Effect on microbial communities in apple orchard soil when exposed short-term to climate change abiotic factors and different orchard management practices.

AIM: We assessed the effect of exposing apple orchard soil to different temperatures and CO2 levels on the resident microbiome of soils from a conventionally managed and an organically managed apple orchard. The key difference between these two orchards was that synthetic fertilizers and pesticides are routinely used in the former one. METHODS AND RESULTS: To investigate the effect of CO2 and temperature, soil samples from each site at two depths were exposed to either elevated temperature (29°C) at either 5000 or 10 000 ppm for five weeks or control conditions (25°C + 400 ppm). Both bacterial and fungal communities were profiled with amplicon-sequencing. The differences between the two orchards were the most significant factor affecting the bacterial and fungal communities, contributing to 53.7-14.0% of the variance in Bray-Curtis ß diversity, respectively. Elevated CO2 concentration and increased temperature affected organic orchard microbial diversity more than the conventionally managed orchard. A number of candidate beneficial and pathogenic microorganisms had differential abundances when temperature and CO2 were elevated, but their effect on the plant is unclear. CONCLUSIONS: This study has highlighted that microbial communities in bulk soils are most significantly influenced by crop management practices compared to the climate conditions used in the study. The studied climate conditions had a more limited effect on microbial community diversity in conventionally managed soil samples than in organically managed soils.

Inoculation of drought-stressed strawberry with a mixed inoculum of two arbuscular mycorrhizal fungi: effects on population dynamics of fungal species in roots and consequential plant tolerance to water deficiency.

The effect of inoculation with two arbuscular mycorrhizal fungi (AMF) on growth and drought tolerance of cultivated strawberry (Fragaria × ananassa) was studied. Three treatments (a single treatment either of Funneliformis mosseae BEG25, Funneliformis geosporus BEG11 or a 50:50 mixed inoculation treatment of both species) were compared to uninoculated plants. Species-specific primers for qPCR quantification of F. geosporus and F. mosseae DNA were developed to quantify the relative abundance of each fungus in roots of strawberry under different conditions of water stress. Co-occupation of the same root by both species was shown to commonly occur, but their relative abundance varied with water stress (reduced irrigation of up to 40%). Greater root colonisation was observed microscopically under water stress, but this increased colonisation was often accompanied with decreased amounts of fungal DNA in the root. F. mosseae tended to become more abundant under water stress relative to F. geosporus. There was significant correlation in the fungal colonisation measurements from the microscopic and qPCR methods under some conditions, but the nature of this relationship varied greatly with AMF inoculum and abiotic conditions. Single-species inoculation treatments gave similar benefits to the host to the mixed inoculation treatment regardless of irrigation regime; here, amount of colonisation was of greater importance than functional diversity. The addition of AMF inocula to plants subjected to reduced irrigation restored plant growth to the same or higher values as the non-mycorrhizal, fully-watered plants. The water use efficiency of plants was greater under the regulated deficit irrigation (RDI) regime and in AMF-inoculated plants, but there were no significant differences between plants inoculated with the single or combined inoculum. This study demonstrated that the increase in plant growth was directly influenced by an increase in root colonisation by AMF when individual plants were examined.

Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor ß (TGFß) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

Tuning the Nanotopography and Chemical Functionality of 3D Printed Scaffolds through Cellulose Nanocrystal Coatings.

In nature, cells exist in three-dimensional (3D) microenvironments with topography, stiffness, surface chemistry, and biological factors that strongly dictate their phenotype and behavior. The cellular microenvironment is an organized structure or scaffold that, together with the cells that live within it, make up living tissue. To mimic these systems and understand how the different properties of a scaffold, such as adhesion, proliferation, or function, influence cell behavior, we need to be able to fabricate cellular microenvironments with tunable properties. In this work, the nanotopography and functionality of scaffolds for cell culture were modified by coating 3D printed materials (DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose nanocrystals (CNCs). This general approach was demonstrated on a variety of structures designed to incorporate macro- and microscale features fabricated using photopolymerization and 3D printing techniques. Atomic force microscopy was used to characterize the CNC coatings and showed the ability to tune their density and in turn the surface nanoroughness from isolated nanoparticles to dense surface coverage. The ability to tune the density of CNCs on 3D printed structures could be leveraged to control the attachment and morphology of prostate cancer cells. It was also possible to introduce functionalization onto the surface of these scaffolds, either by directly coating them with CNCs grafted with the functionality of interest or with a more general approach of functionalizing the CNCs after coating using biotin-streptavidin coupling. The ability to carefully tune the nanostructure and functionalization of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of natural microenvironments, which are key to understanding their impact on cells and developing artificial tissues.

Water-in-PDMS Emulsion Templating of Highly Interconnected Porous Architectures for 3D Cell Culture.

The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500 µm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm3. These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development of 3D cell culture platforms.

The Use of Arbuscular Mycorrhizal Fungi to Improve Strawberry Production in Coir Substrate.

Strawberry is an important fruit crop within the UK. To reduce the impact of soil-borne diseases and extend the production season, more than half of the UK strawberry production is now in substrate (predominantly coir) under protection. Substrates such as coir are usually depleted of microbes including arbuscular mycorrhizal fungi (AMF) and consequently the introduction of beneficial microbes is likely to benefit commercial cropping systems. Inoculating strawberry plants in substrate other than coir has been shown to increase plants tolerance to soil-borne pathogens and water stress. We carried out studies to investigate whether AMF could improve strawberry production in coir under low nitrogen input and regulated deficit irrigation. Application of AMF led to an appreciable increase in the size and number of class I fruit, especially under either deficient irrigation or low nitrogen input condition. However, root length colonization by AMF was reduced in strawberry grown in coir compared to soil and Terragreen. Furthermore, the appearance of AMF colonizing strawberry and maize roots grown in coir showed some physical differences from the structure in colonized roots in soil and Terragreen: the colonization structure appeared to be more compact and smaller in coir.

Ultrastructure of spore development in Scutellospora heterogama.

The ultrastructural detail of spore development in Scutellospora heterogama is described. Although the main ontogenetic events are similar to those described from light microscopy, the complexity of wall layering is greater when examined at an ultrastructural level. The basic concept of a rigid spore wall enclosing two inner, flexible walls still holds true, but there are additional zones within these three walls distinguishable using electron microscopy, including an inner layer that is involved in the formation of the germination shield. The spore wall has three layers rather than the two reported previously. An outer, thin ornamented layer and an inner, thicker layer are both derived from the hyphal wall and present at all stages of development. These layers differentiate into the outer spore layer visible at the light microscope level. A third inner layer unique to the spore develops during spore swelling and rapidly expands before contracting back to form the second wall layer visible by light microscopy. The two inner flexible walls also are more complex than light microscopy suggests. The close association with the inner flexible walls with germination shield formation consolidates the preferred use of the term 'germinal walls' for these structures. A thin electron-dense layer separates the two germinal walls and is the region in which the germination shield forms. The inner germinal wall develops at least two sub-layers, one of which has an appearance similar to that of the expanding layer of the outer spore wall. An electron-dense layer is formed on the inner surface of the inner germinal wall as the germination shield develops, and this forms the wall surrounding the germination shield as well as the germination tube. At maturity, the outer germinal wall develops a thin, striate layer within its substructure.