As part of a multinational project and with the help of other professionals, I gathered and analyzed 110 samples of dental calculus (fossilized plaque) from human remains discovered at Paquimé and the Convento site in the Casas Grandes River valley to identify various microfossils still present in the silica matrix. Once identified, I used the results to reconstruct human/plant relationships present during the Viejo (700-1250 CE) and Medio (1250-1450 CE) periods in and around Paquimé. My data suggest that maize was used throughout both time periods, supplemented by wild plants, and possible marine resources. Further, evidence for cultural food modification methods such as fermentation, roasting, grinding, and nixtamalization (an alkaline treatment of the grain) was present. The data suggest prehistoric plant use went beyond simple subsistence, being modified for use as part of non-subsistence activities.

<p>Fungi display diverse growth forms. Some grow as unicellular yeasts, some grow as multicellular hyphae, while others switch between these two growth forms, i.e., the dimorphic fungi. Dimorphism is found in many pathogenic fungi, and it is thought to be a strategy to maximize their fitness during different stages of life cycles. The corn smut fungus <i>Ustilago maydis</i> serves as a renowned model organism for studying fungal dimorphism and its role in pathogenesis. However, knowledge only from the model species may not be expanded to other species unless multispecies studies have been demonstrated. In this dissertation, I performed comparative analyses to examine if knowledge from <i>U. maydis</i> is translational to other dimorphic fungi. First, a physiological study was conducted to find what can serve as a common signal for dimorphic transition of several Ustilaginomycotina species. I found that the lipid serves as a potential common cue for yeast-to-hyphal transition in most dimorphic species, while alternate types of energy-source carbohydrate do not affect fungal dimorphism. In addition, pectin and high temperature can also trigger filamentous growth in some Ustilaginomycotina species. Second, I performed comparative transcriptomics to determine if a mechanism for yeast-to-hyphal dimorphic transition is conserved across multiple dimorphic species. Three species of Ustilaginomycotina (<i>U. maydis</i>, <i>Tilletiopsis washingtonensis </i>and <i>Meira miltonrushii</i>) plus one species from Ascomycota (<i>Ophiostoma novo-ulmi</i>) were included in the analyses. I found that the similarity of transcriptomic alteration is not dependent on phylogenetic relatedness. Genes in amino acid transport and metabolism, energy production and conversion and cytoskeleton are commonly altered during the dimorphic transition of all studied species. Moreover, I discovered several core genes which can play a conserved role in transducing signals for the dimorphic transition. Finally, I performed comparative analyses of 190 fungal genomes to determine genomic properties that are associated with types of fungal growth form. I found that small genome size is a characteristic for yeast-like fungi. Few indicator genes, such as genes encoding proteins in the NADPH oxidase complex and cytoskeletons, which are predominantly lost in yeast-like fungi in both Ascomycota and Basidiomycota. However, many other genes are associated with types of growth form in a lineage-specific manner. Findings from this dissertation will serve as fundamentals for future research in fungal cell biology, especially in fungal dimorphism. Additionally, results from this study suggest cautions when extrapolating results from model species onto non-model species.</p>

During recent years the principles of Mendelism have been used extensively in the production of the new types of plants possessing resistance to various diseases. Results of this mode of attacking the disease problem have been very favorable. Old varieties are gradually giving way to newer types equal to or exceeding in quality and productivity as well as possessing resistance to one or more diseases. Loose smut (U. tritici) in wheat, while not as serious a problem in Utah as the covered smut (T. tritici), according to Tapke (14) has caused an average annual loss of between 50,000 and 100,000 bushels of wheat. these data shown in figure 1 are for the period 1917 to 1926, inclusive. The various methods advocated for the control of loose smut (U. tritici), with the exception of the use of resistant varieties and hot water treatments, either have been impradtical of application or have been ineffective in control or both (14). The modified hot water treatment of Freeman and Johnson (4), has generally been the method recommended. While this method is effective if properly executed, it is rather complicated and tedious to apply, especially for farmers who are not usually properly equipped. Because of this and the fact that the disease frequently escapes observation, seed treatment for the control of loose smut (U. tritici) is seldom practiced; as a result the smut is allowed to go unchecked. The development of a resistant variety possessing the adaptability and desirable characteristics of our locally-grown spring wheats would be a decided advantage to the farmers in combating the disease.

The fungal disease smut (causal agent: Ustilago scitaminea H. & P. Sydow) and stalk borer Eldana saccharina Walker place major constraints on sugarcane agriculture in South Africa. The best approach for management is the introduction of resistant cultivars; however, conventional field-based screening for pest and disease resistance is a lengthy process. This study evaluated in vitro techniques combined with artificial inoculation of 12 week old in vitro plantlets and 8-10 week old embryogenic calli as rapid screening methods. Preliminary investigations were conducted on cultivars with known field ratings to smut and E. saccharina: NCo376, N26 and N39; and 5 „test‟ cultivars, whose identities were undisclosed until completion of experiments, were used to assess the accuracy of protocols. Infective U. scitaminea sporidia generated from teliospores, were used as inocula. Development of a callus protocol was unsuccessful due to sporidial and mycelial overgrowth, despite addition of a contact fungicide, Dithane M-45® (0.025 g/l) and a biocide/fungicide, PPMTM (5 ml/l), to media. Plantlet inoculation by injection, 1 cm above the apical meristem, resulted in 12% and 20% of smut susceptible NCo376 plantlets producing smut whips after 5 weeks when inoculated with 1 x 106 and 1 x 109 sporidia/ml, respectively. Smut whip production in 5 of the 8 (63%) cultivars inoculated with the lower sporidial concentration correlated with their field resistance ratings. In addition, whips harvested from in vitro plantlets were a valuable source of aseptic teliospores for future research. Ongoing work involves inoculation of NCo376 calli with such teliospores and maintenance on medium with PPMTM - emergence of whips from plantlets remains to be assessed. The E. saccharina screening protocol involved surface decontamination of eggs with 1% sodium hypochlorite (NaOCl) for 15 min. Feeding bioassays were conducted by placement of first instar larvae on in vitro plantlets and calli for 3 and 2 weeks, respectively. Larval mass, length and percentage infestation were recorded. Although greater larval size was expected in susceptible compared with resistant cultivars, the results did not support this. Significant differences in plantlet infestation were observed between susceptible (94-98%) and resistant (72-86%) lines. No significant differences were found in the callus feeding bioassay. However, a 24 h callus choice bioassay which investigated larval preference between callus genotypes compared with NCo376, showed significant differences and correctly discerned cultivar susceptibility according to field ratings. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2010.

A ferrugem e o carvão estão entre as principais doenças da cana-de-açúcar, visto que são consideradas em todos os Programas de Melhoramento Genético (PMG), tanto na seleção de genitores resistentes quanto no comportamento das progênies. Quando a cultura está distribuída em grandes extensões tornam-se importante conhecer a amplitude da variação patogênica na população dos agentes causais. Diante a importância de obter variedades com resistência efetiva e duradoura, o objetivo do presente estudo foi verificar a ocorrência e caracterizar, no Estado de São Paulo, a variabilidade patogênica de Puccinia melanocephala e Ustilago scitaminea. Coletaram-se 12 populações de cada patógeno em 3 regiões canavieiras, Piracicaba, Jaú e Ribeirão Preto. Para os experimentos de ferrugem usaram-se 3 variedades de comportamento conhecido frente à doença, SP70-1143, SP91-1397 e R570, suscetível, intermediária e resistente, respectivamente. Plantas com 20 dias de desenvolvimento foram inoculadas com suspensões de 104 esporos viáveis/mL de cada população, e colocadas em câmara de crescimento a 20 ºC e 12 h de fotoperiodo. Após 14 dias foram avaliados os sintomas na folha +1, quantificando severidade medida em porcentagem de área foliar afetada e incidência, medida em número de pústulas/cm2. Para carvão utilizaram-se 3 variedades de reação conhecida, SP84-2066, suscetível, Co421 intermediária e SP71-8210 resistente. Inocularam-se gemas de cada variedade com suspensões de 1,7 x 106 esporos viáveis/mL das populações. Após do desenvolvimento em casa de vegetação, as plantas foram transplantadas no campo instalando-se um ensaio em delineamento de blocos casualizados com 4 repetições. As avaliações foram realizadas a cada 14 dias durante o ciclo de cana-planta, determinando-se a incidência da doença segundo as porcentagens acumuladas de touceiras doentes por parcelas, obtendo-se também a área abaixo da curva de progresso da doença. Para ferrugem verificou-se que houve diferenças significativas de agressividade entre as populações estudadas. Duas populações de Jaú, J3 e J4, destacaram-se pela maior agressividade em todas as variedades. Além disso, as populações de Jaú mostraram também virulência diferenciada em relação às demais populações. As populações de U. scitaminea apresentaram diferentes níveis de agressividade, porém não houve alterações de virulência entre as populações estudadas. Segundo os parâmetros observados, uma população de Piracicaba e duas de Jaú comportaram-se como as mais agressivas. Dessa forma, ficou comprovada que, tanto para P. melanocephala como para U. scitaminea, existe uma considerável variabilidade patogênica entre as populações destes agentes causais no Estado de São Paulo, Brasil. Os PMG que submetem os materiais a inoculações artificiais com esporos de ferrugem e carvão em alguma etapa do programa necessitam considerar a variação na agressividade dos patógenos determinada neste estudo. / Rust and smut are among the most important diseases of sugarcane, being considered in every breeding program both in parent selection as well as in evaluating progeny behavior. When the crop is distributed to large extensions, it becomes very important to know the amplitude of the pathogenic variation in the population of the causal agent. Considering the importance of obtaining varieties with effective and long lasting resistance, the objective of the present study was to verify the occurrence, in the state of São Paulo, of pathogenic variability in Puccinia melanocephala and Ustilago scitaminea. Twelve populations of each pathogen were collected from three sugarcane producing regions: Piracicaba, Jaú and Ribeirão Preto. For the rust experiments three varieties of known behavior to the disease were used, SP70-1143, SP91-1397 and R570, susceptible, intermediate, and resistant, respectively. Twenty day-old plants were inoculated with 104 viable spores/mL suspensions of each population, and incubated at 20 ºC with 12 h photoperiod. Fourteen days later, symptoms were evaluated in the leaf +1, quantifying severity as the percentage of the leaf area affected and incidence as the number of pustules/cm2. For smut, three varieties with know response to the disease were used, SP84-2066, susceptible, Co421, intermediate and SP71-8210, resistant. Buds of each variety were inoculated with 1,7 x 106 viable spores/mL of each population. After growth in the greenhouse the plants were transplanted to the field in a random block trial with 4 repetitions. Disease was monitored every fourteen days during the plant cane cycle, determining the incidence according to the accumulated percentage of diseased stools per plot, also obtaining the area below the disease progression curve. For rust, it was observed that the populations showed different levels of aggressiveness. Two populations from Jaú, named J3 and J4, behaved as the most aggressive in the three varieties. Moreover, populations from Jaú showed different virulence among studied populations. The populations of U. scitaminea showed distinct aggressiveness levels, however it was not observed changes in the virulence among tested populations. According to the evaluated parameters, one population from Piracicaba and two from Jaú behaved as the most aggressive ones. Thus, it was showed that a considerable pathogenic variability exists among Brazilian populations of both pathogens. The sugarcane breeding programs that perform artificial inoculations with rust and smut spores at some stage in the program must take into consideration the variability in the aggressiveness of the pathogens determined in this study.

O carvão é uma das doenças mais importantes da cana-de-açúcar. Grande parte dos Programas de Melhoramento Genético dessa cultura utiliza inoculações artificiais para selecionar e caracterizar as variedades quanto a sua resistência a doenças. Assim o conhecimento das condições favoráveis para infecção torna-se indispensável. Neste trabalho foram estudadas as condições que afetam a infecção de gemas pelo fungo Sporisorium scitamineum. Do ponto de vista do hospedeiro foi avaliada a incidência de chicotes para diferentes idades das gemas e das brotações das variedades NA56-79, SP71-1406 e SP84-2066. As gemas mais novas mostraram-se mais suscetíveis que as medianas e velhas, porém na variedade mais suscetível, a SP84-2066, não houve diferenças na taxa de infecção das gemas mais novas e velhas. Gemas sem prébrotação e com pré-brotação de 1 dia foram infectadas pelo fungo enquanto que gemas pré-brotadas com 4, 6, 8 e 10 dias não foram infectadas. Para o patógeno, foi avaliada a concentração de teliósporos para a inoculação. As concentrações de 1x106 e 1x 108 foram as únicas que produziram sintomas e sinais, não diferindo a incidência da doença entre elas. Como condições de pós inoculação foram testadas as variáveis temperatura de 25, 28 e 32 °C, umidade de 65, 80 e 95% e períod o de incubação de 24, 48 e 96 horas. Foram realizados dois experimentos em tempos diferentes, sendo que no primeiro foram utilizadas as variedades NA56-79, SP71-1406 e SP84-2066 e no segundo, além das citadas, foram acrescentadas as variedades SP79-2312 e IAC66-6. O tratamento de 28°C, 65% de umidade por um período de 24 horas foi o que promoveu maior índice de infecção, para todas as variedades, exceto a SP84-2066 que foi favorecida por umidade de 95%. Além desses parâmetros foi avaliado o método de inoculação por aspersão de teliósporos e do ferimento seguido por aspersão de teliósporos. A inoculação por ferimento proporcionou maior índice de infecção para as variedades NA56-79, RB72-454, IAC66-6, SP70-1143, mas não teve interferência nas variedades SP79-1011, SP80-185 e SP86-155. Um segundo objetivo do trabalho foi validar um método de diagnose precoce para o carvão. Para isto foram desenvolvidos iniciadores específicos para a detecção de S. scitamineum. Esses iniciadores foram avaliados quanto a sua especificidade e sensibilidade e demonstraram bons resultados. Para a validação prática, plantas de 12 variedades inoculadas tiveram suas folhas amostradas aos 20, 30, 45, 60, 75, 90, 105, 120, 135 e 150 dias após o plantio. Todas as plantas que apresentaram o chicote puderam ser diagnosticadas precocemente com o método criado. As datas de diagnóstico precoce foram de no mínimo 30 dias e no máximo 120 dias antes do aparecimento do sintoma/sinal. Este é um método que pode ser incorporado para os testes de quarentena reduzindo a permanência de plantas na casa de vegetação e reduzindo os custos de manutenção dessas plantas. / Smut is one of the most important diseases of sugarcane. Most part of genetic breeding programs from this culture use artificial inoculation in order to select and characterize the varieties regarding their resistance to diseases. Therefore knowledge of favorable conditions for infection becomes indispensable. This work studied conditions that affect bud infection by the fungus Sporisorium scitamineum. From the standpoint of the host it was assessed the whip incidence for different bud ages and shoot of the varieties NA56-79, SP71-1406 and SP84-2066. The younger buds were more susceptible than median and older ones, but in the susceptible variety, SP84-2066, there were no differences in the younger and older bud infection rates. Buds with and without shoots of 1 day were infected with the fungus while pre-sprouting buds with 4, 6, 8 and 10 days were not infected. For the pathogen it was evaluated teliospore concentration for inoculation. The concentrations 1x106 and 1x 108 were the only ones that produced symptoms and signs, not differing disease incidence among them. As pos-inoculation conditions the variables tested were temperature of 25, 28 and 32 °C, humidity of 65, 80 and 95% and incubation period of 24, 48 and 96 hours. Two experiments were performed at different times, the first one were used NA56-79, SP71- 1406 and SP84-2066 varieties and the second one, besides those mentioned, it was added SP79-2312 and IAC66-6 varieties. The treatment of 28 °C, 65% humidity and for a period of 24 hours was the one that promoted the highest infection rate, for all varieties, except for SP84-2066 that was favored by 95% of humidity. Besides these parameters, it was evaluated the inoculation method by teliospore spraying and wound followed by teliospore spraying. Inoculation by wounding provided higher infection rate for the NA56-79, RB72-454, IAC66-6, SP70-1143 varieties, however it had no interference in SP79-1011, SP80-185 and SP86-155 varieties. A second objective was to validate a method of early diagnosis of smut. Thus, it was developed specific primers to detect S. scitamineum. These primers were evaluated for their specificity and sensibility and showed good results. To practice validation, plants of 12 varieties inoculated had their leaves sampled at 20, 30, 45, 60, 75, 90, 105, 120, 135 and 150 days after planting. All the plants that presented whip could be early diagnosed by the created method. The dates of premature diagnosis were of at least 30 days and a maximum of 120 days before symptom/sign onset. This method can be incorporated for quarantine tests, reducing permanence of plant in greenhouse and reducing maintenance costs of these plants and varieties selection.

Wheat is the most important cereal crop in the world. It is the principal staple food in Russia, United States, China, Canada, India and a number of other countries. Covered smut is one of the most serious disease of wheat all over the world. According to Woolman and Humphrey (82) it was known from very early times and is referred to by Theoprastus and other early Greek Roman writers.

O fungo Sporisorium scitamineum é o agente causador de uma das doenças mais importantes da cultura da cana-de-açúcar, o carvão da cana, e vem sendo alvo de diversos estudos genéticos para a elucidação de seu mecanismo de patogenicidade e interação com o seu hospedeiro. Dentre esses estudos há os que visam acessar a variabilidade genética entre isolados desse fungo utilizando diversos marcadores moleculares, porém todos os experimentos indicaram baixa variabilidade, com exceção de isolados provenientes da Ásia. O primeiro estudo realizado com esse intuito em isolados brasileiros utilizou o tel-RFLP e AFLP, obtendo uma variabilidade de 34 e 3%, respectivamente. O AFLP é muito utilizado para a detecção de variação genética em indivíduos próximos, porém, nesse caso, apresentou baixa variação, o que nos levou a caracterizar, no presente trabalho, os poucos polimorfismos encontrados através dessa técnica. Os fragmentos polimórficos foram reamplificados, sequenciados e caracterizados quanto ao seu contexto genômico. Como essas sequências estão dispersas ao longo do genoma e geralmente estão em regiões intergênicas, foi feita a análise de expressão dos genes vizinhos a esses polimorfismos em três condições distintas. Desses genes, 70% apresentaram maior número de reads mapeadas in planta que em meio de cultura, indicando que são mais importantes durante a interação planta-patógeno. Dessa forma, mesmo havendo uma baixa variabilidade detectada por AFLP, essas diferenças podem ser importantes na regulação desses genes, podendo influenciar na agressividade do patógeno e na sua defesa. / The fungi Sporisorium scitamineum is the causal agent of one of the most important diseases in sugarcane, the sugarcane smut, and it has been the target of several genetic studies aiming the elucidation of pathogenicity and host interaction macanisms. Among these studies are those that aim to access the genetic variability among isolates of this fungus using various molecular markers techniques, however in all of them the experiments indicated low variability, with exception of the Asian isolates. The first work performed with Brazilian isolates was done using tel-RFLP and AFLP, and detected a variability of 34 and 3%, respectively. The AFLP is widely used to detect variability in close related individuals, but in this case, the method revealed low polimorphism. These results led us to characterize in the present work the few polymorphisms detected. The polymorphic fragments were isoladed, reamplified, sequenced and in silico characterized regarding the genomic context. Since these sequences are disperse along the genome and generally they are in intergenic regions, an expression analysis of the polymorfisms neighbor genes was made at three different conditions. Of these genes, 70% presented higher numbers of maped reads in planta than in culture media, which indicates that they are important during the plant-pathogen interaction. Thus, even with low variability detected via AFLP, these differences can be important in the gene regulation and may influence in the pathogen agressivity and in its defense.

O fungo Sporisorium scitamineum é o agente causador de uma das doenças mais importantes da cultura da cana-de-açúcar, o carvão da cana, e vem sendo alvo de diversos estudos genéticos para a elucidação de seu mecanismo de patogenicidade e interação com o seu hospedeiro. Dentre esses estudos há os que visam acessar a variabilidade genética entre isolados desse fungo utilizando diversos marcadores moleculares, porém todos os experimentos indicaram baixa variabilidade, com exceção de isolados provenientes da Ásia. O primeiro estudo realizado com esse intuito em isolados brasileiros utilizou o tel-RFLP e AFLP, obtendo uma variabilidade de 34 e 3%, respectivamente. O AFLP é muito utilizado para a detecção de variação genética em indivíduos próximos, porém, nesse caso, apresentou baixa variação, o que nos levou a caracterizar, no presente trabalho, os poucos polimorfismos encontrados através dessa técnica. Os fragmentos polimórficos foram reamplificados, sequenciados e caracterizados quanto ao seu contexto genômico. Como essas sequências estão dispersas ao longo do genoma e geralmente estão em regiões intergênicas, foi feita a análise de expressão dos genes vizinhos a esses polimorfismos em três condições distintas. Desses genes, 70% apresentaram maior número de reads mapeadas in planta que em meio de cultura, indicando que são mais importantes durante a interação planta-patógeno. Dessa forma, mesmo havendo uma baixa variabilidade detectada por AFLP, essas diferenças podem ser importantes na regulação desses genes, podendo influenciar na agressividade do patógeno e na sua defesa. / The fungi Sporisorium scitamineum is the causal agent of one of the most important diseases in sugarcane, the sugarcane smut, and it has been the target of several genetic studies aiming the elucidation of pathogenicity and host interaction macanisms. Among these studies are those that aim to access the genetic variability among isolates of this fungus using various molecular markers techniques, however in all of them the experiments indicated low variability, with exception of the Asian isolates. The first work performed with Brazilian isolates was done using tel-RFLP and AFLP, and detected a variability of 34 and 3%, respectively. The AFLP is widely used to detect variability in close related individuals, but in this case, the method revealed low polimorphism. These results led us to characterize in the present work the few polymorphisms detected. The polymorphic fragments were isoladed, reamplified, sequenced and in silico characterized regarding the genomic context. Since these sequences are disperse along the genome and generally they are in intergenic regions, an expression analysis of the polymorfisms neighbor genes was made at three different conditions. Of these genes, 70% presented higher numbers of maped reads in planta than in culture media, which indicates that they are important during the plant-pathogen interaction. Thus, even with low variability detected via AFLP, these differences can be important in the gene regulation and may influence in the pathogen agressivity and in its defense.

Loose smut of barley, caused by fungal pathogen Ustilago nuda is one of the major concerns throughout the globe for barley producers. The infection takes place without exhibiting any obvious symptoms and an infected seed lot can only be identified at the heading stage when the fungal teliospores emerge at the place of crop. The percentage losses on yield are directly proportional to the occurrence of infection. Currently available detection methods include seed health testing protocols which are time-consuming and cumbersome. With the globalization of the international market and increased crop demand, development of rapid disease screening methodologies has become an essential focus in the field of plant pathology. The present study sought to develop a rapid probe-based detection method for screening of U. nuda with real-time qPCR. Two U. nuda specific primer pairs were compared using standard PCR alongside optimization of real-time qPCR assay. The advantage of high fidelity DNA polymerase for amplification of U. nuda genomic DNA was recorded. U. nuda genomic DNA was amplified and cloned into a vector which was further used for generation of a quantification curve with a specific probe. The qPCR assay developed in this study was successful in the detection of as little as 43 copies of U. nuda genomic DNA. With studies involving larger sample size and field samples, this assay can be improved for enhanced sensitivity and specificity which can help in monitoring infection from DNA extractions of barley seeds and further improving the current microscopic detection methods.