Ultrastructural and microbial analyses of cellulose degradation in leaf-cutter ant colonies.

Leaf-cutter ants (Atta and Acromyrmex) use fresh leaves to cultivate a mutualistic fungus (Leucoagaricus gongylophorus) for food in underground gardens. A new ant queen propagates the cultivar by taking a small fragment of fungus from her parent colony on her nuptial flight and uses it to begin her own colony. Recent research has shown that the ants' fungus gardens are colonized by symbiotic bacteria that perform important functions related to nitrogen fixation and have been implicated in contributing to plant biomass degradation. Here, we combine bacterial culturing in several media for counts and identification using the 16S rRNA gene with electron microscopy to investigate the process of cellulose degradation in the fungus garden and refuse dumps, and to assess the potential role of symbiotic bacteria. We show through electron microscopy that plant cell walls are visibly degraded in the bottom section of fungus gardens and refuse dumps, and that bacteria are more abundant in these sections. We also consistently isolated cellulolytic bacteria from all sections of fungus gardens. Finally, we show by culture-dependent and electron microscopy analysis that the fungus garden pellets carried by recently mated queens are colonized by fungus garden-associated bacteria. Taken together, our results indicate that cellulose is degraded in fungus gardens, and that fungus garden bacteria that may contribute to this deconstruction are vertically transmitted by new queens.

Production of selenium nanoparticles in Pseudomonas putida KT2440.

Selenium (Se) is an essential element for the cell that has multiple applications in medicine and technology; microorganisms play an important role in Se transformations in the environment. Here we report the previously unidentified ability of the soil bacterium Pseudomonas putida KT2440 to synthesize nanoparticles of elemental selenium (nano-Se) from selenite. Our results show that P. putida is able to reduce selenite aerobically, but not selenate, to nano-Se. Kinetic analysis indicates that, in LB medium supplemented with selenite (1 mM), reduction to nano-Se occurs at a rate of 0.444 mmol L-1 h-1 beginning in the middle-exponential phase and with a final conversion yield of 89%. Measurements with a transmission electron microscope (TEM) show that nano-Se particles synthesized by P. putida have a size range of 100 to 500 nm and that they are located in the surrounding medium or bound to the cell membrane. Experiments involving dynamic light scattering (DLS) show that, in aqueous solution, recovered nano-Se particles have a size range of 70 to 360 nm. The rapid kinetics of conversion, easy retrieval of nano-Se and the metabolic versatility of P. putida offer the opportunity to use this model organism as a microbial factory for production of selenium nanoparticles.

Surface structures involved in plant stomata and leaf colonization by shiga-toxigenic Escherichia coli o157:h7.

Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC. The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE. This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection.

Ultrastructure of the wild rice Oryza grandiglumis (Gramineae) in Costa Rica

Oryza grandiglumis is a wild species of rice endemic to tropical America. This species was first found in 1998 in the wetlands of Caño Negro, located in the northern part of Costa Rica. Twenty five plants of O. grandiglumis were processed for scanning electron microscope. An ultrastructural description of the leaf blade, ligule, auricles, spikelet and caryopsis, with an emphasis on structures of taxonomic value. The leaf blade has a characteristic cuticular wax pattern, composed of dense rod-like structures, and is surrounded by papillae, zipper-like silica cells, abundant bulky prickle trichomes, and hooked trichomes. The blade's edge has three rows of hooked prickle trichomes of various sizes. The auricles wrapped the culm, with long attenuated trichomes at the edges; the base was surrounded by oblong cells. The ligule is a blunt membrane covered by short prickle trichomes. Spikelet morphology is characteristic of the Poaceae family, but the sterile lemmas were nearly as long as the fertile lemmas, and they have an unique crown-like structure of lignified spines between the rachilla and the fertile lemmas. Comparison with Brazilian specimens of O. grandiglumis revealed little differences in the ultrastructural characteristics.

El arroz silvestre Oryza grandiglumis es endémico de América. Se localiza en la zona norte de Costa Rica, principalmente en el humedal de Caño Negro y del Río Medio Queso. Es una planta vigorosa y grande. Su nombre deriva del gran tamaño de las lemas estériles (glumas). Presentamos una descripción ultraestructural de la lámina foliar, lígula, aurículas, espiguilla y cariópside, con énfasis en las estructuras de valor taxonómico, usando el microscopio electrónico de barrido. La lámina foliar se caracteriza por presentar un patrón de cera cuticular en forma de densos bastoncillos. Presenta estomas rodeados de papilas, células de sílice en forma crenada, varias formas de papilas de cera, distribuidas en forma muy regular, y tricomas espinosos abultados en la base y tricomas ganchudos. En el borde de la lámina hay tres hileras de tricomas espinosos ganchudos de diferente tamaño. Las aurículas son envolventes y los bordes presentan tricomas atenuados largos cubiertos en la base por células alargadas. La lígula es truncada y cubierta de tricomas espinosos pequeños. La morfología de la espiguilla es algo similar a las de las otras especies de la familia Poaceae, pero las lemas estériles son casi del mismo tamaño que las fértiles. Además se observó entre la raquilla y las lemas fértiles una corona de espinas lignificadas. Ultraestructuralmente, esta población es similar a las brasileñas.

Ultrastructure of the wild rice Oryza grandiglumis (Gramineae) in Costa Rica.

Oryza grandiglumis is a wild species of rice endemic to tropical America. This species was first found in 1998 in the wetlands of Caño Negro, located in the northern part of Costa Rica. Twenty five plants of O. grandiglumis were processed for scanning electron microscope. An ultrastructural description of the leaf blade, ligule, auricles, spikelet and caryopsis, with an emphasis on structures of taxonomic value. The leaf blade has a characteristic cuticular wax pattern, composed of dense rod-like structures, and is surrounded by papillae, zipper-like silica cells, abundant bulky prickle trichomes, and hooked trichomes. The blade's edge has three rows of hooked prickle trichomes of various sizes. The auricles wrapped the culm, with long attenuated trichomes at the edges; the base was surrounded by oblong cells. The ligule is a blunt membrane covered by short prickle trichomes. Spikelet morphology is characteristic of the Poaceae family, but the sterile lemmas were nearly as long as the fertile lemmas, and they have an unique crown-like structure of lignified spines between the rachilla and the fertile lemmas. Comparison with Brazilian specimens of O. grandiglumis revealed little differences in the ultrastructural characteristics.

Ultrastructure of Oryza glumaepatula, a wild rice species endemic of tropical America

Oryza glumaepatula is a perennial wild rice species,endemic to tropical America, previously known as the Latin American race of Oryza rufipogon .In Costa Rica, it is found in the northern region of the country, mainly in the wetland of the Medio Queso River, Los Chiles, Alajuela. It is diploid, of AA type genome and because of its genetic relatedness to cultivated rice it is included in the O.sativa complex. We describe the ultrastructure of leaf blade, spikelet, ligule and auricles. Special emphasis is given to those traits of major taxonomic value for O.glumaepatula and to those characters that distinguish this species from O. rufipogon and O. sativa . O. glumaepatula has a leaf blade covered with tombstone-shaped, oblong and spheroid epicuticular wax papillae. It has diamond-shaped stomata surrounded by spherical papillae, rows of zipper-like silica cells, bulky prickle trichomes of ca .40 µm in length and small hirsute trichomes of ca. 32 µm in length.The central vein is covered with large,globular papillae of ca. 146 µm in length,a characteristic that distinguishes this species from O.rufipogon and O.sativa. The border of the leaf blade exhibits a row of even-sized bulky prickle trichomes of ca .42.5 µm in length.Auricles have attenuated trichomes of ca .5.5 mm in length on the edges and small bicellular trichomes of 120 µm in length on the surface.The ligule has a large number of short attenuated trichomes on its surface of 100 µm in length.These latter two traits have important taxonomic value since they were found in O.glumaepatula but not found in O.sativa or in O.rufipogon . The spikelet has the typical morphology of the Oryza genus. Fertile lemmas have abundant spines, a trait shared with O.rufipogon but not with O.sativa. The sterile lemmas are wing-shaped with serrated borders,a characteristic that distinguishes this species from O. rufipogon and O.sativa. All the ultrastructure characters observed in O.glumaepatula from Costa Rica are also common to the specimens from Brazil

O.glumaepatula es una especie de arroz silvestre perenne, endémica de América Tropical, conocida anteriormente como la O.rufipogon americana. En Costa Rica se le encuentra en la zona norte del país, principalmente en los humedales del río Medio Queso, Los Chiles, Alajuela. Es una especie diploide de genoma AA y por su cercanía genética con el arroz cultivado se le incluye en el complejo de O.sativa. El propósito de este trabajo es realizar una descripción ultraestructural de la morfología de la lámina foliar, espiguilla, lígula y aurículas. Se enfatizó en aquellas características de valor taxonómico para O.glumaepatula , destacando las características que la distinguen de O.rufipogon y O.sativa. Esta especie presenta la lámina foliar cubierta de papilas de cera en forma de lápida, alargadas y esferoides, tiene estomas romboidales rodeados de papilas esféricas, hileras de células de sílice de forma crenada, tricomas espinosos abultados de ca.40 m m y tricomas hirsutos pequeños de ca.32 m m. La vena central se encuentra cubierta de papilas globulares de ca.146 m m, característica que la distingue de O.rufipogon y O.sativa. El borde de la lámina presenta una hilera de tricomas espinosos abultados de tamaño homogéneo de ca. 42.5 m m. Las aurículas rizoides tienen tricomas atenuados largos en los bordes de ca. 5.5 mm y tricomas bicelulares en la superficie de ca.120 m m, esta última característica es de valor taxonómico. La lígula presenta en su superficie gran cantidad de tricomas atenuados cortos de ca.100 m m. La espiguilla presenta la morfología típica del género, las lemas fértiles tienen espinas abundantes y largas, característica que la comparte con O.rufipogon pero no con O.sativa. Las lemas estériles son aladas, de bordes dentados, característica que la distingue de O.rufipogon y de O.sativa. Las características ultraestructurales observadas en O.glumaepatula son comunes con la O.glumaepatula de Brasil

Ultrastructure of Oryza glumaepatula, a wild rice species endemic of tropical America.

Orv'za gluniaepatula is a perennial wild rice species, endemic to tropical America, previously known as the Latin American race of Orrza rufipogon. In Costa Rica, it is found in the northern region of the country, mainly in the wetland of the Medio Queso River, Los Chiles, Alajuela. It is diploid, of AA type genome and because of its genetic relatedness to cultivated rice it is included in the O. saliva complex. We describe the ultrastructure of leaf blade, spikelet, ligule and auricles. Special emphasis is given to those traits of major taxonomic value for O. glumaepatula and to those characters that distinguish this species from O. rufipogon and O. sativa. O. glumaepatula has a leaf blade covered with tombstone-shaped, oblong and spheroid epicuticular wax papillae. It has diamond-shaped stomata surrounded by spherical papillae, rows of zipper-like silica cells, bulky prickle trichomes of ca. 40 microm in length and small hirsute trichomes of ca. 32 tpm in length. The central vein is covered with large, globular papillae of ca. 146 microm in length, a characteristic that distinguishes this species from O. rufipogon and O. sativa. The border of the leaf blade exhibits a row of even-sized bulky prickle trichomes of ca. 42.5 microm in length. Auricles have attenuated trichomes of ca. 5.5 mm in length on the edges and small bicellular trichomes of 120 microm in length on the surface. The ligule has a large number of short attenuated trichomes on its surface of 100 microm in length. These latter two traits have important taxonomic value since they were found in O. glumaepatula but not found in O. sativa or in O. rufipogon. The spikelet has the typical morphology of the Oryza genus. Fertile lemmas have abundant spines, a trait shared with O. rufipogon but not with O. sativa. The sterile lemmas are wing-shaped with serrated borders, a characteristic that distinguishes this species from O. rufipogon and O. sativa. All the ultrastructure characters observed in O. glumaepatula from Costa Rica are also common to the specimens from Brazil.

[A new species of Euglena (Euglenozoa: Euglenales) isolated from extreme environments in "boiling mudflats" of Rincón de la Vieja volcano, Costa Rica]. / Una nueva especie de Euglena (Euglenozoa: Euglenales) aislada de ambientes extremófilos en las Pailas de Barro del Volcán Rincón de la Vieja, Costa Rica.

A new species of euglena isolated from a hot and acid mud pool located in Las Pailas de Barro, Volcán Rincón de la Vieja, Costa Rica is described. This species inhabits hot and acid environments. Euglena pailasensis sp. nov. main features are: the absence of flagella, the presence filaments like "pilis", the presence of chloroplasts with pyrenoids crossed by several tylakoids, and acid and heat tolerance. Molecular phylogeny studies using 18S rDNA and Gap C genes indicated that the new species is related to E. mutabilis. Its taxonomic characters based on morphology, biology and sequence of the 18S rDNA and Gap C genes are discussed and compared with other closely related species of the genus.

[Ultrastructural description of Euglena pailasensis (Euglenozoa) from Rincón de la Vieja volcano, Guanacaste, Costa Rica]. / Descripción ultraestructural de Euglena pailasensis (Euglenozoa) del Volcán Rincón de la Vieja, Guanacaste, Costa Rica.

The euglenoids are unicellular eukaryotic flagellates living in a diversity of soils and aquatic environments and ecosystems. This study describes the ultrastructure of an euglenoid isolated from the surface of a boiling mud pool with temperatures ranging from 38 to 98 degrees C and pH 2 - 4. The hot mud pool is located in Area de Pailas de Barro, Las Pailas, Rincón de la Vieja Volcano, Guanacaste, Costa Rica. The morphological characterization of the Euglena pailasensis was performed by SEM and TEM. It was determined that, although the euglenoid was obtained from an extreme volcanic environment, the general morphology corresponds to that of a typical member of Euglena of 30-45 microm long and 8-10 microm wide, with membrane, pellicle, chloroplasts, mitochondria, nucleus, pigments and other cytoplasmic organelles. E. pailasensis is delimited by a membrane and by 40 to 90 pellicle strips. It was observed up to 5 elongated chloroplasts per cell. The chloroplast contains several osmiophilic globules and a pyrenoid penetrated by few thylakoid pairs. The nutritious material is reserved in numerous small paramylon grains located at the center of the cell, mitocondria are characterized by the presence of crests in radial disposition toward the interior of the lumen. It was also observed around the external surface "pili" like filaments originating from the pellicle strips. There is no evidence for the presence of flagella in the ampulla (reservoir/canal area), a fact confirmed by negative staining, and a difference regarding other species of Euglena. The observed ultrastructural characteristics are not sufficient to explain the adaptation of this species to acid and hot environments.

Ultrastructural morphologic description of the wild rice species Oryza latifolia (Poaceae) in Costa Rica

The wild rice species Oryza latifolia is endemic to Tropical America, allotetraploid and has a CCDD genome type. It belongs to the officinalis group of the genus Oryza. This species is widely distributed throughout the lowlands of Costa Rica and it is found on different life zones, having great morphologic diversity. The purpose of this research is to perform a morphologic description of O. latifolia samples of three Costa Rican localities (Carara, Liberia and Cañas) and to see if the phenotypic diversity of the species is reflected at the ultra-structure level. Structures such as the leaf blade, ligule, auricles and spikelet were analyzed. Leaf blade morphology of the specimens from the three localities is characterized by the presence of diamond-shaped stomata with papillae, zipper-like rows of silica cells; a variety of evenly distributed epicuticular wax papillae and bulky prickle trichomes. The central vein of the leaf blade from the Cañas populations is glabrous, while those from Carara and Liberia have abundant papillae. There are also differences among the borders of the leaf blade between these locations. Cañas and Liberia present alternating large and small prickle trichomes ca. 81 and 150 microns, while Carara exhibits even sized prickle trichomes of ca. 93 microns. Auricles from Cañas are rectangular and present long trichomes along the surface ca. 1.5 mm, while those of Liberia and Carara wrap the culm and exhibit trichomes only in the borders. The ligule from the plants of Carara has an acute distal tip, while that of Cañas and Liberia is blunt. The Liberia spikelet has large lignified spines while Cañas and Carara show flexible trichomes.

Ultrastructural morphologic description of the wild rice species Oryza latifolia (Poaceae) in Costa Rica.

The wild rice species Oryza latifolia is endemic to Tropical America, allotetraploid and has a CCDD genome type. It belongs to the officinalis group of the genus Oryza. This species is widely distributed throughout the lowlands of Costa Rica and it is found on different life zones, having great morphologic diversity. The purpose of this research is to perform a morphologic description of O. latifolia samples of three Costa Rican localities (Carara, Liberia and Cañas) and to see if the phenotypic diversity of the species is reflected at the ultra-structure level. Structures such as the leaf blade, ligule, auricles and spikelet were analyzed. Leaf blade morphology of the specimens from the three localities is characterized by the presence of diamond-shaped stomata with papillae, zipper-like rows of silica cells; a variety of evenly distributed epicuticular wax papillae and bulky prickle trichomes. The central vein of the leaf blade from the Cañas populations is glabrous, while those from Carara and Liberia have abundant papillae. There are also differences among the borders of the leaf blade between these locations. Cañas and Liberia present alternating large and small prickle trichomes ca. 81 and 150 microns, while Carara exhibits even sized prickle trichomes of ca. 93 microns. Auricles from Cañas are rectangular and present long trichomes along the surface ca. 1.5 mm, while those of Liberia and Carara wrap the culm and exhibit trichomes only in the borders. The ligule from the plants of Carara has an acute distal tip, while that of Cañas and Liberia is blunt. The Liberia spikelet has large lignified spines while Cañas and Carara show flexible trichomes.

[Bacterial present in the xylem of coffee (Rubiaceae: coffea arabica) with "Crespera" disease]. / Presencia de bacterias en el xilema de cafetos (Rubiaceae: Coffea arabica) afectados por la enfermedad conocida como "Crespera".

"Crespera" is an infectious disease of coffee plants that affects both the coffee production and the economy of the coffee producer countries. This disease affects morphologically the plant: long and narrow leaves with wavy borders and marginal necrosis; strong chlorosis results in drying of the leave, and leads to bad conditions of the plant. The internodes are short, producing the appearance of multiple sprouts in the axial sprout, the flowers can turn greenish, and the plant can present branches with severe symptoms, and branches without apparent symptoms at the same time. As a result, the coffee bean production decreases strikingly. The aim of this work was to determine the occurrence of the possible causative agent in the coffee plants using transmission electron microscopy. Normal and infected plants were compared looking at the leaves, central vein, lateral veins and petiole. It was determined that xylematic vessels show the presence of gram negative bacilliform bacteria (of thick-wavy walls), with dimensions of 0.3-0.5 micron diameter and 1-4 microns, length. The control plants did not show bacteria in the xylem.

Presencia de bacterias en el xilema de cafetos (Rubiaceae: Coffea arabica) afectados por la enfermedad conocida como "Crespera" / Bacterial present in the xylem of coffee (Rubiaceae: coffea arabica) by the well-known disease \"Crespera\"

"Crespera" is an infectious disease of coffee plants that affects both the coffee production and the economy of the coffee producer countries. This disease affects morphologically the plant: long and narrow leaves with wavy borders and marginal necrosis; strong chlorosis results in drying of the leave, and leads to bad conditions of the plant. The internodes are short, producing the appearance of multiple sprouts in the axial sprout, the flowers can turn greenish, and the plant can present branches with severe symptoms, and branches without apparent symptoms at the same time. As a result, the coffee bean production decreases strikingly. The aim of this work was to determine the occurrence of the possible causative agent in the coffee plants using transmission electron microscopy. Normal and infected plants were compared looking at the leaves, central vein, lateral veins and petiole. It was determined that xylematic vessels show the presence of gram negative bacilliform bacteria (of thick-wavy walls), with dimensions of 0.3-0.5 micron diameter and 1-4 microns, length. The control plants did not show bacteria in the xylem.

Characterization of a photosynthetic Euglena strain isolated from an acidic hot mud pool of a volcanic area of Costa Rica.

Abstract Conspicuous green patches on the surface of an acidic hot mud pool located near the Rincón de la Vieja volcano (northwestern Costa Rica) consisted of apparently unialgal populations of a chloroplast-bearing euglenoid. Morphological and physiological studies showed that it is a non-flagellated photosynthetic Euglena strain able to grow in defined mineral media at temperatures up to 40 degrees C and exhibiting higher thermotolerance than Euglena gracilis SAG 5/15 in photosynthetic activity analyses. Molecular phylogeny studies using 18S rDNA and GapC genes indicated that this strain is closely related to Euglena mutabilis, another acid-tolerant photosynthetic euglenoid, forming a clade deeply rooted in the Euglenales lineage. To our knowledge this is the most thermotolerant euglenoid described so far and the first Euglenozoan strain reported to inhabit acidic hot aquatic habitats.