Description and Molecular Phylogeny of a Novel Hypotrich Ciliate from the Soil of Marche Region, Italy; Including Notes on the MOSYSS Project

The morphology and morphogenesis during cell division of a new stylonychine hypotrich, Rigidocortex quadrinucleatus n. sp., were investigated using live observation and protargol staining. The new species was isolated from soil samples collected from an organic farm in the Marche Region, Italy, in framework of the MOSYSS project. Rigidocortex quadrinucleatus is characterized as follows: cell size about 180 9 80 lm in vivo; four ellipsoidal macronuclear nodules; 44 adoral membranelles: 18 fronto-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse, and five transverse cirri; dorsal kinety 3 with multiple fragmentation; resting cyst with hyaline ridges. Rigidocortex quadrinucleatus mainly differs from the type species R. octonucleatus in having four (vs. eight) macronuclear nodules. Rigidocortex quadrinucleatus can be easily confused with Sterkiella cavicola since both have a rather similar ventral ciliature; however, they can be separated by the slightly higher number of cirri in the left marginal row that runs along the posterior cell’s margin in R. quadrinucleatus. Morphogenesis on the ventral surface is highly similar to that of Sterkiella species, but differs significantly on the dorsal surface (multiple vs. simple fragmentation of dorsal kinety 3). Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to S. cavicola

Biological indicators of soil health in organic viticultures: A case study in the Verdicchio terroir of Matelica (Italy)

Soil health represents the foundation for the production of healthy food. Soil conditions influence the soil capacity for agricultural production and the provision of key ecosystem services. Healthy soils are key contributors to biodiversity and are a prerequisite for sustainable development. According to van Bruggen & Semenov [1] a healthy soil can be defined as “a stable soil, with resilience to stress, high biological diversity, and high levels of internal cycling of nutrients”. Soil biodiversity constitutes one of the main components of agroecosystems, being involved in the delivery of several essential ecosystem services such as, among others, nutrient cycling, soil formation, pest and pollution control. Thus, soil biodiversity indicators can be used by governments and farmers to monitor soil health and ecosystem functioning under various land uses and management practices. At present, organic wine represents an emerging market that is showing a high potential for growth. There is, in fact, a growing worldwide interest and attention for environmental friendly products and sustainable agricultural practices which may ensures both the increase of profitability and the improvement of life and food safety. In this scenario, the aim of our study was to assess the long-term effects of organically managed vineyards on soil health by means of two bioindicators: ciliated protozoa and microarthropods. Ciliated protozoa are eukaryotic microorganisms which constitutes an essential component of the soil microbial loop. By feeding on bacterial biomass ciliates play an essential role in the liberation of nutrients in the plant rhizosphere [2]. Soil microarthropods play crucial roles in regulating ecosystem processes and functions by influencing organic matter decomposition (pre-decomposers) and facilitating the microbial activity, soil porosity and water infiltration [3]. Both ciliates and microarthropods are very sensitive to changes in their habitat and fluctuations in their communities can affect the food web and the energy transfer within the soil ecosystem. Thus, the monitoring of the structure of microarthropod and ciliate communities represents a valuable tool to assess soil health and functioning. The study was realized in the terroir of Verdicchio di Matelica (Marche, Italy), on 3 vineyard plots belonging to the commercial wine farm Collestefano, that were organically managed since 1992 (V92, 19 years), 1998 (V98, 13 years) and 2009 (V09, 2 years) respectively. In each vineyard, soil samples (0-10 cm depth) were taken every month from March to October 2011. For microarthropods, the measured biological parameters were: the Soil Biological Quality (QBS-ar) index [4], abundances of biological and euedaphic forms (BF & EF) and diversity indices. Soil samples were collected in both disturbed (tillage) and not-disturbed (no-tillage) inter-rows. In this regards, the wine farm adopted an alternate management of the tractor-rows on either side of the vine row with tillage and non-tillage annual cover crop species (clover, common vetch and oats). For ciliates: abundances and diversity indices were measured and soil samples were randomly collected in the whole sampling area for a total of 3 sampling (May, June and July 2011). For ciliates, the results of the multivariate analysis (Cluster Analysis and Non-metric Multidimensional scaling) seem to indicate that the most stable habitat for ciliates is represented by the older V92 followed by the V98 and the V09 vineyards. For microarthropods, the comparison between tilled and no-tilled inter-rows in the vineyards V92, V98 & V09 by mean of QBS-ar values and abundances of FB and EF, shows that exsclusively in the oldest V92 vineyard no significant differences were detected between the tilled and no-tilled inter-rows with respect to QBS, EF and BF values by the non parametric Kruskal-Wallis (K-W) test. On the contrary, significant differences between the two differently managed inter-rows were detected for the V98 & V09 vineyards. Thus, and in agreement with ciliate indications, in the V92 the microartrhopod communities remain more stable than in the other vineyards irrespective of the type of disturbance applied (tillage). Overall, our analysis shows that the biological communities experience less fluctuations (>stable) in the “older” vineyards compared with the “younger” V09 vineyard (V92>V98>V09). As reported also by other authors [4-5], this effect may be due to the greater resilience of the soils of the vineyards V92 and V98 possible achieved during the long term organic management. In final, this study helps in evaluating the long term effects of common organic vineyard floor management practices as well as, in the definition of possible biotic baseline values to evaluate soil health in vineyards. Furthermore, and in agreement with the guidelines of the European Commission and the Food and Agriculture Organization (FAO) of the United Nations, our study contribute to raising awareness among stakeholders and policy makers on the importance of soil biodiversity in preserving soil health (and food safety) and in assisting them to select, promote and stimulate adequate sustainable farming practices by which to reduce the environmental impact of agriculture and move the world toward a more sustainable food future. References [1] A.H.C. van Bruggen, A.M. Semenov. In search of biological indicators for soil health and disease suppression. App. Soil Ecol. 2000, 15, 13-24 [2] W. Foissner, W. Soil protozoa as bioindicators in agroecosystems, with enphasis on farming practices, biocides and biodiversity. Agric, Ecos. and Env. 1997, 62:,93-103. [3] C. Menta. Soil Fauna Diversity, Function, Soil Degradation, Biological Indices, Soil Restoration in "Biodiversity Conservation and Utilization in a Diverse World" edited by Gbolagade Akeem Lameed, InTech Publishing, 2012, 59-94 [4] V. Parisi, C. Menta. C. Gardi, C. Jacomini, E. Mozzanica.. Microarthropod Communities as a Tool to Assess Soil Quality and Biodiversity: a new Approach in Italy. Agric. Ecos and Env. 2005, 105, 323-333. [4] L.F. Elliot, JM Lynch. Biodiversity and soil resilience in “Soil Resilience and Sustainable Land Use”. D.J. Greenland and I. Szabolcs Eds CAB International, Wallingford UK, 1994, 353-364 [5] F. Stagnari, G. Perpetuini, R. Tofalo, G. Campanelli, F. Leteo, U.D. Vella, M.Schirone, G. Suzzi, M. Pisante. Long-term impact of farm management and crops on soil microorganisms assessed by combined DGGE and PLFA analyses. Frontiers in Microbiology, 2014, 1-1

ASSESSMENT OF BIOLOGICAL SOIL QUALITY IN LONG-TERM ORGANICALLY MANAGED VINEYARDS IN THE VERDICCHIO TERROIR OF MATELICA (MARCHE, ITALY)

Soil biodiversity constitutes one of the main component of agroecosystems, being involved in the delivery of several essential ecosystem services such as, among others, nutrient cycling, soil formation, pest and pollution control. Thus, soil biodiversity indicators can be used by governments and farmers to monitor soil quality and ecosystem functioning under various land uses and management practices. At present, organic wine represents an emerging market that is showing potential for growth. There is, in fact, a growing worldwide interest and attention for environmental friendly products and sustainable agricultural practices. Organic farming employs a set of farming practices that contribute in preserving soil quality. In this scenario, the aim of our study was to assess the long-term effects of organically managed vineyards on soil quality by means of two bioindicators: ciliated protozoa and microarthropods. Ciliated protozoa are eukaryotic microorganisms which constitutes an essential component of the soil microbial loop. By feeding on bacterial biomass ciliates play an essential role in the liberation of nutrients in the plant rhizosphere. Soil microarthropods contribute to the organic matter degradation and dispersal, affect the soil porosity, aeration, water infiltration, modifying and improving soil fertility. Both ciliates and microarthropods are very sensitive to changes in their habitat and fluctuations in their communities can affect the food web and the energy transfer within the soil ecosystem. Thus, the monitoring of the structure of microarthropod and ciliate communities represent a valuable tool to assess soil quality and functioning. The study was realized in the terroir of “Verdicchio di Matelica” (Marche, Italy), on three vineyards that were organically managed since 1992, 1998 and 2009 respectively. In each vineyard, soil samples (0-10 cm depth) were taken every month from March to October 2011. In addition, soil chemical-physical (texture, soil moisture, pH, NPK, OM, C/N, Cu), were measured in each site. For microarthropods, the measured biological parameters were: the Soil Biological Quality (QBS-ar) index, abundances of biological (BF) and euedaphic forms (EF) and diversity indices. Soil samples were collected in both disturbed (tillage) and not-disturbed (no-tillage) inter-rows. For ciliates: abundances and diversity indices were measured and soil samples were randomly collected in the whole sampling area. The results of the multivariate data analysis (Cluster Analysis, CA; non-metric Multi-Dimensional Scaling, nMDS) and diversity indices (H’,J,d) indicate that the most stable habitat for ciliates and microarthropods is represented by the “older” (V92) followed by the V98 and the “younger” V09 vineyards. Collectively, the data seem to indicate that the long-term organic management of the soil contributes to global soil quality in vineyards at least in this particular pedoclimatic area and for the investigated bioindicators. Furthermore, this study helps in the definition of possible biotic baseline values to evaluate soil quality and health in vineyards

Soil biodiversity and sustainable vineyards: hints from the analysis of microarthropods and ciliated protozoa communities

Soil biodiversity constitutes one of the main components of agroecosystems, being involved in the delivery of several essential ecosystem services such as, among others, nutrient cycling, soil formation, pest and pollution control. Thus, soil biodiversity indicators can be used by governments and farmers to monitor soil quality and ecosystem functioning under various land uses and management practices. In this scenario, the aim of our study was to assess the long-term effects of organically managed vineyards on soil quality by means of two bioindicators: protozoan ciliates and microarthropods. The study was realized in the terroir of Verdicchio di Matelica (Marche, Italy), on three vineyards that were organically managed since 1992, 1998 and 2009 respectively. In each vineyard, soil samples (0-10 cm depth) were taken every month from March to October 2011. In addition, soil chemical-physical (texture, soil moisture, pH, NPK, OM, C/N, Cu), were measured in each site. For microarthropods, the measured biological parameters were: the Soil Biological Quality (QBS-ar) index, abundances of Biological (BF) and Euedaphic forms (EF) and diversity indices. Soil samples were collected in both disturbed (tillage) and not-disturbed (no-tillage) inter-rows. For ciliates: abundances and diversity indices were measured and soil samples were randomly collected in the whole sampling area. The results of the multivariate data analysis (Cluster Analysis, CA; non-metric Multi-Dimensional Scaling, nMDS) and diversity indices (H’,J,d) indicate that the most stable habitat for ciliates and microarthropods is represented by the “older” (V92) followed by the V98 and the “younger” V09 vineyards. Collectively, the data seem to indicate that the long-term organic management of the soil contributes to global soil quality in vineyards at least in this particular pedoclimatic area and for the investigated bioindicators. Furthermore, this study helps in the definition of possible biotic baseline values to evaluate soil quality/health in vineyards

THE BIOPRINT PILOT PROJECT: CILIATED PROTOZOA COMMUNITIES AS A TOOL TO ASSESS SOIL QUALITY IN AGROECOSYSTEMS AND NATURAL SITES OF MARCHE REGION (ITALY)

In the recent years the potential of ciliated protozoa, as indicators of soil quality has been highlighted by several studies. Thus, in the framework of the BioPrint Pilot Project and for the first time in Italy, we have investigated the biodiversity and the community structure of soil ciliates from agroecosystems and natural sites of Marche Region. The aims of our study were: i) to evaluate the capacity of ciliates to discriminate between different types of land uses; and ii) farming management practices; iii) to assess relationships among ciliate community and abiotic parameters. Soil samples were collected twice from 10 sites (5 natural sites: FORest; and 5 arable field: 3 ORGanic and 2 CONventional). Ciliate communities were studied by means of qualitative (non-flooded Petri dish) and quantitative methods. Soil chemical-physical (texture, CEC NPK, OM, C/N, soil moisture and temperature) parameters were measured. Qualitative ciliate analysis allowed us to identify a total of 59 species representing 29 genera and 12 orders (plus 10 new species for the science). ORG sites were the richest in species followed by CON and FOR. The mean values for H’ (2.6), d (3.4) and J (0.8) were significantly higher in ORG than in CON (H’=2.1; d=2.7; J=0.7) and FOR (H’=1.7; d=1.95; J=0.6) supporting the intermediate disturbance hypothesis. Multivariate analysis showed statistically significant differences between natural sites (FORest) and agricultural sites, as well as between the ORGanic and CONventional management farming systems. CCA analysis showed correlations between the distribution of species with environmental parameters indicating the importance of these parameters in shaping the ciliate communities in the different type sites. Altogether, these results showed the bioindicative potential of ciliate communities in discriminating between natural sites (FORests) and agroecosystems, as well as their capacity to discriminate, at least preliminary, between different management systems (ORG vs CON)

Shedding light on the polyphyletic behavior of the genus Sterkiella: The importance of ontogenetic and molecular phylogenetic approaches

Present study, investigates a poorly known species of the genus Sterkiella, i.e., S. tricirrata, based on two populations isolated from soil samples collected from the Colfiorito Regional Park, Umbria Region, Italy and from the Silent Valley National Park, India. Both populations showed a highly similar morphology, however different ontogenetic pattern in between. The study confirms the validity of the species S. tricirrata which was considered to be a species within the Sterkiella histriomuscorum complex. The main ontogenetic difference between S. tricirrata and other species of the genus Sterkiella is the different mode of formation of anlagen V and VI of the proter in the former. In the phylogenetic analyses, Sterkiella tricirrata clusters with Sterkiella sinica within the stylonychine oxytrichids, in a clade away from the type species (Sterkiella cavicola) of the genus Sterkiella. The study highlights the importance of ontogenetic as well as molecular data in shedding light on the polyphyletic behavior of the genus Sterkiella. A detailed description of S. tricirrata based on morphology, ontogenesis and molecular phylogenetic methods is presented. Further, the improved diagnosis has been provided for the genus Sterkiella and the poorly known species S. tricirrata

Ciliate diversity and behavioural observations from the chemoautotrophic cave ecosystem of Frasassi, (Marche region, Italy)

Chemoautotrophic cave organisms require specific adaptations to tolerate the stress of living in extreme environmental conditions, such as darkness, nutrient and energy limitations, low temperatures (12-13°C), highly variable sulphide concentrations (from 0 up to 415 μM H2S) and toxic levels of gases (H2S, CO2, CH4). To date, due to the difficulties in sampling in such harsh environment, very few studies were performed in order to describe the ciliate communities from caves; these have remained largely unexplored. Thus, the main aims of our study were to characterize for the first time, the ciliates from the sulfide-rich Frasassi cave complex (Marche region, Italy) and to observe possible behavioural differences with their non-cave-dwelling counterpart ciliate species. Four main sampling sites within Frasassi caves were selected: Pozzo dei Cristalli, Lago Verde, Ramo Solfureo and Grotta Solfurea. The ciliate diversity from the site Pozzo dei Cristalli was studied in greater detail for its spatio-temporal distribution, since it is highly diversified and includes several microhabitats represented by small sulfidic (H2S-rich) ponds, streams and springs as well as, deep and shallow muddy, stagnant lakes. Periodic sampling was realised from 2009 to 2011 in the form of water-sediments, picked up by scraping the surface. Classical culturing, silver staining methods and 18S rRNA gene (for some selected species) for phylogenetic analysis were employed. A total of 31 species belonging to 9 classes, 15 orders and 23 genera were identified. Fluctuation of the ciliate communities were mainly recorded at the Pozzo dei Cristalli sites during various sampling occasions, this could be due to changing environmental conditions (mainly H2S concentrations and water levels). Interestingly, it was observed that some species e.g. Urocentrum turbo, Coleps hirtus hirtus, Oxytricha sp, Euplotes sp, showed adaptation for the cave environment (photo-sensitivity, sulphur tolerance, feeding behaviour, morphological difference). Overall, these results provide a platform for various in-depth studies of ciliates to understand potential role in aquatic microhabitats, nature of chemical compounds secreted, dispersal pattern, and adaptations to cave environment