ARN de interferencia (ARNi): una herramienta eficaz en agrobiotecnología / RNA interference (RNAi): an effective tool in agrobiotechnology

RESUMEN El ARN de interferencia (ARNi) es un mecanismo evolutivamente conservado en la mayoría de las células eucariotas que permite silenciar genes mediante la degradación de ARN mensajero (ARNm) y la supresión de la síntesis de proteínas. En plantas, las moléculas de ARNi están involucradas en mecanismos de defensa contra patógenos y transposones, en la respuesta adaptativa al estrés, y en la expresión de genes relacionados con su crecimiento. El ARNi se considera una herramienta biotecnológica eficaz para silenciar la expresión de genes de microorganismos fitopatógenos, esto permite el diseño de bioplaguicidas ambientalmente seguros con una afinidad y selectividad, en muchos casos superior a la de los plaguicidas químicos. En esta revisión se señalan los últimos avances en la aplicación del ARNi en el contexto agrícola y su efectividad en el control biológico de fitopatógenos e insectos plaga. Asimismo, se presentan diversos ensayos experimentales cuyos resultados pueden ser la base para futuros bioproductos, además de algunos ejemplos disponibles en el mercado. Por último, se abordan aspectos de bioseguridad y consideraciones regulatorias necesarias para la aceptación y uso de esta tecnología a nivel global.

ABSTRACT RNA interference (RNAi) is an evolutionarily conserved mechanism in most eukaryotic cells that allows genes to be silenced by degradation of messenger RNA (mRNA) and suppression of protein synthesis. In plants, RNAi molecules are involved in defense mechanisms against pathogens and transposons, in the adaptive response to stress, and in the expression of genes related to their growth. RNAi is an effective biotechnological tool to silence the expression of specific genes which are essential for the survival of phytopathogenic microorganisms, thus allowing the design of environmentally safe biopesticides with affinity and selectivity, in many cases greater than chemical pesticides. This review describes the latest advances in the application of RNAi in the agricultural context and its effectiveness in the biological control of phytopathogens and pest insects. Likewise, various experimental trials are presented, the results of which may be the basis for future bioproducts, as well as some examples available on the market. Finally, biosafety aspects and regulatory considerations necessary for the acceptance and use of this technology at a global level are presented.

Hongos detectados en la entomofauna asociada a Espeletia pycnophylla / Fungi detected in insects associated to Espeletia pycnophylla

RESUMEN Algunas especies del género Espeletia sp. (Asteraceae) son afectados por hongos fitopatógenos e insectos en páramos de los Andes del Norte, amenazando su permanencia y la prestación de servicios que se les atribuye, como la regulación del ciclo hídrico. La escaza información sobre la afectación en el páramo de Paja Blanca (Nariño, Colombia), dificulta la comprensión de la dinámica del fenómeno y la formulación de estrategias de manejo. El objetivo de esta investigación fue identificar cambios en la composición de las comunidades de insectos asociados a E. pycnophylla durante el declive de las poblaciones de frailejones y evaluar si reflejan modificaciones en los hongos, asociados a dichos insectos. Para ello, se colectaron insectos en plantas de E. pycnophylla sanas y afectadas, a los que se aplicó índices ecológicos, para identificar posibles modificaciones en la diversidad y la composición; además, se aislaron e identificaron morfológica y molecularmente hongos, a partir de estructuras corporales de los insectos. Como resultado, se identificó una diversidad de insectos considerable y que no hay modificación en su composición de insectos entre los dos estados de frailejón analizados. Se logró aislar hongos, como Fusarium oxysporum, Botrytis sp., Epicoccum nigrum, Cladosporium sp., a partir de estructuras de los géneros de insectos Neomyopites sp., Diabrotica sp., Bradysia sp. y Dyscolus sp. Este estudio aporta información sobre la entomofauna presente en E. pycnopylla del páramo de Paja Blanca y hongos asociados a estructuras corporales, como el canal alimentario y aparato bucal de los insectos.

ABSTRACT Some species of the genus Espeletia sp. (Asteraceae) are affected by phytopathogenic fungi and insects in the paramos of the Northern Andes, threatening their permanence and the provision of services attributed to them such as the regulation of the water cycle. The scarce information about the impact on the Paja Blanca páramo (Nariño, Colombia) makes it difficult to understand the dynamics of the phenomenon and the formulation of management strategies. The goal of this research was to identify changes in the composition of the insect communities associated to E. pycnophylla during the decline of the frailejon populations and to evaluate if these reflect modifications in the fungi associated with those insects. For this, insects were collected in healthy and affected E. pycnophylla plants to which ecological indices were applied to identify possible changes in diversity and composition, Also fungi were isolated and identified morphologically and molecularly from body structures of insects. As result, a considerable diversity of insects was identified and no modification in their composition of insects between the two states of frailejón were found. It was possible to isolate fungi such as Fusarium oxysporum, Botrytis sp., Epicoccum nigrum, Cladosporium sp. from structures of the insect genera Neomyopites sp., Diabrotica sp., Bradysia sp. and Dyscolus sp. This study provides information about the entomofauna present in E. pycnopylla from the Paja Blanca páramo and fungi associated with body structures such as the alimentary canal and the oral system of insects.

Identificación morfológica y comprobación de patogenicidad de agentes biológicos asociados Tabebuia rosea (Bertol) en etapa de vivero en Morelia (Risaralda) / Morphological identification of pathogenic agents associated with Tabebuia rosea (Bignoniaceae) in nursery stage in morelia (Risaralda)

Introducción. Las susceptibilidades de las plantaciones al ataque de organismos dañinos pueden ocasionar efectos nocivos para su desarrollo, cuando se considera que el nivel de daños ocasionado no se puede tolerar, es indispensable determinar acertadamente la naturaleza del agente causal, sea éste tipo biótico o abiótico; la acertada determinación del agente de un problema fitosanitario en un vivero o plantaciones forestale, se fundamenta en un análisis detallado de los factores que la pueden estar afectando. Tabebuia rosea o Guayacán rosado es una especie forestal nativa de amplia aceptación en el mercado, que presenta durante su etapa de vivero, limitantes en las fases de germinación, emergencia y sobrevivencia, por lo anterior el objetivo de esta investigación era la de Identificar morfológicamente los posibles agentes patógenos asociados a Tabebuia rosea en etapa de vivero y comprobar su patogenicidad. Metodología. Se tomaron plántulas de vivero, las cuales se les realizo un análisis de los signos y los síntomas para el aislamiento y siembra de zonas afectadas en agar papa dextrosa. En el cultivo mixto se identificó los posibles agentes patógenos, los cuales fueron sembrados para obtener cultivos puros, y utilizados en la prueba de patogenicidad, para la inoculación de las estructuras sanas (hojas, peciolos y tallos) y su respectivo control. Resultados. Se identificaron tres géneros de hongos Colletotrichum, Mucor, y Candida, y un género de nematodo denominado Meloidogyne. La prueba de patogenicidad para Colletotrichum y Candida mostraron relaciones significativas en el proceso de infección principalmente en estructuras foliares. Se concluye en este trabajo dos nuevos posibles géneros patógenos para Tabebuia rosea evidenciados en la prueba de patogenicidad. Y se corrobora que el género Meloidogyne es un patógeno severo en el guayacán rosado que ocasiona anomalías morfológicas en la raíz; cabe resaltar que es el primer reporte para el departamento de Risaralda

Introduction. The susceptibilities of plantations to attack by harmful organisms can cause harmful effects for their development, when it is considered that the level of damage caused cannot be tolerated, it is essential to correctly determine the nature of the causal agent, be it biotic or abiotic; The correct determination of the agent of a phytosanitary problem in a nursery or forest plantations is based on a detailed analysis of the factors that may be affecting it. Tabebuia rosea or pink Guayacán is a native forest species widely accepted in the market, which presents, during its nursery stage, limitations in the germination, emergence and survival phases, therefore the objective of this research was to morphologically identify the possible pathogens associated with Tabebuia rosea in the nursery stage and check its pathogenicity. Methodology. Nursery seedlings were taken, which were analyzed for signs and symptoms for the isolation and sowing of affected areas on potato dextrose agar. In the mixed culture, possible pathogens were identified, which were sown to obtain pure cultures, and used in the pathogenicity test, for the inoculation of healthy structures (leaves, petioles and stems) and their respective control. Results. Three fungal genera Colletotrichum, Mucor, and Candida, and a nematode genus named Meloidogyne were identified. The pathogenicity test for Colletotrichum and Candida showed significant relationships in the infection process mainly in leaf structures. Two new possible pathogenic genera for Tabebuia rosea evidenced in the pathogenicity test are concluded in this work. And it is corroborated that the genus Meloidogyne is a severe pathogen in the pink guayacán that causes morphological anomalies in the root; It should be noted that it is the first report for the department of Risaralda

Growth supression of plant pathogenic fungi using bryophite extracts / Supressão de crescimento de fungos patogênicos de plantas usando extratos de briófitas

Chemicals are often used in attempts to control diseases caused by plant pathogenic fungi during food production. However, chemicals can have adverse effects not just on food, but they also remain active for a long time within ecosystems, and thus are not environmentally friendly. Therefore,development of bio-treatment and avoiding use of chemicals are urgently needed. With the aim of studying and developing new environmentally-friendly treatments, we tested extracts from selected bryophyte species(Porella platyphylla, Cinclidotus fontinaloides and Anomodon viticulosus) on five plant pathogenic fungi under controlled conditions. The fungi (Botryosphaeria dothidea, Phomopsis viticola, Calosphaeria sp., Colletotrichum acutatum and Monilinia laxa) were selected based on common diseases they cause on fruits and grapevine. They were isolated in cultures and treated with bryophyte extracts. Bryophyte extracts were shown to be effective in suppression of certain plant pathogenic fungi growth and to have a huge potential in development of novel biotechnological treatments and biofungicides. The best results were achieved in inhibition of B. dothidea, P. viticola and Calosphaeria sp.

Fungos fitopatogênicos são controlados com produtos químicos para combater doenças causadas por eles durante a produção de alimentos. Tais produtos são ruins não apenas para a alimentação, mas também podem permanecer por muito tempo nos ecossistemas, portanto, não são ecologicamente corretos. Desta forma, os biotratamentos e a prevenção de usos químicos são urgentemente necessários no futuro próximo. Com o objetivo de estudar e desenvolver nova alternative decontrole de doenças de plantas, testamos os extratos de espécies de briófitas selecionadas (Porella platyphylla, Cinclidotus fontinaloides e Anomodon viticulosus) em cinco fungos fitopatogênicos (Botryosphaeria dothidea, Phomopsis viticola, Calosphaeria sp., Colletotrichum acutatum e Monilinia laxa) em condição controlada. Estes fungos, selecionados com base nas doenças que causam em frutos e videiras, foram isolados em culturas puras e tratados com os respectivos extratos de briófitas. Os extratos de briófitas demonstraram ser eficazes na supressão de certos fungos fitopatogênicos e têm um enorme potencial no desenvolvimento de novos biofungicidas e tratamentos biotecnológicos. Os mais interessantes resultados foram obtidos na inibição de B. dothidea, P. viticola eCalosphaeria sp.

Evaluación de la actividad antifúngica del extracto de Usnea laevis en hongos fitopatógenos / Evaluation of the antifungal activity of the lichenic extract of Usnea laevis in phytopathogenic fungi

Los líquenes son producto de una asociación simbiótica entre un hongo y alga y/o cianobacteria; esta simbiosis produce una serie de metabolitos secundarios o sustancias liquénicas únicas, las mismas que son aisladas a partir de sus extractos y que han presentado una marcada actividad antibiótica y antifúngica. Considerando que en el Ecuador no se tienen antecedentes acerca de este tipo de estudios, el objetivo planteado fue el evaluar la actividad antifúngica del extracto liquénico de Usnea laevis frente a hongos fitopatógenos. En el presente estudio las sustancias liquénicas almacenadas en el talo de Usnea laevis fueron extraídas con metanol y etanol. La actividad antifúngica in vitro de estos extractos fue probado frente a géneros de hongos fitopatógenos previamente aislados (Aspergillus, Penicillium y Rhizopus stolonifer); añadiéndolo al medio de cultivo Agar Papa Dextrosa (PDA) a una concentración de 0,5%; determinándose el porcentaje de inhibición. Los datos fueron analizados estadísticamente y se realizó la clasificación del extracto en base a su porcentaje de inhibición según la OILB. El extracto metanólico y etanólico de Usnea laevis mostró una mayor efectividad frente a Rhizopus stolonifer, inhibiendo más del 50% de su crecimiento, a diferencia de Penicillium y Aspergillus cuyo porcentaje de inhibición fue mucho menor y mostraron diferencias estadísticamente significativas. El extracto liquénico fue moderadamente tóxico para Rhizopus stolonifer, ligeramente tóxico para Aspergillus sp 1 y Penicillium sp e inocuo para Aspergillus sp 2.

A lichen is an organism product of a symbiotic association between a fungus and algae and / or cyanobacteria; this symbiosis produces many secondary metabolites or unique lichen substances, which are isolated from their extracts and show a marked antibiotic and antifungal activity. Considering there is no background on this type of studies in Ecuador, the aim of this study was to evaluate the antifungal in vitro activity of Usnea laevis extract on phytopathogenic fungi. The lichenic substances stored in the thallus of Usnea laevis were extracted with methanol and ethanol. The in vitro antifungal activity of these extracts was tested against phytopathogenic fungal genera previously isolated (Aspergillus, Penicillium and Rhizopus stolonifer) . They were adding to Potato Dextrose Agar (PDA) culture medium at a concentration of 0.5%, and the inhibition percentage was determined. Data were statistically analyzed and the extract was classified based on its inhibition percentage according to the OILB. Methanolic and ethanolic extracts of Usnea laevis showed greater effectiveness against Rhizopus stolonifer, inhibiting more than 50% of its growth, unlike Penicillium and Aspergillus, whose inhibition percentage was lower and showed significant differences. The liquid extract was also moderately toxic to Rhizopus stolonifer, slightly toxic to Aspergillus sp 1 and Penicillium sp and harmless to Aspergillus sp 2.

Microorganismos potencialmente fitopatógenos en aguas de riego proveniente de la cuenca media del rio Bogotá / Potentially phytopathogenic microorganisms irrigation water from the media Bogota river basin

Resumen Objetivo. Identificar microorganismos fitopatógenos presentes en las aguas utilizadas para riego en la cuenca media del rio Bogotá. Métodos. Las muestras fueron tomadas en doce fincas de la cuenca media y analizadas con la técnica de filtración por membrana. Las bacterias se identificaron por bioquímica y los hongos por claves dicotomas. Resultados. El agua de riego analizada contenía bacterias como: E.coli, P. vulgaris, A. urinae, E. cloacae, B.cereus, S. marcescens, B. brevis, y en cuanto a los hongos se encontraron: Aspergillus fumigatus, Aspergillus flavus., Mucor sp., Rhizopus sp., Penicillium sp. y Fusarium sp. La presencia de estos potenciales fitopatógenos en el agua indican un alto riesgo de contaminación y dispersión de éstos en los cultivos.

Abstract Objective. Identify phytopathogenic microorganisms present in the water used for irrigation in the middle basin of the river Bogota. Methods. Samples were taken at 12 farms of the middle basin and analyzed by the membrane filtration technique. The bacteria were identified by biochemical and fungi by dichotomies keys. Results. The irrigation water contained bacteria analyzed as E. coli, P. vulgaris, A. urinae, E. cloacae, B. cereus, S. marcescens, B. brevis, and as for the mushrooms found: A. fumigatus, A. flavus, Mucor sp., Rhizopus sp., Penicillium sp. and Fusarium sp. The presence of these potential pathogens in water indicate a high risk of contamination and dispersion thereof in crops.

Caracterización química y evaluación de la actividad antifúngica del aceite esencial foliar de Lippia alba contra Colletotrichum gloeosporioides

El objetivo de este estudio fue caracterizar y evaluar in vitro la eficiencia del aceite esencial foliar de Lippia alba contra Colletotrichum gloeosporioides. Se extrajo el aceite esencial a partir de las hojas mediante el método de hidrodestilación asistida por microondas (MWHD) y se identificaron sus metabolitos mediante una cromatografía de gases acoplada a espectrometría de masas (GC/MS). La evaluación in vitro se realizó empleando 4 concentraciones del aceite esencial (500, 1000, 3000 y 10000 ppm) diluidas en acetona; se utilizó un control positivo con Benomil 1 g/L, un control negativo con acetona y un testigo absoluto; la eficiencia del aceite se midió mediante el porcentaje de índice antifúngico (%I.A). El componente mayoritario en el aceite esencial fue el citral (34.62 a 40.03%) y que el mayor %I.A se encontró a la concentración de 10000 ppm (97.8%), muy similar a la del Benomil (100%). Lo anterior demostraría la eficacia del aceite esencial de L. alba para controlar a C. gloeosporioides, y su posible uso como fungicida biológico.

The aim of this study was to characterize and evaluate the in vitro efficiency of leaf essential oil of Lippia alba against Colletotrichum gloeosporioides. Essential oil from the leaves was extracted by the method of assisted microwave hydrodistillation (MWHD) and metabolites were identified by gas chromatography coupled to mass spectrometry (GC/ MS). The in vitro evaluation was performed using 4 concentrations of essential oil (500, 1000, 3000 and 10000 ppm) diluted in acetone; positive control with Benomyl 1 g /L, a negative control with acetone and absolute control was used; oil efficiency was measured by the percentage of antifungal index (% IA). It was found that the major component was in the essential oil citral (34.62 to 40.03%) and the highest %I.A was found to 10000 ppm concentration (97.8%) very similar to Benomyl (100%). This would demonstrate the efficacy of the L. alba essential oil to control C. gloeosporioides, and its possible use as a biological fungicide.

Bases bioquímicas de la tolerancia al estrés osmótico en hongos fitopatógenos: el caso de Macrophomina phaseolina (Tassi) Goid / Biochemical basis of tolerance to osmotic stress in phytopathogenic fungus: The case of Macrophomina phaseolina (Tassi) Goid

El hongo Macrophomina phaseolina (Tassi) Goid., agente causal de la enfermedad denominada «pudrición carbonosa¼, provoca pérdidas significativas en la producción de cultivos como maíz, sorgo, soya y frijol en México. Este hongo, parásito facultativo, muestra amplia capacidad de adaptación a ambientes estresantes, donde existen altas temperaturas y deficiencia hídrica, condiciones frecuentes en gran parte de la agricultura de dicho país. En este trabajo se describen algunos aspectos básicos de la etiología y la epidemiología de M. phaseolina. Asimismo, se revisa la importancia que guardan las respuestas de este hongo a ambientes estresantes, particularmente la deficiencia hídrica, de acuerdo con caracteres morfológicos y del crecimiento, así como fisiológicos, bioquímicos y de patogenicidad. Finalmente, se presentan algunas perspectivas de estudio del género, que enfatizan la necesidad de mejorar su conocimiento, con base en la aplicación de herramientas tradicionales y de biotecnología, y de dilucidar mecanismos de tolerancia al estrés ambiental, extrapolables a otros microorganismos útiles al hombre.

Fungus Macrophomina phaseolina (Tassi) Goid. is the causative agent of charcoal rot disease which causes significant yield losses in major crops such as maize, sorghum, soybean and common beans in Mexico. This fungus is a facultative parasite which shows broad ability to adapt itself to stressed environments where water deficits and/or high temperature stresses commonly occur. These environmental conditions are common for most cultivable lands throughout Mexico. Here we describe some basic facts related to the etiology and epidemiology of the fungus as well as to the importance of responses to stressed environments, particularly to water deficits, based on morphology and growth traits, as well as on physiology, biochemistry and pathogenicity of fungus M. phaseolina. To conclude, we show some perspectives related to future research into the genus, which emphasize the increasing need to improve the knowledge based on the application of both traditional and biotechnological tools in order to elucidate the mechanisms of resistance to environmental stress which can be extrapolated to other useful organisms to man.

Extracts of Caesalpinia ferrea and Trichoderma sp. on the control of Colletotrichum sp. transmission in Sideroxylon obtusifolium seeds / Extratos de Caesalpinia ferrea e Trichoderma spp. no controle da tramissibilidade de Colletotrichum sp. em sementes de Sideroxylon obtusifolium (Sapotaceae)

ABSTRACT Recent research reports the importance of preserving plants in Brazilian semiarid regions, in this context, the scientific literature has reported different pharmacological studies from plant extracts with an antifungal potential, coming from forest species that can contribute as a control and management strategy in the transmission of phytopathogens. This study aimed to evaluate the effect of biotech treatments in controlling the transmission of Colletotrichum sp. in seeds of S. obtusifolium. In this study, 100 seeds were subjected to the following preventive treatments: fungicide Captan®, extract of Caesalpinia ferrea Mart. Ex. Tul., and biological control with Trichoderma spp. The biological control with Trichoderma spp. and the alternative control using C. ferrea extract provided a greater protection to seeds and seedlings of S. obtusifolium facing the transmissibility of Colletotrichum sp.The treatment based on plant extract is more efficient for this purpose only in large seeds and does not interfere on the germination percentage and speed. Therefore it is necessary to perform other studies with Trichoderma spp. and C. ferrea extract to test different doses of these products.

RESUMO Recentes pesquisas relatam a importância da preservação de plantas do semiárido brasileiro. Neste contexto, a literatura científica tem relatado diferentes estudos farmacológicos com extratos vegetais com potencial antifúngico proveniente de espécies florestais que podem contribuir como estratégia de controle e gerenciamento na transmissão de fitopatógenos. No presente estudo o objetivo foi avaliar o efeito de tratamentos biotecnológicos no controle da transmissibilidade de Colletotrichum sp. em sementes de S. obtusifolium. Neste estudo foram utilizadas 100 sementes submetidas aos seguintes tratamentos preventivos: fungicida Captan®, extrato de Caesalpinia ferrea Mart. Ex. Tul. e controle biológico com Trichoderma spp. O controle biológico com Trichoderma spp. e o alternativo com extrato de C. ferrea proporcionam maior proteção às sementes e plântulas S. obtusifolium quanto a transmissibilidade do Colletotrichum sp. O tratamento à base de extrato vegetal foi o mais eficiente para este fim, apenas em sementes de maior tamanho, por não interferir na porcentagem e velocidade de germinação. Portanto, faz-se necessário à realização de outros trabalhos com Trichoderma spp. e extrato de C. ferrea para testar doses diferentes desses produtos.

Efecto antibacteriano in vitro de exudados foliares de tabaco contra dos bacterias fitopatógenas / In vitro antibacterial effect of tobacco leaf exudates against two bacterial plant pathogens

Los productos naturales son una alternativa para el control de microorganismos que ocasionan enfermedades en los cultivos. Este trabajo tuvo como objetivo evaluar diferentes solventes para la obtención de extractos crudos a partir de exudados foliares de líneas de tabaco, y el efecto in vitro de estos extractos contra dos bacterias fitopatógenas: Xanthomonas campestris (Xc) y Pectobacterium carotovorum (Pc). Se evaluaron solventes con polaridades entre 3.1 y 6.2 (diclorometano, n-butanol, acetato de etilo, metanol y etanol 90 %). El etanol 90 % se seleccionó como mejor solvente y como sustituto del diclorometano por su mayor rendimiento. Los extractos etanólicos crudos se obtuvieron a partir de exudados foliares de diez líneas de tabaco seleccionadas. La diversidad de la composición química de los extractos etanólicos se reveló por cromatografía en capa delgada. La actividad antibacteriana se evaluó por el método de difusión en agar con discos de papel de filtro y la medición del diámetro del halo de inhibición. Se observó inhibición para todos los extractos contra Xc destacándose los correspondientes a las líneas Nic 1061 "TI 1738" y Nic 1016 "Incekara" hasta 5 µg de extracto crudo seco /disco, con un mayor rendimiento para la línea Nic 1061. El extracto de la línea Nic 1015 fue el único con actividad contra Pc hasta 5 µg de extracto crudo seco por disco. Estos resultados sugieren un uso potencial de los extractos crudos de las líneas Nic 1061 y Nic 1015 "TI 1341" como un agente efectivo para la protección de cultivos contra estas bacterias.

Natural products are an alternative to control microorganisms that cause diseases in crops. This work aimed to evaluate different solvents for obtaining crude extracts from tobacco leaf exudates and to determine in vitro effect of these extracts against two phytopathogenic bacteria: Xanthomonas campestris(Xc) and Pectobacterium carotovorum(Pc). Crude extracts from ten tobacco lines using solvents with polarities between 3.1 and 6.2 (dichloromethane, n-butanol, ethyl acetate, methanol and ethanol 90%) were obtained. Ethanol 90% was selected as the best solvent for obtaining extracts from tobacco leaf exudates and as a substitute of dichloromethane due to the best yield. The chemical composition diversity of the ethanolic extracts was revealed by thin-layer chromatography. The antibacterial activity was evaluated by agar disk diffusion method recording the inhibition zones. Growth inhibition was observed for all extracts against Xc, and the better activity corresponded to the lines Nic 1061"TI 1738" and Nic 1016 "Incekara" until a minimal amount of 5 µg/ disc, with higher yield in case of the line Nic1061 . Only the extract of the line Nic 1015 was able to inhibit the growth of Pc until a minimal inhibitory concentration of 5 µg/disc. These results suggest a potential use of crude extracts from lines Nic 1061 and Nic 1015 "TI 1341" as an effective agent for the crop protection against Xc and Pc respectively.

Composição química e atividade in vitro de três óleos essenciais sobre Colletotrichum gloeosporioides do maracujazeiro / Chemical composition and in vitro activity of three essential oils on Colletotrichum gloeosporioides from passion fruit

Objetivou-se avaliar a ação de diferentes concentrações de óleos essenciais de três plantas medicinais sobre o crescimento micelial, a produção e a germinação de conídios de Colletotrichum gloeosporioides (Penz.). Utilizou-se os óleos essenciais de Lippia sidoides Cham., Cymbopogon citratus (DC) Stapf.) e Ocimum gratissimum L. em cinco concentrações (0 µL mL-1, 1 µL mL-1, 3 µL mL-1, 5 µL mL-1 e 7 µL mL-1) com 4 repetições no delineamento inteiramente casualizado, em esquema fatorial 5x3. Os óleos foram analisados por cromatografia gasosa acoplada com espectrometria de massas. Para todas as características verificou-se efeito significativo da interação "Óleo" versus "Concentração". O óleo essencial de C. citratus proporcionou a maior inibição do crescimento micelial em todas as concentrações testadas. Os outros óleos não se diferenciaram, exceto a partir da concentração de 5 µL mL-1, onde o óleo de L. sidoides foi superior ao óleo de O. gratissimum. Na produção e germinação de conídios, o óleo de C. citratus, foi superior aos óleos de L. sidoides e de O. gratissimum nas concentrações de 1 µL mL-1 e 3 µL mL-1, inibindo completamente a produção e germinação a partir da concentração de 3 µL mL-1. As concentrações influenciam em todas as variáveis, ajustando-se à equação do modelo linear. Observa-se o decréscimo nessas, à medida em que se aumentam as concentrações dos óleos testados. Os óleos essenciais testados possuem efeito fungitóxico e fungistático.

The objective of this study was to evaluate the action of different concentrations of essential oils of three medicinal plants on the mycelial growth, production and conidial germination of Colletotrichum gloeosporioides (Penz.). We used a completely randomized design, in a 5x3 factorial design, being five concentrations (0 µL mL-1, 1 µL mL-1, 3 µL mL-1, 5 µL mL-1 e 7 µL mL-1) x three oils from Lippia sidoides Cham., Cymbopogon citratus (DC) Stapf. and Ocimum gratissimum L., with four replications. The oils were analyzed by gas chromatography with mass spectrometry. There was a significant interaction of concentration versus species on all analyzed traits. The essential oil from C. citratus yielded the highest mycelial growth inhibition at all concentrations. The other oils were not differentiated, except at the concentration of 5µL mL-1, in which the oil of L. sidoides was more efficient that the oil of O. gratissimum. In conidial production and germination, the oil of C. citratus was superior to the oil of L. sidoides and O. gratissimum at the concentrations of 1 µL mL-1 and 3 µL mL-1, as it completely inhibited the production and germination of conidia at the concentrations of 3 µL mL-1 and above. The concentrations influenced all the variables, adjusting themselves to the linear model equation. We observed a decrease in the variables, as the concentrations of the oils tested are increased. The essential oils used have antifungal and fungicidal effect.

Clonagem e análise da expressão de genes de proteínas de mamão papaia com atividade inibitória sobre poligalacturonases fúngicas / Cloning and expression analysis of papaya genes encoding proteins with inhibitory activity against fungal polygalacturonases

As proteínas inibidoras de poligalacturonases (PGIPs) presentes na parede celular são capazes de limitar o potencial destrutivo da poligalacturonase (PG) fúngica e, assim, constituem um tipo importante dentre os diversos sistemas de defesa do tecido vegetal frente à infecção fúngica. No mamão, o ataque fitopatogênico é o principal causador de danos pós-colheita, e sua alta susceptibilidade pode estar relacionada com a baixa eficácia ou pouca abundância dos meios de defesa anti-fitopatogênica. Uma vez que isso pode estar relacionado com as PGIPs e nada se conhece sobre o papel dessas proteínas nesse fruto, o objetivo do trabalho foi clonar os genes das PGIPs de mamoeiro e definir seu padrão de expressão em diferentes órgãos e tecidos e ao longo do amadurecimento. Para tanto, foram identificadas no genoma do mamoeiro, a partir de critérios que definem a identidade de uma PGIP, duas prováveis sequências dentre 13 candidatas iniciais. Ambas foram clonadas a partir das sequências genômicas e de cDNA, sequenciadas e sua identidade confirmada, sendo denominadas Cppgip4 e Cppgip6. As análises de expressão relativa em diversos tecidos e idades fisiológicas do mamoeiro demonstraram que os dois genes apresentaram diminuição da expressão com o desenvolvimento dos frutos, sendo que com a polpa apresentou redução dos níveis de expressão relativa de Cppgip4 em até 18 vezes dos 30 dias pós-antese (DPA) ao 9 dias pós-colheita (DPC). Na casca também houve redução significativa da expressão com o desenvolvimento. Para a expressão absoluta, nos frutos, sementes, caules, raízes e folhas, o número de cópias de ambos os transcritos decresceu com o desenvolvimento, sendo cerca de cem mil vezes mais abundante para Cppgip6 que para Cppgip4. As tentativas de expressão de proteínas recombinantes em Pichia pastoris não geraram resultado positivo, provavelmente em virtude das condições ideais de indução ainda não terem sido estabelecidas corretamente para o ensaio. A atividade de PGIPs extraídas diretamente do tecido foi medida por análise de difusão em ágar empregando pectinase de Aspergillus niger e revelou uma tendência à diminuição da porcentagem de inibição à medida que os frutos se desenvolveram, em concordância com os resultados da análise por qPCR. O conjunto de resultados sugere que a expressão varia com o estádio de desenvolvimento do fruto e é tecido-específica, possivelmente em resposta à diferente susceptibilidade dos tecidos ao ataque fitopatogênico, indicando que menores níveis de transcritos e atividade no amadurecimento, período de maior susceptibilidade, poderiam sinalizar para a regulação do processo degradativo marcando o início da senescência

Polygalacturonase inhibiting proteins (PGIPs) present in plant cell walls are able to inhibit the destructive action of fungal polygalacturonase (PG). In this way, they constitute an important type of plant defense system against fungal infections. In papaya fruit, the pathogenic attack is the main cause of post harvesting loss, and its high susceptibility may be related to the low efficiency or low abundance of anti-phytopathogenic defense. Since this fact could be related to PGIPs expression and little is known about the response of these proteins in the fruit, the aim of the present work was to clone the genes of PGIPs papaya fruit and set their expression pattern in different organs and tissues throughout fruit ripening. Thus, two probable PGIP sequences among 13 initial candidates were identified in the papaya genome by using specific criteria. Both sequences were cloned from cDNA and genomic samples, sequenced and confirmed its identity, and then being named Cppgip4 and Cppgip6. Analysis of relative expression in various tissues at different physiological stages demonstrated that both genes were down regulated during fruit development. The relative expression levels of Cppgip4 in papaya pulp was reduced by 18 times from the 30 days post-anthesis (DPA) to the 9 days post-harvest (DPH). Similarly, gene expression in papaya peel was significant down regulated during fruit development. Absolute expression analysis revealed gene expressions in the fruit pulp, seed, stem, root and leaf were also down regulated within development. Moreover, Cppgip6 gene expression was a hundred thousand times more abundant than Cppgip4. The recombinant protein expression in Pichia pastoris did not result positive, probably because of the ideal conditions of induction have not been properly established the yet. The activity of PGIPs extracted directly from the tissue was measured by the agar diffusion assay using pectinase from Aspergillus niger and showed decrease of inhibition during fruit developed in accordance with the results of the qPCR analysis. Based on the results it is possible to suggest the expression of these genes varies temporally with the developmental stage of the fruit and is tissue-specific, possibly in response to the different susceptibility of tissues to pathogenic attack. In addition, the lowest levels of PGIP expression were achieved at the fruit ripening, when the susceptibility to fungal infection is high and could signal for regulating the degradation process characterized by the onset of senescence

Efeito de extratos e óleos essenciais de plantas na germinação de urediniósporos de Phakopsora pachyrhizi / Effect of plant extracts and essential oils on the germination of urediniospores of Phakopsora pachyrhizi

Métodos alternativos para o controle de doenças e pragas de plantas vêm sendo pesquisados buscando causar menores danos ao ambiente e a saúde humana. Diante disso, o objetivo deste estudo foi identificar extratos de plantas e óleos essenciais com possíveis efeitos sobre Phakopsora pachyrhizi, agente etiológico da ferrugem-asiática da soja. Assim, 61 extratos de plantas da flora nativa da região do Alto Rio Grande, Minas Gerais e cinco óleos essenciais obtidos comercialmente foram utilizados com a finalidade de testar os efeitos na germinação de urediniósporos do referido fungo. Foram utilizadas placas de Petri de 6 cm, com meio ágar-água, adicionado com 150 µL de extrato de planta ou 6 µL do óleo essencial misturado ao meio de cultura. Como testemunhas foram utilizadas 150 µL de Tween 20 a 0,7% e 150 µL de água destilada. Após, foram adicionados 50µL de uma suspensão de urediniósporos, na concentração de 2mg.5mL-1 e espalhados com alça de Drigalsky. As placas foram colocadas em BOD, a 23(±2ºC), por 4 horas. A germinação foi paralisada com adição de lactoglicerol. A avaliação da percentagem de esporos germinados foi realizada em microscópio de luz. Dentre os extratos vegetais testados, dez apresentaram percentual de germinação abaixo de 15%, tendo os extratos obtidos das plantas Pelargonium sp., Salvia officinalis, Lavandula officinalis, Mentha pulegium e M. arvensis apresentado germinação de esporos de 3,0%, 3,5%, 3,0%, 2,0% e 4,0%, respectivamente. Todos os óleos essenciais avaliados foram efetivos na inibição da germinação, com destaque para o óleo essencial de Caryophilus aromaticus, que apresentou percentagem de germinação de 5%, resultando num percentual de inibição de 50,0%.

Alternative methods to control plant diseases and plagues have been studied, in order to cause less damages to the environment and human health. The purpose of this study was to identify plant extracts and essential oils as possible alternative products that affect the urediniospore germination of Phakopsora pachyrhizi, the causal agent of the Asian soybean rust. Plant extracts (61) from the native flora of the Alto Rio Grande region, State of Minas Gerais, Brazil, as well as five commercial essential oils were used with the purpose of testing their effects on the germination of urediniospores of the referred fungus. 6cm Petri dishes were used, with water-agar medium, added with 150 µL of plant extract or 6 µL of essential oil mixed to the culture medium. As control, 150 µL of Tween 20 at 0.7% and 150 µL of distilled water were used. Then, 50 µL of a suspension of urediniospores were added, at a concentration of 2mg5mL-1 and spread with Drigalsky spatula. Then, the plates were put in BOD, at 23(±2ºC), for 4 hours. Germination was paralyzed by adding lactoglicerol. The percentage of spore germination was evaluated in a light microscope. Among the vegetal extracts tested, ten of them presented a germination percentage below 15%. The extracts from the Pelargonium sp., Salvia officinalis, Lavandula officinalis, Mentha pulegium and M. arvensis plants presented 3.0%, 3.5%, 3.0%, 2.0% and 4.0% of urediniospore germination, respectively. All the essential oils evaluated were effective in inhibiting germination, especially the Caryophilus aromaticus essential oil that presented 5.0% of germination, with 50.0% of inhibition.

Hongos asociados al manchado del grano en la variedad de arroz INCA LP-5 (Oryza sativa L.) en Cuba / Fungi associated to grain spotting in the INCA LP-5 (Oryza sativa L.) variety of rice in Cuba

Se aislaron e identificaron los hongos presentes en granos de arroz manchados, de la variedad INCA LP-5, cosechados durante las épocas lluviosas de los años 2003 y 2005, en áreas de la Estación Experimental del Arroz “Los Palacios”, Pinar del Río, perteneciente al Instituto Nacional de Ciencias Agrícolas (INCA). Se determinó la incidencia y se realizaron las pruebas de patogenicidad mediante los postulados de Koch. Se identificaron 35 especies, correspondientes a 19 géneros de hongos asociados a granos manchados de la variedad INCA LP-5, y entre ellas se señalaron, como nuevos registros asociados al manchado del grano de arroz para Cuba a: Bipolaris maydis, Fusarium merismoides, F. solani, F. subglutinans, Penicillium itallicum, Phoma medicaginis var. pinodella y Syncephalastrum racemosum. Los hongos de mayor incidencia fueron Bipolaris oryzae, Sarocladium oryzae y especies de los géneros Alternaria, Fusarium, Curvularia y Phoma. Se confirmó que los hongos F. subglutinans, B. oryzae, A. padwickii, Alternaria sp., F. verticillioides, C. lunata, C. lunata var. aeria, C. pallescens, C. senegalensis, Curvularia sp., Phoma sp. y S. oryzae, son agentes causales del manchado del grano de arroz.

Isolation and identification of the fungi present in spotted rice grains of the INC LP-5 variety, harvested during the rainy seasons of the years 2003 and 2005, was done at sites of the Experimental Rice Station “Los Palacios”, Pinar del Río, belonging to the Instituto Nacional de Ciencias Agrícolas (INCA). Incidence was determined, and pathogenicity tests were carried out according to Koch’s postulates. Thirty-five species corresponding to 19 fungi genus associated to spotted grains of the INCA-LP-5 variety were identified, and among them, the following strains were indicated as new registrations associated to spotting of rice grains in Cuba: Bipolaris maydis, Fusarium merismoides, F. solani, F. subglutinans, Penicillium itallicum, Phoma medicaginis var. pinodella and Syncephalastrum racemosum. The fungi with the highest incidence were Bipolaris oryzae, Sarocladium oryzae and species of the genus Alternaria, Fusarium, Curvularia and Phoma. It was confirmed that the fungi F. subglutinans, B. oryzae, A. padwickii, Alternaria sp., F verticilloides, C. lunata, C.lunata var. aeria, C. pallescens, C. senegalensis, Curvularia sp., and S. oryzae, are causative agents of the spotting of rice grains.

Efecto del control biológico por antagonistas sobre fitopatógenos en vegetales de consumo humano / Biological control effect by antagonist on plant pathogens in vegetables for human consumption

Tomando en consideración el efecto adverso que provocan los pesticidas al medio ambiente y la salud y la posibilidad de al menos minimizar su uso, se investigó el efecto del control biológico de tres antagonistas microbianos (Trichoderma viride, Pseudomonas aeruginosa y Bacillus subtilis), sobre fitopatógenos causantes de enfermedades en vegetales como: tomate, cebolla y pimentón. Los ensayos se realizaron en semillas de estos tres rubros infectadas experimentalmente con tres fitopatógenos (Phytophthora spp, Fusarium monilliforme y Rhyzoctonia solani). Inicialmente, el efecto de los antagonistas sobre los fitopatógenos se determinó en medios de cultivo Sabouraud sólido (agar) y líquido (caldo). Posteriormente, 35 semillas de cada una de las especies vegetales seleccionadas se sometieron a diferentes tratamientos: I-Grupo control, sin ninguna adición; II-Grupo tratado con los antagonistas y III-Grupo infectado con los fitopatógenos. Todos los grupos se sembraron en tierra abonada, a cielo abierto y a temperatura ambiente y regados dos veces diariamente con agua. Adicionalmente, al grupo III se le agregó diariamente un inóculo de 0,1 mL de antagonista (10(4) UFC/mL). El desarrollo de las plantas se monitoreo por dos semanas determinándose el número de semillas germinadas, la longitud de las plántulas y el follaje. Los resultados se analizaron a través de Statistix 8.0, pruebas Tukey y Kruskal-wallis. Los antagonistas inhibieron la proliferación de los fitopatógenos en medio Sabouraud. Los experimentos con semillas demostraron una mayor estimulación de la germinación y tamaño de las plántulas en el grupo tratado con fitopatógenos y antagonistas respecto al grupo control (p<0,1).

Taking in account the negative actions of pesticides exert on natural environment and health and due to the possibility to minimize/eliminate its use, the effect of microbial antagonist (Trichoderma viride, Pseudomonas aeruginosa and Bacillus subtilis), were evaluated on tomato, onion and pimento seeds experimentally infected with three known phytopatogens (Phytophthora spp, Fusarium monilliforme and Rhyzoctonia solani). Initially, the antagonist effect on the phytopatogens were studied on Sabouraud solid (agar) and liquid (broth) media. Further 35 seeds of each vegetable were treated according to the following experimented protocol group I (control): no addition and water, group II (treated): antagonist and water, group III (infected): antagonist, water and phytopathogens. All groups were grown on soil, under sun light, at room temperature and water was added twice a day. Group III was daily infected with 0,1 mL of the antagonist suspensions (10(4) UFC/mL). The plant development was followed for 2 weeks, determining number of germinated seeds (germination index), plantule length and foliage. The results obtained was analyzed with Statistix 8.0, Tukey and Kruskal-Wallis. The antagonist inhibited the proliferation of the phytopathogens in Sabouraud media. The seed treated and infected showed a higher index of germination and plantule respect to the control and treated groups (p<0,1).

Defensinas de paantas y su uso potencial como controladores de plagas en la agricultrra / Plant Defensins and Their Potential Use as Pest Control in Agriculture

RESUMEN Las plantas, al igual que todos los organismos de la naturaleza, poseen elaborados sistemas de defensa contra patógenos, que pueden ser físicos y químicos, y producirse de forma constitutiva e inducida. Dentro de las barreras químicas inducidas se encuentra el grupo de proteínas de bajo peso molecular denominadas péptidos antimicrobianos (AMPs), al cual pertenecen las defensinas, péptidos con peso molecular entre 5 a 7 kDa, punto isoeléctrico de 9, y longitud de 45 a 55 aminoácidos; que tienen la capacidad de inhibir efectivamente el crecimiento de microorganismos fitopatógenos, en su mayoría hongos, y además, generan resistencia a condiciones abióticas de estrés en plantas. Este texto pretende realizar una descripción clara y actual de las características e investigaciones recientes con relación a las defensinas de plantas y sus más destacados usos en el control de patógenos en cultivos de importancia económica. Se plantea además la necesidad de profundizar en el conocimiento de dichas proteínas para su uso en estrategias de control tales como la producción de plantas y microorganismos transgénicos.

ABSTRACT Plants, as all organisms in nature, have elaborate systems of defense against pathogens; which can be physical or chemical and produced in a constitutive and induced way. Among the induced chemical barriers, there is a group of low molecular weight proteins, known as antimicrobial peptides (AMPs). These peptides include defensins, which are peptides with a molecular weight about 5 to 7 kDa, isoelectric point of 9, and length of about 45 to 55 amino acids. Likewise, they have the ability to avoid the growth of phytopathogenic microorganisms, mainly funguses. Moreover, these peptides create resistance to abiotic conditions of stress in plants. This manuscript seeks to make a clear and current description about the recent characteristics and researches related to plant defensins and their most significant uses in pathogens management in crops of economical relevance. It also intends to go deep into the study of such proteins in order to use them as a control strategy, such as production of transgenic plants and microorganisms.

Efecto del quitosano en el desarrollo in vitro de rhizopus stolonifer ehrenb: fr) vuill en dos medios de cultivo / Effect of chitosan on the in vitro developtment of rhizopus stolonifer (ehrenb: fr) vuill on two culture medium

En este trabajo se evaluó el efecto antifúngico del quitosano (0; 0,5; 1,0; 1,5; 2,0 mg mL-1) en el desarrollo in vitro (crecimiento micelial, formación de cuerpos fructíferos, esporulación, germinación y liberación de proteínas) de Rhizopus stolonifer en dos medios de cultivo (papa dextrosa agar y medio mínimo). Los resultados obtenidos demostraron que el quitosano inhibió el crecimiento micelial de R. stolonifer en ambos medios de cultivo. El mayor índice antifúngico se observó en el medio papa dextrosa agar. El quitosano no afectó la formación de los cuerpos fructíferos de R. stolonifer en los medios estudiados. La esporulación y la germinación de las esporas se afectaron en ambos medios de cultivo por efecto de quitosano, siendo más notable en el medio medio mínimo. Se demostró la liberación de proteínas por efecto del quitosano en medio mínimo y caldo papa dextrosa. En general, en este estudio se evidenció el efecto antifúngico del quitosano en el desarrollo in vitro de Rhizopus stolonifer con independencia del medio de cultivo empleado. Sin embargo, en medio medio mínimo podrían observarse mejor los efectos antifúngicos del quitosano.

In this work the antifungal effect of chitosan (0, 0.5, 1.0, 1.5, 2.0 mg mL-1) on the in vitro development (mycelial growth, fruit bodies formation, sporulation, germination and proteins release) of Rhizopus stolonifer on two culture medium (Potato Dextrose Agar and Minimal medium) was evaluated. The obtained results demonstrated that chitosan inhibited the mycelial growth of R. stolonifer in both culture medium. The highest antifungal effect was observed on potato dextrose agar medium. Chitosan was not affected the fruit bodies formation of R. stolonifer on the studied medium. Sporulation and spore germination were affected in both culture mediun by effect of chitosan, it was more noticeable in minimal medium. It was demonstrated proteins release by effect of chitosan in minimal medium and potato dextrose broth medium. In general, in this study it was showed the antifungal effect of chitosan on in vitro development of Rhizopus stolonifer regardless of the culture medium used. However, in minimal medium could be observed best the antifungal effects of chitosan.

Actividad antifúngica de Burkholderia cepacia aislada de maíz amarillo (Zea mays L.) bajo diferentes condiciones de cultivo / Antifungal activity of Burkholderia cepacia isolated from yellow corn (Zea mays L.) under different culture conditions

Se evaluó la actividad antifúngica in vitro de 4 cepas de Burkholderia cepacia aisladas de rizósfera de maíz amarillo (Zea mays L.) de diferentes plantaciones del municipio Montes, estado Sucre, Venezuela, contra hongos fitopatógenos. Se comparó la actividad antagónica con una cepa control de origen ambiental CVCM 626 (ATCC 25416) y un aislado clínico CVCM 1282, empleando el método directo de enfrentamientos duales y un método indirecto de difusión con discos de agar. Las pruebas se realizaron por triplicado, se midió el diámetro de los halos de inhibición formados y se aplicó un análisis de varianza de una vía, así como la prueba Tukey. Las cuatro cepas aisladas (SR2, ER3, LG12 y N10) mostraron una actividad antifúngica similar sobre Trichoderma viride y ocasionaron una inhibición total o parcial de la esporulación en hongos como Fusarium moniliforme, Fusarium solani, Aspergillus niger y Penicillium expansum. Los mayores halos de inhibición fueron producidos por la cepa control CVCM 626, observándose un promedio de 9,9 mm. No se evidenció ningún nivel de antagonismo utilizando el aislado clínico CVCM 1282. B. cepacia mostró un aumento de la actividad antagónica en presencia de algunas fuentes de carbono como xilosa, maltosa, manosa y fructosa y de aminoácidos como cistina, valina y leucina, en un rango de pH ácido (4 a 5,5), y a una temperatura de 35 ºC.

The antifungal activity against phytophatogenic fungi of four Burkholderia cepacia strains isolated from yellow corn (Zea mays L.) from different plantations at the Montes Municipality, Sucre State, Venezuela, was evaluated in vitro. The antagonist activity was compared by using a control strain of environmental origin CVCM 626 (ATCC 25416) and one clinical isolate CVM 1282, with the direct dual confrontation method, and an indirect method with agar-diffusion disks. The tests were done in triplicate, measuring the inhibition halos that occurred, and applying a one way variance analysis, as well the Tukey test. The four isolated strains (SR2, ER3, LG12 and N10) showed similar antifungal activity over Trichoderma viride and produced a total or partial inhibition of sporulation of fungi such as Fusarium moniliforme, Fusarium solani, Aspergillus niger and Penicillium expansum. The largest inhibition halos were produced by the control strain CVCM 626, with a 9,9 mm average. There was no evidence of any antagonism level using the clinical isolate CVCM 1282. Burkholderia cepacia showed an increase of antagonist activity in presence of some carbon sources such as xilose, maltose, mannose, and fructose and of amino-acids such as cystine, valine and leucine, in an acid pH range (4 to 5.5), and a 35ºC temperature.

Potencial fungitóxico do óleo essencial de Piper hispidinervum (pimenta longa) sobre os fungos fitopatogênicos Bipolaris sorokiniana, Fusarium oxysporum e Colletotrichum gloeosporioides / Fungitoxic potential of the essential oil the Piper hispidinervum (long-pepper) against phytopathogenic fungi Bipolaris sorokiniana, Fusarium oxysporum e Colletotrichum gloeosporioides

O objetivo deste trabalho foi avaliar a atividade fungicida in vitro do óleo essencial das folhas de Piper hispidinervum sobre Bipolaris sorokiniana, Fusarium oxysporum e Colletotrichum gloeosporioides. Para os ensaios biológicos, empregou-se o teste bioanalítico in vitro utilizando as concentrações de 100, 200, 500, 1000, 1500 e 2000 µg.mL-1 do óleo essencial. Estas foram incorporadas no meio de cultura BDA (batata-dextrose-ágar) para avaliação do crescimento ou inibição micelial. O delineamento estatístico utilizado foi o inteiramente casualizado, com quatro repetições. Na concentração de 200 µg.mL-1, observou-se uma inibição total do fitopatógeno Bipolaris sorokiniana enquanto que, para o Fusarium oxysporum e o Colletotrichum gloeosporioides esta ocorreu na concentração de 1000 µg.mL-1.

The objective of this study was to evaluate the in vitro antifungal activity of the essential oil of the leaves of Piper hispidinervum against Bipolaris sorokiniana, Fusarium oxysporum and Colletotrichum gloeosporioides. For the biological tests, using the bioanalitic test in vitro the concentrations of 100, 200, 500, 1000, 1500 and 2000 µg.mL-1 the essential oil. This were incorporated into PDA (potato dextrose agar) medium in order to evaluate fungal mycelial growth or inhibition. The statistic design used was completely randomized, with four replicates. In the concentration of 200 µg.mL-1, observed inhibited complete the phytopathogens Bipolaris sorokiniana while the Fusarium oxysporum and Colletotrichum gloeosporioides this is occurred in the concentration of 1000 µg.mL-1.

IDENTIFICACIÓN DEL AGENTE CAUSAL DE UNA BACTERIOSIS EN ÑAME (Dioscorea alata L.) / ISOLATION AND IDENTIFICATION OF A PHYTOPATHOGENIC BACTERIA FROM WATER YAM (Dioscorea alata L.) / IDENTIFICAÇÃO DO AGENTE CAUSAL DE UMA BACTERIOSE NO INHAME (Dioscorea alata L.)

El ñame (Dioscorea alata L.), ampliamente usado como fuente de calorías en África, Asia y el Caribe, es una planta herbácea y trepadora que se reproduce generalmente de forma vegetativa y produce un tubérculo comestible de gran tamaño con alto contenido de almidón. Varios microorganismos fitopatógenos, incluyendo virus, hongos y bacterias, afectan al cultivo. Plantas enfermas de la zona de Guarataro, estado Bolívar, Venezuela, fueron colectadas en el 2003, año en que hubo ~80% de pérdidas en la producción de ñame de la zona a causa de una enfermedad desconocida. Hojas colectadas de plantas enfermas con lesiones acuosas y necróticas fueron esterilizadas superficialmente y transferidas a solución salina para permitir la difusión de bacterias desde los bordes del tejido y ser luego sembradas en medio LB agar. Como controles se emplearon agua y medio LB. Colonias amarillas, brillantes y lisas, fueron visibles a las 24-48h de incubación a 30ºC. Varios aislados fueron inoculados en las pruebas de hipersensibilidad en tabaco y de patogenicidad en plántulas de ñame cultivadas in vitro. Varios grados de respuesta de hipersensibilidad en tabaco fueron observados. Las plántulas de ñame inoculadas mostraron lesiones acuosas en la lámina foliar, las cuales no se observaron en los controles. Aislados bacterianos fueron observados mediante microscopía de luz, revelando bacterias bacilares Gram negativas. Varios flagelos peritricos fueron observados por microscopía electrónica. Los resultados de las pruebas bioquímicas y fisiológicas indicaron que los aislados corresponden a Pantoea agglomerans (Enterobacteriacea). Se discuten diferentes metodologías para la identificación de bacterias.

Water yam (Dioscorea alata L.), a staple crop in Africa, Asia and the Caribbean, is a herbaceous twining vine plant generally reproduced by vegetative sections and highly affected by several phytopathogenic organisms including virus, fungi and bacteria. Diseased plants were collected from Guarataro, Bolívar State, Venezuela, in 2003. That year an unknown disease led to ~80% loss of water yam production. The leaves from diseased plants have watery or necrotic lesions on the leaf lamina. Pieces of necrotic tissue were surface sterilized, then transferred to saline solution and the bacteria allowed to diffuse into the solution from the edge of the dissected tissue. The bacteria were isolated on LB agar media. Yellow, shining, smooth colonies with regular margins were visible within 24-48h of incubation at 30ºC. Several isolates were inoculated on tobacco plants. Water and LB media were used as negative controls. Several degrees of hypersensitivity response resulted from the inoculation of tobacco. However, inoculation of in vitro water yam plants showed watering lesions on the leaf lamina inoculated with bacteria cultures and no lesions on water and media inoculated leaves. Bacterial cells observed by light microscopy showed Gram-negative regular rods. Several peritricous flagella were observed under EM. Data from biochemical and physiological tests indicated that the isolates belong to the Enterobacteriaceas family specifically to Pantoea agglomerans. Different methodologies for bacterial identification are discussed.

O inhame (Dioscorea alata L.), amplamente usado como fonte de calorias na África, Ásia e no Caribe, é uma planta herbácea e trepadeira que se reproduz geralmente de forma vegetativa e produz um tubérculo comestível de grande tamanho com alto conteúdo de amido. Vários microorganismos fitopatógenos, incluindo vírus, fungos e bactérias, afetam ao cultivo. Plantas enfermas da zona de Guarataro, estado Bolívar, Venezuela, foram coletadas em 2003, ano em que houve ~80% de perdas na produção de inhame dessa zona a causa de uma enfermidade desconhecida. Folhas coletadas de plantas enfermas com lesões aquosas e necróticas foram esterilizadas superficialmente e transferidas a uma solução salina para permitir a difusão de bactérias desde as bordas do tecido e ser logo plantadas em meio LBA. Como controles foram empregados água e meio LBA. Colônias amarelas, brilhantes e lisas, foram visíveis às 24-48h de incubação a 30ºC. Vários isolados foram inoculados nas provas de hipersensibilidade em tabaco e de patogenicidade em plântulas de inhame cultivadas in vitro. Vários graus de resposta de hipersensibilidade em tabaco foram observados. As de plântulas de inhame inoculadas mostraram lesões aquosas na lâmina foliar, as quais não se observaram nos controles. Isolados bacterianos foram observados mediante microscopia de luz, revelando bactérias bacilares Gram negativas. Vários flagelos perítricos foram observados por microscopia eletrônica. Os resultados das provas bioquímicas e fisiológicas indicaram que os isolados correspondem a Pantoea agglomerans (Enterobacteriacea). Discutem-se diferentes metodologias para a identificação de.