Nodulation and nitrogen fixation in Medicago truncatula strongly alters the abundance of its root microbiota and subtly affects its structure.

The plant common symbiosis signalling (SYM) pathway has shared function between interactions with rhizobia and arbuscular mycorrhizal fungi, the two most important symbiotic interactions between plants and microorganisms that are crucial in plant and agricultural yields. Here, we determine the role of the plant SYM pathway in the structure and abundance of the microbiota in the model legume Medicago truncatula and whether this is controlled by the nitrogen or phosphorus status of the plant. We show that SYM mutants (dmi3) differ substantially from the wild type (WT) in the absolute abundance of the root microbiota, especially under nitrogen limitation. Changes in the structure of the microbiota were less pronounced and depended on both plant genotype and nutrient status. Thus, the SYM pathway has a major impact on microbial abundance in M. truncatula and also subtly alters the composition of the microbiota.

Multimodal correlative imaging and modelling of phosphorus uptake from soil by hyphae of mycorrhizal fungi.

Phosphorus (P) is essential for plant growth. Arbuscular mycorrhizal fungi (AMF) aid its uptake by acquiring P from sources distant from roots in return for carbon. Little is known about how AMF colonise soil pore-space, and models of AMF-enhanced P-uptake are poorly validated. We used synchrotron X-ray computed tomography to visualize mycorrhizas in soil and synchrotron X-ray fluorescence/X-ray absorption near edge structure (XRF/XANES) elemental mapping for P, sulphur (S) and aluminium (Al) in combination with modelling. We found that AMF inoculation had a suppressive effect on colonisation by other soil fungi and identified differences in structure and growth rate between hyphae of AMF and nonmycorrhizal fungi. Our results showed that AMF co-locate with areas of high P and low Al, and preferentially associate with organic-type P species over Al-rich inorganic P. We discovered that AMF avoid Al-rich areas as a source of P. Sulphur-rich regions were found to be correlated with higher hyphal density and an increased organic-associated P-pool, whilst oxidized S-species were found close to AMF hyphae. Increased S oxidation close to AMF suggested the observed changes were microbiome-related. Our experimentally-validated model led to an estimate of P-uptake by AMF hyphae that is an order of magnitude lower than rates previously estimated - a result with significant implications for the modelling of plant-soil-AMF interactions.

Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans.

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Postnatal prebiotic supplementation in rats affects adult anxious behaviour, hippocampus, electrophysiology, metabolomics, and gut microbiota.

We have shown previously that prebiotic (Bimuno galacto-oligosacharides, B-GOS®) administration to neonatal rats increased hippocampal NMDAR proteins. The present study has investigated the effects of postnatal B-GOS® supplementation on hippocampus-dependent behavior in young, adolescent, and adult rats and applied electrophysiological, metabolomic and metagenomic analyses to explore potential underlying mechanisms. The administration of B-GOS® to suckling, but not post-weaned, rats reduced anxious behavior until adulthood. Neonatal prebiotic intake also reduced the fast decay component of hippocampal NMDAR currents, altered age-specific trajectories of the brain, intestinal, and liver metabolomes, and reduced abundance of fecal Enterococcus and Dorea bacteria. Our data are the first to show that prebiotic administration to rats during a specific postnatal period has long-term effects on behavior and hippocampal physiology. The study also suggests that early-life prebiotic intake may affect host brain function through the reduction of stress-related gut bacteria rather than increasing the proliferation of beneficial microbes.

Genomic Diversity of Pigeon Pea (Cajanus cajan L. Millsp.) Endosymbionts in India and Selection of Potential Strains for Use as Agricultural Inoculants.

Pigeon pea (Cajanus cajan L. Millsp. ) is a legume crop resilient to climate change due to its tolerance to drought. It is grown by millions of resource-poor farmers in semiarid and tropical subregions of Asia and Africa and is a major contributor to their nutritional food security. Pigeon pea is the sixth most important legume in the world, with India contributing more than 70% of the total production and harbouring a wide variety of cultivars. Nevertheless, the low yield of pigeon pea grown under dry land conditions and its yield instability need to be improved. This may be done by enhancing crop nodulation and, hence, biological nitrogen fixation (BNF) by supplying effective symbiotic rhizobia through the application of "elite" inoculants. Therefore, the main aim in this study was the isolation and genomic analysis of effective rhizobial strains potentially adapted to drought conditions. Accordingly, pigeon pea endosymbionts were isolated from different soil types in Southern, Central, and Northern India. After functional characterisation of the isolated strains in terms of their ability to nodulate and promote the growth of pigeon pea, 19 were selected for full genome sequencing, along with eight commercial inoculant strains obtained from the ICRISAT culture collection. The phylogenomic analysis [Average nucleotide identity MUMmer (ANIm)] revealed that the pigeon pea endosymbionts were members of the genera Bradyrhizobium and Ensifer. Based on nodC phylogeny and nod cluster synteny, Bradyrhizobium yuanmingense was revealed as the most common endosymbiont, harbouring nod genes similar to those of Bradyrhizobium cajani and Bradyrhizobium zhanjiangense. This symbiont type (e.g., strain BRP05 from Madhya Pradesh) also outperformed all other strains tested on pigeon pea, with the notable exception of an Ensifer alkalisoli strain from North India (NBAIM29). The results provide the basis for the development of pigeon pea inoculants to increase the yield of this legume through the use of effective nitrogen-fixing rhizobia, tailored for the different agroclimatic regions of India.

Poor Competitiveness of Bradyrhizobium in Pigeon Pea Root Colonization in Indian Soils.

Pigeon pea, a legume crop native to India, is the primary source of protein for more than a billion people in developing countries. The plant can form symbioses with N2-fixing bacteria; however, reports of poor crop nodulation in agricultural soils abound. We report here a study of the bacterial community associated with pigeon pea, with a special focus on the symbiont population in different soils and vegetative and non-vegetative plant growth. Location with respect to the plant roots was determined to be the main factor controlling the bacterial community, followed by developmental stage and soil type. Plant genotype plays only a minor role. Pigeon pea roots have a reduced microbial diversity compared to the surrounding soil and select for Proteobacteria, especially for Rhizobium spp., during vegetative growth. While Bradyrhizobium, a native symbiont of pigeon pea, can be found associating with roots, its presence is dependent on plant variety and soil conditions. A combination of 16S rRNA gene amplicon survey, strain isolation, and co-inoculation with nodule-forming Bradyrhizobium spp. and non-N2-fixing Rhizobium spp. demonstrated that the latter is a much more successful colonizer of pigeon pea roots. Poor nodulation of pigeon pea in Indian soils may be caused by a poor Bradyrhizobium competitiveness against non-nodulating root colonizers such as Rhizobium. Hence, inoculant strain selection of symbionts for pigeon pea should be based not only on their nitrogen fixation potential but, more importantly, on their competitiveness in agricultural soils. IMPORTANCE Plant symbiosis with N2-fixing bacteria is a key to sustainable, low-input agriculture. While there are ongoing projects aiming to increase the yield of cereals using plant genetics and host-microbiota interaction engineering, the biggest potential lies in legume plants. Pigeon pea is a basic food source for a billion low-income people in India. Improving its interactions with N2-fixing rhizobia could dramatically reduce food poverty in India. Despite the Indian origin of this plant, pigeon pea nodulates only poorly in native soils. While there have been multiple attempts to select the best N2-fixing symbionts, there are no reliable strains available for geographically widespread use. In this article, using 16S rRNA gene amplicon, culturomics, and plant co-inoculation assays, we show that the native pigeon pea symbionts such as Bradyrhizobium spp. are able to nodulate their host, despite being poor competitors for colonizing roots. Hence, in this system, the establishment of effective symbiosis seems decoupled from microbial competition on plant roots. Thus, the effort of finding suitable symbionts should focus not only on their N2-fixing potential but also on their ability to colonize. Increasing pigeon pea yield is a low-hanging fruit to reduce world hunger and degradation of the environment through the overuse of synthetic fertilizers.

Conditional sanctioning in a legume-Rhizobium mutualism.

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nodule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later developmental stages, sanctioned nodules contained fewer viable bacteria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners.

Engineering rhizobacteria for sustainable agriculture.

Exploitation of plant growth promoting (PGP) rhizobacteria (PGPR) as crop inoculants could propel sustainable intensification of agriculture to feed our rapidly growing population. However, field performance of PGPR is typically inconsistent due to suboptimal rhizosphere colonisation and persistence in foreign soils, promiscuous host-specificity, and in some cases, the existence of undesirable genetic regulation that has evolved to repress PGP traits. While the genetics underlying these problems remain largely unresolved, molecular mechanisms of PGP have been elucidated in rigorous detail. Engineering and subsequent transfer of PGP traits into selected efficacious rhizobacterial isolates or entire bacterial rhizosphere communities now offers a powerful strategy to generate improved PGPR that are tailored for agricultural use. Through harnessing of synthetic plant-to-bacteria signalling, attempts are currently underway to establish exclusive coupling of plant-bacteria interactions in the field, which will be crucial to optimise efficacy and establish biocontainment of engineered PGPR. This review explores the many ecological and biotechnical facets of this research.

Agricultural Selection of Wheat Has Been Shaped by Plant-Microbe Interactions.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

Influence of Plant Fraction, Soil, and Plant Species on Microbiota: a Multikingdom Comparison.

Plant roots influence the soil microbiota via physical interaction, secretion, and plant immunity. However, it is unclear whether the root fraction or soil is more important in determining the structure of the prokaryotic or eukaryotic community and whether this varies between plant species. Furthermore, the leaf (phyllosphere) and root microbiotas have a large overlap; however, it is unclear whether this results from colonization of the phyllosphere by the root microbiota. Soil, rhizosphere, rhizoplane, and root endosphere prokaryote-, eukaryote-, and fungus-specific microbiotas of four plant species were analyzed with high-throughput sequencing. The strengths of factors controlling microbiota structure were determined using permutational multivariate analysis of variance (PERMANOVA) statistics. The origin of the phyllosphere microbiota was investigated using a soil swap experiment. Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root itself more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. A reciprocal soil swap experiment shows that the phyllosphere is colonized from the soil in which the plant is grown.IMPORTANCE Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root fraction more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined, indicating conserved adaptation of microbial communities to plants. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. We observed a remarkable similarity in the structure of a plant's phyllosphere and root microbiotas and show by reciprocal soil swap experiments that both fractions are colonized from the soil in which the plant is grown. Thus, the phyllosphere is continuously colonized by the soil microbiota.

Casing microbiome dynamics during button mushroom cultivation: implications for dry and wet bubble diseases.

The casing material required in mushroom cultivation presents a very rich ecological niche, which is inhabited by a diverse population of bacteria and fungi. In this work three different casing materials, blonde peat, black peat and a 50 : 50 mixture of both, were compared for their capacity to show a natural suppressive response against dry bubble, Lecanicillium fungicola (Preuss) Zare and Gams, and wet bubble, Mycogone perniciosa (Magnus) Delacroix. The highest mushroom production was collected from crops cultivated using the mixed casing and black peat, which were not significantly different in yield. However, artificial infection with mycoparasites resulted in similar yield losses irrespective of the material used, indicating that the casing materials do not confer advantages in disease suppression. The composition of the microbiome of the 50 : 50 casing mixture along the crop cycle and the compost and basidiomes was evaluated through next-generation sequencing (NGS) of the V3-V4 region of the bacterial 16S rRNA gene and the fungal ITS2 region. Once colonized by Agaricus bisporus, the bacterial diversity of the casing microbiome increased and the fungal diversity drastically decreased. From then on, the composition of the casing microbiome remained relatively stable. Analysis of the composition of the bacterial microbiome in basidiomes indicated that it is highly influenced by the casing microbiota. Notably, L. fungicola was consistently detected in uninoculated control samples of compost and casing using NGS, even in asymptomatic crops. This suggests that the naturally established casing microbiota was able to help to suppress disease development when inoculum levels were low, but was not effective in suppressing high pressure from artificially introduced fungal inoculum. Determination of the composition of the casing microbiome paves the way for the development of synthetic casing communities that can be used to investigate the role of specific components of the casing microbiota in mushroom production and disease control.

Absolute quantitation of microbiota abundance in environmental samples.

BACKGROUND: Microbial communities (microbiota) influence human and animal disease and immunity, geochemical nutrient cycling and plant productivity. Specific groups, including bacteria, archaea, eukaryotes or fungi, are amplified by PCR to assess the relative abundance of sub-groups (e.g. genera). However, neither the absolute abundance of sub-groups is revealed, nor can different amplicon families (i.e. OTUs derived from a specific pair of PCR primers such as bacterial 16S, eukaryotic 18S or fungi ITS) be compared. This prevents determination of the absolute abundance of a particular group and domain-level shifts in microbiota abundance can remain undetected. RESULTS: We have developed absolute quantitation of amplicon families using synthetic chimeric DNA spikes. Synthetic spikes were added directly to environmental samples, co-isolated and PCR-amplified, allowing calculation of the absolute abundance of amplicon families (e.g. prokaryotic 16S, eukaryotic 18S and fungal ITS per unit mass of sample). CONCLUSIONS: Spikes can be adapted to any amplicon-specific group including rhizobia from soils, Firmicutes and Bifidobacteria from human gut or Enterobacteriaceae from food samples. Crucially, using highly complex soil samples, we show that the absolute abundance of specific groups can remain steady or increase, even when their relative abundance decreases. Thus, without absolute quantitation, the underlying pathology, physiology and ecology of microbial groups may be masked by their relative abundance.

Understanding the holobiont: the interdependence of plants and their microbiome.

The holobiont is composed by the plant and its microbiome. In a similar way to ecological systems of higher organisms, the holobiont shows interdependent and complex dynamics [1,2]. While plants originate from seeds, the microbiome has a multitude of sources. The assemblage of these communities depends on the interaction between the emerging seedling and its surrounding environment, with soil being the main source. These microbial communities are controlled by the plant through different strategies, such as the specific profile of root exudates and its immune system. Despite this control, the microbiome is still able to adapt and thrive. The molecular knowledge behind these interactions and microbial '-omic' technologies are developing to the point of enabling holobiont engineering. For a long time microorganisms were in the background of plant biology but new multidisciplinary approaches have led to an appreciation of the importance of the holobiont, where plants and microbes are interdependent.

Role of root microbiota in plant productivity.

The growing human population requires increasing amounts of food, but modern agriculture has limited possibilities for increasing yields. New crop varieties may be bred to have increased yields and be more resistant to environmental stress and pests. However, they still require fertilization to supplement essential nutrients that are normally limited in the soil. Soil microorganisms present an opportunity to reduce the requirement for inorganic fertilization in agriculture. Microorganisms, due to their enormous genetic pool, are also a potential source of biochemical reactions that recycle essential nutrients for plant growth. Microbes that associate with plants can be considered to be part of the plant's pan-genome. Therefore, it is essential for us to understand microbial community structure and their 'metagenome' and how it is influenced by different soil types and crop varieties. In the future we may be able to modify and better utilize the soil microbiota potential for promoting plant growth.

Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition.

We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alternative community compositions rather than a single distinctive community. Organisms selected in rhizospheres can have positive or negative effects. Two abundant bacteria are shown to promote plant growth, but in Brassica the pathogen Olpidium brassicae came to dominate the fungal community. So plants exert strong selection on the rhizosphere microbiota but soil composition is critical to its stability. microbial succession/ plant-microbe interactions/rhizosphere microbiota/selection.