Preservation of microorganisms

Cultures of bacteria, yeasts and fungi were grown on common agar media in normal culture tubes. About 1 week after maximum growth the cotton plugs of the tubes were replaced by sterile rubber seals. The cultures were stored in the dark at room temperature. Tests for viability of cultures were made after periods between 1 and 10 years. The results of this simple method show long survival periods of many bacteria and yeasts.     

Evaluation of Media for Selective Isolation of Yeasts from Oral, Rectal, and Burn Wound Specimens

Six media were evaluated to determine their ability to isolate yeasts and inhibit bacteria. The media included the following: Snyder, Snyder tellurite, Sabouraud tellurite, Littman-gentamicin, molybdate, and Mycosel (BBL). Doses of mixed intestinal gram-negative bacilli and enterococci were most effectively inhibited by Snyder tellurite agar. Klebsiella pneumoniae was the most common bacterial contaminant of the other media. All six media were comparable in isolating yeasts while preventing the growth of the oral bacterial flora. The selection of a basal fungal growth medium for tellurite incorporation to inhibit bacteria but permit growth of yeasts was affected by pH. The bacteriostatic effect of tellurite was decreased with increasing pH of media while fungistatic action was increased. The arbitrary selection of Snyder and Littman agars to isolate yeast from burn wound cultures demonstrated the need to include a selective medium for these specimens. Blood, phenylethyl alcohol blood agar, and Columbia CN blood agar were all inadequate for isolating yeasts from burns. Growth of a variety of filamentous saprophytic and pathogenic dimorphic fungi grew adequately on four of five selective media tested.     

温室黄瓜叶部微生物区系

采用稀释分离法和消毒叶片研磨液培养法对温室黄瓜叶围和内生微生物进行了分离,共分离到248个菌株,初步鉴定出13个属的叶围真菌,其中链格孢属(Alternaria)和青霉属(Penicillium)真菌为优势类群;鉴定出4个属的内生真菌,其中曲霉属(Aspergillus)真菌为优势类群;10个属的叶围细菌,其中芽孢杆菌属(Bacillus)和黄单胞菌属(Xanthomonas)细菌为优势类群;6个属的内生细菌,其中芽孢杆菌属和假单胞菌属(Pseudomonas)细菌为优势类群;6个属的叶围酵母菌,其中隐球酵母属(Cryptococcus)为优势类群;已鉴定出2个属的叶围放线菌,分别为链霉菌属(Streptomyces)和小多孢菌属(Micropolpspora).未分离到内生酵母菌和放线菌.     

Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C

We developed a procedure to culture microorganisms below freezing point on solid media (cellulose powder or plastic film) with ethanol as the sole carbon source without using artificial antifreezes. Enrichment from soil and permafrost obtained on such frozen solid media contained mainly fungi, and further purification resulted in isolation of basidiomycetous yeasts of the genera Mrakia and Leucosporidium as well as ascomycetous fungi of the genus Geomyces. Contrary to solid frozen media, the enrichment of liquid nutrient solutions at 0 degrees C or supercooled solutions stabilized by glycerol at -1 to -5 degrees C led to the isolation of bacteria representing the genera Polaromonas, Pseudomonas and Arthrobacter. The growth of fungi on ethanol-microcrystalline cellulose media at -8 degrees C was exponential with generation times of 4.6-34 days, while bacteria displayed a linear or progressively declining curvilinear dynamic. At -17 to -0 degrees C the growth of isolates and entire soil community on 14C-ethanol was continuous and characterized by yields of 0.27-0.52 g cell C (g of C-substrate)(-1), similar to growth above the freezing point. The 'state of maintenance,' implying measurable catabolic activity of non-growing cells, was not confirmed. Below -18 to -35 degrees C, the isolated organisms were able to grow only transiently for 3 weeks after cooling with measurable respiratory and biosynthetic (14CO2 uptake) activity. Then metabolic activity declined to zero, and microorganisms entered a state of reversible dormancy.     

Saccharide-mediated antagonistic effects of bark beetle fungal associates on larvae

Bark beetles are among the most destructive of pine forest pests and they form close symbiotic relationships with ophiostomatoid fungi. Although some fungi are considered to be mutualistic symbionts of bark beetles with respect to the supply of nutrients, detrimental effects of fungal symbionts on larval growth have also been frequently reported. The mechanisms of such antagonistic effects are hypothesized to be a decrease in nutritional resources caused by competition for saccharides by the fungi. Here, we provide experimental evidence that three beetle-associated fungi modify the nutritional content of an artificial phloem diet, leading to a detrimental effect on the growth of Dendroctonus valens larvae. When larvae were fed a diet of pine phloem in agar medium colonized with any of these fungi, feeding activity was not affected but weight significantly decreased. Additional analysis showed that fungi depleted the fructose and glucose concentrations in the phloem media. Furthermore, these detrimental effects were neutralized by supplementing the media with fructose or glucose, suggesting that fungi may affect larval growth by modifying diet saccharide contents. These data indicate that fungus-induced nutritional changes in bark beetle diet can affect larval growth, and that the mechanism involves fungus-induced saccharide depletion from the larval diet.     

pH dependence and thermostability of lipases from cultures from the ARS culture collection

Summary Previously we used a simple, sensitive agar plate method to screen lipase activity from 1229 selected cultures including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi covering many genera and species. About 25% of the cultures tested were lipase-positive. These lipase-positive strains were further classified as good, moderate or weak enzyme producers. We have expanded our screening method to focus specifically on the pH dependence and thermostability of these lipase activities. The lipases exhibited various pH sensitivities and were divided into three groups: (i) lipases which are active at pH 5.5 but not at pH 7.5—produced by 36 bacteria, 23 yeasts and four actinomycetes; (ii) lipases which are active at pH 7.5 but not at pH 5.5—produced by 17 bacteria, four yeasts, two actinomycetes and one fungus; and (iii) lipases which are active at both pH 5.5 and pH 7.5—produced by 112 bacteria, 90 yeasts, 15 actinomycetes and five fungi. By screening at 60°C and pH 9.0, we further identified 50 bacteria and 26 yeasts that produce thermostable alkali-tolerant lipases. Product analyses confirmed our screening results. Lipases with specific pH dependency and thermostability have potential to be developed into industrial enzymes.     

Studies on Antiviral and Antitumor Antibiotics

A screening for antiviral antibiotics was carried out using paper-disc agar-diffusion method. The microorganisms tested were unidentified soil fungi and the type cultures of our laboratory including actinomycetes, fungi, yeasts and bacteria. Mycelia or cells were extracted with acetone and the antiviral activity of the acetone extracts was determined. The extracts of actinomycetes mycelia showed the highest frequency of the appearance of antiviral activity against Newcastle disease virus. The frequencies of the appearance of antiviral activity in fungal and bacterial type cultures were the same degree and that of yeasts was low. Antiviral activity of the principles thus obtained was studied by microscopic observation in tube cultures using HeLa cells as a host.     

Antagonistic activity of soil acidophilic actinomycetes

It has been shown that soil acidophilic actinomycetes (mycelial prokaryotes with a growth optinum between pH 3 and 7) markedly differ from neutrophilic actinomycetes in antimicrobial activity: the former are more active against fungi and yeasts, whereas the latter effectively suppress Gram-positive bacteria. Acidophilic streptomycetes actively inhibit the growth of phytopathogenic fungi, especially on acidic media.     

Absorption and Transport of Fluorescent Brighteners by Microorganisms

The absorption of brighteners by living cells and their transport to subsequent growth is described. Brighteners are highly fluorescent, ultraviolet-absorbing compounds which appear to be essentially nontoxic, stable biological markers. They have been effectively absorbed by growing cultures of bacteria, yeasts, actinomycetes, and higher fungi, with active growth centers evidencing the greatest flourescence.     

Quantitative Studies of Total Lipids of Pathogenic Fungi

Ten species of dermatophytes and four of the systemic fungi were assayed for total lipids, acetone-soluble fraction, and phospholipid content in different types of cultures. The yeast phase of each of the systemic fungi grown on solid medium exhibited a higher total lipid content than did the mycelial growth in liquid medium, either shake or still. Shake cultures, in all the fungi tested, produced the least lipids. The yeasts were consistently higher also in the acetone-soluble fraction. Histoplasma duboisii in the yeast phase and Microsporum gypseum produced the greatest amount of phospholipid, and Blastomyces dermatitidis in the yeast phase and M. canis produced the largest acetone-soluble fraction among the systemic fungi and dermatophytes, respectively.     

Distribution of psychrophilic microorganisms in soils of Terra Nova Bay and Edmonson Point, Victoria Land and their biosynthetic capabilities

Microbiota of soil samples from Terra Nova Bay and Edmonson Point, Antarctica was observed by dilutions spread plate method. Variety of mesophilic and psychrophilic microorganisms was detected and isolated. Bacteria, actinomycetes, fungi, and microalgae occurred. Fungi genera Penicillium, Aspergillus, Paecilomyces, Cladosporium, Mortierella, Candida, Rhodotorula were found. By morphology and cell wall aminoacid composition the actinomycete genus Streptomyces was characterized. The bacteria and actinomycetes were screened for biologically active products. Some cultures formed enzymes, glycolipids and antibiotics. Psychrophilic strain Streptomyces sp. no. 8 was studied more detail and was established that it produced following antibiotics: azalomycin B, nigericin and non-polyenic macrolide antibiotic composed from two components that inhibited the growth of Gram-positive bacteria, yeasts, and phytopathogenic fungi.     

Debaryomyces mycophilus sp. nov., a siderophore-dependent yeast isolated from woodlice

Four strains of an ascogenous yeast were isolated from the guts of the woodlice species Armadillidium vulgare (Latreille). This yeast differed from all known yeasts by its inability to grow in culture without the presence of a metabolite produced by some common soil fungi such as Cladosporium cladosporioides, Aspergillus alliaceus, and Penicillium spp. Phylogenetic analysis based on 18S rDNA and 26S rDNA (domain D1/D2) sequences indicated that the yeast represents a new taxon in the genus Debaryomyces. The new species Debaryomyces mycophilus is thus proposed. It was, furthermore, shown that the fungal metabolite necessary for growth of D. mycophilus did not provide the yeast with carbon, nitrogen or vitamins. The active compound was partially purified and it was shown that it is a siderophore used by the yeast as a source of iron. The addition of ferrichrome or high concentrations of FeCl(3) to growth media replaced the obligate dependence on a fungal metabolite. Symbiosis among fungi, based on the availability and utilization of iron, is an aspect of mycology that has not previously been recognized. The addition of chelated iron to isolation media could lead to the discovery of many unknown yeasts and fungi.     

Evidence for 1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non-saline conditions

The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns. They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls. The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions. We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol. Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography. This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.     

Orientation of chlorophyll in vivo. Studies with magnetic field oriented chlorella

The streptomycetes have a bacterial-type cell wall and a mycelial growth habit. Although the exact taxonomic relationship to yeasts or fungi and the bacteria is unknown, a great similarity to bacteria is assumed. $\text{Streptomyces erythreus}$ CA340 (Abbott) is found to possess a multienzyme complex making fatty acids. The crude enzyme complex is stimulated by FMN; is inhibited by iodoacetamide; is excluded and partially purified (ca. 70-fold) by Sephadex G-200; is most active at pH 7.5; is most stable in 0.5 M phosphate buffer. Thus, $\text{S. erythreus}$ CA340 and possibly all streptomycetes have a major biochemical similarity to yeasts and fungi.     

Spores of the mycorrhizal fungus <Emphasis Type="Italic">Glomus mosseae</Emphasis> host yeasts that solubilize phosphate and accumulate polyphosphates

The present paper reports the presence of bacteria and yeasts tightly associated with spores of an isolate of Glomus mosseae. Healthy spores were surface disinfected by combining chloramine-T 5%, Tween-40, and cephalexin 2.5 g L⁻¹ (CTCf). Macerates of these spores were incubated on agar media, microorganisms were isolated, and two yeasts were characterized (EndoGm1, EndoGm11). Both yeasts were able to solubilize low-soluble P sources (Ca and Fe phosphates) and accumulate polyphosphates (polyPs). Sequence analysis of 18S ribosomal deoxyribonucleic acid showed that the yeasts belong to the genera Rhodotorula or Rhodosporidium (EndoGm1) and Cryptococcus (EndoGm11). Results from inoculation experiments showed an effect of the spore-associated yeasts on the root growth of rice, suggesting potential tripartite interactions with mycorrhizal fungi and plants.     

Succession and activity of microorganisms in stored bagasse

Summary Fresh sugarcane bagasse was fermented under defined conditions and investigated regarding a microbial succession during fermentation, in view of the enzyme activities of microorganisms against the main bagasse components: sucrose, pectin, hemicellulose, cellulose, and lignin.Altogether, 400 pure cultures of microorganisms were obtained from 8 g bagasse during 6.5 days of storage. This flora consists of bacteria (74%), actinomycetes (6%), yeasts (13%), and fungi (7%). The yeasts dominate in early fermentation, followed by bacteria, and then by actinomycetes and fungi.This succession coincides with the enzymic activities of the isolated organisms during fermentation. At first, residual sugar is consumed predominantly by the yeasts. Then the bacteria degrade the pectin, the hemicellulose, and in parts, the cellulose. Later, the actinomycetes and the fungi imperfecti attack the hemicellulose, the cellulose, and, partly, the lignin within the bagasse fiber.These results are corroborated by investigations using bagasse from bulk storage.     

Population dynamics of Pythium Irregulare Buis. In container-plant production as influenced by physical structure of media

Summary Helleri holly were grown in 100% sand, 50% bark-50% sand, and 100% bark media and inoculated with Pythium irregulaye Buis. Pythium populations were determined at 4, 10 and 17 cm depths at planting date and every two weeks thereafter. After 7 weeks fresh weight of plants and populations of bacteria, actinomycetes, and total fungi were determined. P. irregulare was highest at the 4 and 10 cm depths, with bark media reaching their highest Pythium levels 4 weeks after inoculation. Pythium peaked in 100% sand 6 weeks after inoculation and declined thereafter.Total bacteria increased with an increase in percent bark and were the predominant microbes. Actinomycetes comprised a greater proportion of the microbial population in sand and bark-sand media than in 100% pine bark. Total fungi were greatest in the 50% bark-50% sand medium. Fresh weight of Helleri holly increased with an increase in the percent bark in the medium.Portion of dissertation submitted in partial fulfillment of the requirements of Ph.D. in Plant Science.Graduate Assistant, Associate Horticulturist, and Associate Plant Pathologist, respectively.     

Interactions among endophytic bacteria and fungi: Effects and potentials

Plants benefit extensively by harbouring endophytic microbes. They promote plant growth and confer enhanced resistance to various pathogens. However, the way the interactions among endophytes influence the plant productivity has not been explained. Present study experimentally showed that endophytes isolated from rice (Oryza sativa) used as the test plant produced two types of interactions; biofilms (bacteria attached to mycelia) and mixed cultures with no such attachments. Acidity, as measured by pH in cultures with biofilms was higher than that of fungi alone, bacteria alone or the mixed cultures. Production of indoleacetic acid like substances (IAAS) of biofilms was higher than that of mixed cultures, fungi or bacteria. Bacteria and fungi produced higher quantities of IAAS than mixed cultures. In mixed cultures, the potential of IAAS production of resident microbes was reduced considerably. There was a negative relationship between IAAS and pH of the biofilms, indicating that IAAS was the main contributor to the acidity. However, such a relationship was not observed in mixed cultures. Microbial acid production is important for suppressing plant pathogens. Thus the biofilm formation in endophytic environment seems to be very important for healthy and improved plant growth. However, it is unlikely that an interaction among endophytes takes place naturally in the endophytic environment, due to physical barriers of plant tissues. Further, critical cell density dependant quorum sensing that leads to biofilm formation may not occur in the endophytic environment as there is a limited space. As suchin vitro production and application of beneficial biofilmed inocula of endophytes are important for improved plant production in any agro-ecosystem. The conventional practice of plant inoculation with monocultures or mixed cultures of effective microbes may not give the highest microbial effect, which may only be achieved by biofilm formation.     

Roles of bacteria in the bark beetle holobiont – how do they shape this forest pest?

Bark beetles are well‐known forest pests, some species inducing massive attacks on trees, resulting in the devastation of entire woodlands. Bark beetles are associated with microorganisms, forming an entity known as ‘holobiont’. Beetles and fungi are the best‐studied members of this multipartite symbiosis. However, recent studies have shown that bacteria may play important roles in the bark beetle holobiont, such as providing certain nutrients, promoting the growth of beneficial fungi, detoxifying the environment by lowering the levels of phenolic compounds synthesised by the host tree or by inhibiting the growth of antagonistic fungi whereas some bacterial symbionts have the potential to kill beetles under certain conditions. Therefore, bacteria probably greatly affect the life cycle of bark beetles; hence, more research is needed to clarify the extent to which a bacterial associate is implicated in a bacterial bark beetle symbiosis and how much it determines host's performance. This review summarises all of the known activities of bacteria present in the bark beetle holobiont, indicates some important gaps in the knowledge of this symbiosis and provides some guidance for overcoming the difficulties in investigating this relationship in future studies.     

Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity

A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.