Atualmente, a produtividade da cultura da batata (Solanum tuberosum L.) pode ser reduzida devido à ocorrência de doenças em todo o território nacional, destacando-se a pinta preta, causada por Alternaria spp. Historicamente se acreditava que A. solani (AS) era a principal causadora mas, atualmente, descobriram outras espécies associadas, sendo predominante hoje, em território nacional a espécie A. grandis (AG). O objetivo deste trabalho foi avaliar a sensibilidade in vitro de três isolados de A. grandis e um isolado de A. solani frente a onze fungicidas amplamente utilizados no manejo desta doença em campo. Foi avaliado o efeito dos fungicidas sobre o crescimento micelial dos isolados, utilizando-se do método da incorporação do fungicida no meio de cultura fundente. As diluições dos fungicidas foram realizadas em água destilada e esterilizada, de acordo com a metodologia descrita por Camargo (2013) e Ishizuka (2016). Para os fungicidadas com mistura de ingrediente ativo, considerou-se a soma das concentrações no cálculo. Inicialmente, as concentrações do meio de cultura, foram 100, 10, 1, 0,1, 0,01 e 0,001 mg L-1. Após, as doses foram ajustadas conforme a CI50 de cada fungicida. Quantificou-se também a inibição sobre a germinação dos conídios nas contrações discriminatórias de 0; 0,1; 1; 10 e 100 mg.L-1 .24 horas após o preparo do meio as placas foram observadas em microscópio óptico, determinando-se a germinação dos conídios em 300 unidades observadas por placa de Petri. Todos os isolados testados foram altamente sensíveis aos ingredientes ativos fluazinam, pirimetanil, procimidona e piraclostrobina. Não houve diferenças de comportamento de sensibilidade entre as espécies de A. solani e A grandis que possam caracterizar uma perda de sensibilidade por conta da predominância da espécie de A. grandis associada à pinta preta da batata no Brasil. Encontraram-se evidências de insensibilidade dos isolados de A. grandis e A. solani aos ingredientes ativos Clorotalonil e Boscalida. / Currently, the productivity of potato crop (Solanum tuberosum L.) may be reduced due to the occurrence of disease throughout the country, especially due to early blight, caused by Alternaria spp. Historically it was believed that A. solani (AS) was the main cause, most currently was founded others associated species, being predominant today the occurrence of the disease associated with the species A. grandis (AG). The objective of this study was to evaluate the in vitro susceptibility of three isolates of A. grandis and one isolate of A. solani for eleven fungicides widely used in the management of this disease in the field, was evaluated the effect of fungicides on mycelial growth using the fungicidal method that incorporate it in the medium culture. Dilutions of fungicides were performed in sterile distilled water according to the methodology described by Camargo (2013) and Ishizuka (2016). For the fungicidates with active ingredient mixture, was considered the sum of the both concentrations in the calculation.The concentrations of the culture medium were 100, 10, 1, 0.1, 0.01 and 0.001 mg L-1. The doses were adjusted according to the CI50 of each fungicide, was quantified also the inhibition of spore germination, and the fungicides were incorporated into the agar medium - water in discriminatory contractions of 0; 0.1; 1; 10 and 100 mg.L-1 . Then, 24 hours after preparation of the medium the plates were observed under an optical microscope, determining the spore germination observed at 300 units per plate. All tested isolates were highly sensitive to the active ingredients fluazinam, pyrimethanil, procymidone and pyraclostrobin. There was no sensitivity behavioral differences between species of A. solani and A. grandis which may characterize a loss of sensitivity due to the predominance of species of A. grandis associated with early blight in Brazil, was found evidences of insensitivity of isolates of A. grandis and A. solani to the active ingredients Chlorothalonil and Boscalida.

As viroses causadas pelo Tomato severe rugose virus (ToSRV) e Tomato chlorosis virus (ToCV) têm sido relatadas com frequência em cultivos de tomateiro e outras solanáceas no Brasil. Por compartilharem o mesmo vetor, a mosca-branca B. tabaci MEAM1, hospedeiros comuns e estarem presentes nas principais regiões produtoras, infecções mistas desses dois vírus são comuns em tomateiros. Em uma infecção mista, as diferentes espécies de vírus podem interagir, resultando em sinergismo, antagonismo ou neutralismo. Estudos referentes às interações destes dois vírus quando em infecções mistas, ainda são escassos. Este trabalho teve como objetivo avaliar o efeito da prévia aquisição do ToSRV por B. tabaci MEAM1 na subsequente taxa de transmissão e no período de retenção do ToCV pelo inseto. Além disso, avaliou-se o efeito dos princípios ativos cloridrato de cartape, ciantraniliprole e flupiradifurone no controle das transmissões primária e secundária desses vírus por B. tabaci para tomateiros. Para avaliar o efeito do begomovírus em parâmetros de transmissão do crinivírus por B. tabaci MEAM1 foram comparados os seguintes tratamentos: 1) plantas inoculadas com insetos que adquiriram apenas o ToSRV; 2) plantas inoculadas com insetos que adquiriram somente o ToCV; 3) plantas inoculadas com insetos que adquiriram inicialmente o ToSRV e em seguida o ToCV (ToSRV → ToCV); 4) plantas inoculadas com insetos que adquiriram os vírus simultaneamente de uma mesma planta fonte (ToSRV+ToCV) e 5) plantas inoculadas com insetos livres de vírus (controle). Quarenta dias após, as plantas foram avaliadas por RT-PCR e RCA-PCR para a detecção do ToCV e do ToSRV, respectivamente. Nos ensaios para avaliar a eficiência dos inseticidas no controle da simulação da transmissão primária dos vírus, insetos virulíferos foram liberados em gaiolas individuais contendo plantas de tomate sadias pulverizadas com os inseticidas de acordo com cada tratamento. A simulação da transmissão secundária foi feita através da liberação de adultos de B. tabaci MEAM1 livres de vírus, em gaiolas contendo tomateiros sadios e infectados por ToSRV+ToCV pulverizados com cada inseticida. Da mesma forma, aos 40 dias após a primeira liberação, amostras de todas as plantas foram coletadas e posteriormente avaliadas por RT-PCR e RCA-PCR para detecção do ToCV e ToSRV, respectivamente. O ToSRV previamente ou simultaneamente adquirido pela B. tabaci MEAM1 não interferiu significativamente na taxa de transmissão do ToCV para tomateiros, 24 h após a aquisição. No entanto, quando o vetor adquiriu os vírus simultaneamente, a taxa de transmissão do ToCV foi incrementada no 2° e 3° dias após a aquisição por B. tabaci. O período de retenção do crinivírus foi de três dias conforme relatado anteriormente. Nenhum dos inseticidas foi eficiente em controlar as simulações das transmissões primária e secundária do ToSRV+ToCV por B. tabaci MEAM1 para tomateiros. Os resultados obtidos neste trabalho demostram a importância em conhecer as interações vírus-vetor-planta para que as estratégias de manejo sejam melhor delineadas e efetivas. Demostram também que o uso de inseticidas deve fazer parte de um manejo integrado com outras medidas que devem ser adotadas em larga escala. / Virus diseases caused by Tomato severe rugose virus (ToSRV) and Tomato chlorosis virus (ToCV) have been reported frequently in tomato and other solanaceous crops in Brazil. By sharing the same vector, the whitefly B. tabaci MEAM1, common hosts and present in the major producing regions, tomato plants infected with both viruses is common. In a mixed infection, different virus species may interact, resulting in synergism, antagonism or neutralism. Studies regarding the interactions between these two viruses when in mixed infections are still scarce. The objective of this work was to evaluate the effect of the previous acquisition of ToSRV by B. tabaci MEAM1 on the subsequent transmission rate and on the retention period of ToCV by the adult whitefly. In addition, the effect of the insecticides cartape chloridate, cyantraniliprole and flupiradifurone on the control of the simultaneous transmission of these viruses by B. tabaci to tomatoes was evaluated. To evaluate the effect of the begomovirus on the transmission parameters of the crinivirus by B. tabaci, the following treatments were compared: 1) plants inoculated with whiteflies that only acquired ToSRV; 2) plants inoculated with whiteflies that only acquired ToCV; 3) plants inoculated with whiteflies that acquired ToSRV followed by ToCV (ToSRV → ToCV); 4) plants inoculated with whiteflies that acquired both viruses simultaneously (ToSRV + ToCV) and 5) plants inoculated with virus-free whitelflies (control). Forty days after inoculation all plants were evaluated by RT-PCR and RCA-PCR for the detection of ToCV and ToSRV, respectively. In assays to evaluate the efficiency of insecticides in controlling the simulation of the primary transmission of ToSRV+ToCV, viruliferous insects were released into individual cages containing healthy tomato plants sprayed with the insecticides according to each treatment. Simulation of the secondary transmission was done by the release of virus free B. tabaci in cages containing ToSRV + ToCV infected and healthy tomato plants sprayed with each insecticide. In the same way, at 40 days after the first whiteflies release, samples from all plants were collected and evaluated by RT-PCR and RCA-PCR for the detection of ToCV and ToSRV, respectively. When B. tabaci MEAM1 acquired ToCV simultaneously or after ToSRV acquisition, the gegomovirus did not interfere with the ToCV transmission rate in tomato plants 24 h after aquisition. When the vector acquired the virus simultaneously, the ToCV transmission rate was increased on the 2nd and 3rd days after B. tabaci virus acquisition. The retention period of the crinivirus was three days, as previously reported. None of the insecticides were efficient in controlling the simulations of the primary and secondary transmissions of ToSRV+ToCV by B. tabaci MEAM1 to tomato plants. The results obtained in this work demonstrate the importance of knowing the virus-vector-plant interactions, so that the management strategies are better delineated and effective. They also show that the use of insecticides should be part of integrated management with other measures that should be adopted on a large scale.

O Tomato chlorosis virus (ToCV), família Closteroviridae, gênero Crinivirus é um vírus de RNA de fita simples, senso positivo, transmitido de maneira semi-persistente por espécies da família Aleyrodidae, dos gêneros Bemisia e Trialeurodes. Possui uma gama de hospedeiros considerável que inclui plantas domesticadas e ervas daninhas das famílias Alzoaceae, Amaranthaceae, Apiaceae, Apocynaceae, Asteraceae, Plumbaginaceae, e Solanaceae. No estado de São Paulo, Brasil, foi relatado pela primeira vez em 2008, causando clorose internerval nas folhas de tomateiros. A importância desta doença emergente tem incrementado nos últimos anos e, no entanto, até o momento não existem estimativas dos danos causados nem alternativas adequadas para o manejo da doença no campo. Diante disso, esse trabalho teve como objetivos (i) avaliar a resistência de genótipos de tomateiro à infecção com o ToCV, (ii) avaliar a tolerância de alguns dos genótipos à doença e (iii) estimar o dano produzido em campo protegido. Para isso, 57 genótipos, incluindo espécies selvagens, linhagens avançadas e cultivares comerciais de tomateiro foram inicialmente avaliados quanto à resistência à infecção. Plantas jovens, produzidas em bandejas de poliestireno expandido, protegidas por gaiola recoberta com tecido de voil foram inoculadas por meio da liberação massal de B. tabaci MEAM1 virulífera para o ToCV. A incidência de plantas infectadas por genótipo foi determinada mediante observação dos sintomas e a detecção do vírus por RT-PCR. Alguns dos genótipos também foram avaliados quanto à tolerância à doença causada pelo crinivírus. Plantas sadias e sabidamente infectadas com o ToCV foram transplantadas no campo no interior de telados protegidos com tecido de voil. As plantas foram avaliadas quanto ao peso de frutos produzidos. No fim do ensaio, todas as plantas foram cortadas na região do colo e avaliaram-se os pesos fresco e seco da parte aérea. Em dois ensaios independentes de avaliação da resistência à infecção com o ToCV por meio da liberação massal de B. tabaci virulífera constatou-se que em condições de livre chance de escolha dos insetos os acessos Solanum peruvianum LA 444-1 e S. habrochaites PI 127826 e PI 134417 e as linhagens avançadas IAC 14-2-49+14-2-85 (somente no primeiro ensaio) e IAC 68F-22-2-24-1 não tiveram plantas infectadas, sugerindo alto grau de resistência à infecção pelo crinivírus. Para os demais genótipos avaliados a reação das plantas à infecção com o ToCV variou de moderadamente resistente à altamente suscetível. Dois ensaios independentes para avaliar a tolerância dos diferentes genótipos de tomateiro ao amarelão causado pelo ToCV, com base no desenvolvimento e na produção das plantas mostrou resultados bastante variáveis. Os resultados desse trabalho fornecerão subsídios para futuros trabalhos de melhoramento genético para o desenvolvimento de cultivares resistentes/tolerantes ao ToCV. / Tomato chlorosis virus (ToCV), family Closteroviridae, genus Crinivirus is a single-stranded, positive sense RNA virus, transmitted semi-persistently by species of the family Aleyrodidae, belonging to the genus Bemisia and Trialeurodes. ToCV infects several species including domesticated and weed plants belonging to the families Alzoaceae, Amaranthaceae, Apiaceae, Apocynaceae, Asteraceae, Plumbaginaceae and Solanaceae. In São Paulo, Brazil, this crinivirus was first reported in 2008, causing chlorosis in the leaves of tomato plants. The importance of this emerging disease has increased in recent years and yet, so far there are no estimates of the damage, nor suitable alternatives for the management of the disease in the field. Therefore, this study aimed to (i) evaluate the resistance of tomato genotypes to infection with ToCV, (ii) to evaluate the tolerance of some genotypes to the disease and (iii) estimate the damage produced in infected plants. Fifty seven genotypes, including wild species, hybrids and commercial tomato cultivars were initially evaluated for resistance to infection. Seedlings produced in expanded polystyrene trays protected by cage covered with voile fabric were inoculated through the mass release of ToCV viruliferous B. tabaci MEAM1. The incidence of infected plants per genotype was determined by observation of symptoms and virus detection by RT-PCR. Some of the genotypes were also evaluated for tolerance to the disease caused by the crinivirus. Healthy and ToCV infected plants were separately transplanted in the field, in cages protected with voile fabric. Weight of harvested fruits of the plants were evaluated. At the end of the test, all the plants were cut out and their fresh and dry weights were measured. Results from two independent trials showed that the accesses Solanum peruvianum LA 444-1, and S. habrochaites PI 127826 and PI 134417, and the hybrids IAC 14249+14285 and IAC 68F-22-2-24-1 did not have infected plants, suggesting a high degree of resistance to infection by the crinivirus. For all other genotypes the response of the plants to infection with ToCV ranged from moderately resistant to highly susceptible. Results from two independent trials to assess the tolerance of different tomato genotypes to the disease caused by ToCV, based on the development and production of the plants were widely variable. These findings provide insights for future breeding programs for the development of cultivars resistant and/or tolerant to ToCV.

O Tomato chlorosis virus (ToCV) é uma espécie do gênero Crinivirus que causa danos, principalmente na cultura do tomateiro (Solanum lycopersicum). Foi primeiramente isolado e descrito em 1998, nos Estados Unidos, e em seguida foi reportado em doze países. No Brasil, foi constatado primeiramente no Estado de São Paulo, na região de Sumaré, em 2008, e posteriormente nos Estados da Bahia, Espírito Santo, Goiás, Minas Gerais e Rio de Janeiro. Há evidência da sua presença também nos Estados do Paraná e Santa Catarina. O ToCV pode infectar outras solanáceas além do tomateiro e, recentemente, foi observado infectando plantas de batata (Solanum tuberosum) no Brasil. Esse crinivirus é transmitido no Brasil principalmente pelo aleirodídeo (mosca branca) Bemisia tabaci MEAM1. Considerando o patossistema batateira/ToCV, não há estudos sobre a ocorrência, sintomatologia em diferentes variedades e danos provocados por esse crinivirus. Também não há antissoro policlonal para o isolado brasileiro do ToCV para uso na diagnose da doença em solanáceas. Esse trabalho teve por objetivos: purificar o ToCV e produzir antissoro policlonal; avaliar a reação de genótipos de batateira à infecção com o ToCV; avaliar o dano provocado por esse vírus em duas variedades de batateira. A purificação do vírus a partir de folhas de tomateiro e a produção de antissoro policlonal em coelho foram satisfatórias. No entanto, o antissoro não foi eficiente em ELISA, mas sim em dot-blot e somente na diluição de 1:20. Foi avaliada a reação de 21 genótipos de batateira à infecção com o ToCV, por meio da inoculação com B. tabaci MEAM1, com chance de escolha do vetor. Nenhum genótipo exibiu resistência à infecção; enquanto a variedade Camila foi assintomática e não apresentou alteração na fotossíntese. Plantas de batateira das variedades Ágata e Asterix sadias foram inoculadas com o ToCV, por meio da B. tabaci MEAM1 e ao final foram avaliadas a massa fresca da parte aérea, peso e número dos tubérculos colhidos. Em dois experimentos independentes, as reduções médias no peso fresco da parte aérea foram de 60,1% para Ágata e 46% para Asterix. Porém, as reduções nas produções dessas variedades, no primeiro experimento foram de 99,5% e 98,1%, respectivamente; enquanto no segundo os valores foram de 82,3% e 56,2%, respectivamente. / Tomato chlorosis virus (ToCV) is a species of the genus Crinivirus, which is causing considerable losses mainly on tomato crop (Solanum lycopersicum). It was first isolated and described on 1998 in the United States and subsequently reported in twelve countries. In Brazil, it was first reported in São Paulo State, in Sumaré region in 2008, and after that on the states of Bahia, Espírito Santo, Goiás, Minas Gerais and Rio de Janeiro. There is evidence of the presence of ToCV on the states of Paraná and Santa Catarina. ToCV can also infect other solanaceae and more recently, it was reported infecting potato plants (Solanum tuberosum) in Brazil. This crinivirus is transmitted by Bemisia tabaci MEAM1. Considering the patosystem potato/ToCV, there are no studies on the occurrence, symptomatology in different varieties, and damages caused by this crinivirus. In addition, there is no polyclonal antiserum for the Brazilian isolate of ToCV for use in diagnosis. The objectives of the present work were: to purify the virus and produce a polyclonal antiserum; to evaluate the reaction of potato genotypes to ToCV infection; to evaluate the yield loss caused by this crinivirus on two potato cultivars. The virus purification from tomato leaves and the production of polyclonal antiserum in rabbit were satisfactorily accomplished. However, the antiserum was not efficient on ELISA test, but in dot-blot, only when diluted 1:20. The reaction of 21 potato genotypes to infection with ToCV was evaluated by inoculation with B. tabaci MEAM1, with chance of choice for the vector. All genotypes were infected with ToCV and Camila was the only one asymptomatic. Plants of cultivars Ágata and Asterix were inoculated with ToCV, by means of viruliferous vector, and at the end were evaluated for the fresh mass of the aerial part, weight and number of harvested tubers. In two independent experiments, average reductions in aerial fresh weight were 60.1% for Ágata and 46% for Asterix. However, reductions in yield of these varieties in the first experiment were 99.5% and 98.1%, respectively; while in the second the values were 82.3% and 56.2%, respectively.

Uma das alternativas para reduzir os efeitos negativos das altas temperaturas nas plantas e, consequentemente a perda na produção e qualidade é a utilização de indutores de tolerância. Dentro deste contexto, a L-arginina, se apresenta como uma alternativa, por ser um dos aminoácidos mais funcionais e precursora de importantes compostos na regulação metabólica das plantas, com efeito na atenuação do estresse abiótico. O objetivo deste trabalho foi avaliar o efeito da aplicação foliar de diferentes doses de L-arginina (0; 0,15; 0,25; 0,5; 1,0 e 2,0 g L-1) na fisiologia, produção e qualidade do tomate cultivar Pizzadoro cultivado em ambiente protegido no período de verão. Para avaliação do efeito da L-arginina na fisiologia, foram feitas análises de indicadores de estresse dos compostos malonaldeído (MDA) e peróxido de hidrogênio (H2O2), das enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT) e ascorbato peroxidase (APX), teores de aminoácidos livres e teores de nutrientes nas folhas de tomateiro. Na produção foram determinadas a produção total (PT), a produção com presença de defeitos (PRDEF), a produção não comercial (PRNC) e a produção comercial (PC) com a classificação de calibre pequeno (PROP), médio (PROM) e grande (PROG). Além disso, foram determinados o número de frutos totais (NFT), número de frutos com presença de defeitos (NDEF), número de frutos não comerciais (NFNC) e número de frutos comerciais (NFC) com classificação de calibre pequeno (NFP), médio (NFM) e grande (NFG). Amostras de frutos maduros foram colhidas para a determinação de sólidos solúveis (SST), pH da polpa, ácido ascórbico (AA) e acidez total titulável (ATT) para determinação da qualidade do tomate. A aplicação de 1,0 g L-1 de L-arginina diminuiu o teor de MDA, e aumentou a atividade das enzimas CAT e APX. Além disso, essa dose do aminoácido aumentou a PC, PT, NFC, NFT e diminuiu a produção de NFNC. / Tolerance inductor products are one of the alternatives to reduce the negative effects of abiotic factors in plants, for instance the decrease of crop yield and quality. In this context, L-arginine is one option of this products to mitigate abiotic plant stress, due to its importance as a functional amino acid and precursor of metabolic plant compounds. Hence, the project objective was to evaluate the exogenous foliar application of different rates of L-arginine (0; 0.15; 0.25; 0.5; 1.0 e 2.0 g L-1) on physiology, yield and quality of tomato cultivar Pizzadoro growth in protected enviroment during Summer. Physiological effect were analized by markers of oxidative stress, as malondialdehyde (MDA) and hydrogen peroxide; activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) enzymes; free amino acids and nutrient contents in tomato leaves. Yield was estimated by total production (TP), production of defective fruit (PDF), non-commercial fruit production (NCFP) and commercial fruit production (CP). CP was classified as small (SPRO), medium (MPRO) and large (LPRO) sizes. Number of total fruit (NTF), number of defective fruit (NDF), number of non-commercial fruit (NNCF) and number of commercial fruit (NCF) were classified as small (NSF), medium (NMF) and large (NLF) sizes. Sample of mature tomatoes were harvested to assess quality, by content of soluble solids (SST), pH of tomato pulp, ascorbic acid (AA) and titratable acidity (TA). Application of L-arginine at rate of 1.0 g L-1 reduced MDA and increased CAT and APX. Besides physiological aspects, this level of amino acid increased CP, TP, NCF, NTF and decreased NNCF.

Solanum paludosum Moric. popularmente conhecida como jurubeba roxa, é uma espécie arbustiva encontrada no Brasil, utilizada na medicina popular principalmente para tratar distúrbios do fígado. Embora alguns trabalhos tenham descrito o perfil fitoquímico de S. paludosum, não existem estudos, até o presente, avaliando as propriedades biológicas desta planta. Assim, dois procedimentos de extração, maceração e extração supercrítica foram utilizados para obter diferentes extratos desta planta. Estes extratos foram comparados no que diz respeito ao perfil cromatográfico dos seus compostos fenólicos e avaliados quanto a citotoxicidade contra duas linhagens de células de mamíferos e quanto a atividade antioxidante in vitro utilizando três sistemas: o método de DPPH, o sistema xantina/xantina oxidase/luminol e a atividade inibidora da peroxidação lipídica. Além disso, a possibilidade de utilizar extratos de S. paludosum como uma nova fonte de inibidores de bombas de efluxo contra cepas de Staphylococcus aureus foi investigada. A maceração e a SFE levaram a obtenção de três extratos com diferentes perfis cromatográficos: SP-EtOHB, com uma composição química complexa, SP-SFECO2, enriquecido com flavonóides metoxilados, e SP-EtOHM, contendo principalmente polifenóis polares. No que diz respeito à atividade citotóxica, SP-EtOHB e SP-EtOHM mostraram similar baixa toxicidade contra as células de fibroblasto, enquanto o SP-SFECO2 foi mais citotóxico. A SP-EtOHB apresentou o melhor efeito citotóxico contra células de melanoma. SP-SFECO2 não demonstrou capacidade de reduzir o radical DPPH enquanto que os extratos SP-EtOHB e SP-EtOHM foram altamente antioxidantes (CI50 19,3 µg/mL). Todos os extratos apresentaram atividade inibitória do sistema xantina/XOD/luminol de maneira dependente de concentração (28 % a 97% em 4,7 a 298,5 µg/mL). A inibição da peroxidação lipídica aumentou com o aumento das concentrações dos extratos sem diferença entre SP-SFECO2 e SP-EtOHM (CI50 56,76 µg/mL). Curiosamente, a maior potência anti lipoperoxidativa foi com relação ao extrato SP-EtOHB (CI50 3,23 µg/mL), o que pode indicar um efeito sinérgico de compostos com mecanismos diferentes de ação antioxidante. Nenhum dos extratos testados apresentaram atividade antibacteriana. Por outro lado, todos os extratos apresentaram acentuada atividade moduladora nas cepas estudadas, principalmente a SP-SFECO2, que reduziu a CIM dos antibióticos em até oito vezes. Esta atividade pode estar relacionada com a elevada lipofilicidade de flavonóides polimetoxilados em SP-SFECO2, visto que a lipofilicidade é uma característica comum de diversos inibidores da bomba de efluxo. Em conclusão, os dois métodos de extração foram adequados para obter extratos com diferentes atividades biológicas de acordo com seu padrão de compostos fenólicos, o que contribue para aumentar o valor agregado de Solanum paludosum Moric. / Solanum paludosum Moric., popularly known as purple jurubeba, is a srhub found in Brazil, used in folk medicine mainly to treat liver disorders. Although some works have described the phytochemical profile of S. paludosum, there are no studies, until now, evaluating the biological properties of this plant. Thus, two procedures of extraction, maceration and supercritical fluid extraction, were used to obtain different extracts of this plant. These extracts were compared as regard the chromatographic profile of its phenolic compounds and evaluated for citotoxicity against two mammalian cell lines and for in vitro antioxidant activity using three systems: the DPPH method, the xanthine/xanthine oxidase/luminol assay and the lipid peroxidation inhibitory activity. Also, the possibility of using S. paludosum extracts as a new source of inhibitors of efflux pumps against Staphylococcus aureus strains has been investigated. The maceration and SFE led the obtaining of three extracts with distinct chromatographic profiles: SP-EtOHCRUDE, with a complex chemical composition, SP-SFECO2, enriched with methoxylated flavonoids, and SP-EtOHMARC, containing mainly polar polyphenols. As regard the cytotoxic activity, extracts SP-EtOHCRUDE and SP-EtOHMARC showed a similar low toxicity against fibroblast cells while the SP-SFECO2 was more cytotoxic. The SP-EtOHCRUDE presented the best cytotoxic effect against melanoma cells. SP-SFECO2 showed no capacity to reduce the radical DPPH while SP-EtOHCRUDE and SP-EtOHMARC were highly antioxidants (IC50 19,3 g/mL). All extracts exhibited inhibitory activity of the xanthine/XOD/luminol system in a dose-dependent manner (28 % to 97 % at 4.7298.5 g/ml). The inhibition of lipid peroxidation increased with increasing concentrations of extracts, with no difference between SP-SFECO2 and SP-EtOHMARC (IC50 56.76 g/ml). Interestingly, the highest anti-lipoperoxidative potency was with regard to SP-EtOHCRUDE (IC50 3.23 g/ml), which may indicate a synergistic effect of compounds with different mechanisms of antioxidant action. None of the extracts tested showed significant antibacterial activity. On the other hand, all extracts showed marked modulator activity in the studied strains, mainly the SP-SFECO2 which reduced the MIC of antibiotics up to eight fold. This activity may be related to the high lipophilicity of polymethoxylated flavonoids in SP-SFECO2, since lipophilicity is a common feature of several efflux pump inhibitors. In conclusion, both methods of extraction were adequate for obtain extracts with different biological activities according to its pattern of phenolic compounds, contributing to increase the aggregate value of Solanum paludosum Moric.

Made available in DSpace on 2014-08-20T14:33:07Z (GMT). No. of bitstreams: 1 Tese_Clarissa_ Melo_ Cogo.pdf: 408988 bytes, checksum: f45ceadefc0fe4f19f36b57bade21cbd (MD5) Previous issue date: 2009-03-23 / The goal of this work was to determine the effect of different nutritive solution concentration about the growth, yield and consuption of nutritive solution of this crop using rusk rice substrate in natura. Two experiments were carried in two periods, in spring/summer and summer/autumn in a plastic greenhouse in the Department of Fitotecny of University Federal of Pelotas. Treatments consisted of five concentrations of nutritive solution, in bifactorial scheme and casualized design randomized. The testemmun (T3) was the nutritive solution with concentration standarized and the other treatments were concentrations of 0,9 (T1); 1,6 (T2); 3,0 (T4) and 3,7 (T5) dS m-1. The determination of transpirated volumes in treatments with concentrations 1,6, 2,3, 3,0 e 3,7 dS m-1 was made through estimative of nutritive solution volume decreased in recipient of similar form of methodology used by Valandro (1999). The leaf area was determined through a measured image equipment (LI-COR 3100). The temperature and solar radiation were measured daily. The absorved volumes were converted for unity of solar radiation and leaf area. The substrate was placed in bags of 10 L drilled in the base and put in density 2,81 pl m-2. Fresh and dry vegetative mass, fresh and dry fruit mass and also leaf area in three plants in each repetition were determined. The dry vegetative mass, the dry fruit mass and the total dry mass showed polinomial effect of nutritive solution concentration. The leaf area evolution of crop in two periods, adjusted similar polinomial tendency observed in fresh and dry mass. The results show values for EC of 2,3 and 3,0 dS m-1, like the ones that maximize fruit production in spring/summer and summer/autumn, respectively. There was a decrease of leaf area after the EC 2,3 and 3,0 dS m-1 in spring/summer and summer/autumn, respectively. A polinomial regression was adjusted between EC and consumo of nutritive solution in spring/summer. Solanum esculentum, eletrical condutivity, substrate / O objetivo do trabalho foi determinar o efeito de diferentes concentrações da solução nutritiva sobre o crescimento, produtividade e consumo de solução nutritiva pelo tomateiro empregando como substrato a casca de arroz in natura. Dois experimentos foram conduzidos, na primavera/verão e verão-outono, em ambiente protegido, no Departamento de Fitotecnia, do Campus da Universidade Federal de Pelotas. Os tratamentos foram constituídos por cinco concentrações de solução nutritiva, no delineamento inteiramente casualizado, em esquema unifatorial. A testemunha (T3) foi a solução nutritiva com a concentração padrão (T3) e os demais tratamentos foram concentrações de 0,9 (T1); 1,6 (T2); 3,0 (T4) e 3,7 (T5) dS m-1 A determinação dos volumes transpirados nos tratamentos com concentrações 1,6; 2,3; 3,0 e 3,7 dS m-1 foi feita através da estimativa da diminuição do volume de solução nutritiva nos reservatórios de solução nutritiva, de forma similar à metodologia empregada anteriormente por Valandro (1999). A área foliar (AF) foi determinada através de um equipamento medidor de imagens (LI-COR, modelo 3100). Diariamente foi medida a temperatura do ar e radiação solar. Os volumes absorvidos foram convertidos por unidade de radiação solar e de área foliar (AF). O substrato foi posto em sacos plásticos de 10 L perfurados na base e dispostos na densidade de 2,81 pl m-2 Foram determinadas a matéria fresca e seca vegetativa e de frutos e também a área foliar em três plantas escolhidas aleatoriamente em cada repetição. A massa seca vegetativa, de frutos e total mostrou efeito polinomial da concentração da solução nutritiva. A evolução da área foliar da cultura nas duas épocas ajustou-se a mesma tendência polinomial observada na massa fresca e seca. Os resultados indicam valores de condutividade elétrica (CE) de 2,3 dS m-1 e de 3,0 dS m-1 como aquelas que maximizam a produção de frutos nos cultivos de primavera-verão e verão-outono, respectivamente. Houve decréscimo da AF após as CE 2,3 e 3,0 dS m-1 , nos cultivos primavera-verão e verãooutono, respectivamente. Uma regressão polinomial se ajustou entre a CE e o consumo de solução nutritiva na primavera-verão.

General Abstract There has been a steady increase in the use of medicinal plants in the last two decades in both developed and developing countries for prevention, management and treatment of diseases. This increase has been due to reasons such as ease of access, better cultural acceptability and compatibility, cost effectiveness and also the bid to “go natural”. Unfortunately, the notion that herbal medicines are relatively safe because they are natural has led to serious and potentially fatal consequences in phytotherapy. The lack of rigorous research to prove the effectiveness and safety of many medicinal plants is of great concern to the health care system. This thesis therefore addresses not just the efficacy, but also the safety of the extracts of the berry of Solanum aculeastrum - a medicinal plant used, among other things, for the treatment of breast cancer in the Eastern Cape Province of South Africa. Particular attention was paid to the possible effect of different processing methods of the berry extracts on inflammation, cytotoxicity, and toxicity. In studying the comparative effects of various processing methods, four different preparations of the extracts were investigated. These include fresh, dried, boiled fresh, and boiled dried berries. While the effect of processing on the anti-inflammatory properties of the extracts was not dose dependent, the percentage reduction in inflammation was highly significant and more prominent in both concentrations of the boiled fresh berries than the reference drug, indomethacin. Furthermore, the four extracts varied in their ability to act either centrally or peripherally in their effect on pain. Assessment of the analgesic response using the formalin test showed that, at both concentrations tested, none of the extracts inhibited the first phase of the formalin test. Furthermore, it was observed that boiling had differing results on the activity of the iii fresh and dried extracts. While boiling of the dried berries reduced pain in the rats, the opposite trend was observed with the boiled fresh berries. Results of the influence of processing of the berries on cytotoxicity indicated that the extracts are potent inhibitors of human breast, cervical and colonic carcinoma cells and the non-cancerous cells (both the actively dividing and confluent Chang liver cells). Although, in terms of relative potency, the fresh berries appeared to be the most active of the extracts, processing of the berries caused an increase in apoptotic cells and a subsequent decrease in the necrotic cells. The effect of processing on the safety of the berry of S. aculeastrum on the rats fed for 28 days was also investigated. The various doses (1, 10 and 25 mg/kg body weight) of all the four extracts did not alter the activity and the weight of the animals throughout the period of treatment. A reduction in organ to body weight ratio of the heart, kidney, liver and spleen was observed in all the extracts. Regarding the haematological parameters, different patterns of effect were observed between the extracts and within the treated doses. The observed alterations in the biochemical parameters by the various extract of Solanum aculeastrum berries at all the doses may have consequential effects on the normal functioning of these organs. In conclusion, this study has shown that there is some justification for the traditional use of the berries of Solanum aculeastrum in the treatment of inflammation related ailments and cancer. However, the medicinal use of the plant also poses considerable health risks. Investigation conducted into the plant’s safety revealed that the berry extracts were nephrotoxic, hepatotoxic, haematotoxic and at higher doses, fatal. Another concern with regard to the plant’s safety is the non-selectivity of its extracts in the inhibition of carcinoma, actively dividing and un-dividing cells. Assessment of the effect of the processing on the berry’s efficacy and safety as herbal iv remedy produced mixed results. On the one hand, processing seemed to improve the extract’s anti-inflammatory and analgesic activity, while reducing its cytotoxic potential. On the other hand, a reduction in the toxicity was observed on the processed extracts compared to the fresh ones. This may be an indication that processing has an overall beneficial effect on the medicinal properties of the plant and should thus be considered as a method of making the berries of Solanum aculeastrum safer for use as a herbal remedy.

Leaves are responsible for the majority of the fixed carbon in most plant species. Along leaf development, the photosynthetic capacity increases until the organ reaches maturity. Consequently, at the onset of senescence the leaves have the highest photosynthetic activity, then, as the chloroplasts are dismantled and the photosynthetic machinery is degraded, leaves gradually lose the rate of carbon assimilation. Although the capacity to fix carbon declines as senescence progresses, nutrient remobilization from macromolecule degradation nourishes the developing sink organs. In this regard, delaying leaf senescence stands out as a promising strategy to increase plant yield as extends the window of time with maximum carbon fixation rate. Another approach that is receiving much attention is the manipulation of chlorophyll degradation once it potentially regulates photosynthetic capacity and affects the nutritional quality of harvestable organs. As chlorophyll is degraded, the released phytol is recycled and can be either stored (i.e. as fatty acid phytyl esters), used for chlorophyll synthesis or be incorporated in tocopherol biosynthesis. Tocopherols have high nutraceutical value due to their antioxidant properties. However, the majority of the studies regarding senescence and chlorophyll degradation were carried out in the model plant Arabidopsis thaliana or grasses, creating a knowledge gap about these processes in fleshy fruit-bearing plants of human diet interest. In this regard, the tomato, Solanum lycopersicum, is an excellent model not only for the genetic and genomic resources, but also for its agronomic and nutritional importance. Thus, this project aims to extend what is known about the effects of chlorophyll degradation and senescence manipulation over the metabolism and yield of tomato plants, as well as fruit nutritional quality. In order to evaluate the consequences of alteration in chlorophyll degradation, first the enzymes chlorophyllase and pheophytinase, both capable of dephytylating the chlorophyll molecule, were identified and characterised. An extensive phylogenetic, evolutive and transcriptional analysis allowed the identification of two groups of chlorophyllases, one putatively involved in the response to different stimuli, while the other may act in chlorophyll homeostasis. As for pheophytinase, only one group was identified, being related to physiologically programmed processes that trigger chlorophyll degradation (i.e. leaf senescence and fruit ripening). Given this scenario, pheophytinase was chosen to be constitutively knocked-down in order to evaluate the effects over the metabolism of leaves and fruits. As consequence of this manipulation, transgenic plants were impaired in the leaf senescence-associated chlorophyll breakdown, but, although with an initial delay, fruit ripening-associated degreening was not compromised. Several photosynthetic and biochemical parameters were signs of photoinhibition, possibly due to a deficiency in chlorophyll recycling in leaves. This led to an increase in sugar exportation towards fruits, ultimately increasing soluble sugar content of ripe fruits. However, as a consequence, carotenoid levels were reduced, what, at least partially, it was compensated by an increase in tocopherol content. The results indicated that pheophytinase plays a role beyond senescence-associated chlorophyll degradation and its manipulation led to the development of fruit with increased soluble sugars and tocopherols at the cost of lowering carotenoid levels. Thus, these evidences support the manipulation of chlorophyll breakdown as a strategy for improvement of fleshy fruit plants. In order to address the effects of senescence over yield and fruit quality, the transcription factor ORESARA1, which has been widely characterised in A. thaliana and is considered a key regulator of senescence initiation, was targeted. After a comprehensive phylogenetic analysis and the characterization of the regulatory mechanisms, one putative ortholog was selected to be silenced. As consequence of this manipulation, leaves displayed increased chlorophyll content. Moreover, as senescence was delayed, the extent of photosynthetic activity of the leaves was also expanded. As the number of fruits was increased in the knockdown lines, this reflected in an increment in the harvest index. Ripe fruits accumulated more soluble sugars and tocopherols. Collectively, the results support the manipulation of leaf senescence as a strategy for tomato yield improvement. Altogether, data obtained enhance the knowledge about the impacts of chlorophyll degradation and leaf senescence over the metabolism of fleshy-fruit plants, providing strategies to increase yield and nutritional quality of fruits / As folhas, para a maioria das espécies vegetais, são o principal órgão responsável pela fixação de carbono. Durante o desenvolvimento foliar, o potencial fotossintético aumenta até a folha atingir a sua maturidade. Consequentemente, no momento que o programa de senescência se inicia, a folha apresenta a maior taxa de fotossíntese, a qual passa então a declinar conforme o cloroplasto se desorganiza e a maquinaria fotossintética é degradada. Apesar da redução na fixação de carbono, o catabolismo de macromoléculas possibilita a remobilização de nutrientes para os órgãos dreno em desenvolvimento. Neste contexto, atrasar a senescência destaca-se como uma promissora estratégia para aumento da produtividade, uma vez que estende o período de máxima fixação de carbono das folhas. Outra estratégia que tem recebido atenção por, potencialmente, regular a capacidade fotossintética e afetar a qualidade nutricional dos órgãos coletáveis é a manipulação da degradação da clorofila. Durante o catabolismo deste pigmento, o fitol liberado é reciclado podendo ser armazenado (i.e. na forma de ésteres de fitil com ácidos graxos), ser utilizado na síntese de novas moléculas de clorofila ou ser incorporado na rota biossintética de tocoferóis. Estes últimos compostos, por seu potencial antioxidante, possuem alto valor nutracêutico. No entanto, a maior parte dos estudos sobre senescência e degradação de clorofila foi realizada na planta modelo Arabidopsis thaliana ou em gramíneas, tornando escassas as informações relativas a plantas com frutos carnosos de interesse para a dieta humana. Nesse âmbito, o tomateiro, Solanum lycopersicum, é um excelente modelo de estudo não apenas pela disponibilidade de recursos genético e genômicos, mas também pela importância agronômica e nutricional desta espécie. Assim, este trabalho pretende expandir o conhecimento acerca dos efeitos da manipulação da degradação de clorofila e da senescência sobre o metabolismo e produtividade do tomateiro, bem como sobre a qualidade nutricional dos frutos. De modo a se avaliar as consequências de alterações na degradação de clorofila, iniciou-se por identificar e caracterizar em tomateiro as enzimas clorofilase e feofitinase, as quais catalisam a defitilação da molécula de clorofila. Uma vasta análise filogenética, evolutiva e transcricional permitiu a identificação de dois grupos de clorofilases, um dos quais estaria envolvido na plasticidade de respostas a estímulos e o outro na homeostase dos níveis de clorofila. Já para feofitinase, somente um grupo foi identificado, o qual está relacionado a processos fisiologicamente programados que levam à degradação de clorofila (i.e. senescência foliar e amadurecimento de frutos). Dado o panorama obtido, a feofitinase foi escolhida para ser constitutivamente silenciada de modo a se avaliar as consequências para o metabolismo de folhas e frutos. Como consequência do silenciamento, as linhagens transgênicas mostraram-se incapazes de degradar clorofila durante a senescência, mas, embora com um atraso nas etapas iniciais, a degradação ao longo do amadurecimento de frutos não foi comprometida. Diversos parâmetros fotossintéticos e bioquímicos foram indicativos de fotoinibição, possivelmente em virtude de uma deficiência na reciclagem da clorofila em folhas. Isto acarretou em um aumento na exportação de açúcares para frutos, incrementando a concentração de açúcares solúveis nos frutos maduros, que, em contrapartida, resultou na queda nos teores de carotenoides. A queda nestes compostos antioxidantes foi, ao menos parcialmente, compensada por um aumento nos níveis de tocoferóis. Os resultados indicaram que a feofitinase possui um papel além da degradação de clorofila associada à senescência, e que sua manipulação leva ao desenvolvimento de frutos com maior teor de açúcares solúveis e de tocoferóis ao custo da redução no de carotenoides. Desta forma, estas evidências suportam a manipulação da clorofila como estratégia para o melhoramento de frutos carnosos. Para investigar o efeito da senescência sobre a produtividade e qualidade de frutos foi escolhido o fator de transcrição ORESARA1, o qual está amplamente caracterizado em A. thaliana e é considerado um regulador chave no desencadeamento deste processo. A partir de uma extensa análise filogenética e da caracterização de sua regulação, um putativo ortólogo foi selecionado como alvo para silenciamento. Como consequência desta manipulação, folhas apresentaram os níveis de clorofila incrementados. Além disto, taxas fotossintéticas maiores que as do genótipo controle foram mantidas por maior tempo indicando que a iniciação da senescência foi retardada. Assim, estas plantas produziram um maior número de frutos, consequentemente, aumentando o índice de colheita dessas linhagens. Os frutos maduros apresentaram maiores teores de açúcares solúveis e de tocoferóis. Os resultados demostraram que o retardo do início da senescência é uma estratégia efetiva para aumento da produtividade de tomateiro. Coletivamente, os resultados obtidos aprofundam o conhecimento acerta dos impactos da degradação de clorofila e senescência sobre o metabolismo de plantas com frutos carnoso, além de prover estratégias para se incrementar a produtividade e a qualidade nutricional de frutos

Among viral diseases affecting cultivated tomato, Tomato yellow leaf curl disease (TYLCD) is one of the most devastating. This disease is caused by a complex of viruses of which Tomato yellow leaf curl virus (TYLCV) is regarded as the most important species. Current control strategies to fight viral diseases in tomato are mainly based on genetic resistance derived from wild relatives. In the present thesis, resistance derived from S. chilense and S. peruvianum has been exploited in breeding for resistance to TYLCD. In a previous study, TYLCV-resistant breeding lines derived from LA1932, LA1960 and LA1971 S. chilense accessions were developed. Therefore, the first objective of this thesis was to study the genetic control of the resistance derived from these accessions. With this aim, response to viral infection was assayed in segregating generations derived from the aforementioned resistant lines. The results obtained were compatible with a monogenic control of resistance. Resistance levels were higher in LA1960- and LA1971-derived F2 generations, as shown by slighter symptoms in the resistant plants and a higher number of asymptomatic plants compared with the results obtained in the LA1932-derived F2 generation. It is noteworthy that the level of resistance present in our materials is comparable to or even higher than the levels found in tomato lines homozygous for Ty-1. The response in plants heterozygous for the resistance gene was comparable to the response in homozygous plants for all three sources employed. This implies that the resistance genes derived from all three sources seem to be almost completely dominant. This effect was stronger for LA1971-derived resistance. The results were similar when comparing viral accumulation, as was expected, since a positive correlation was found in these families between viral accumulation and symptom scores. This has important implications in breeding, since the resistance will be used mostly for hybrid development. Our second objective was to map the loci associated with the major resistance genes identified. A total of 263 markers were screened, 94 of them being polymorphic between both species. Recombinant analysis allowed the resistance loci to be localized on chromosome 6, in a marker interval of 25 cM. This interval includes the Ty-1/Ty-3 region, where two S. chilense-derived TYLCD resistance loci were previously mapped. In order to test if the resistance genes identified in our populations were allelic to Ty-1 and Ty-3, further fine mapping was carried out. A total of 13 additional molecular markers distributed on chromosome 6 allowed 66 recombinants to be identified, and the resistance region to be shortened to a marker interval of approximately 950 kb, which overlaps with the Ty-1/Ty-3 region described previously by other authors. Therefore, the results obtained indicate that closely linked genes or alleles of the same gene govern TYLCV resistance in several S. chilense accessions. The third objective of the present thesis was to start the construction of a set of introgression lines (ILs) derived from Solanum peruvianum accession PI 126944 into the cultivated tomato genetic background. Once this collection of ILs is developed, it will represent a powerful tool for exploiting the resistance to different pathogens found in this particular accession in addition to other possible characters of interest. The starting plant material consisted of several segregating generations that were derived from two interspecific hybrids previously obtained by our group. Many crosses and embryo rescue were required to obtain subsequent generations due to the high sexual incompatibility that exists between tomato and PI 126944. Several mature fruits from the most advanced generations produced a few viable seeds, although embryo rescue was also employed to obtain progeny. As only a few plants were obtained by direct backcrossing, additional crosses were made in order to increase the number of descendants. A high degree of incompatibility was also found in crosses between sib plants. A total of 263 molecular markers were tested in some generations, 105 being polymorphic between tomato and PI 126944. Available generations were genotyped with these polymorphic markers in order to determine which alleles of S. peruvianum were already introgressed. On average, 79, 78 and 84 % of the S. peruvianum genome was represented in the pseudo-F2, pseudo-F4 and pseudo-F5 generations, respectively, for the markers analyzed. A reduction in the S. peruvianum genome was observed in more advanced generations, such as BC1 (56 %), pseudo-F2-BC1 (60 %) and pseudo-F3-BC1 (70 %). A greater reduction was observed in the pseudo-F3-BC2 generation (33 %). As a consequence of the reduction in the S. peruvianum genome, a loss of incompatibility was observed in some cases. The S. peruvianum genome was almost completely represented among the different plants of the most advanced generations. An evaluation for resistance to TYLCD and Tomato spotted wilt virus (TSWV) was carried out in some of the advanced generations, some of which were resistant to one or both viruses. In conclusion, we have conducted a successful and deeper exploitation of two wild species with proved resistance to TYLCD, S. chilense and S. peruvianum, identifying and fine mapping new genes of resistance. / Julián Rodríguez, O. (2014). Exploitation of Solanum chilense and Solanum peruvianum in tomato breeding for resistance to Tomato yellow leaf curl disease [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36867 / TESIS