Covering ground: insights into soil ecology by molecular monitoring of nematode assemblages

Soil performs numerous functions, which allow us to produce food and feed and provide us with clean freshwater. These functions rely on the high diversity of organisms residing in soils. Within the high complexity of the soil food web, nematodes, worm-shaped animals belonging to the phylum Nematoda, are an informative group for assessing the status of a soil-dwelling community due to their ubiquity, abundance and trophic diversity. Although nematodes also possess several other assets favourable for a biological indicator of soil ecosystems (e.g. easy extractability, differential sensitivities to disturbances, ecological interpretability), their microscopic identification demands a considerable amount of expertise and time because of their relatively conserved morphology. Hence, the use of a molecular method for the identification and quantification of nematode assemblages has the potential to lift practical limitations and allows for more intensive sampling schemes. The aim of the research described in this thesis was to assess the suitability of molecular taxon-specific assays, developed on the basis of a phylum-wide molecular framework of 2,400 full-length small subunit ribosomal DNA sequences, for the monitoring of nematode assemblages in field experiments. The method was applied to monitor the impact of different types of disturbances on the soil food web, i.e. agricultural practices, invasive plant species and the effects of genetically modified crop (potato). The second chapter of this thesis presents the background of the molecular method and the results of its first field application. It demonstrates the suitability of this method for use in extensive field experiments and the results of this study reveal distinct seasonal fluctuations between nematode genera classified to belong to the same feeding type group. A distinct response of nematode genera within trophic groups – taxonomically diverse groups of nematodes having the nature of their main food source as a common denominator – was also observed in the study described in Chapter 3. In this chapter, a study was conducted to investigate the impact of an invasive plant species called Giant goldenrod (Solidago gigantea, native to North America) on the plant community as well as on different trophic levels of the soil food web. In addition to monitoring the nematode community, pH and fungal biomass were measured in plots invaded or uninvaded by Giant goldenrod in two contrasting habitats. The results revealed that, in addition to outcompeting native plant species, this invader also reduced pH and increased fungal biomass in the soil of both habitats. Based on the results concerning the nematode community, the impact on the soil food web seemed to be selective since the local increase of fungal biomass appeared to benefit only one fungivorous nematode lineage of the three present in the field. This suggests that invasion by Giant goldenrod only stimulates one part of the fungal community. Contrary to the preceding chapters, the effects of different disturbances on the nematode community were studied in an arable setting in chapters 4 and 5. Chapter 4 describes a field experiment in which the impact of biofumigation, a pest control measure, on the nematode community was assessed. Biofumigation is considered as an alternative for the use of synthetic fumigants and entails the incorporation of mulched brassicaceous plant material, which, upon de-compartmentalisation, releases general biocides called isothiocyanates. In our experiment, these compounds as well as their precursors could not be related to the effects observed for the nematode community. Therefore, changes in nematode assemblages are more likely to be related to the intense mechanical disturbance and green manure – the addition of a large quantity of fresh plant material to the topsoil – rather than the release of isothiocyanates from the plant material Chapter 5 presents a field experiment in which the possible belowground side effects of a waxy starch GM potato, a genetically modified plant blocked in its amylose biosynthesis, were investigated. The nematode community was monitored during the growing season of this GM variety, its parental line and four other conventional potato cultivars in two experimental fields. Although we observed clear effects of location and time, no GM-related effects were observed on the nematode community. Our results, in line with previous studies concerning the microbial community, indicate there are no observable, non-transient effects related to this particular GM trait on the soil food web during the growing season. Overall, the results presented in this thesis demonstrate that, first of all, the developed molecular approach is suitable as a tool for the quantitative monitoring of nematode assemblages in field experiments, and, secondly, how a molecular monitoring method based on nematode taxon-specific DNA motifs can be exploited to get new insights into the ecology of terrestrial nematodes and – more in general – into the ecological functioning of this obscure, highly biodiverse and poorly understood habitat below our feet.</p

Розумний будинок на платформі Arduino

Проекти «розумних» будинків на даний момент дуже активно обговорюються і реалізуються в усьому світі. Мета проекту «Розумний будинок» розробити систему для автоматичного керування освітленням, температурою, вологістю, сигналізацією в кімнаті, квартирі, гаражі, теплиці, системи автополиву і т.д

SSU Ribosomal DNA-Based Monitoring of Nematode Assemblages Reveals Distinct Seasonal Fluctuations within Evolutionary Heterogeneous Feeding Guilds

Soils are among the most complex, diverse and competitive habitats on Earth and soil biota are responsible for ecosystem services such as nutrient cycling, carbon sequestration and remediation of freshwater. The extreme biodiversity prohibits the making of a full inventory of soil life. Hence, an appropriate indicator group should be selected to determine the biological condition of soil systems. Due to their ubiquity and the diverse responses to abiotic and biotic changes, nematodes are suitable indicators for environmental monitoring. However, the time-consuming microscopic analysis of nematode communities has limited the scale at which this indicator group is used. In an attempt to circumvent this problem, a quantitative PCR-based tool for the detection of a consistent part of the soil nematofauna was developed based on a phylum-wide molecular framework consisting of 2,400 full-length SSU rDNA sequences. Taxon-specific primers were designed and tested for specificity. Furthermore, relationships were determined between the quantitative PCR output and numbers of target nematodes. As a first field test for this DNA sequence signature-based approach, seasonal fluctuations of nematode assemblages under open canopy (one field) and closed canopy (one forest) were monitored. Fifteen taxa from four feeding guilds (covering ~ 65% of the free-living nematode biodiversity at higher taxonomical level) were detected at two trophic levels. These four feeding guilds are composed of taxa that developed independently by parallel evolution and we detected ecologically interpretable patterns for free-living nematodes belonging to the lower trophic level of soil food webs. Our results show temporal fluctuations, which can be even opposite within taxa belonging to the same guild. This research on nematode assemblages revealed ecological information about the soil food web that had been partly overlooked

Ideas to improve the efficacy and the robustness of bio-fumigation

Plants use at least two main strategies to protect themselves against pathogens and herbivores: by a physical barrier making the plant content inaccessible to the invader and/or by the production of appetite spoiling or even toxic components (or the precursors thereof). Apart from shaping the habitus of plants, cell walls, a rigid and tightly interwoven fabric consisting mainly of polysaccharides, constitute a formidable physical barrier. The primary cell wall is a relatively thin layer made up of carbohydrates and structural proteins. The secondary cell wall is a thicker layer rich in cellulose and lignin, polymers that greatly contribute to the physical strength of the plant cell wall. Combined the two cell wall elements constitute a compact protective layer. On top of this physical barrier, plants may produce secondary metabolites to reduce its attractiveness as a food source for pathogens and herbivores. Some plant families are well known by the production of specific categories of metabolites that reduce their attractiveness for herbivores. The plantain family (Plantaginaceae) harbours a wide diversity of genera including Plantago (about 200 species). Some Plantago species are known for their anti-toxic and antimicrobial characteristics. Iridoid glucosides such as aucubin and catalpol are terpenoid secondary metabolites produced by a range of plantain species, and the components make these plants unattractive for a wide spectrum of herbivores. Upon cleaving of the sugar residue by beta-glucosidases either from the plant or from the digestive tract of the invading organism, the resulting aglycon acts un-specifically by cross linking proteins and the inhibitions of enzymes. Iridoids are a class of compounds with as a core a cyclopentane fused to a 6-C oxygen heterocycle. All by all, iridoid glucosides are precursors of defence molecules against a broad range of pathogens and herbivores (Dobler et al., 2011). Members of the buttercup family (Ranunculaceae) produce another category of non-specific anti-feedants. The genus Ranunculus L. (buttercups) comprises about 600 herbaceous species, and fresh buttercup plant parts are poisonous for a wide range of organisms. This can be explained by a secondary metabolite named ranunculin. Upon de-compartimentation (chewing, maceration etc.), the enzyme beta-glucosidase, normally stored in the vacuole, comes into contact with ranunculin, and as a result this glucoside is broken down to form the volatile lactone protoanemonin (C5H4O2). This highly reactive component has anti-microbial properties, and ingestion by herbivorous mammals results in erythema and blistering (Sedivy et al., 2012; Martin et al., 1990). Yet another plant family that produces relatively harmless glycosides that are turned into toxic compounds upon the mechanical damaging of plant tissues is the mustard family (Brassicaceae). Over 100 types of glucosinolates are produced by members of this family. This class of secondary metabolites are the most important flavour compounds in edible representatives such as cabbage, 2 mustard and horseradish. The actual reason why these compounds are produced is (again) the defence against pathogens and herbivores. Just like we’ve seen for the Plantaginaceae and the Ranunculaceae, Brassicaceae produce precursors that are converted into toxic components upon de-compartimentation. The activating enzyme myrosinase (a thioglucoside glucohydrolase) is stored in the vacuole. Upon damaging or maceration of the plant tissue, myrosinase cleaves off the thio-linked glucose from its substrate, and the resulting chemically-unstable aglycone is converted into toxic isothiocyanate or related components (Textor &amp; Gershenzon, 2009). The ban of certain categories of soil fumigantia and the severe restrictions with regard to the application of those fumigants that are still on the market to control soil-borne diseases including plant-parasitic fungi and nematodes have been the driving factors behind a search to find effective environmentally acceptable alternatives. One of the options was the use specific types of green manure. Incorporation in the topsoil of plant material harbouring compounds (or precursors thereof) that are toxic for a range of soil borne pathogens is called biofumigation. As compared to aglycones such as protoamonin from buttercups or iridoids from plantain species, the effective products released upon mechanically damaging Brassicaceous plant parts are relatively stable and volatile. Hence it will spread easily throughout the topsoil. In fact, the incorporation of mulched Brassicaceous material could be considered as a natural replacement of the soil fumigant metam sodium (Matthiessen &amp; Kirkegaard, 2006). Recently we investigated in a field experiment in the western part of Germany the impact of Indian mustard (Brassica juncea) on plant parasitic and free-living nematode taxa (Vervoort et al., 2014). Just before the incorporation of plant material in the topsoil, aliquots of above and below plant parts were sampled and the glucosinolate content was determined both qualitatively and quantitatively. As a positive control 2-propenyl ITC was directed applied to the control plots. Although the impact of the incorporation of mulched Indian mustard material on the nematode community was clear and significant, this impact could not be attributed the effect of isothiocyanates on the nematode community. The severe physical disturbance of the soil in combination with addition of a large quantity of green manure alone sufficed to explain the observed changes. Even more remarkable: the positive control treatments by direct application of 2-propenyl ITC (two concentrations, the highest being two times the concentration predicted for the highest producing Indian mustard cultivar) had no significant effect on the nematode taxa under investigation. We cannot rule out the possibility that local circumstances such as soil type, organic matter type and content, moisture content and/or temperature of the soil at the day of biofumigation can be explanatory factors for the absence of a clear glucosinolate-related response. Our results suggest that a more widely acceptance of bio-fumigation as a tool to manage soil borne pathogens would require an increase in the efficacy and the robustness of this tool. In this paper we pay attention to some other plant families that a producing broad-spectrum toxic components (aglycones) upon maceration and/or physical damage. It could be worthwhile to investigate the impact of the simultaneous release of multiple plant-derived aglycones on plant pathogenic soil biota. The two ‘other’ plant families highlighted in this paper, the Plantaginaceae and the Ranunculaceae, should just be considered as examples (not necessarily the best ones) of families producing distinct broad-spectrum anti-microbial aglycones. We hypothesize that simultaneous exposure of soil pathogens to multiple plant defense-related aglycones could contribute to an improved efficacy and robustness of biofumigation

Assessment of a GM-crop impact on soil systems using the DNA barcode-based tool for nematode community analysis

The RIVM has developed with the Wageningen University (WUR) a new technique by which the soil quality can be determined accurately, the so-called nematode DNA barcode tool. This molecular method provides faster and more detailed information about disturbances in soil quality and the possible causes. This can be done because this novel information is combined with data on the overall processes by which crops are grown. Examples are the use of pesticides and effects on the soil systems of agricultural techniques such as ploughing and fertilizing. In this way a better understanding of the influences on soil quality of agricultural practices, such as genetically modified (GM) crops, can be achieved

Assessment of a GM-crop impact on soil systems using the DNA barcode-based tool for nematode community analysis

The RIVM has developed with the Wageningen University (WUR) a new technique by which the soil quality can be determined accurately, the so-called nematode DNA barcode tool. This molecular method provides faster and more detailed information about disturbances in soil quality and the possible causes. This can be done because this novel information is combined with data on the overall processes by which crops are grown. Examples are the use of pesticides and effects on the soil systems of agricultural techniques such as ploughing and fertilizing. In this way a better understanding of the influences on soil quality of agricultural practices, such as genetically modified (GM) crops, can be achieved

Data from: The differential impact of a native and a non-native ragwort species (Senecioneae) on the first and second trophic level of the rhizosphere food web

Whereas the impact of exotic plant species on above-ground biota is relatively well-documented, far less is known about the effects of non-indigenous plants on the first and second trophic level of the rhizosphere food web. Here, rhizosphere communities of the invasive narrow-leaved ragwort Senecio inaequidens and the native tansy ragwort Jacobaea vulgaris, co-occurring in three semi-natural habitats are compared. For both species, two life stages were taken into consideration. Quantitative PCR assays for the analyses of bacterial and fungal communities at a high taxonomic level were optimized, and it was investigated whether changes in the primary decomposer community were translated in alterations in bacterivorous and fungivorous nematode communities. In contrast to J. vulgaris, small but significant reductions were observed for Actinobacteria and Bacteroidetes (both p < 0.05) in case of the invasive S. inaequidens. More pronounced changes were detected for the overall nematode community density, and, more specifically, for the bacterivorous genus Anaplectus and the family Monhysteridae (both p < 0.05), as well as the necromenic Pristionchus (p < 0.001). At high taxonomic level, no differences were observed in fungal rhizosphere communities between native and non-native ragwort species. The impact of plant developmental stages on rhizosphere biota was prominent. The overall bacterial and fungal biomasses, as well as a remarkably consistent set of constituents (Actinobacteria, α- and β-Proteobacteria and Bacteroidetes) were negatively affected by plant stage for both ragwort species. Although later developmental stages of plants generally coincided with lower levels for individual nematode taxa, densities of the fungivorous genera Diphtherophora and Tylolaimophorus remain unaltered. Hence, even at a high taxonomic level, differential effects of native and non-native ragwort could be pinpointed. However, plant developmental stage has a more prominent impact and this impact was similar in nature for both native and non-native ragwort species

The differential impact of a native and a non-native ragwort species (Senecioneae) on the first and second trophic level of the rhizosphere food web

Whereas the impact of exotic plant species on above-ground biota is relatively well-documented, far less is known about the effects of non-indigenous plants on the first and second trophic level of the rhizosphere food web. Here, rhizosphere communities of the invasive narrow-leaved ragwort Senecio inaequidens and the native tansy ragwort Jacobaea vulgaris, co-occurring in three semi-natural habitats are compared. For both species, two life stages were taken into consideration. Quantitative PCR assays for the analyses of bacterial and fungal communities at a high taxonomic level were optimized, and it was investigated whether changes in the primary decomposer community were translated in alterations in bacterivorous and fungivorous nematode communities. In contrast to J. vulgaris, small but significant reductions were observed for Actinobacteria and Bacteroidetes (both p < 0.05) in case of the invasive S. inaequidens. More pronounced changes were detected for the overall nematode community density, and, more specifically, for the bacterivorous genus Anaplectus and the family Monhysteridae (both p < 0.05), as well as the necromenic Pristionchus (p < 0.001). At high taxonomic level, no differences were observed in fungal rhizosphere communities between native and non-native ragwort species. The impact of plant developmental stages on rhizosphere biota was prominent. The overall bacterial and fungal biomasses, as well as a remarkably consistent set of constituents (Actinobacteria, α- and β-Proteobacteria and Bacteroidetes) were negatively affected by plant stage for both ragwort species. Although later developmental stages of plants generally coincided with lower levels for individual nematode taxa, densities of the fungivorous genera Diphtherophora and Tylolaimophorus remain unaltered. Hence, even at a high taxonomic level, differential effects of native and non-native ragwort could be pinpointed. However, plant developmental stage has a more prominent impact and this impact was similar in nature for both native and non-native ragwort species