Orientador: Paulo Cezar Ceresini / Resumo: Na primeira etapa da nossa pesquisa propusemos e testamos a utilização de um método baseado em leitor automatizado de microplacas para detectar resistência a fungicidas em populações do fungo hemibiotrófico fitopatogênico Pyricularia graminis-tritici (Pygt) associado à brusone do trigo no Brasil. A disponibilidade de um método mais rápido, acurado e eficiente para detecção de resistência a fungicidas facilitaria a tomada de decisão sobre o manejo químico da brusone do trigo no país. Como há indícios da ineficácia de fungicidas triazóis (DMI) no controle da brusone do trigo e evidências da ocorrência generalizada de resistência a estrobilurinas (QoI) em populações de Pygt no Brasil, inicialmente, testou-se a aplicação do método de microplacas para detecção de resistência a estes dois grupos de fungicidas. O método de microplaca foi acurado em discriminar a variação fenotípica na sensibilidade entre isolados do patógeno aos fungicidas DMI tebuconazol e epoxiconazol e ao fungicida QoI azoxistrobina. Economicamente, ambos os métodos representam um alto investimento inicial. Portanto, é necessário analisar, de forma integral, as necessidades de cada laboratório ou grupo de pesquisa quanto à aquisição dos equipamentos necessários para a fenotipagem visando detecção de resistência a fungicidas em atividades de rotina. Na segunda etapa do nosso estudo buscou-se evidências de resistência ao fungicida SDHI fluxapiroxade em populações de Pygt. Para se estabelecer o baseline de populaçõe... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by PRISCILA SANTOS CASADO null (pri.casado@hotmail.com) on 2017-10-22T18:46:39Z No. of bitstreams: 1 Dissertação Final - Priscila Casado.pdf: 1866790 bytes, checksum: 8d4b39875813bd43bc592acd4b71faef (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-10-26T15:50:29Z (GMT) No. of bitstreams: 1 casado_ps_me_ilha.pdf: 1866790 bytes, checksum: 8d4b39875813bd43bc592acd4b71faef (MD5) / Made available in DSpace on 2017-10-26T15:50:29Z (GMT). No. of bitstreams: 1 casado_ps_me_ilha.pdf: 1866790 bytes, checksum: 8d4b39875813bd43bc592acd4b71faef (MD5) Previous issue date: 2017-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na primeira etapa da nossa pesquisa propusemos e testamos a utilização de um método baseado em leitor automatizado de microplacas para detectar resistência a fungicidas em populações do fungo hemibiotrófico fitopatogênico Pyricularia graminis-tritici (Pygt) associado à brusone do trigo no Brasil. A disponibilidade de um método mais rápido, acurado e eficiente para detecção de resistência a fungicidas facilitaria a tomada de decisão sobre o manejo químico da brusone do trigo no país. Como há indícios da ineficácia de fungicidas triazóis (DMI) no controle da brusone do trigo e evidências da ocorrência generalizada de resistência a estrobilurinas (QoI) em populações de Pygt no Brasil, inicialmente, testou-se a aplicação do método de microplacas para detecção de resistência a estes dois grupos de fungicidas. O método de microplaca foi acurado em discriminar a variação fenotípica na sensibilidade entre isolados do patógeno aos fungicidas DMI tebuconazol e epoxiconazol e ao fungicida QoI azoxistrobina. Economicamente, ambos os métodos representam um alto investimento inicial. Portanto, é necessário analisar, de forma integral, as necessidades de cada laboratório ou grupo de pesquisa quanto à aquisição dos equipamentos necessários para a fenotipagem visando detecção de resistência a fungicidas em atividades de rotina. Na segunda etapa do nosso estudo buscou-se evidências de resistência ao fungicida SDHI fluxapiroxade em populações de Pygt. Para se estabelecer o baseline de populações do patógeno da brusone do trigo ao fungicida fluxapiroxade, determinou-se a distribuição de frequência de padrões de sensibilidade, com base na EC50 (concentração suficiente para inibir 50% do crescimento micelial fúngico) de cada isolado. Fenótipos extremos de sensibilidade e resistência a fluxapiroxade foram detectados nas diferentes populações do patógeno. Considerando que os fungicidas da segunda geração de SDHI são de alto risco para desenvolvimento de resistência em populações de fipatógenos, é necessária a avaliação do registro e recomendação de uso contínuo do fluxapiroxade, como único princípio ativo numa formulação para o manejo da brusone e de outras doenças do trigo no Brasil. Como estratégia anti-emergência para evitar resistência a SDHI, esse fungicida só poderia ser registrado em co-formulações com outro princípio ativo de baixo risco para resistência. / In the first part of our study, we proposed and tested the use of an automated microplate reader to detect resistance to fungicides in populations of the plant pathogenic hemibiotrophic fungus Pyricularia graminis-tritici (Pygt) associated with the wheat blast disease in Brazil. The availability of a faster, more accurate and efficient method for detecting resistance to fungicies would facilitate the decision making on the chemical management of wheat blast in the country. Because there is indication of inefficacy of triazole fungicides (DMI) for wheat blast control and evidence of the widespread occurrence of strobilurin (QoI) resistance in pathogen populations in Brazil, initially, we tested the application of the microplate method for detecting resistance to these two fungicide groups. The microplate method was accurate in discriminating the phenotypic variation in sensitivity between isolates of the pathogen to the fungicides DMI tebuconazole and epoxiconazole and the fungicide QoI azoxystrobin. Economically, both methods require a high initial investment. Therefore, it is necessary to fully analyze the needs of each laboratory or research group regarding the acquisition of the equipments necessary for phenotyping to detect resistance to fungicides in routine activities. In the second part of our study, using the microplate method, we searched for evidence of resistance to the SDHI fungicide fluxapiroxade in populations of Pygt. To establish the baseline of populations of the wheat blast pathogen to fluxapiroxade, the frequency distribution of sensitivity patterns was determined based on the EC50 (concentration sufficient to inhibit 50% mycelial fungal growth) of each isolate. Extreme phenotypes of sensitivity and resistance to fluxapiroxade were detected in the different pathogen populations. Whereas the second generation of SDHI fungicides are at high risk for development of resistance in populations of plant pathogens, it is necessary to review the labeling and recommendation of continuous application of fluxapyroxad, as the sole active ingredient in any formulation for chemical management of wheat blast and other wheat diseases in Brazil. As an antiemergence strategy to avoid the resistance to SDHI, this fungicide could only be labeled in co-formulations with another active ingredient of low risk for resistance.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Mancozeb has been the protective fungicide with a multi-site mechanism widely used associated with fungicide programs in soybean cultivation. This compound has provided significant increases in the systemic fungicides performance in the soybean rust control. Besides the obvious fungicide effect, mancozeb has provided also effects on physiological parameters in plants promoting benefits such as fungicide phytotoxicity reduction and increases in plant vigor. The studies aimed to understand the interaction of mancozeb associated to systemic fungicides against the soybean rust control, in addition to determine the physiological plants responses from the product versus plant relation. The experiments were conducted in greenhouse and field, divided in four chapters in manuscript format. The first study investigates the effect of the mancozeb interaction with commercial mixtures of QoI + DMI based on two methodologies. From this, it was possible to verify that mancozeb increases significantly the control efficiency when associated with less efficient mixtures. In such interactions was obtained a control synergistic response. However it is expected at least an additive response, when associated to mixtures with high efficiency, the increase in control is lower. The second study consisted of the enzymes activity linked to the antioxidant response, the photosynthetic pigments concentration and lipid peroxidation investigation in plants exposed to the fungicide trifloxystrobin + prothioconazole, with potential to cause phytotoxicity, associated or not with mancozeb. Antioxidant enzymes activity increase, reduction in pigment concentration and cell damage due to increased lipid peroxidation were observed in plants exposed to systemic fungicide. These damages were attenuated when the systemic fungicide was associated with mancozeb. Moreover, photosynthesis and chlorophyll a fluorescence parameters were analyzed in plants exposed to mancozeb and trifloxystrobin + prothioconazole, constituting the third study. Decreased in the photosynthetic efficiency was verified in plants treated with systemic fungicide characterizing a significant phytotoxic effect. The parameters evaluated elucidate a beneficial effect of mancozeb reducing the damages caused by the systemic fungicide. The fourth study aimed to evaluate changes in the leaves soybean nutritional status exposed to mancozeb. Macro and micronutrients concentration were determined on 4th and 8th days exposure. Significant changes in concentration of S, Mn and Zn were observed. Such changes were related to “green effect” on plants after mancozeb exposure. All data provide new information regarding the recent use of the mancozeb molecule in soybean. The contribution is original and presents data that can contribute to the definition of strategies in anti-resistance management and increase site-specific systemic fungicides effective life commonly used in soybean. In addition, the results present important physiological responses on plants induced Figure 1by mancozeb that can aggregate on development, vigor and productivity. / Mancozebe tem sido o fungicida protetor com mecanismo multi-sítio amplamente utilizado associado aos programas fungicidas na cultura da soja. Este composto tem proporcionado incrementos significativos no desempenho de fungicidas sistêmicos no controle da ferrugem da soja. Além do efeito evidente como fungicida, tem sido relatado efeitos sobre parâmetros fisiológicos nas plantas conferindo benefícios como redução de fitotoxicidade de outros fungicidas e incrementos no vigor de plantas. Nossos estudos objetivaram compreender a interação de mancozebe associado a fungicidas sistêmicos frente ao controle da ferrugem da soja, além de determinar respostas fisiológicas das plantas oriundas da relação produto versus planta. Foram conduzidos ensaios em casa de vegetação e a campo, aqui divididos em quatro capítulos no formato de manuscritos. Em um primeiro estudo o efeito da interação de mancozebe com misturas comerciais de IQo + IDM foi investigado com base em duas metodologias. A partir disso foi possível verificar que mancozebe agrega mais significativamente a eficácia de controle quando associado a misturas menos eficientes. Em tais interações obteve-se uma resposta sinérgica de controle. Quando associado a misturas com alta eficácia o incremento no controle foi menor, entretanto no mínimo uma resposta aditiva é esperada. Em um segundo estudo a atividade de enzimas ligadas à resposta antioxidante, a concentração de pigmentos fotossintetizantes e a peroxidação lipídica foi investigada em plantas expostas ao fungicida trifloxistrobina + protioconazol, com potencial de causar fitotoxidade, estando este associado ou não ao mancozebe. Aumento na atividade de enzimas antioxidantes, redução no conteúdo de pigmentos e danos celulares evidenciadas pela maior peroxidação de lipídios foram verificadas em plantas expostas ao fungicida sistêmico. Tais danos foram atenuados quando o fungicida sistêmico foi associado ao mancozebe. Nesse mesmo sentido, parâmetros de fotossíntese e fluorescência da clorofila a foram analisados em plantas expostas a mancozebe e trifloxistrobina + protioconazol, sendo este o terceiro estudo aqui apresentado. Decréscimo na eficiência fotossintética foi verificado em plantas tratadas com o fungicida sistêmico caracterizando um efeito fitotóxico significativo. Os parâmetros avaliados elucidam um efeito benéfico de mancozebe reduzindo os danos causados pelo fungicida sistêmico. Um quarto e último estudo visou avaliar alterações no estado nutricional de folhas de soja expostas a mancozebe. Concentração de macro e micronutrientes foram determinadas aos 4 e 8 dias após exposição. Alterações significativas na concentração de S, Mn e Zn foram observadas. Tais alterações foram relacionadas a incrementos na coloração verde da cultura após exposição à mancozebe. Os dados trazem informações novas em relação ao recente uso da molécula mancozebe em soja. A contribuição é original e apresenta dados que podem contribuir na definição de estratégias no manejo anti-resistência e aumento da vida efetiva dos fungicidas sistêmicos sítio-específico comumente utilizados em soja. Além disso, os resultados apresentam uma série de respostas fisiológicas importantes de mancozebe sobre as plantas que podem agregar em termos de desenvolvimento, vigor e produtividade.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Botrytis cinerea Pers. Fr. [teleomorph Botryotinia fuckeliana (de Bary) Whetzel] causes serious losses of over 200 crops worldwide, including rooibos seedlings and pears. This pathogen is characterized by morphological, physiological and genetic diversity. The genetic diversity and population structure have not been investigated for B. cinerea populations in South Africa. Botrytis cinerea collected from rooibos seedlings and in pear orchards in the Western Cape of South Africa were investigated in the present study. The study was done with the aid of microsatellite markers, the amplification of mating type alleles MAT1-1 and MAT1-2 and determination of resistance towards various fungicides. Population dynamics was inferred and a similar picture emerged in both production systems. Botrytis cinerea annually causes severe losses of rooibos seedlings (Aspalathus linearis) in nurseries situated in the Clanwilliam region. Sampling was done in five nurseries and the cryptic species status of the isolates obtained was determined through restriction enzyme digestion of the Bc-hch gene. All but one (206 out of 207) of the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the B. cinerea Group II population, using seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.67. Two of the nurseries populations’ sample sizes were severely limited after clone correction, yet 100 genotypes were discerned among the 206 isolates genotyped. The percentage of maximal genotypic diversity (G) ranged between 16 and 68 for the five populations, with a total value of 17 for the 100 genotypes. One genotype, represented by 27 clones, was isolated from four nurseries. Relatively low but significant population differentiation was observed in total between nurseries (mean FST = 0.030, P = 0.001). The distribution of mating types MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population plus two of the nurseries’ populations. Three nursery populations had an equal mating type distribution. The index of association (IA) analyses suggests that the populations are asexually reproducing. Analysis of molecular variance (AMOVA) indicated that 97% of the total genetic variation is distributed within subpopulations. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates displayed highly varying levels of resistance amongst the five nurseries. The mean total incidence of resistance towards iprodione was 43%, ranging from 0% to 81% for the five nurseries. Baseline sensitivity towards pyrimethanil yielded an average EC50 value of 0.096 mg/L. Botrytis cinerea isolates were collected from pear blossoms (Pyrus communis) in four orchards. Two orchards in the Ceres area and two in the Grabouw area were sampled from. A total of 181 isolates were collected from the four orchards. Incidence of blossom infection in the orchards ranged from 3% to 17%. Overall, there was a high incidence of isolates that had only the Boty transposable element (74%) compared to those harbouring both (Boty and Flipper), simultaneously (transposa, 24%). One isolate examined had the Flipper element only. Cryptic species status according to restriction enzyme digestion of the Bc-hch gene indicated that all the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the Group II population, through the use of seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Although two of the subpopulations displayed a high clonal proportion, overall 91 genotypes were discerned among the 181 isolates. The percentage of maximal genotypic diversity (G) ranged between 18 and 33 for the four populations, with a total value of 14 for the 91 genotypes. One genotype, represented by 27 clones, was isolated from all orchards. Moderate, but significant population differentiation was present in total among orchards (mean FST = 0.118, P = 0.001). The distribution of the mating types, MAT1-1 and MAT1-2, did not differ significantly from a 1:1 ratio for the total population as well as the subpopulations. Index of association (IA) analyses, on the other hand, suggests that the populations reproduce asexually. Analysis of molecular variance (AMOVA) indicated that 88% of the total genetic variation is distributed within subpopulations, 9% between subpopulations and only 3% between production areas. Fungicide resistance frequency against fenhexamid, iprodione and benomyl varied, with the highest levels of resistance present against benomyl and low levels of resistance seen towards iprodione and fenhexamid. In conclusion, this study has shown that there exist within the studied populations of B. cinerea, obtained from rooibos nurseries and pear orchards, an adaptive capacity to overcome current means of control. The use of population genetics to further our understanding of how plant pathogens interact and spread throughout a given environment is of cardinal importance in aiding the development of sustainable and integrated management strategies. Knowledge of the dispersal of B. cinerea in the two studied cropping systems has shed light on the inherent risk that it poses, and this together with knowledge of the levels of resistance that occurs should serve as an early warning to help divert possible loss of control in future. / AFRIKAANSE OPSOMMING: Botrytis cinerea Pers. Fr. [teleomorf Botryotinia fuckeliana (de Bary) Whetzel] veroorsaak ernstige verliese van meer as 200 gewasse wêreldwyd, insluitende rooibossaailinge en pere. Hierdie patogeen word deur morfologiese, fisiologiese, asook genetiese diversiteit gekenmerk. Die genetiese diversiteit en populasie-struktuur van B. cinerea populasies wat in Suid-Afrika voorkom, is nog nie ondersoek nie. Botrytis cinerea verkryg vanaf rooibossaailinge en in peerboorde in die Wes-Kaap van Suid-Afrika is ondersoek. Hierdie studie is met behulp van mikrosatellietmerkers, amplifikasie van die twee paringstipe gene (MAT1-1 en MAT1-2), asook die bepaling van weerstandsvlakke teenoor verskeie swamdoders, uitgevoer. Populasie-dinamika is afgelei en ‘n soortgelyke tendens is in beide produksie-sisteme waargeneem. Botrytis cinerea veroorsaak jaarliks ernstige verliese van rooibossaailinge (Aspalathus linearis) in kwekerye in die Clanwilliam-area. Monsters is in vyf kwekerye versamel en die kriptiese spesiestatus van die verkrygde isolate is deur restriksie-ensiemvertering van die Bc-hch geen bepaal. Almal behalwe een (206 uit 207) isolaat het aan Groep II of B. cinerea ‘sensu stricto’ behoort. Analise van die B. cinerea Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om die genetiese populasiestruktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.67. Alhoewel twee van die kwekerye se monstergrootte erg ingeperk is ná kloonverwydering, is daar nogtans 100 genotipes onder die 206 isolate wat geïsoleer is, waargeneem. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 16 en 68, vir die vyf populasies, gewissel, met ‘n totaal van 17 vir die 100 genotipes. Een genotipe, verteenwoordig deur 27 klone, is uit vier kwekerye geïsoleer. Relatief lae dog noemenswaardige populasie-differensiasie is in totaal tussen kwekerye waargeneem (gem. FST = 0.030, P = 0.001). Die verspreiding van die twee paringstipes (MAT1-1 en MAT1-2) het beduidend verskil van ‘n 1:1 verhouding vir die totale populasie, asook twee van die kwekerye se populasies. Die drie oorblywende kwekerye se populasies het egter ‘n gelyke verdeling van die twee paringstipes getoon. Die indeks van assosiasie (IA) analises toon dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 97% van die totale genetiese variasie binne die subpopulasies versprei is. Hoogs variërende vlakke van weerstand tussen die vyf kwekerye teenoor die swamdoder iprodioon, is vir die 198 isolate wat getoets is, gevind. Die totale gemiddelde frekwensie van weerstand teenoor iprodioon was 43%, wat tussen 0% en 81% vir die vyf kwekerye gevarieer het. Fondasie-vlak-sensitiwiteit vir pyrimethanil het ‘n gemiddelde EC50 waarde van 0.096 mg/L opgelewer. Botrytis cinerea isolate is ook vanuit peerbloeisels (Pyrus communis L.) vanuit vier boorde versamel, twee uit elk van die Ceres- en Grabouw-areas. In totaal is 181 isolate vanuit die vier boorde versamel. Die frekwensie van bloeiselinfeksie het tussen 3% en 17% gewissel. Oor die algemeen was daar ‘n hoë frekwensie van isolate wat slegs die Boty transponeerbare element teenwoordig gehad het (74%) in vergelyking met dié wat tegelykertyd beide (Boty en Flipper) teenwoordig gehad het. Een isolaat het slegs die Flipper element gehad. Bepaling van die kriptiese spesiestatus met behulp van restriksie-ensiemvertering van die Bc-hch geen het aangedui dat alle versamelde isolate tot Groep II of B. cinerea ‘sensu stricto’ behoort het. Analise van die Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om genetiese populasie-struktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.69 oor alle populasies. Alhoewel twee subpopulasies ‘n hoë klonale fraksie getoon het, is 91 genotipes tussen die 181 isolate wat verkry is, onderskei. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 18 en 33 vir die vier populasies gewissel, met ‘n totale waarde van 14 vir die 91 genotipes. Een genotipe, verteenwoordig deur 27 klone, was in al vier boorde teenwoordig. Gematigde dog beduidende populasie differensiasie was in totaal tussen boorde teenwoordig (gem. FST = 0.118, P = 0.001). Die verspreiding van die paringstipes (MAT1-1 en MAT1-2) het nie betekenisvol van ‘n 1:1 verhouding vir die totale populasie, insluitende die subpopulasies, verskil nie. Indeks van assosiasie (IA) analises het egter aangedui dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 88% van die totale genetiese variasie in subpopulasies te vinde was, 9% tussen subpopulasies en slegs 3% tussen produksie-areas. Frekwensie van swamdoder weerstandbiedendheid vir fenhexamid, iprodioon en benomyl het gewissel, met die hoogste vlakke teenoor benomyl waargeneem, maar baie lae vlakke teenoor fenhexamid en iprodioon. Samevattend het hierdie studie getoon dat die populasies van B. cinerea wat in hierdie twee produksie-sisteme, op rooibossaailinge en in peer boorde, ondersoek is, ‘n aanpasbaarheid toon om huidige metodes van beheer te oorkom. Die gebruik van populasiegenetika as ‘n hulpmiddel om ons kennis van patogeen-interaksies en -verspreiding te verbreed, is van kardinale belang in die ontwikkeling van geïntegreerde en volhoubare beheermaatreëls. Kennis van die verspreiding van B. cinerea in die bestudeerde gewasproduksiestelsels, werp lig op die inherente risiko wat dié patogeen inhou. Dít, tesame met kennis van die weerstandsvlakke wat voorkom, kan as ‘n vroegtydige waarskuwing dien ten einde moontlike verlies van beheer in die toekoms te help teenwerk.