Einfluß von Tricyclazol auf die Entwicklung und Melaningehalte der Sklerotien von Botrytis cinerea

Effect of tricyclazole on production and melanin contents of sclerotia of Botrytis cinerea Tricyclazole retarded production of sclerotia of Botrytis cinerea on agar medium more severely than mycelium growth. At a concentration range (50–200 mg/l) that did not control Botrytis on grape leaves, sclerotia production was significantly reduced. There was a negative relation between the bleaching duration of sclerotia and the tricyclazole concentrations in the medium on which they were formed. Light microscopical studies showed that sclerotia from tricyclazole-containing medium contained a significantly poorer developed outer melanin layer than that from the control medium. Ultrastructural studies with 5 days old untreated sclerotia revealed intense electron impermeable deposits in the intercellular spaces and a small electron dense layer in the outer cell walls, on the other hand treated sclerotia of the same age showed only sporadic small pigmented deposits between the cells and the pigmentation of the outer cell wall was absent.     

Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth,but not pathogenicity,in Sclerotinia sclerotiorum

The fungus Sclerotinia sclerotiorum is a necrotrophic plant pathogen causing significant damage on a broad range of crops. This fungus produces sclerotia that serve as the long‐term survival structures in the life cycle and the primary inoculum in the disease cycle. Melanin plays an important role in protecting mycelia and sclerotia from ultraviolet radiation and other adverse environmental conditions. In this study, two genes, SCD1 encoding a scytalone dehydratase and THR1 encoding a trihydroxynaphthalene reductase, were disrupted by target gene replacement, and their roles in mycelial growth, sclerotial development and fungal pathogenicity were investigated. Phylogenetic analyses indicated that the deduced amino acid sequences of SCD1 and THR1 were similar to the orthologues of Botrytis cinerea. Expression of SCD1 was at higher levels in sclerotia relative to mycelia. THR1 was expressed at similar levels in mycelia and sclerotia at early stages, but was up‐regulated in sclerotia at the maturation stage. Disruption of SCD1 or THR1 did not change the pathogenicity of the fungus, but resulted in slower radial growth, less biomass, wider angled hyphal branches, impaired sclerotial development and decreased resistance to ultraviolet light.     

A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea

Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in ∆BcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that ∆BcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.     

Decay of Sclerotia of Botrytis cinerea by Trichoderma spp. at Low Temperatures

In vitro, tests were conducted at 10°C and 5°C against sclerotia of Botrytis cinerea with 58 isolates of Trichoderma spp., highly antagonistic at 24°C but differing in their cold tolerance. Some isolates macerated and colonized sclerotia even at 5 °C. With 19 isolates of Trichoderma spp. less than 10 % of the sclerotia remained viable after 42 d at 5 °C. Conidia ol some Trichoderma spp. germinated at 5 °C within a few days and reached germination rates higher than 80 %. It seems to be feasible to use selected isolates of Trichoderma spp. for biological control of sclerotia of ß. cinerea also during the colder season.     

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.     

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.     

The signalling mucin Msb2 regulates surface sensing and host penetration via BMP1 MAP kinase signalling in Botrytis cinerea

Botrytis cinerea is a necrotrophic fungus that infects a wide range of fruit, vegetable and flower crops. Penetration of the host cuticle occurs via infection structures that are formed in response to appropriate plant surface signals. The differentiation of these structures requires a highly conserved mitogen‐activated protein (MAP) kinase cascade including the MAP kinase BMP1. In yeast and several plant‐pathogenic fungi, the signalling mucin Msb2 has been shown to be involved in surface recognition and MAP kinase activation. In this study, a B. cinerea msb2 mutant was generated and characterized. The mutant showed normal growth, sporulation, sclerotia formation and stress resistance. In the absence of nutrients, abnormal germination with multiple germ tubes was observed. In the presence of sugars, normal germination occurred, but msb2 germlings were almost unable to form appressoria or infection cushions on hard surfaces. Nevertheless, the msb2 mutant showed only a moderate delay in lesion formation on different host plants, and formed expanding lesions similar to the wild‐type. Although the wild‐type showed increasing BMP1 phosphorylation during the first hours of germination on hard surfaces, the phosphorylation levels in the msb2 mutant were strongly reduced. Several genes encoding secreted proteins were found to be co‐regulated by BMP1 and Msb2 during germination. Taken together, B. cinerea Msb2 is likely to represent a hard surface sensor of germlings and hyphae that triggers infection structure formation via the activation of the BMP1 MAP kinase pathway.     

Effects of NaCl on the response of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> callus to <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

Callus of the halophyte Mesembryanthemum crystallinum was used to study the effect of NaCl on the response to Botrytis cinerea infection. The fungus easily colonized the callus surface and the intercellular spaces. However, in the NaCl-adapted tissues the incidence of penetration was 67 % lower than in the inoculated control tissue. The modification of the infection pattern found in the salt-adapted callus could be related to metabolic adaptations to salinity. This was manifested by the enhanced antioxidant potential of ascorbate, the up-regulated activities of ascorbate peroxidase, as well as guaiacol and syringaldazine peroxidases together with the increased detoxification capacity of glutathione transferase in the NaCl-adapted callus. The post-inoculation changes in NaCl-adapted and non-adapted calli were roughly similar and supported the prooxidative nature of B. cinerea infection.     

Effect of temperature on growth and nitrogen mineralization of fungi and fungal-feeding nematodes

Dish and column microcosms containing alfalfa-sand medium were established to determine effect of temperature on growth and N mineralization ability of two fungi (Rhizoctonia solaniand Botrytis cinerea) and two nematodes (Aphelenchus avenaeand Aphelenchoides composticola). The microcosms were incubated at 15, 20, 25 and 29 °C for 21 days. In the dish microcosms, hyphal growth rates of both fungal species increased with temperature in the range of 15–25 °C. Above that temperature range, the growth rate of R. solani remained almost constant while that of B. cinereadecrease considerably. The population growth rate of A. avenae increased with temperature between 15 and 29 °C on colonies of R. solani and B. cinerea in dish microcosms. The growth rate of A. composticola also increased in the range of 15–25 °C but decreased greatly beyond that temperature range independent of the fungal species as food source. Inorganic N (NH₄ ⁺ + NO₃ ^–) was collected from each column microcosm by leaching every 3 days. In the columns containing R. solani, there was a significant effect of temperature on the amount of N detected in the fungus+A avenaeor A. composticolabut not in the fungus alone columns. The total amount of N was greatest at 29 °C for A. avenaeand at 20 °C for A. composticola columns, concurrent with the population growth rates of the nematodes. In the columns containing B. cinerea, the effect of temperature on the amount of inorganic N was not significant in either the fungus alone or fungus+nematode columns, although the population growth rates of the both nematode species were highest at 20 °C. For B. cinerea, the N amount across temperatures was the same or larger for the fungus alone as for the fungus+nematode columns. In general, the contribution of fungal-feeding nematodes to N mineralization was small in any combinations of fungus and nematode species at any temperature. Similarity in C/N ratio of the fungal and nematode biomass, organic substrate C/N ratios too low for measurable increase in net mineralization by the nematodes and small reproduction of the nematodes in the column microcosms were probable contributory factors.     

Carbon sources,growth, sclerotium formation and carbohydrate composition of Sclerotinia sclerotiorum

Summary Sclerotinia sclerotiorum (Lib.) D By. was grown in stationary liquid mineral-salts medium, pH 4.3, containing various carbon sources and the weight of mycelia and sclerotia was determined at regular intervals. When grown on various glucose concentrations (0–24 g of C/l), more sclerotia were produced at 8–12 g of C/l. Sclerotia were not usually formed in shake cultures. The ability of the fungus to use other carbon sources for growth and sclerotium formation was tested at 12 g of C/l in the stationary mineral-salts medium. The highest weights of mycelia and sclerotia occurred with raffinose, sucrose, maltose, lactose, d-mannose, d-glucose, d-fructose or l-arabinose. Good growth but decreased sclerotium production were found on cellobiose and d-xylose. Reduced or poor growth, a long lag period and few or no sclerotia occurred on trehalose, melibiose, l-sorbose, l-rhamnose, d-ribose, d-arabinose, l-xylose or 8 polyols. No growth was observed with erythritol or i-inositol. A combination of glucose plus trehalose or polyols resulted in increased growth and the formation of sclerotia. Organic acids supported little or no growth and no sclerotia were produced. Generally culture filtrates which supported growth and formation of sclerotia became acid (about pH 3.5). The pH of the culture filtrate usually increased slowly during the growth period when the fungus grew poorly and no sclerotia were formed. The alcoholsoluble sugars and polyols present in culture filtrates, mycelia and sclerotia were determined by paper and thin-layer chromatography. Regardless of the carbon source, mannitol was usually present in culture filtrates. The occurrence of other compounds in the filtrates depended on the carbon source. Trehalose, mannitol and usually small quantities of glucose or fructose were present in mycelia and sclerotia from all carbon sources. Galactitol or pentitols occurred in mycelia and sclerotia when the fungus grew on galactose and oligosaccharides containing galactose or the corresponding pentose, sugars. Acid hydrolyzates of the alcohol-insoluble fraction of mycelia or sclerotia contained glucose, smaller amounts of galactose and mannose and traces of ribose and rhamnose.     

Reserve substances inPaxillus involutus sclerotia

Summary The ectomycorrhizal fungus,Paxillus involutus, produces sclerotia in culture. These can be induced to form on agar medium by exposing mycelium grown at 25°C to various temperatures between6°C and 15°C. Sclerotia formed at 10°C and above were large and covered with drops of exudate, while those formed at 6°C or 8°C were very small and did not produce an exudate. Mature sclerotia were bounded by a compact rind and contained abundant storage reserves. Histochemistry of the larger sclerotia showed large quantities of protein stored as protein bodies in the cytoplasm, lipid present as small droplets, glycogen granules stored in the cytoplasm and polyphosphate present as small granules in the cytoplasm and in the protein bodies. Energy dispersive X-ray microanalysis confirmed the presence of phosphate in the granules and was used to map its distribution throughout the sclerotium. The smaller sclerotia induced at 8°C and below on the same medium had the same basic structure and composition, but lacked the complex phenolic cell network found in large sclerotia, and had abundant extracellular polysaccharides. The rind was not well developed and these small sclerotia are interpreted to have been arrested at an early stage of development.     

Ultrastructure and Development of Sclerotia of Botrytis cinerea Pers. in vitro

The development of sclerotia of Botrytis cinerea was examined at four stages during their maturation. The surface structure developed a network of profusely branched hyphae through their coalescence to a compact sclerotial body which was maturated by the deposition of melanin pigment. A characteristic feature of the hyphal cells of B. cinerea during the later stages of development was the presence of paramural bodies (plasmalemmasomes and lomasomes). Electrondense bodies with a limiting double-membrane congregated against the transverse septa of hyphal cells as sclerotia matured and may migrate from cell to cell through septal pores. We suggest that these and the lipid bodies found in hyphal cells may have a storage function in the resting sclerotia.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

The small GTPase BcCdc42 affects nuclear division, germination and virulence of the gray mold fungus Botrytis cinerea

The small GTPase Cdc42 plays a central role in various processes in eukaryotic cells including growth, differentiation and cytoskeleton organization. Whereas it is essential in the yeast Saccharomyces cerevisiae, its role in filamentous fungi differs, due to the complementing, partly overlapping function of Rac. We analyzed the role of the Cdc42 homologue in the necrotrophic, broad host range pathogen Botrytis cinerea. Deletion mutants of bccdc42 showed various growth abnormalities; the mutants had reduced growth rate and hyphal branching, they produced fewer conidia, which were enlarged and misshapen and had germination defects. Additionally, the mutants were impaired in sclerotia development. Cytological studies indicate that at least part of this phenotype could be attributed to disturbed control of nuclear division: conidia and hyphae of the mutant showed twofold higher nucleus/cytoplasm ratio compared to wild type cells. Apart from these effects on vegetative growth and differentiation, Δbccdc42 strains were attenuated in penetration and colonization of host tissue, confirming that BcCdc42 – though being not essential like in yeast – is involved in important developmental processes in B. cinerea.     

Morphological characteristics of sclerotia formed from hyphae of <Emphasis Type="Italic">Grifola umbellata</Emphasis> under artificial conditions

The morphological characteristics of sclerotia were induced in cultures of the fungus Grifola umbellata by introducing an unidentified companion fungus were studied by light microscope, scanning and transmission electron microscope (SEM and TEM). Light microscope and SEM investigations of developing sclerotia revealed that aerial mycelial hyphae diminished with age, and mature sclerotia had two tissue layers, the rind and medulla. The medulla was comprised of thin and thick-walled hyphae of varying diameter. The thick-walled cells always formed below the hyphal tips. Retraction of the cytoplasm was accompanied by the thickening of cell wall. There were crystalline initials in the newly formed sclerotium. Crystalline initials were always formed in the tip of medullary hyphae and were not of regular shape. A series of changes occurred in the cells in which the crystalline initials would be formed, such as enlargement of size, formation of one or several large vacuoles. Crystalline initials developed via amorphous materials in the cytoplasm deposited in the vacuoles. At last crystalline initials was released by degradation of the cell wall.     

Sclerotia formed by citric acid producing strains of Aspergillus niger: Induction and morphological analysis

Some strains of Aspergillus niger have been previously reported to produce sclerotia under certain conditions. Sclerotia are aggregations of hyphae which can act either as survival or as sexual structures in species related to A. niger. In this study, we were able to induce the formation of sclerotia in the progenitor of the industrial citric acid producing strains of A. niger, ATCC 1015, and in pyrG mutants derived from it. Sclerotia can be stably formed by ATCC 1015 on malt extract agar medium supplemented with raisins, showing a spatial differentiation of the fungus dependent on the addition and on the position of the fruits into the medium. On other media, including malt extract agar, pyrG auxotrophs also form abundant sclerotia, while the complementation of this gene reverses this phenotype. Additionally, a macro- and microscopical analysis of the sclerotia is reported. Our results show that the sclerotia formed by A. niger are similar to those formed by other fungi, not only in their morphology but also in their ability to germinate and regenerate the organism.     

Baseline sensitivity of Botrytis cinerea to fluazinam and cross‐resistance

Grey mould, caused by the fungus Botrytis cinerea Pers ex Fr., is a very destructive and important disease worldwide. Fluazinam is a phenylpyridinamine fungicide with broad‐spectrum activities. The baseline sensitivity of B. cinerea to fluazinam is yet to be established in Henan Province, China. In this study, a total of 117 field isolates of B. cinerea were collected from 49 commercial greenhouses in different locations of Henan Province, in 2016, and the sensitivities of these isolates to fluazinam were determined based on mycelial growth. The effective concentration for 50% (EC₅₀) values ranged from 0.0038 to 0.0441 μg/ml, and the mean EC₅₀ value was 0.0201 ± 0.0081 μg/ml for mycelial growth. The frequency distribution range presented a unimodal curve. To define the cross‐resistance relationships, the linear correlation coefficients of the EC₅₀ values between fluazinam and carbendazim, procymidone, pyrimethanil or boscalid were analysed. The results showed that no correlation was observed between fluazinam and the other tested fungicides. These results provide important information to growers for the prevention and control of grey mould.     

Mycopappus quercus sp. nov., causing frosty mildew inQuercus acutissima

The causal fungus of frosty mildew inQuercus acutissima was taxonomically studied. Brown spots with conical white to yellowish tufts of the causal, fungus developed on the leaves, followed by successive defoliation. The fungus was regarded as a new species ofMycopappus, M. quercus, from the morphology of the multicellular propagules. The ascogenous stage and true conidia of the fungus were not observed, although sclerotia were formed on the fallen diseased leaves.