1,720,964 research outputs found
Botrytis cinerea transcriptomic response to the active substance eugenol and role of Snf1 kinase in fungal pathogenesis
Get PDFBotrytis cinerea is a fungal pathogen causing grey mould on more than 240 dicotyledonous plants resulting huge economic losses. To control the disease in the field and to prevent post-harvest damages, frequent fungicidal treatments are required, thus increasing the risk of spread of resistant strains to fungicides and the negative effects on environment and human health. Therefore, new molecules with different mode of action or alternatives to fungicides should be introduced to better manage the disease, to better preserve the environment and to increase the food safety.
Several groups of natural substances can be used as alternatives to synthetic fungicides and among these compounds, plant essential oils are largely experimented for their well known antimicrobial and antifungal activity. Eugenol is an effective compound of several essential oils, the main component of clove oil, which is extracted from clove buds and used as local antiseptic in traditional medicine. The mechanism of the antifungal effect of eugenol was examined on Saccharomyces cerevisiae and on some plant pathogenic fungi. The main described effects of this phenolic component seem to be the disruption of integrity of fungal cell membrane and cell wall; however the precise mode of action of eugenol is unclear.
To address the mode of action of eugenol, transciptome analysis was performed using NimbleGen 4x72K arrays. Cultures of B. cinerea were treated with eugenol at the effective concentration (EC80 ) of 250 µg ml-1, and control (untreated) and treated mycelia were collected after 6 h and 12 h. The transcriptional response of B. cinerea to eugenol was statistically analyzed and the differentially expressed genes were evaluated. Significant enrichment of down-regulated genes was found in the ergosterol biosynthesis, DNA replication factors class, glutathione pathway, transmembrane transport, and botrydial toxin biosynthesis, while up-regulated genes were detected in several functional categories belonging to stress response.
A modern approach to fungicidal development is a rational design of drugs mirrored to target fundamental node of the complex cellular network.
In yeast, the Snf1 protein kinase is a global regulator and important in a glucose signalling pathway controlling the response to nutritional and environmental stresses. In order to characterize the role of B. cinerea Snf1 kinase, deletion mutants of the encoding gene were obtained and analyzed in vitro and in vivo. Δsnf1 mutants grew slower with a reduction of 20-80 % on simple sugars, polysaccharides, and on lipidic carbon sources at pH 5 and mostly at pH 7. The enzyme activity produced by a mutant strains was also measured and a significant reduction of xylanase activity and an increase of polygalacturonase activity were observed in comparison with the wild type. These results suggest that Snf1 kinase does not act as general activator of B. cinerea cell wall degrading enzymes but seems to be involved in a possible pH dependent regulation. In pathogenicity test, the virulence of the Δsnf1 mutants was reduced by 60-90 % in comparison with the wild type. Δsnf1 mutants were impaired in sporulation as well; in fact, they did not produce normal conidiophores with macroconidia, but only microconidia.
In conclusion, B. cinerea Snf1 protein kinase regulates conidiation, carbon source utilization and strongly affects virulence; therefore it could be a possible target for new fungicide
SNF1 protein kinase is important for growth and full virulence of Botrytis cinerea.
No full textAt early stages of infection, Botrytis cinerea secretes a wide spectrum of plant cell wall degrading enzymes (CWDEs) to facilitate penetration into host. So far, only the polygalacturonases PG1 and PG2 and the xylanase XYN11A were proved by reverse genetics to be required for virulence. Verification of the function of single members of the different CWDE classes is indeed difficult, due to gene redundancy in multigenic families.
In various plant pathogenic fungi, production of these enzymes is under catabolic repression and positively regulated by the snf1 (sucrose non-fermenting 1) gene which is expressed when glucose is depleted.
To examine the function of the snf1 gene in B. cinerea, knock-out mutants were obtained by targeted mutagenesis. The growth of the Δsnf1 mutants was compared with the wild type strain on minimal medium enriched with different simple and complex carbon sources (fructose, sucrose, glucose, xylan, xylose, pectin, polygalacturonic acid, cellulose). A significant reduction of growth was observed on some carbon sources except with glucose and xylan. Microscopic studies verified, that the sporulation of the mutants was almost abolished and unusually shaped mycelia were found. Pathogenicity tests performed on apple fruits displayed a strong defect in colonization by the Δsnf1 mutants with a 60% decrease in virulence.
Pathogenicity tests on plant systems are in progress together with the characterization of possible effects on secretion and expression of CWDEs
Fungitoxic activity of eugenol against Botryitis cinerea may be mediated by fungal peroxidase.
No full textPlant essential oils have shown antifungal activity against several plant pathogenic fungi in pre- and post-harvest. This activity is due to their monoterpenic constituents like eugenol. However, eugenol mechanism of action is not completely clear.
We studied the effect of eugenol on Botrytis cinerea (strain B05.10). The effective concentration of eugenol in inhibiting mycelium growth and conidia germination was 1-2 mM and the minimum effective dose of eugenol to protect grape leaves from B. cinerea infection was about 10 mM.
Experimental data showed that eugenol causes a release in the B. cinerea culture filtrate of potassium ions and of intracellular material, indicating a damage to cell membrane possibly due to an oxidative stress. However, in comparison to untreated control, no accumulation of reactive oxygen species (measured by xylan orange assay) was observed in B. cinerea mycelia treated with eugenol, while the concentration of antioxidant molecules such as glutathione and cysteine decreased.
Since B. cinerea produces high level of peroxidase activity and eugenol is a good competitive substrate of cellular antioxidants, the eugenol activity may be ascribed to the release of hydroxyl radical, a product of the reaction of eugenol with hydrogen peroxide. Moreover, the oxidized eugenol showed the same fungitoxic activity of the not oxidized eugenol
Inhibitory activity of essential oils components against the fungal plant pathogen Botrytis cinerea
No full textBotrytis cinerea is the fungus responsible of the grey mould
disease of grape and other fruits. Chemical control is necessary
when environmental conditions are favorable to the disease.
In order to protect plants from disease the European Union by
directive 2009/128/EC gives priority to non-chemical methods
and encourages the use of natural non-toxic substances. Natural
compounds are used as an alternative to chemical fungicides
mostly to control post-harvest diseases. The major aim of this
work is to compare the fungicidal activity against B. cinerea of
11 natural compounds that are produced in plants through
the shikimate/phenylalanine pathway. In a spore germination
assay six compounds (eugenol, thymol, carvacrol, isoeugnol,
cinnamaldehyde and vanillin) completely inhibited the germination
of conidia. These natural products also reduced the viability of the
spores and inhibited the growth of B. cinerea mycelium (strain
PM10) even if with different efficiency. These compounds are the
major constituents of essential oils of many aromatic species.
Some chemical characteristics, like the high lipophilic and the low
value of polar surface area, are common among these compounds
indicating an high affinity for phospholipids compartment and
their difficult to overcome the external layer of the cell
Effect of structurally related essential oil components on growth of Botrytis cinerea.
No full textEssential oils are used as an alternative to chemical fungicides mostly to control post-harvest diseases, included grape grey mould. Main components of some essential oils are structurally related but may differ in fungicide activity. The major aim of this work was to compare the fungicidal activity against Botrytis cinerea Pers. of some related components of essential oils.
The compounds, compared by a plate growth assay, showed the following order of decreasing activity: eugenol, thymol, cinnamaldehyde, isoeugenol, carvacrol, vanillin and guaiacol. The EC50 for eugenol was about 100 μg/ml for the strain B05.10 but was even less against the PM10 strain.
Treatment with eugenol affected also conidia germination, in particular a MIC of 500 μg/ml completely inhibited spore germination of the PM10 strain also at 60 hpi.
The same eugenol concentration was used in a leaf assay and was effective in preventing the infection of both strains of B. cinerea for the entire duration of experiment (10 days). Treatment of grape berries was also performed and confirmed the effectiveness of eugenol in delaying B. cinerea infection. However, the effect was influenced by the grapevine cv; in fact, treated cv. Cabernet Sauvignon was more sensitive to B. cinerea infection than cv. Merlot, although both cvs were similarly infected in the untreated controls.
Although eugenol is known to be an antioxidant compound and increases bacterial membrane permeabilization, and it is reported as a strong inhibitor of soybean lipoxygenase activity, the mechanism by which it affects B. cinerea spore germination and growth is not yet known.
In order to better understand the targets of eugenol and the fungal response to this phenolic compound, transcriptomic analysis of B. cinerea mycelium grown for 72 h and then treated for 6 and 12 hours with eugenol by using EC50 and EC80 concentrations is currently under investigation
The activity of the Botrytis cinerea endo-polygalacturonase PG1 is detected in berry skins and is required for full virulence during grape infection.
No full textThe necrotrophic fungal pathogen Botrytis cinerea is the causal agent of grey mould or Botrytis bunch rot in grapes. During the infection process this fungus secretes several cell-wall degrading enzymes, in particular endo-polygalacturonases (PGs) which are involved in the depolymerization of pectin, the main constituent of primary cell wall and middle lamella. The genome of B. cinerea contains six endo-PG encoding genes. Aim of the present work was to characterize the role of these enzymes during infection of grape berries. First, we studied the expression of the corresponding genes on berries of cv. Pinot blanc and Pinot noir. Among the genes analyzed by qPCR, only Bcpg2 was not expressed, while Bcpg1, encoding a basic isoform, showed the highest transcript levels at all time points analyzed and in both cvs. We also analyzed the PG activity produced by B. cinerea during grape berries infection by loading on a gel activity assay grinded berry skins: the BcPG1 isoform was detected but only as a weak band compared to other PGs produced by the fungus. However, since the BcPG1 has been previously demonstrated to be an important virulence factor in several host tissues although its role has never been investigated on grape berries, we performed infection experiments with a ΔBcPG1 knock-out mutant. A 20% reduction of symptoms caused by the ΔBcPG1 mutant was observed on both Pinot cv. and also on cv. Italia table grape, thus indicating that BcPG1 is required for full virulence on grape berries
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
