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The olive tree is a hallmark crop in the Mediterranean region. Its cultivation is characterized by an enormous variability in existing genotypes and geographical areas. As regards the associated microbial communities of the olive tree, despite progress, we still lack comprehensive knowledge in the description of these key determinants of plant health and productivity. Here, we determined the prokaryotic, fungal and arbuscular mycorrhizal fungal (AMF) microbiome in below- (rhizospheric soil, roots) and above-ground (phyllosphere and carposphere) plant compartments of two olive varieties ‘Koroneiki’ and ‘Chondrolia Chalkidikis’ grown in Southern and Northern Greece respectively, in five developmental stages along a full fruit-bearing season. Distinct microbial communities were supported in above- and below-ground plant parts; while the former tended to be similar between the two varieties/locations, the latter were location specific. In both varieties/locations, a seasonally stable root microbiome was observed over time; in contrast the plant microbiome in the other compartments were prone to changes over time, which may be related to seasonal environmental change and/or to plant developmental stage. We noted that olive roots exhibited an AMF-specific filtering effect (not observed for bacteria and general fungi) onto the rhizosphere AMF communities of the two olive varieties/locations/, leading to the assemblage of homogenous intraradical AMF communities. Finally, shared microbiome members between the two olive varieties/locations include bacterial and fungal taxa with putative functional attributes that may contribute to olive tree tolerance to abiotic and biotic stress.

AimsThis study aims to identify main factors that influence the tripartite association of legumes with arbuscular mycorrhiza fungi (AMF) and nitrogen-fixing rhizobia.Methods and ResultsConcurrent inoculations with Mesorhizobium loti and four AMF strains were performed on the model legume Lotus japonicus. Nodulation was significantly enhanced by all AMF strains, under normal conditions, and by specific AMF strains under heat-stress conditions. The impact of rhizobia on mycorrhizal colonization was AMF strain dependent. Co-inoculation trials, where either AMF or rhizobia were restricted outside the root, showed that the symbiotic phenotypes are not influenced by microbial interactions at the pre-symbiotic stage. External application of nutrients showed that P enhances nodulation, while N application does not enhance mycorrhizal colonization.ConclusionsNodulation and mycorhization affect one another during advanced stages of the symbiosis. AMF strains may enhance nodulation under both normal and high environmental temperatures. Rhizobium-AMF compatibility is critical, as rhizobium may positively affect specific AMF strains, an effect that does not derive from increased N uptake.

Dicyandiamide (DCD) and nitrapyrin (NP) are nitrification inhibitors (NIs) used in agriculture for over 40 years. Recently, ethoxyquin (EQ) was proposed as a novel potential NI, acting through its derivative quinone imine (QI). Still, the specific activity of these NIs on the different groups of ammonia-oxidizing microorganisms (AOM), and mostly their effects on other soil microbiota remain unknown. We determined the impact of QI, and comparatively of DCD and NP, applied at two doses (regular versus high), on the function, diversity, and dynamics of target (AOM), functionally associated (nitrite-oxidizing bacteria-NOB), and off-target prokaryotic and fungal communities in two soils mainly differing in pH (5.4 versus 7.9). QI was equally effective to DCD but more effective than NP in inhibiting nitrification in the acidic soil, while in the alkaline soil QI was less efficient than DCD and NP. This was attributed to the higher activity of QI toward AOA prevailing in the acidic soil. All NIs induced significant effects on the composition of the AOB community in both soils, unlike AOA, which were less responsive. Beyond on-target effects, we noted an inhibitory effect of all NIs on the abundance of NOB in the alkaline soil, with

Cultivation of olive trees covers large coastal areas of land in Mediterranean regions, many of them characterized by low soil fertility and exposed to salinity and seasonal drought. In this frame, we developed mixed community inocula of arbuscular mycorrhizal fungi (AMF) derived from the extreme, seasonally arid environments of six Mediterranean sand dunes and evaluated their effects, in the form of community inocula, on rooted semi-woody olive tree cuttings (Olea europaea cv. Koroneiki). The plantlets were grown in the greenhouse for 10 months under 50 mM and 100 mM concentrations of NaCl, successively applied to induce osmotic stress. Inoculation had a positive effect on plant growth and nutrient uptake. However, the three best-performing inocula in early colonization and in plant growth enhancement also resulted in high plant sensitivity to high salinity, which was not observed for the other three inocula. This was expressed by decreased nutrient uptake and drastically lower plant growth, plant photosynthesis, and stomatal conductance (generally an over 50% reduction compared to no salinity application). Amplicon sequencing analysis of the olive plants under salinity stress showed that the AMF communities in the roots were clearly differentiated by inoculation treatment. We could not, however, consistently associate the plant responses observed under high salinity with specific shared AMF community membership or assembly attributes. The observed physiological overreaction to osmotic stress may be an adaptation trait, potentially brought about by host selection coupled to abiotic environmental filtering, in the harsh conditions from which the AMF inocula were derived. The overreaction may, however, be undesirable if conveyed to allochthonous plants at an agronomic level.

Sand dunes of the Mediterranean region constitute drought-stressed, low-fertility ecosystems. Arbuscular mycorrhizal fungi (AMF) are regarded as key components of their biota, that contribute to plant host adaptation and fitness. However, AMF community assembly rules in the roots of the psammophilous plants of coastal sand dunes have not been investigated. We studied the root colonizing AMF communities of four characteristic native plants of eastern Mediterranean coastal foredunes, in nine locations in Greece. Host specificity (plant identity) was the major driver of AMF community assembly in the plant roots, while geographical distance between locations was not related to differences in the AMF communities. Additionally, colonizer AMF communities were characterized by overdominance of a single OTU which was remarkably host-specific among locations. Wider dissimilarity in AMF communities was observed in small and disturbed (SD) sites compared to large and undisturbed (LU) sites, a trait that may be attributed to relaxed environmental filtering and facilitated AMF dispersal/immigration in SD sites from surrounding habitats. Overall, our results indicate that the assembly of root-colonizing AMF communities in the eastern Mediterranean sand dunes is characterized by strong biotic filtering (host identity), suggesting that co-adaptation processes may be more pronounced than previously proposed, under extreme environmental conditions.

The phenolic profile of the grape berries is a key quality factor for the red grapevine varieties and several techniques have been applied to improve it. An innovative technique is the application of resistance elicitors and phytohormones. In the present study, leaves and berries of a Greek red indigenous variety (Mouhtaro) sprayed with two elicitors, benzothiadiazole and chitosan and a plant hormone abscisic acid, during veraison. Physicochemical and phenolic characteristics of the berries and microbial communities of rhizosphere, phyllosphere and carposphere were analyzed at harvest. Differences in the microbial communities on different plant compartments were observed after the application of the plant activators. Chitosan treatment increased the abundance of the beneficial lactic acid bacteria, while the abscisic acid treatment decreased the presence of spoilage fungi on the carposphere. Treatments differentiate total phenolics, anthocyanins and in the chemical characteristics of grape must with chitosan treated grapes had increased anthocyanins and skin-derived phenolics that correlated positively with the microbial taxa that was discriminant by LefSe analysis. This research provides an overview of the effect of plant activators on the microbial ecology and grape quality of the Greek variety Mouhtaro and presents the potential of new and innovative approaches in the field of sustainable viticulture.

Mutualistic relationships of legume plants with, either bacteria (like rhizobia) or fungi (like arbuscular mycorrhizal fungi), have been investigated intensively, usually as bi-partite interactions. However, diverse symbiotic interactions take place simultaneously or sequentially under field conditions. Their collective, but not additive, contribution to plant growth and performance remains hard to predict, and appears to be furthermore affected by crop species and genotype, non-symbiotic microbial interactions and environmental variables. The challenge is: (i) to unravel the complex overlapping mechanisms that operate between the microbial symbionts as well as between them, their hosts and the rhizosphere (ii) to understand the dynamics of the respective mechanisms in evolutionary and ecological terms. The target for agriculture, food security and the environment, is to use this insight as a solid basis for developing new integrated technologies, practices and strategies for the efficient use of beneficial microbes in legumes and other plants. We review recent advances in our understanding of the symbiotic interactions in legumes roots brought about with the aid of molecular and bioinformatics tools. We go through single symbiont-host interactions, proceed to tripartite symbiont-host interactions, appraise interactions of symbiotic and associative microbiomes with plants in the root-rhizoplane-soil continuum of habitats and end up by examining attempts to validate community ecology principles in the legume-microbe-soil biosystem.

Arbuscular mycorrhizal fungi (AMF) are soilborne microorganisms that establish a mutualistic symbiotic association with most of land plants. To investigate the effects of AMF symbiosis under different water status conditions, we grew AMF-inoculated and non-inoculated tomato plants in the greenhouse under two irrigation regimes, 70% and 30% of growth-substrate water holding capacity. Two different AMF inoculation strains, Funneliformis mosseae and Rhizophagus irregularis, were applied as single inocula. AMF colonization significantly enhanced plant vegetative growth by 40% and 50–60%, under normal and reduced irrigation respectively. In the presence of the AMF, phosphorous concentrations in the leaves were increased under both watering regimes while K, Ca, Mg, Zn, and Mn were also increased under limited watering to levels similar to those of non-stressed plants. Transpiration and stomatal conductance increased by an average 80% and 65% respectively in the presence of the AMF under full watering, but were kept stable and coupled to reduced leaf area-to-leaf biomass ratios and to increased metabolic water use efficiency under limited irrigation. This indicates a different mode of action induced by AMF colonizers, prioritizing water conservation in tomato plants under drought stress.

There is a renewed interest in recent years on the ecological processes (stochastic vs selective) driving the assembly of microbial communities. Such information could potentially improve our understanding on ecosystem functioning and resilience to disturbances, ecosystem response to environmental shifts, and adoption of sustainable soil management practices. Herein, employing a suite of existing methodologies, we show that stochastic processes have an important role on the assembly of soil bacterial communities at a Mediterranean watershed. Moreover, we document that the relative contribution of assembly processes varies over the years. The observed intensification of stochastic processes was accompanied by a decrease in the contribution of variable selection in favor of homogeneous selection and dispersal and this trend was only marginally affected by land use (natural vs agricultural lands) or soil depth. Our study also revealed a high inter-annual turnover of soil microbial communities that was likely stimulated by the weak environmental selection and the prevailing environmental conditions (drying-wetting cycles) in Mediterranean landscapes, implying potential impacts on ecosystem functioning and our ability to predict soil response to environmental shifts. Using nitrogen mineralization rate (NMR) as a representative function we document highly variable NMR over the sampling years, land uses and soil depths and lack of significant associations with the monitored environmental variables and individual taxa. In summary, our study provides novel insights on the organization and functioning of microbial communities at Mediterranean ecosystems and sets directions towards a more advanced understanding of the relationships among environmental factors, microbial community structure, and ecosystem functioning that could contribute to sustainable management of these severely degraded ecosystems.

This work investigates the effect of plant species (Eucalyptus camaldulensis vs. Arundo donax) on carbon (C) turnover during wastewater application to the land. The study was carried out in 40-liter pots under field conditions and plant species were treated either with pre-treated municipal wastewater or freshwater. Plant species had a strong effect on soil organic matter with pots planted with E. camaldulensis showing greater values than pots planted with A. donax. In accordance, greater respiration rates were measured in E. camaldulensis pots compared to those planted with A. donax. The respiration rate followed a decreasing trend with the progress of the season for both species. These findings suggest differences in soil microbial community composition and/or activity in the rhizosphere of plant species. Minor effects of plant species or effluent were observed in dissolved organic carbon, protein, and hexoses content. In conclusion, the results of the present study reveal an important role of plant species on C cycling in terrestrial environments with potential implications on the sequestration of C and release of nutrients and pollutants.

This work investigated the effect of plant species (Eucalyptus camaldulensis vs Arundo donax) on N-turnover during land application of pre-treated municipal wastewater. The study was carried out in 40-L pots under field conditions and revealed strong effects of plant species on N uptake, potential nitrification rate (PNR), and gaseous losses of N. E. camaldulensis accumulated 38% more N in its biomass than A. donax. With regard to the soil N, there was no effect of plant species on total Kjeldahl N content. The lower PNR measured in pots planted with A. donax could not be explained by differences in archaeal or bacterial amoA gene copies; suggesting that plant species affected the activity of nitrifiers.The loads of NH4+-N applied were not found to have delayed the oxidation of NH4+-N, despite the differences in PNR, as indicated by soil solution and soil NH4+-N concentrations in the rhizosphere of the two plant species throughout the period of the study. However, decreased concentrations of NO3−-N were measured in the pots planted with A. donax from the end of June onwards. This finding implies increased losses of N through denitrification and reveals a direct effect of plant species on the activity of denitrifiers since differences in copy numbers of denitrification genes (nirK, nirS, nosZ) were observed only early in the season. Our findings reveal a critical role of plant species on N cycling in terrestrial environments with important implications for the management/restoration of N-polluted areas, such as riparian zones, and for land treatment systems and constructed wetlands.

Summarization: Single-stage constructed wetlands (CWs) are characterized by a low potential for N removal. Understanding the pathways regulating N cycling as well as their dependence on environmental variables might improve the potential of CWs for N removal and results in more accurate simulation tools. In this study we employed qPCR targeting marker functional genes (amoA, nirK, nirS, clade I and II nosZ) or microorganisms (anammox) regulating key pathways of N cycling to unravel their relative importance. Furthermore, the influence of plant species on treatment performance was studied. Our findings indicated nitrification-denitrification as the principal route of N removal in CWs, while anammox did not have a strong contribution. Evidence was also arisen that ammonia oxidizing archaea (AOA) contributed on NH3 oxidation. Overall, plant species had a weak effect on the abundance of N functional genes (amoA of AOA), but it strongly affected the performance of CWs in terms of N removal in the following order: unplanted < Phragmites communis < Typha latifolia. These findings suggest that plant species stimulate N removal by upregulating the rates that the responsible biochemical pathways operate, probably by increasing O2 supply. In addition, our study revealed differences in indicators linked to N2O emissions. The abundance of clade II nosZ genes remained low across the season scaling down a strong contribution in the reduction of the emitted N2O. The increasing ratios of nosZ/σnir and nirS/nirK with the progress of season indicate a shift in the composition of denitrifiers towards strains with a lower genetic potential for N2O release. Similar trends were observed among the treatments but the mechanisms differed. The planted treatments stimulated an increase in the σnosZ/σnir ratio, while the unplanted an increase in the nirS/nirK ratio. Presented on: Water Research

To date only few studies have dealt with the biogeography of microbial communities at large spatial scales, despite the importance of such information to understand and simulate ecosystem functioning. Herein, we describe the biogeographic patterns of microorganisms involved in nitrogen (N)-cycling (diazotrophs, ammonia oxidizers, denitrifiers) as well as the environmental factors shaping these patterns across the Koiliaris Critical Zone Observatory, a typical Mediterranean watershed. Our findings revealed that a proportion of variance ranging from 40 to 80% of functional genes abundance could be explained by the environmental variables monitored, with pH, soil texture, total organic carbon and potential nitrification rate being identified as the most important drivers. The spatial autocorrelation of N-functional genes ranged from 0.2 to 6.2 km and prediction maps, generated by cokriging, revealed distinct patterns of functional genes. The inclusion of functional genes in statistical modeling substantially improved the proportion of variance explained by the models, a result possibly due to the strong relationships that were identified among microbial groups. Significant relationships were set between functional groups, which were further mediated by land use (natural versus agricultural lands). These relationships, in combination with the environmental variables, allow us to provide insights regarding the ecological preferences of N-functional groups and among them the recently identified clade II of nitrous oxide reducers.

Data on soil microbial community distribution at large scales are limited despite the important information that could be drawn with regard to their function and the influence of environmental factors on nutrient cycling and ecosystem services. This study investigates the distribution of Archaea, Bacteria and Fungi as well as the dominant bacterial phyla ( Acidobacteria , Actinobacteria , Bacteroidetes , Firmicutes ) , and classes of Proteobacteria ( Alpha- and Betaproteobacteria ) across the Koiliaris watershed by qPCR and associate them with environmental variables. Predictive maps of microorganisms distribution at watershed scale were generated by co-kriging, using the most significant predictors. Our findings showed that 31–79% of the spatial variation in microbial taxa abundance could be explained by the parameters measured, with total organic carbon and pH being identified as the most important. Moreover, strong correlations were set between microbial groups and their inclusion on variance explanation improved the prediction power of the models. The spatial autocorrelation of microbial groups ranged from 309 to 2.226 m, and geographic distance, by itself, could explain a high proportion of their variation. Our findings shed light on the factors shaping microbial communities at a high taxonomic level and provide evidence for ecological coherence and syntrophic interactions at the watershed scale.

This study investigated the cycling of C and N following application of olive mill wastewater (OMW) at various rates (0, 42, 84, and 168 m(3)/ha). OMW stimulated respiration rate throughout the study period, but an increase in soil organic matter was observed only at the highest rate. Soil phenol content decreased rapidly within 2 weeks following application but neither phenol oxidase and peroxidase activity nor laccase gene copies could explain this response. Soil NH4 (+)-N content increased in response to OMW application rate, while an opposite trend observed for NO3 (-)-N, which attributed to immobilization. This decrease was in accordance with amoA gene copies of archaeal and bacterial ammonia oxidizers in the first days following OMW application. Afterwards, although amoA gene copies and potential nitrification rates recovered to values similar to or higher than those in the non-treated soils, NO3 (-)-N content did not change among the treatments. A corresponding increase in denitrifying gene copies (nirK, nirS, nosZ) during that period indicates that denitrification, stimulated by OMW application rate, was responsible for this effect; a hypothesis consistent with the decrease in total Kjeldahl nitrogen content late in the season. The findings suggest that land application of OMW is a promising practice for OMW management, even at rates approaching the soil water holding capacity.

This study investigated the effect of municipal solid waste (MSW) compost (0, 50, and 100 t/ha) on N cycling and the microorganisms involved in it, in a clay-loam soil. After a release of nitrates (NO3 −-N) in the first 6 days after compost incorporation, soil NO3 −-N content remained constant in all the treatments until day 62, suggesting N immobilization induced by the soil used in this study. Then, soil NO3 −-N content increased in all treatments and especially in the highest compost dose, providing evidence that immobilization effect has been at least partially relieved. amoA gene copies of ammonia-oxidizing archaea (AOA) and bacteria (AOB) followed the overall pattern of soil NO3 −-N content; however, no differences were found in amoA gene copies among treatments, except in the last sampling, an effect attributed to the slight differences in the potential nitrification rate among them. Ammonia oxidizer pattern provided evidence that both groups were involved in ammonia oxidation and changes in their abundance can be used as ‘indicator’ to predict changes in soil nitrification status. Moreover, the strong correlation between AOA and AOB amoA copies (R 2 = 0.94) and the high slope (13) of the curve suggest that AOA had probably an important role on ammonia oxidation. Denitrifying genes (nirS, nirK, nosZ) also followed the general pattern of soil NO3 −-N, and they were strongly correlated with both groups of ammonia oxidizers, and particularly AOA, suggesting strong interrelationships among them. Losses of N through denitrification, as they were estimated by total nitrogen, were inversely related to soil NO3 −-N content. Similar to ammonia oxidizers, denitrifying gene copies did not differ among compost treatments an effect that could be probably explained by the low availability of organic-C in the MSW compost and hence the competition with aerobic heterotrophs.