Fungal Assciations: II. Cultural Studies on Rhizoctonia solani Kuhn, Fusariutn solani Snyder and Hansen, and Other Fungi, and Their Interactions

Several types of interaction between Rhizoctonia solani, Fusariumsolani, and Phoma foveata were found when these fungi were grownon potato-dextrose agar. After being used by Rhizoctonia a potatomash medium gave better growth of Rizoctonia and Fusarium thanit did when the medium was initially used by Fusarium; and thiswas so whether the reaction of the spent medium was readjustedor not. It is suggested that potato mash medium used by Fusariumcontains a thermostable factor(s) affecting the subsequent growthof Rhizoctonia or Fusarium. The range of pH values suitable for Rhizoctonia growth was narrowerthan that for Fusarium, optimum values being approximately 5•9for the former and 7•8 for the latter. In mixed culturesof the two fungi on potato-dextrose agar adjusted to differentpH values, the fungus for which the reaction of the medium wasmore suitable usually became visually predominant after sometime. A study of various carbon sources showed that poor growth ofRhizoctonia was obtained when pectin was used as the sole sourceof carbon. On a pectin-agar medium, the rate of growth of aRhizoctonia colony increased on the sector which lay towardsan adjacent Fusarium colony; also, after the two fungi camein contact, there was more rapid growth of Rhizoctonia roundthe Fusarium colony than elsewhere. On a synthetic liquid mediumwith pectin as the carbon source better Rhizoctonia growth wasobtained when Fusarium-spent medium was added to it than whenRMzoctoma-spent medium was added. Rhizoctonia showed partial deficiencies in thiamine, biotin,and inositol. Both the extract of Fusarium mycelium, grown onvitamin-free medium, and the Fusarium-spent medium, stimulatedthe growth of Rhizoctonia on vitamin-free medium.     

The Fungus-culturing Behavior of Ants

A colony of attine ants begins with a recently fecundated femalecarrying hyphae from the parental garden in a pellet in an infrabuccalpocket. All future food of the colony will be derived from thisnucleus. She digs a cavity in the ground, ejects this pelletand manures it with her liquid excrement. As the hyphae proliferate,eggs are laid on them and the colony is launched. She continuallylicks both the hyphae and the brood. Thus, both salivary andanal excretions play a vital role in the beginning of a colonyand this pattern is repeated by the resulting workers. About60–65% of them in Atta are the minima and these are intimatelyinvolved in brood and fungus care. Their excretions are disproportionatelylarge. About 1/3 of the workers in Atta are 4–6 mm mediaand these cut and prepare the substrate. The 7–9 mm maximasizes and the soldiers (over 9 mm) are less directly involvedin culturing the fungus. The effectiveness of fungus culturing is shown by the rapidbuild-up of gardens. The ants maintain their garden despitesurrounding contamination after a fragment with ants is introducedto a plate of sterile nutrient agar.     

Studies on Pyricularia oryzae Cav. in Sierra Leone: Morphological and Physiological Variability of Some Isolates

The morphological and physiological variability of six isolatesof Pyricularia oryzae, the causal organism of rice blast disease,were investigated. The rate of growth, colony characters, andtime of sporulation were found to vary with the different isolatesthough not by appreciable amounts. Each of the isolates showedconsistently better growth on Takahashi's B medium than on Czapek-Dox'smedium although the growth trend was the same in both media.The colony characters developed by each isolate are not dependenton the medium on which it is growing—a pointer to thefact that such characters may be genetically controlled. Germinationwas faster in distilled water than in 2 per cent agar. All theisolates produced appresoria in vivo and in vitro; those producedin vivo were, however, considerably larger than those producedin vitro. On the basis of appresorial types, the isolates werefound to fall into two physiological races—smooth-walledand rough-walled. Each isolate produced consistently only oneappresorial type in vitro from the apical or basal cell of theconidium. The utilization of carbon and nitrogen compounds variedfrom one isolate to the other, carbon compounds being generallybetter utilized than nitrogen compounds. Pyricularia oryzaecan metabolize a wide range of carbon compounds. However, mannose,sucrose, glucose, fructose, and maltose proved to be most suitablecarbon sources. The variations in the utilization of the variouscarbon and nitrogen compounds seem to reflect inherent biochemicaland physiological differences among the isolates.     

Improvement of human melanoma colony formation in soft agar using boiled instead of autoclaved agar

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.     

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

Dark Formation of Chlorophyll in Cultured Tobacco Cells

Dark accumulation of chlorophyll was demonstrated in calluscells of Nicotiana glutinosa. The chlorophyll content of dark-growncallus cells was dependent on the agar concentration in theculture medium: the content was significantly higher at lowerconcentrations of agar. Some properties of the dark-formed chlorophyllare described. (Received June 25, 1983; Accepted December 2, 1983)     

Inhibition of agar colony formation by partially purified granulocyte extracts (chalone)

Granulocytic extracts (GE) of different sources, presumably containing the granulocytic chalone, were prepared in different laboratories and purified to some extent. They specifically inhibited the formation of granulocyte and macrophage colonies in agar. The effect was however most pronounced on granulocyte and mixed granulocyte-macrophage colonies, and less on macrophage types. Addition of GE to bone marrow cells at the time of plating in agar, as well as short incubation of the cells together with GE prior to plating, inhibited subsequent colony formation. The inhibitory effect could easily be reversed by washing the cells with an excess of medium prior to plating during the first hour of preincubation, but not after five hours. Increasing the doses of colony stimulating activity (CSA) (at low doses of GE) released the inhibitory effect, but not at high doses of GE. The inhibitory effect of GE on colony formation was dose dependent down to almost 100% inhibition. No apparent cytotoxic effect of GE on bone marrow cells could be found and lymphoblastic cells were not inhibited. Extracts containing a specific inhibitor of erythropoiesis (EIF) stimulated myelopoietic colony formation in agar.     

Optimum conditions for growth of cyanobacteria on solid media

The colony forming ability of single cells or very short filaments of 7 strains of cyanobacteria was tested on media solidified by agar or by agar substitutes (Gel Gro or Gel Rite). In addition, the effect of various methods for preparation of agar media on colony forming ability was measured. High efficiency colony formation for most of the strains required that the agar be autoclaved separately from the salts in the medium. The addition of thiosulfate, but not buffer, significantly increased the plating efficiency of most strains.     

Growth kinetics by scanning of granulocytic cell colonies in glass capillaries.

Pure granulocytic colonies were cultivated from mouse bone marrow cells in agar contained in glass capillary tubes using mouse embryo conditioned medium as colony stimulating activity. A random distribution of colonies along the agar gels was achieved under controlled conditions. Only 3 capillaries were needed for a coefficient of variation around 5% provided at least 104 cells were seeded per capillary. The daily growth of single colonies within an agar capillary was followed, using the light scattering properties of the colonies for automatic scanning. The position of the colonies in the capillary greatly affected the scan signal; the consequences of positional changes were studied in detail. Using the mean peak height as growth parameter, the onset of measureable granulocytic colony growth was found between day 2 and 3, the maximum colony size was reached between day 7 and 9, after which the colonies decayed. Other parameters such as colony count and total peak are were determined and their sinificance discussed.     

Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous γ-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after busulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells (“committed” stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.     

Zoospore Structure and Colony Formation in Pediastrum Spp. and Hydrodictyon reticulatum (L.) Langerheim

The process of daughter colony formation in Pediastrum biradiatumMeyen, P. tetras (Ehren-berg) Ralfs, P. duplex Meyen, and Hydrodictyonreticulatum (L.) Lagerheim has been investigated using lightmicroscopy and electron microscopy of thin sections. The precisedisposition of the organelles confers upon the zoospores ofthese algae a special symmetry of a type unusual for the Chlorophyta.Apart from this, the detailed ultrastructure of the zoosporeand young vegetative cells resembles that of other green algaepreviously investigated. A common sequence of events has beenfound to occur during colony formation in both Pediastrum andHydrodictyon, with a correlation between zoospore symmetry andthat of the colony produced. The changes involved in the transitionfrom a motile to a non-motile cell are considered, and appearto have features in common with this process in some other greenalgae. The early stages in wall deposition involve the simultaneousformation of two distinct layers and serve to stick togetherthe cells which by this time have collectively assumed the basicmorphological characteristics of the mature coenobia.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10⁶ ml⁻¹) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10⁶ ml⁻¹). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10⁶ ml⁻¹) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10⁶ ml⁻¹) or tetraclycline (1.5×10⁶ ml⁻¹). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10² ml⁻¹) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO₄ to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.     

Structure of the Cells, Cell Division and Colony Formation in the Diatoms Isthmia enervis Ehr. and I. nervosa Ktz

The two common species of Isthmia have been studied mainly byscanning electron microscopy. The occurrence of differing cellshape in the same species is an extremely unusual feature amongstdiatoms. This is coupled with heteropolar valves. The formationof colonies and the peculiar attachment of cells to one anotheris recorded. A particularly intricate attachment of the firstgirdle band (valvocopula) to the valve is recorded. Isthmia, diatom, cell division, colony formation     

Scanning Electron Microscopy of Bacterial Colonies

A technique is described for observing bacterial colony growth. Bacillus cereus, B. subtilis, and B. cereus var. mycoides were grown on strips of dialysis membrane layered on nutrient agar. Microcolonies of the organisms on strips were fixed in Formalin vapor in situ; the strips then were removed from the agar and secured to scanning microscope specimen stubs without markedly disturbing the cellular arrangement. Scanning electron micrographs clearly depict morphology of individual cells, as well as the spatial orientation of cells within the colony. This technique is reproducible, adaptable, and simple.     

Mitosis, Cytokinesis and Colony Formation in Pediastrum boryanum

Uninucleate cells of Pediastrum become multinucleate by a seriesof synchronous mitoses. Mitotic nuclei are enclosed by a perinuclearenvelope of endoplasmic reticulum. Cytoplasmic cleavage of themultinucleate cells leads to the production of uninucleate,biflagellate zoospores (zooids) which are subsequently releasedinto a lenticular vesicle through a rupture in the outer layerof the parental cell wall. Within the vesicle, presumably derivedfrom part of the inner layer of parental wall, the zooids swarmactively before aggregating in a planar array. Bands of microtubulesunderlie the plasmalemma of the zooids which, when the zooidsaggregate, are usually coplanar with the newly formed colony.The role of microtubules in patterned colony formation and inthe development of the characteristic horns on peripheral cellsof colonies of Pediastrum is discussed.     

Formation of colonies of P-388 leukemic cells in semisolid agar

Substrain P-388/A2 adapted to cultivation of agar gel in the form of compact colonies was obtained as a result of alternating passages of cells of ascitic mouse leukemia P-388 in the primary semifluid agar culture and in the mouse abdominal cavity. The efficacy of colony formation and the size of the colonies depended on the initial density of the cell suspension. In case of introduction into the agar medium of 100 cells/ml the planting efficacy constituted 20%, and the number of cells in the colony by the 8th--10th days of cultivation reached 13 000.     

Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue

Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.     

An efficient method of selecting photoautotrophic cells from cultured heterogeneous cells

A new and efficient method was demonstrated for the establishmentof photoautotrophic cultures of plant cells. Leaf segments ofAtropa belladonna, Datura stramonium and Hyoscyamus niger wereinoculated on sugar-free Linsmaier-Skoog agar medium then aeratedwith 1% CO₂ enriched air under 3,000 to 5,000 lux of illumination.Under these regulated conditions we could select photoautotrophicgreen cells efficiently, and these cells subsequently have grownwell under photoautotrophic conditions. ¹Department of Horticulture and Agriculture, Faculty of Agriculture,Kobe University, Kobe 657, Japan (Received June 12, 1980; )