Aspergillus fumigatus exoβ(1‐3)glucanases family GH55 are essential for conidial cell wall morphogenesis

The cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched β(1‐3)glucan, to which the chitin and the galactomannan are covalently bound. Softening of the cell wall is an essential event during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosyl hydrolases. In this study, we characterised the role of the glycosyl hydrolase GH55 members in A. fumigatus fungal morphogenesis. We showed that deletion of the six genes of the GH55 family stopped conidial cell wall maturation at the beginning of the development process, leading to abrogation of conidial separation: the shape of conidia became ovoid, and germination was delayed. In conclusion, the reorganisation and structuring of the conidial cell wall mediated by members of the GH55 family is essential for their maturation, normal dissemination, and germination.     

Permeabilization method for in-situ investigation of fungal conidia on surfaces

Aims:  4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces.
Methods and Results:  We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and β-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples.
Conclusions:  The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol.
Significance and Impact of the Study:  The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.     

蓖麻根腐病抗性鉴定及其SSR标记的初步建立

蓖麻根腐病是茄腐镰孢菌(Fusarium solani)引起的根部病害,严重影响蓖麻产量。抗源缺乏制约了抗病品种的选育。为寻找抗病种质、建立抗性分子标记,该研究对252份蓖麻材料的抗性进行了表型和分子标记鉴定。结果表明:(1)浓度为1×10⁶个·mL^-1的孢子悬浮液灌根是一种有效的接种方法; 以接种后枯萎天数为基础的5级评价法,可作为鉴定标准。(2)鉴定出130份抗病材料,其中高抗为105份。(3)野生材料中抗病材料比率(66%)远高于栽培材料(35%),建议将野生材料,尤其是中国华南野生材料的研究利用作为今后抗病育种的重要方向。(4)初步建立了8个与抗性关联的SSR标记。该研究结果提供了有效的根腐病抗性鉴定方法和评价标准,筛选出了一批育种迫切需要的抗病基因资源,初步建立了可用于辅助选择的SSR标记,为蓖麻抗根腐病育种奠定了重要基础。     

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims: To determine the effect of water activity (a_w = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. Methods and Results: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a_w values (glycerol being added to adjust the a_w). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a_w, temperature and the a_w × temperature interaction on mycelial growth (P < 0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a_w and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a_w and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. Conclusions: Our results confirm the previous finding that a_w has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. Significance and Impact of the Study: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.     

The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

Aims:  To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia.
Methods and Results:  The model system was Penicillium discolor , a food spoilage fungus. Filipin resulted in permeabilization of germinating conidia for the fluorescent probes TOTO-1 and FM4-64, but not for ferricyanide ions. Nystatin caused influx of all these compounds while natamycin did not. Untreated germinating conidia internalize the endocytic marker FM4-64. Pretreatment of germinating conidia with natamycin showed a dose and time dependent inhibition of endocytosis as judged by the lack of formation of early endosomal compartments.
Conclusions:  The results obtained from this study indicated that, unlike nystatin and filipin, natamycin is unable to permeabilize germinating conidia, but interferes with endocytosis in a dose and time dependent manner.
Significance and Impact of the Study:  Natamycin acts via a different mode of action than other polyene antimycotics. These results offer useful information for new strategies to prevent fungal spoilage on food products and infection on agricultural crops. For laboratory use, natamycin can be used as a specific inhibitor of early endocytosis in fungal cells.     

Immunocytochemical localization of stachylysin in Stachybotrys chartarum

Stachylysin is a proteinaceous hemolytic agent that is produced by Stachybotrys chartarum. Stachylysin was found, using immunohistochemical and immunocytochemical methods, to be localized in S. chartarum spores/mycelia primarily in the inner wall suggesting that it is constitutively produced. Spores instilled in mouse or rat lung tissues resulted in granuloma formation, which showed the highest stachylysin concentration in the inner wall of the spore and near the spore, with less at distance indicating that it had diffused out from the spore. The in vitro high stachylysin producing strain (58-06) was also highest in vivo, based on immunohistochemistical staining. More stachylysin was observed in the mouse lung tissue at 72 h than at 24 h indicating that production/release is a relatively slow process. The localization of stachylysin in macrophage phagolysosomes suggests that these cells may be involved with hemolysin inactivation. This would be consistent with what is known about asp-hemolysin produced by Aspergillus fumigatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

Survival of Microorganisms under the Extreme Conditions of the Atacama Desert

Spores of Bacillus subtilis, conidia of Aspergillus niger, versicolor and ochraceus andcells of Deinococcus radiodurans have been exposed in the dark at two locations (at about 23°S and 24°S) in the Atacama Desert for up to 15 months. B. subtilis spores (survival 15%) and A. niger conidia (survival 30%) outlived the other species. The survival of the conidiaand spores species was only slightly poorer than that of thecorresponding laboratory controls. However, the Deinococcus radiodurans cells did not survive the desertexposure, because they are readily inactivated at relativehumidities between 40 and 80% which typically occurduring desert nights. Cellular monolayers of the dry sporesand conidia have in addition been exposed to the full sunlight for up to several hours. The solar fluences causing 63% loss in viability (F₃₇-values) have been determined.These F₃₇-values are compared with those determined atother global locations such as Punta Arenas (53°S), Key Largo (25°N) or Mainz (50°N) during the same season. Thesolar UVB radiation kills even the most resistantmicroorganisms within a few hours due to DNA damages. Thedata are also discussed with respect to possible similaritiesbetween the climatic conditions of the recent Atacama Desertand the deserts of early Mars.     

The hyphomycete Sorosporetta — Syngliocladium from mole cricket,Scapteriscus vicinus

The hyphomycete Sorosporella sp. was isolated from the mole cricket, Scapteriscus vicinus collected in Alachua Co., Florida. Scanning (SEM) and/or transmission (TEM) electron microscopy were used to study the chlamydospores characteristic of this genus and the conidial production in the alternate state, Syngliocladium. Brick-red chlamydospores, which occur in clusters in insect cadavers, have a fibrous cell wall as revealed by TEM. Pores often occur between walls of adjacent cells. Lipids, including a large central lipid droplet and smaller droplets along the periphery of the cell, are abundant in the cytoplasm. Most organelles were difficult to distinguish due to the density of the cytoplasmic material. Chlamydospores germinate on water or media. On Sabouraud maltose agar, germinating chlamydospores produce a white mycelial mat; synnematous-type growth was sometimes observed. Mycelia may bear conidiophores and ellipsoid conidia.Florida Agricultural Experiment Station Journal Series No. 7789.