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Effect of some compounds on the decay of strawberry fruits caused by Botrytis cinerea Pers.

Field experiments were conducted in the years 1997-2000 to determine the effect of various sprayings strawberry plants on the percentage of infected fruits caused by Botrytis cinerea Pers. The effect of garlic's extract at concentration 1%, water solution of microcrystalline chitosan (3% polymer content) at concentration 0.2% and iprodione (255 g active form in 1000 cm3 of water) was estimated. The experiment was conducted on cultivar Senga-Sengana. During the growing season strawberry plants were sprayed three times--at the beginning of blooming, during full blooming and at the end of blooming. Ripped fruits were stored for 24 hours in a cooling chamber. Then the strawberry fruits were weighted and the percentage of infected fruits was estimated. In the first year of cultivation there was found no effect of used plant protection on the decay of strawberry fruits. Intensification of the disease was very low because of the lack of rainfalls and good weather. These conditions did not propitious the infection. In the second and in the third year of cultivation the best efficacy in control grey mould had iprodione which limited decay of strawberry fruits to 9% (in the second year) and 11.1% (in the third year). In the second year the chitosan water solution had protective effect and the decay of strawberry fruits was diminished to 15%. The highest development of infection was in combination where garlic extract was used for spraying both in the second 23% and in the third year 32.5% as well.

Aminoimidazole Carboxamide↗

Biological control of grey mould (Botrytis cinerea) with the antagonist Ulocladium atrum.

The competitive saprophytic fungus Ulocladium atrum was selected on the basis of its antagonistic potential for the control of grey mould caused by the necrotrophic pathogen Botrytis cinerea. Field trials were carried out to asses the efficacy of U. atrum as a biocontrol agent against B. cinerea in grapevine. The results demonstrated that under moderate disease pressure U. atrum had the potential to control grey mould, whereas under high disease pressure the efficacy was not sufficient to substitute the use of fungicides completely.

Amides↗

Activity of some chemicals in the control of Botrytis cinerea on roses.

In the control of grey mould (Botrytis cinerea) on rose petals all 15 tested fungicides used as a spray, suppressed very effectively development of necrosis. But the best results in suppression of necrosis development, more than 90% effectiveness gave: Amistar 250 SC (azoxystrobin), Bravo 500 SC (chlorothalonil), Discus 500 WG (kresoxim methyl), Euparen 50 WP (dichlofluanid) Euparen Multi 50 WG (tolyfluanid), Folpan 80 WG (folpet), Kaptan zaw. 50 WP (captan), Penncozeb 80 WP (mancozeb), Ronilan 50 WP (vinclozolin), Rovral Flo 255 SC (iprudione), Sumilex 500 SC (procymidone) and Teldor 500 SC (fenhexamid). When Dithane M-45 80 WP (mancozeb), Sarbrawit 530 SC (chlorothalonil + carbendazim) or Topsin M 70 WP (thiophanate methyl) were used, the diameter of spots was about 6-times smaller than on control flowers (about 84% effectiveness). All tested fungicides used for spraying of flower petals with abundant visible sporulation of B. cinerea were ineffective in the inhibition of spore germination. But when Petri dishes with potato-dextrose-agar were sprayed with tested fungicides and inoculated with spore suspension, Euparen 50 WP, Euparen Multi 50 WG, Folpan 80 WG, Kaptan zaw. 50 WP and Penncozeb 80 WP completely suppressed conidia germination.

Botrytis↗

In situ development and application of cDNA-AFLP to isolate genes of Candida oleophila (strain O) potentially involved in antagonistic properties against Botrytis cinerea.

The yeast Candida oleophila (strain O) presents a high level of protective activity against Botrytis cinerea (gray mold) on postharvest apples. The cDNA-AFLP technique allows the comparison of mRNA populations extracted from cells grown in different conditions. In order to isolate yeast genes potentially involved in biological control properties, that technique was applied on strain O cells growing on apple wounds. The biological control properties of 8 C. oleophila strains and strain O were assessed in order to compare the gene expression of a non antagonistic strain against gene expression of strain O. In the absence of a non-antagonistic strain, an other comparison model was designed. It was based on the growth of strain O in different in situ conditions: strain O applied on apple wounds (O), strain O applied on apple wounds in presence of B. cinerea (B) and B. cinerea alone on apple wounds (F). A recovering technique, based on the washing of cells in the wound and a RNA extraction method followed by a DNase treatment were optimised before cDNA-AFLP application. Thirteen primer pairs were used. Their application resulted in an average of 54 and 55 bands for O and B respectively whereas no bands were observed for F. Among these bands, 8 were expressed more intensely in presence of the pathogen (1.1% of the fragments).

Botrytis↗

Selection of antagonists of postharvest apple parasites: Penicillium expansum and Botrytis cinerea.

The objectives of this study were to constitute a collection of pathogenic agents of economic importance which cause losses of apple fruits after harvest namely Botrytis cinerea and Penicillium expansum and to select in vivo efficient antagonistic strains able to protect fruits against both pathogens at 5 degrees C (P. expansum) and 25 degrees C (P. expansum & B. cinerea). Twenty strains of P. expansum and ten strains of B. cinerea have been isolated from infected apple fruits. Potential antagonistic micro-organisms (thirty three isolates) belonging to yeast, bacteria and fungi have been isolated from apple surface. Six of them (strains Ach1-1, Ach2-1, Ach2-2 belonging to Aureobasidium pullulans (De Bary) Arnaud, and strains 1112-3, 1113-10 and 1113-5 belonging to Aureobasidium pullulans (de Bary) Am. v. pullulans) showed a high level of protection (more than 80%) at 25 degrees C. once inoculated with P. expansum or B. cinerea for 5 days. The highest level of protection against P. expansum (96%) was observed with the application of Ach 2-1. Six days after inoculation of B. cinerea, strains Ach 2-2 and Ach 2-1 insured 100% and 96% of protection, respectively. At lower temperature (5 degrees C), first symptoms of P. expansum appeared 13 days after its inoculation. Percentages of protection observed after apple treatment with one of the six antagonistic strains were ranged from 78% to 94% 20 days after P. expansum inoculation. Strains labelled Ach showed a protective level higher than 90% against this pathogen, followed by strain 1113-10 (90%), strain 1113-5 (89%) and strain 1112-3 (82%). At 26 days post-inoculation, levels of protection decreased but remained higher than 60% (more than 80% with strain Ach2-2 and strain 1113-5, 75% with strain Ach2-1 and 1113-10, 72% with ach1-1, 61% for the other strains). Strain Ach2-2 and 1113-10 were retained as the best antagonists for the subsequent studies.

Antibiosis↗

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

Electrophoresis, Gel, Two-Dimensional↗

Lipid Peroxidation and Membrane Disruption by Vinclozolin in Dicarboximide-Susceptible and -Resistant Isolates of Botrytis cinerea

A dicarboximide-susceptible (DS) isolate and a dicarboximide-resistant (DR) isolate of Botrytis cinerea were compared with regard to spore germination, mycelial growth, cellular leakage, and lipid peroxidation upon treatment with the dicarboximide fungicide vinclozolin. The fungicide inhibited spore germination and mycelial growth of the DS isolate, but not those of the DR isolate. The inhibitory effect of the fungicide was greater on mycelial growth than on spore germination of the DS isolate. Significant cellular leakage from the fungicide-treated DS or DR isolate began to increase after 24 hr incubation, depending on the concentration of the fungicide and the duration of incubation time. However, the magnitude of cellular leakage was much greater from the DS isolate than from the DR isolate. Vinclozolin caused considerable lipid peroxidation in the DS isolate, whereas little or no lipid peroxidation occurred in the DR isolate treated with the fungicide. Lipid peroxidation preceded cellular leakage from the DS isolate following fungicide treatment. The effects of the fungicide on mycelial growth, cellular leakage, and lipid peroxidation of the DS isolate were all reversed by the addition of alpha-tocopherol to the incubation medium. These results demonstrate that vinclozolin causes significant lipid peroxidation and subsequent cellular leakage from a DS isolate, but not from a DR isolate. Furthermore, they suggest that the mechanism of action of dicarboximide fungicides is associated with membrane lipid peroxidation.

Journal Article↗

Transformation of Botrytis cinerea with the hygromycin B resistance gene, hph.

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), conferring hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis, showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2-10 transformants per micrograms of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.

Drug Resistance, Microbial↗

Variations in ploidy among isolates of Botrytis cinerea: implications for genetic and molecular analyses.

Field isolates and laboratory strains of Botrytis cinerea, an ascomycetous fungus causing considerable economic losses, e.g., as "grey mould" of vine, were compared for differences in ploidy level by determining their DNA content per nucleus. Strain SAS56, an ascospore line used routinely for genetic analyses, is probably polyploid, since treatment with benomyl causes a significant reduction in DNA content per nucleus. This conclusion is substantiated by the increased sensitivity of the putative haploid derivatives to mutagens (UV and EMS). Molecular analyses (RAPD) of the haploidized strains indicate a very limited degree of heterozygosis of the parent strain SAS56. Analysis of field isolates of B. cinerea showed that their DNA content per nucleus varied considerably, indicating that aneuploidy/polyploidy is a widespread phenomenon in this species. This can explain both the variability and phenotypic instability of many field isolates of this fungus and the unusual difficulties faced by researchers in recovering stable recessive laboratory mutants. Since the haploid derivatives of SAS56 resemble the parent strain in their parasitic and physiological properties they should provide a good basis for classical and molecular genetic studies.

Base Sequence↗

Floral homeotic gene expression defines developmental arrest stages in Brassica oleracea L. vars. botrytis and italica.

Brassica oleracea L. vars, italica (broccoli) and botrytis (cauliflower) both undergo developmental arrests which result in heading phenotypes. We characterized these arrested tissues at the morphological and molecular levels, and defined the developmental changes that ensure after arrest has been broken. We found that the order of floral organ initiation and the positions of resulting floral organ primordia in this species in some respects from that of Arabidopsis, which is a member of the same family, Brassicaceae. We also cloned homologs of the Arabidopsis floral homeotic genes APETALA1 (AP1) and APETALA3 (AP3) from B. oleracea and characterized their expression patterns. We found that the AP1 homolog was expressed in some of the meristems of arrest-stage cauliflower, providing evidence that this tissue is florally determined. In broccoli, both the AP1 and AP3 homologs were expressed. However, the spatial pattern of expression of the broccoli AP1 homolog differed from that of Arabidopsis. In addition we identified a homolog of the CAULIFLOWER (CAL) gene, BoiCAL, from broccoli. The predicted amino acid sequence indicated that the BoiCAL gene product does not contain the mutation thought to be responsible for the early arrest exhibited in cauliflower (Kempin et al. 1995), but contains other changes that might play a role in the broccoli heading phenotype.

Amino Acid Sequence↗

Purification and properties of an extracellular lipase from the fungus Botrytis cinerea.

An extracellular lipase (EC 3.1.1.3) from the fungus Botrytis cinerea has been purified to homogeneity and characterized. The purification included ammonium sulfate fractionation and sequential column chromatography. The purification of the preparation was 31-fold and recovery yield was 21%. The purified enzyme was associated with esterase activity according to activity staining on polyacrylamide gel. The molecular weight was determined as 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and estimated at 72 kDa using gel filtration, which suggests that the enzyme may be a monomer. The isoelectric point was 6.5, and optimal activity was obtained at 38 degrees C and pH 6.0. This lipase showed a high specificity for synthetic substrates containing long-chain unsaturated fatty acids using umbelliferone esters. The effect of beta-cyclodextrin on the hydrolysis of olive oil has been studied. The specific activity was 25 mumole/min/mg in the absence of beta-cyclodextrin and 132 mumole/min/mg in its presence.

Enzyme Induction↗

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination.

The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea.

Cloning, Molecular↗

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues.

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23-29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M(r) of 25,500. The c26-2 insert is a 5' truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3' untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin gamma-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products.

Amino Acid Sequence↗

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems.

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methylesterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls.

Brassica↗

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea.

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.

Amino Acid Sequence↗

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis).

The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of beta-carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as "membranous" chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.

Brassica↗

Polygalacturonase of Botrytis cinerea E-200 Pers.

A polygalacturonase (poly(1,4-alpha-D-galacturonide)glycanohydrolase, EC 3.2.1.15) was purified from the culture fluid of Botrytis cinerea. The polygalacturonase preparation, homogeneous on the basis of disc-gel electrophoresis also showed pectinesterase activity. Some properties of the purified polygalacturonase were studied. It had a molecular weight about 69 000. It was inactivated by p-chloromercuribenzoate, tetranitromethane and urea. A 50% loss in viscosity of sodium polypectate solution occurred when 4.6% of the glycosidic bonds were hydrolyzed. The only end product of sodium polypectate and oligogalacturonides hydrolysis was monogalacturonic acid.?

Chloromercuribenzoates↗

Structural properties of native and sonicated cinerean, a beta- (1-->3) (1-->6)-D-glucan produced by Botrytis cinerea.

Cinerean, the extracellular beta-(1-->3) (1-->6)-D-glucan of the fungus Botrytis cinerea was studied. Electron micrographs of the native polysaccharide revealed quasi-endless fibrils with an estimated diameter of ca. 1.5 nm. A particle mass of 10(9)-10(10) daltons was determined from dilute solutions by low-angle laser light scattering. Sonication of increasing duration led to fragmentation of the native polymer with an approximately exponential decrease of mass in the range of average molecular masses between 250,000 and 50,000 daltons. Shadowed by platinum, cinerean fibril fragments with a weight-average molecular mass of 172,000 +/- 3000 daltons could be characterized from electron micrographs as a distribution of rods of most probable length of 45 nm and an average length of 72 nm. Small-angle X-ray scattering confirmed the fibrillar structure of the native cinerean and the rodlike structure of sonicated cinerean. A rod diameter of 1.9 +/- 0.2 nm and a mass per unit length of 2250 +/- 490 daltons/nm were found. The latter is in agreement with the value of 1830 daltons/nm calculated from the length distribution determined from the electron micrographs. These data-especially the mass per unit length-suggest a quaternary structure for the polysaccharide. Such a structure would explain the rigidity of the rods which, in turn, is responsible for the characteristic phase separation behaviour in aqueous solutions observed by nephelometry and viscometry.

Carbohydrate Sequence↗