In vitro antifungal activity of Myrcia ovata essential oils and their major compounds against pathogens of citrus, sweet potato, and coconut / Atividade antifungica in vitro de óleos essenciais de myrcia ovata e seus compostos majoritários sobre patógenos de citros, batata-doce e coqueiro

Myrcia ovata, an endemic species to the Brazilian Atlantic Forest, presents antifungal properties. The phytopathogens Colletotrichum acutatum, Plenodomus destruens,and Thielaviopsis paradoxa are responsible for the diseases citrus postbloom fruit drop, sweet potato foot rot, and coconut stem bleeding, respectively. The antifungal activity of the essential oils of five M. ovata chemotypes (MYRO-159, nerolic acid chemotype; MYRO-180, nerolic acid + linalool chemotype; MYRO-388, geraniol chemotype; MYRO-157, citral + (E)-nerolidol chemotype; and MYRO-174, isopulegol + linalool chemotype), four major compounds (nerolic acid, nerolic acid + linalool, geraniol, and citral + (E)-nerolidol), and threepure compounds (citral, (E)-nerolidol, and linalool) against the fungi C. acutatum, P. destruens,and T. paradoxawere evaluated. For this, in vitro tests were conducted in a completely randomized design with three replications, testing concentrations (v/v) ranging from 0.01 to 1.0 µL.mL-1. All treatments presented toxicity at different levels to the three fungi. For C. acutatum,the essential oil from the individual MYRO-180 (nerolic acid + linalool chemotype) and its major compound showed the lowest Minimal Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of 0.03 and 0.1 µL.mL-1, respectively. For P. destruens, the essential oil from the individual MYRO-159 (nerolic acid chemotype) presented the lowest MIC of 0.05 µL.mL-1. The nerolic acid + linalool chemotype and its major compound presented an MFC of 0.07 µL.mL-1. For T. paradoxa, the major compound citral + (E)-nerolidol stood out with the lowest MIC and MFC of 0.03 and 0.2 µL.mL-1, respectively. Linalool presented the lowest toxicity to the three tested fungi.

Myrcia ovata, uma espécie nativa de fitofisionomia de Restinga, possui atividade antifúngica. Os fitopatógenos Colletotrichum acutatum, Plenodomus destruens e Thielaviopsis paradoxa são responsáveis pelas doenças podridão floral de citros, mal-do-pé da batata doce e resinose do coqueiro, respectivamente. A atividade antifúngica de cinco quimiotipos de M. ovata (MYRO-159, quimiotipo ácido nerólico; MYRO-180, ácido nerólico + linalol; MYRO-388, quimiotipo geraniol; MYRO-157, quimiotipo citral + (E)-nerolidol; e, MYRO-174, quimiotipo isopulegol + linalol), quatro compostos majoritários (ácido nerólico, ácido nerólico + linalol, geraniol e citral + (E)-nerolidol) e três compostos isolados (citral, (E)-nerolidol e linalol) foram avaliados sobre os fungos C. acutatum, P. destruens e T. paradoxa. Testes in vitro foram conduzidos em delineamento inteiramente casualizado com três repetições e concentrações (v/v), que variaram de 0,01 a 1,0 µL.mL-1. Todos os tratamentos testados apresentaram atividade antifúngica. Para o fungo C. acutatum, o óleo essencial do indivíduo MYRO-180, de quimiotipo ácido nerólico + linalol, e seu composto majoritário apresentaram menores Concentração Mínima Inibitória (CMI) e Concentração Mínima Fungicida (CMF) de 0,03 e 0,1 µL.mL-1, respectivamente. Para o fungo P. destruens, o óleo essencial do indivíduo MYRO-159, de quimiotipo ácido nerólico, apresentou menor CMI de 0,05 µL.mL-1, e o quimiotipo ácido nerólico + linalol e seu composto majoritário apresentaram a menor CMF de 0,07 µL.mL-1. Para o fungo T. paradoxa,a combinação de citral + (E)-nerolidol destacou-se com CMI e CMF de 0,03 e 0,2 µL.mL-1, respectivamente. Linalol foi o menos tóxico sobre os três fungos testados.

A diagramatic scale to quantify severity of antracnose in Rubus glaucus Benth / Uma escala diagramática para quantificar a severidade da antracnose em Rubus glaucus Benth

ABSTRACT: Rubus glaucus Benth, commonly referred to as mora de Castilla, is affected by Colletotrichum acutatum, as it induces anthracnose in many of the plant organs. Generally, it affects the fruits during the post-harvest phase and damages them, causing economic losses due to the poor crop quality. At present, no standardized methods are available for this pathosystem that can be used to characterize quantitatively the epidemic and to permit the prediction and comparison of the disease management techniques. In this research, we proposed a logarithmic diagrammatic scale of the severity of anthracnose induced by C. acutatum in the fruits of the thornless variety of R. glaucus Benth. This scale is constructed on the adjustment of the Weber-Fechner law and includes six classes, viz., 0%, 1-6%, 7-25%, 26-50%, 51-75%, 76-100%. The scale was validated using 14 evaluators, which entailed measuring the affected fruits with and without utilizing the scale; this improved the precision, accuracy and reproducibility of the calculations whenever the scale was used. We concluded that the scale proposed can be used to assess the severity of anthracnose induced by the fungus in the R. glaucus Benth fruits.

RESUMO: Mora de Castilla (Rubus glaucus Benth) é afetado por Colletotrichum acutatum, causando antracnose em órgãos diferentes da planta. Nos frutos geralmente é em pós-colheita, causando danos que geram perdas econômicas relacionadas com a qualidade das culturas. Ainda não existem para esse patossistema métodos padronizados para caracterizar quantitativamente a epidemia e que pode prever e comparar os métodos de manejo da doença. Por esta razão, neste trabalho foi criada uma escala logarítmica diagramática da severidade da antracnose causada pelo fungo C. acutatum em frutos de R. glaucus Benth com base na lei de Weber-Fechnere composto por 6 niveis: 0%, 1-6%, 7-25%, 26-50%, 51-75% e 76-100%. A escala foi validada por 14 evaluadores, fazendo medições de frutos afetados com e sem o uso da escala, que mostrou melhor precisão, exatitude e reproducibilidade nas avaliações em que o uso da escala foi feito. Isto permite concluir que a escala proposta pode ser usada na estimação da severidade da antracnose causada em frutos de R. glaucus Benth.

Metabolism and antifungal activity of safrole, dillapiole, and derivatives against botryodiplodia theobromae and colletotrichum acutatum / Metabolismo y actividad antifúngica de safrol, dilapiol, y derivados contra botryodiplodia theobromae y colletotrichum acutatum

The direct in vitro fungitoxicity and metabolism of safrole and dillapiole (isolated from Piper auritum and Piper holtonii, respectively) by Botryodiplodia theobromae and Colletotrichum acutatum were investigated. Higher values of mycelial growth inhibition for both fungi were obtained for dillapiole, as compared with safrole. B. theobromae was able to metabolize both compounds to their respective vicinal diols, reaching 65 percent relative abundance during the biotransformation of dillapiole; while C. acutatum only transformed safrole to various metabolites with relative abundances under 5 percent. According to the low antifungal activity of the major metabolic products (< 5 percent for vicinal diols), a detoxification process was implied. Studies on the influence of some substituents in the aromatic ring of safrole and dillapiole on the antifungal activity against B. theobromae were also carried out. As result, the safrole nitrated derivative, 6-nitrosafrole, showed a fungitoxicity level similar to that displayed by the commercial fungicide Carbendazim® under the conditions used. In light of this, safrole and dillapiole could be suggested as feasible structural templates for developing new antifungal agents.

Se investigó la fungitoxicidad directa in vitro y el metabolismo de safrol y dilapiol (obtenidos desde Piper auritum and Piper holtonii, respectivamente) por Botryodiplodia theobromae y Colletotrichum acutatum. Los valores mayores de inhibición del crecimiento micelial de ambos hongos se obtuvieron para dilapiol, en comparación con safrol. B. theobromae metabolizó ambos compuestos a sus respectivos dioles vecinales, alcanzando abundancias relativas del 65 por ciento durante la biotransformación del dilapiol; mientras que C. acutatum solo transformó safrol en varios metabolitos con abundancias relativas menores al 5 por ciento. De acuerdo con la baja actividad antifúngica de los productos metabólicos mayoritarios (< 5 por ciento para los dioles vecinales), se sugiere un proceso de desintoxicación. Adicionalmente, se evaluó la influencia de algunos sustituyentes en el anillo aromático de safrol y dilapiol sobre la actividad antifúngica contra B. theobromae. Como resultado, el derivado nitrado del safrol, el 6–nitro safrol, presentó un nivel de fungitoxicidad similar al exhibido por el fungicida comercial Carbendazim® bajo las condiciones usadas. A la luz de lo anterior, safrol y dilapiol podrían ser sugeridos como plantillas estructurales adecuadas para el desarrollo de nuevos agentes antifúngicos.

Application of Rhizobacteria for Plant Growth Promotion Effect and Biocontrol of Anthracnose Caused by Colletotrichum acutatum on Pepper

In vitro and greenhouse screening of seven rhizobacterial isolates, AB05, AB10, AB11, AB12, AB14, AB15 and AB17, was conducted to investigate the plant growth promoting activities and inhibition against anthracnose caused by Colletotrichum acutatum in pepper. According to identification based on 16S rDNA sequencing, the majority of the isolates are members of Bacillus and a single isolate belongs to the genus Paenibacillus. All seven bacterial isolates were capable of inhibiting C. acutatum to various degrees. The results primarily showed that antibiotic substances produced by the selected bacteria were effective and resulted in strong antifungal activity against the fungi. However, isolate AB15 was the most effective bacterial strain, with the potential to suppress more than 50% mycelial growth of C. acutatum in vitro. Moreover, antibiotics from Paenibacillus polymyxa (AB15) and volatile compounds from Bacillus subtilis (AB14) exerted efficient antagonistic activity against the pathogens in a dual culture assay. In vivo suppression activity of selected bacteria was also analyzed in a greenhouse with the reference to their prominent in vitro antagonism efficacy. Induced systemic resistance in pepper against C. acutatum was also observed under greenhouse conditions. Where, isolate AB15 was found to be the most effective bacterial strain at suppressing pepper anthracnose under greenhouse conditions. Moreover, four isolates, AB10, AB12, AB15, and AB17, were identified as the most effective growth promoting bacteria under greenhouse conditions, with AB17 inducing the greatest enhancement of pepper growth.

INDUCCIÓN DE ACTIVIDAD PEROXIDASA Y DE FENOLES TOTALES COMO RESPUESTA DEL FRUTO DE LULO (Solatium quitoense L.) AL PATÓGENO CAUSAL DE LA ANTRACNOSIS / INDUCTION OF PEROXIDASE AND TOTAL PHENOLS AS A RESPONSE OF LULO (Solanum quitoense L.) FRUIT TO PATHOGEN CAUSING ANTHRACNOSE / INDUÇÃO DA ACTMDADE PEROXIDASA E FENÓIS TOTAIS COMO RESPOSTA DO FRUTO DE LULO (Solanum quitoense L.) AO PATÓGENO QUE CAUSA ANTRACNOSE

Se evaluó la inducción de peroxidasa en frutos de lulo con el fin de determinar su participación en las respuestas bioquímicas hacia el patógeno Colletotrichum acutatum, causante de la antracnosis. Se establecieron como mejores condiciones para su extracción y para determinación de la actividad: buffer fosfatos 100 mM pH 7, 1% SDS y 1 % PVPP; sustrato guayacol 15 mM, peróxido de hidrógeno 10 mM, pH 6,5, 55 °C y 30 µL de extracto. Se realizó un ensayo in vivo usando frutos verdes, pintones y maduros, inoculados con el hongo o con agua estéril. Se determinó la actividad peroxidasa a diferentes horas a partir de la inoculación, encontrándose una respuesta diferencial con el tiempo por efecto de la presencia del patógeno, y según el estado de madurez de los frutos. En lulos verdes inoculados con el hongo se observó aumento en la actividad al cabo de 6 y 144 horas. En lulos pintones no se observó efecto notable, mientras que en maduros el aumento en actividad fue prácticamente a todos los tiempos. Los resultados del contenido de fenoles totales mostraron que hubo acumulación a 96 y 144 horas por efecto del patógeno, para lulos en estado verde y maduro, mientras que para pintones, en los que se presentaron más rápido y con mayor severidad los síntomas de la antracnosis, no se observó aumento a ninguno de los tiempos. En los frutos más enfermos, el cambio en actividad peroxidasa y en contenido total de fenoles fue menos evidente, por lo que se sugiere una relación inversa de estos con el desarrollo de la antracnosis.

The induction of peroxidase in lulo fruits was evaluated in order to determine its participation in the biochemical responses towards the pathogen Colletotrichum acutatum, which causes the antrachnosis disease. For extraction and activity determination, these conditions were established as the best: buffer phosphates 100 mM pH 7, 1% SDS y 1 % PVPP; substrate guaiacol 15 mM, hydrogen peroxide 10 mM, pH 6,5, 55 °C and 30 µL of the extract. An in vivo test was developed using unripe, semi-ripe and ripe fruits, inoculated with the fungus or sterile water. The peroxidase activity was measured at different hours starting from the inoculation, finding a time differential response caused by the pathogen presence and according to the maturity state of fruits; in unripe lulo fruits inoculated with the fungus, an increase of the activity was observed after 6 and 144 hours. In semi-ripe fruits no considerable effect was seen, while in ripe fruits the increase of the activity was found practically at all times. The results of the measured total phenol content, showed accumulation at 96 and 144 hours as a result of the pathogen presence in unripe and ripe fruits, while for semi-ripe fruits, in which the antrachnosis symptoms were noticed faster and more severely, no phenol increase was found at any time. The less evident changes seen in peroxidase and phenol content, using severely affected fruits by the disease, suggest an inverse relationship between these parameters and the development of the antrachnosis.

A indução da peroxidasa foi avaliada na casca de frutos de lulo com a finalidade de determinar a sua possível participação em reacções de defesa contra o patógeno Colletotrichum acutatum, causador da doença antracnose. Foram estabelecidas as melhores condições para a sua extracção e para medir a actividade da enzima extraída, encontrando-se que com tampão fosfato 100 mM pH 7, 1% SDS y 1% PVPP, substrato guaicol 15 mM, peróxido de hidrogeno 10 mM, pH 6,5, 55 °C y 30 µL de extracto enzimático se conseguiram as actividades enzimáticas mais elevadas. Foi realizado um ensaio in vivo usando frutos verdes, em processo de maduração e maduros, inoculados com o fungo ou com água estéril. A actividade peroxidasa foi determinada a diferentes horas a partir da inoculação encontrando-se uma resposta diferencial com o tempo pelo efeito da presença ao patógeno e de acordo ao estado de madurez dos frutos. Em lulos verdes observou-se um aumento da actividade nos frutos inoculados com o fungo ao fim de 6 e 144 horas. Em lulos em processo de maduração, o patógeno não teve maior efeito, enquanto que em lulos maduros o aumento na actividade da enzima, como resposta à presença do patógeno, foi praticamente a todos os tempos avaliados. Os resultados do conteúdo de fenóis totais mostraram que ouve uma acumulação significativa a 96 e 144 horas por efeito da inoculação com o patógeno para lulos em estado verde e maduro, enquanto que para os lulos em processo de maduração, nos quais se apresentaram mais rápido e com maior severidade os sintomas de antracnose, não se observou aumento em nenhum dos tempo avaliados. Nos frutos mais afectados pelos sintomas, a mudança na actividade POD e no conteúdo total de fenóis foi menos evidente, motivo pelo qual se sugere uma relação inversa de estes com o desenvolvimento de antracnose.

Occurrence of Anthracnose on Highbush Blueberry Caused by Colletotrichum Species in Korea

A total of 82 isolates of Colletotrichum species were obtained from anthracnose symptoms of highbush blueberry trees grown in the Gochang area of Korea during a disease survey in 2008. Out of the isolates, 75 were identified as Colletotrichum gloeosporioides and the others as C. acutatum based on their morphological and cultural characteristics. Twenty six of C. gloeosporioides isolates produced their teleomorph Glomerella cingulata in PDA culture. Three isolates of each C. gloeosporioides and C. acutatum caused anthracnose symptoms on the leaves by artificial inoculation, which were similar to what was observed in the orchards. Previously in Korea, only C. gloeosporioides has been reported as causing anthracnose in blueberries. This is the first report that C. acutatum causes anthracnose in the highbush blueberry in Korea.

INDUCCIÓN DE POLIFENOLOXIDASA EN FRUTOS DE LULO (Solanum quitoense) COMO RESPUESTA A LA INFECCIÓN CON Colletotrichum acutatum / Polyphenol Oxidase Induction in Lulo Fruits (Solanum quitoense) Infected by Colletotrichum acutatum

Se evaluó la actividad polifenoloxidasa (PFO) en corteza de frutos de lulo con el fin de determinar su participación en respuestas hacia el patógeno Colletotrichum acutatum, causante de la antracnosis. Se estudiaron condiciones para la adecuada extracción de esta enzima, encontrándose que con buffer fosfatos 100 mM pH 7, 1% SDS y 1% PVPP se logran las mayores actividades. Se determinaron como mejores parámetros para medir la actividad de la enzima extraída, sustrato catecol 40 mM, pH 7,0, 23 °C y 30 µL de extracto. Para determinar su posible inducción en la interacción con el patógeno, se realizó un ensayo in vivo usando frutos verdes, pintones y maduros, inoculados con el hongo o con agua estéril. A nueve tiempos postinoculación se determinó la actividad PFO encontrándose que hay una respuesta diferencial con el tiempo y la madurez de los frutos y por efecto del patógeno. Se obtuvo aumento de actividad en lulos verdes a 48, 96 y 144 horas postinoculación (hpi) y en maduros a la mayoría de los tiempos evaluados, siendo éste estado en el que se presentó la respuesta más notable de inducción. En pintones aumentó solo a 72 y 144 hpi. Los mayores valores se registraron en general para frutos en estado verde. Los frutos respondieron al estrés ocasionado por la herida activando también esta enzima. La inducción de actividad se presentó a tiempos más rápidos en los frutos menos afectados por la enfermedad (verdes y maduros), por lo que se puede postular una relación positiva entre inducción de PFO y respuesta de tolerancia a la antracnosis.

Polyphenol oxidase (PFO) activity induction was evaluated in lulo fruits to determine the role of this enzyme in resistance responses towards the pathogen Colletotrichum acutatum which causes anthracnose disease. We studied the experimental conditions to obtain the enzyme, using lulo peel, and found that extraction with phosphates buffer 100 mM pH 7, 1% SDS y 1% PVPP showed higher activities. The adequate parameters for activity measurement was also evaluated and established as cathecol 40 mM, pH 7,0, 23 °C and 30 µL of extract. Then, we performed an in vivo assay using lulo fruits in three maturity stages, green, semimature and mature, which were inoculated with the fungus or with sterile water. Enzymatic induction of this protein was studied at nine postinoculation times, and it was found that the induction was differential according to the time, the maturity stage, and as consequence of pathogen presence. The PFO activity increased in green fruits at 48, 96 y 144 (hours postinoculation (hpi), and in mature lulos for the majority of times studied, with the most significant induction response at this stage. In semimature lulo, the induction was observed only at 72 and 144 hpi. The highest nominal value of activity was found in green fruits. Fruits responded to incision with enzyme activation. The increase in the activity of the enzyme was faster in the fruits with the minor anthracnose symptoms than the ones that were more affected. Therefore, it is possible to postulate a positive relation between PFO induction and tolerance to anthracnose symptoms.

Occurrence of Anthracnose on Fruits of Asian Pear Tree Caused by Colletotrichum acutatum

Anthracnose symptoms often occurred on fruits of Asian pear trees grown in Anseong, Naju, Seonghwan and Pyeongtaek in Korea during the harvesting period from 2000 to 2005. A total of 28 isolates of Colletotrichum sp. were obtained from the anthracnose symptoms. All the isolates were identified as Colletotrichum acutatum based on their morphological and cultural characteristics. Four isolates of the fungus were tested for pathogenicity to fruits of Asian pear tree by artificial inoculation. All the isolates induced anthracnose symptoms on the fruits by wound inoculation but not by unwound inoculation. The anthracnose symptoms induced by artificial inoculation were similar to those observed in the orchard. This is the first report of anthracnose of Asian pear tree caused by Colletotrichum acutatum.

Contribuição ao estudo da atividade antifúngica de Maytenus ilicifolia Mart. ex Reiss., Celastraceae / Study of antifungal activity of Maytenus ilicifolia Mart. ex Reiss., Celastraceae

Maytenus ilicifolia Mart. ex Reiss, Celastraceae, espinheira-santa, é nativa em muitas partes da América do Sul, sendo encontrada no sul do Brasil. É utilizada no tratamento de gastrite, úlceras e outras desordens do estômago, na forma de decocção das folhas ou extrato liofilizado em cápsulas. Neste trabalho, estudou-se a atividade antifúngica do seu extrato etanólico sobre o crescimento micelial dos fungos fitopatogênicos Colletotrichum acutatum, Fusarium oxysporum e Cylindrocladium spathulatum. Na comparação do crescimento micelial de isolados desses fungos em BDA (Batata Dextrose Agar), adicionados do extrato etanólico da espinheira-santa, foi observado que o mesmo inibiu em mais de 10 por cento o crescimento micelial de Fusarium oxysporum, nas três concentrações avaliadas (0,2; 0,4 e 0,6mg/mL), estimulou o crescimento micelial de Colletotrichum acutatum em mais de 30 por cento na concentração de 0,2mg/mL, e algumas de suas frações inibiram o desenvolvimento de Cylindrocladium spathulatum.

Study of antifungal activity of Maytenus ilicifolia Mart. ex Reiss., Celastraceae. Maytenus ilicifolia Mart. ex Reiss., Celastraceae, known as espinheira-santa, is native to South America, and in southern Brazil its leaves decoction is traditionally used for the treatment of gastritis, ulcers and other gastrointestinal disorders. The objective of this work was to investigate the effect of the crude ethanolic extract of espinheira-santa on the mycelial growth of three phytopathogenical fungi: Colletotrichum acutatum, Fusarium oxysporum and Cylindrocladium spathulatum. It was observed that this extract inhibited in more than 10 percent the mycelial growth of Fusarium oxysporum at all used concentrations (0.2; 0.4 and 0.6 mg/mL), stimulated the Colletotrichum acutatum mycelial growth in more than 30 percent at 0.2 mg/mL and some of its fractions inhibited the Cylindrocladium spathulatum development.