Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (M_r 133,000 and 146,000). Reductions in the M_r of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM extracellular matrix - BPA Bauhinia purpurea agglutinin - BSA bovine serum albumin - DIC differential interference contrast - FITC fluorescein isothiocyanate - GNL Galanthus nivalis lectin - GSI-B₄ Griffonia simplicifolia isolectin B₄ - HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - IIF indirect immunofluorescence - IPC isopycnic centrifugation - MAb monoclonal antibody - PEG polyethylene glycol - PBS phosphate buffered saline - PNGase peptideN-glycosidase - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCS tissue culture supernatant - TEM transmission electron microscopy - TFMS trifluoromethane sulphonic acid     

Effects of Rehydration on the Conidial Viability of Metarhizium flavoviride Mycopesticide Formulations

The rehydration of dried conidia of Metarhizium flavoviride was investigated in an attempt to increase speed of kill of locusts and grasshoppers by formulations of this fungus. Conidia were dried to 4-5% moisture content with no apparent adverse effects on viability, but rapid rehydration (by putting dried conidia directly in free water) reduced viability. Rehydration in an atmosphere of high humidity allowed dry conidia to absorb sufficient moisture to avoid imbibition damage. Rehydrating and pre-germinating conidia prior to spraying (in an oil-based formulation) on to the desert locust, Schistocerca gregaria, did not decrease the time to death, suggesting that moisture uptake by dry conidia on the desert locust cuticle is easily achieved.     

中国被毛孢分生孢子形成过程显微结构的研究

分生孢子是中国被毛孢(H sinensis)生活史中极为重要的一个环节,其在冬虫夏草侵染和形成过程中有着十分关键的作用.本实验以中国被毛孢纯培养菌种为实验材料,采用显微镜观察了其菌丝体经营养生长产分生孢子过程中显微结构变化,结果表明:中国被毛孢在固体培养条件下,可以形成分生孢子,其形态和产生方式与微循环产孢过程中形成的分生孢子一致,同时,在产孢过程中,还存在瓶梗结构纵裂形成对生分生孢子的现象.     

Polyphyly,asexual reproduction and dual trophic mode in Buchwaldoboletus

Most Boletaceae form ectomycorrhizas but species in two sister groups (subfamily Chalciporoideae and the genus Pseudoboletus) have unclear trophic modes and are putatively mycoparasitic. Chalciporoideae is the earliest diverging lineage in Boletaceae and includes Chalciporus and Buchwaldoboletus. Buchwaldoboletus species fruit on wood and were thought to be saprobes, but the type species Buchwaldoboletus lignicola is mycoparasitic on the wood decay fungus Phaeolus schweinitzii. However, the nutritional mode and host range of B. lignicola and other Buchwaldoboletus remain unclear. We obtained fresh collections and axenic cultures of B. lignicola, B. hemichrysus and B. sphaerocephalus from the southeastern USA. We use cultures to examine the phylogenetic relationships among taxa in Chalciporoideae, elucidate the trophic mode of Buchwaldoboletus species and test the effect that Buchwaldoboletus species have on wood decay. Our analysis supports the hypothesis that the Chalciporus lineage is sister to all other Boletaceae but suggests that Buchwaldoboletus is not monophyletic. Confrontation experiments on agar suggest that Buchwaldoboletus species might parasitize a wide range of decay fungi. Buchwaldoboletus hemichrysus and B. sphaerocephalus produce abundant conidia in culture whereas the more distantly related B. lignicola produces sclerotia. Wood decay assays show that Buchwaldoboletus species did not have a significant effect on the decay caused by wood decay fungi. Interestingly, when B. lignicola or B. hemichrysus were inoculated alone on wood both species decayed wood, producing a brown rot that can cause 35–65% mass loss over 6 months. Our results suggest that Buchwaldoboletus species have a dual trophic mode as saprobes and mycoparasites.     

Survival of Botrytis cinerea in Soil in South-Eastern Spain

The survival of Botrytis cinerea in sterile and unsterile soil at different temperatures and relative air humidities was investigated in south‐eastern Spain. Conidia survived only 7 days at 40^°C but, depending on relative humidity, for 30–90 days at 22^°C. High air humidity (95%) was needed to maintain soil humidity (8%) at a level that favoured conidial survival. Conidia survived better in sterile soil than in unsterile soil, probably because of the presence in the latter of soil microorganisms antagonistic to B. cinerea. Survival of conidia in environmental conditions simulating those in a greenhouse was less than 28 days. Results showed that B. cinerea conidia cannot survive over summer in south‐eastern Spain, and other primary sources of inocula are discussed.     

Airborne fungal spores in urban and rural environments in Poland

The concentrations of airborne fungal spores were measured during 2001–2002 in two sites in Poland—one in the city and the other in the countryside. The sites differed in habitat characteristics, such as urbanisation level, vegetation and microclimate. The aim of the study was to determine if, and in which way, land use type would affect spore occurrence. The volumetric method was used, and ten easily identifiable spore types were sampled and anyalysed: Alternaria, Botrytis, Cladosporium, Epiccocum, Ganoderma, Pithomyces, Polythrincium, Stemphylium, Torula and Drechslera. The season of spore occurrence was determined using the 90% method. The fungal spores studied were very frequent in the air (in most instances at a frequency higher than 50%). The most common spores were those of Cladosporium, with a frequency range of 83.1–90.5%. In both years the proportion of Cladosporium spores was statistically significantly higher in the city. In both 2001 and 2002 the total seasonal sum of all the spores was higher in the countryside than in the city as was the Seasonal Fungal Index (SFI) values and average concentrations of Botrytis, Ganoderma and Torula. These latter three genera are usually represented as pathogens of plants. The mean spore concentrations of most taxa were significantly higher in the rural environment. Correlation coefficients between daily concentrations at both sites for most of the taxa studied were significant, but with lower correlation values between variables. Such results indicate that the values from the sites are weakly interdependent. The study confirms that land use type may very likely have an impact on the course of spore occurrence, the mean daily concentrations of spores as well as SFI values.     

Some Nutritional Factors Affecting Production of Biomass and Antifungal Metabolites of Coniothyrium minitans

The ability of the mycoparasite Coniothyrium minitans to utilize a range of C and N sources and vitamins for growth, pycnidial formation and antifungal metabolite production was examined using a defined liquid medium. Coniothyrium minitans was able to use all the C sources tested, with the exception of D -xylose, and all the N sources tested, although growth was generally better on organic N sources rather than NO 3 -N. Increasing C:N ratios from 9:1-202:1 with N constant (2.0 g L -l L -alanine) resulted in steadily increasing yields, whereas increasing C:N ratios with C constant (40.0 g L -l D -glucose) gradually decreased yield. Addition of thiamine to the glucose-alanine basal medium resulted in the greatest increase in growth but biomass was still less than that achieved using an undefined molasses-yeast medium. Pycnidial production was generally low or failed to occur in the basal medium + C + N sources in the absence of vitamins, but addition of thiamine consistently led to abundant pycnidial formation. Molasses-yeast static culture provided greater biomass and conidial yields than molasses-yeast shaken culture. Incorporation of C. minitans culture medium into potato dextrose broth (10% v/v) resulted in consistent reduction in growth of Sclerotinia sclerotiorum irrespective of C, N or vitamin content of the basal medium or whether molasses-yeast medium was used. This is the first report of consistent production of antifungal metabolites by C. minitans .     

The oxygen concentration in cultures modulates protein expression and enzymatic antioxidant responses in Metarhizium lepidiotae conidia

Conidia from Metarhizium spp. are used for integrated pest control; however, environmental factors diminish the effectivity of these programs. Several approaches tried to improve conidia resistance to overcome this limitation, although little is known about the mechanisms involved in this effect. Here we measured the activity of antioxidant enzymes and conidia virulence, comparing the proteomic profiles of Metarhiziumlepidiotae CP-OAX conidia produced under normal (21% O₂) and high oxygen atmospheres (pulses with 30% O₂). We detected a higher virulence against Tenebrio molitor larvae, in addition to an increase in ultraviolet light tolerance in conidia produced under 30% O₂, which correlates with increased glutathione reductase activity. Two-dimensional gel electrophoresis (2D SDS–PAGE) of proteins extracted in conidia harvested from both experimental conditions revealed a group of proteins that was observed only in conidia from oxidant atmospheres. Some of those proteins were directly involved in oxidative stress responses, whereas others were involved in conidial virulence, thermo-tolerance, and the central metabolism. Thus, a high atmospheric oxygen concentration (30%) activates antioxidant defence and general stress response mechanisms involved in conidia resistance to adverse environmental factors, which can ultimately translate into higher effectivity for the use of entomopathogenic fungi conidia in pest control.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.