The chemical landscape of leaves as a driver for microbial community structure and metabolic interactions

The intercellular space of leaves (e.g., the apoplast) is critical for plants, functioning in nutrient, water and photosynthate transport as well as cell homeostasis. It is also where important microbiota colonize, presumably by utilizing host resources. This environment is truly dynamic; the fluctuation of nutrients and a large array of defense molecules suggests it is a challenging niche for microbial colonization, where microbe-microbe interactions may play an important role. Thus, linking leaf resources to assembly of the leaf microbial community would have major impacts on understanding plant-microbe interactions. In the frame of this thesis, an infiltration-centrifugation method was optimized to recover apoplastic fluid wash (AFW) from leaves of diverse plant species. Closely related Flaveria species could be differentiated based on the AFW metabolomic profile, specifically on the quantities of valuable amino acids that are costly for microorganisms to synthesize. The AFW could also be used to assess bacterial diversity in the leaf apoplast. Amplicon sequencing analyses revealed that more alpha diversity could be recovered from AFW samples than from crushed leaves (the conventional method to assess leaf microbial diversity) and that beta diversity differences were clearer when using AFW. The optimized method was then used to track diurnal oscillations of metabolites and microbial communities in leaves of three Flaveria species with different carbon fixing strategies. Although additional work is needed to overcome low bacterial reads at some time points, the results suggested that enrichment of taxa over the day corresponded with increasing metabolite complexity of the AFW in a species-dependent manner. Considering the differences found in levels of amino acids between F. robusta and F. trinervia, an in vitro assay was established. Leaf bacteria from these two species were enriched with either sucrose as a sole carbon source or with addition of amino acids

Soil inoculation with Pseudomonas fluorescens, Azospirillum oryzae, Bacillus subtilis and mountain microorganisms (MM) and its effect on a soybean-tomato crop rotation system under greenhouse conditions.

Se evaluó un sistema de rotación soyatomate, con incorporación de biomasa verde y aplicación de inóculos microbianos individuales y en mezcla sobre el crecimiento de las plantas y propiedades edáficas; para ello se evaluaron en invernadero por 24 meses los siguientes 9 tratamientos: solo tomate (T); rotación tomate-soya (TS); rotación tomate-soya con inoculaciones individuales de Azospirillum oryzae (A); de Pseudomonas fluorescens (P); de Bacillus subtilis (B); de microorganismos de montaña (MM); y las inoculaciones en mezcla de B. subtilis y P. fluorescens (BP); de B. subtilis, P. fluorescens y A. oryzae (BPA); de B. subtilis, P. fluorescens, Azospirillum sp. y MM (BPAMM). Se evaluaron las variables físicas: densidad aparente y de partículas; conductividad hidráulica; poros totales; estabilidad de agregados; resistencia a la penetración (RP); las variables químicas: pH; conductividad eléctrica; contenido de N y C; relación C/N; contenido de nutrientes en suelos y foliares; las variables biológicas: respiración de suelos, unidades formadoras de colonias de hongos, bacterias y actinomicetos y el peso fresco y seco foliar. Las variables físicas no fueron afectadas por los tratamientos, con excepción de RP, que fue mayor en el tratamiento T. Las variables biológicas y químicas fueron sensibles a los tratamientos, con valores significativamente más altos en presencia de MM. Destaca el incremento del P en solución de suelo en tratamientos a los que se adicionó MM: pasó de 6 a 20 mg.l-1; esta condición se reflejó además en la cantidad de P en el tejido foliar al final del ensayo. Se determinó que el pH, CE y la respiración del suelo fueron afectados por la interacción entre los tratamientos aplicados y el tiempo transcurrido; los mayores valores se obtuvieron al final del ensayo y en los tratamientos con MM.The effect, on growth of plants and soil properties, of incorporating green biomass and applying individual and compound microbial inocula was evaluated in a soybean-tomato crop rotation system during 24 months in the greenhouse. Nine different conditions were evaluated: tomato alone (T); tomato-soybean rotation (TS); tomato-soybean rotation with individual inoculations of Azospirillum oryzae (A); of Pseudomonas fluorescens (P); of Bacillus subtilis (B); of mountain microorganisms (MM); and tomato-soybean rotation with mixtures of B. subtilis and P. fluorescens (BP); of B. subtilis, P. fluorescens, and A. oryzae (BPA); of B. subtilis, P. fluorescens, Azospirillum sp., and mountain microorganisms (BPAMM). The following physical and chemical soil indicators (variables) were evaluated: apparent density; particle density; hydraulic conductivity; pore volume (porosity); aggregate stability; penetration resistance (PR); pH; electrical conductivity (EC); carbon and nitrogen content; C/N ratio; and soil and foliar nutrient content. Biological indicators were also assessed: soil respiration; colony-forming units (CFU) of fungi, bacteria and actinomycetes; fresh and dry foliar weight. The physical variables were not affected by the treatments, except for PR, which was higher in treatment T. Chemical and biological variables were sensitive to the treatments, with significantly higher values in the presence of MM. The rise of P in soil solution in treatments with MM was of special importance: it increased from 6 to 20 mg.l-1; this condition was also reflected in the concentration of P in foliar tissue at the end of assay. It was determined that pH, EC and soil respiration were affected by the interaction among the treatments applied and the time elapsed; the highest values were obtained at the end of the assay and in treatments with MM.Universidad de Costa Rica/[733-B1-129]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro de Investigaciones Agronómicas (CIA

Obtaining deeper insights into microbiome diversity using a simple method to block host and nontargets in amplicon sequencing

Abstract Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce “blocking oligos”, a low‐cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A . thaliana ‐specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots ( Bromus erectus , Plantago lanceolata , Lotus corniculatus , Amaranth sp., Arabidopsis thaliana ). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant‐derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A . thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems‐based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package

Production of renewable fuel and value-added bioproducts using pineapple leaves in Costa Rica

Pineapple, Ananas comosus, is one of the most important cash crops in Costa Rica with more than 44,500 ha of plantation. The pineapple industry contributes approximately 1.7% of the gross domestic product (GDP) of Costa Rica. Pineapple cultivation generates a large amount of plant residues (250 metric tons per hectare of wet plant residues mainly leaves). Current practices of the field residue handing include direct burning, in situ decomposition and removal of residue before planting, which are neither economically sound nor environmentally friendly. New approaches are urgently needed to utilize the residues and improve sustainability of pineapple production in Costa Rica. This study developed a simple, efficient process to convert the pineapple plant leaves into bioethanol, spent yeast proteins, and fibrous material (pulp). The residue was first treated by a mechanical extruder to generate juice and fibrous material. The juice was fermented by a yeast, Kluyveromyces marxianus, to produce ethanol and spent yeast proteins. Under the selected process conditions, the plant leaves (125 tons fresh weight per year) from 1 ha can generate 2.1 tons of bio-ethanol, 1.55 tons of spent yeast biomass, and 11.65 tons of dry fibrous material. The mass and energy balance analysis concluded that using the studied process, the pineapple plant leaves from 44,500 ha of pineapple plantation in Costa Rica can produce 93,043, 68,975, and 518,425 tons of bioethanol, spent yeast, and fibrous material per year, respectively. The amount of bioethanol is able to replace approximately 8.51% of transportation fossil fuel consumption in Costa Rica.Michigan State University/[]/MSU/Estados UnidosNational Natural Science Foundation of China/[31701533]/NSFC/ChinaProgram of Study Abroad for Young Scholars/[gxgwfx 2018036]//Estados UnidosUCR::Vicerrectoría de Docencia::Ingeniería::Facultad de Ingeniería::Escuela de Ingeniería de Biosistema

Responses of anaerobic microorganisms to different culture conditions and corresponding effects on biogas production and solid digestate quality

Microbial communities of anaerobic digestion have been intensively investigated in the past decades. Majority of these studies focused on correlating microbial diversity with biogas production. The relationship between microbial communities and compositional changes of the solid digestate (AD fiber) has not been comprehensively studied to date. Therefore, the objective of this study was to understand the responses of microbial communities to different operational conditions of anaerobic co-digestion and their influences on biogas production and solid digestate quality. Two temperatures and three manure-to-food waste ratios were investigated by a completely randomized design. Molecular analyses demonstrate that both temperature and manure-to-food waste ratio greatly influenced the bacterial communities, while archaeal communities were mainly influenced by temperature. The digestion performance showed that biogas productivity increased with the increase of supplemental food wastes, and there were no significant differences on carbohydrate contents among different digestions. The statistical analyses conclude that microbes changed their community configuration under different conditions to enhance digestion performance for biogas and homogenized solid digestate production.U.S. Department of State West Hemisphere Affairs/[S-LMAQM-11-GR-075]//Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM

INOCULACIÓN AL SUELO CON Pseudomonas fluorescens, Azospirillum oryzae, Bacillus subtilis Y MICROORGANISMOS DE MONTAÑA (MM) Y SU EFECTO SOBRE UN SISTEMA DE ROTACIÓN SOYA-TOMATE BAJO CONDICIONES DE INVERNADERO

Se evaluó un sistema de rotación soya- tomate, con incorporación de biomasa verde y aplicación de inóculos microbianos individuales y en mezcla sobre el crecimiento de las plantas y propiedades edáficas; para ello se evaluaron en invernadero por 24 meses los siguientes 9 trata - mientos: solo tomate (T); rotación tomate-soya (TS); rotación tomate-soya con inoculaciones individuales de Azospirillum oryzae (A); de Pseudomonas fluorescens (P); de Bacillus sub - tilis (B); de microorganismos de montaña (MM); y las inoculaciones en mezcla de B. subtilis y P. fluorescens (BP); de B. subtilis, P. fluorescens y A. oryzae ( BPA); d e B. subtilis , P. fluorescens , Azospirillum sp. y MM (BPAMM). Se evalua - ron las variables físicas: densidad aparente y de partículas; conductividad hidráulica; poros totales; estabilidad de agregados; resistencia a la penetración (RP); las variables químicas: pH; conductividad eléctrica; contenido de N y C; relación C/N; contenido de nutrientes en suelos y foliares; las variables biológicas: respiración de suelos, unidades formadoras de colonias de hongos, bacterias y actinomicetos y el peso fres - co y seco foliar. Las variables físicas no fueron afectadas por los tratamientos, con excepción de RP, que fue mayor en el tratamiento T. Las varia - bles biológicas y químicas fueron sensibles a los tratamientos, con valores significativamente más altos en presencia de MM. Destaca el incremento del P en solución de suelo en tratamientos a los que se adicionó MM: pasó de 6 a 20 mg.l -1 ; esta condición se reflejó además en la cantidad de P en el tejido foliar al final del ensayo. Se determi - nó que el pH, CE y la respiración del suelo fueron afectados por la interacción entre los tratamientos aplicados y el tiempo transcurrido; los mayores valores se obtuvieron al final del ensayo y en los tratamientos con MM

What can myxomycetes tell us about floricolous microbial systems?

The study described herein was conducted in Costa Rica in an effort to generate baseline data on the association between tropical inflorescences and a group of microorganisms known as myxomycetes. The assemblage structure of myxomycetes was tested in three canopy cover classes between two independent study areas subject to different management strategies. One study area was an isolated patch in a suburban location while the other was located in a protected area with extensive connectivity to several national parks. Twenty-four species were recorded with only small differences in species occurrence and diversity between the two study areas. However, differences in diversity estimators were found for assemblages among the canopy cover classes within each study area. Intermediate and open canopy cover classes were the most dissimilar for the complete investigation, with those in the area with high forest connectivity and lack of plant manipulation being the ones responsible for the majority of the differences. Differences in assemblage structure among canopy cover categories were associated with differences in pH values. This study found a strong connection between monocot plants and myxomycetes in tropical environments and provides additional evidence for the existence of a guild of floricolous myxomycetes. The results suggest that vector-driven colonization of inflorescences by myxomycetes may be partially responsible for their occurrence in this microhabitat, but additional studies are required. Our study showed a resilient system of interaction that would naturally occur in different environmental conditions, suggesting a strong and stable relationship.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ingeniería::Instituto Investigaciones en Ingeniería (INII)UCR::Vicerrectoría de Docencia::Ingeniería::Facultad de Ingeniería::Escuela de Ingeniería de BiosistemasUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Inoculación al suelo con <i>Pseudomonas fluorescens, Azospirillum oryzae, Bacillus subtilis</i> y microorganismos de montaña (mm) y su efecto sobre un sistema de rotación soya-tomate bajo condiciones de invernadero

Se evaluó un sistema de rotación soyatomate, con incorporación de biomasa verde y aplicación de inóculos microbianos individuales y en mezcla sobre el crecimiento de las plantas y propiedades edáficas; para ello se evaluaron en invernadero por 24 meses los siguientes 9 tratamientos: solo tomate (T); rotación tomate-soya (TS); rotación tomate-soya con inoculaciones individuales de <em>Azospirillum oryzae</em> (A); de <em>Pseudomonas fluorescens</em> (P); de <em>Bacillus subtilis</em> (B); de microorganismos de montaña (MM); y las inoculaciones en mezcla de <em>B. subtilis</em> y <em>P. fluorescens</em> (BP); de <em>B. subtilis</em>, <em>P. fluorescens</em> y <em>A. oryzae</em> (BPA); de <em>B. subtilis</em>, <em>P. fluorescens</em>, <em>Azospirillum sp</em>. y MM (BPAMM). Se evaluaron las variables físicas: densidad aparente y de partículas; conductividad hidráulica; poros totales; estabilidad de agregados; resistencia a la penetración (RP); las variables químicas: pH; conductividad eléctrica; contenido de N y C; relación C/N; contenido de nutrientes en suelos y foliares; las variables biológicas: respiración de suelos, unidades formadoras de colonias de hongos, bacterias y actinomicetos y el peso fresco y seco foliar. Las variables físicas no fueron afectadas por los tratamientos, con excepción de RP, que fue mayor en el tratamiento T. Las variables biológicas y químicas fueron sensibles a los tratamientos, con valores significativamente más altos en presencia de MM. Destaca el incremento del P en solución de suelo en tratamientos a los que se adicionó MM: pasó de 6 a 20 mg.l-1; esta condición se reflejó además en la cantidad de P en el tejido foliar al final del ensayo. Se determinó que el pH, CE y la respiración del suelo fueron afectados por la interacción entre los tratamientos aplicados y el tiempo transcurrido; los mayores valores se obtuvieron al final del ensayo y en los tratamientos con MM

Biogas Production and Microbial Communities of Mesophilic and Thermophilic Anaerobic Co-Digestion of Animal Manures and Food Wastes in Costa Rica

Biomass generated from agricultural operations in Costa Rica represents an untapped renewable resource for bioenergy generation. This study investigated the effects of two temperatures and three mixture ratios of manures and food wastes on biogas production and microbial community structure. Increasing the amount of fruit and restaurant wastes in the feed mixture significantly enhanced the productivity of the systems (16% increase in the mesophilic systems and 41% in the thermophilic). The methane content of biogas was also favored at higher temperatures. Beta diversity analysis, based on high-throughput sequencing of 16S rRNA gene, showed that microbial communities of the thermophilic digestions were more similar to each other than the mesophilic digestions. Species richness of the thermophilic digestions was significantly greater than the corresponding mesophilic digestions (F = 40.08, p = 0.003). The mesophilic digesters were dominated by Firmicutes and Bacteroidetes while in thermophilic digesters, the phyla Firmicutes and Chloroflexi accounted for up to 90% of all sequences. Methanosarcina represented the key methanogen and was more abundant in thermophilic digestions. These results demonstrate that increasing digestion temperature and adding food wastes can alleviate the negative impact of low C:N ratios on anaerobic digestion

Biogas production and microbial communities of mesophilic and thermophilic anaerobic co-digestion of animal manures and food wastes in Costa Rica

Biomass generated from agricultural operations in Costa Rica represents an untapped renewable resource for bioenergy generation. This study investigated the effects of two temperatures and three mixture ratios of manures and food wastes on biogas production and microbial community structure. Increasing the amount of fruit and restaurant wastes in the feed mixture significantly enhanced the productivity of the systems (16% increase in the mesophilic systems and 41% in the thermophilic). The methane content of biogas was also favored at higher temperatures. Beta diversity analysis, based on high-throughput sequencing of 16S rRNA gene, showed that microbial communities of the thermophilic digestions were more similar to each other than the mesophilic digestions. Species richness of the thermophilic digestions was significantly greater than the corresponding mesophilic digestions (F = 40.08, p = 0.003). The mesophilic digesters were dominated by Firmicutes and Bacteroidetes while in thermophilic digesters, the phyla Firmicutes and Chloroflexi accounted for up to 90% of all sequences. Methanosarcina was the key methanogen and was more abundant in thermophilic digestions. These results demonstrate that increasing digestion temperature and adding food wastes can alleviate the negative impact of low C:N ratios on anaerobic digestion.Universidad de Costa Rica/[733-A1-821]/UCR/Costa RicaUniversidad de Michigan/[]/UMICH/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro de Investigaciones Agronómicas (CIA)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Electroquímica y Energía Química (CELEQ