Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit

Abstract Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

Resumo As perdas de alimentos por contaminação causada por fungos são de grande impacto negativo para a agricultura, gerando altos prejuízos econômicos. Para evitar as perdas de diversos produtos alimentícios pela contaminação fúngica são utilizados fungicidas sintéticos e naturais. As espécies do gênero Capsicum são usadas há muitos anos para auxiliar na conservação de alimentos por possuírem substâncias químicas com ação antifúngica entre outras. Neste contexto, o objetivo deste estudo foi identificar alguns compostos fenólicos por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (LC-ESI-MS/MS) presentes nos extratos acetato de etila (EAE) e metanólico (ME) dos frutos maduros da pimenta biquinho (C. chinense) e avaliar atividade antifúngica de EAE e ME contra os fungos Sclerotinia sclerotiorum, Rhizopus stolonifer e Colletotrichum gloeosporioides. Os extratos foram obtidos de forma sequencial, utilizando o procedimento de maceração. A atividade antifúngica foi avaliada seguindo a metodologia de difusão em disco, nas doses de 25 µL, 50 µL, 100 µL e 200 µL de cada extrato diluído. A análise química evidenciou a presença de ácido protocatequico, ácido gentisico, ácido vanílico, kaempferol-3-O-robinobiosídeo e naringenina em ambos os extratos. EAE revelou maior poder de inibição do crescimento micelial nas doses de 100µL e 200µL contra os três fungos testados, enquanto ME exibiu fraca atividade inclusive na maior dose investigada. Entretanto, estudos mais aprofundados ainda são necessários para consolidar seu uso e aplicação prática na área agronômica.

Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit / Compostos fenólicos e atividade antifúngica dos extratos acetato de etila e metanólico dos frutos maduros de Capsicum chinense Jacq

Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

As perdas de alimentos por contaminação causada por fungos são de grande impacto negativo para a agricultura, gerando altos prejuízos econômicos. Para evitar as perdas de diversos produtos alimentícios pela contaminação fúngica são utilizados fungicidas sintéticos e naturais. As espécies do gênero Capsicum são usadas há muitos anos para auxiliar na conservação de alimentos por possuírem substâncias químicas com ação antifúngica entre outras. Neste contexto, o objetivo deste estudo foi identificar alguns compostos fenólicos por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (LC-ESI-MS/MS) presentes nos extratos acetato de etila (EAE) e metanólico (ME) dos frutos maduros da pimenta biquinho (C. chinense) e avaliar atividade antifúngica de EAE e ME contra os fungos Sclerotinia sclerotiorum, Rhizopus stolonifer e Colletotrichum gloeosporioides. Os extratos foram obtidos de forma sequencial, utilizando o procedimento de maceração. A atividade antifúngica foi avaliada seguindo a metodologia de difusão em disco, nas doses de 25 µL, 50 µL, 100 µL e 200 µL de cada extrato diluído. A análise química evidenciou a presença de ácido protocatequico, ácido gentisico, ácido vanílico, kaempferol-3-O-robinobiosídeo e naringenina em ambos os extratos. EAE revelou maior poder de inibição do crescimento micelial nas doses de 100µL e 200µL contra os três fungos testados, enquanto ME exibiu fraca atividade inclusive na maior dose investigada. Entretanto, estudos mais aprofundados ainda são necessários para consolidar seu uso e aplicação prática na área agronômica.

Hexane Extract from Tradescantia pallida (Rose) D.R. Hunt (Commelinaceae): Its Volatile Constituents and in vitro Antifungal and Cytotoxic Activities

Abstract: Tradescantia pallida (Commelinaceae) has shown promising antibacterial, antioxidant and anticancer activities. This study aimed at extracting hexane from T. pallida (HE-TP) aerial parts to identify and quantify its volatile compounds by GC-MS and GC-FID and at evaluating its antifungal and antiproliferative activities. (E)-4-Methoxycynnamic acid (50.2%), 2,5-di-tert-butyl-1,4-benzoquinone (13.7%) and epijuvabione (10.4%) were the major components identified in HE-TP. HE-TP was incorporated into PDA medium, poured into Petri dishes and transferred to mycelial discs of pathogens. Percentages of inhibition of fungal growth were determined. HE-TP showed remarkable antifungal potential at the dose of 400 µL since it inhibited 100% of Penicillium digitatum and Sclerotinia sclerotiorum growth and 92.6% of Rhizopus stolonifer growth. Besides, HE-TP demonstrated cytotoxic activity against different human tumor cell lines with IC50 values between 231.43 and 428.76 µg/mL. Therefore, results showed that HE-TP has potential against fungi of agronomic interest and tumor cells.

Atividade antifúngica, in vitro, do óleo de café verde / Antifungal activity, in vitro, of the green coffee oil

O objetivo deste trabalho foi avaliar o efeito do contato direto e da fração volátildo óleo de café verde, testado nas concentrações de 500, 1.000, 1.500 e 2.000 µL L-1, sobre o crescimento micelial e a esporulação dos fungos Penicillium roqueforti e Rhizopus stolonifer. O óleo essencial de cravo-da-índia na concentração de 800 µL L-1 foi utilizado para comparação. Nas concentrações de 1.500 e 2.000 µL L-1, o óleo de café verde em contato direto proporcionou redução da esporulação do fungo R. stolonifer , sendo estatisticamente semelhante ao óleo de cravo-da-índia. Na fração volátil do óleo de café verde, observou-se redução significativa da esporulação de P. roqueforti e R. stolonifer na concentração de 2.000 µL L-1. O óleo de café verde, em contato direto ou por volatilização, reduziu significativamente o crescimento micelial e a esporulação de ambos os fungos em comparação com a testemunha.(AU)

The objective of this study was to evaluate the effect of direct contact and volatile fraction of from green coffee oil, tested at concentrations of 500, 1,000, 1,500 and 2,000 µL L-1, on mycelial growth and sporulation of Penicillium roqueforti and Rhizopus stolonifer . The essential oil of clove at a concentration of 800 µL L-1 was used for comparison. At concentrations of 1,500 and 2,000 µL L-1, the green coffee oil in direct contact caused a reduction of sporulation for R. stolonifer, similar to clove oil. In the volatile fraction of the green coffee oil, there was a significant reduction in sporulation of P. roqueforti and R. stolonifer at a concentration of 2,000 µL L-1. The green coffee oil, in direct contact or by volatilization, significantly reduced the mycelial growth and sporulation of both fungi compared to the control.(AU)

Determining the effectiveness of Candida guilliermondii in the biological control of Rhizopus stolonifer in postharvest tomatoes / Determinación de la efectividad del control biológico de Rhizopus stolonifer en tomates poscosecha utilizando candida guilliermondii / Determinação da efetividade do controlo biológico de Rhizopus stolonifer em tomates poscosecha utilizando candida guilliermondii

Las alteraciones de frutos de tomate poscosecha por causas microbiológicas conllevan a su deterioro fisiológico produciendo su descomposición. En este estudio, Candida guilliermondii fue examinada en ensayos in vitro e in vivo para determinar su eficacia en el control biológico contra Rhizopus stolonifer en tomates (Lycopersicon esculentum). El efecto antagónico se llevo a cabo usando biomasa de la levadura y sus metabolitos los cuales se identificados en un cromatógrafo gases-masas. Los ensayos in vitro mostraron que la única fase con respuesta antagónica al patógeno fue la fase acuosa sin filtrar. En los ensayos in vivo, la biomasa de Candida guilliermondii presento el mayor porcentaje de protección contra el patógeno (87 %) en comparación con la fase acuosa sin filtrar y la fase orgánica sin concentrar que presentaron el 77 y 80 % de protección respectivamente. Estos resultados son de gran importancia para evitar las perdidas económicas además de garantizar un tiempo de vida mas largo del tomate asegurando una mejor distribución del producto y evitando sus perdidas en estos tiempos de crisis alimentaria en especial en países en desarrollo.

Post-harvest losses in tomato crops can be accredited to a number of causes, which include biological causes that produce physiological deterioration; infection caused by diseases and pests that cause rotting. In this study, we assayed, in vitro and in vivo, the yeast Candida guilliermondii to determine its effectiveness as a biocontrol to reduce the rotting produced by Rhizopus stolonifer on tomatoes (Lycopersicon esculentum). The antagonistic effect was tested using a yeast biomass suspension; gas chromatography and a mass selective detector were used to identify its metabolites. According to the in vitro test, the only phase with an antagonistic response against Rhi%opus stolonifer was the unfiltered water phase. In the in vivo tests, the biomass of Candida guilliermondii presented the highest percentage of protection of the tomato (87 %) compared with the unfiltered aqueous phase and the unconcentrated organic phase, which reached only 77 and 80 % respectively. These results are essential to avoid economic losses caused by fungi and to increase the shelf life of tomatoes improving the distribution of the product and preventing postharvest losses in this time of food crises, especially in developing countries.

As perdas pós-colheita de frutos de tomate são creditadas em várias fontes, incluindo causas biológicas que levam ao deterioro fisiológico e infecção por doenças e pragas que podem levar à sua decomposição. Neste estudo, a levedura Candidaguilliermondii foi examinada in vitro e in vivo para determinar a sua eficácia no controlo biológico de Rhizopus stolonifer em tomates (Lycopersicon esculentum). O efeito antagonista realizou-se utilizando biomassa de levedura e os seus metabolitos, os quais foram identificados por cromatografia gasosa acoplada a um detector selectivo de massa. Os ensaios in vitro mostraram que a única fase com resposta antagonista a Rhizopus stolonifer foi a fase aquosa não filtrada. Nos ensaios in vivo, a biomassa Candida guilliermondii teve a maior percentagem de protecção contra o patógeno (87%) em comparação com a fase aquosa não filtrada e a fase orgânica não concentrada apresentando 77 e 80 % de protecção, respectivamente. Estes resultados são muito importantes para evitar perdas económicas causadas por fungos, além de garantir ao tomate uma vida útil mais longa, o que assegura uma melhor distribuição do produto e evita perdas pós-colheita neste momento de crise alimentar, especialmente nos países em desenvolvimento.

Actividad antifúngica del quitosano y aceites esenciales sobre Rhizopus stolonifer -Ehrenb: Fr Vuill, agente causal de la pudrición blanda del tomate / Antifungal activity of chitosan and essential oils on Rhizopus stolonifer Ehrenb: Fr Vuill causal agent of soft rot of tomato

Rhizopus stolonifer es el agente causal de la pudrición blanda, enfermedad poscosecha que ocasiona pérdidas económicas importantes. Se han empleado fungicidas sintéticos como el dicloran para controlar a este microorganismo, sin embargo, se ha demostrado que los fungicidas representan un riesgo para el ambiente y la salud humana. Actualmente se buscan alternativas naturales para el control de las pudriciones poscosecha. Se evaluó in vitro e in situ el efecto antifúngico del quitosano y de los aceites esenciales de canela (Cinnamomum zeylanicum), clavo (Syzygium aromaticum) y tomillo (Thymus vulgaris) y dicloran sobre Rhizopus stolonifer. Los tratamientos más efectivos para inhibir in vitro a Rhizopus stolonifer fueron obtenidos con quitosano a 10 mg mL-1, con los tres aceites esenciales probados a la concentración de 0,3 mg mL-1, las mezclas de quitosano a 10 mg mL-1 con los aceites a 0,3 mg mL-1 y el dicloran a 1 mg mL-1. Los experimentos in situ mostraron que el tratamiento individual con quitosano fue el mejor para reducir el porcentaje de infección de los frutos de tomate (Lycopersicon esculentum) inoculados con Rhizopus stolonifer y que la mezcla de quitosano con aceites esenciales no mejora la actividad antifúngica. El quitosano y el dicloran fueron los mejores tratamientos para reducir la pérdida de peso de los frutos. Los tratamientos individuales con quitosano representan una alternativa natural para controlar la pudrición blanda en frutos de tomate.

Rhizopus stolonifer is the causal agent of soft rot, postharvest disease that causes important economic losses. Synthetic fungicides such as dichloran have been used to control this microorganism; however, it has been shown that fungicides represent a risk for the environment and human health. Actually, natural alternatives are looked for the control of postharvest rotting. In vitro and in situ experiments the antifungal effect of chitosan, essential oils of cinnamon (Cinnamomum zeylanicum), clove (Syzygium aromaticum), and thyme (Thymus vulgaris) and dichloran on Rhizopus stolonifer were evaluated. The most effective treatments for in vitro inhibition of Rhizopus stolonifer were obtained by quitosano to 10 mg mL-1, with the three essential oils proved to the concentration of 0.3 mg mL-1, the mixtures chitosan to 10 mg mL-1 with the oils at 0.3 mg mL-1 and dichloran at 1 mg mL-1. In situ experiments showed that the individual treatment with chitosan was the best to reduce the infection percentage of the tomato (Lycopersicon esculentum) fruits inoculated with Rhizopus stolonifer and chitosan mixture with essential oils did not improve its antifungal activity. Chitosan and dichloran were the best treatments to reduce the weight loss of the fruits. Individual treatments with chitosan represent a natural alternative for the control of soft rot on tomato fruits.

Screening of Functional Rhizopus stolonifer for Alcohol Fermentation and Production of High Quality Korean Traditional Rice Wine

Different strains of mold were screened for the production of high quality Korean traditional rice wine with anti-hypertension and good acceptability. We isolated 867 nuruk mold strains and selected 24 for further study based on measurement of amylase activity. Among them, mold No. 17 showed high ethanol production upon fermentation with Saccharomyces cerevisiae as well as anti-hypertensive properties. The No. 17 strain was therefore selected as the functional mold and later identified as Rhizopus stolonifer based on molecular biological characteristics. Optimal fermentation conditions for the brewing of anti-hypertensive traditional rice wine comprised the addition of R. stolonifer No. 17 koji at a concentration of 35 sp/g and a fermentation period of 10 days at 25degrees C using S. cerevisiae.

Rhizopus Soft Rot on Pear (Pyrus serotina) Caused by Rhizopus stolonifer in Korea

Rhizopus soft rot caused by Rhizopus stolonifer occurred on pears (Pyrus serotina) in the Jinju City Agricultural Products Wholesale Market in Korea from 2004 to 2005. The infection usually started from wounds due to cracking at harvest time. The lesions started as water-soaked, rapidly softened, then gradually expanded. The mycelia grew vigorously on the surface of the fruits and formed stolons. Colonies on potato dextrose agar at 25degrees C were white cottony to brownish black. Sporangia were globose, black and 90~120 microm in size. Sporangiophores were light brown and 480~2600 x 12~18 microm in size. Sporangiospores were globose to oval, brownish, streaked, and 8~14 x 6~10 microm in size. Columella were light brownish gray, hemispherical and 70~80 microm in size. On the basis of these symptoms, mycological characteristics and pathogenicity tests on host plants, the fungus was identified as Rhizopus stolonifer (Ehrenb.) Vuill. This is the first report of rhizopus soft rot on pear (P. serotina) caused by R. stolonifer in Korea.

Rhizopus Soft Rot on Cherry Tomato Caused by Rhizopus stolonifer in Korea

A soft rot of fruits caused by Rhizopus stolonifer occurred on cherry tomato in Jinju City Agricultural Products Wholesale Market, Korea. The disease infection usually started from wounding after cracking of fruits. At first, the lesions started with water soaked and were rapidly softened and diseased lesion gradually expanded. The mycelia grew vigorously on the surface of fruits and formed stolons. Colonies on potato dextrose agar at 25degrees C were white cottony at first, becoming heavily speckled by the presence of sporangia and the browinish black, and spreading rapidly by means of stolons fired at various points to the substrate by rhizoids. Sporangia were 82.7x196.7 microm in size and globose or sub-globose with somewhat flattened base. The color of sporangia was white at first and then turned black with many spores, and never overhanging. Sporangiophores were 2.6~5.8x12.3~24.2 microm in width, smooth-walled, non-septate, light brown, simple, long, arising in groups of 3~5 from stolons opposite rhizoids. Sporangiospores were 8.2~18.8 microm long, irregular, round, oval, elongate, angular, and browinish-black streaked. Columella was 64.1x136.3 microm. brownish gray, and umberella-shaped when dehisced. The causal organism was identified to be R. stolonifer. This is the first report of Rhizopus soft rot on cherry tomato caused by R. stolonifer in Korea.