Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

Generation and characterization of activation tagged potato mutants could aid in functional genomic studies. Morphological and molecular studies were conducted to compare potato cv. Bintje, its two mutants, underperformer (up), and nikku generated using the activation tagging vector pSKI074, and nikku revertant plants. Mutant up exhibited a dwarf phenotype (plant height 42 cm vs. 73 cm in cv. Bintje), abundant axillary shoot growth (3.1 shoots/plant compared to 0.7 shoots/plant in cv. Bintje; in vitro plants), greater tuber yield, altered tuber traits and early senescence compared to wild-type Bintje under in vitro conditions. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant were consistent, but the tuber yield of up was less (250 g/plant compared to 610 g/plant in wild-type Bintje) and had fewer axillary shoots compared to wild-type (1.9 shoots/plant in up vs. 4.7 shoots/plant in Bintje). Mutant nikku plants exhibited an extremely dwarf phenotype (plant height 2 cm in nikku vs. 6 cm in Bintje), had small hyponastic leaves, were rootless, and infrequently produced small tubers when compared to cv. Bintje. The overall nikku phenotype was suggestive of a constitutive stress response, which was further supported by the higher expression levels of several stress-responsive genes in nikku. The nikku revertant plants exhibited near normal stem elongation, larger leaves and consistent rooting, and it was a case of partial reversion. Southern blot analyses indicated the presence of single T-DNA insertions on chromosome 10 in the up and on chromosome 12 in the nikku mutant. The reversion in the nikku plants was not associated with the loss of enhancer copies from the original nikku mutant. Reverse transcriptase PCR analyses indicated transcriptional activation/repression of several genes in the up and nikku mutants, suggesting pleiotropic effects. In revertant, the expression levels of several genes which were differentially regulated in the nikku mutant were similar to Bintje. The gene immediately flanking the right border of the T-DNA insertion, which encoded a novel BTB/POZ (Broad complex, Tramtrac, Bric a brac; also known as Pox virus and Zinc finger) domain-containing protein, was highly up-regulated in the up mutant. This protein domain plays an important role in several important developmental, transcriptional and regulatory pathways. The mRNA-seq analyses resulted in 1,632 genes that were differentially expressed between mutant up and Bintje and the total number of up-regulated genes (661) were less than the number of genes down-regulated (971 genes) in the up mutant. Further analyses indicated that a variety of biological processes including decreased cell division, cell cycle activity, and abiotic stress responses were modified in the up mutant. In the nikku mutant, two potato genes, encoding an Acyl-CoA N-acyltransferases (NAT) superfamily protein, and a predicted major facilitator superfamily protein (MFS) were identified and overexpression lines Bintje/35S::NAT1 and Bintje/35S::PMT1 were created for recapitulation of the nikku mutant phenotype. Methylated DNA-PCR between the nikku and the revertant indicated a change in methylation status of the 35S enhancers, suggesting that the nikku revertant phenotype may be associated with some epigenetic modification. / Ph. D.

A Solanum lycocarpum St. Hil (Solanaceae) é uma planta nativa do cerrado brasileiro, cujos frutos possuem os glicoalcalóides esteroidais solasonina e solamargina. É possível que a ingestão de frutos que contenham esses glicoalcalóides possam interferir no equilíbrio do sistema endócrino. O presente estudo foi realizado com o objetivo de avaliar os possíveis efeitos tóxicos provenientes da ingestão diária de frutos verdes da S. lycocarpum (10% na ração) por ratas prenhes, a partir do 6° dia da gestação (DG 06) ao 7° dia pós-natal (PN 07). Os princípios ativos isolados dos frutos da planta mostraram a presença de 0,6% de solamargina e 0,9% de solasonina. A exposição das mães à planta alterou alguns parâmetros do desempenho reprodutivo; foram observados redução tanto do peso como do comprimento corporal das ninhadas experimentais ao nascimento, um ou dois filhotes mortos ao nascimento em 20% das ninhadas experimentais e menor capacidade em adquirir peso do nascimento à vida adulta. A prole experimental adulta fêmea apresentou degeneração de células epiteliais uterinas no dia PN 60, não sendo mais observada esta alteração no dia PN 90. A avaliação do comportamento sexual de fêmeas (PN 10O) mostrou redução do coeficiente de lordose das fêmeas experimentais quando comparado com o coeficiente de lordose das fêmeas do grupo controle. Na prole experimental masculina (PN 60 e PN 90) observou-se importante degeneração de células germinativas dos duetos testiculares. Por outro lado, o comportamento sexual desta prole não mostrou-se alterado. Na avaliação de neuroaminas cerebrais, a prole feminina apresentou maior atividade do sistema noradrenérgico estriatal, caracterizada por redução dos níveis de noradrenalina, sem alteração dos níveis de seu metabólito ácido vanilmandélico (VMA), e reduzida atividade do sistema serotoninérgico hipotalâmico, caracterizada por elevação dos níveis de serotonina (5-HT) sem alteração dos níveis de seu metabólito ácido 5-hidroxiindolacético (5 HIAA). Os filhotes machos apresentaram concentrações reduzidas de noradrenalina, VMA e ácido homovanílico (HV A). Ambos, machos e fêmeas, apresentaram níveis estriatais diminuídos de dopamina e seu metabólito ácido dihidroxifenilacético (DOPAC), sem, contudo, alterar a atividade do sistema dopaminérgico. Não foram observadas alterações hormonais tanto nas mães, quanto na prole, avaliadas por meio da mensuração não invasiva de metabólitos dos hormônios progesterona, estradiol e testosterona nas fezes. Estes dados tomados em conjunto indicam que a planta promoveu leve toxicidade materna e apresentou leve efeito fetotóxico. A avaliação das proles do nascimento à vida adulta indicou ainda que os glicoalcalóides esteroidais presentes nos frutos provocaram efeitos diferenciados nas proles feminina e masculina e possivelmente apresentam atividade estrogênica. / Solanum lycocarpum St. Hil (Solanaceae) is a native shrub of the Brazilian savanna. The fruits contain steroidal glycoalkaloids, solasonine and solamargine, that can disrupt the endocrine system once in the body by ingestion of S. lycocarpum fruit. The present study was performed in order to determine the possible toxic effects of S. lycocarpum fruit ingestion (10% added in the diet) by pregnant rats from gestation day (GD) 06 to post-natal day (PND) 07. The unripe fruits employed contain 0.021 % of solamargine and 0.031 % of solasonine. S. lycocarpum 10% in the diet, during pregnancy and beginning of lactation, did not impair gestation but alterations in some parameters of the reproductíve performance were observed: lower body weight and body size of the experimental litters at PND 01, one or two stillbirths were observed in 20% of the experimental litters and reduced weight gain of the pups from birth to adulthood. Female offspring (PN 60) presented reversible uterine epithelial cel! degeneration, and at PND 100, presented impaired sexual behavior caracterized by reduced lordosis quotient. Male offspring (PN 60 and PND 90) showed irreversible degeneration of testis germinative cells and normal sexual behavior. The central neuroamine systems avaliation presented, in the female exposed offspring, increased activity of striatal norepinephrine system, with reduced levels of norepinephrine (NOR) and normal levels of vanilmandelic acid (VMA), and reduced activity of the hypothalamic serotoninergic system, with increased serotonin (5-HT) and normal 5-hydroxindolacetic acid (5-HIAA) levels. Exposed male offspring presented reduced hypothalamic NOR, VMA and homovanilic acid (HV A) levels, and both males and females presented striatal dopamine (DA) and dihydroxylphenylacetic acid (DOPAC) reduced levels, without alterations in the activity of the dopaminergic system. The sexual hormones were also evaluated by a non invasive method with feces extraction. However no alterations were observed in progesterone, estradiol and testosterone metabolite levels in dams and offspring feces. Present data showed light maternal toxicity and light fetotoxic effects. The evaluation of the offspring from birth to adult age indicate that the steroidal glycoalkaloids present in the fruit act by different ways in male and female fetuses, and perhaps presents estrogenic activity.

A importância do cultivo de espécies do gênero Capsicum vem crescendo nos últimos anos no Brasil, e consequentemente, a ocorrência de doenças torna-se um problema relevante. A antracnose é a mais comum e destrutiva doença de Capsicum no Brasil e em vários países, podendo ocasionar perdas de até 100%. Seu controle usando resistência genética seria altamente desejável. O objetivo deste trabalho foi (i) caracterizar a reação à antracnose de 90 acessos de Capsicum annuum, 30 de C. baccatum, 16 de C. chinense e um de C. frutescens; (ii) estabelecer a correlação da reação à antracnose entre plântulas e frutos de Capsicum e; (iii) avaliar a reação à antracnose das progênies F3RC1 (C.annuum x C. chinense - resistente) x C. annuum e F2RC2 [(C. annuum x C. chinense) x C. annuum] x C. annuum. Para as inoculações, foram utilizados quatro isolados do fungo Colletotrichum sp. provenientes de pimentão e um isolado de pimenta, obtidos de folhas e frutos doentes. As suspensões de inóculo foram preparadas na concentração de 1 x 106 conídios/ml, separadamente para cada isolado. Após ajustada a concentração das suspensões, estas foram misturadas, a fim de reduzir efeitos de possível variabilidade patogênica dos isolados. O delineamento utilizado foi inteiramente casualizado com 20 repetições para plântulas e 15 repetições para frutos verdes destacados. Os ensaios foram conduzidos duas vezes. Nas inoculações das plântulas, foi adicionado 5% de caldo de cana à suspensão, como fonte exógena de energia ao patógeno. A inoculação nas plântulas foi realizada no estádio de primeira folha verdadeira completamente expandida, pela aspersão da suspensão de esporos até o ponto de escorrimento. As plântulas foram mantidas em câmara úmida por 24 horas antes e 72 horas após a inoculação. O ensaio foi instalado em sala climatizada com temperatura de 26 +/- 2°C e fotoperíodo de 12 horas. Foi avaliada a severidade de doença, usando uma escala de notas e também a incidência da doença, com base na percentagem de plântulas doentes. A inoculação nos frutos foi realizada pela deposição de uma gota de 20 µl da suspensão de esporos na parte mediana dos frutos. Posteriormente, com o auxílio de uma agulha entomológica, foi realizado um ferimento abaixo da gota. Os frutos inoculados foram mantidos em câmara úmida durante o período de avaliação. As avaliações foram realizadas pela mensuração do diâmetro médio das lesões, período latente, velocidade de crescimento da lesão e incidência. Baseando-se na avaliação dos frutos para classificação da reação, identificou-se apenas um acesso de C. annuum como resistente (n°233 - Jalapeno), um de C. baccatum (n°222 - BGH 4176) e dois de C. chinense (n°105 - Bode e n°141 - Pimenta n°2). Os demais acessos foram suscetíveis. Não houve correlação entre a reação de plântulas e frutos. As plântulas apresentaram menor quantidade de doença, em relação aos frutos. As progênies F3RC1 (3G, 4A, 4B, 4F, 4H, 5H, 6B, 6F, 8B, 8E, 8F, 9B, 9D, 9E, 9J, 10A, 13D, 13F, 14A, 14C, 16D, 16I, 17B, 19B) e as progênies F2RC2 (33F e 40G) não apresentaram lesões no estádio de frutos. Avançou-se geração nas progênies com reação de resistência em frutos, e suas populações F4RC1 e F3RC2 foram avaliadas no estádio de plântulas. Constatou-se variabilidade para a reação de antracnose nestas populações, com expressão de menor severidade e possibilidade de obter cultivares de pimentas e pimentões resistentes à antracnose. / Capsicum cultivation is an increasing activity in Brasil, and, in conseqüence, disease occurence is becoming more frequent. Anthracnose is the most common and desctructive disease of Capsicum in Brasil and other countries. Losses of 100% due to the disease are very frequent. Control of anthracnose using genetic resistance can be very suitable. The aim of this work was (i) to describe the reaction of 90 accesses of Capsicum annuum, 30 of C. baccatum, 16 of C. chinense and one of C. frutescens to anthracnose (ii) to establish the correlation of the reaction to anthracnose between seedlings and fruits of Capsicum and (iii) to evaluate the reaction to anthracnose of the progenies F3RC1 (C.annuum x C. chinense - resistant) x C. annuum and F2RC2 [(C. annuum x C. chinense - resistant) x C. annuum] x C. annuum. Four isolates of Colletotrichum sp. from pepper and one isolate from hot pepper were used for the inoculations, at a concentration of 1 x 106 spores/ml. After adjustment of the concentration of each suspension, they were mixed, in order to reduce possible effects of pathogenic variability among the isolates. The experimental design used was of completely randomized blocks with 20 and 15 replications for seedlings and green-ripe fruits inoculations, respectively. For the inoculation in seedlings, sugarcane juice at 5% was added to the spore suspensions, which were sprayed on the leaves of the seedlings at one-leaf stage. The seedlings were kept in a moist chamber for 24 hours before and 72 hours after the inoculation, at 26 +/-2°C and 12 hours photoperiod, inside a growth chamber. To evaluate anthracnose on the inoculated plants, disease incidence and disease severity were considered. The last variable was rated based on a 1 to 5 scale, according to the amount of disease on the seedlings. The fruits were inoculated with a 20 µl droplet of the spore suspensions. After depositing the inoculum, the fruits were wounded with a needle at the inoculation point and kept at 24 +/- 2°C, inside covered plastic containers. The evaluation was done based on lesions diameter, latent period, growth rate of lesions and incidence. The fruits evaluations were used for classifying the accesses reactions. Only one access of C. annuum (n°233 - Jalapeno), one of C. baccatum (n°222 - BGH 4176) and two of C. chinense (n°105 - Bode and n°141 - Pimenta n°2) were considered resistant. The remaining accesses varied in degrees of susceptibility. Positive correlation between levels of disease in seedlings and fruits were not significant. The seedlings showed less amount of disease in relation to fruits. The progenies 3G, 4A, 4B, 4F, 4H, 5H, 6B, 6F, 8B, 8E, 8F, 9B, 9D, 9E, 9J, 10A, 13D, 13F, 14A, 14C, 16D, 16I, 17B, 19B (population F3RC1) and 33F and 40G (F2RC2) did not show lesions at the fruit stage. Generations were advanced in this population and populations F4RC2 and F3RC2 were evaluated at seedling stage. These seedlings showed variability for reaction to anthracnose, with lower severity, suggesting possibility of selection for resistance in Capsicum varieties.

A Solanum lycocarpum St. Hil (Solanaceae) é uma planta nativa do cerrado brasileiro, cujos frutos possuem os glicoalcalóides esteroidais solasonina e solamargina. É possível que a ingestão de frutos que contenham esses glicoalcalóides possam interferir no equilíbrio do sistema endócrino. O presente estudo foi realizado com o objetivo de avaliar os possíveis efeitos tóxicos provenientes da ingestão diária de frutos verdes da S. lycocarpum (10% na ração) por ratas prenhes, a partir do 6° dia da gestação (DG 06) ao 7° dia pós-natal (PN 07). Os princípios ativos isolados dos frutos da planta mostraram a presença de 0,6% de solamargina e 0,9% de solasonina. A exposição das mães à planta alterou alguns parâmetros do desempenho reprodutivo; foram observados redução tanto do peso como do comprimento corporal das ninhadas experimentais ao nascimento, um ou dois filhotes mortos ao nascimento em 20% das ninhadas experimentais e menor capacidade em adquirir peso do nascimento à vida adulta. A prole experimental adulta fêmea apresentou degeneração de células epiteliais uterinas no dia PN 60, não sendo mais observada esta alteração no dia PN 90. A avaliação do comportamento sexual de fêmeas (PN 10O) mostrou redução do coeficiente de lordose das fêmeas experimentais quando comparado com o coeficiente de lordose das fêmeas do grupo controle. Na prole experimental masculina (PN 60 e PN 90) observou-se importante degeneração de células germinativas dos duetos testiculares. Por outro lado, o comportamento sexual desta prole não mostrou-se alterado. Na avaliação de neuroaminas cerebrais, a prole feminina apresentou maior atividade do sistema noradrenérgico estriatal, caracterizada por redução dos níveis de noradrenalina, sem alteração dos níveis de seu metabólito ácido vanilmandélico (VMA), e reduzida atividade do sistema serotoninérgico hipotalâmico, caracterizada por elevação dos níveis de serotonina (5-HT) sem alteração dos níveis de seu metabólito ácido 5-hidroxiindolacético (5 HIAA). Os filhotes machos apresentaram concentrações reduzidas de noradrenalina, VMA e ácido homovanílico (HV A). Ambos, machos e fêmeas, apresentaram níveis estriatais diminuídos de dopamina e seu metabólito ácido dihidroxifenilacético (DOPAC), sem, contudo, alterar a atividade do sistema dopaminérgico. Não foram observadas alterações hormonais tanto nas mães, quanto na prole, avaliadas por meio da mensuração não invasiva de metabólitos dos hormônios progesterona, estradiol e testosterona nas fezes. Estes dados tomados em conjunto indicam que a planta promoveu leve toxicidade materna e apresentou leve efeito fetotóxico. A avaliação das proles do nascimento à vida adulta indicou ainda que os glicoalcalóides esteroidais presentes nos frutos provocaram efeitos diferenciados nas proles feminina e masculina e possivelmente apresentam atividade estrogênica. / Solanum lycocarpum St. Hil (Solanaceae) is a native shrub of the Brazilian savanna. The fruits contain steroidal glycoalkaloids, solasonine and solamargine, that can disrupt the endocrine system once in the body by ingestion of S. lycocarpum fruit. The present study was performed in order to determine the possible toxic effects of S. lycocarpum fruit ingestion (10% added in the diet) by pregnant rats from gestation day (GD) 06 to post-natal day (PND) 07. The unripe fruits employed contain 0.021 % of solamargine and 0.031 % of solasonine. S. lycocarpum 10% in the diet, during pregnancy and beginning of lactation, did not impair gestation but alterations in some parameters of the reproductíve performance were observed: lower body weight and body size of the experimental litters at PND 01, one or two stillbirths were observed in 20% of the experimental litters and reduced weight gain of the pups from birth to adulthood. Female offspring (PN 60) presented reversible uterine epithelial cel! degeneration, and at PND 100, presented impaired sexual behavior caracterized by reduced lordosis quotient. Male offspring (PN 60 and PND 90) showed irreversible degeneration of testis germinative cells and normal sexual behavior. The central neuroamine systems avaliation presented, in the female exposed offspring, increased activity of striatal norepinephrine system, with reduced levels of norepinephrine (NOR) and normal levels of vanilmandelic acid (VMA), and reduced activity of the hypothalamic serotoninergic system, with increased serotonin (5-HT) and normal 5-hydroxindolacetic acid (5-HIAA) levels. Exposed male offspring presented reduced hypothalamic NOR, VMA and homovanilic acid (HV A) levels, and both males and females presented striatal dopamine (DA) and dihydroxylphenylacetic acid (DOPAC) reduced levels, without alterations in the activity of the dopaminergic system. The sexual hormones were also evaluated by a non invasive method with feces extraction. However no alterations were observed in progesterone, estradiol and testosterone metabolite levels in dams and offspring feces. Present data showed light maternal toxicity and light fetotoxic effects. The evaluation of the offspring from birth to adult age indicate that the steroidal glycoalkaloids present in the fruit act by different ways in male and female fetuses, and perhaps presents estrogenic activity.

A importância do cultivo de espécies do gênero Capsicum vem crescendo nos últimos anos no Brasil, e consequentemente, a ocorrência de doenças torna-se um problema relevante. A antracnose é a mais comum e destrutiva doença de Capsicum no Brasil e em vários países, podendo ocasionar perdas de até 100%. Seu controle usando resistência genética seria altamente desejável. O objetivo deste trabalho foi (i) caracterizar a reação à antracnose de 90 acessos de Capsicum annuum, 30 de C. baccatum, 16 de C. chinense e um de C. frutescens; (ii) estabelecer a correlação da reação à antracnose entre plântulas e frutos de Capsicum e; (iii) avaliar a reação à antracnose das progênies F3RC1 (C.annuum x C. chinense - resistente) x C. annuum e F2RC2 [(C. annuum x C. chinense) x C. annuum] x C. annuum. Para as inoculações, foram utilizados quatro isolados do fungo Colletotrichum sp. provenientes de pimentão e um isolado de pimenta, obtidos de folhas e frutos doentes. As suspensões de inóculo foram preparadas na concentração de 1 x 106 conídios/ml, separadamente para cada isolado. Após ajustada a concentração das suspensões, estas foram misturadas, a fim de reduzir efeitos de possível variabilidade patogênica dos isolados. O delineamento utilizado foi inteiramente casualizado com 20 repetições para plântulas e 15 repetições para frutos verdes destacados. Os ensaios foram conduzidos duas vezes. Nas inoculações das plântulas, foi adicionado 5% de caldo de cana à suspensão, como fonte exógena de energia ao patógeno. A inoculação nas plântulas foi realizada no estádio de primeira folha verdadeira completamente expandida, pela aspersão da suspensão de esporos até o ponto de escorrimento. As plântulas foram mantidas em câmara úmida por 24 horas antes e 72 horas após a inoculação. O ensaio foi instalado em sala climatizada com temperatura de 26 +/- 2°C e fotoperíodo de 12 horas. Foi avaliada a severidade de doença, usando uma escala de notas e também a incidência da doença, com base na percentagem de plântulas doentes. A inoculação nos frutos foi realizada pela deposição de uma gota de 20 µl da suspensão de esporos na parte mediana dos frutos. Posteriormente, com o auxílio de uma agulha entomológica, foi realizado um ferimento abaixo da gota. Os frutos inoculados foram mantidos em câmara úmida durante o período de avaliação. As avaliações foram realizadas pela mensuração do diâmetro médio das lesões, período latente, velocidade de crescimento da lesão e incidência. Baseando-se na avaliação dos frutos para classificação da reação, identificou-se apenas um acesso de C. annuum como resistente (n°233 – Jalapeno), um de C. baccatum (n°222 – BGH 4176) e dois de C. chinense (n°105 – Bode e n°141 – Pimenta n°2). Os demais acessos foram suscetíveis. Não houve correlação entre a reação de plântulas e frutos. As plântulas apresentaram menor quantidade de doença, em relação aos frutos. As progênies F3RC1 (3G, 4A, 4B, 4F, 4H, 5H, 6B, 6F, 8B, 8E, 8F, 9B, 9D, 9E, 9J, 10A, 13D, 13F, 14A, 14C, 16D, 16I, 17B, 19B) e as progênies F2RC2 (33F e 40G) não apresentaram lesões no estádio de frutos. Avançou-se geração nas progênies com reação de resistência em frutos, e suas populações F4RC1 e F3RC2 foram avaliadas no estádio de plântulas. Constatou-se variabilidade para a reação de antracnose nestas populações, com expressão de menor severidade e possibilidade de obter cultivares de pimentas e pimentões resistentes à antracnose. / Capsicum cultivation is an increasing activity in Brasil, and, in conseqüence, disease occurence is becoming more frequent. Anthracnose is the most common and desctructive disease of Capsicum in Brasil and other countries. Losses of 100% due to the disease are very frequent. Control of anthracnose using genetic resistance can be very suitable. The aim of this work was (i) to describe the reaction of 90 accesses of Capsicum annuum, 30 of C. baccatum, 16 of C. chinense and one of C. frutescens to anthracnose (ii) to establish the correlation of the reaction to anthracnose between seedlings and fruits of Capsicum and (iii) to evaluate the reaction to anthracnose of the progenies F3RC1 (C.annuum x C. chinense - resistant) x C. annuum and F2RC2 [(C. annuum x C. chinense - resistant) x C. annuum] x C. annuum. Four isolates of Colletotrichum sp. from pepper and one isolate from hot pepper were used for the inoculations, at a concentration of 1 x 106 spores/ml. After adjustment of the concentration of each suspension, they were mixed, in order to reduce possible effects of pathogenic variability among the isolates. The experimental design used was of completely randomized blocks with 20 and 15 replications for seedlings and green-ripe fruits inoculations, respectively. For the inoculation in seedlings, sugarcane juice at 5% was added to the spore suspensions, which were sprayed on the leaves of the seedlings at one-leaf stage. The seedlings were kept in a moist chamber for 24 hours before and 72 hours after the inoculation, at 26 +/-2°C and 12 hours photoperiod, inside a growth chamber. To evaluate anthracnose on the inoculated plants, disease incidence and disease severity were considered. The last variable was rated based on a 1 to 5 scale, according to the amount of disease on the seedlings. The fruits were inoculated with a 20 µl droplet of the spore suspensions. After depositing the inoculum, the fruits were wounded with a needle at the inoculation point and kept at 24 +/- 2°C, inside covered plastic containers. The evaluation was done based on lesions diameter, latent period, growth rate of lesions and incidence. The fruits evaluations were used for classifying the accesses reactions. Only one access of C. annuum (n°233 – Jalapeno), one of C. baccatum (n°222 – BGH 4176) and two of C. chinense (n°105 – Bode and n°141 – Pimenta n°2) were considered resistant. The remaining accesses varied in degrees of susceptibility. Positive correlation between levels of disease in seedlings and fruits were not significant. The seedlings showed less amount of disease in relation to fruits. The progenies 3G, 4A, 4B, 4F, 4H, 5H, 6B, 6F, 8B, 8E, 8F, 9B, 9D, 9E, 9J, 10A, 13D, 13F, 14A, 14C, 16D, 16I, 17B, 19B (population F3RC1) and 33F and 40G (F2RC2) did not show lesions at the fruit stage. Generations were advanced in this population and populations F4RC2 and F3RC2 were evaluated at seedling stage. These seedlings showed variability for reaction to anthracnose, with lower severity, suggesting possibility of selection for resistance in Capsicum varieties.

Buzz-pollination or pollination by vibration occurs in several families of angiosperms including some important commercial crops such as potatoes and tomatoes. Buzz-pollinated flowers release pollen via small pores or slits on the anther’s tip that require the use of vibrations by specialized pollinators, usually bees, to remove the pollen. Some buzz-pollinated species have elaborate floral morphologies including dimorphic anthers within the same flower (heteranthery), and mirror-image flowers (enantiostyly) where the style is reciprocally deflected to either the left or right side of the floral axis. The complex floral morphology and buzz-pollination syndrome seen in these species require a close physical interaction between the sexual organs of the flowers and the bodies of insect visitors. Despite the broad taxonomic distribution of buzz-pollination in angiosperms (more than 60 families are buzz-pollinated) relatively few studies have described the pollination ecology of these species under natural conditions. The main goal of the present work was to characterize the pollination biology, mating patterns and antagonistic interactions (e.g. pollen theft) in natural populations of a buzz-pollinated species. For this purpose, I studied Solanum rostratum (Solanaceae), a buzz-pollinated, self-compatible, annual weed with complex floral morphology (both enantiostylous and heterantherous flowers). This species usually grows in disturbed areas in its native range (Mexico) and has become invasive around the world. My research was divided into three components. First, I characterized the pollination and reproductive biology of natural populations in Mexico. I performed floral manipulations in six natural populations of S. rostratum to estimate fruit and seed set. In three of these populations, I carried out 115 hours of pollinator observations and quantified the incidence of pollinators versus pollen thieves. I also measured the efficiency of a subset of floral visitors in triggering fruit set after single visits. Second, I investigated whether morphological correspondence between the size of the pollinator’s body and floral morphology influences pollen transfer. In experimental arrays, I exposed flowers of S. rostratum that varied in the distance between their sexual organs, to bumblebees (Bombus terrestris) of different sizes, and recorded pollen deposition and fruit and seed production. Finally, I determined the mating system (i.e. the proportion of self- and cross-fertilized offspring) of natural populations in Mexico and of introduced populations in the United States of America, using newly developed microsatellite markers. My results show that S. rostratum is visited by a wide range of bees of different sizes (0.9–9.8 mm in thorax diameter), but that only a small subset of these visitors act as pollinators. Most visitors act as pollen thieves, consuming pollen while effecting little or no pollination. I also found that correspondence between a pollinator’s size and the separation of the S. rostratum sexual organs determines pollen deposition and fruit production; pollen deposition decreased when bees were small relative to the distance between the sexual organs visited the flowers. My genetic analyses show that natural populations of S. rostratum maintain a relatively high outcrossing rate (tm = 0.75 ± 0.03) across the native and introduced range. Furthermore, genetic diversity is reduced in invasive populations, but this is not accompanied by changes in mating system. My work shows that the morphological fit between the pollinator and the flowers is important in determining the dynamics of pollen transfer and fruit production in this buzz-pollinated plant. Distinguishing between pollinators and pollen thieves in buzz-pollinated plants is essential for understanding the evolution of buzz-pollination, as pollen theft could be a major selective force for these species.

Ralstonia solanacearum est une bactérie vasculaire d'origine tellurique dévastatrice chez les Solanacées maraîchères. L'une des difficultés pour la sélection variétale réside dans le contournement de la résistance, en raison de la grande variabilité et plasticité génotypique du pathogène. Trente accessions de tomate, aubergine et piment (Core-TEP) testées vis-à-vis de 12 souches représentatives des phylotypes I, II et III de R. solanacearum (Core-Rs2) ont été classées de très sensibles à très résistantes. Des groupes de souches définis en fonction de leur virulence et agressivité ont été nommés pathoprofils, d'une part, lorsque établis sur les 3 espèces et pathotypes, d'autre part, lorsque établis sur chacune des trois espèces. Aucune résistance universelle n'a pu être mise en évidence chez aucune des trois espèces. Le déterminisme génétique de la résistance à R. solanacearum chez l'aubergine a été étudié sur une population de lignées recombinantes F6 ainsi que sur les générations issues du croisement intraspécifique S. melongena MM738 (S) x AG91-25 (R), testées vis-à-vis de quatre souches du phylotype I. Une carte génétique composée de 119 marqueurs AFLP, SSR et SRAP, positionnés sur 18 groupes de liaisons, pour une longueur totale de 884 cM, a été construite. Les analyses génétiques montrent que la résistance aux souches CMR134, PSS366, GMI1000 est contrôlée par un même gène majeur expliquant jusqu’à 87% de la variance phénotypique. Celui-ci n'est pas détecté vis-à-vis de la souche virulente PSS4 qui contourne cette résistance. Dans ce cas, seul un QTL à effet intermédiaire expliquant jusqu’à 38 % de la variance phénotypique est trouvé sur un autre groupe de liaison. / The plant pathogenic soil-borne bacteria Ralstonia solanacearum causes heavy damage to solanaceous crops. One of the dilemmas for varietal breeding of solanaceous crops is the circumvention of the resistance to R. solanacearum due to the genotypic variability and plasticity of this pathogen. Thirty accessions of Tomato, Eggplant, and Pepper (Core-TEP), screened with a collection of 12 representatives strains of R. solanacearum phylotype I, II and III (Core-Rs2) within the species complex ranked from highly resistant to highly susceptible. The strains were grouped according to there profile of virulence on all three solanaceous, and by considering each species individually. We refer to these groups respectively, as pathoprofiles and pathotypes. No universal resistance was found for the three host species. Genetic analysis of resistance to R. solanacearum in eggplant was conducted against four strains of phylotype I. We used a population of recombinant lines F6 as well as the generations derived from the intraspecific cross between S. melongena MM738 (S) and AG91-25 (R). A genetic map was built with 119 markers a combination of AFLPs, SSRs and SRAPs positioned on 18 linkage groups, for a total length of 884 cM. The genetic analysis revealed that resistance to strains CMR134, PSS366, and GMI1000 was controlled by one single major gene, which explained up to 87% of the phenotypic variation. When the resistance against the highly virulent PSS4 strain is tested, this gene is not detected. In this case, a significant QTL with intermediate effect that explains up to 38% of phenotypic variation was found on another linkage group for this strain.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-27T13:57:34Z No. of bitstreams: 1 texto completo.pdf: 2630530 bytes, checksum: 112de5671b9d3e521f0446f6d6d7237e (MD5) / Made available in DSpace on 2017-04-27T13:57:34Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2630530 bytes, checksum: 112de5671b9d3e521f0446f6d6d7237e (MD5) Previous issue date: 2004-03-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Alguns aspectos da interação entre o gene de resistência Sw-5 e os tospovírus, foram analisados neste trabalho. A capacidade de Sw-5 conferir resistência em Nicotiana benthamiana foi avaliada em plantas transformadas com uma construção na qual a ORF e a região 3' do gene estavam sob controle do promotor 35S. As plantas transformadas foram resistentes à infecção por tospovírus. A comparação do espectro da resistência destas plantas com o observado para outras solanáceas , indica que as vias de sinalização e as respostas de defesa ativadas por Sw-5 estão conservadas nesta família, e que o polimorfismo genético nos componentes das vias de transdução de sinais pode resultar em diferentes níveis de resistência. A fim de identificar o gene de avirulência dos tospovírus, os genes N, NSm e NSs , foram expressos isoladamente ou em combinações, por meio do vetor viral PVX, em plantas com Sw-5. A expressão destes genes não foi capaz de desencadear a resposta de hipersensibilidade e tampouco interferiu na infecção da planta por PVX. Portanto, outro componente dos tospovírus deve ser responsável pelo desencadeamento da reação de resistência. Independentemente da presença do gene Sw-5, a expressão do gene NSs por meio do PVX , agravou os sintomas provocados por este vírus em algumas solanáceas, o que pode ter relação com a capacidade desta proteína de suprimir silenciamento gênico. Em plantas com Sw-5, a co-expressão da região 5' deste gene por meio do vetor PVX , favoreceu a infecção sistêmica por tospovírus. Uma vez que o efeito foi observado tanto para expressão senso quanto anti-senso, a redução dos níveis de mRNA de Sw-5 provocada por silenciamento gênico , poderia ser a causa desta interferência na resistência, embora a análise de Northern blot não tenha demonstrado tal redução. Uma seqüência homóloga a Sw-5 contendo uma ORF sem deleções ou interrupções prematuras foi clonada a partir do acesso LA371-20 de Lycopersicon peruvianum. Análises moleculares demonstraram que esta seqüência é originada do loco Sw-5 ou de região próxima a este e que segrega com a resistência a tospovírus. A capacidade deste e de outros três homólogos oriundos de distintos acessos de tomateiro de conferir resistência a tospovírus, foi avaliada em plantas transgênicas de tomateiro e tabaco. As plantas transformadas foram suscetíveis ao vírus. A não-funcionalidade destes homólogos pode ser devido à estrutura das construções utilizadas na transformação. Alternativamente, outros homólogos presentes nestes acessos de tomateiro podem ser os responsáveis pela resistência. / In this work some aspects of the tospovirus - Sw-5 interaction was analyzed. The capacity of the Sw-5 to confer resistance in Nicotiana benthamiana was evaluated in transgenic plants transformed with Sw-5 ORF containing its own 3 ́ UTR region under 35S promoter control. Transgenic plants were resistant to tospovirus infection. Comparisons of the resistance spectrum with other members of the Solanaceae suggest that the signal transduction pathways and resistance responses triggered by Sw-5 are conservated in solanaceae and that the genetic polymorphism in the signal transduction components may result in different resistance levels. The N, NSm and NSs genes isolated or in combination were expressed by PVX vector in plants harboring Sw-5 in order to detect the tospovirus avirulence gene. These genes were not able to trigger the hypersensitive response and to affect PVX infection in Sw-5 plants, which suggest that another tospovirus component is the elicitor of the resistance response. Independently of the Sw-5 gene, PVX clones harboring NSs gene induced more severe symptoms in some solanaceae plants. Gene silencing may be the cause of this symptoms. In transgenic plants harboring Sw-5 gene, the co-expression of the 5 ́ region of this gene by PVX favored tospovirus infection. As this effect was observed both for 5 ́ sense and anti- sense constructions it is possible that it has been caused by reduction on mRNA levels by gene silencing, although Northern blot analysis did not agree with this hypothesis. An Sw-5 homolog was cloned from LA371-20 accession. This homolog is localized in or near of Sw-5 locus and segregated with tospovirus resistance. The capacity of this and other three homologs originated from others accessions to confer tospovirus resistance was evaluated in tobacco and tomato transgenic plants. All the transformants were susceptible to the virus. Other homologs presents in the different accessions evaluated may be responsible for the resistance, although problems in the structure of the constructions can not be discarded. / Tese importada do Alexandria

Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-07-06T18:48:11Z No. of bitstreams: 1 Dissertacao Ana Leticia Stedelle Schneider.pdf: 1754890 bytes, checksum: 06963edc7521fb74eabf56c7d74e8823 (MD5) / Made available in DSpace on 2015-07-06T18:48:11Z (GMT). No. of bitstreams: 1 Dissertacao Ana Leticia Stedelle Schneider.pdf: 1754890 bytes, checksum: 06963edc7521fb74eabf56c7d74e8823 (MD5)