Tomato (Solanum lycopersicum L.) and Fusarium oxysporum f. sp. lycopersici interaction. A review / Interacción entre tomate (Solanum lycopersicum L.) y Fusarium oxysporum f. sp. Lycopersici. Una revisión

ABSTRACT The interaction between plants and pathogens is a very dynamic and complex relationship that also includes a high degree of specificity, and it is precisely this last characteristic which triggers such important responses in the survival of one or the other. The pathosystem formed by tomato (Solanum lycopersicum L.) and Fusarium oxysporum f. sp. lycopersici (Fol) has been the subject of multiple studies due to the importance of the vegetable worldwide and for the economic and ecological impact of the fungus responsible for the vascular wilt disease in tomato, causing losses that go up to 100%. One way to find alternatives for the management of any pathosystem is to know the actors involved and the mechanisms that govern the interaction through technological and scientific advances that clearly show how the interaction develops on a genetic level. This review collects the information from different scientific sources with focus on the knowledge of the fungus, tomato cultivation and plant defense applied to this pathosystem, as well as the molecular mechanisms.

RESUMEN La interacción entre plantas y patógenos es una relación muy dinámica y compleja, que conlleva un alto grado de especificidad y es esta última característica, la que desencadena respuestas tan importantes en la supervivencia de uno u otro. El patosistema formado por tomate (Solanum lycopersicum L.) y Fusarium oxysporum f. sp. lycopersici (Fol) ha sido objeto de múltiples estudios, debido a la importancia de la hortaliza, a nivel mundial y por el impacto económico y ecológico del hongo, responsable de la marchitez vascular, provocando pérdidas que llegan hasta el 100%. Una forma de encontrar alternativas para el manejo de cualquier patosistema es conocer los actores involucrados y los mecanismos que rigen la interacción, a través de avances tecnológicos y científicos, que muestren, claramente, cómo se desarrolla la interacción, a nivel genético. Esta revisión recoge la información de fuentes científicas con énfasis en el conocimiento del hongo, el cultivo del tomate y la defensa vegetal, aplicada a este patosistema, así como los mecanismos moleculares.

Fructosan form Paenibacillus kribbensis PS04 enhance disease resistance against Rhizoctonia solani and tobacco mosaic virus

BACKGROUND: Rice sheath blight (caused by Rhizoctonia solani) and tobacco mosaic virus are very important plant diseases, causing a huge loss in global crop production. Paenibacillus kribbensis PS04 is a broad-spectrum biocontrol agent, used for controlling these diseases. Previously, extracellular polysaccharides (EPS) from P. kribbensis PS04 had been purified and their structure was inferred to be fructosan. This study aimed to evaluate the effects of exogenous EPS treatment on plant­pathogen interactions. RESULTS: Plant defense genes such as phenylalanine ammonia-lyase, catalase, chitinase, allene oxide synthase, and PR1a proteins were significantly induced by exogenous EPS treatment. Moreover, subsequent challenge of EPSpretreated plants with the pathogens (R. solani or tobacco mosaic virus) resulted in higher expression of defenseassociated genes. Increased activities of defense-associated enzymes, total phenols, and flavonoids were also observed in EPS pretreated plants. The contents of malondialdehyde in plants, which act as indicator of lipid peroxidation, were reduced by EPS treatment. CONCLUSIONS: This study comprehensively showed that EPS produced from P. kribbensis PS04 enhances disease resistance in plants by the activation of defense-associated genes as well as through the enhancement of activities of defense-related enzymes.

Phytophagous mites on genetically modified maize with Bacillus thuringiensis genes / Ácaros fitófagos em milho geneticamente modificado com genes de Bacillus thuringiensis

ABSTRACT: The study of non-target organisms such as mites, can provide information about the possible effects of transgenic maize cultivars on the arthropod community. This study aimed to evaluate the abundance of phytophagous mites in the area of Bt maize and evaluate the instantaneous population growth rate, and food preference of phytophagous mites on Bt maize. We registered the species occurrence and the number of adult mite individuals on four maize cultivars, one non-Bt 30F35, and three containing the proteins Cry1Ab (30F35 Yg), Cry1F (30F35 Hx) and Vip3a (Impacto Viptera). Cry proteins disrupt the midgut epithelium of insect pests. The food preference and instantaneous population growth rate (ri) were evaluated using the two-spotted spider mite Tetranychus urticae as model. The species Catarhinus tricholaenae and Aceria zeala were recorded. The Bt maize cultivars did not significantly affect the average number of C. tricholaenae and A. zeala mites compared to conventional cultivar. The population growth rates of T. urticae were similar for the different maize cultivars. T. urticae showed no preference between the leaf of Bt or conventional cultivars. Bt maize did not affect the abundance of species in phytophagous mite.

RESUMO: O estudo de organismos não-alvo, tais como ácaros, pode fornecer informações sobre os possíveis efeitos de cultivares de milho transgênico na comunidade de artrópodos. Este estudo teve como objetivo avaliar a abundância de ácaros fitófagos na área de milho Bt e avaliar a taxa de crescimento populacional instantânea e preferência alimentar de ácaros fitófagos em milho Bt. Registramos a ocorrência de espécies e número de indivíduos adultos de ácaros em quatro cultivares de milho, um não-Bt 30F35, e três contendo a proteína Cry1Ab (30F35 Yg), Cry1F (30F35 Hx) e VIP3A (Impacto Viptera). A preferência alimentar e taxa instantânea de crescimento populacional foram avaliadas utilizando fêmeas adultas do ácaro-rajado Tetranychus urticae. As espécies Catarhinus tricholaenae e Aceria zeala foram registradas. As cultivares de milho Bt não afetaram significativamente o número médio de ácaros C. tricholaenae e A. zeala em relação ao cultivar convencional. As taxas de crescimento populacional de T. urticae foram similares para os diferentes cultivares. T. urticae não mostrou preferência entre a folha de milho Bt ou cultivares convencionais. Sugere-se, então, que o milho Bt não afeta a abundância de espécies, preferência alimentar e taxa de crescimento de ácaros fitófagos.

New Assay Method for Allene Oxide Cyclase, an Important Enzyme in Jasmonic Acid Biosynthesis.

Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method. Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo- (10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC. Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan. Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system. Results: We found in the presence of 50 nmol of purified AOS together with 20 M 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system. Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.

Mecanismos de defensa y respuestas de las plantas en la interacción micorrícica: una revisión / Plant defense mechanisms and responses in the arbuscular mycorrhizal symbiosis: a review

El establecimiento de la simbiosis planta-hongos formadores de micorrizas Arbusculares (HFMA) requiere procesos armónicos a nivel espacio-temporal, que dependen de señales para el reconocimiento, colonización e intercambio bidireccional de nutrientes. Las plantas presentan respuestas de defensa frente a posibles organismos invasores; sin embargo, frente a HFMA estas son débiles, localizadas y no impiden la colonización del hongo. Los beneficios de la simbiosis generalmente se asocian a nutrición vegetal, aunque, también está relacionada con el incremento de la tolerancia-resistencia de plantas a los estreses bióticos. La resistencia inducida HFMA (MIR) es importante en el control de patógenos foliares, comedores de hojas y necrótrofos, encontrándose protección de plantas micorrizadas tanto a nivel local como sistémico, relacionada con los niveles de ácido jasmónico en tejidos. Un mecanismo en la MIR está asociado con el “priming”, que permite una rápida y eficiente respuesta de defensa de plantas micorrizadas. Se han planteado posibles mecanismos de atenuación de las respuestas de defensa, considerando: activación de supresores de defensa; plantas que producen respuestas de defensa frente a HFMA y otras que no las producen, y plantas que suprimen las respuestas de defensa en la simbiosis. Aunque el control de la simbiosisestá regulado básicamente por la planta, aún se desconoce el papel de los HFMA en el debilitamiento de las respuestas de defensa. Recientemente, se ha dado un avance importante en entender los mecanismos mediante los cuales se establece y mantiene la biotrofía del hongo, al describirse la proteína SP7 que interactúa con el factor de transcripción PR, ERF19 en el núcleo de la célula vegetal. Se ha sugerido que SP7 es un efector que actúa oponiéndose al programa de inmunidad de la planta.

Harmonic processes between plant and arbuscular mycorrhyzal fungi (AMF) are required for the symbiosis formation between the two organisms. These processes depend on specific signalling for the plant-fungus recognition, colonisation and bidirectional nutrient exchange. Plants show defence responses against invasive organisms, however they react weakly and localised when challenged by AMF. The benefits derived from the mycorrization are described for the nutritional aspect; however, it is known that mycorrhized plants are more tolerant to biotic stresses. Mycorrhizal induced resistance (MIR) is especially important for the control of foliar pathogens, leaf cutters and necrotrophs. There has also been found that mycorrhizal plants are protected both locally and systemically and their protection is related with jasmonic acid levels at their tissues. One of the most important mechanisms for MIR is the so called “priming” that allows plants to exert a fast and efficient defence response. Possible mechanisms to unravel mycorrhizal plants lower defence systems include: defence suppressor activation, differential plants response towards AMF from inexistent to low, and plant defence response suppression during the AMF symbiosis. The symbiosis control is known to be regulated by the plant, however, no role has been assigned to the AMF for the weakening of the plant defence system. Recently, a big step towards understanding of the fungal role has been made. A protein SP7 that interacts with a PR transcription factor ERF19, in the plant nucleus, has been described. This discovery indicates a possible mechanism to establish and maintain the biotrophic status of the AMF counteracting the immune plant system.

INDUCCIÓN DIFERENCIAL DE LA ENZIMA PEROXIDASA Y SU RELACIÓN CON LIGNIFICACIÓN EN LOS MECANISMOS DE DEFENSA DEL CLAVEL (Dianthus caryophyllus L.) DURANTE SU INTERACCIÓN CON Fusarium oxysporum f. sp. Dianthi / DIFFERENTIAL INDUCTION OF PEROXIDASE ENZYME AND ITS RELATIONSHIP WITH LIGNIFICATION IN CARNATION DEFENSE (Dianthus caryophyllus L.) MECHANISM AGAINST Fusarium oxysporum f. sp. Dianthi / INDUCAO DIFERENCIAL DA ENZIMA PEROXIDASEEASUARELACAO COM A LENHIFICACAO NOS MECANISMOS DE DEFESA DE CRAVO (Dianthus caryophyllus L.) DURANTE A SUAINTERACCAO COM Fusarium oxysporum f. sp. Dianthi

Se evaluó la actividad enzimática peroxidasa (POD) y el contenido de lignina en tallos de esquejes de clavel (Dianthus cayophyllus L.) inoculados con el hongo Fusarium oxysporum f.sp. dianthi (Fod) raza 2, con el fin de determinar su posible participación en la respuesta de defensa y en la resistencia de la planta al marchitamiento vascular. Inicialmente se seleccionaron las condiciones para la extracción y determinación de la actividad enzimática. Se encontró que el tratamiento con buffer citrato 100 mM pH 5,0 con 3% de PVPP presentó los mejores resultados de actividad peroxidasa para la extracción de la enzima a partir de tallos de clavel. Se determinaron las mejores condiciones para la medida de actividad enzimática POD a través de la reacción de oxidación de o-dianisidina con H2O2 seguida a 460 nm. Una mezcla de reacción con una concentración 0,6 mM de o-dianisidina y 2,0 mM de H2O2 en buffer citrato 100 mM a pH 5,5 y 45°C, presentó los mejores resultados para la cuantificación de la enzima. Una vez establecidas las condiciones, esquejes de clavel de una variedad tolerante y una susceptible al marchitamiento vascular fueron inoculados con el patógeno y se evaluaron los niveles de actividad de POD y de acumulación de lignina a diferentes tiempos pos-inoculación. La variedad tolerante (Bárbara), presentó inducción de dicha enzima a las 8 y 48 h pos-inoculación y aumento en el contenido de lignina a 48 y 72 h. A su vez, en la variedad susceptible (Delphi) no se encontraron cambios significativos en los niveles de POD ni en el contenido de lignina. Se estableció, por tanto, que la inducción de la actividad POD está asociada a la lignificación, que a nivel del tallo, hace parte de los mecanismos asociados a defensa en el modelo clavel-Fusarium oxysporum f. sp. dianthi.

Peroxidase (POD) enzymatic activity and lignin content in carnation stems (Diant-hus cayophyllus L.) infected with the fungus Fusarium oxysporum f.sp. dianthi race 2 were evaluated with the purpose of determining the possible enzyme participation in the plant defense response and in the resistance to the vascular wilting. Initially, the conditions for extraction and determination of enzymatic activity were selected. It was found that when citrate buffer 100 mM pH 5.0 with 3% PVPP was employed, the results of POD activity in enzyme extract obtained from carnation stems were higher. Best conditions were set for POD activity measure through oxidation reaction of o-dianisidine and H2O2 at 460 nm. A reaction mixture with a concentration of o-dianisidine 0,6mM and H2O2 2.0 mM in citrate buffer 100 mM pH 5.5 at 45 ºC displayed the best results for enzyme quantification. Once conditions were established, tolerant and susceptible vascular wilting carnation cuttings were inoculated with the pathogen. In each treatment at stem level, different postinoculation times were evaluated for enzyme activity levels and lignin accumulation. Tolerant variety (Barbara) showed a POD induction at 8 and 48 h pos-inoculation and an increase in lignin content at 48 and 72 h. At the same time, in susceptible variety (Delphi) there were no significant changes in POD levels or lignin contents. Therefore, it was established that POD activity induction is associated to lignification which, at stem level, is part of the mechanisms associated to defense in carnation (Dianthus caryophyllus L.) against Fusarium oxysporum f. sp. dianthi.

Foram avaliadas a actividade enzimática peroxidasa (POD) e o conteúdo de lignina em talos de estacas de cravo (Dianthus cayophyllus L.) inoculados com o fungo Fusarium oxysporum f.sp. dianthi raça 2, com o propósito de determinar a sua possível participação na resposta de defensa e à resistência da planta ao murchamento vascular. Inicialmente foram seleccionadas as condiciones para a extracção e determinação da actividade enzimática. Foi encontrado que o tratamento com buffer citrato 100 mM pH 5,0 com 3% de PVPP, apresentou os melhores resultados de actividade peroxidasa na extracção da enzima a partir de talos de cravo. Foram determinadas as melhores condições para medir a actividade enzimática POD a través da reacção de oxidação de o-dianisidi na com H2O2 e com seguimento a 460 nm. Uma mistura de reacção com uma concentração 0,6 mM de o-dianisidina e 2,0 mM de H2O2 em buffer citrato 100 mM a pH 5,5 e 45 °C, apresentou os melhores resultados para a quantificação da enzima. Uma vez estabelecidas as condições, estacas de cravo de uma variedade tolerante e uma susceptível ao murchamento vascular foram inoculadas com o fungo patogénico, e se avaliaram a diferentes tempos pos-inoculação os níveis de actividade desta enzima e de acumulação de lignina. A variedade tolerante (Bárbara), apresentou uma indução de POD às 8 e 48 h pos-inoculação e um aumento no conteúdo de lignina às 48 y 72 h. Por outro lado, na variedade susceptível (Delphi) não se encontraram mudanças significativas nos níveis de POD nem no conteúdo de lignina. Se estabeleceu, por tanto, que a indução da actividade POD está associada à lignificação que a nível de talo faz parte dos mecanismos associados à defesa no modelo cravo-Fusarium oxysporum f. sp. dianthi raça 2.

Biotechnological approaches for plant viruses resistance: from general to the modern RNA silencing pathway

Virus diseases are significant threats to modern agriculture and their control remains a challenge to the management of cultivation. The main virus resistance strategies are based on either natural resistance or engineered virus-resistant plants. Recent progress in understanding the molecular mechanisms underlying the roles of resistance genes has promoted the development of new anti-virus strategies. Engineered plants, in particular plants expressing RNA-silencing nucleotides, are becoming increasingly important and are likely to provide more effective strategies in future. A general discussion on the biotechnology of plant responses to virus infection is followed by recent advances in engineered plant resistance.

As viroses são problemas importantes para a agricultura moderna e o seu controle representa um desafio para o manejo de áreas cultivadas. As principais estratégias de resistência a vírus se baseiam em mecanismos naturais ou em engenharia genética. Recentemente, a maior compreensão dos mecanismos moleculares envolvidos na função de genes de resistência da planta facilitou o desenvolvimento de novas estratégias antivirais. Plantas modificadas geneticamente, em particular aquelas expressando a via de silenciamento de RNA, são alvo de interesse crescente e representam a possibilidade de estratégias futuras mais efetivas. Neste trabalho são discutidos diferentes aspectos relacionados à resistência a viroses em plantas. Adicionalmente, a perspectiva de aplicação biotecnológica das diferentes vias de resistência é apresentada.