GENETIC VARIABILITY OF SUGARCANE-ASSOCIATED DIAZOTROPHIC BACTERIA CAPABLE OF INORGANIC PHOSPHATE SOLUBILIZING

The sugarcane is a culture of great importance for the Brazilian agriculture. Every year this culture consumes great amounts of nitrogen and phosphate fertilizers. However, the use of plant growth-promoting bacteria can reduce the use of the chemical fertilizers, contributing to the economy and the environment conservation. So, the goal of this study was to select sugarcane-associated diazotrophic bacteria able to solubilize inorganic phosphate and to evaluate the genetic diversity of these bacteria. A total of 68 diazotrophic bacteria, leaf and root endophytic and rizoplane, of three sugarcane varieties. The selection of inorganic phosphate solubilizing diazotrophic bacteria was assayed by the solubilization index (SI) in solid medium containing insoluble phosphate. The genetic variability was analyzed by the BOX-PCR technique. The results showed that 74% of the diazotrophic strains were able to solubilize inorganic phosphate, presenting classes of different SI. The results showed that the vegetal tissue and the genotype plant influenced in the interaction between phosphate solubilizing diazotrophic bacteria and sugarcane plants. BOX-PCR revealed high genetic variability among the strains analyzed. So, sugarcane-associated diazotrophic bacteria express the capacity to solubilize inorganic phosphate and they present high genetic diversity.

IMPACTS OF CLIMATE CHANGE ON SOIL MICROORGANISMS IN NORTHERN HARDWOOD FORESTS

As global climate continues to change, it becomes more important to understand possible feedbacks from soils to the climate system. This dissertation focuses on soil microbial community responses to climate change factors in northern hardwood forests. Two soil warming experiments at Harvard Forest in Massachusetts, and a climate change manipulation experiment with both elevated temperature and increased moisture inputs in Michigan were sampled. The hyphal in-growth bag method was to understand how soil fungal biomass and respiration respond to climate change factors. Our results from phospholipid fatty acid (PLFA) analyses suggest that the hyphal in-growth bag method allows relatively pure samples of fungal hyphae to be partitioned from bacteria in the soil. The contribution of fungal hyphal respiration to soil respiration was examined in climate change manipulation experiments in Massachusetts and Michigan. The Harvard Forest soil warming experiments in Massachusetts are long-term studies with 8 and 18 years of +5 °C warming treatment. Hyphal respiration and biomass production tended to decrease with soil warming at Harvard Forest. This suggests that fungal hyphae adjust to higher temperatures by decreasing the amount of carbon respired and the amount of carbon stored in biomass. The Ford Forestry Center experiment in Michigan has a 2 x 2 fully factorial design with warming (+4-5 °C) and moisture addition (+30% average ambient growing season precipitation). This experiment was used to examine hyphal growth and respiration of arbuscular mycorrhizal fungi (AMF), soil enzymatic capacity, microbial biomass and microbial community structure in the soil over two years of experimental treatment. Results from the hyphal in-growth bag study indicate that AMF hyphal growth and respiration respond negatively to drought. Soil enzyme activities tend to be higher in heated versus unheated soils. There were significant temporal variations in enzyme activity and microbial biomass estimates. When microbial biomass was estimated using chloroform fumigation extractions there were no differences between experimental treatments and the control. When PLFA analyses were used to estimate microbial biomass we found that biomass responds negatively to higher temperatures and positively to moisture addition. This pattern was present for both bacteria and fungi. More information on the quality and composition of the organic matter and nutrients in soils from climate change manipulation experiments will allow us to gain a more thorough understanding of the mechanisms driving the patterns reported here. The information presented here will improve current soil carbon and nitrogen cycling models.

Different Land Use Intensities in Grassland Ecosystems Drive Ecology of Microbial Communities Involved in Nitrogen Turnover in Soil

Understanding factors driving the ecology of N cycling microbial communities is of central importance for sustainable land use. In this study we report changes of abundance of denitrifiers, nitrifiers and nitrogen-fixing microorganisms (based on qPCR data for selected functional genes) in response to different land use intensity levels and the consequences for potential turnover rates. We investigated selected grassland sites being comparable with respect to soil type and climatic conditions, which have been continuously treated for many years as intensely used meadows (IM), intensely used mown pastures (IP) and extensively used pastures (EP), respectively. The obtained data were linked to above ground biodiversity pattern as well as water extractable fractions of nitrogen and carbon in soil. Shifts in land use intensity changed plant community composition from systems dominated by s-strategists in extensive managed grasslands to c-strategist dominated communities in intensive managed grasslands. Along the different types of land use intensity, the availability of inorganic nitrogen regulated the abundance of bacterial and archaeal ammonia oxidizers. In contrast, the amount of dissolved organic nitrogen determined the abundance of denitrifiers (nirS and nirK). The high abundance of nifH carrying bacteria at intensive managed sites gave evidence that the amounts of substrates as energy source outcompete the high availability of inorganic nitrogen in these sites. Overall, we revealed that abundance and function of microorganisms involved in key processes of inorganic N cycling (nitrification, denitrification and N fixation) might be independently regulated by different abiotic and biotic factors in response to land use intensity.

Biotic and Abiotic Properties Mediating Plant Diversity Effects on Soil Microbial Communities in an Experimental Grassland

Plant diversity drives changes in the soil microbial community which may result in alterations in ecosystem functions. However, the governing factors between the composition of soil microbial communities and plant diversity are not well understood. We investigated the impact of plant diversity (plant species richness and functional group richness) and plant functional group identity on soil microbial biomass and soil microbial community structure in experimental grassland ecosystems. Total microbial biomass and community structure were determined by phospholipid fatty acid (PLFA) analysis. The diversity gradient covered 1, 2, 4, 8, 16 and 60 plant species and 1, 2, 3 and 4 plant functional groups (grasses, legumes, small herbs and tall herbs). In May 2007, soil samples were taken from experimental plots and from nearby fields and meadows. Beside soil texture, plant species richness was the main driver of soil microbial biomass. Structural equation modeling revealed that the positive plant diversity effect was mainly mediated by higher leaf area index resulting in higher soil moisture in the top soil layer. The fungal-to-bacterial biomass ratio was positively affected by plant functional group richness and negatively by the presence of legumes. Bacteria were more closely related to abiotic differences caused by plant diversity, while fungi were more affected by plant-derived organic matter inputs. We found diverse plant communities promoted faster transition of soil microbial communities typical for arable land towards grassland communities. Although some mechanisms underlying the plant diversity effect on soil microorganisms could be identified, future studies have to determine plant traits shaping soil microbial community structure. We suspect differences in root traits among different plant communities, such as root turnover rates and chemical composition of root exudates, to structure soil microbial communities.

Soil characteristics and abundance of archaea in samples from the Ross Sea Region, Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (>99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.

(Supplemental Table 2) Plant characteristics, GHG fluxes, and soil chemical and microbial properties in experimental treatments in Alexandra Fiord

We evaluated above- and belowground ecosystem changes in a 16 year, combined fertilization and warming experiment in a High Arctic tundra deciduous shrub heath (Alexandra Fiord, Ellesmere Island, NU, Canada). Soil emissions of the three key greenhouse gases (GHGs) (carbon dioxide, methane, and nitrous oxide) were measured in mid-July 2009 using soil respiration chambers attached to a FTIR system. Soil chemical and biochemical properties including Q10 values for CO2, CH4, and N2O, Bacteria and Archaea assemblage composition, and the diversity and prevalence of key nitrogen cycling genes including bacterial amoA, crenarchaeal amoA, and nosZ were measured. Warming and fertilization caused strong increases in plant community cover and height but had limited effects on GHG fluxes and no substantial effect on soil chemistry or biochemistry. Similarly, there was a surprising lack of directional shifts in the soil microbial community as a whole or any change at all in microbial functional groups associated with CH4 consumption or N2O cycling in any treatment. Thus, it appears that while warming and increased nutrient availability have strongly affected the plant community over the last 16 years, the belowground ecosystem has not yet responded. This resistance of the soil ecosystem has resulted in limited changes in GHG fluxes in response to the experimental treatments.

Abundance and community structures of heterotrophic and oligotrophic bacteria from the Sierra Leone Abbyssal Plain

Colony counts on high and low-nutrient agar media incubated at 2 and 20 °C, Acridine Orange Direct Counts and biomasses are reported for sediments of the Sierra Leone Abyssal Plain. All isolates from low-nutrient agars also grew in nutrient-rich seawater broth (100 % SWB). However, a greater proportion of the 2 °C than of the 20 °C isolates grew in 2.5% SWB, containing 125 mg/l peptone and 25 mg/l yeast extract. Only 14 strains or 12.7% of the 2 °C isolates, but none of the 20 °C isolates, grew in 0.25 % SWB. Psychrophilic bacteria with maximum growth temperatures below 12 °C, isolated at 2 °C, were predominant among the cultivable bacteria from the surface layer. They required seawater for growth and belonged mainly to the Gram-negative genera Alteromonas and Vibrio. In contrast to the earlier view that psychrophily is connected with the Gram-negative cell type, it was found that cold-adapted bacteria of the Gram-positive genus Bacillus predominated in the 4 to 6 cm layer. The 20 °C isolates, however, were mostly Gram-positive, mesophilic, not dependent on seawater for growth, not able to utilize organic substrates at 4 °C, and belonged mainly to the genus Bacillus and to the Gram-positive cocci. The majority of the mesophilic bacilli most likely evolved from dormant spores, but not from actively metabolizing cells. It can be concluded that only the strains isolated at 2 °C can be regarded as indigenous to the deep-sea.

Nematodes as indicators of the effect of soil fumigants on soil functioning

Debido al futuro incierto de la mayor parte de los fumigantes edáficos usados actualmente en la Unión Europea, que pueden implicar riesgos para la salud humana/animal y el medio ambiente, es necesario desarrollar programas de manejo integrado para el control de plagas de cultivos. Estos programas se incluyen como obligatorios en el Reglamento (EC) No. 1107/2009. De acuerdo con este Reglamento, es obligatoria la evaluación del riesgo asociado al uso de productos fitosanitarios sobre los organismos edáficos no diana y sus funciones, además de llevar a cabo ensayos con diferentes especies indicadoras para obtener datos de toxicidad que puedan ser usados posteriormente en la evaluación de riesgo. Sin embargo, la baja representatividad de algunas de estas especies indicadoras en el área Mediterránea supone una gran limitación. En esta situación, el Panel Científico de Productos Fitosanitarios y sus Residuos de la Autoridad Europea en Seguridad Alimentaria (EFSA), ha señalado la necesidad de modificar los datos ecotoxicológicos requeridos para evaluar los efectos adversos de los productos fitosanitarios de una manera más integrada, incluyendo criterios funcionales y estructurales mediante organismos como bacterias, hongos, protozoos y nematodos. De este modo, la EFSA ha recomendado el uso de los nematodos en la evaluación de la funcionalidad y estructura del suelo. Los nematodos están globalmente distribuidos y son morfológicamente diversos; esto junto con su gran abundancia y diversidad de respuestas a las perturbaciones edáficas, los convierte en indicadores adecuados del estado del suelo. Puesto que los nematodos interaccionan con muchos otros organismos que participan en diferentes eslabones de la red trófica edáfica, jugando papeles importantes en procesos edáficos esenciales en los agroescosistemas, la diversidad de nematodos es, a menudo, usada como indicador biológico de los efectos de las prácticas agrícolas en el estado del suelo. En los últimos años, diferentes índices basados en la comunidad nematológica han facilitado la interpretación de datos complejos sobre la ecología del suelo. Los índices de la red trófica edáfica, basados en la abundancia de grupos funcionales definidos como grupos C-P y grupos tróficos, permiten la evaluación de la funcionalidad de la red trófica edáfica. Por otra parte, la dificultad en la identificación taxonómica de nematodos para explicar su uso limitado como indicadores ecológicos, es ampliamente discutida, y existe cierta controversia en cuanto a la eficacia de los diferentes métodos de identificación de nematodos. Se argumenta que la identificación morfológica es difícil y puede llevar mucho tiempo debido a la falta de expertos especializados, y se afirma que las técnicas moleculares pueden resolver algunas limitaciones de las técnicas morfológicas como la identificación de juveniles. Sin embargo, los métodos de identificación molecular tienen también limitaciones; la mayoría de las bases de datos de secuencias de ADN están fuertemente orientadas hacia los nematodos fitoparásitos, los cuales representan sólo una parte de la comunidad edáfica de nematodos, mientras que hay poca información disponible de nematodos de vida libre a pesar de representar la mayoría de los nematodos edáficos. Este trabajo se centra en el estudio de los efectos de fumigantes edáficos en la funcionalidad del suelo a través del uso de diferentes indicadores basados en la comunidad de nematodos, como los índices de la red trófica, índices de diversidad, abundancia de los taxones más relevantes etc. También se han analizado otros indicadores funcionales relacionados con la supresividad edáfica, el ciclo de nutrientes o la actividad de la microfauna del suelo. En el capítulo 1, la diversidad de nematodos estudiada en una explotación comercial de fresa y sus alrededores durante dos campañas consecutivas en el suroeste español, fue baja en los suelos fumigados con fumigantes químicos ambas campañas y, aunque se observó una recuperación a lo largo de la campaña en la zona tratada, los suelos fumigados mostraron una condición perturbada permanente. La comunidad de nematodos estuvo más asociada al ciclo de nutrientes en la zona sin cultivar que en los suelos cultivados, y se observó poca relación entre la biomasa de las plantas y la estructura de la comunidad de nematodos. Los surcos sin tratar dentro de la zona de cultivo funcionaron como reservorio tanto de nematodos fitoparásitos como beneficiosos; sin embargo estas diferencias entre los surcos y los lomos de cultivo no fueron suficientes para mantener la supresividad edáfica en los surcos. Los suelos tratados fueron menos supresivos que los suelos sin tratar, y se observaron correlaciones positivas entre la supresividad edáfica y la estructura de la red trófica edáfica y la diversidad de nematodos. En el capítulo 2, se evaluaron los efectos de dos pesticidas orgánicos con efecto nematicida y dos nematicidas convencionales sobre las propiedades físico químicas del suelo, la diversidad de nematodos y la biomasa de las plantas en condiciones experimentales en dos tipos de suelo: suelos agrícolas poco diversos y suelos provenientes de una zona de vegetación natural muy diversos. El mayor efecto se observó en el tratamiento con neem, el cual indujo un gran incremento en el número de dauerlarvas en los suelos pobres en nutrientes, mientras que el mismo tratamiento indujo un incremento de poblaciones de nematodos bacterívoros, más estables y menos oportunistas, en los suelos del pinar ricos en materia orgánica. En el capítulo 3, se comparó la eficacia de métodos moleculares (TRFLP, Terminal Restriction Fragment Length Polymorphism) y morfológicos (microscopía de alta resolución) para la identificación de diferentes comunidades denematodos de España e Irlanda. Se compararon estadísticamente las diferencias y similitudes en la diversidad de nematodos, otros indicadores ecológicos y de la red trófica edáfica. Las identificaciones mediante el uso de TRFLP sólo detectó un porcentaje de los taxones presentes en las muestras de suelo identificadas morfológicamente, y los nematodos omnívoros y predadores no fueron detectados molecularmente en nuestro estudio. Los índices calculados en base a los nematodos micróboros mostraron más similitud cuando se identificaron morfológica y molecularmente que los índices basados en grupos tróficos más altos. Nuestros resultados muestran que, al menos con la técnica usada en este estudio, la identificación morfológica de nematodos es una herramienta fiable y más precisa que la identificación molecular, puesto que en general se obtiene una mayor resolución en la identificación de nematodos. En el capítulo 4, se estudiaron también los efectos de los nematicidas químicos sobre la comunidad de nematodos y la biomasa de las plantas en condiciones experimentales de campo, donde se aplicaron en una rotación de cultivo judía-col durante un ciclo de cultivo. Se aplicaron dos tipos de enmiendas orgánicas con el objetivo de mitigar el efecto negativo de los productos fitosanitarios sobre la diversidad edáfica. El efecto de los nematicidas sobre las propiedades del suelo y sobre la comunidad de nematodos fue más agudo que el efecto de las enmiendas. La incorporación de los restos de cosecha al final del ciclo de cultivo de la judía tuvo un gran efecto sobre la comunidad de nematodos, y aunque el número total de nematodos incrementó al final del experimento, se observó una condición perturbada permanente de la red trófica edáfica a lo largo del experimento. ABSTRACT Due to the uncertain future of the soil fumigants most commonly used in the EU, that might involve risks for human/animal health and the environment, there is a need to develop new integrated pest management programs, included as mandatory in the Regulation (EC) No. 1107/2009, to control crop diseases. According to this Regulation, evaluating the risk associated to the use of the plant production products (PPP) on non-target soil fauna and their function, and developing assays with different indicator species to obtain toxicity data to be used in the risk evaluation is mandatory. However, the low representativeness of some of these indicator species in the Mediterranean area is a relevant limitation. In this situation, the Scientific Panel of Plant Protection Products and their Residues of the European Food Safety Authority (EFSA) has pointed out the necessity of modifying the ecotoxicological data set required to evaluate non-target effects of PPP in a more integrated way, including structural and functional endpoints with organism such as bacteria, fungi, protists and nematodes. Thus, EFSA has recommended the use of nematodes in the assessment of the functional and structural features of the soil. Nematodes are globally distributed and morphologically diverse, and due to their high abundance and diversity of responses to soil disturbance, they are suitable indicators of the soil condition. Since nematodes interact with many other organisms as participants in several links of the soil food web, playing important roles in essential soil processes in agroecosystems, nematode diversity is often used as a biological indicator of the effects of agricultural practices on soil condition. In the last years, various indices based on soil nematode assemblages, have facilitated the interpretation of complex soil ecological data. Soil food web indices based on the abundances of functional guilds defined by C-P groups and trophic groups, permit evaluating soil food web functioning. On the other hand, the difficulty of nematode taxonomical identification is commonly argued to explain their limited used as ecological indicators, and there is a certain controversy in terms of the efficacy of various nematode identification methods. It is argued that the morphological identification is difficult and time consuming due to the lack of specialist knowledge, and it is claimed that molecular techniques can solve some limitations of morphological techniques such as the identification of juveniles. Nevertheless, molecular identification methods are limited too, since most of the DNA-based databases are strongly oriented towards plant-parasitic nematodes that represent only a fraction of the soil nematode community, while there is little information available on free-living nematodes, which represent most soil nematodes. This work focuses on the study of the effects of soil fumigants on soil functioning through the use of different indicators based on soil nematode community as soil food web indices, diversity indices, the abundance of more relevant taxa etc. Other functional indicators related to soil suppressiveness, nutrient cycling, or the activity of soil microfauna have been also studied. In chapter 1, nematode diversity assessed in a commercial strawberry farm and its surroundings for two consecutive growing seasons in southern Spain, was low in fumigated soils with chemical pesticides throughout both seasons and, although yearly recovery occurred within the treated fields, fumigated soils showed a permanent perturbed condition. The nematode community was more closely associated to nutrient cycling in the non-cropped than in the cropped soils, and the link between plant biomass and nematode community structure was weak. Non-treated furrows within the treated fields were a reservoir of both beneficial and plant-parasitic nematodes, but such difference between furrows and beds was not enough to maintain more suppressive soil assemblages in the furrows. Treated soils were less suppressive than unmanaged soils, and there was a positive and significant correlation between soil suppressiveness and soil food web structure and diversity. In chapter 2, the effects of two organic pesticides with nematicide effect and two chemical nematicides on soil physicalchemical properties, soil nematode diversity and plant biomass in experimental conditions were assessed in two types of soils: low diversity soils from an agricultural farm, and high diversity soils from a natural vegetation area. The larger effect was observed on the neem treatment, which induced a large boost of dauer juveniles in the nutrient-depleted soil, while the same treatment induced the increase of more stable, less opportunistic, populations of generalist bacterivore nematodes in the pine forest soil, rich in organic matter. In chapter 3, comparison of the efficiency of molecular (TRFLP, Terminal Restriction Fragment Length Polymorphism) and morphological (microscopy at high magnification) identification methods was carried out in different nematode communities from five sites of different land uses in Spain and Ireland. Differences and similarities on nematode diversity and other ecological and soil food web indices assessed by both methods, were statistically compared. Molecular identification with TRFLP only detected a percentage of the taxa present in the soil samples identified morphologically, and omnivores and predators were not detected molecularly in our study. Indices involving microbial feeding nematodes were more similar between identification methods than indices involving higher trophic links. Our results show that, at least with the technique used in this study, identifying nematodes morphologically is a reliable and more precise identification tool than molecular identification, since a higher taxonomic resolution is in general obtained compared to TRFLP. In chapter 4, the effect of chemical nematicides on nematode community descriptors and plant biomass was also studied in field conditions in an experimental area in which dazomet and dimethyl disulfide was applied in a bean-cabbage rotation system for a single season. Organic amendments were incorporated into the soil with the aim of mitigate the negative effect of the pesticides on soil diversity. The effect of the nematicides was much more noticeable than the effect of the amendments on soil properties and nematode community descriptors. The incorporation of bean crop residues into the soil at the end of bean crop cycle affected soil nematode community descriptors to a great extent, and although total number of nematodes increased at the end of the experiment, a permanent perturbed soil food web condition was observed along the experiment.

Three-dimensional tracking of motile bacteria near a solid planar surface.

Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers. In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface. Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria. The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al. [Ramia, M., Tullock, D. L. & Phan-Tien, N. (1993) Biophys J. 65,755-778], which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate. The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces. The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.