Draft genomes of two phylotype I and II Ralstonia solanacearum species complex (RSSC) isolates causing bacterial wilt in tomato plants from Costa Rica.

Bacteria from RSSC hold agricultural significance as they are the causal agents of bacterial wilt. Here, we report the draft genomes of two bacteria extracted from vascular tissues of infected tomato plants. Isolate RALF-MA was classified as Ralstonia pseudosolanacearum (phylotype I) and RALSA-MA as Ralstonia solanacearum (phylotype II).

First insights into the prokaryotic community structure of Lake Cote, Costa Rica: Influence on nutrient cycling.

Prokaryotic diversity in lakes has been studied for many years mainly focusing on community structure and how the bacterial assemblages are driven by physicochemical conditions such as temperature, oxygen, and nutrients. However, little is known about how the composition and function of the prokaryotic community changes upon lake stratification. To elucidate this, we studied Lake Cote in Costa Rica determining prokaryotic diversity and community structure in conjunction with physicochemistry along vertical gradients during stratification and mixing periods. Of the parameters measured, ammonium, oxygen, and temperature, in that order, were the main determinants driving the variability in the prokaryotic community structure of the lake. Distinct stratification of Lake Cote occurred (March 2018) and the community diversity was compared to a period of complete mixing (March 2019). The microbial community analysis indicated that stratification significantly altered the bacterial composition in the epi-meta- and hypolimnion. During stratification, the Deltaproteobacteria, Chloroflexi, Bacteroidetes, Nitrospirae, and Euryarchaeota were dominant in the hypolimnion yet largely absent in surface layers. Among these taxa, strict or facultative anaerobic bacteria were likely contributing to the lake nitrogen biogeochemical cycling, consistent with measurements of inorganic nitrogen measurements and microbial functional abundance predictions. In general, during both sampling events, a higher abundance of Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and Cyanobacteria was found in the oxygenated layers. Lake Cote had a unique bacterial diversity, with 80% of Amplicon Sequence Variant (ASV) recovered similar to unclassified/uncultured strains and exhibits archetypal shallow lake physicochemical but not microbial fluctuations worthy of further investigation. This study provides an example of lake hydrodynamics impacts to microbial community and their function in Central American lakes with implications for other shallow, upland, and oligotrophic lake systems.

Novel Cyanobacterial Diversity Found in Costa Rican Thermal Springs Associated with Rincon de la Vieja and Miravalles Volcanoes: A Polyphasic Approach.

Central America is one of the most important biodiversity hot spots in the world, and Costa Rican microbial communities from thermal springs are the best characterized in the isthmus. Miravalles is an inactive quaternary stratovolcano, and the Rincón de la Vieja is a unique active volcano, in whose slopes diverse hydrothermal springs, such as Las Lilas, are located. These springs harbor extensive microbial mats, whose diversity has been studied. Based on their importance as primary producers, in this study we focused on cultured cyanobacterial diversity from two geothermal environments of northern Costa Rica. Several cultural, molecular and taxonomic techniques were employed to maximize the results of a polyphasic approach. Sample collection sites were physicochemically described, and strains were isolated and characterized by light and electron microscopy. Phylogenetic analysis was performed using 16S rRNA gene sequences and amplified ribosomal DNA restriction analysis (ARDRA). Fifty-six phylotypes were isolated and classified into 21 morphotypes and identified in 14 genera, some of them might be new species within these genera. Furthermore, according to phylogenetic analysis, there are three possible new genera in our collection. Miravalles and Las Lilas thermal springs are reservoirs of novel phylogeographic lineages of phototrophic microorganisms. This study is the first report of strains that belong to the genera Gloeocapsa, Stanieria, Microseira, Klisinema and Oculatella isolated from thermal springs and growing at temperatures above 50°C. We also obtained isolates assigned to Synechococcus, Leptolyngbya spp., and Fischerella, which are considered typical strains in these environments.

Tolerance and sorption of Bromacil and Paraquat by thermophilic cyanobacteria Leptolyngbya 7M from Costa Rican thermal springs.

We studied the adsorption ability and tolerance of the thermophilic filamentous cyanobacteria Letolyngbya 7M towards Paraquat and Bromacil. Adsorption isotherms at pH = 7.0 showed an adsorption capacity of 24.4 mg/g and 66.8 mg/g, respectively, and a good fit to the Langmuir model (R2 = 0.97 and 0.99, respectively). To evaluate the effect of both herbicides on photosynthetic pigments and viability of cyanobacteria, cell autoflorescence and esterase activity was determined using flow cytometry. Autofluorescence was less sensitive to changes in cell viability, as it was only slightly reduced at high Paraquat and Bromacil concentrations. Herbicide effect on esterase activity is dose-dependent. Bromacil did not cause a significant effect on either chlorophyll a content or cell viability. This study demonstrates the potential of Leptolyngbya 7M to remove Paraquat and Bromacil herbicides from aqueous solution under laboratory conditions.

The influence of temperature and pH on bacterial community composition of microbial mats in hot springs from Costa Rica.

We used the 16S rRNA gene pyrosequencing approach to investigate the microbial diversity and community composition in several Costa Rican hot springs alongside the latitudinal axis of the country, with a range of temperatures (37-63°C), pH (6-7.5) and other geochemical conditions. A principal component analyses of the physicochemical parameters showed the samples were separated into three geochemically distinct habitats associated with the location (North, Central, and South). Cyanobacteria and Chloroflexi comprised 93% of the classified community, the former being the most abundant phylum in all samples except for Rocas Calientes 1, (63°C, pH 6), where Chloroflexi and Deinococcus-Thermus represented 84% of the OTUs. Chloroflexi were more abundant as temperature increased. Proteobacteria, Bacteriodetes and Deinococcus-Thermus comprised 5% of the OTUs represented. Other Phyla were present in very small percentages (<1%). A LINKTREE analysis showed that the community structure of the mats was shaped primarily by pH, separating samples with pH > 6.6 from samples with pH < 6.4. Thus, both pH and temperature were relevant for community composition even within the moderate ranges of variables studied. These results provide a basis for an understanding of the physicochemical influences in moderately thermophilic microbial mats.

Effects of coffee processing residues on anaerobic microorganisms and corresponding digestion performance.

The objective of this study was to delineate the effects of different coffee processing residues on the anaerobic microbes and corresponding digestion performance. The results elucidated that mucilage-rich feed enhanced the accumulation of methanogens, which consequently led to better digestion performance of biogas production. Fifty percent more methane and up to 3 times more net energy (heat and electricity) output were achieved by the digestion of the mucilage-rich feed (M3). The microbial community and statistical analyses further elucidated that different residues in the feed had significant impact on microbial distribution and correspondingly influenced the digestion performance.

Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed on different diets.

As primary consumers of foliage, caterpillars play essential roles in shaping the trophic structure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH is exceptionally alkaline and contains high concentrations of toxic compounds derived from the ingested plant material (secondary compounds or allelochemicals) and from the insect itself. The midgut, therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxa have been recovered from caterpillar midguts, but little is known about the impact of these microorganisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and to investigate their functions. As a first step, different diet formulations were evaluated for rearing two species of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primers hybridizing broadly to sequences from the bacterial domain, 16S rRNA gene libraries were constructed with midgut DNA extracted from caterpillars reared on different diets. Amplified rDNA restriction analysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliage were more diverse than those from caterpillars fed on artificial diet. Sequences related to Methylobacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar libraries regardless of diet, but were not detected in a library constructed from the diet itself. Furthermore, libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for vertical transmission of midgut symbionts. Taken together, these results suggest that microorganisms associated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologically important insects.

Characterization of true-branching cyanobacteria from geothermal sites and hot springs of Costa Rica.

Costa Rica is at the centre of the Mesoamerican biodiversity hotspot. Little is known about cyanobacteria from this region so far. Here, four isolates of the order Stigonematales (section V) were characterized in a polyphasic approach. All strains were isolated from geothermal sites and hot springs of Costa Rica. However, one of them, identified as Westiellopsis sp. Ar73, did not grow at more than 40 degrees C. Based on its identical 16S rRNA to several previously isolated Westiellopsis sp. and Fischerella muscicola strains, a ubiquitous distribution throughout tropical and subtropical regions can be implied. In contrast, the isolates MV9, MV11 and RV14 grew well up to 50-55 degrees C. Based on morphologic, ultrastructural, molecular and physiologic data, MV9, MV11 and RV14 were identified to belong to the genus Fischerella. Two distinct intergenic transcribed spacer (ITS) types, with or without tRNA genes, were detected for all Stigonematales analysed here, indicating ITS polymorphism as a characteristic feature of heterocystous cyanobacteria. In phylogenetic trees, these Fischerella spp. formed a new and distinct clade within the wider lineage of thermophilic Fischerella (Mastigocladus cf. laminosus), which might represent a geographic lineage. Thus, geographic isolation may be an underestimated aspect of microbial evolution. The strains presented here are suitable as new models to study this group of cyanobacteria.

Steinernema costaricense n. sp. and S. puntauvense n. sp. (Rhabditida: Steinernematidae), two new entomopathogenic nematodes from Costa Rica.

Steinernema costaricense n. sp. and S. puntauvense n. sp. were recovered during a survey for native entomopathogenic nematodes in Costa Rica. Morphological data, molecular (28S rDNA sequence data) studies and cross-hybridisation tests were used for diagnostic and identification purposes. Additionally, 28S rDNA sequence data were used to assess the evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. costaricense n. sp. include: the body size of the infective juvenile (av. 1,696); the presence of protruding 'horn-like' cephalic papillae; the position of the excretory pore in the infective juvenile (av. 77 microm) and the first generation male (av. 117 microm); the D% value of the infective juvenile (av. 53) and the first generation male (av. 56); the E% value of the infective juvenile (av. 85); and the morphology of the spicules and gubernaculum of the first generation male. Diagnostic traits for S. puntauvense n. sp. are: the position of the excretory pore in the infective juveniles (av. 25 microm); the shape and size of the spicules and gubernaculum of the first generation male; and the shape of the tail of the first generation female. In addition to these traits, 28S rDNA sequences analysis and hybridisation tests showed that both new Steinernema species are distinct and unique entities.

Actividades enzimáticas en aislamientos bacterianos de tractos digestivos de larvas y del contenido de pupas de Automeris zugana y Rothschildia lebeau (Lepidoptera: Saturniidae)

Enzymatic activities of bacteria isolated from the digestive tract of caterpillars and the pupal content of Automeris zugana and Rothschildia lebeau (Lepidoptera: Saturniidae). The enzymatic activities of bacteria isolated from the digestive tracts of caterpillars and the pupal contents of Automeris zugana and Rothschildia lebeau was studied. This digestive tract represents an extreme microenvironment due to its high pH and presence of antimicrobial substances secreted by the insect or derived from ingested plant tissue. At the same time, it contains large amounts of nutrient-rich food, for which microbes may compete among themselves and with the caterpillar. There is little information about the microbiota associated with tropical caterpillar guts, although bacteria from different genera have been isolated from gut and pupae samples. The study of the enzymatic activities generated by these organisms constitutes a starting point to understand their metabolic and physiological relationships with their hosts, and to find enzymes that have potential biotechnological applications. In this study we evaluated several enzymatic activities in two collections of bacteria isolated from caterpillar guts and pupae of the tropical lepidopteran species A. zugana and R. lebeau. Bacteria grown under aerobic conditions were tested for an array of enzymes, including gelatinases, caseinases, lipases, esterases, cellulases, xylanases, amylases and chitinases. Both collections displayed similar patterns of enzymatic activity. No isolate showed activity for all enzymatic tests, but as a whole, at least some bacteria in each collection were able to degrade each substrate tested. Isolates with the same taxonomic identification obtained from caterpillar guts and pupae had almost the same enzymatic activities. In both collections, it was possible to group bacterial isolates according to their enzyme activity pattern. In addition to a heterogeneous ensemble of isolates exhibiting two or less enzymatic activities, there were two groups with at least five activities that showed an apparent specialization for the substrates they were able to use. The first consisted exclusively of isolates of the family Enterobacteriaceae, which were positive for lipolytic and chitinolytic activities, but completely lacked amylasic, cellulolytic and xylanolytic activities. The second group, composed mainly of Gram-positive rods, exhibited the opposite pattern: they were positive for amylasic, cellulolytic and xylanolytic activities, lacked chitinolytic activity and had few isolates with lipolytic activity. This work forms the foundation for future research to explore the biotechnological potential of bacterial isolates from caterpillar guts. Rev. Biol. Trop. 55 (2): 401-415. Epub 2007 June, 29.

El tracto digestivo de orugas constituye un microambiente extremo, debido a su elevado pH y presencia de sustancias antimicrobianas secretadas por el insecto o derivadas del tejido vegetal ingerido. Al mismo tiempo, el intestino alberga gran cantidad de alimento, por el cual los microorganismos presentes podrían competir entre sí y con su hospedero. Existe poca información sobre la microbiota asociada con el intestino de orugas tropicales, aunque se ha demostrado la presencia de bacterias de diversos géneros tanto en el intestino como en el interior de pupas. El estudio de las actividades enzimáticas de estos microorganismos constituye un punto de partida en la comprensión de la posible relación metabólica y fisiológica que establecen con sus hospederos, a la vez que permite investigar enzimas con potenciales aplicaciones biotecnológicas. En este trabajo se evaluó la presencia de actividades gelatinolítica, caseinolítica, esterásica, lipolítica, quitinolítica, amilásica, celulolítica y xilanolítica en dos colecciones de aislamientos bacterianos provenientes de tractos digestivos de orugas y de pupas de los lepidópteros Automeris zugana y Rothschildia lebeau. Se utilizaron ensayos bioquímicos tradicionales para detectar enzimas secretadas en condiciones aerobias, en las que ambas colecciones exhibieron un comportamiento enzimático similar. Ningún aislamiento produjo un resultado positivo en todas las pruebas, pero como conjunto ambas colecciones fueron capaces de utilizar todos los sustratos evaluados. Los aislamientos obtenidos de pupas presentaron prácticamente las mismas actividades que sus homólogos provenientes de intestinos. En ambas colecciones fue posible agrupar los aislamientos de acuerdo con su patrón de producción de enzimas. Además de un conjunto heterogéneo de aislamientos poco activos (dos o menos actividades), se destacan dos grupos muy activos (al menos cinco actividades), que manifiestan una aparente especialización en los sustratos que utilizan. El primero de ellos está constituido exclusivamente por miembros de la familia Enterobacteriaceae, los cuales exhibieron un alto porcentaje de positividad en actividades lipolítica y quitinolítica, pero no demostraron la expresión de las actividades amilásica, celulolítica ni xilanolítica. El segundo grupo, formado en su gran mayoría por bacilos Gram-positivos, presenta la situación opuesta: alta positividad en actividades amilásica, celulolítica y xilanolítica, no detección de actividad quitinolítica y pocos aislamientos con actividad lipolítica. Este trabajo pretende ser la base de futuras investigaciones que exploren el potencial biotecnológico de aislamientos bacterianos provenientes del tracto digestivo de orugas.

[Enzymatic activities of bacteria isolated from the digestive tract of caterpillars and the pupal content of Automeris zugana and Rothschildia lebeau (Lepidoptera: Saturniidae)]. / Actividades enzimáticas en aislamientos bacterianos de tractos digestivos de larvas y del contenido de pupas de Automeris zugana y Rothschildia lebeau (Lepidoptera: Saturniidae).

The enzymatic activities of bacteria isolated from the digestive tracts of caterpillars and the pupal contents of Automeris zugana and Rothschildia lebeau was studied. This digestive tract represents an extreme microenvironment due to its high pH and presence of antimicrobial substances secreted by the insect or derived from ingested plant tissue. At the same time, it contains large amounts of nutrient-rich food, for which microbes may compete among themselves and with the caterpillar. There is little information about the microbiota associated with tropical caterpillar guts, although bacteria from different genera have been isolated from gut and pupae samples. The study of the enzymatic activities generated by these organisms constitutes a starting point to understand their metabolic and physiological relationships with their hosts, and to find enzymes that have potential biotechnological applications. In this study we evaluated several enzymatic activities in two collections of bacteria isolated from caterpillar guts and pupae of the tropical lepidopteran species A. zugana and R. lebeau. Bacteria grown under aerobic conditions were tested for an array of enzymes, including gelatinases, caseinases, lipases, esterases, cellulases, xylanases, amylases and chitinases. Both collections displayed similar patterns of enzymatic activity. No isolate showed activity for all enzymatic tests, but as a whole, at least some bacteria in each collection were able to degrade each substrate tested. Isolates with the same taxonomic identification obtained from caterpillar guts and pupae had almost the same enzymatic activities. In both collections, it was possible to group bacterial isolates according to their enzyme activity pattern. In addition to a heterogeneous ensemble of isolates exhibiting two or less enzymatic activities, there were two groups with at least five activities that showed an apparent specialization for the substrates they were able to use. The first consisted exclusively of isolates of the family Enterobacteriaceae, which were positive for lipolytic and chitinolytic activities, but completely lacked amylasic, cellulolytic and xylanolytic activities. The second group, composed mainly of Gram-positive rods, exhibited the opposite pattern: they were positive for amylasic, cellulolytic and xylanolytic activities, lacked chitinolytic activity and had few isolates with lipolytic activity. This work forms the foundation for future research to explore the biotechnological potential of bacterial isolates from caterpillar guts.

First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica.

A survey of entomopathogenic nematodes was conducted in the north Pacific (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identified as Heterorhabditis indica and the three Steinernema isolates were identified as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing.

Does a polyphagous caterpillar have the same gut microbiota when feeding on different species of food plants?

We used classical culture techniques to explore gut bacteria and changes associated with dietary change in the highly polyphagous, tropical caterpillar Automeris zugana (Saturniidae). Fifty-five third instar wild-caught sibs feeding on Annona purpurea (Annonaceae) in the Area de Conservación Guanacaste (ACG) in northwestern Costa Rica were divided into eight groups. Each of seven groups was reared to the ultimate instar on another species of food plant normally used by A. zugana. Some pupae were also analyzed for the presence of bacteria. Aerobic bacterial cultures were obtained from all 33 caterpillar guts and the eight pupae inventoried. There was no clear pattern in species composition of cultivated bacteria among the eight diets, and each caterpillar on a given food plant carried only a small fraction of the total set of species isolated from the set of caterpillars feeding on that food plant. Taken as a whole, the larvae and pupae contained 22 species of cultivable bacteria in 12 genera. Enterobacter, present in 81.8% of the samples, was the genus most frequently isolated from the caterpillars, followed by Micrococcus and Bacillus. Bacillus thuringiensis was isolated from 30.3% of the dissected caterpillars, but found in caterpillars feeding on only half of the species of food plants.

Does a polyphagous caterpillar have the same gut microbiota when feeding on different species of food plants

We used classical culture techniques to explore gut bacteria and changes associated with dietary change in the highly polyphagous, tropical caterpillar Automeris zugana (Saturniidae). Fifty-five third instar wild-caught sibs feeding on Annona purpurea (Annonaceae) in the Area de Conservación Guanacaste (ACG) in northwestern Costa Rica were divided into eight groups. Each of seven groups was reared to the ultimate instar on another species of food plant normally used by A. zugana. Some pupae were also analyzed for the presence of bacteria. Aerobic bacterial cultures were obtained from all 33 caterpillar guts and the eight pupae inventoried. There was no clear pattern in species composition of cultivated bacteria among the eight diets, and each caterpillar on a given food plant carried only a small fraction of the total set of species isolated from the set of caterpillars feeding on that food plant. Taken as a whole, the larvae and pupae contained 22 species of cultivable bacteria in 12 genera. Enterobacter, present in 81.8 of the samples, was the genus most frequently isolated from the caterpillars, followed by Micrococcus and Bacillus. Bacillus thuringiensis was isolated from 30.3 of the dissected caterpillars, but found in caterpillars feeding on only half of the species of food plants.