The fungus Penicillium variabile Sopp 1912 isolated from permafrost deposits as a producer of rugulovasines

It has been established that relict fungi Penicillium variabile Sopp can synthesize clavine alkaloids, rugulovasines A and B, which are revealed in this species for the first time. Submerged cultivation of the strain-producer revealed several microcycles of conidia formation. The synthesis of alkaloids was also of a cyclic character. The synchronism of cyclic rugulovasine biosynthesis and conidia formation was revealed. Zinc ions stimulated fungal growth but had a negative effect on the biosynthesis of rugulovasines.     

Growth and biosynthesis of rugulovasines in Penicillium variabile Sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

Growth and biosynthesis of rugulovasines in <Emphasis Type="Italic">Penicillium variabile</Emphasis> sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

Association of ergot alkaloids with conidiation in Aspergillus fumigatus

Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festucla-vine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (brlA), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation.     

Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.     

Abundant Respirable Ergot Alkaloids from the Common Airborne Fungus Aspergillus fumigatus

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.     

Secondary metabolites in taxonomy of the <Emphasis Type="Italic">Penicillium</Emphasis> fungi

A correlation was established between species specificity and synthesis of specific secondary metabolites by the Penicillium fungi. Strains of the subgenus Aspergilloides usually synthesize metabolites of polyketide nature. Most strains of the subgenus Furcatum produce clavine ergot alkaloids and metabolites of diketopiperazine nature. The only clavine ergot alkaloids and diketopiperazine alkaloids produced by strains of the subgenus Biverticillium are rugulovasines and rugulosuvines, respectively. Species designations of the strains of the subgenus Penicillium isolated from permafrost soil, the Mir orbital complex, and sites undergoing anthropogenic load were refined based on the marker secondary metabolites. Changes in the taxonomic position of some strains in the genus Penicillium are suggested.     

Penicillium rubrum and Penicillium biforme, new sources of rugulovasines A and B.

An isolate of Penicillium biforme Thom produced rugulovasines A and B. An isolate of Penicillium rubrum Stoll produced rugulovasines A and B and also chlororugulovasines A and B. Both fungi represent new sources of the rugulovasines.     

黑星病菌在苹果叶片上的发育过程研究

采用电子显微镜技术,研究了苹果黑星病菌在苹果叶片上发育过程。扫描电子显微镜观察结果表明,接种后12 h 分生孢子即可萌发并形成附着胞,统计结果显示其孢子萌发率在6 h和12 h分别为83% 和95%,附着胞形成率在12 h和24 h 分别为93% 和95%。透射电子显微镜观察结果表明,黑星病菌侵入以后在寄主角质层下和表皮细胞之间扩展、定殖并可形成子座。接种后12d,病菌开始从子座上产生分生孢子梗和分生孢子,分生孢子梗顶端每产生一个单生的分生孢子就形成一个环痕并延伸其长度。分生孢子梗和分生孢子主要沿叶脉形成,在叶片上呈网状扩展,此时叶片表现明显的病害症状。     

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Formation of conidia in a saprophytic strainClaviceps paspali producing simple lysergic acid derivatives

A non-mutant saprophytic strain C. paspali which forms conidia both on a solid medium and during submerged fermentation is described. Conidiation proceeded in parallel with culture growth and production of alkaloids. The effect of composition of culture media on the intensity of conidiation is described.     

Individual and combined effect of (±)-α-hydrastine and (±)-β-hydrastine on spore germination of some fungi

(+/-)-alpha-Hydrastine and (+/-)-beta-hydrastine were isolated from Corydalis longipes; both exhibited considerable efficacy against spore germination of some saprophytic and phytopathogenic fungi. While (+/-)-alpha-hydrastine was effective against most of the fungi, Helminthosporium echinoclova was least affected even at the highest dose (150 ppm). (+/-)-beta-Hydrastine was equally effective against several fungi. Mixture of both compounds was more effective than each one individually. Helminthosporium species were again the most resistant toward the mixture. The effect of both alkaloids independently on germination and development of E. pisi conidia on excised pea leaves was also shown. After pre-inoculation with (+/-)-alpha-hydrastine, the effect was more pronounced than the addition post-inoculation; maximum inhibition occurred at 200 ppm. (+/-)-beta-Hydrastine also reduced germination of conidia but was less effective than (+/-)-alpha-hydrastine. The number of primary and secondary branches of conidia and number of appressoria were not affected significantly by either compound.     

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.     

Adhesive knob formation by conidia of the nematophagous fungusDrechmeria coniospora

We studied conidiogenesis and adhesive knob formation (maturation) by newly developed conidia of the nematophagous fungusDrechmeria coniospora. Upon conidiogenesis on infected nematodes or during saprophytic growth of the fungus in axenic cultures compact clusters of conidia developed. Less than 10% of such clustered conidia matured; mature conidia were invariably located on the periphery of the clusters.The kinetics and rate of maturation of conidia were studied inin vitro systems and in soil. In both cases adhesive knobs were formed; the rate at which knobs were formed appeared to be determined by the age of the conidia, the temperature and the soil moisture. In addition, knob formation was suppressed at increasing conidial densities. Under favorable conditions, however, over 90% of the conidia matured within a period of 3 days. The rate of knob formation was neither influenced by the presence of nematodes nor by that of exogenous nutrients, which suggests that maturation is an autonomous process. Electron-microscopical analysis indicated that budding of the conidia at the initial stage of maturation occurred simultaneously with the deposition of the sticky, adhesive layer around the wall of the developing knob.The ecological significance of the time- and spatially separated maturation of conidia after conidiogenesis is discussed with respect to survival of the conidia.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids.

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

Effects of temperature,light, carbon and nitrogen nutrition on reproduction in Phoma medicaginis

Reproduction by three isolates ofPhoma medicaginis growing on potato dextrose agar was studied. The formation of pycnidia was optimum at 30°C whereas the optimum for the formation of conidia was 20°C. Light consistently increased the numbers of pycnidia and conidia over those formed in darkness and more conidia were produced in light at 23°C than at 30°C. The effects of carbon and nitrogen sources on reproduction were studied using modified Richard's medium as the basal medium. Fourteen carbohydrates, 11 amino acids, 9 inorganic nitrogen sources and urea were evaluated by replacing the carbohydrate or nitrogen in Richard's medium with the test substance. Generally, the monosaccharides and disaccharides were about alike and superior to polysaccharides for the production of pycnidia. The carbon sources were about equally useful for production of conidia, but the polysaccharides were superior to the other two classes of carbon sources when the number of conidia/pycnidium was calculated. Generally, the formation of pycnidia and conidia was favored by nitrate more than by ammonium nitrogen sources. The average number of conidia/pycnidium was greatest, however, when the nitrogen source was NH₄NO₃. All amino acids tested appeared to be useful nitrogen sources for production of pycnidia but none were especially good for conidia production. L-isoleucine was superior to the other amino acids tested. Of three isolates used, Illinois 23 consistently produced more pycnidia and conidia that did isolates Minnesota 2 and Missouri 5. Usually the significant interactions between isolates and other treatments were due to a greater response by isolate Ill. 23. It was concluded that the reproduction ofP. medicaginis varies significantly with the isolates of the fungus, the environment, and nutrients as well as with interactions among these factors, and conclusions about the influence of a particular factor will depend on whether the formation of pycnidia, conidia or conidia/pycnidium is being studied.Paper No. 7425, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection

The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.