Production of B-group vitamins by mycorrhizal fungi and actinomycetes isolated from the root zone of pine (Pinus sylvestris L.)

Summary Studies were carried out on synthesis of B-group vitamins by mycorrhizal fungi and actinomycetes (Streptomyces sp.) derived from soil, rhizosphere and mycorrhizosphere of pine.None of the fungal isolates produced biotin. The vitamin produced in largest amounts by the mycorrhizal fungi was thiamin.In general more actinomycetes isolated from the rhizosphere than from the root free soil produced B-group vitamins. This was particularly true for thiamin.The amount of vitamins produced was higher in actinomycetes than the amounts produced by the mycorrhizal fungi.This research was carried out under problem MR.II. 16 coordinated by the Institute of Dendrology, Polish Academy of Sciences.     

Production of vitamins by Azospirillum brasilense in chemically-defined media

The production of vitamins by Azospirillum brasilense was studied in chemically-defined media amended with malate, gluconate and fructose. The liberation of vitamins was significantly affected by the presence of different carbon sources and the age of the culture. Thiamine, niacin and pantothenic acid were produced in large amounts. Thiamine and riboflavin were produced only in culture containing malate or fructose. Biotin was not detected in the supernatants of the culture media.     

Uptake and accumulation of B-group vitamers in Saccharomyces cerevisiae in ethanol-stat fed-batch culture

The uptake and accumulation of the B-group vitamins thiamine, riboflavin, nicotinamide, pantothenic acid and pyridoxine in Saccharomyces cerevisiae was studied by gradually increasing the specific dosage of vitamins in an ethanol-stat fed-batch culture. Thiamine, nicotinamide, pantothenic acid, and pyridoxine were almost completely taken up at low vitamin dosages. Thiamine was determined to be the major accumulating form of vitamin B₁ while most of the assimilated nicotinamide and pantothenic acid accumulated in cofactor forms. Despite the obvious uptake of pyridoxine, accumulation of B₆ vitamers was not observed. In contrast with the other vitamins studied, riboflavin began accumulating in the culture medium immediately after vitamin addition was initiated. By the end of the experiment, the apparent uptake of all vitamins exceeded their accumulation in the cells. Variations in the growth rate of yeast at different vitamin dosages demonstrate the importance of balancing the vitamins in the media during cultivation.     

High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B₁ and B₆ and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH₂PO₄ (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B₁ deficiency increased urinary levels of all 2-oxo acids, while vitamin B₆ deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.     

Studies on the distribution of alkalophilic and alkali-tolerant soil fungi I

Soil fungi were isolated from two different soil types using alkaline and slightly acidic media (alkaline cornmeal agar (AC-MA), pH 9.7; cornmeal agar (CMA), pH 6.0) to study their distribution. Different species were obtained on each isolation medium. The number of species ofAcremonium andFusarium increased on ACMA, though many species growing well in acidic conditions were not detected on ACMA. Most of the fungi isolated on ACMA, especially from the alkaline soils, were alkalophiles or alkali-tolerants that can grow at pH 10.Acremonium alternatum, A. furcatum, Acremonium sp. 6,Gliocladium cibotii (YBLF 575),Phialophora geniculata, Stachylidium bicolor andStilbella annulata were alkalophilic, of whichAcremonium sp. 6 was the most pronounced alkalophile. Ability to grow under alkaline conditions, as well as under acidic conditions, was common in manyAcremonium species. The use of alkaline medium facilitates the isolation of alkalophilic soil fungi.     

Plant root excretions in relation to the rhizosphere effect

Summary The root exudates from seedlings of ten plant species grown under conditions of controlled environment and nutrition were biassayed for six vitamins of the B-group. Biotin was consistently present in the exudates in amounts sufficient to influence the growth of rhizosphere micro-organisms. Pantothenate and niacin were generally present, but usually at low levels unlikely to influence the microflora; riboflavin and thiamine were occasionally found in traces; pyridoxine was not detected in any root exudate.The vitamin content of the exudate varied with plant species. Field pea released large quantities of biotin, pantothenate, and niacin, but other plants including legumes, produced exudates medium to low in vitamin content and varying in relative amounts of each. Subterranean clover produced moderate amounts of vitamins, and from seed samples of graded size exuded vitamins in quantities unrelated to seed size. A comparison of five species of clover showed distinct differences in patterns of exudation in closely related plant species.Raising temperature and reducing light intensity by shading, produced only small effects upon vitamin exudation. Improved nutrient status produced marked increases in plant growth, but only small increases in amount of vitamin exuded, with pantothenate an exception tending to be released in greater amounts under unfavourable growing conditions. The presence of a root microflora caused sharp reduction in vitamin concentration of the culture solution.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Effects of pH and heavy metal concentrations in solution culture on the proton release,growth and elemental composition ofAlyssum murale andRaphanus sativus L.

The proton release by a species that can hyperaccumulate nickel (Alyssum murale) and by a non-accumulator (Raphanus sativus L.) was studied at different pH and heavy metal concentrations in solution culture. Both factors influenced the growth and composition of the plants.A. murale was more sensitive than radish to a decrease of pH from 7.0 to 6.0 in the growth medium; plant yield and proton production diminished with decreasing pH. However, yields and proton production of radish only decreased at pH 5.5. The differences in the amounts of protons produced between the hyperaccumulator species and radish were not large enough to conclude that decreasing pH in the rhizosphere ofA. murale is a mechanism for heavy metal solubilization.Nickel concentrations inA. murale followed the typical pattern of an accumulator plant — more Ni was accumulated in the shoots than in the roots. Lower concentrations of Zn and Cd occurred in the shoots than in roots ofA. murale, and also of Ni in radish. The concentrations of Co inA. murale shoots were increased when Zn, Ni and Cd were absent from the nutrient solution. However, Co concentrations in radish shoots were independent of the concentrations of other heavy metals in the growth medium.     

Volatile acid production from threonine,valine, leucine and isoleucine by clostridia

The amounts of the volatile acids produced from thereonine, valine, leucine and isoleucine by growing cultures of clostridia have been measured. The species used were Clostridium sporogenes; C. caloritolerans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. botulinum proteolytic type F; C. botulinum proteolytic type G; C. putrificum; C. difficile; C. ghoni; C. bifermentans; C. sordellii; C. mangenoti; C. cadaveris; C. lituseburense; C. propionicum; C. sticklandii; C. scatologenes; C. subterminale; C. putrefaciens; C. histolyticum; C. tetanomorphum; C. limosum; C. lentoputrescens; C. tetani; C. melanomenatum; C. cochlearium; C. sporospheroides. Most of the species tested gave increased yields of propionic acid when grown in the threonine medium; in addition, some species resembled C. propionicum and produced n-butyric acid when grown in this medium. C. histolyticum produced only acetic acid in the basal medium; all seven strains of this species produced more acetic acid when grown in the threonine medium than in the basal medium. Species which oxidize valine to iso-butyric acid also oxidize leucine to 3-methyl butyric acid and isoleucine to 2-methylbutyric acid. The iso-caproic fraction produced by some species is shown to be derived from leucine. The identitity of the branched-chain acids produced by C. sporogenes has been confirmed by gas liquid chromatography/mass spectrometry.Abbreviations GLC gas liquid chromatography - RCM reinforced clostridial medium - VFA volatile fatty acid     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Phosphatase production byAspergillus ficuum

Summary Effects of nutritional and cultural conditions on cell growth and phosphatase production byAspergillus ficuum were studied.A. ficuum produced high levels of phosphatases when grown on a basal medium that contained a minimal amount (2 mg/100 ml) of phosphorus in an acidic growth medium. The organism produced a nonspecific acid phosphomonoesterase rather than phytin-specific phosphatase. The enzyme hydrolyzed a variety of phosphates and produced orthophosphate. The rate of phosphate hydrolysis was dependent on the pH of the reaction, where the pH optimum for acid phosphatase was 2.5 and that for phytase was 5.0. The organism slowly released the phosphatase, and the enzyme activity in the growth medium increased continually during a one-month growth period. For a high level of phosphatase production, low levels (1–5 mg%) of initial phosphorus were necessary and polyphosphates were the desired form rather than the monophosphate. The addition of surfactants, such as polyoxyethylene ethers and sodium oleate, to fungal culture medium markedly increased the level of phosphatase production.     

Production of B--vitamins by heterotrophic planktonic bacteria isolated from littoral zone of lake Jeziorak.

Different genera and groups of heterotrophic planktonic bacteria of the littoral zone of the lake Jeziorak produced different B-group vitamins. Most numerous among the planktonic bacteria of this zone of the lake were biotin producers and least numerous were organisms synthesizing riboflavin and nicotinic acid. Most bacteria produced one or two vitamins. Three or more vitamins were produced by only a few strains. In autumn no strain produced all five vitamins studied in this work.     

The Influence of pH and Temperature on Cellulolytic and Pectolytic Activity of Cylindrocarpon destructans (Zins.) Scholt.

In studies on the effect of pH and temperature on cellulolytic and pectolytic activity of C. destructans, it was found that the isolates used produced only endoglucanases. The temperature and pH affected the synthesis of these enzymes. Fungi cultured at 26°C produced more of these enzymes than those grown at the two other temperatures. At 10°C, only one isolate produced minute amounts of endoglucanases. None of fungi studied exhibited cellulolytic activity in cultures grown at 20°C. Cellulolytic activity was found only in acidic media (pH 5.0). The fungi studied exhibited higher pectolytic than cellulolytic activity. In the post culture liquids of these organisms, both types of pectolytic enzymes (exo- and endo-PMG) were detected. Different temperature and pH values affected the production of these enzymes differently in various isolates.     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.     

The cell content and secretion of water-soluble vitamins by several freshwater algae

Three green algae, Chlamydomonas reinhardii, Chlorella vulgaris and Scenedesmus obliquus, and one blue-green alga, Anabaena cyclindrica, were grown in chemically defined media. All the algac examined contained folates, -carotene and vitamins C and E; several of the B-vitamins and vitamin A were found in varying amounts in some but not in all the algae examined. All the green algae secreted significant amounts of folate and biotin and all but Scenedesmus secreted pantothenate into their growth medium; Anabaena secreted folate and pantothenate.This work was done with the support of grant BMS 74-08918 from the National Science Foundation     

Effects of pH and temperature on growth ofSaprolegnia diclina andS. parasitica isolated from various sources

Saprolegnia diclina andS. parasitica isolated from three sources could germinate in strong acidic conditions. Growth ability correlated with the species of fungi rather than with the sources from which they were isolated.S.diclina isolates appeared to germinate at a pH condition as low as 3.5, whereasS. parasitica isolates could not germinate at below pH 3.8.S. parasitica isolates from visceral mycoses still showed good growth at 30°C, whereas other isolates did not. Also,S. parasitica isolates from visceral mycoses produced more abundant motile zoospores, and continued to do so for a longer period of time (28 d), thanS. parasitica isolates from external saprolegniasis andS. diclina isolates.     

Ammonia fungi of iriomote Island in the southern Ryukyus,Japan and a new ammonia fungus,Hebeloma luchuense

Species composition and fruiting season of ammonia fungi were investigated in Iriomote Island of the southern Ryukyus, Okinawa Prefecture, Japan.Castanopsis andPinus forests were surveyed and 10 species of ammonia fungi were collected, including one new agaric species,Hebeloma luchuense sp. nov. This new fungus is characterized by having a rooting, squamulose-scaly stipe and cortinate veil and forms ectomycorrhizae withCastanopsis cuspidata var.sieboldii. Although the general mushroom season in theCastanopsis forest in Iriomote island was very short and restricted to summer, ammonia fungi were observed to fruit throughout the year in urea-treated plots.     

石灰土和酸性土生境下金花茶组植物叶片钙形态差异

为探究不同生境下金花茶组植物的叶片钙形态特征，该研究以10种石灰土生境和4种酸性土生境的金花茶为对象，测定了其生境土壤的钙含量和pH值，以及该生境下金花茶组植物叶中的硝酸钙和氯化钙、水溶性有机酸钙、果胶酸钙、磷酸钙和碳酸钙、草酸钙、硅酸钙和总钙的含量。结果表明：(1)石灰土生境的土壤钙含量和土壤pH均极显著(P<0.01)高于酸性土。(2)在石灰土生境中，金花茶组植物的叶钙形态以草酸钙(41.17%)为主，而在酸性土生境中则以果胶酸钙(43.10%)为主，除硝酸钙和氯化钙、果胶酸钙外，石灰土金花茶的各叶钙形态和总钙含量均极显著(P<0.01)高于酸性土金花茶。(3)相关性分析结果显示，大部分叶钙形态含量与土壤pH和土壤钙含量呈极显著(P<0.01)正相关，表明土壤环境对金花茶组植物叶钙形态特征具有重要影响。(4)单因素方差分析结果显示，各叶钙形态含量在物种间存在极显著(P<0.01)差异，表明金花茶组植物在物种分化过程中叶钙形态特征具有多样性。(5)基于叶钙形态特征的聚类分析显示，14种金花茶可归为3大类。总体而言，不同生境背景下金花茶组植物的叶钙形态差异可能是...     

Bioactivity of intra and extracellular substances from cyanobacteria and lactic acid bacteria on “wood blue stain” fungi

Cyanobacteria (photoautotrophic prokariota) have potential for the control of pathogenic bacteria and fungi. The effect of intra and extracellular products from cyanobacterial strains on the growth of fungi isolated from “wood blue stain,” was tested. Extracellular products were obtained by concentration and sterilization of the culture medium where cyanobacteria were grown. Cyanobacterial substances promoted or inhibited fungal growth according to the fungal and cyanobacterial strains tested. Extracellular products from Nostoc muscorum 79a and the methanolic extract from Microchaete tenera 84b biomass inhibited growth of Sphaeropsis sapinea 2157 (64.7 and 775.6%, respectively). Extracellular products of Nostoc piscinale 59 and biomass methanolic extract from N. muscorum 79a produced the highest growth promotion of Trichoderma boningii 452 (105.0%) and T. viride 993 (136.7%). Extracellular products of the heterotrophic lactic acid bacterium Streptococcus termophilus were also tested and strongly inhibited (64–92%) all the fungal strains. The tested fungi have different sensitivity to the bioactive substances present in the biomass and/or the culture medium of the studied cyanobacteria and lactic acid bacterium. N. muscorum 79a, M. tenera 84b, and S. termophilus have potential to control the wood blue stain fungi by a friendly environmental alternative.     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.