Disposition of Mycophenolic Acid, Brevianamide A, Asperphenamate, and Ergosterol in Solid Cultures of Penicillium brevicompactum

When Penicillium brevicompactum was grown between layers of dialysis membrane on Czapek-Dox agar for 3 days, no synthesis of mycophenolic acid, brevianamide A, asperphenamate, or ergosterol occurred. After removal of the uppermost membrane layer, aerial mycelium developed and all four metabolites were formed. The bulk of the mycophenolic acid that was formed was excreted into the medium, the bulk of the brevianamide A and asperphenamate was found in the aerial hyphae, and ergosterol was found in both vegetative and aerial mycelia.     

Mycophenolic Acid Production by Penicillium brevicompactum on Solid Media

When grown on Czapek-Dox agar, Penicillium brevicompactum produced mycophenolic acid after a vegetative mycelium had been formed and as aerial hyphae were developing. Nutrients were still plenteous in the agar when the synthesis began. If aerial hyphal development was prevented by placing a dialysis membrane over the growing fungus, no mycophenolic acid was produced. When the dialysis membrane was peeled back and, as a consequence, production of aerial hyphae began, mycophenolic acid biosynthesis was observed. We concluded that mycophenolic acid was produced only by P. brevicompactum colonies that possessed an aerial mycelium.     

6-Methylsalicylic Acid Production in Solid Cultures of Penicillium patulum Occurs Only When an Aerial Mycelium Is Present

When Penicillium patulum was grown on Czapek-Dox agar, 6-methylsalicylic acid was produced as an aerial mycelium was forming. Nutrients were often plentiful in the medium when biosynthesis began. If the formation of an aerial mycelium was prevented by growing the fungus between two sheets of dialysis membrane, no 6-methylsalicylic acid was produced even when nutrients were completely consumed. If the upper sheet of dialysis membrane was stripped off cultures of the latter type, an aerial mycelium formed; concomitantly, 6-methylsalicylic acid biosynthesis was observed. We conclude that 6-methylsalicylic acid was produced only by P. patulum colonies that possessed an aerial mycelium.     

Viability of Heterodera glycines Exposed to Fungal Filtrates

Filtrates from nematode-parasitic fungi have been reported to be toxic to plant-parasitic nematodes. Our objective was to determine the effects of fungal filtrates on second-stage juveniles and eggs of Heterodera glycines. Eleven fungal species that were isolated from cysts extracted from a soybean field in Florida were tested on J2, and five species were tested on eggs in vitro. Each fungal species was grown in Czapek-Dox broth and malt extract broth. No toxic activity was observed for fungi grown in Czapek-Dox broth. Filtrates from Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, and Fusarium solani grown in malt extract broth were toxic to J2, whereas filtrates from Exophiala pisciphila, Fusarium oxysporum, Gliocladium catenulatum, Pyrenochaeta terrestris, Verticillium chlamydosporium, and sterile fungi 1 and 2 were not toxic to J2. Filtrates of P. lilacinus, S. heteroderae, and N. vasinfecta grown in malt extract broth reduced egg viability, whereas F. oxysporum and P. terrestris filtrates had no effect on egg viability.     

Improvement of lipid production from an oil-producing filamentous fungus, <Emphasis Type="Italic">Penicillium brevicompactum</Emphasis> NRC 829, through central composite statistical design

In the present study, 13 filamentous fungi were screened for their lipid production and an oleaginous fungus, Penicillium brevicompactum NRC 829, was found to be the highest lipid producer. Screening of various agro-industrial residues was performed and sunflower oil cake proved to be the best substrate for lipid production. A central composite design was employed to investigate the optimum concentrations of the most significant medium components required to improve the lipid production by P. brevicompactum. The results clearly revealed that the maximal lipid production of 8.014 ± 0.06 gL^?1 (representing 57.6% lipid/dry biomass) was achieved by the fungus when grown for 6 days at 30 °C under static condition in a medium containing sunflower oil cake, NaNO₃ and KCl at final concentrations of 8, 0.75 and 0.25 gL^?1, respectively. Gas chromatography-mass spectrometry analysis of P. brevicompactum lipid indicated that linoleic acid (LA) (C18:2–6, 9) was the most abundant fatty acid, accounting for up to 62% of the total fatty acid profile, followed by palmitoleic acid (C16:1, 16%) and linolenic acid (C18:3, 8%). These results suggest that P. brevicompactum NRC 829 may have potential for commercial development for the production of LA by fermentation using cheap raw material.     

Fascitis necrotizante por Saksenaea vasiformis en una paciente inmunocompetente tras un accidente de tráfico

BackgroundCutaneous mucormycosis (zygomycosis), with subcutaneous spreading and dissemination, in immunocompetent patients is an uncommon disease caused by species belonging to the fungal genera Apophysomyces, Rhizopus and Saksenaea, among others.Case reportA case of necrotising fasciitis by Saksenaea vasiformis in an immunocompetent woman is described. The infection was acquired through a car accident resulting in multiple injuries affecting mainly her right arm. After the surgical reduction of fractures, skin lesions worsened and led to necrosis. The patient quickly developed a severe necrotising fasciitis with negative cultures at first. Despite the extensive surgical debridement and the aggressive antifungal treatment, the patient died. The histopathological study showed a fungal infection due to a fungus belonging to the Mucorales order, which was confirmed by culturing the clinical sample on Sabouraud agar, and identifying the species by cultures on Czapek-Dox agar, and sequencing of the ITS region of the ribosomal DNA.ConclusionsThis case confirm the presence of this fungus in Spain, the value of histopathology for the mucormycosis diagnosis, as well as the need to perform special cultures to facilitate their isolation and identification to the species level by the combined use of Czapek-Dox agar and sequencing of the ITS region.     

Assessment of the antifungal activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Pediococcus</Emphasis> spp. for use as bioprotective cultures in dairy products

Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum O_VI9 and Lactobacillus rhamnosus BIO_III28 at initial concentrations of 10⁵ and 10⁷ CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 10⁵ CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIO_III28 at 10⁷ CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.     

Effect of potassium sorbate,sodium benzoate,and sodium nitrite on biosynthesis of cyclopiazonic and mycophenolic acids and citrinin by fungi of the <Emphasis Type="Italic">Penicillium</Emphasis> genus

The effect of potassium sorbate, sodium benzoate, and sodium nitrite used as preservatives in the food industry in the production of such mytotoxins as citrinin cyclopiazonic and mycophenolic acids by the contaminating fungi Penicillium citrinum, P. commune, and P. brevicompactum, respectively, was investigated. It was shown that the effect of preservatives used at concentrations relevant to the food industry on the synthesis of mycotoxins depended on the species-specific biochemical and physiological features of the cultures. The growth of P. brevicompactum was inhibited to the highest degree by sodium nitrite and potassium sorbate, and the growth of P. commune was so inhibited by sodium benzoate. It was established that the introduction of 0.015% sodium nitrite into the medium resulted in 1.3- and 1.4-fold reductions of the production of citrinin and mycophenolic acid, respectively, while the production of cyclopiazonic acid did not change in comparison with the control. The introduction of 0.015% sodium benzoate caused a more than 1.5-fold increase of the concentration of citrinin, cyclopiazonic, and mycophenolic acids, and the addition of 0.02% potassium sorbate increased the production of cyclopiazonic and mycophenolic acids by 1.7 and 2.6 times, respectively.     

In vitro production of zoospores by the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) on solid media

Reliable, large-scale production of Lagenidium giganteum zoospores was obtained on solid media. The fungus was grown for 7 days in a liquid medium of wheat germ, hemp seed, yeast extract, and glucose, then placed onto hemp-seed agar. Zoosporogenesis was induced on agar by immersing the fungal cultures into water. Zoospore production began 10 hr postimmersion, peaked at 18 hr, and ceased by 36 hr. A single, 10-cm Petri dish of fungus on hemp-seed agar produced 1.7?3.8 × 10⁷ zoospores during the 26 hr of zoosporogenesis. Optimal zoospore production occurred with 4- to 7-day-old cultures; cultures older than 10 days produced few zoospores. The temperature range for zoosporogenesis was 15–35°C. The extent of zoosporogenesis was directly related to the volume of water used to induce zoospore formation and inversely proportional to agar thickness. Bioassay of zoospores against second instar Culex quinquefasciatus larvae yielded an LD₅₀ of 400 zoospores/ml.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Biology of Azospirillum-Sugarcane Association: Enhancement of Nitrogenase Activity

Azospirillum brasilense was reisolated from associations with callus tissue cultures of sugarcane and compared with stock cultures of the inoculated bacterium and related strains. Although the reisolate had a growth rate similar to stock cultures, it exhibited a severalfold increase in maximum specific activity of nitrogenase. The reisolate and the parent culture had similar ultrastructure. The general ultrastructure of Azospirillum is described. The bacterium was capsulated when grown on nitrogen-free nutrient agar plates and on callus, but was not capsulated when growing in a subsurface zone in N-free semisolid nutrient agar, except rarely in aging cultures. Capsulation may be a protective mechanism against unfavorable pO₂ under dinitrogen-fixing conditions. Pleomorphism occurred in capsulated forms, and the ultrastructure of these forms is described.     

Ergosterol as an indicator of mould growth on wood in relation to culture age, humidity stress and nutrient level

Changes in ergosterol content in cultures of Penicillium brevicompactum and Aspergillus versicolor on wood with time, changes in humidity or addition of glucose solutions to wood were studied with HPLC. Lowering of the humidity level caused a very large decline in ergosterol content of cultures of P. brevicompactum on wood over a 10 day period, although small amounts remained after this time. After an initial increase, up to an inoculation time of 45 days, reductions were also observed in control samples maintained at 100% RH, but these were smaller. The amount of ergosterol decreased to very low levels in wood impregnated with low levels of glucose during a 93 day incubation period. Ergosterol concentration in hyphae produced in surface liquid cultures was shown to be higher in mycelia growing on media enriched with nitrogen or with more available nutrients. The concentration of ergosterol in the mycelia of P. brevicompactum in surface liquid cultures varied by a factor of 5 from 2 to 10 mg g⁻. The results clearly show that ergosterol present in solid materials in mainly related to active biomass. With certain prerequisites, ergosterol determinations could also be used for total fungal biomass estimations on wood.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Tapé fermentation

Microorganisms isolated from ragi, originally obtained from Indonesia, were selected for their ability to convert steamed glutinous rice into tapé, an Indonesian fermented food. A mixture of Chlamydomucor oryzae and Endomycopsis fibuliger had good fermentation characteristics. Prepared starters, produced by growing pure cultures on rice and drying them, were as active as pure cultures grown for 4 days on Difco mycological agar slants at 30 C.     

Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.     

Tapé Fermentation

Microorganisms isolated from ragi, originally obtained from Indonesia, were selected for their ability to convert steamed glutinous rice into tapé, an Indonesian fermented food. A mixture of Chlamydomucor oryzae and Endomycopsis fibuliger had good fermentation characteristics. Prepared starters, produced by growing pure cultures on rice and drying them, were as active as pure cultures grown for 4 days on Difco mycological agar slants at 30 C.     

Discolored Red Seaweed <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> with Low Commercial Value Is a Novel Resource for Production of Agar Polysaccharides

The red seaweed Pyropia yezoensis has been demonstrated to be a novel resource for the production of high-quality agar. P. yezoensis is grown for the food industry in large-scale Japanese mariculture operations. However, discolored P. yezoensis is mostly discarded as an industrial waste, although it has some kind of utility values. Here, we evaluated the utility of discolored P. yezoensis as a resource for agar production. The quality of agar from the discolored seaweed was comparable to that from normal seaweed. In addition, as a distinguishing characteristic, agar yield was higher from discolored seaweeds than from normal types. Moreover, we successfully used agar from discolored P. yezoensis for bacterial plate media and DNA electrophoresis gels without agarose purification. Thus, our results demonstrate that discolored P. yezoensis is suitable for agar production and use in life science research. Diverting discolored P. yezoensis from disposal to agar production provides a solution to the current industrial waste problem in mariculture, as well as a secure source of agar for research purposes.     

Morphological changes of conidiophores and mycelium of some hyphomycetes

The formation of enlarged cells and other abnormalities of mycelium and conidiophores occurring inAspergillus flavus Link,Penicillium chrysogenum Thom,Penicillium notatum Westling,Penicillium variabile Sopp.,Scopularipsis brevicaulis Bainier,Fusarium moniliforme Sheld.,Botrytis cinera Pers. ex Fr.,Botrytis allii Rud.,Humicola brunnea var.africana Fass. andTritirachium heimii var.grisea Fass. under the influence of uranium acetate, radioactive sediment from P?íbram mines (uranium mines in P?íbram, Bohemia), fungicidal compounds (fungizon, fungistatin, nitrofungin), antibiotic nystatin and antifungal bacteriumBacillus subtilis Cohn emend. Prazmowski were investigated. Enlarged phialides or phialides with extended orifices and collarettes and occasionally a conversion of phialides to spherical enlarged cells were observed inAspergillus flavus grown on a Sabouraud agar supplemented with uranium acetate. InPenicillium chrysogenum some conidiophores were completely reduced to enlarged cells. Less abnormalities were observed on the Czapek-Dox agar. The radioactive sediment as a component of malt agar caused either complete reduction of conidiophores inAspergillus flavus to dichotomously branched hyphae or sometimes only an enlargement of phialides. The antifungal effect ofBacillus subtilis was demonstrated in all studied strains. Enlarged cells instead of whole conidiophores were formed under the influence of the bacterium. This effect was observed inBotrytis allii, Botrytis cinerea and to a lesser extent inPenicillium notatum, Aspergillus flavus andFusarium moniliforme. The enlarged cells could still germinate and can be considered as reduced conidiophores. The used fungicidal compounds and the antibiotic nystatin inhibited to a certain degree the studied strains, however, their application did not bring about any abnormalities of mycelium and conidiophores.     

Influence on plant growth of the breakdown of organic phosphorus compounds by micro-organisms

Summary Micro-organisms which break down lecithin or phytin have been isolated from soil by enrichment cultures. Only those organisms were investigated which on an agar medium containing lecithin or phytin as the sole source of P (in the case of lecithin, also as a source of N and energy), produced a clear area around their colonies. Certain of these organisms were inoculated into sterile cultures of radish plants grown on sand or agar substrates containing lecithin or phytin as the source of P, and the growth and N- and P-uptakes of the plants were compared with those of similar plants grown in non-sterile sand cultures or supplied with KH₂PO₄. Evidence was obtained that both lecithin and phytin can serve as P-sources for higher plants even under sterile conditions, phytin producing a greater effect than lecithin. Inoculation, however, had only in the phytin-containing medium sometimes a slight effect on the P-nutrition of the plants and further work in this field is therefore necessary.