Biodegradation of eucalyptus urograndis wood by fungi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assessment of fungi in soils of sugarcane crops and their potential for production of biomass-degrading enzymes

Soil management practices are konwn to affect the biomass and enzyme activities of microbial soil communities. To assess whether burning of sugarcane prior to harvesting affects the community of soilborne fungi, we collected soil simples in two sites: burned sugarcane culture prior harvesting (BS) and non-burned sugarcane culture (NBS). A total of 75 filamentous fungal isolates were recovered from soils in both sites. Trichoderma was the most prevalent genus in both sites, followed by Fusarium, Cunninghamella and Aspergillus. The Sorensen's index (0.60) suggested a slight difference in fungi associated with both areas, with high number of fungal isolates found on BB soil. The abundance of Trichoderma isolates in NBS soil was higher than BS soil; however, the abundance of Fusarium, Aspergillus and Cunninghamella was higher in the latter type of soil. In addition, fungi isolated from BS soil showed the highest production of xylanase and laccase in comparision with fungi isolated form NBS soil. Our results indicate that the different types of sugarcane harvesting apparently did not interfere with the diversity of fungal communnities as revealed by culture-dependent methods. In addition, our data indicates the potencial of fungi from soils of sugarcane crops to produce relevant enzymes related to biomass conversion.

Biodegradation of PP and PE blended with PHBV in soil samples

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PHOSPHORUS DOSES DETERMINE THE PREVALENCE OF NATIVE ARBUSCULAR MYCORRHIZAL FUNGI IN Araucaria angustifolia

A greenhouse experiment was installed with bait cultures to extract the AMF species present in a rhizosphere soil sample of a native Araucaria angustifolia forest in Campos do Jordao, Brazil. The experimental design was completely randomized, with four increasing phosphorus doses (0, 20, 50, and 150 mg kg(-1), as triple superphosphate), with five replicates, the bait plant was araucaria, and all pots were inoculated with 100 g of rhizospheric soil collected in an araucaria forest. After twelve months the spores were extracted, counted and identified, and the percent root colonization was also determined. When taking all four P doses into account, eleven AMF species could be identified: Acaulospora bireticulata, Acaulospora morrowiae, Acaulospora sp., Entrophospora colombiana, Gigaspora margarita, Glomus diaphanum, Glomus etunicatum, Glomus macrocarpum, Scutellospora calospora, Scutellospora gilmorei, and Scutellospora pellucida. There was no effect of the P dose on the total amount of spores neither on the percent root colonization. However, the correspondence analysis showed that the different AMF species were selectively associated mostly to either one or another P dose.

ISOLATION OF MICROSPORUM GYPSEUM IN SOIL SAMPLES FROM DIFFERENT GEOGRAPHICAL REGIONS OF BRAZIL, EVALUATION OF THE EXTRACELLULAR PROTEOLYTIC ENZYMES ACTIVITIES (KERATINASE AND ELASTASE) AND MOLECULAR SEQUENCING OF SELECTED STRAINS

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Differences in Soil Fungal Communities between European Beech (Fagus sylvatica L.) Dominated Forests Are Related to Soil and Understory Vegetation

Fungi are important members of soil microbial communities with a crucial role in biogeochemical processes. Although soil fungi are known to be highly diverse, little is known about factors influencing variations in their diversity and community structure among forests dominated by the same tree species but spread over different regions and under different managements. We analyzed the soil fungal diversity and community composition of managed and unmanaged European beech dominated forests located in three German regions, the Schwäbische Alb in Southwestern, the Hainich-Dün in Central and the Schorfheide Chorin in the Northeastern Germany, using internal transcribed spacer (ITS) rDNA pyrotag sequencing. Multiple sequence quality filtering followed by sequence data normalization revealed 1655 fungal operational taxonomic units. Further analysis based on 722 abundant fungal OTUs revealed the phylum Basidiomycota to be dominant (54%) and its community to comprise 71.4% of ectomycorrhizal taxa. Fungal community structure differed significantly (p≤0.001) among the three regions and was characterized by non-random fungal OTUs co-occurrence. Soil parameters, herbaceous understory vegetation, and litter cover affected fungal community structure. However, within each study region we found no difference in fungal community structure between management types. Our results also showed region specific significant correlation patterns between the dominant ectomycorrhizal fungal genera. This suggests that soil fungal communities are region-specific but nevertheless composed of functionally diverse and complementary taxa.

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.

Soil property and management effects on grassland microbial communities across a latitudinal gradient in Germany

There is much interest in the identification of the main drivers controlling changes in the microbial community that may be related to sustainable land use. We examined the influence of soil properties and land-use intensity (N fertilization, mowing, grazing) on total phospholipid fatty acid (PLFA) biomass, microbial community composition (PLFA profiles) and activities of enzymes involved in the C, N, and P cycle. These relationships were examined in the topsoil of grasslands from three German regions (Schorfheide-Chorin (SCH), Hainich-Dun (HAI), Schwabische Alb (ALB)) with different parent material. Differences in soil properties explained 60% of variation in PLFA data and 81% of variation in enzyme activities across regions and land-use intensities. Degraded peat soils in the lowland areas of the SCH with high organic carbon (OC) concentrations and sand content contained lower PLFA biomass, lower concentrations of bacterial, fungal, and arbuscular mycorrhizal PLFAs, but greater enzyme activities, and specific enzyme activities (per unit microbial biomass) than mineral soils in the upland areas of the HAI and ALB, which are finer textured, drier, and have smaller OC concentrations. After extraction of variation that originated from large-scale differences among regions and differences in land-use intensities between plots, soil properties still explained a significant amount of variation in PLFA data (34%) and enzyme activities (60%). Total PLFA biomass and all enzyme activities were mainly related to OC concentration, while relative abundance of fungi and fungal to bacterial ratio were mainly related to soil moisture. Land-use intensity (LUI) significantly decreased the soil C:N ratio. There was no direct effect of LUI on total PLFA biomass, microbial community composition, N and P cycling enzyme activities independent of study region and soil properties. In contrast, the activities and specific activities of enzymes involved in the C cycle increased significantly with LUI independent of study region and soil properties, which can have impact on soil organic matter decomposition and nutrient cycling. Our findings demonstrate that microbial biomass and community composition as well as enzyme activities are more controlled by soil properties than by grassland management at the regional scale. (C) 2013 Elsevier B.V: All rights reserved.

Are ectomycorrhizal fungi alleviating or aggravating nitrogen limitation of tree growth in boreal forests?

•Symbioses between plant roots and mycorrhizal fungi are thought to enhance plant uptake of nutrients through a favourable exchange for photosynthates. Ectomycorrhizal fungi are considered to play this vital role for trees in nitrogen (N)-limited boreal forests. •We followed symbiotic carbon (C)–N exchange in a large-scale boreal pine forest experiment by tracing 13CO2 absorbed through tree photosynthesis and 15N injected into a soil layer in which ectomycorrhizal fungi dominate the microbial community. •We detected little 15N in tree canopies, but high levels in soil microbes and in mycorrhizal root tips, illustrating effective soil N immobilization, especially in late summer, when tree belowground C allocation was high. Additions of N fertilizer to the soil before labelling shifted the incorporation of 15N from soil microbes and root tips to tree foliage. •These results were tested in a model for C–N exchange between trees and mycorrhizal fungi, suggesting that ectomycorrhizal fungi transfer small fractions of absorbed N to trees under N-limited conditions, but larger fractions if more N is available. We suggest that greater allocation of C from trees to ectomycorrhizal fungi increases N retention in soil mycelium, driving boreal forests towards more severe N limitation at low N supply.

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.

Initial vegetation, and spore density, diversity and evenness of different arbuscular mycorrhizal fungi morphotaxa 29,36 and 39 months after experimental vegetation removal, central Argentina

1. Dominant plant functional types (PFTs) are expected to be primary determinants of communities of other above- and below-ground organisms. Here, we report the effects of the experimental removal of different PFTs on arbuscular mycorrhizal fungi (AMF) communities in a shrubland ecosystem in central Argentina. 2. On the basis of the biomass-ratio hypothesis and plant resource use strategy theory, we expected the effect of removal of PFTs on AMF colonization and spores to be proportional to the biomass removed and to be stronger when more conservative PFTs were removed. The treatments applied were: undisturbed control (no plant removed), disturbed control (mechanical disturbance), no shrub (removal of deciduous shrubs), no perennial forb (removal of perennial forbs), no graminoid (removal of graminoids) and no annual forb (removal of annual forbs). AMF colonization was assessed after 5,17 and 29 months. Total density of AMF spores, richness and evenness of morpho-taxa, and AMF functional groups were quantified after 5,17,29,36 and 39 months. 3. Five months after the initial removal we found a significant reduction in total AMF colonization in all plots subjected to PFT removals and in the disturbed control plots, as compared with the undisturbed controls. This effect disappeared afterwards and no subsequent effect on total colonization and colonization by arbuscules was observed. In contrast, a significant increase in colonization by vesicles was observed in months 17 and 29, mainly in no graminoid plots. In general, treatments did not significantly affect AMF spores in the soil. On the other hand, no annual forb promoted transient (12-18 months) higher ammonia availability, and no shrub promoted lower nitrate availability in the longer term (24-28 months). 4. Synthesis. Our experiment, the first to investigate the effects of the removal of different PFTs on AMF communities in natural ecosystems, indicates that AMF communities are resilient to changes in the soil and in the functional composition of vegetation. Furthermore, it does not provide consistent evidence in support of the biomass-ratio hypothesis or differential trait-based direct or indirect effects of different PFTs on AMF in this particular system.

Does the natural "microcosm" created by Tuber aestivum affect soil microarthropods? A new hypothesis based on Collembola in truffle culture

microarthropods play an important role in fungi dispersion, but little is still known about the interaction between truffle and soil microarthropods. The aim of this study was to investigate the ability of the truffle Tuber aestivum to modify soil biogeochemistry (i.e. create a zone of scarce vegetation around the host plant, called a burn or brûlé) and to highlight the effects of the brûlé on the soil fauna community. We compared soil microarthropod communities found in the soil inside versus outside the T. aestivum brûlé with the chemistry of soil collected inside versus outside the brûlé. The study was carried out in three Mediterranean areas, two in Italy and one in Spain. The results confirmed the ability of T. aestivum to modify soil biogeochemistry in the brûlé: pH was higher and total organic carbon tended to be lower inside the brûlé compared to outside. Soil fauna communities showed some interesting differences. Some groups, such as Symphyla and Pauropoda, adapted well to the soil; some Collembolan families, and biodiversity and soil quality indices were generally higher outside the brûlé. Folsomia sp. showed higher abundance in the soil of the brûlé compared to outside. The results suggest that some Collembola groups may be attracted by the fungal metabolites produced by T. aestivum, while other Collembola and other microarthropods may find an unfavourable environment in the soil of the brûlé. The next steps will be to confirm this hypothesis and to extend the study to other keys groups such as nematodes and earthworms and to link fluctuations of soil communities with the biological phases of truffle growth.

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.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

A PCR based method to detect Russula spp. in soil samples and Limodorum abortivum roots in Mediterranean environments

Aim of study. Orchidaceae has the largest number of species of any family in the plant kingdom. This family is subject to a high risk of extinction in natural environments, such as natural parks and protected areas. Recent studies have shown the prevalence of many species of orchids to be linked to fungal soil diversity, due to their myco-heterotrophic behaviour. Plant communities determine fungal soil diversity, and both generate optimal conditions for orchid development. Area of study. The work was carried out in n the two most important natural parks in Alicante (Font Roja and Sierra Mariola), in South-eastern of Spain. Material and Methods. We designed a molecular tool to monitor the presence of Russula spp. in soil and orchids roots, combined with phytosociological methods. Main results. Using a PCR-based method, we detected the presence in the soil and Limodorum abortivum orchid roots of the mycorrhizal fungi Russula spp. The species with highest coverage was Quercus rotundifolia in areas where the orchid was present. Research highlights. We present a useful tool based on PCR to detect the presence of Russula spp. in a natural environment. These results are consistent with those obtained in different studies that linked the presence of the mycorrhizal fungi Russula spp. in roots of the species Limodorum and the interaction between these fungal species and Quercus ilex trees in Mediterranean forest environments.