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Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L.

The essential oil extracted by steam distillation from the capitula of Indian Tagetes patula, Asteraceae, was evaluated for its antifungal properties and analyzed by gas chromatography and gas chromatography-mass spectrometry. Thirty compounds were identified, representing 89.1% of the total detected. The main components were piperitone (24.74%), piperitenone (22.93%), terpinolene (7.8%), dihydro tagetone (4.91%), cis-tagetone (4.62%), limonene (4.52%), and allo-ocimene (3.66%). The oil exerted a good antifungal activity against two phytopathogenic fungi, Botrytis cinerea and Penicillium digitatum, providing complete growth inhibition at 10 microl/ml and 1.25 microl/ml, respectively. The contribution of the two main compounds, piperitone and piperitenone, to the antifungal efficacy was also evaluated and ultrastructural modifications in mycelia were observed via electron microscopy, evidencing large alterations in hyphal morphology and a multisite mechanism of action.

Antifungal Agents↗

Mode of action and antifungal properties of two cold-adapted chitinases.

The mode of action of two chitinases from the Antarctic Arthrobacter sp. strain TAD20 on N-acetyl-chitooligomers and chitin polymers has been elucidated. Identification of the length of chitin oligomers following enzymatic hydrolysis was verified by using HPLC-based analysis. It was observed that the length of the oligomer is important for enzyme action. The enzymes cannot effectively hydrolyze chitin oligomers with a degree of polymerization lower than four. ArChiA is an endochitinase which hydrolyzes chitin substrates randomly, whereas ArChiB is an exochitinase which degrades chitin chains and N-acetyl-chitooligomers from the nonreducing end, releasing N- N'-diacetyl-chitobiose. ArChiB (100 microg/ml) inhibited spore germination and hyphal elongation of the phytopathogenic fungus Botrytis cinerea by 15% and 30%, respectively. A more pronounced effect was observed with ArChiA (100 microg/ml) resulting in 70% inhibition of spore germination and 60% inhibition of germ tube elongation. A slight additive effect was observed, when the two enzymes were used in combination, only on the inhibition of germ tube elongation.

Antifungal Agents↗

CASAR82A, a pathogen-induced pepper SAR8.2, exhibits an antifungal activity and its overexpression enhances disease resistance and stress tolerance.

Pepper SAR8.2 gene (CASAR82A) was previously reported to be locally or systemically induced in pepper plants by biotic and abiotic stresses. In this study, the physiological and molecular functions of the pepper SAR8.2 protein in the plant defense responses were investigated by generating Arabidopsis transgenic lines overexpressing the CASAR82A gene. The transgenic Arabidopsis plants grew faster than the wild-type plants, indicating that the CASAR82A gene was involved in plant development. The ectopic expression of CASAR82A in Arabidopsis was accompanied by the expression of the Arabidopsis pathogenesis-related (PR)-genes including AtPR-1, AtPR-4 and AtPR-5. CASAR82A overexpression enhanced the resistance against infections by Pseudomonas syringae pv. tomato, Fusarium oxysporum f.sp. matthiolae or Botrytis cinerea. The transgenic plants also exhibited increased NaCl and drought tolerance during all growth stages. Moreover, the methyl viologen test showed that the transgenic plants were tolerant to oxidative stress. The purified recombinant CASAR82A protein and crude protein extracts of the transgenic plants exhibited antifungal activity against some phytopathogenic fungi, indicating that the enhanced resistance of the transgenic plants to fungal pathogen infection may be due to the antifungal effect of SAR8.2 protein.

Antifungal Agents↗

Transcriptional activator TSRF1 reversely regulates pathogen resistance and osmotic stress tolerance in tobacco.

Increasing evidences show that ethylene-responsive factor (ERF) proteins regulate plant stress response and the interaction of different stress responsive pathways through interacting with different cis-acting elements, even other transcription factors. Here, we report a transcriptional activator TSRF1, which was previously demonstrated to regulate plant resistance to Ralstonia solanacearum, reversely regulates pathogen resistance and osmotic stress tolerance in tobacco. Sequence analysis revealed that TSRF1 contains a putative transcriptional activation domain. Using yeast two hybrid system we evidenced that this activation domain is essential for activating the expression of reporter gene. To confirm the broad-spectrum pathogen resistance of TSRF1 we observed that over-expressing TSRF1 enhances the resistance to Pseudomonas syringae and Botrytis cinerea in both tobacco and tomato plants, but RNA interference of TSRF1 in tomato plants decreases the resistance to these pathogens, unraveling the positive regulation of TSRF1 in plant pathogen infections. The expression of TSRF1 in response to NaCl and mannitol suggests the possible functions of TSRF1 in osmotic stress responses, but the physiological tests indicate that expressing TSRF1 in tobaccos decreases tolerance to NaCl or mannitol during germination and seedling root development, and this result was consistent with PEG6000 treatment with mature tobacco seedlings, indicating the negative modulation of TSRF1 in osmotic stress response. Therefore, our research reveals that transcriptional activator TSRF1 reversely regulates plant pathogen resistance and osmotic stress response.

Amino Acid Sequence↗

Determination of pathogen-related enzyme action by mass spectrometry analysis of pectin breakdown products of plant cell walls.

An analytical approach using matrix-assisted laser desorption/ionization mass spectrometry for the structural characterization and assessment of the degree of polymerization of cell wall pectin-derived oligosaccharides (PDOs) in three regions of Botrytis cinerea-infected tomato fruit tissue is described. The PDOs were isolated from lesion centers (extensively macerated tissue), the area just beyond visible lesion margins, and healthy and intact tissue of an inoculated fruit, sampled at a distance from developing lesions. PDO mixtures were directly analyzed by mass spectrometry without chromatographic separation, after minimum cleanup by membrane drop dialysis. The structures identified implied the action of three different pathogen pectin-modifying enzymes. Modifications such as methyl esterification were identified by determination of exact PDO molecular masses and tandem mass spectrometry via collision-induced dissociation. We have identified four PDO series that were generated through the breakdown of homogalacturonan pectins. The decayed and lesion edge areas had fewer and less diverse PDOs than healthy tissues, possibly due to metabolic by-products of the pathogen. This analytical technique provides a simple and rapid method to characterize the pectin-derived oligosaccharides produced by in vivo digestion during pathogen infection.

Botrytis↗

Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology.

Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase chain reaction (PCR)-amplified fluorescently labeled DNA extracted from pure culture and from diseased plant tissue. The probes easily distinguished these pathogens from each other without cross reaction. Thickness of the agarose layer and length of the sample DNA were important factors affecting hybridization efficiency of immobilized probe to PCR product. These factors did not affect hybridization with short complementary oligonucleotide. Probes fixed on agarose-coated slides could differentiate samples as readily as probes on nylon but with potentially higher spot density and gave much better signal than probes on silylated slides. The use of plain glass slides, agarose, and a manual arrayer makes this technique useful for developing specialized and inexpensive DNA microarrays on a solid rigid substrate.

Ascomycota↗

Microwave-assisted, one-pot syntheses and fungicidal activity of polyfluorinated 2-benzylthiobenzothiazoles.

Polyfluorinated 2-benzylthiobenzothiazoles 3a-l are prepared via a microwave-assisted, one-pot procedure. The advantages, such as good to excellent yields, shorter reaction time (14-21min), readily available starting material, and simple purification procedure, distinguish the present protocol from other existing methods used for the synthesis of 2-benzylthiobenzothiazoles. Bioassay indicated that most of the compounds showed significant fungicidal activity against Rhizoctonia solani, Botrytis cinereapers, and Dothiorella gregaria at a dosage of 50microg/mL. Interestingly, compared to the control of commercial fungicide, triadimefon, compound 3c exhibited much higher activities against R. solani, B. cinereapers, and D. gregaria, which showed that the polyfluorinated 2-benzylthiobenzothiazoles can be used as lead compound for developing novel fungicides.

Antifungal Agents↗

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens.

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

Apoptosis↗

The G protein alpha subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and differential production of antifungal metabolites.

Trichoderma mycoparasitism includes recognition, attack, overgrowth and lysis of the host fungus accompanied by morphological changes and secretion of hydrolytic enzymes and antibiotics. Studying the underlying signal transduction pathways, the tga1 gene encoding a Galpha subunit of Trichoderma atroviride P1 was analysed. A Deltatga1 mutant showed continuous sporulation and elevated internal steady-state cAMP levels. tga1 gene deletion resulted in a complete loss of mycoparasitic overgrowth and lysis of Rhizoctonia solani, Botrytis cinerea, and Sclerotinia sclerotiorum during direct confrontation, although infection structure formation was unaffected. The reduced mycoparasitic abilities were reflected by strongly decreased chitinase activities and reduced nag1 and ech42 gene transcription. Furthermore, production of 6-pentyl-alpha-pyrone and of metabolites with sesquiterpene structure was reduced in the Deltatga1 mutant. Regardless of these deficiencies, the mutant displayed an enhanced growth inhibition of the host fungi by over-producing other low molecular weight antifungal metabolites, suggesting opposite roles of Tga1 in regulating the biosynthesis of different antifungal substances in T. atroviride.

Antifungal Agents↗

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum.

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.

Amino Acid Motifs↗

Antifungal limonoids from the fruits of Khaya senegalensis.

Investigation of the fruits of Khaya senegalensis resulted in the isolation of three new mexicanolide limonoids containing a rare conjugated diene lactone system named seneganolide A (1), 2-hydroxyseneganolide A (2) and 2-acetoxyseneganolide A (3). Two known limonoids, 3-deacetyl-7-deacetoxy-7-oxokhivorin (4) and methyl 6-hydroxyangolensate (5), were also found. The structures of the new compounds were elucidated on the basis of spectral methods. The antifungal activity of compounds 1, 3 and 5 was tested against the fungus Botrytis cinerea.

Antifungal Agents↗

Antifungal effect of some spice hydrosols.

The antifungal effects of rosemary, cumin, sater (savory), basil and pickling herb hydrosols were investigated against Rhizoctonia solani, Fusarium oxysporum f. sp tulipae, Botrytis cinerea and Alternaria citri. Hydrosols of sater and pickling herb showed the most relevant fungicidal activity.

Alternaria↗

Inhibition of phytopathogenic fungi by essential oil, hydrosol, ground material and extract of summer savory (Satureja hortensis L.) growing wild in Turkey.

The antifungal activities of the essential oil, hydrosol, ground material and extract of summer savory (Satureja hortensis L.) on mycelial growth of Alternaria mali Roberts and Botrytis cinerea Pers. were determined. All doses of extract inhibited 100% the mycelial growth of both fungi, and exhibited a fungicidal effect. The 15% level of hydrosol and the 1.0% level of ground material had a 100% effect on B. cinerea. The other doses showed weak inhibition on mycelial growth of the fungi, and antifungal activity of the essential oil varied depending on concentrations. While the levels of essential oil show fungistatic effect, the increasing doses of hydrosol and ground material showed a fungicidal effect against B. cinerea and A. mali. While the ground material had not showed any fungicidal activity against mycelial growth of A. mali, the 1% and 1.5% levels of the ground material exhibited a fungicidal effect on B. cinerea. The results obtained from this study may contribute to the development of environmentally safer alternatives to protect the spoilage of food products from pathogenic and saprophytic fungi.

Alternaria↗

Antifungal activity of Tagetes patula extracts on some phytopathogenic fungi: ultrastructural evidence on Pythium ultimum.

Methanol extract, obtained from Tagetes patula plant, was assayed against three phytopathogenic fungi: Botrytis cinerea, Fusarium moniliforme and Pythium ultimum. The antifungal activity was tested both in the dark and in the light, using two different lighting systems. The data showed that the extract proved to have a dose-dependent activity on all the fungi with a marked difference between treatments in the light than in the dark. Good growth inhibition was observed in fungi only when these were treated with the highest dose of the extract and irradiated, whereas the same dose gave only a modest inhibition when the experiment was conducted in the dark. At 5 and 10 microg/ml in the dark, growth increased. The results indicated that the presence of a luminous source enhances the antifungal activity, with small differences between UV-A and solar spectrum light. SEM and TEM observations on Pythium ultimum revealed that the Tagetes patula extract induced alterations on cell fungal membranes with a photoactivation mechanism possibly involving the production of free radicals and leading to a premature aging of the mycelium.

Antifungal Agents↗

Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli.

Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover.

Antifungal Agents↗

Actinchinin, a novel antifungal protein from the gold kiwi fruit.

An antifungal protein designated actinchinin, with an N-terminal sequence different from that of the thaumatin-like antifungal protein from green kiwi fruit, was isolated from the gold kiwi fruit. The antifungal protein, unlike its counterpart from green kiwi fruit, did not exert antifungal activity against Botrytis cinerea, but was active against Fusarium oxysporum which was unresponsive to thaumatin-like protein from green kiwi fruit. Actinchinin was isolated using a protocol that comprised ion exchange chromatography on CM-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. Actinchinin was adsorbed on CM-cellulose, Affi-gel blue gel and Mono S. It was devoid of mitogenic activity toward mouse splenocytes. In contrast to thaumatin-like protein from green kiwi fruit, actinchinin lacked HIV-1 reverse transcriptase inhibiting activity.

Actinidia↗

An antifungal protein from flageolet beans.

A protein with antifungal and hemagglutinating activities was isolated from dried flageolet beans (Phaseolus vulgaris cv. 'Flageolet Bean'). The protein was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. The protein demonstrated antifungal activity against Mycophaerella arachidicola with an IC50 of 9.8 microM, but was inactive toward Fusarium oxysporum and Botrytis cinerea. Its hemagglutinating activity could not be inhibited by a variety of the sugars tested. The activity was stable up to 60 degrees C. At 70 degrees C, 75% of the hemagglutinating activity remained while no activity was discernible at and above 100 degrees C. The hemagglutinating activity was stable in the presence of a variety of monovalent, divalent and trivalent chlorides, and also when the ambient pH changed from 3 to 12. It did not exert any mitogenic activity on mouse splenocytes in vitro. Neither did it inhibit HIV-1 reverse transcriptase. It inhibited [3H-methyl]-thymidine incorporation into leukemia L1210 cells with an IC50 of about 4 microM.

Animals↗

Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum.

A 15-kDa antifungal protein, designated ganodermin, was isolated from the medical mushroom Ganoderma lucidum. The isolation procedure utilized chromatography on DEAE-cellulose, Affi-gel blue gel, CM-Sepharose and Superdex 75. Ganodermin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. Ganodermin inhibited the mycelial growth of Botrytis cinerea, Fusarium oxysporum and Physalospora piricola with an IC50 value of 15.2 microM, 12.4 microM and 18.1 microM, respectively. It was devoid of hemagglutinating, deoxyribonuclease, ribonuclease and protease inhibitory activities.

Amino Acid Sequence↗