Extração de DNA e identificação molecular de Olivea neotectonae (T.S. Ramakrishnan & K. Ramakrishnan) isoladas de folhas de teca (Tecto-na grandis L.f)

Phytosanitary problems can pose a threat in different crops. And, for this, the first step is to identify and know the main diseases and their impacts on forest stands, such as teak (Tectona grandis L.f). Currently, the rust caused by Olivea neotectonae  (T.S. Ramakrishnan & K. Ramakrishnan) is one of the main fungal diseases that affect the species. The objective of this work was to characterize O. neotectonae isolated from teak leaves in the state of Tocantins. The urediniospores of O. neotectonae were collected from naturally infected teak leaves in the city of Gurupi-TO. Three different methods were tested, to verify the one that best provided obtaining DNA with sufficient quantity and adequate quality for the correct identification of the pathogen. The method that resulted in the best quantity and quality of DNA from isolates was that described by Alessio and collaborators. The extracted DNA was subjected to amplification of the ITS region and sequenced. The nucleotide sequence of O. neotectonae shared a similarity of about 70% with sequences from the ITS region of other fungi deposited in the GenBank, since until now there are no available sequences of the species O. neotectonae.Problemas fitossanitários podem representar uma ameaça em diferentes cultivos. E, para isso, o primeiro passo é identificar e conhecer as principais doenças e seus impactos nos povoamentos florestais, como no cultivo da teca (Tectona grandis L.f). Atualmente, a ferrugem causada por Olivea neotectonae (T.S. Ramakrishnan & K. Ramakrishnan) é uma das principais doenças fúngicas que acometem a espécie. Objetivou-se com este trabalho realizar a caracterização do fungo O. neotectonae isolado de folhas de teca no estado do Tocantins, Brasil. Os urediniósporos de O. neotectonae foram coletados em folhas de teca infectados naturalmente, na cidade de Gurupi-TO. Foram testados três diferentes métodos, para verificar o que melhor proporcionasse obtenção de DNA com quantidade suficiente e qualidade adequada para a correta identificação do patógeno. O método que resultou em melhor quantidade e qualidade de DNA de isolados foi o descrito por Alessio e colaboradores. O DNA extraído foi submetido a amplificação da região ITS e sequenciados. A sequência de nucleotídeos de O. neotectonae compartilhou uma similaridade de cerca de 70% com sequências da região ITS de outros fungos depositados no GenBank, visto que até o momento não se encontra sequencias disponíveis da espécie O. neotectonae

Botanical fungicides in the control of soybean leaf diseases / Fungicidas botânicos no controle de doenças foliares na soja

The soybean cultivation (Glycine max (L.) Merrill) is responsible for the highest pesticides use in agriculture in Brazil. There is an environmental and social need to reduce the use of these substances in crops. The alternative products applied in agriculture such as plant extracts and essential oils, becomes necessary and indispensable, mainly in disease control. Among the plants studied, the Noni (Morinda citrifolia L.), has stood out in some studies, where relevant fungitoxic results have been demonstrated, however, there are still few works that prove its viability in the diseases management in field. Thus, this work aimed to evaluate the soybean diseases alternative control through the aqueous extracts and noni essential oil application, in plantings high and low disease pressure. Two field experiments were implemented, with soybean culture, evaluating leaf, fruit aqueous extract and noni essential oil as a fungicidal action. Foliar application of noni extracts and essential oil did not differ from fungicide in Asian Rust and Anthracnose control, in the 2016/17 crop, in both experiments. Soybean productivity was similar in treatments that received leaf extract (1748,8 Kg ha-1), essential oil (1762,5 Kg ha-1) and fungicides (2031,7 Kg ha-1). Where there was no large disease pressure all agronomic characteristics were equivalent, regardless of treatment. 

Caracterização morfológica, molecular e controle alternativo da ferrugem da teca (Olivea neotectonae) com óleos essenciais

The area planted with teak (Tectona grandis L.f) has increased in Brazil and in recent years in the state of Tocantins. However, research related to phytosanitary problems of the species have not yet been developed, especially for the fungus Olivea neotectonae causal agent of rust teak, one of the fungal diseases of major economic importance in this forest species. Given this fact, the work was developed with the following objectives: In Chapter I, the morphological and molecular characterization of O. neotectonae, causal agent of teak rust was conducted. To this end, teak sheets featuring yellow and powdery pustules on the abaxial surface, characteristic of the teak rust were collected in the municipalities of Gurupi and Lagoa da Confusão in the state of Tocantins. In the observation by an optical microscope, they were viewed and measured fungal structures by scanning electron microscopy was observed pathogen structures and the penetration mode through the stomata. PCR amplification of the ITS region of rDNA O. neotectonae using pairs of primers ITS1-ITS4 was successful, obtaining a 600 bp fragment. Direct sequencing of the PCR product from ITS O. neotectonae region, suggested it is a species belonging to the family Chaconiaceae. Based on these results and of morphology observed was confirmed O. neotectonae as the causal agent of teak rust. In Chapter II, due to the absence of fungicides registered for the control of this disease in teak and the search for alternative methods of disease control that are not harmful to the environment, to evaluate the effect of the essential oils of lemongrass (Cymbopogon citratus L.), citronella (Cymbopogon nardus L.), mastruz (Chenopodium ambrosioides L.) and noni (Morinda citrifolia L.) on germination urediniospores O. neotectonae; assess the phytotoxic effect of these essential oils in teak seedlings; and evaluate the use of essential oils in control of the teak rust when applied preventive and curative manner. It was found that essential oils of noni and lemongrass inhibited 100% germination of urediniospores. However, when applied to plants at higher doses, the essential noni and lemongrass oils phytotoxicity caused plants. The majority chemical constituents were identified in the essential oil of lemongrass leaves the geranial (41.46%) and neral (32.43%), the grass citronella citronellal (30.07%) in matruz the α- terpineol (41.97%) and essential oil extracted from mature noni fruit was octanoic acid (64.03%). The preventive control was more effective in controlling the disease than curative application, both for lemongrass oil as for noni. When comparing the two oils in the curative control were found lower values of the Area Under the Rust Progress Curve when using the essential oil of lemongrass.A área cultivada com teca (Tectona grandis L. f) tem aumentado no Brasil e também nos últimos anos no estado do Tocantins. Entretanto, as pesquisas relacionadas aos problemas fitossanitários da espécie ainda não foram desenvolvidas, especialmente para o fungo Olivea neotectonae, agente causal da ferrugem da teca, uma das doenças fúngicas de maior importância econômica nesta espécie florestal. Diante deste fato, o trabalho foi desenvolvido com os seguintes objetivos: No capítulo I, foi realizado a caracterização morfológica e molecular de O. neotectonae, agente causal da ferrugem da teca. Para tal, folhas de teca apresentando pústulas amarelas e pulverulentas na face abaxial, característicos da ferrugem da teca, foram coletadas nos municípios de Gurupi e Lagoa da Confusão, no estado do Tocantins. Na observação em microscópio ótico, foram visualizadas e medidas as estruturas fúngicas. Através da microscopia eletrônica de varredura foi possível observar as estruturas do patógeno e o modo de penetração através dos estômatos. A amplificação por PCR da região ITS do rDNA de O. neotectonae utilizando os pares de iniciadores ITS1-ITS4 foi bem sucedida, obtendo um fragmento de 600 pb. O sequenciamento direto do produto do PCR da região ITS de O. neotectonae, sugeriu se tratar de uma espécie pertencente à família Chaconiaceae. Com base nestes resultados e das características morfológicas observadas, foi possível confirmar O. neotectonae como sendo o agente causal da ferrugem da teca. No capítulo II, em razão da ausência de fungicidas registrados para o controle desta doença em plantas de teca e a busca por métodos alternativos de controle de doenças que não sejam prejudiciais ao meio ambiente, objetivou avaliar o efeito dos óleos essenciais de capim-limão (Cymbopogon citratus L.), citronela (Cymbopogon nardus L), mastruz (Chenopodium ambrosioides L.) e noni (Morinda citrifolia L.) na germinação de urediniósporos de O. neotectonae; avaliar o efeito fitotóxico destes óleos essenciais sob as mudas de teca; e avaliar o uso dos óleos essenciais no controle da ferrugem da teca quando aplicados de forma preventiva e curativa. Verificou-se que os óleos essenciais de noni e capim-limão inibiram 100% da germinação dos urediniósporos. Porém, quando aplicados nas mudas em doses mais elevadas, os óleos essenciais de noni e capim-limão causaram fitotoxidez às plantas. Foram identificados os constituintes químicos majoritários no óleo essencial de folhas de capim-limão o geranial (41,46%) e neral (32,43%), no capim-citronela o citronelal (30,07%), no matruz o α–terpinol (41,97%), e no óleo essencial extraído a partir de frutos de noni maduros foi o ácido octanóico (64,03%). O controle preventivo foi mais eficaz no controle da ferrugem do que a aplicação curativa, tanto para o óleo de capim-limão quanto para o noni. Quando se comparou os dois óleos no controle curativo foram encontrados menores valores da área abaixo da curva de progresso da ferrugem quando se utilizou o óleo essencial de capim-limão

Essential Oils for Alternative Teak Rust Control

ABSTRACT The objectives of this study were to evaluate the effect of lemon grass, citronella grass, Mexican-tea and noni essential oils on urediniospore germination of Olivea neotectonae , the agent responsible for rust in Teak (Tectona grandis L.f.); to evaluate the phytotoxic effect of these essential oils on teak seedlings; and to evaluate the use of essential oils to control rust in teak plants when preventively and curatively applied. We found that the noni and lemon grass essential oils inhibited 100% of urediniospore germination. On the other hand, the essential oils from noni and lemon grass caused phytotoxicity when applied to seedlings at concentrations of 2000 and 1500 μL L-1, respectively. The major constituents found in lemon grass essential oil were Geranial and Neral, while Octanoic Acid was found in noni oil. Lower values in the area below the rust progress curve were observed with the preventive application of lemon grass and noni essential oils

Essential Oils for Alternative Teak Rust Control

<div><p>ABSTRACT The objectives of this study were to evaluate the effect of lemon grass, citronella grass, Mexican-tea and noni essential oils on urediniospore germination of Olivea neotectonae , the agent responsible for rust in Teak (Tectona grandis L.f.); to evaluate the phytotoxic effect of these essential oils on teak seedlings; and to evaluate the use of essential oils to control rust in teak plants when preventively and curatively applied. We found that the noni and lemon grass essential oils inhibited 100% of urediniospore germination. On the other hand, the essential oils from noni and lemon grass caused phytotoxicity when applied to seedlings at concentrations of 2000 and 1500 μL L-1, respectively. The major constituents found in lemon grass essential oil were Geranial and Neral, while Octanoic Acid was found in noni oil. Lower values in the area below the rust progress curve were observed with the preventive application of lemon grass and noni essential oils.</p></div