ABSTRACT. This study aimed to investigate the potential of rhizobacteria isolated from tomato plants to control Sclerotinia sclerotiorum and induce the activity of pathogenesis-related enzymes in Micro-Tom tomato plants. Three rhizobacterial isolates were evaluated to determine the most efficient antagonist agent, which was later identified by gene sequencing as Bacillus amyloliquefaciens PKM16. The antagonistic effects of B. amyloliquefaciens against S. sclerotiorum were assessed in vivo and in vitro using live and autoclaved cultures at concentrations of 0% (control), 20%, 30%, and 40% (v/v). The residual effects of four treatments (20% live culture, 20% autoclaved culture, a Bacillus subtilis-based commercial product, and autoclaved distilled water) on tomato plants inoculated with S. sclerotiorum were determined. The same treatments were also used to assess the myceliogenic germination of sclerotia and induction of plant defense enzymes (peroxidase, catalase, polyphenol oxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase) in tomato plants. The live culture had a residual effect for 4 days and inhibited sclerotial germination by approximately 30%. Furthermore, live and autoclaved bacterial growth cultures stimulated enzyme activity. Therefore, B. amyloliquefaciens PKM16 was antagonistic to S. sclerotiorum, effectively inhibiting mycelial growth and activating defense mechanisms in Micro-Tom tomato plants.

This study was aimed at developing a script in R to assess fungal growth in Petri dishes using computer vision. The script was developed to aid studies in agricultural phytopathology. The fungi used were Elsinoe ampelina, Fusarium oxysporum and Fusarium verticillioides. The images were analyzed with R and the EBImage package. The command computeFeatures.shape was used to calculate the area and the mean diameter of the colony and the label in square pixels. The script was run in a loop to automate the analysis of all images in sequence. The script developed in R with the package EBImage was able to recognize and measure the fungus colonies examined. Simultaneously with photographing, the mean diameter of colonies was obtained by calculating the arithmetic mean of the diameter measured with two straight perpendicular lines using a ruler. Using the mean diameter, the area of the colony was calculated as a circle. The coefficient of determination and the root mean square error were calculated using the measurements and the values obtained with the program. Based on the coefficient of determination, the measurements obtained with the software are similar to those made using a ruler. The script reduces the image analysis time and increases accuracy, especially for non-uniform colonies.

Este estudio tuvo como objetivo el desarrollo de un script en R para evaluar el crecimiento de hongos en placas de Petri utilizando la visión por ordenador. El script fue desarrollado para ayudar a los estudios de fitopatología agrícola. Los hongos utilizados fueron Elsinoe ampelina, Fusarium oxysporum y Fusarium verticillioides. Las imágenes se analizaron a continuación con R y EBImage. La función computeFeatures.shape se utilizó para calcular el área y el diámetro medio de la colonia y de la etiqueta en píxeles cuadrados. La secuencia de comandos se ejecuta en un bucle para automatizar el análisis de todas las imágenes en secuencia. El script desarrollado en R con EBImage era capaz de reconocer y medir las colonias de hongos examinadas. Simultáneamente a fotografiar, el diámetro medio de las colonias se obtuvo mediante el cálculo de la media aritmética del diámetro medido con dos líneas perpendiculares rectas utilizando una regla. Utilizando el diámetro medio, el área de la colonia se calculó como un círculo. El coeficiente de determinación y el error cuadrado medio de la raíz se calcularon utilizando las medidas y los valores obtenidos con el programa. Con base en el coeficiente de determinación, las mediciones obtenidas con el software son similares a las realizadas utilizando una regla. La secuencia de comandos reduce el tiempo de análisis de imágenes y aumenta la precisión, especialmente para colonias no uniformes.

The bacterial spot of tomato, caused by Xanthomonas spp., is a very important disease, especially in the hot and humid periods of the year. The chemical control of the disease has not been very effective for a number of reasons. This study aimed to evaluate, under greenhouse conditions, the efficacy of leaf-spraying chemicals (acibenzolar-S-methyl (ASM) (0.025 g.L−1), fluazinam (0.25 g.L−1), pyraclostrobin (0.08 g.L−1), pyraclostrobin + methiran (0.02 g.L−1 + 2.2 g.L−1), copper oxychloride (1.50 g.L−1), mancozeb + copper oxychloride (0.88 g.L−1 + 0.60 g.L−1), and oxytetracycline (0.40 g.L−1)) on control of bacterial spot. Tomatoes Santa Clara and Gisele cultivars were pulverized 3 days before inoculation with Xanthomonas perforans. The production of enzymes associated with resistance induction (peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, β-1,3-glucanase, and protease) was quantified from leaf samples collected 24 hours before and 24 hours after chemical spraying and at 1, 2, 4, 6, and 8 days after bacterial inoculation. All products tested controlled bacterial spot, but only ASM, pyraclostrobin, and pyraclostrobin + metiram increased the production of peroxidase in the leaves of the two tomato cultivars, and increased the production of polyphenol oxidase and β-1,3-glucanase in the Santa Clara cultivar.

Plant extracts for the control of plant disease are emerging as promising alternatives to the use of synthetic fungicides. The objective of this work was to study the effect of plant extracts and essential oils on naturally infected apple fruits with Penicillium spp. causal agent of blue mold. Apple fruits were immersed in citric and Azadirachta indica extracts at concentrations of 0.5 and 1.0% (v/v), stored at room temperature (25 °C and 90% RH) and analyzed at 21 days after the application of the treatments. Evaluated also the effect of fumigation of apple fruits with essential oils of Thymus vulgaris, Origanum vulgaris, Mentha piperita and Melaleuca alternifolia. Treated fruits were stored at 25±2 °C and 90±5% relative humidity for 20 days. Treated fruit with distilled water constituted the group controls. The higher values of incidence (100%), severity (99.98%) and the lowest percentage of disease control (4.56%) were observed in apple fruits treated with distilled water (group control). All treatments exhibited inhibitory effect on Penicillium spp. in apple fruits, however, A. indica and citric extracts, at concentrations of 1.0%, had a disease control of98.33% and 99.57%, respectively. There were no significant differences between the oils applied; except between these and the control. However, the treatments with essential oils were that exhibited the least incidence, severity and control of the disease. The results showed that essential oils showed good results, above 98.1% of control of blue mold disease in postharvest. It was demonstrated that extracts the different concentration and essential oils have antifungal activities and that they have a potential use as an alternative control in relation to chemical methods.

El uso de los extractos vegetales con actividad antimicrobiana para controlar enfermedades en pre o poscosecha es una alternativa promisoria frente al uso de fungicidas sintéticos. El objetivo de este estudio fue evaluar el efecto de extractos vegetales y aceites esenciales en manzanas infectadas naturalmente con Penicillium spp., agente causal del moho azul. Se sumergieron manzanas en extractos de cítricos y de neem (Azadirachta indica) en concentraciones de 0,5 y 1,0% (v/v) y se almacenaron a 25 °C y 90% de humedad relativa. Después de 21 días se midió incidencia y severidad de la enfermedad. También se evaluó el efecto de los aceites esenciales de Thymus vulgaris, Origanum vulgaris, Mentha piperita y Melaleuca alternifolia por fumigación. La fruta se almacenó a 25±2 °C y 90±5% de humedad relativa durante 21 días. Frutos tratados con agua destilada estéril constituyeron el tratamiento control. Los frutos sin tratar presentaron los valores más altos de incidencia (100%) y severidad (99,98%) de moho azul, así como el menor porcentaje de control (4,56%). Todos los tratamientos mostraron efecto antifúngico de Penicillium spp, sin embargo, los extractos cítricos y de A. indica en la concentración de 1,0% proporcionaron los mayores niveles de control de la enfermedad (99,57% y 98,33%, respectivamente). Los frutos tratados con aceites esenciales mostraron menor incidencia, menor severidad y mayor control de la enfermedad. Se ha demostrado que el extracto y aceites esenciales estudiados tienen actividad antifúngica y potencial para uso en poscosecha como una alternativa al control químico.

The objective of this work was to evaluate in vitro and in vivo the bioactivity of the essential oil of Cymbopogon citratus on the mycelial growth and sporulation of Alternaria solani, and also some biochemical and structural mechanisms of resistance induction on tomato plants. The plants were treated with essential oils at 0, 250, 500, 750, 1000 and 1500 μL L-1 72 hrs before the fungi inoculation. Foliar discs were collected at 0, 12, 24 and 48 h after the inoculation to verify the activity of peroxidase, polyphenol oxidase and β-1,3 glucanase. Spore germination, appressorium formation, and the mechanisms of structural resistance were evaluated 48 h after the inoculation. The in vitro responses showed direct toxic activities unlike the ones observed in vivo, where the essential oils had no fungitoxic effects on the fungi spores. In vitro, the peroxidase and polyphenol oxidase enhanced both local and systemic activities, unlike the β-1,3-glucanase which enhanced only local activities at the highest concentrations. The essential oils of C. citratus have, therefore, potential to induce resistance in tomato plants.

El objetivo de este trabajo fue evaluar la bioactividad in vitro e in vivo del aceite esencial de Cymbopogon citratus sobre el crecimiento micelial y la esporulación de Alternaria solani, y también algunos mecanismos bioquímicos y estructurales de la inducción de resistencia en plantas de tomate. Las plantas fueron tratadas con aceite esencial a 0, 250, 500, 750, 1000 y 1500 μL-1 72 horas antes de inocular los hongos. Se recogieron discos foliares a 0, 12, 24 y 48 h después de la inoculación para verificar la actividad de la peroxidasa, polifenol oxidasa y β-1, 3 glucanasa. Se evaluó la germinación de esporas, formación de apresorios, y los mecanismos de resistencia estructural 48 horas después de la inoculación. La respuesta in vitro mostró actividad tóxica directa, a diferencia de lo observado in vivo, donde los aceites esenciales no tuvieron efectos fungitóxicos sobre las esporas de hongos. In vitro, la peroxidasa y polifenol oxidasa mejoro su actividad, tanto local como sistêmica, a diferencia de la β-1, 3-glucanasa que aumentó solo su actividad local en las concentraciones más altas. Los aceites esenciales de C. citratus tienen, por lo tanto, el potencial para inducir resistencia en plantas de tomate.

The present study aims to evaluate the effect of fungicides and antibiotics to control bacterial spot (Xanthomonas perforans) in tomato, and the activation of pathogenesis-related proteins. Hybrid tomato AP 529 was used to assess the severity of disease. The treatments consisted of spraying with acibenzolar-S-methyl, fluazinam, pyraclostrobin, pyraclostrobin + methiran, copper oxychloride, copper oxychloride and mancozeb + oxytetracycline, and inoculated and non-inoculated controls. After three days of treatment, all plants were inoculated with X. perforans (10(6) CFU / mL). Leaf discs were collected for assessment of peroxidase, polyphenol oxidase, β-1,3 glucanase, phenylalanine ammonia lyase and protease. The area under the disease progress curve (AUDPC) was calculated with the data of severity. All treatments had reduced AUDPC compared to the inoculated control. Fungicides acibenzolar-S-methyl, pyraclostrobin, and pyraclostrobin + methiran had more satisfactory results in reducing the severity of bacterial spot on tomato. The products based on pyraclostrobin together with acibenzolar-S-methyl induced enzymatic activities of peroxidase, polyphenoloxidase and β-1,3 glucanase, indicating that these products may be related to the induction of resistance to bacterial spot on tomato plants.

O presente trabalho teve por objetivo avaliar a ação de fungicidas e antibiótico no controle da mancha bacteriana (Xanthomonas perforans) em tomate e na ativação de proteínas relacionada à patogênese. Para avaliação da severidade da doença foi utilizada o híbrido de tomate "AP529". Os tratamentos consistiram da pulverização com acibenzolar-S-metil, fluazinam, piraclostrobina, piraclostrobina + methiran, oxicloreto de cobre, oxicloreto de cobre + mancozeb e oxitetraciclina, além de uma testemunha inoculada e uma testemunha não inoculada. Após três dias do tratamento, as plantas foram inoculadas com a bactéria X. perforans (10(6) UFC/mL). Discos foliares foram coletadas para avaliação das enzimas peroxidase, polifenoloxidase, β-1,3 glucanase, fenilalanina amonia liase e protease. Com os dados da severidade foi calculada a Área Abaixo da Curva de Progresso da Doença (AACPD). Todos os tratamentos apresentaram redução da AACPD em relação à testemunha inoculada. Os fungicidas acibenzolar-S-metil, piraclostrobina e piraclostrobina + methiran tiveram resultados mais satisfatórios na redução da severidade da mancha bacteriana em tomateiro. Os produtos à base de piraclostrobina juntamente com acibenzolar-S-metil induziram as atividades enzimáticas de peroxidase, polifenoloxidase e β-1,3 glucanase, evidenciando que estes produtos podem estar relacionados à indução de resistência à mancha bactéria em plantas de tomateiro.

O objetivo foi verificar se a fungigação via gotejamento é uma alternativa ao método convencinal de pulverização no controle da pinta-preta do tomate. Plantas de tomate (var Santa Clara) foram cultivadas em vasos instalados no interior de uma casa-de-vegetação. Aos 50 dias após o transplantio, foram inoculadas com Alternaria solani e tratadas com quatro diferentes fungicidas: azoxystrobina (8 g 100 L-1), difeconazole (50 mL 100 L-1), metiram+piraclostrobin (200 g 100 L-1) e tebuconazole (100 mL 100 L-1), em duas formas de aplicação: pulverização convencional e fungigação por gotejamento. Uma testemunha não recebeu aplicação de fungicidas. Avaliou-se a severidade da doença através de escala de notas expressa em área abaixo da curva de progresso da doença (AACPD) e fatores de produção, como número, peso e diâmetro médio dos frutos e produtividade. O delineamento experimental foi 4 x 2 + 1, com oito repetições. Cada parcela constou de uma planta em um vaso. Houve redução da severidade da doença de 27% em comparação com a testemunha. Não houve diferença significativa entre os métodos de aplicação. O número de frutos não diferiu estatisticamente entre os tratamentos. O peso médio e diâmetro dos frutos foram maiores nos tratamentos com fungicidas em comparação a testemunha, o que refletiu no aumento da produtividade. A fungigação por gotejamento é alternativa de aplicação de fungicida sistêmico por pulverização na cultura do tomate.

The aim was to verify if the fungigation via drip irrigation is an alternative to the conventional method of spraying on tomato for controlling early blight. Tomato plants (variety Santa Clara) were grown in pots inside a greenhouse. Fifty days after transplanting, the plants were inoculated with Alternaria solani and treated with four different fungicides: azoxystrobin (8 g 100 L-1), difeconazole (50 mL 100 L-1), metiram+piraclostrobin (200 g 100 L-1) and tebuconazole (100 mL 100 L-1) using two applications methods: conventional spraying and fungigation dripping. The control plants did not receive fungicide application. To assess the severity of the disease, we used a rating scale expressed as the area under the disease progress curve (AUDPC) and production factors, such as number, weight and average diameter of the fruit and its productivity. The experimental design was completely randomized in factorial scheme 4 x 2 + 1 with eight replicates. Each plot had one plant in one pot. A 27% reduction in disease severity was observed when compared with the control plants, with no significant difference noted regarding the application method. The number of fruits did not statistically differ between the treatments. The average weight and diameter of the fruits were superior in the plants that had fungicide application compared to the control plant, reflecting an increase in productivity. Fungigation through water dripping is an alternative to the conventional method of spraying cultured tomatoes.