Occurrence and ultrastructural characterization of bacteria in association with and isolated from Azolla caroliniana.

The occurrence and ultrastructure of bacteria in leaf cavities of symbiotic Azolla caroliniana were examined by transmission electron microscopy. Bacteria were observed in all leaf cavities of Azolla cultures. Five ultrastructurally distinct types of bacteria were observed in each individual leaf cavity. Features used to characterize the bacteria included morphology, cell wall structure, and cytoplasmic organization. At least one gram-positive and as many as four gram-negative types of bacteria reside in leaf cavities of A. caroliniana. The morphological and ultrastructural characteristics of the gram-positive bacterium suggest that it is an Arthrobacter sp. The gram-negative bacteria could not be cultured; therefore, they have not been classified further. Bacterial cell shape and cell wall structure were similar in leaf cavities of different ages, but cell size and cytoplasmic composition varied. The relative contributions of each bacterial type to the total community within individual leaves was determined. Ultrastructural characteristics of bacterial isolates cultured from A. caroliniana in a free-living state were also examined.     

The occurrence of coryneform bacteria in the leaf cavity of Azolla

Coryneform bacteria were found associated with the nitrogen fixing blue-green alga, Anabaena azollae in the leaf cavity of Azolla caroliniana. Plate counts indicated ca. 7,400±1,900 bacterial cells per mature leaf cavity or approximately 1 bacterial cell for every algal cell. No other type of bacterium was found in these cavities.     

Immunocytochemical localization of nitrogenase in bacteria symbiotically associated with Azolla spp.

In situ immunogold labeling and transmission electron microscopy were used to detect nitrogenase in bacteria (bactobionts) symbiotically associated with leaf cavities of Azolla caroliniana and Azolla filiculoides. In A. caroliniana, the Fe protein of the nitrogenase complex was detected in a subset of the distinct bactobiont types present in leaf cavities of all ages. Similar results were obtained for the bactobionts of A. filiculoides with antisera against both the Fe and MoFe subunits of nitrogenase.     

Immunocytochemical localization of nitrogenase in bacteria symbiotically associated with Azolla spp.

In situ immunogold labeling and transmission electron microscopy were used to detect nitrogenase in bacteria (bactobionts) symbiotically associated with leaf cavities of Azolla caroliniana and Azolla filiculoides. In A. caroliniana, the Fe protein of the nitrogenase complex was detected in a subset of the distinct bactobiont types present in leaf cavities of all ages. Similar results were obtained for the bactobionts of A. filiculoides with antisera against both the Fe and MoFe subunits of nitrogenase.     

Sub-cellular distribution of nitrogen compounds inAzolla andAnabaena by ESI and EELS analysis

Summary The sub-cellular localization of some nitrogen compounds within the leaf cavities ofAzolla filiculoides Lam. was obtained by means of electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The analyses were performed on ultrathin unstained sections of differentAzolla leaf cavities which contain epidermal hairs,Anabaena azollae Strasb. and bacteria. Net nitrogen distributions were visualized by image analysis, and nitrogen peaks were evidenced in spectra recorded in the same areas. Different distributions of nitrogen compounds were observed within the leaf cavities along the stem, in particular inside the epidermal hairs ofAzolla and the vegetative cells and heterocysts ofA. azollae.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

Thermohydrogenium kirishiense gen. nov. and sp. nov., a new anaerobic thermophilic bacterium

Six strains of a new anaerobic thermophilic non-sporeforming bacterium were isolated in pure culture from industrial yeast biomass. Cells were rod-shaped (0.4–0.8×1.0–11.0 m), non-motile. They stained gram-negative, but outer membrane was not present. The growth occurred between 45–75 °C, the optimal temperature is 65°. Optimal pH value was 7.0–7.4. The bacterium utilized for growth several sugars, starch and yeast extract. The best source of nitrogen was peptone. The main fermentation products of glucose were ethanol, acetate, H₂ and CO₂. As minor products isopropanol, butanol, butyrate and lactate were found. Glucose was metabolized via the Embden-Meyerhoff pathway. Cytochromes and quinones were not found. DNA-base composition was 33.2–34.0 mol%. The DNA-DNA hybridization and 5S rRNA nucleotide sequences showed distantly related of isolated stains to phenotypical similar bacteria. It was proposed to consider the isolated bacterium as Thermohydrogenium kirishiense gen. nov. and sp. nov.     

Localisation of the amathamide alkaloids in surface bacteria of Amathia wilsoni Kirkpatrick, 1888 (Bryozoa: Ctenostomata)

The marine bryozoan Amathia wilsoni contains several brominated secondary metabolites, the alkaloid amathamides A–F. Energy dispersive X-ray analysis coupled with scanning electron microscopy was used to localise bromine in sections of Amathia wilsoni. Bromine concentrations higher than background levels were only found on the surface of the bryozoan and not within any of the different internal cell types. The correlation between bromine levels and a rod-shaped bacterium, ubiquitous to the tip region, points to the bacterium being closely associated with the range of amathamides found in Amathia wilsoni.     

BRYOPHYTE LEAF EPIFLORA: AN SEM AND TEM STUDY OF POLYTRICHUM COMMUNE HEDW.

Epiphytes on leaves of Polytrichum commune Hedw. were observed and studied with SEM and TEM. A unicellular green alga, a fungus, and a rod-shaped bacterium were present on both leaf surfaces. Culturing experiments led to the identification of the alga as Chlorococcum sp. and the fungus as Trichoderma viride Lk. ex Fr. (Deuteromycetes). No attempt was made to culture and identify the bacterium. Terminal cells of the leaf lamellae form a ventral pseudo-epidermis which provides a microhabitat for the epiphytes. Fungal hyphae were seen intracellularly as well as penetrating cell walls between lamellar cells. The alga and bacterium were not observed intracellularly. Chlorococcum sp. displayed a distinctive wall morphology which consisted of short (0.3–0.5 μm), blunt papillae. The rod-shaped bacterium may be similar to the Agrobacterium-like isolates reported recently from Polytrichum commune. Host-epiphyte associations relative to potential beneficial and/or pathological aspects of growth require further investigation.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

Nematicidal and allelopathic potential of Argemone mexicana,a tropical weed

Argemone mexicana L. (Papaveraceae), a tropical annual weed, is phytotoxic to many crop species. This study was designed to examine the allelochemical and nematicidal potential of A. mexicana and to better understand the role of this weed in the ecosystem. A methanol-soluble extract of the leaf material caused greater juvenile mortality of Meloidogyne javanica than did ethyl acetate or hexane extracts indicating the polar nature of the toxins. Decomposing tissues of A. mexicana in soil at 50 g kg^–1 were highly deleterious causing 80% mortality of tomato plants. At 10 g kg^–1 plant growth was enhanced, while at 30 g kg^–1 plant growth was substantially retarded. M. javanica population densities in the rhizosphere and in roots, and gall formation were significantly suppressed when 10, 30 or 50 g kg^–1 A. mexicana was allowed to decompose in the soil. To establish whether decomposition was necessary to produce phytotoxic symptoms, or whether the shoot extract alone could interfere with plant growth, an aqueous shoot extract was applied to soil. Whereas a 50% extract promoted plant growth, a 100% (100 g/500 mL distilled water) concentration significantly reduced plant height, and fresh weights of shoot and root. In general, decomposing plant material caused greater phytotoxicity compared to the aqueous extract. Addition of N as NH₄NO₃ partially alleviated the phytotoxic action of A. mexicana,and also reduced severity of root-knot disease. Adding Pseudomonas aeruginosa to soil amended with A. mexicana resulted in decreased density of M. javanicain the rhizosphere and in tomato roots, suppressed galling rates and enhanced plant growth.     

Fructose-induced dark germination ofAnabaena akinetes

Standard sporulation medium was used to produce akinetes from vegetative cells ofAnabaena variabilis and culturedAnabaena azollae isolated fromAzolla mexicana. Singlecelled, axenic akinetes were incubated on BG-11 medium supplemented with 1% Bacto-agar. Three different sources of combined nitrogen–KNO₃ (5 mM), NH₄Cl(5 mM), and glutamine (5 mM)–were added singly or in combination with fructose (50 mM) to the BG-11 plates. The akinetes were incubated under continuous light (5500 lux) or in the dark at 25±2°C. Akinetes of both species germinated in the dark when BG-11 was supplemented with fructose. Akinetes incubated in the dark on plates containing NH₄Cl and fructose germinated less than those germinated in the presence of fructose alone. Akinetes of both species germinated in the light.     

Spheroplast induction inAnabaena variabilis Kütz andA. azollae stras

Summary Spheroplasts were obtained by lysozyme treatment of 48 hour (4– 8cells) akinete germlings of the cultured cyanobacteriaAnabaena variabilis andA. azollae originally isolated from the leaf cavity of the fernAzolla pinnata. The osmotic stabilizer was 0.5 M sucrose. At least 50% of the cells in a short filament became spheroplasts after 1–4 hours in lysozyme (1 mg/ml) in incubation medium at 34 °C, with greater than 75% viability after 2 hours. The spheroplasts were osmotically fragile and showed intense chlorophyll autofluorescence in UV light. In phase microscopy, treated cells appeared larger, became spherical and lost some of their optical refraction. Transmission electron microscopy confirmed the loss of the peptidoglycan layer and the partial remains of the outer membrane after lysozyme exposure. We previously obtained protoplasts ofAzolla fern leaf cells so that we now can study the recognition sites in both members of theAzolla/Anabaena nitrogen fixing symbiosis during cell wall degradation and regeneration.     

Isolation and molecular characterization of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> from coffee plants in Costa Rica

Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica’s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as “crespera” disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 μm in width and 1.5 to 3.0 μm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.     

Taxonomic re-evaluation of the Azolla genus in Portugal

ABSTRACT

The Portuguese and the European Flora refer to the presence of two or three Azolla species in Portugal: A. filiculoides Lam., A. caroliniana Willd. and/or Azolla mexicana Presl., the latter included in the last edition of Flora Europaea. In the present work, the taxonomy of Azolla species is reviewed using the two most important characters that can distinguish between these two/three species: papillae in the dorsal leaf lobe and perine architecture of the megaspore apparatus. Other characteristics, such as the hyaline border cells of the dorsal leaf lobes and the number of glochidia septa in microsporangium massulae, are also used. All the Azolla specimens, collected from several locations in Portugal, were identified as Azolla filiculoides Lam. This identification disagrees with previous published reports on Azolla taxonomy in Portugal as well as with herbarium identification.     

Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell

Cell shape in most eubacteria is maintained by a tough external peptidoglycan cell wall. Recently, cell shape determining proteins of the MreB family were shown to form helical, actin-like cables in the cell. We used a fluorescent derivative of the antibiotic vancomycin as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria. In the rod-shaped bacterium B. subtilis, synthesis of the cylindrical part of the cell wall occurs in a helical pattern governed by an MreB homolog, Mbl. However, a few rod-shaped bacteria have no MreB system. Here, a rod-like shape can be achieved by a completely different mechanism based on use of polar growth zones derived from the division machinery. These results provide insights into the diverse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.     

“Candidatus Paraholospora nucleivisitans”, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological–botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.     

The response of rice to the growth and nitrogen fixation inAzolla caroliniana andAzolla pinnata at varying rates of phosphate fertilization

The response of rice toAzolla caroliniana, newly introduced in India, was compared with the reponse to the local isolate ofAzolla pinnata at varying rates of phosphate fertilizer (4.4–8.8 kg P ha^–1) during a wet and a dry season.Fresh weight, dry weight and fixed N were more for both species 21 DAI (days after inoculation) than 14 DAI, but acetylene reduction activity (ARA) was higher 14 DAI than 21 DAI. Dry weight of Azolla and fixed N were less 14 DAI forA. caroliniana than forA. pinnata during the wet season. Twenty-one DAI, fresh weight ofA. caroliniana was 62.1 and 27.6% higher than that ofA. pinnata during the wet and dry season, respectively. However, dry weight and fixed N were more 21 DAI inA. caroliniana than inA. pinnata during only the wet season. The ARA was higher inA. caroliniana both 14 and 21 DAI, irrespective of season. The presence of either species in the rice field increased grain yield, straw yield, number of panicles m^–2, number of grains per panicle and reduced percentage sterility during both the wet and the dry season. Phosphate application significantly increased fresh weight, dry weight, ARA and fixed N for both species as well as grain and straw yields of rice. The responses to phosphate fertilizer were similar for both Azolla species and for rice grown with either one of the Azolla species.     

Gram positivity induced in Escherichia coli by chloroform-methanol treatment

Summary Extraction of lipid by chloroform-methanol from Escherichia coli induces Gram positivity in the cells of this bacterium. It has been observed that approximately 1.5 times more lipid is extracted from Escherichia coli than from Staphylococcus aureus by this treatment. An increase of 1.5–1.8 fold in the retention of dye has been evident in the case of S. aureus where as this was 70–90 fold in the case of E. coli, which is 1/4th of the normal retention by S. aureus. The more lipid present in the cell wall or cell membrane, the less retention of dye seems to be exhibited by the bacteria. This also substantiates our model for Gram reaction presented in a previous communication.