Above- and below-ground microbiome in the annual developmental cycle of two olive tree varieties.

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.

Interplay between rhizobial nodulation and arbuscular mycorrhizal fungal colonization in Lotus japonicus roots.

AIMS: This study aims to identify main factors that influence the tripartite association of legumes with arbuscular mycorrhiza fungi (AMF) and nitrogen-fixing rhizobia. METHODS AND RESULTS: Concurrent 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. CONCLUSIONS: Nodulation 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.

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota.

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 Nitrobacter being more sensitive than Nitrospira. QI, unlike the other NIs, induced significant changes in the composition of the bacterial and fungal communities in both soils. Our findings have serious implications for the efficiency and future use of NIs on agriculture and provide unprecedented evidence for the potential off-target effects of NIs on soil microbiota. IMPORTANCE NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms. Here, we studied the behavior of a new potent NI, QI, compared to established NIs. We show that (i) the variable efficacy of NIs across soils with different pH reflects differences in the inherent specific activity of the NIs to AOA and AOB; (ii) beyond AOM, NIs exhibit negative effects on other nitrifiers, like NOB; (iii) QI was the sole NI that significantly affected prokaryotic and fungal diversity. Our findings (i) highlight the need for novel NI strategies that consider the variable sensitivity of AOM groups to the different NIs (ii) identify QI as a potent AOA inhibitor, and (iii) stress the need for monitoring NIs' impact on off-target soil microorganisms to ensure sustainable N fertilizers use and soil ecosystem functioning.

Unraveling negative biotic interactions determining soil microbial community assembly and functioning.

Microbial communities play important roles in all ecosystems and yet a comprehensive understanding of the ecological processes governing the assembly of these communities is missing. To address the role of biotic interactions between microorganisms in assembly and for functioning of the soil microbiota, we used a top-down manipulation approach based on the removal of various populations in a natural soil microbial community. We hypothesized that removal of certain microbial groups will strongly affect the relative fitness of many others, therefore unraveling the contribution of biotic interactions in shaping the soil microbiome. Here we show that 39% of the dominant bacterial taxa across treatments were subjected to competitive interactions during soil recolonization, highlighting the importance of biotic interactions in the assembly of microbial communities in soil. Moreover, our approach allowed the identification of microbial community assembly rule as exemplified by the competitive exclusion between members of Bacillales and Proteobacteriales. Modified biotic interactions resulted in greater changes in activities related to N- than to C-cycling. Our approach can provide a new and promising avenue to study microbial interactions in complex ecosystems as well as the links between microbial community composition and ecosystem function.

Multi-species relationships in legume roots: From pairwise legume-symbiont interactions to the plant - microbiome - soil continuum.

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.

Pathways regulating the removal of nitrogen in planted and unplanted subsurface flow constructed wetlands.

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.

Environmental drivers of the distribution of nitrogen functional genes at a watershed scale.

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.

Environmental drivers of soil microbial community distribution at the Koiliaris Critical Zone Observatory.

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.

Effects of olive mill wastewater on soil carbon and nitrogen cycling.

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.