Made available in DSpace on 2014-06-11T19:28:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-05Bitstream added on 2014-06-13T19:58:04Z : No. of bitstreams: 1 motta_rr_me_jabo.pdf: 906417 bytes, checksum: 313df4fc7f074ff43b3f9ee2d69280ca (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este trabalho teve como objetivos monitorar a quantidade de resíduo existente em frutos de laranjeira ‘Valência’ dos fungicidas oxicloreto de cobre, tiofanato metílico e pyraclostrobin, como também determinar a capacidade protetora e fungistática (oxicloreto de cobre) e a capacidade preventiva e curativa (tiofanato metílico e pyraclostrobin) no controle da mancha preta dos frutos cítricos (MPC). No primeiro experimento, aproximadamente 1000 frutos, previamente ensacados, foram primeiramente imersos nas caldas fungicidas e posteriormente inoculados com conídios de Guignardia citricarpa em intervalos regulares de 1, 7, 14, 21, 28 e 35 dias após a aplicação do fungicida, para o oxicloreto de cobre e de 1, 7, 14, 21, 28, 35, 42, 49 e 56 dias após a aplicação do fungicida, para o tiofanato metílico e pyraclostrobin. No segundo experimento, aproximadamente 1000 frutos, previamente ensacados, foram primeiramente inoculados e posteriormente tratados por imersão com os respectivos fungicidas nos mesmos intervalos regulares. Foram utilizados 16 frutos por período de inoculações e oito frutos por período de aplicação do fungicida. Para o primeiro experimento, oito frutos foram utilizados para a realização das análises de resíduo e outros oito frutos deixados na planta até a época da colheita. Para o segundo experimento, somente avaliou-se a incidência da doença e severidade na colheita. As análises de resíduos foram realizadas por técnicas de Espectrofotometria de Absorção Atômica na superfície dos frutos, para o oxicloreto de cobre e de Cromatografia Líquida de Alta Eficiência acoplada ao detector de massas/massas (CLAE - EM/EM) na superfície e casca dos frutos, para o tiofanato metílico e na superfície, casca e polpa dos frutos para o pyraclostrobin. Na época da colheita, os frutos foram avaliados quanto à incidência, severidade... / The objectives of this study were: (i) measure the amount of residues of copper oxychloride, methyl thiophanate and pyraclostrobin in 'Valencia’ sweet orange fruits after their application; (ii) to evaluate the protective and fungistatic capacity of copper oxychloride and (iii) to evaluate the preventive and curative capacity of systemic fungicides methyl thiophanate and pyraclostrobin for the control of citrus black spot (CBS). Two experiments were carried out, in the first on 1.000 fruits were first immersed in the fungicides and then inoculated with conidia of Guignardia citricarpa at regular intervals of 1, 7, 14, 21, 28 and 35 days, for the copper oxychloride and 1, 7, 14, 21, 28, 35, 42, 49 and 56 days for methyl thiophanate and pyraclostrobin. In the second experiment, on 1.000 fruits were first inoculated with conidia of G. citricarpa and then treated by immersion with the fungicides in the same regular intervals as described. Sixteen fruits were used for each period of inoculations and eight fruits per application. For the first experiment, eight fruits were used for analysis of residues and eight fruits were left in the plant until harvesting to evaluate disease incidence and severity. For the second experiment it was evaluate only disease incidence and severity in the harvest. Analysis of residues were performed by Atomic Absorption Spectrometry on the fruits surface, for copper oxychloride and High Performance Liquid Chromatography coupled to the detector mass/mass (HPLC - MS / MS) on the fruits surface and peel for the methyl thiophanate, and on the fruits surface, peel and pulp for the fungicide pyraclostrobin. Under conditions of high inoculum pressure, copper oxychloride showed limited protective capacity and no fungistatic capacity. Residues found on the fruits surface were directly related to the fruits growth. Methyl thiophanate was converted on its metabolite... (Complete abstract click electronic access below)

Submitted by Andréia Fujimoto null (andreia.fujimoto@gmail.com) on 2017-07-18T04:41:49Z No. of bitstreams: 1 Tese_Andréia_Fujimoto.pdf: 4726485 bytes, checksum: c5c04fef91dcac42292a2f2a70af9a32 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-07-19T17:16:00Z (GMT) No. of bitstreams: 1 fujimoto_a_dr_jabo.pdf: 4726485 bytes, checksum: c5c04fef91dcac42292a2f2a70af9a32 (MD5) / Made available in DSpace on 2017-07-19T17:16:00Z (GMT). No. of bitstreams: 1 fujimoto_a_dr_jabo.pdf: 4726485 bytes, checksum: c5c04fef91dcac42292a2f2a70af9a32 (MD5) Previous issue date: 2017-06-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O setor citrícola é o maior produtor de frutos no mundo, porém, encontra-se ameaçado por diversos patógenos, dentre esses, o fungo Phyllosticta citricarpa, agente causal da mancha preta dos citros. Como alternativa à utilização de fungicidas sintéticos para o controle do patógeno, o controle biológico, utilizando bactérias do gênero Bacillus, apresenta destaque. Tais microrganismos possibilitam o biocontrole pela produção de compostos orgânicos voláteis ou não voláteis. Sendo assim, este trabalho teve por objetivos: identificar os isolados de Bacillus spp. por meio de técnicas moleculares; avaliar a produção de compostos orgânicos voláteis produzidos por Bacillus spp. em diferentes meios de cultura; verificar os seus efeitos na evolução dos sintomas da doença em frutos de laranja, na morfologia de P. citricarpa em lesões de mancha preta e na indução de resistência, identificar os compostos voláteis por cromatografia gasosa unidimensional e finalmente, avaliar o efeito de diferentes meios de cultura no antagonismo e metabolismo secundário dos isolados de Bacillus. Os isolados bacterianos foram identificados como pertencentes às espécies de Bacillus subtilis, B. amyloliquefaciens e B. methylotrophicus. Os resultados mostraram que houve maior controle do patógeno, em decorrência da produção de voláteis, quando se utilizou os meios triptona de soja ágar (TSA) e caldo de triptona (TSB) para cultivo da bactéria, apresentando valores de inibição da colônia do fungo de até 73%. Com relação ao efeito dos compostos voláteis na evolução dos sintomas da doença, verificou-se que os melhores resultados, em termos de inibição (86%) de manchas sardentas que evoluíram para manchas duras, foram obtidos com o ACB-65 e ACB-73, quando cultivados em meio de cultivo TSB. Tais antagonistas causaram deformações nas hifas do patógeno, que podem explicar a não evolução dos sintomas da doença, como relatado anteriormente. Os voláteis de Bacillus spp. não foram capazes de ativar a atividade de enzimas indicativas de indução de resistência em frutos de laranja. As frações voláteis produzidas pelas bactérias foram identificadas e apresentaram álcoois, cetonas, aminas, éteres, aldeídos, fenóis e ácidos carboxílicos que podem servir como arsenal contra o fitopatógeno. Em relação ao efeito de diferentes meios de cultivo no antagonismo microbiano e no metabolismo secundário dos isolados de Bacillus, não houve diferença significativa entre os meios testados, porém proporcionaram redução significativa da colônia do fungo. Os metabólitos difundidos no meio de cultura foram identificados e apresentaram dois tipos de antibióticos, as iturinas e as surfactinas. O presente trabalho demonstrou o potencial de compostos orgânicos voláteis e não voláteis produzidos por Bacillus spp. no controle de P. citricarpa in vitro e in vivo, sendo uma alternativa ao uso de fungicidas sintéticos para o controle da mancha preta dos citros. / The citrus sector is the largest producer of fruits in the world, but it is threatened by several pathogens, among them Phyllosticta citricarpa fungus, causal agent of the citrus black spot. As an alternative to the use of synthetic fungicides to control the pathogen, the biological control, using bacteria of the genus Bacillus, is highlighted. Such microorganisms enable biocontrol by the production of volatile or non-volatile organic compounds. Therefore, this work had as objectives: to identify the isolates of Bacillus spp. by means of molecular techniques; to evaluate the production of volatile organic compounds produced by Bacillus spp. in different culture media; to verify its effects on the evolution of orange disease symptoms, P. citricarpa morphology in black spot lesions and induction of resistance, to identify the volatile compounds by one-dimensional gas chromatography and, finally, to evaluate the effect of different culture in the antagonism and secondary metabolism of Bacillus isolates. The bacterial isolates were identified as belonging to the species of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus methylotrophicus. The results showed that there was greater control of the pathogen, due to the volatile production, when tryptone broth (TSA) and tryptone broth (TSB) were used to culture the bacteria, presenting inhibition values of the fungus colony up to 73%. With regard to the effect of volatile compounds on the evolution of disease symptoms, the best results in terms of inhibition (86%) of freckles that progressed to hard spots were obtained with ACB-65 and ACB-73, when cultured in TSB culture medium. Such antagonists have caused deformations in the hyphae of the pathogen, which may explain the non-evolution of the disease symptoms, as previously reported. The volatiles of Bacillus spp. were not able to activate the enzyme activity indicative of induction of resistance in orange fruits. The volatile fractions produced by the bacteria were identified and showed alcohols, ketones, amines, ethers, aldehydes, phenols and carboxylic acids that can serve as arsenal against the phytopathogen. In relation to the effect of different culture media on the microbial antagonism and the secondary metabolism of the Bacillus isolates, there was no significant difference between the media tested, but provided a significant reduction of the fungus colony. The metabolites diffused in the culture medium were identified and presented two types of antibiotics, iturins and surfactins. The present work demonstrated the potential of volatile and non-volatile organic compounds produced by Bacillus spp. In the control of P. citricarpa in vitro and in vivo, being an alternative to the use of synthetic fungicides for the control of citrus black spot.

Orientador: Antonio de Goes / Banca: Edson Luiz Furtado / Banca: Marcel Bellato Spósito / Resumo: Embora o Brasil seja o maior produtor de laranja a nível mundial, a produção citrícola ressente-se de vários problemas de natureza fitossanitária, entre estes a mancha preta dos frutos cítricos, causada por Guignardia citricarpa. Esse fungo além de depreciar comercialmente os frutos, pode levar à queda precoce de mais de 80% da produção. O controle baseia-se no uso de fungicidas, sendo importante determinar o período curativo em que estes produtos estão sendo mais eficientes. Neste trabalho foi determinado o período curativo do pyraclostrobim e do carbendazim, em duas etapas fisiológicas do desenvolvimento dos frutos, sendo a primeira na fase de de queda de pétalas até a 8a semana, e a segunda repetindo-se o tratamento, porém a partir da 9a semana após a queda das pétalas. Para tal, procedeu-se o tratamento dos frutos com os fungicidas, os quais foram aplicados sob a forma de imersão, durante 1 minuto, sendo posteriormente ensacados com sacos de papel cristal, e mantidos assim até a 42a semana. Na primeira etapa, os níveis de incidência e severidade nos frutos tratados com fungicidas foram baixos até a 4a semana de exposição às infecções naturais, não diferindo estatisticamente entre si. Na segunda etapa, constatou-se maior eficiência do fungicida carbendazim, a qual se estendeu até a 6a semana de exposição. O pyraclostrobin, embora tenha se mostrado eficiente no controle da doença, manteve sua efetividade de forma mais consistente nas duas primeiras semanas de exposição. Admite-se que os fungicidas testados apresentaram excelente efeito curativo, resultados esses coerentes às respostas que têm sido observadas sob condições naturais de infecção. / Abstract: Although Brazil is the largest world-wide orange producer, its production presents several disease problems, among these is the Citrus Black Spot (CBS) caused by Guignardia citricarpa. This fungs besides depreciating the fruits commercially can cause premature fall of more than 80% of the production. The control is based on the use of fungicides, being important to determine the healing period, in wich these products are more efficient. In the present work the healing period of pyraclostrobin and carbendazim was determined on two physiological stages of the fruits development, being the first in the petals fall stage until the 8th week and the second repeating the treatment, however starting from the 9th week after the petals fall. The fruits were treated with the fungicides, wich were applied on immersion form, during 1 minute, being after sacked with crystal paper bags and maintained like this until the 42nd weekend. In the first stage, the incidence levels and severity on the treated fruits were low until the 4th week of exposure to natural infections, presenting no statistic differences between then. In the second stage, the efficiency of carbendazim was larger, wich extended for 6 weeks of exposure. The pyraclostrobin, although has shown efficient in the disease control, maintained its effectiveness in a consistent form on the first two weeks. It is admitted that the tested fungicides presented excellent healing effect, corresponding to the results observed under natural conditions of infection. / Mestre

Master of Science

Submitted by DANILO FRANCO null (franco.danilo@gmail.com) on 2018-01-08T23:48:13Z No. of bitstreams: 1 Tese_Danilo_Franco.pdf: 1663923 bytes, checksum: 2d8e6e2bc505a1017e531d2fb608942f (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-01-09T09:37:21Z (GMT) No. of bitstreams: 1 franco_d_dr_jabo.pdf: 1663923 bytes, checksum: 2d8e6e2bc505a1017e531d2fb608942f (MD5) / Made available in DSpace on 2018-01-09T09:37:21Z (GMT). No. of bitstreams: 1 franco_d_dr_jabo.pdf: 1663923 bytes, checksum: 2d8e6e2bc505a1017e531d2fb608942f (MD5) Previous issue date: 2017-11-13 / Neste estudo, foram realizados três experimentos (E1, E2 e E3). Os dois primeiros experimentos (E1 e E2) referem-se a avaliações da eficiência dose-resposta dos fungicidas hidróxido de cobre (HC), oxicloreto de cobre (OC) e óxido cuproso (OCp), aplicados nos estádios F1 e F2, no controle de Phyllosticta citricarpa, agente causal da mancha preta dos citros (MPC). E, no terceiro experimento (E3), foi avaliada a influência da aplicação de piraclostrobina em consonância com eventos chuvosos, no controle da MPC. Em E1 e E2 os fungicidas e dosagens avaliadas foram (g de cobre metálico 100 L-1 de calda): HC (43,7; 35,0; e 26,2; OC (100,8; 90,7; e 68,0) e; OCp (90,0; 56,3; e 42,2). No experimento E1 foram utilizadas plantas de laranjeira ‘Pera’, sob condições naturais de infecção, em Olímpia/SP. Foi adotado o delineamento experimental de blocos ao acaso, com quatro repetições. Cada parcela foi constituída de 33 plantas. Os fungicidas foram aplicados mediante turbopulverizador e vazão de 135 mL de calda m-3 de copa. Posteriormente, em todos os tratamentos, a partir do estádio F4 foram realizadas quatro aplicações de azoxistrobina a 75 g de i.a. 2.000 L-1, em intervalo de 42 dias. Foram realizadas quatro avaliações mensais, de agosto a novembro, amostrando-se casualmente 100 frutos nas seis plantas centrais da parcela, determinando-se a incidência (I) e severidade dos sintomas mediante escala de notas. A partir dos dados de severidade dos sintomas foi determinado o índice de doença (ID). Com os dados de ID, ao longo do tempo, calculou-se a área abaixo da curva de progresso da doença (AACPD). Em E2 foram empregadas plantas de laranjeira ‘Pera‘, também em Olímpia/SP. Os tratamentos avaliados foram semelhantes aos anteriormente citados, sendo, porém, aplicados com pulverizador manual do tipo “De Vilbs”, com volume suficiente para cobertura uniforme dos frutos. Foi adotado o delineamento de blocos ao acaso, em arranjo fatorial 10 tratamentos, sendo nove combinações de fungicidas cúpricos e uma testemunha, sem fungicida) x 5 (épocas de inoculação) e 4 (repetições). Cada parcela foi constituída por 20 frutos previamente ensacados com papel semi-kraft, no estádio F1. Posteriormente, no estádio F4, procedeu-se remoção dos sacos de papel, seguido do tratamento dos frutos exclusivamente com fungicidas cúpricos. O tratamento dos frutos deu-se mediante sua imersão, durante x segundos, na calda fungicida acrescida de óleo mineral a x%. Posteriormente, após secagem da calda procedeu-se o reensacamento dos frutos. A seguir, em intervalo semanal, entre 0 a 28 dias após o tratamento, foi realizada inoculação mediante aspersão de suspensão contendo 104 conídios mL-1. Na avaliação, realizada quando da colheita da maturação dos frutos, foram determinados os valores de I e ID. O experimento E3 foi realizado em pomar comercial de laranjeira ‘Folha Murcha’, no município de Paraíso/SP. Inicialmente, nos estádios F1 e F2 foram realizadas duas aplicações de oxicloreto de cobre (90 g de cobre metálico 100 L-1 de calda). Os tratamentos avaliados foram: aplicação do fungicida piraclostrobina em intervalo de 14 dias entre si, na dose de 25 g 2000 L-1; aplicação de piraclostrobina, na dose de 75 g 2000L-1 após o primeiro evento de chuva ocorrido 14 dias após a última aplicação do tratamento; aplicação de piraclostrobina, na dose de 75 g 2000L-1 após o primeiro evento de chuva, ocorrido 35 dias após a última aplicação do tratamento; aplicação de piraclostrobina, com intervalo de 42 dias entre si nas doses de 50 e 75 g 2000L-1. Esses tratamentos foram comparados com o tratamento padrão de azoxistrobina, na dose de 75 g 2000L-1, aplicado em intervalo de 42 dias. Foi utilizado o delineamento de blocos casualizados, e cada parcela foi constituída por 66 plantas. As condições operacionais adotadas para aplicação dos fungicidas foram semelhantes às adotadas em E1, porém com 150 mL de calda m-3 de copa. Os critérios de avaliações foram semelhantes aos citados em E1, em intervalos mensais, de setembro a janeiro. Concluiu-se que os fungicidas HC, OC e OCp nas doses de 43,7, 90,7 e 56,3 g de cobre metálico 100 L-1, respectivamente, aplicados nos estádios F1 e F2, complementado por aplicações de azoxistrobina, foram igualmente eficientes no controle de MPC. A eficiência do efeito protetor dos fungicidas cúpricos foi constatada por pelo menos 28 dias após a sua aplicação. Porém, a maior eficiência dos fungicidas cúpricos deu-se quando da sua aplicação previamente à inoculação de P. citricarpa. Aplicações de piraclostrobina a 75 g de i.a. 2000L-1 em intervalo de no mínimo 14 dias entre eventos chuvosos resultaram em maior controle da doença, quando comparado aos demais intervalos e doses de piraclostrobina e a azoxistrobina, na dose de 75 g 2.000 L-1, em intervalo fixo de 42 dias, com consequente redução da incidência e severidade de sintomas de MPC. / In this study, three experiments were performed (E1, E2 and E3). The first two experiments refer to the rate-response efficiency of the fungicides copper hydroxide (HC), copper oxychloride (OC) and cuprous oxide (OCp) in the control of Phyllosticta citricarpa, causal agent of citrus black spot (CBS). And the third (E3) the influence of pyraclostrobin application was evaluated in consonance with rainy events. In E1 and E2 the evaluated rates were: HC (43.7; 35.0; and 26.2 g of metallic copper 100L-1); OC (100.8, 90.7, and 68.0 g of metallic copper 100L-1); OCp (90.0, 56.3, and 42.2 g of metallic copper 100L-1). The experiment (E1) was conducted under natural infection by P. citricarpa in 'Pera' sweet orange planted in Olímpia/SP. A randomized blocks design was used, with four replications. Each plot was represented by 33 plants. The fungicides were applied in stages F1 and F2 by turbosprayer and 135 ml m-3 of the tree canopy. Subsequently, in F4 stage, all treatments were treated with azoxystrobin at 75 g 2000 L-1, four times in a 42-day interval. Four monthly evaluations were performed, from August to November, evaluating 100 fruits to determining the incidence and symptoms severity. Data were used to determined area under disease progress curve (AUDPC). In E2, a 'Pera' sweet orange plants were used, also located in Olímpia/SP. The treatments evaluated were similar those previously in E1, but were applied by a manual "De Vilbs" sprayer, with sufficient volum for uniform fruit coverage. A randomized blocks design were used in a factorial arrangement of 10 treatments (9 copper fungicides treatments and one untreated check) x 5 (inoculation times) and 4 (replicates). Each plot was formed by 20 pre-bagged fruits with semi-kraft paper in the F1 stage. In the F4 stage the bags were removed and the fruits sprayed exclusively with copper fungicides. Subsequently, in a weekly interval from 0 to 28 days after application at inoculation with suspension spray containing 104 conidia mL-1 was performed. In the harvest time an evaluation was performed and the values of I and DI were determined. E3 was carried out in a commercial 'Folha Murcha' sweet orange orchard, in Paraíso/SP. Initially, in stages F1 and F2, two applications of copper oxychloride based on 90 g of metallic copper 100 L-1 were perfomed. The treatments evaluated were: application with 14 days fixed interval in the pyraclostrobin rate of 25 g 2000L-1; application of 75 g 2000L-1 after the first rain event occurred 14 days after the last fungicide application; application of 75 g 2000L-1 after the first rain event occurred 35 days after the last fungicide application; application with 42 days fixed interval of 50 and 75 g 2000L-1. These treatments were compared with the standard treatment with 42 days fixed interval of azoxystrobin at rate 75 g 2000L-1. A randomized blocks design was used with 66 plants per plot. The fungicides applications were similar that in E1, but with 150 mL m-3 of canopy volume. The evaluations were similar that in E1, in monthly intervals, from September to January, in the ten central plants per plots. It was concluded that the fungicides HC, OC and OCp in the rates of 43.7, 90.7 and 56.3 g of metallic copper 100 L-1, respectively, applied in stages F1 and F2, complemented by applications of azoxystrobin, were equally efficient to control CBS. The protective effect of the copper fungicides was maintained until 28 days after treatment. However, the best response was obtained when inoculating P. citricarpa immediately after copper fungicides treatment. The 75 g 2000L-1 of pyraclostrobin application after the first rainy event occurred at least 14 days from the last fungicide application resulted in a greater reduction in the incidence and severity of CBS symptoms.

<p align="justify">Les glioblastomes sont les tumeurs cérébrales malignes les plus répandues chez l’adulte et l’enfant, et les plus agressives avec une médiane de survie inférieure à quinze mois. Deux caractéristiques biologiques rendent leur traitement particulièrement difficile. Tout d’abord, la capacité d’invasion du parenchyme cérébral par les cellules gliales tumorales rend la résection chirurgicale totale impossible. De plus, ces cellules développent une résistance aux stimuli pro-apoptotiques rendant la plupart des agents chimiothérapeutiques peu efficaces contre ce type de tumeur. Actuellement, le seul agent chimiothérapeutique efficace dans le traitement des glioblastomes est le témozolomide. Malheureusement des mécanismes de résistance acquise peuvent apparaître chez des patients traités avec cette molécule. Nous avons donc étudié ces mécanismes moléculaires de résistance acquise au témozolomide et dans un deuxième temps nous avons tenté de trouver de potentielles molécules susceptibles de lutter contre les gliomes. </p align="justify"><p><p align="justify">Nous avons rendu résistants deux modèles de cellules gliales tumorales au témozolomide par un traitement incrémental durant plusieurs mois. Grâce à une analyse de type Affymétrix, nous avons comparé l’expression de l’ensemble des gènes entre les lignées normales et les lignées rendues résistantes. Deux familles de gènes sont ressorties comme impliquées dans ce processus de résistance :les transporteurs glucose et les aldo-kéto réductase 1C (akr1c). Pour les transporteurs glucose, il se pourrait que leur implication se fasse au niveau du métabolisme des cellules résistantes en permettant d’apporter plus de substrat énergétique et ainsi contourner les effets pro-autophagiques du témozolomide. Dans le cas des akr1c, il semblerait que leur surexpression ait pour but d’augmenter la prolifération des cellules tumorales résistantes. </p align="justify"><p><p align="justify">Nous avons, par la suite, étudié les propriétés anticancéreuses de métabolites fongiques, les phyllostictines, extraites du champignon Phyllosticta cirsii. Quatre métabolites naturels et deux dérivés hémisynthétiques ont été testés démontrant des activités plus ou moins importantes sans toutefois montrer in vitro de biosélectivité entre cellules tumorales et cellules normales. Les phyllostictines semblent se lier au groupement thiol libre comme nous l’avons déterminé sur le glutathion ou la cystéine. Il reste à déterminer la cible des phyllostictines dans les cellules tumorales et à améliorer la sélectivité de ces molécules.</p align="justify"> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Viticulture is a multibillion-dollar market based on grapes, one of the most valuable fruits in agriculture, but it is also characterized by the highest uses of fungicides per hectare for disease management. In fact, despite the availability of genetic resources resistant to the most diffuse fungal pathogens, those cover less than a fifth of the worldwide vine area. The remaining 80% is represented mainly by susceptible Vitis vinifera L. varieties. Instead, re-sistance traits are known to derive from other Vitis species, due to their coevolution with the pathogens, and their exploitation in breeding is a valuable strategy to reduce pesticide treat-ments. Black rot (BR) is a destructive disease caused by the ascomycete, Phyllosticta ampeli-cida, whose telomorphic form was known as Guignardia bidwellii. In the European continental area in the last two decades its pressure increased due to the concomitant decreased usage of chemicals and the occurrence of mild-rainy summer because of the climate change. This work was established to fight the spread of BR by means of the introduction of BR resistance in susceptible genetic background. The study consisted of (i) the reconstruction of BR resistant donor pedigree based on the collection of historical phenotypic data, (ii) the eval-uation of parental lines and selections of the breeding program of the Edmund Mach Founda-tion (Italy), (iii) the microscopic inspection of disease progression, and (iv) QTL analysis of the segregating population (N=146) ‘Merzling’ (hybrid, resistant) × ‘Teroldego’ (V. vinifera, suscep-tible). The outcomes revealed a large unexplored pool of disease resistance donors of 148 va-rieties belonging to 14 different species, that permitted to evaluate and identify five new promising genotypes readily exploited for breeding. Concurrently, a new inoculation method based on spore production from fresh infected leaves was developed. Ex vivo (detached leaves) inoculation did not reveal significant differences among the assessed genotypes and highlight-ed the tendency of spores to accumulate and germinate near leaf hairs, while disease progres-sion did not occur. The screening of the segregating population, both under greenhouse and field conditions, allowed the discrimination on chromosome 14 between two distinct QTLs as-sociated with leaf/shoot and bunch resistance. The first QTL confirmed the Resistance to Gui-gnardia bidwellii (Rgb)1 locus previously identified by three studies and the high-density link-age mapping allowed to reduce it from 2.4 to 0.7 Mb along the PN40024.v4 reference genome. The region resulted enriched in genes belonging to phloem dynamics and mitochondrial pro-ton transfer. The second QTL associated with bunch resistance was designated Rgb3. Located at 9 cM (6 Mb) upstream Rgb1, it was characterized by a cluster of Germin-like protein 3 genes and lipid transfer and localization, notably known to be linked to broad spectrum disease re-sistance and stress response. No resistance (R)-genes have been annotated in the region under-lying the QTL in the PN40024 reference genome. In conclusion, this work provided new insights for grapevine breeding programs by the identification of previously unknown BR resistance donors, the development of protocols and good practices towards large-scale resistance screening as well as the discovery of a novel QTL associated with bunch resistance. Marker validation in different genetic backgrounds is ongoing for its routinary implementation in marker-assisted breeding and further studies are planned to dissect BR resistance mechanism by the sequencing of the genomic regions, the study of the Germin-like 3 gene cluster and the cell wall characterization.