Boosting species evenness, productivity and weed control in a mixed meadow by promoting arbuscular mycorrhizas.

Lowland meadows represent aboveground and belowground biodiversity reservoirs in intensive agricultural areas, improving water retention and filtration, ensuring forage production, contrasting erosion and contributing to soil fertility and carbon sequestration. Besides such major ecosystem services, the presence of functionally different plant species improves forage quality, nutritional value and productivity, also limiting the establishment of weeds and alien species. Here, we tested the effectiveness of a commercial seed mixture in restoring a lowland mixed meadow in the presence or absence of inoculation with arbuscular mycorrhizal (AM) fungi and biostimulation of symbiosis development with the addition of short chain chito-oligosaccharides (CO). Plant community composition, phenology and productivity were regularly monitored alongside AM colonization in control, inoculated and CO-treated inoculated plots. Our analyses revealed that the CO treatment accelerated symbiosis development significantly increasing root colonization by AM fungi. Moreover, the combination of AM fungal inoculation and CO treatment improved plant species evenness and productivity with more balanced composition in forage species. Altogether, our study presented a successful and scalable strategy for the reintroduction of mixed meadows as valuable sources of forage biomass; demonstrated the positive impact of CO treatment on AM development in an agronomic context, extending previous observations developed under controlled laboratory conditions and leading the way to the application in sustainable agricultural practices.

Impact of Phosphatic Nutrition on Growth Parameters and Artemisinin Production in Artemisia annua Plants Inoculated or Not with Funneliformis mosseae.

Artemisia annua L. is a medicinal plant appreciated for the production of artemisinin, a molecule used for malaria treatment. However, the natural concentration of artemisinin in planta is low. Plant nutrition, in particular phosphorus, and arbuscular mycorrhizal (AM) fungi can affect both plant biomass and secondary metabolite production. In this work, A. annua plants were ino- culated or not with the AM fungus Funneliformis mosseae BEG12 and cultivated for 2 months in controlled conditions at three different phosphatic (P) concentrations (32, 96, and 288 µM). Plant growth parameters, leaf photosynthetic pigment concentrations, artemisinin production, and mineral uptake were evaluated. The different P levels significantly affected the plant shoot growth, AM fungal colonization, and mineral acquisition. High P levels negatively influenced mycorrhizal colonization. The artemisinin concentration was inversely correlated to the P level in the substrate. The fungus mainly affected root growth and nutrient uptake and significantly lowered leaf artemisinin concentration. In conclusion, P nutrition can influence plant biomass production and the lowest phosphate level led to the highest artemisinin concentration, irrespective of the plant mineral uptake. Plant responses to AM fungi can be modulated by cost-benefit ratios of the mutualistic exchange between the partners and soil nutrient availability.

Directional turnover towards larger-ranged plants over time and across habitats.

Species turnover is ubiquitous. However, it remains unknown whether certain types of species are consistently gained or lost across different habitats. Here, we analysed the trajectories of 1827 plant species over time intervals of up to 78 years at 141 sites across mountain summits, forests, and lowland grasslands in Europe. We found, albeit with relatively small effect sizes, displacements of smaller- by larger-ranged species across habitats. Communities shifted in parallel towards more nutrient-demanding species, with species from nutrient-rich habitats having larger ranges. Because these species are typically strong competitors, declines of smaller-ranged species could reflect not only abiotic drivers of global change, but also biotic pressure from increased competition. The ubiquitous component of turnover based on species range size we found here may partially reconcile findings of no net loss in local diversity with global species loss, and link community-scale turnover to macroecological processes such as biotic homogenisation.

Quantitative methods in microscopy to assess pollen viability in different plant taxa.

High-quality pollen is a prerequisite for plant reproductive success. Pollen viability and sterility can be routinely assessed using common stains and manual microscope examination, but with low overall statistical power. Current automated methods are primarily directed towards the analysis of pollen sterility, and high throughput solutions for both pollen viability and sterility evaluation are needed that will be consistent with emerging biotechnological strategies for crop improvement. Our goal is to refine established labelling procedures for pollen, based on the combination of fluorescein (FDA) and propidium iodide (PI), and to develop automated solutions for accurately assessing pollen grain images and classifying them for quality. We used open-source software programs (CellProfiler, CellProfiler Analyst, Fiji and R) for analysis of images collected from 10 pollen taxa labelled using FDA/PI. After correcting for image background noise, pollen grain images were examined for quality employing thresholding and segmentation. Supervised and unsupervised classification of per-object features was employed for the identification of viable, dead and sterile pollen. The combination of FDA and PI dyes was able to differentiate between viable, dead and sterile pollen in all the analysed taxa. Automated image analysis and classification significantly increased the statistical power of the pollen viability assay, identifying more than 75,000 pollen grains with high accuracy (R2 = 0.99) when compared to classical manual counting. Overall, we provide a comprehensive set of methodologies as baseline for the automated assessment of pollen viability using fluorescence microscopy, which can be combined with manual and mechanized imaging systems in fundamental and applied research on plant biology. We also supply the complete set of pollen images (the FDA/PI pollen dataset) to the scientific community for future research.

Size matters: three methods for estimating nuclear size in mycorrhizal roots of Medicago truncatula by image analysis.

BACKGROUND: The intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis. RESULTS: We here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita. All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins. On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization. Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation. CONCLUSIONS: In conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.

Behavioural response of European starlings exposed to video playback of conspecific flocks: effect of social context and predator threat.

We studied the behavioural response of European starlings to a socially mediated predation event. Adult starlings were exposed to either a video of a flock attacked by a peregrine falcon or a video of a flock not under attack. We investigated whether the social condition affected the anti-predator response under the hypothesis that in such a gregarious species singletons would increase their anti-predator behaviour more than individuals in groups, to compensate for potential increased risk. The video of the flock under attack caused an increase in immobility and vigilance, more marked in singletons, both during and after the exposure. The video of the non-threatened flock caused an increase in activity levels, especially during the exposure. Furthermore, we observed a marked increase in comfort activities in singletons as well as in social interactions and vocalizations in mini-flocks. Only birds in mini-flocks vocalized, which may be explained by an audience effect, a process of social cognition mediated by the social context, and not only by the stimulus. The results are in line with previous field studies, which showed that isolated starlings are exposed to a higher risk of predation compared to individuals in flocks.