Iron and copper on Botrytis cinerea: new inputs in the cellular characterization of their inhibitory effect.

Certain metals play key roles in infection by the gray mold fungus, Botrytis cinerea. Among them, copper and iron are necessary for redox and catalytic activity of enzymes and metalloproteins, but at high concentrations they are toxic. Understanding the mechanism requires more cell characterization studies for developing new, targeted metal-based fungicides to control fungal diseases on food crops. This study aims to characterize the inhibitory effect of copper and iron on B. cinerea by evaluating mycelial growth, sensitivity to cell wall perturbing agents (congo red and calcofluor white), membrane integrity, adhesion, conidial germination, and virulence. Tests of copper over the range of 2 to 8 mM and iron at 2 to 20 mM revealed that the concentration capable of reducing mycelial growth by 50% (IC50) was 2.87 mM and 9.08 mM for copper and iron, respectively. When mixed at equimolar amounts there was a significant inhibitory effect mostly attributable to copper. The effect of Cu50, Fe50, and Cu50-Fe50 was also studied on the mycelial growth of three wild B. cinerea strains, which were more sensitive to metallic inhibitors. A significant inhibition of conidial germination was correlated with adhesion capacity, indicating potential usefulness in controlling disease at early stages of crop growth. Comparisons of the effects of disruptive agents on the cell wall showed that Cu, Fe, and Cu-Fe did not exert their antifungal effect on the cell wall of B. cinerea. However, a relevant effect was observed on plasma membrane integrity. The pathogenicity test confirmed that virulence was correlated with the individual presence of Cu and Fe. Our results represent an important contribution that could be used to formulate and test metal-based fungicides targeted at early prevention or control of B. cinerea.

Fungi associated with Paspalum guenoarum seeds: their impact on physiology and control / Fungos associados com sementes de Paspalum guenoarum: seus impactos na fisiologia e controle

ABSTRACT: Of the natural pastures grown in southern Brazil, those of the genus Paspalum are the most important. One of the factors that hinder their cultivation is the availability of quality seeds, that are often compromised by the presence of fungi. This study determined the in vitro sensitivity of Paspalum guenoarum ecotype azulão seed-associated fungus to certain fungicides and to measure the efficiency of chemical treatments for fungal control and seed physiological performance. Bipolaris micropus, Epicoccum sorghinum, Curvularia geniculata and Fusarium incarnatum associated with seeds were tested in vitro against Carbendazim; Tiram, Fludioxonil; Metalaxil-M, Carboxina; Tiram and Tiofanato-metílico at 0, 1, 2.5, 5, 10 and 30 µg/mL. This experiment was conducted in a completely randomized design (factorial 4 × 4 × 6). Qualitative variables were compared using the Tukey test (P ≤ 0.05) and quantitative variables were subjected to regression analysis. Carbendazim; Tiram and Fludioxonil; Metalaxil-M had the best fungicidal performances, each inhibiting three of the four fungi with LD50s below 1 µg/mL. Subsequently, these two fungicides were used alone or in combination in the in vivo test. A completely randomized design was used and the means were compared using the Tukey test (P < 0.05). The chemical treatment of the seeds resulted in improvement of five of the six evaluated physiological parameters. The identification of the primary fungi associated with Paspalum seeds reported in this research, as well as damage done to them, can be diminished using appropriate measures such as seed treatments.

RESUMO: Das pastagens naturais cultivadas no sul do Brasil, as do gênero Paspalum são as mais importantes. Um dos fatores que dificultam o cultivo é a disponibilidade de sementes de qualidade, muitas vezes comprometidas pela presença de fungos. Os objetivos deste estudo foram determinar a sensibilidade in vitro de fungos associados a sementes de Paspalum guenoarum ecótipo azulão a determinados fungicidas, medir a eficiência de tratamentos químicos para controle desses fungos e o desempenho fisiológico das sementes. Os fungos associados às sementes, Bipolaris micropus, Epicoccum sorghinum, Curvularia geniculata e Fusarium incarnatum foram testados in vitro contra Carbendazim; Tiram, Fludioxonil; Metalaxil-M, Carboxina; Tiram e Tiofanato-metílico em 0, 1, 2,5, 5, 10 e 30 µg/mL. Este experimento foi conduzido em um delineamento inteiramente casualizado (fatorial 4 × 4 × 6). As variáveis ​​qualitativas foram comparadas pelo teste de Tukey (P ≤ 0,05) e as variáveis ​​quantitativas foram submetidas à análise de regressão. Carbendazim; Tiram e Fludioxonil; Metalaxil-M tiveram os melhores desempenhos fungicidas, cada um inibindo três dos quatro fungos com LD50 abaixo de 1 µg/mL. Posteriormente, esses dois fungicidas foram utilizados isoladamente ou em combinação no teste in vivo. O delineamento experimental foi inteiramente casualizado e as médias comparadas pelo teste de Tukey (P < 0,05). O tratamento químico das sementes resultou na melhoria de cinco dos seis parâmetros fisiológicos avaliados. A identificação dos fungos primários associados às sementes de Paspalum relatadas neste trabalho, bem como os danos causados ​​por eles, podem ser diminuídos usando medidas apropriadas, como tratamento de sementes.

Total Syntheses of Xiamycins†A, C, F, H and Oridamycin†A and Preliminary Evaluation of their Anti-Fungal Properties.

Divergent and enantiospecific total syntheses of the indolosesquiterpenoids xiamycins A, C, F, H and oridamycin A have been accomplished. The syntheses, which commence from (R)-carvone, employ a key photoinduced benzannulation sequence to forge the carbazole moiety characteristic of these natural products. Late-stage diversification from a common intermediate enabled the first syntheses of xiamycins C and F, and an unexpected one-pot oxidative decarboxylation, which may prove general, led to xiamycin H. All synthetic intermediates and the natural products were tested for anti-fungal activity. Xiamycin H emerged as an inhibitor of three agriculturally relevant fungal pathogens.

Secondary metabolites produced by Sardiniella urbana, a new emerging pathogen on European hackberry.

In this study the production of secondary metabolites by a virulent strain of Sardiniella urbana, a recently described pathogen originally found on declining European hackberry trees in Italy, was investigated for the first time. Chemical analysis of the culture filtrate extracts led to the isolation of three well known compounds as R-(-)-mellein and (3R,4R)-and (3R,4S)-4-hydroxy melleins which were identified by spectroscopic methods (essentially NMR and ESIMS). The isolated compounds were tested for their phytotoxic, antifungal and zootoxic activities. Among them, only R-(-)-mellein was found to be active.

Chemical composition and antifungal activity of propolis on Aspergillus flavus / Composição química e atividade antifúngica de própolis sobre Aspergillus flavus

Research has indicated the antifungal activity of ethanol extracts from propolis (EEP) on fungi of the genera Phakopsora, Colletotrichum, and Cercospora. Here, chemical compositions and antioxidant activity of three EEP (propolis from Scaptotrigona polysticta stingless bee and two types produced by Apis mellifera - red and brown) were evaluated and their action against Aspergillus flavus was investigated. Ash, dry extract, total phenolic and total flavonoid contents were determined. Phenolic composition was evaluated by high performance liquid chromatography, with using the following reference substances: gallic acid, caffeic acid, -coumaric acid, ferulic acid, quercetin, kaempferol, and apigenin. Tests on mycelial growth, sporulation, and germination of spores were performed to assess the biological activity of the EEP on A. flavus. All EEP showed low dry extract content (<11%) with adequate amounts of ash (<5%). The red EEP (Apis mellifera) showed the highest contents of total phenolic and total flavonoid (5.38 and 2.77 g 100 g-1), while the highest recorded antioxidant activity was exhibited by brown EEP (92.9%). The EEP of S. polysticta presented higher levels of -coumaric acid (10.99 mg g-1), while red and brown extracts from A. mellifera stood out with the highest levels of quercetin (27.26 mg g-1) and gallic acid (5.88 mg g-1), respectively. No extract was effective in inhibiting mycelial growth and sporulation of A. flavus, but red EEP inhibited spore germination. On the basis of these results, it is suggested that the inhibitory effect of red EEP on spore germination may be associated with increased levels of flavonoids found in it as compared with the other extracts investigated here.

Outros estudos apontaram a ação antifúngica dos extratos etanólicos de própolis (EEP) sobre fungos do gênero Phakopsora, Colletotrichum e Cercospora. Neste estudo, avaliou-se a composição química e a atividade antioxidante de três EEP (própolis produzido pela abelha sem ferrão Scaptotrigona polysticta e dois tipos de própolis produzida pela Apis melífera: marrom e vermelha) e sua ação contra o Aspergillus flavus. Foram determinados os teores de cinzas, extrato seco, fenólicos e flavonoides totais nos EEP. A composição fenólica foi determinada por cromatografia líquida de alta eficiência, empregando-se as seguintes substâncias de referência: ácido gálico, ácido p-cumárico, ácido ferrúlico, quercitina, kaempferol e apigenina. Para avaliar a atividade biológica dos EEP sobre A. flavus foram realizados os testes de crescimento micelial, esporulação e germinação de esporos. Todos os EEP apresentaram baixos teores de extrato seco (< 11%), porém quantidades de cinzas adequadas (< 5%). O EEP vermelha apresentou os maiores teores de fenólicos e flavonoides totais (5,38 e 2,77 g 100g-1), enquanto que a maior atividade antioxidante registrada foi do EEP marrom (92,9%). O EEP de S. polysticta apresentou maiores teores de ácido p-cumárico (10,99 mg g-1), enquanto que os de A. mellifera, vermelha e marrom, destacaram-se com os maiores níveis de quercetina (27,26 mg g-1) e de ácido gálico (5,88 mg g-1), respectivamente. Nenhum extrato foi eficaz na inibição do crescimento micelial e esporulação de A. flavus, porém observou-se inibição na germinação de esporos pelo EEP vermelha. Sugere-se que o efeito inibitório na germinação de esporos pode estar relacionado com os maiores níveis de flavonoides encontrados na própolis vermelha, quando comparado com os outros extratos investigados.

Evaluation of Mentha arvensis essential oil and its major constituents for fungitoxicity.

Essential oil and major constituents of menthe were evaluated for fungicidal activities. Gas chromatography-mass spectrometry (GC-MS) of essential oil from leaves of Mentha arvensis cv. CIM-Saryu revealed that menthol was found in highest amount (77.94%) followed by isomenthone (5.24%), neomenthyl acetate (5.18%) and menthone (5.00%). Menthol and menthone were extracted from the essential oil by column chromatography. Essential oil, menthol and menthone were screened for their fungicidal activity against Rhizoctonia solani and Fusarium moniliforme. Menthol was highly effective as compared to essential oil as well as menthone. All of them exhibited less activity than standard bavistin at all the tested concentrations.

STUDIES ON CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITIES OF BIOACTIVE MOLECULES FROM DATE PALM (PHOENIXDACTYLIFERA L.) POLLENS AND SEEDS.

BACKGROUND: Natural resources have been the crucial origin of chemical elements. They have been used in many traditions as alternative medicines. The chemical profiling of some plant extracts and essential oils related to different plants were followed to unveil their most active components. In this paper, Phoenix dactilyfera L was selected as a host plant to investigate the composition of different organs with different cultivars. MATERIALS AND METHOD: The antibacterial and antifungal activities of the extracts have been tested using different techniques, including optical density and GC/MS analyses of the natural extracts. RESULTS: GC/MS analysis revealed the presence of abundant oleic (36.69%) and lauric (20.49%) acids in date seeds. However, the pollen contains a high amount of palmitic (22.27 %), linoleic (33.4%) and linolenic (17.055%) acids. Moreover, the largest inhibition zone is obtained with the organic extract of Deglet Nour which showed a strong antibacterial activity against Escherichia coli and pollen extract showed also a strong inhibition against Escherichia coli, Staphylococcus aureus, S. aureus MRSA and Enterococcus faecalis. Aqueous extracts of date palm seeds of and pollen seem to have a fongitoxique activity from a concentration of 6 mg.mL-1 and 12mg.mL-1 of cyanidine; as well as the organic extracts of pollen with a concentration of 90 µg.mL"1 induce an inhibition to the growth of five special forms of Fusarium oxysporum. CONCLUSION: The bioactive compounds of date palm can be used for drug development and in the food industry.

Pre-illumination of rice blast conidia induces tolerance to subsequent oxidative stress.

Many environmental factors, alone or combined, affect organisms by changing a pro-/antioxidant balance. Here we tested rice blast fungus (Magnaporthe oryzae) for possible cross-adaptations caused by relatively intense light and protecting from artificially formed reactive oxygen species (ROS) and ROS-dependent fungitoxic response of the host plant. Spore germination was found to be suppressed under 4-h and, to larger extent, 5-h illumination. The effect was diminished by antioxidants and, therefore, suggests involvement of ROS. One-hour of light did not affect spore germination, but stimulated their chemically assayed superoxide production. The illuminated spores were more tolerant (than non-illuminated ones) to artificially generated H(2)O(2), O(2)(-), or OH or to toxic diffusate of rice leaf. They also caused more severe disease symptoms if applied to leaves of the susceptible rice cultivar at low concentration. Spore diffusates decomposed hydrogen peroxide. They detoxified exogenous H(2)O(2) and superoxide radical as well as leaf diffusates. Spore illumination increased some of these protective effects. It is suggested that short-term light led to mild oxidative stress, which induced spore antioxidant capacity, enhancing spore tolerance to subsequent stronger oxidative stress and its aggressiveness in planta. Such tolerance depends partly on the antidotal action of spore extracellular compounds, which may also be light-stimulated. Therefore, a certain ROS-related environmental factor may adapt a fungus to other factors and so modulate its pathogenic properties.

Sensibilidade de Stenocarpella macrospora a fungicidas / Sensibility of Stenocarpella macrospora to fungicide

A aplicação de fungicidas nos órgãos aéreos é estratégia de controle para mancha-de-macrospora causada pelo fungo Stenocarpella macrospora. O objetivo do trabalho foi determinar a sensibilidade de S. macrospora a fungicidas pela inibição do crescimento do micélio (CM) e germinação de conídios (GC). Foram avaliados 12 fungicidas pertencentes aos grupos químicos dos benzimidazóis, estrobilurinas e triazóis, seis concentrações e dois isolados do fungo (SC e MT). Os fungicidas foram diluídos em água destilada e esterilizada e adicionados ao meio de cultura de batata-dextrose-ágar (micélio) e ágar-água (conídios) após a esterilização. A porcentagem de inibição do CM e GC foi calculada em relação à testemunha, estimando-se valores de concentração inibitória de 50% (CI50). Constatou-se que os fungicidas testados foram altamente fungitóxicos na inibição do CM, sendo que a CI50 foi menor que 1 ppm para todos os fungicidas, não havendo diferença entre isolados. Na inibição da GC, as estrobilurinas apresentaram maior fungitoxicidade, pois a CI50 ficou entre 0,0035 e 0,03 ppm, sendo o isolado SC mais sensível aos fungicidas. Os valores de CI50 para os diferentes fungicidas específicos para S. macrospora são úteis no monitoramento da sensibilidade do fungo em regiões com demanda intensa de fungicidas no milho.

The application of fungicides in the aerial organs is control strategy to macrospora spot caused by fungus Stenocarpella macrospora. The objective of this study was to determine the sensitivity of S. macrospora to fungicides by inhibition of mycelial growth (MG) and conidial germination (CG). It was evaluated 12 fungicides belonging to the chemical groups of the benzimidazoles, triazoles and strobilurins, six concentrations and two isolates of the fungus (SC and MT). The fungicides were diluted in sterile distilled water and added to the culture medium of potato dextrose agar (mycelium) and water-agar (spore) after sterilization. The percentage of inhibition of MC and CG was calculed in comparison with control, estimating of 50% inhibitory concentration (IC50). The fungicides tested were effective in inhibiting the MC. The IC50 was less than 1 ppm for all fungicides. There was no difference between isolates. The inhibition of CG had higher fungitoxicity strobilurins, and the IC50 was between 0.0035 and 0.03 ppm, and the isolated SC showed the higher sensitivity to the fungicides. The IC50 values obtained for fungicides and specific S.

Compatibility of Beauveria bassiana with acaricides / Compatibilidade de Beauveria bassiana com acaricidas

Visando avaliar a compatibilidade in vitro, entre o fungo entomopatogênico Beauveria bassiana (Bals.) Vuil. e acaricidas, 12 formulações com os ingredientes ativos clorfenapir, fenpiroximate, amitraz, acrinatrim, hexitiazox, abamectina, piridine, dimetoato, piridaphetion, óxido de fenbutatim, azociclotim e cihexatim, foram testadas em três concentrações (concentração Média - CM, metade daCM e duas vezes a CM). Para os parâmetros germinação de conídios, crescimento vegetativo e esporulação, apresentaram diferenças significativas na compatibilidade entre o fungo e as formulações utilizadas. As formulações mais compatíveis com o fungo, para os parâmetros avaliados, foram as pertencentes ao grupo químico da avermectina e piretróide. As formulações pertencentes ao grupoquímico do organofosforado e organostânico afetaram drasticamente a germinação de conídios, bem como o crescimento vegetativo e a esporulação.

The in vitro compatibility of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuil. with 12 acaricide formulations was evaluated. The active ingredients Clorfenapyr, Fenpyroximate,Amitraz, Acrinathrin, Hexythiazox, Abamectin, Pyridine, Dimethoate, Pyridaphethion, Fenbutatin Oxide, Azocyclotin and Cyhexatin were tested in three different concentrations (mean concentration - MC, half MC and twice the MC). The formulations tested affected conidial germination, vegetative growth and sporulation of the fungus in different levels. The formulations more compatible with B. bassiana were Avermectin and the pyrethroids. The acaricides belonging to the organophosphate and organostanic chemical groups, however, drastically affected conidial germination as well as vegetative growth and sporulation.