de tangerina a MMA. (iii) Dente os genótipos avaliados, foram observados 10 materiais genéticos resistentes a verrugose. Dentre estes materiais, observou-se que os genótipos Commune, Caffin, Bruno, Burgess, Peau Lisse, Zanzibar, Beuty of Glen Retreat, Rode King e Encore também se apresentaram resistentes a MMA. Estes materiais são promissores para o cultivo de tangerinas em áreas de ocorrência destas doenças, e poderão contribuir com futuros trabalhos de melhoramento genético para resistência a MMA e verrugose. / The aims of this work were: (i) select tangerine genotypes resistant to Alternaria alternata infection and classify relative degrees of disease severity among leaves and fruits, in order to ultimately control the pathogen; (ii) evaluate metabolites contribuition on mature leaves of tangerines to A. alternata; (iii) select tangerine genotypes resistant to Elsinoe fawcettii. For such experiments 22 tangerines genotypes from germoplasm bank at Estação experimental de Citricultura de Bebedouro, Bebedouro-SP were used. (i) Among the resistant genetic materials, four clementines genotypes (Citrus clementina), six mandarines, being two of C. reticulata, two C. tangerina, one C. deliciosa and one C. nobilis; one tangelo (C. tangerina x C. paradisi); two mandarines hybrids, being one resulted between C. nobilis x C. deliciosa crossing, and other at C. clementina x C. reticulata; one tangor hybrid (C. clementina x C. sinensis) and two satsumas hybrids (C. unshiu x C. deliciosa) were found. A consistent relationship was noted among level of susceptibility in leaves (as evaluated following either natural infection or artificial inoculation), severity and susceptibility of fruit, suggesting a new methodology for diagnosis, identification and selection of Citrus spp. varieties resistant to A. alternata. (ii) At the end of experiments, it was not found any relationship between the presence of these metabolites and resistance to MMA on tangerines mature leaves. (iii) Among the genotypes evaluated, 10 genetic materials resistant to scab were observed. Finally, Commune, Caffin, Bruno, Burgess, Peau Lisse, Zanzibar, Beuty of Glen Retreat, Rode King and Encore cultivars, being resistant to A. alternata and also to E. fawcettii, exhibited good agronomic characteristics and, consequently, show a promising economic exploration.

La modélisation de la dynamique épidémiologique des ascomycètes parasites des arbres fruitiers présente deux aspects très importants : l’évaluation de la quantité d'inoculum et de son évolution, et l’identification des évènements climatiques donnant lieu à une contamination. Le travail présenté aborde ces deux aspects au travers de deux exemples : la tavelure du pommier, et la maladie de l’œil de paon de l’olivier, parasites majeurs pour les filières concernées.L’acquisition des connaissances sur la biologie des deux maladies a été abordée uniquement sur la base de données acquises sur le terrain, ce qui est original car généralement les modèles ont été réalisés sur la base de connaissances établies en conditions contrôlées.Dans le cas de Venturia inaequalis, agent de la tavelure du pommier, la vitesse de développement journalier de l’inoculum a été établie par optimisation numérique, sur la base d’observations réalisées en Provence. Un modèle de dynamique de projection des ascospores incluant la suspension de l’évolution de l’inoculum primaire lors des périodes sèches a été réalisé puis validé sur un jeu de données indépendantes. La transposabilité du modèle a été ensuite étudiée pour une autre région (Aquitaine). Dans le cas de Fusicladium oleagineum, agent de la maladie de l’oeil de paon, les conditions de contamination ont été établies en fonction de la température et de l’hygrométrie, d’une part par la réalisation d’un abaque sur la base de points sélectionnés par dires d’expert, et d’autre part de façon automatique à l’aide d’un système par apprentissage (réseau de neurones). Un modèle, liant la température moyenne après la contamination à l’apparition des symptômes a été ensuite réalisé. / Apple scab, caused by Venturia inaequalis, and peacoq leaf spot, cause by Fusicladiumoleagineum, are key diseases respectively for apple and olive growers. These disease usuallyrequire a large number of treatments. Adequate protection need a good evaluation ofquantitative disponibility of inoculum, and estimation of infection conditions.In this thesis, these two problems were studied using only field data. This is an originalaprroach to obtain knowledge about biology of fungi : most previous works were realised byregression of laboratory data obtained under controled conditions.In the case of Venturia inaequalis, primary inoculum consists of pseudothecia present in leaflitter. Treatments agains ascospore release period is the cornerstone of the strategy againstapple scab. However, the existing forecasting models are not reliable, and are all based ondegree-day time scale, proposed in 1982.Here, using a corpus of data acquired between 1996 and 2013, including observations ofascospore release and weather data, we assessed the daily rate of development of primaryinoculum by fitting generic new time scale functions. Further improvements were then studiedto take into account elements reported in litterature about the incidence of rain or wetness.Different methods were tested and adapted for the parameterisation of models by numericaloptimisation. Some forcasting models were proposed and adapted to the area where the studywas conducted, with parameters including rain and temperature. The validity was tested, andfurther developements of the forecasting tool was then proposed.In the case of Fusicladium oleagineum, a field-operational model predicting disease outbreakswas established as a function of temperature and relative humidity. First with the help ofpoints selected by experts, Secondly automaticaly using a neural network. A model defininglatent period as a function of average temperature after contamination was then realised.

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Fusarium head blight (FHB) is one of the most economically important diseases of small grains and continues to impact crops when environmental conditions are favorable to Gibberella zeae (Fusarium graminearum), the causal agent of the disease. Corn residues are considered to be primary sources of inoculum for epidemics of FHB. Therefore, knowledge of the movement of Gibberella zeae from a local source of infested corn residue is critical to the management of FHB in wheat and barley. Previous research made significant progress in defining the spatial dissemination of inoculum sources of G. zeae within agricultural fields, but was unable to clearly distinguish between within-field and background sources. Using amplified fragment length polymorphism, released clones of G. zeae were tracked within wheat and barley fields. This strategy allowed the distinction between the contributions of released clones to FHB, compared to that of background inocula. Corn residue infested with clones of G. zeae was placed into small replicated plots in winter wheat fields in New York and Virginia in 2007 and 2008 and wheat spikes were collected at 0, 3, 6, and ≥24 m from the inoculum sources. Recovery of released clones decreased an average of 90% between 3 and 6 m from inoculum sources. Various amounts of corn residue infested with a single clone of G. zeae were placed into small replicated plots in winter wheat and barley fields in Virginia from 2008 to 2010. The use of minimal or conventional tillage and a moderately resistant cultivar of wheat or barley may reduce the contribution of within-field inocula to FHB; however, environmental conditions play an important role in the effectiveness of these management strategies. With the increase of corn production due to incentives for ethanol-based fuel, overwintering sites for G. zeae on corn residue are likely to increase. Our work contributes to an increased understanding of the influence of overwintered corn residue to FHB which will also direct future research on how to reduce the inoculum potential from within-field sources. / Ph. D.