Phaseolin Induction on Common-Bean Cultivars and Biological Control of Colletotrichum lindemuthianum 89 Race by Baccharis trimera (Less) Dc

Abstract The objective of this work was to evaluate the fungitoxic effect of the aqueous extracts of Baccharis trimera on the mycelial growth of Colletotrichum lindemuthianum 89 race, as well as its effect on the accumulation of phaseolin in hypocotyls of different cultivars and common bean varieties. It was obtained 20% aqueous extract from plants collected in municipalities of the Western Region of Paraná. Blocks containing C. lindemuthianum mycelium were transferred to Petri dishes containing medium with the different extracts and incubated at 25 °C. The colonies diameter was measured until the 12th day. Effects of aqueous extracts on phaseolin production was evaluated in hypocotyls of Carioca, Cnpf 8104, Soberana, Tibatã, Uirapurú cultivars, as well as Rosinha and Vermelho varieties. Each one cultivar and variety hypocotyl was transferred separately to test tubes containing 500 μL of 20% aqueous extracts. Sterile water, Bion®, and UV was used as controls. The phaseolin production was measured in spectrophotometer [280 nm]. Results of the evaluation of the antifungal activity of aqueous extracts of Baccharis sp. specimens collected indicate that approximately 50% of the samples presented capacity to reduce between 74 and 92% of C. lindemuthianum growth. Cultivar Tibatã and Vermelho variety showed greater sensitivity over the applied treatments. Results of fungal filtrates and vegetal aqueous extracts presented a low capacity to induce the production of phaseolin in hypocotyls.

Influência de diferentes extratos aquosos de plantas medicinais no desenvolvimento de Colletotrichum gloeosporioides e de Fusarium moniliforme / Influence of different aqueous extracts of medicinal plants in the development of Colletotrichum gloeosporioides and Fusarium moniliforme

O objetivo deste trabalho foi avaliar a influência dos extratos aquosos das plantas medicinais alecrim, alho, cravo-da-índia, sálvia, capim-limão, orégano ou pimenta-do-reino no desenvolvimento in vitro de Colletotrichum gloeosporioides e de Fusarium moniliforme. Os extratos foram obtidos pela infusão de 60 g de cada planta medicinal em 200 mL de água fervente. Cada extrato aquoso foi fracionado em concentrações de 0, 5, 10 e 20% (p:v) e incorporado ao meio de cultivo BDA (batata-dextrose-ágar) antes da esterilização em autoclave. Posteriormente, um disco de 8 mm de diâmetro de micélio fúngico de cada patógeno foi transferido para o centro de placas de Petri. Após 24, 48 e 96 horas de incubação em câmara de crescimento a 22 ± 2 ºC e fotoperíodo de 12 horas avaliou-se o crescimento micelial de F. moniliforme e de C. gloesporioides. No último período de incubação, também se quantificou o número de conídios de cada fungo. Para o teste de germinação adicionou-se nas cavidades de placas de teste Elisa, uma alíquota de 40 µL de cada extrato nas concentrações de 0, 5, 10 e 20%, e outra alíquota, da suspensão de conídios de cada patógeno. Após 24 horas a 22 ± 2 ºC, no escuro, a germinação dos conídios foi paralisada com a adição de 20 µL de lactofenol; avaliou-se então a porcentagem de germinação de conídios. Os experimentos foram conduzidos no delineamento inteiramente casualizado em esquema fatorial 7 x 4 (extratos de plantas medicinais x concentrações) com quatro repetições. Para ambos os patógenos o extrato aquoso de alho e cravo-da-índia apresentaram maior ou total inibição do crescimento micelial, respectivamente, quando comparado com os demais extratos. Para C. gloeosporioides, o extrato de cravo-da-índia apresentou menor número de conídios em todas as concentrações testadas, e para o extrato de alho a 20%, também não foi observada a germinação de conídios. O extrato de alho foi eficiente em reduzir o número e a germinação dos conídios de F. moniliforme na concentração de 20%. Os extratos de alecrim, cravo-da-índia, orégano e pimenta-do-reino, nas maiores concentrações, tiveram efeito positivo na redução da produção de conídios deste mesmo fungo.

The objective of this study was to evaluate the influence of aqueous extracts of the medicinal plants rosemary, garlic, clove, sage, lemongrass, oregano and black pepper in the in vitro development of Colletotrichum gloeosporioides and Fusarium moniliforme. The extracts were obtained by infusing 60 g of each medicinal plant in 200 mL of boiling water. Each aqueous extract was fractionated in the concentrations of 0, 5, 10 and 20% (w:v) and incorporated into the PDA (potato dextrose agar) culture medium before sterilization by autoclaving. Later, an 8 mm diameter disc of each pathogen mycelium was transferred to the center of the Petri dishes. After 24, 48 and 96 hours of incubation in a growth chamber at 22 ± 2 ºC and a photoperiod of 12 hours, we evaluated the mycelial growth of F. moniliforme, and C. gloesporioides. In the last period of incubation, we quantified the production of conidia of each fungus. For the germination test, we added, into the wells of an ELISA test plates, a 40 µL aliquot of each extract at the concentrations of 0, 5, 10 and 20% and another aliquot of a suspension of conidia of each pathogen. After 24 hours at 22 ± 2 ºC in the dark, the germination of the fungi was stopped with the addition of 20 µL of lactophenol. Then, we evaluated the germination of conidia. The experiments followed a completely randomized 7 x 4 factorial design (medicinal plants x concentrations) with four replications. For both pathogens, the aqueous extract of garlic and clove showed a greater or total inhibition of the mycelial growth, when compared to the other extracts. For the C. gloeosporioides, the clove extract showed a lower number of conidia at all concentrations tested, and the garlic extract at 20% showed not conidial germination. The garlic extract was efficient to reduce the conidial number and germination of F. moniliforme at 20%. Extracts of rosemary, clove, oregano and black pepper, in the highest concentrations, had positive effect in reducing the production of spores of the same fungus.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Crecimiento y Desarrollo de Colletotrichum gloeosporioides f. alatae durante su cultivo en medios líquidos / Growth and Development of Colletotrichum gloeosporioides f. alatae During Culture in Liquid Medium

Algunas características descritas como factores de virulencia para el género Colletotrichum sp. tales como la masa del micelio producido, la esporulación, la actividad poligalacturonasa y el pH del medio, fueron evaluadas durante el cultivo de C. gloeosporioides f. alatae en tres medios líquidos (Czapeck, caldo Martin y caldo papa), utilizados comúnmente para el desarrollo de hongos y en el medio de cultivo Czapeck adicionado con extracto de tubérculo de ñame como única fuente de carbono. Al cabo de 17 días de crecimiento, se obtuvieron los niveles máximos de los parámetros mencionados, al utilizar este último medio, respecto a los otros medios de cultivo evaluados. El medio de cultivo implementado con extracto de tubérculo de ñame, suministró los requerimientos nutricionales del hongo para el desarrollo de factores relacionados con los mecanismos de infección que pueden participar en su patogenicidad.

Ensayos de inoculación in vitro de Colletotrichum gloeosporioides aislado de hojas asintomáticas de Citrus limon / In vitro inoculation assays of Colletotrichum gloeosporioidesisolated from symptomless leaves of Citrus limon

Los hongos endofíticos pueden colonizar la parte interna de los tejidos sin causar daños aparentes al hospedador. Colletotrichum gloeosporioides, agente causal de la antracnosis, Es la especie más frecuentemente aislada de plantas sanas de Citrus limon en Tucumán. El objetivo de este trabajo fue estudiar, mediante técnicas de inoculación in vitro, la capacidad de estos aislamientos de producir infecciones sintomáticas o asintomáticas en plantines de Citrange ‘Troyer’ (Poncirus trifoliata x Citrus sinensis) y C. Limon. Se determinó el poder germinativo de conidios de loa aislamientos Nº328 y 797 obtenidos de hojas asintomáticas, y el patogénico Nº. N1. Platines en tubos de ensayo y macetas fueron inoculados con una suspensión de 105 conidios/ml y mantenidos en cámara de crecimiento a 25-28ºC. Los conidios de todas las cepas alcanzaron el máximo de germinación (100 por ciento) entre 6 y 7 horas de la siembra. Los aislamientos Nº 328 y 797 indujeron síntomas de antracnosis (clorosis de las hojas, necrosis del tallo y hojas, y defoliación) únicamente en los platines en tubos de ensayo en macetas no desarrollaron la enfermedad. El aislamiento patogénico produjo síntomas de enfermedad en los platines en tubos y macetas. La frecuencia de infección asintomática de los platines en tubo de ensayo fue del 12,5 por ciento para las cepas Nº 328 y 797. Los síntomas de antracnosis producidos por estas 2 cepas en platines en tubos y no en macetas, indicarían que la falta de preservación de condiciones naturales, favorables al hongo, no resultan las apropiadas para determinar el tipo de simbiosis que ocurre en la naturaleza. Consideramos que la utilización de mutantes endofíticas y la realización de los ensayos en plantas de limón a campo, permitirían estudiar mejor el comportamiento de estos hongos en los tejidos de la planta.

In vitro antagonistic potential of trichoderma spp. against colletotrichum gloeosporioides agent of anthracnose in the passion fruit

Diecisiete cepas de trichoderma aisladas del suelo de plantaciones de pasionaria, fueron seleccionadas por su tasa de crecimiento y capacidad de esporulación, evaluándose su potencial antagónico sobre un aislamiento de c. gloeosporioides, agente de la antracnosis de la pasionaria. Mediante la utilización de la técnica de cultivos apareados se comprobó que todas las cepas de trichoderma causaron alteraciones morfofisiológicas en las hifas del fitopatógeno. Este tipo de actividad fue evidenciado por plasmolisis, vacuolización y eroscamiento de hifas. En 13 apareamiento hubo sobreposición de trichoderma sp., causando inhibición del crecimiento micelial de las colonias del fitopatógeno, no sobreviviendo este en 10 apareamientos. De las tres especies evaluadas t. koningii reunión el mayor número de aislamientos con características favorables para su utilización en el control biológico de c. gloeosporioides, lo que sugiere la posibilidad de controlar la antracnosis de esta planta en el campo