Humic acid-vitamin agar,a new medium for the selective isolation of soil actinomycetes

A new medium, designated HV agar, containing soil humic acid as the sole source of carbon and nitrogen was developed.The HV agar was superior to other currently used media, including colloidal chitin agar, glycerol-arginine agar and starch-casein-nitrate agar, for the isolation and enumeration of soil actinomycetes: It allowed the growth of the largest numbers of actinomycete colonies belonging to each genus of Streptomyces, Micromonospora, Microbispora, Streptosporangium, Nocardia, Dactylosporangium, Microtetraspora and Thermomonospora on the plate, while restricting the development of true bacteria. The HV agar supported adequate growth and good sporulation for these actinomycetes.Even when spore suspensions were used as the inoculum, the HV agar produced remarkably larger numbers of actinomycetes, especially strains of the genera Micromonospora, Microbispora, Streptosporangium, Dactylosporangium and Saccharomonospora, than did glycerol-arginine agar. It was found that the spores of these actinomycetes were activated upon germination by treatment at 20°C for 30 min with a O.2% solution of humic acid prior to incubation.     

Regulation of hyphal growth rate ofHypoxylon mammatum by amino acids: Stimulation by proline

Variation among isolates ofHypoxylon mammatum caused by amino acids added as sole nitrogen sources demonstrated genetic heterogeneity among the population in the mechanisms regulating hyphal growth rates. Isolates were obtained by a random sample consisting of 22 single ascospore isolates from a local population of hypoxylon cankers onP. tremuloides. Hyphal growth rates were determined from colony diameter measurements on defined media containing either alanine, asparagine, glycine, leucine, lysine, or proline. All isolates but two grew faster on proline than the other amino acids tested. The mean growth rate of the population sample was 3.9 mm day⁻¹ on proline compared to 1.96 mm day⁻¹ on asparagine, the second fastest mean growth rate. Growth rates of the 22 isolates on these six amino acids were largely uncorrelated, indicating independent mechanisms of regulation. Only asparagine gave significant correlations with more than two other amino acids. A more detailed examination of selected isolates representing a range of growth rates on proline showed that the rapid growth rates were also dependent on other materials present in Difco Bacto-Agar. Growth of the proline stimulated isolates was considerably slower on media gelled with Noble agar. Other nitrogen sources, especially glutamate, added with proline stimulated the growth of these isolates. Isolate dependent variation in the stimulatory effect of the N source added with proline was demonstrated. Stimulation of growth rate by proline may be related to drought stress-enhanced canker elongationin vivo, since drought stress caused proline accumulation in the host,Populus tremuloides.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media.

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Nitrogen utilization in bacterial isolates from the equine cecum.

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

Studies on the microbial populations of the rhizosphere of big sagebrush (<Emphasis Type="Italic">Artemisia tridentata</Emphasis>)

Prolonged use of broad-spectrum antibiotics has led to the emergence of drug-resistant pathogens, both in medicine and in agriculture. New threats such as biological warfare have increased the need for novel and efficacious antimicrobial agents. Natural habitats not previously examined as sources of novel antibiotic-producing microorganisms still exist. One such habitat is the rhizosphere of desert shrubs. Here, we show that one desert shrub habitat, the rhizosphere of desert big sagebrush (Artemisia tridentata) is a source of actinomycetes capable of producing an extensive array of antifungal metabolites. Culturable microbial populations from both the sagebrush rhizosphere and nearby bulk soils from three different sites were enumerated and compared, using traditional plate-count techniques and antibiotic activity bioassays. There were no statistical differences between the relative numbers of culturable non-actinomycete eubacteria, actinomycetes and fungi in the rhizosphere versus bulk soils, but PCR amplification of the 16S rRNA gene sequences of the total soil DNA and denaturing gradient gel electrophoresis showed that the community structure was different between the rhizosphere and the bulk soils. A high percentage of actinomycetes produced antimicrobials; and the percentage of active producers was significantly higher among the rhizosphere isolates, as compared with the bulk soil isolates. Also, the rhizosphere strains were more active in the production of antifungal compounds than antibacterial compounds. 16S rRNA gene sequence analysis showed that sagebrush rhizospheres contained a variety of Streptomyces species possessing broad spectrum antifungal activity. Scanning electron microscopy studies of sagebrush root colonization by one of the novel sagebrush rhizosphere isolates, Streptomyces sp. strain RG, showed that it aggressively colonized young sagebrush roots, whereas another plant rhizosphere-colonizing strain, S. lydicus WYEC108, not originally isolated from sagebrush, was a poor colonizer of the roots of this plant, as were two other Streptomyces isolates from forest soil. These results support the hypothesis that the rhizosphere of desert big sagebrush is a promising source of habitat-adapted actinomycetes, producing antifungal antibiotics.     

Nitrogen utilization in bacterial isolates from the equine cecum

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

A chitinolytic actinomycete complex in black soil

A chitinolytic actinomycete complex in chernozem soil has a specific taxonomic composition, which differs from that of the actinomycete complex which is typically isolated on standard nutrient media containing sugars and organic acids as carbon sources. The actinomycete complex that was isolated by using nutrient media with chitin as the source of carbon and nitrogen was dominated by representatives of the genus Streptosporangium, and the actinomycete complex that was isolated by using nutrient media with sugars and organic acids as the carbon sources was dominated by representatives of the genus Streptomyces. The confirmation to the ability of actinomycetes to utilize chitin as a sole source of carbon and nitrogen came from the augmented length and biomass of the mycelium, the increased number and biomass of the actinomycete spores, the production of carbon dioxide, and the accumulation of NH4+ ions in the culture liquid of the actinomycetes that are grown in the nutrient media with chitin.     

The utilization of organosulphonates by soil and freshwater bacteria

Utilization of the biogenic aliphatic organosulphonates taurine, isethionate, sulphoacetaldehyde and sulphoacetate was investigated in 100 soil and freshwater bacteria isolated on modified complete mineral salts medium. More than 90% could use all the compounds as sole sulphur sources, and some 10% used taurine and isethionate as sole carbon and energy, or sole carbon, energy and sulphur sources. None could mineralize sulphoacetaldehyde or sulphoacetate; however, two isolates capable of growth on sulphoacetate as sole carbon, energy and sulphur source were obtained by enrichment culture. The results suggest that in the majority of environmental bacteria the pathways of organosulphonate biodegradation may be independently controlled by the supply of carbon and sulphur to the cell, and that a number of routes may exist for cleavage of the organosulphonate C–S bond.     

Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Azotobacter vinelandii strain ATCC 12837 and A. chroococcum strain H23 (CECT 4435) were able to grow on N-free or NH4Cl-amended chemically-defined (Burk's) media, with protocatechuic acid (1-2 mmol 1(-1)) or sodium p-hydroxybenzoate (1-10 mmol 1(-1)) as sole carbon (C) sources. At a concentration of 2 mmol 1(-1), both substrates supported nitrogen fixation (acetylene reduction assay) at similar or higher rates than bacteria grown in control media amended with 2 mmol 1(-1) sodium succinate as C source. The two strains produced the B-group vitamins niacin, pantothenic acid, thiamine, riboflavin and biotin after 72 h of growth in chemically-defined media with 2 mmol 1(-1) protocatechuic acid, sodium phydroxybenzoate or sodium succinate as sole C source, either in N-free media or in media amended with 0.1% NH4Cl. Quantitative production of all vitamins was affected by the use of the different C and N substrates.     

Directed isolation of Micromonospora generic cultures on a selective medium with gentamycin

The results of using selective media with gentamicin for directed isolation of Micromonospora are presented. It was shown that the use of the selective media with gentamicin for isolation of actinomycetes from soils of usual humidity levels markedly increased the frequency of Micronomonospora detection. The use of the selective media with gentamicin for plating out silt substrates containing mainly Micromonospora had practically no effect on the increase in the number of the Micromonospora cultures grown. The number of antibiotic-producing Micromonospora isolated on the media with gentamicin was 3 times higher than that on the control media. The use of the selective media with gentamicin provided directed isolation of Micromonospora.     

Growth of Certain Aquatic Oomycetes on Amino Acids II. Apodachlya, Aphanomyces, and Pythium

Four aquatic fungi —Apodachlya brachynema and A. minima (Leptomitales), Aphanomyces laevis (Saprolegniales), and Pythium ultimum (Peronosporales) —were tested for growth in synthetic media containing one of a variety of carbon sources. Apodachlya brachynema readily utilized five amino acids — alanine, glutamate, aspartate, proline and leucine — as well as glucose and acetate. Growth on sucrose as a carbon source was slight. Apodachlya minima differed from A. brachynema in that it could not utilize proline and leucine. Aphanomyces laevis grew well on only three of the substrates tested — glucose, alanine and glutamate. Pythium ultimum utilized glucose, sucrose, maltose, cellobiose, alanine, glutamate, aspartate, proline, asparagine, ornithine, and serine, but not eight other amino acids. All of these fungi hydrolyzed gelatin. Radioactively labeled carbon dioxide was released during incubation of Aphanomyces laevis in media containing labeled leucine, proline, or phenylalanine. These data provide evidence of some catabolism of the three substrates although none of these substrates can support the growth of Aphanomyces laevis as a sole source of carbon and nitrogen.     

Selective media for three biovars of Agrobacterium

Three selective media for the isolation of the three known biovars of Agrobacterium are described. Selectivity was based on carbon and nitrogen sources; L(—)-arabitol for biovar 1, erythritol for biovar 2 and a combination of tartrate and D-glutamate for biovar 3. The new media were compared with existing selective media. Recovery of agrobacteria from soil was very efficient and discrimination between the three biovars was satisfactory except for tartrate-utilising biovar 1 isolates which grew on the biovar 3 medium. Some strains of Pseudomonas sp. isolated from crown galls on vine could utilise octopine as a source of carbon and nitrogen.     

Utilization of capsaicin and vanillylamine as growth substrates by Capsicum (hot pepper)-associated bacteria

Capsaicin contributes to the organoleptic attributes of hot peppers. Here, we show that capsaicin is utilized as a growth nutrient by certain bacteria. Enrichment cultures utilizing capsaicin were successfully initiated using Capsicum-derived plant material or leaves of tomato (a related Solanaceae) as inocula. No other sources of inoculum examined yielded positive enrichments. Of 25 isolates obtained from enrichments: all utilized 8-methylnonanoic acid; nine were found capable of degrading capsaicin as sole carbon and energy source; 11 were found capable of utilizing vanillylamine; but only two strains could use either of these latter two compounds as sole nitrogen source. Phylogenetic analysis of capsaicin degraders revealed them to be strains of Variovorax and Ralstonia, whereas the vanillylamine degraders were strains of Pseudomonas and Variovorax. Neither of the two strains isolated from one enrichment culture originally inoculated with dried pepper fruit was capable of using capsaicin as sole carbon and nitrogen source. However, good growth was achieved under such conditions when the two isolates, a strain of Variovorax paradoxusThat degraded capsaicin when provided with ammonium, and a vanillylamine degrading strain of Pseudomonas putida, were cultured together. A cross-feeding of capsaicin-derived carbon and nitrogen between members of pepper-associated consortia is proposed.     

The specificity of 1-naphthol oxygenases from three bacterial isolates, Pseudomonas spp. (NCIB 12042 and 12043) and Rhodococcus sp. (NCIB 12038) isolated from garden oil

Abstract Three bacterial isolates which appeared to use the insecticide, carbaryl (1-naphthyl, N -methyl-carbamate) as their sole carbon and nitrogen sources were originally selected from garden soil. Only one isolate, Pseudomonas sp. (NCIB 12043) could metabolise carbaryl rapidly to 1-naphthol and methylamine. The other two isolates, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038) relied on slow chemical hydrolysis of carbaryl to 1-naphthol and methylamine. All three isolates used 1-naphthol as their sole carbon source; however, their ability to use naphthalene and a range of mono- and dihydroxy-substituted naphthalene compounds varied. NCIB 12038 and NCIB 12043 showed little or no growth on naphthalene, 2,3-dihydroxynaphthalene or 1,3-dihydroxynaphthalene as sole carbon sources and their 1-naphthol oxygenases had little activity with these substrates. In contrast, NCIB 12042 could use these compounds as sole carbon sources and its 1-naphthol oxygenase also showed activity with them. We conclude that 1-naphthol oxygenase from NCIB 12042 is a relatively non-specific dioxygenase, whereas the 1-naphthol oxygenases from NCIB 12038 and NCIB 12043 are relatively specific monooxygenases requiring hydroxylated naphthalene compounds as substrates.     

Molecular cloning of a coding sequence of Bordetella pertussis filamentous hemagglutinin gene

Abstract The halophilic phototrophic bacterium Rhodospirillum salexigens was tested for growth on a variety of organic and inorganic nitrogenous compounds as sole nitrogen sources. In media containing acetate as carbon source, the amino acids glutamate, proline, and aspartate supported good growth of R. salexigens ; several other amino acids or ammonia did not support growth. Attempts to grow R. salexigens on ammonia led to the discovery that this organism excretes a highly basic substance under certain nitrogen nutritional conditions which raises the pH above that supporting growth. Cultures of R. salexigens transferred to media containing both pyruvate and acetate as carbon sources grew on ammonia as sole nitrogen source and the culture pH did not rise. Dual substrate experiments showed that R. salexigens utilized glutamate in preference to ammonia when both were present at equimolar concentrations.     

嗜酸丝状放线菌的选择性分离与多样性

摘要:【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16S rRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为：土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸（各50 mg/L）的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属：链霉菌属(Streptomyces)、小单孢菌属(Micromonospora) 、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea) 、韩国生工属(Kribbella) 、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora) 和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。     

Bacterial Utilization of Ether Glycols

A soil bacterium capable of using oligo- and polyethylene glycols and ether alcohols as sole sources of carbon for aerobic growth was isolated. The effects of substituent groups added to the ether bonds on the acceptability of the compounds as substrates were studied. Mechanisms for the incorporation of two-carbon compounds were demonstrated by the observation that acetate, glyoxylate, ethylene glycol, and a number of the tricarboxylic acid cycle intermediates served as growth substrates in minimal media. The rate of oxidation of the short-chained ethylene glycols by adapted resting cells varied directly with increasing numbers of two-carbon units in the chains from one to four. The amount of oxygen consumed per carbon atom of oligo- and polyethylene glycols was 100% of theoretical, but only 67% of theoretical for ethylene glycol. Resting cells oxidized oligo- and polyethylene glycols with 2 to 600 two-carbon units in the chains. Longer chained polyethylene glycols (up to 6,000) were oxidized at a very slow rate by these cells. Dehydrogenation of triethylene glycol by adapted cells was observed, coupling the reaction with methylene blue reduction.     

A Chitinolytic Actinomycete Complex in Chernozem Soil

A chitinolytic actinomycete complex in chernozem soil has a specific taxonomic composition, which differs from that of the actinomycete complex typically isolated on standard nutrient media containing sugars and organic acids as carbon sources. The actinomycete complex that was isolated by using nutrient media with chitin as the source of carbon and nitrogen was dominated by representatives of the genus Streptosporangium, and the actinomycete complex that was isolated by using nutrient media with sugars and organic acids as the carbon sources was dominated by representatives of the genus Streptomyces. The confirmation of the ability of actinomycetes to utilize chitin as a sole source of carbon and nitrogen came from the augmented length and biomass of the mycelium, the increased number and biomass of the actinomycete spores, the production of carbon dioxide, and the accumulation of NH₄ ⁺ ions in the culture liquid of the actinomycetes grown in the nutrient media with chitin.     

Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.