Host control of the rhizosphere assembly under different phosphorus levels and sources
Among different factors impacting the rhizosphere assembling, the host exerts a significant effect on modulating the rhizosphere community structure. A previous study comparing the assembly of bacterial communities in wild and modern common bean, showed that plant genotype explained 31.2% of the variation observed in the rhizosphere microbial community when considering microbiome abundance distances . Here, considering only P-dependent recruitment cues, was observed a significant rhizosphere effect of 4% of IAC Imperador [P-efficient], and 6% of Dor-364 [P-inefficient] compared to bulk soil, and a genotype effect of 3% regarding abundance distances between the two evaluated plant genotypes, and the interaction between the different management (P source) and different genotypes also have significant impacts in the rhizosphere, about 6% considering abundance distance matrices.
Long term rock fertilization showed significant changes in the rhizosphere of maize compared to TSP fertilization . Different sources of P caused an effect in the rhizosphere that was host genotype dependent. The P-efficient plant genotype showed distinct bacterial communities’ structures under different sources of P (Supplementary Fig. 6), while the P-inefficient genotype did not. Possibly the efficient genotype is better adapted to soluble sources of P, recognizing RPB as P depleted condition and requiring a different assembly of bacterial community structure with RPB. On the other hand, the P-inefficient genotype is more dependent on its rhizosphere microbiome resulting in similar communities’ structures in both sources of P.
Deficiency of P in plants might impact the cell permeability, leading to modified exudation patterns ; consequently, P availability is also important in the rhizosphere microbial composition. When considering each source of P separately, we observe that it has a significant effect of the level. A small addition of P (both from TSP or RPB) exerted a priming effect in the rhizosphere of Dor-364 [P-inefficient] not observed in IAC Imperador [P-efficient] (Fig. 1b, c, d, e). Possibly, the plant stress caused by the lack of P limited the richness and diversity of microorganisms in the rhizosphere of Dor-364 [P-inefficient] under P depleted conditions (Supplementary Fig. 5).
Castrillo  showed in Arabidopsis that genotypic changes in the hosts regarding the response to P stressed conditions showed significant differences in the rhizosphere assembly, being more correlated with bacterial community structure than with the level of inorganic P stored in the plant. This finding indicates that the rhizosphere community did not respond to the levels of P fertilizations as the plant phenotype does, suggesting that the P-efficient plant genotype appears to be less dependent on the rhizosphere microbiome for P uptake.
Rhizosphere taxonomical assembly under phosphorus depleted conditions
While the P-efficient common bean responds better to P amendments in terms of plant development, the P-inefficient common bean is more responsive in terms of changing the rhizosphere microbiome structure. How changes in the rhizosphere microbial community affect plant physiology and development is far from being completely elucidated, therefore there is no consensus of what consists in a specialized microbiome for P efficiency. Bergkemper et al., (2016), suggested that the microbial community in P depleted conditions is assembled to increase the levels of P in forest soil, and then identified that Solibacteriales, Acidobacteriales and Actinomycetales, showed important role in the P cycling processes. Here, we also found that Acidobacteriales was predominantly enriched in the P-inefficient plant genotype specially when RPB was used for P amendment (TSP enriched 40 OTUs and RPB 48 enriched OTUs).
In a study aiming to identify the effects of long-term fertilization in the P cycle, Grafe et al  identified that Verrucomicrobiaceae, Sphingomonadaceae, Anaerolinaceae, Planctomycetaceae, Chitniphagaceae, Acidibacteriaceae and Bradyrhizobiaceae are involved in the regulation of P cycling, mobilization of organic or inorganic phosphates and uptake. In the same study other families like Rhodocyclaceae, Chlorobiaceae, Geobacteriaceae, Flavobacteriaceae, Opitutaceae, Verrucomicrobiaceae and Solibacteriaceae have mainly copiotrophic behaviour, and are involved only in the uptake of P and do not apply energy to mobilize it from the soil. Here, the rhizosphere bacteria community structure of IAC Imperador [P-efficient] under P depleted conditions showed consistently (when compared to the rhizosphere amended with both sources of P, Fig. 2a,b) an increase in the phyla Alphaproteobacteria (Sphingomonadaceae) and Bacteroidetes (Chitinophagaceae). The family Sphingomonadaceae was described to be pioneers colonizers of biofilms [43, 44], suggesting a oligotrophic life strategy, also, a genus of this family was described to promote plant growth in endophytic conditions (Khan et al., 2014). Its role in P cycle occurs since the regulation, mobilization and acquisition in soil amended with nitrogen and organic matter . The family Chitinophagaceae is also involved in different phases of P cycle, which is a feature of oligotrophic organisms, unlike those organisms that do not disposal energy in P mobilization .
In Dor-364 [P-inefficient] the consistent enrichment under P depleted conditions, when compared to the rhizosphere amended with both sources of P, besides the enrichment of Bacteroidetes (Chitinophagaceae), an enrichment of Actinobacteria (Micrococcaceae) was also observed. Previous studies correlated the Micrococcaceae family to higher concentrations of carbon and nitrogen , however, its role in P cycle are still poorly explored , and some authors attribute an copiotrophic growth strategy to this group [48, 49]. In IAC Imperador [P-efficient], Alphaproteobacteria was enrichment in P depleted conditions, especially when compared to RPB amended treatments, the enriched bacterial families were Beijerinckiaceae, Bradyrhizobiaceae, Caulobacteraceae, Hyphomicrobiaceae, Methylobacteriaceae, Phyllobacteriaceae, Rhizobiaceae, Sphingomonadaceae and Xanthobacteraceae. The bacterial families, Burkholderiaceae, Comamonadaceae and Oxalobacteriaceae, belonging to Betaproteobacteria, were also enriched in P depleted conditions when compared to RPB amendment treatment. Most of these bacterial families were previously reported as P mobilizing bacteria [9, 50]. This suggests that the amendment with phosphate of difficult solubilization (RPB) selected only a few bacterial groups able to use this resource, favouring the enrichment of a smaller number of differentially enriched OTU when compared to treatments amended with a readily available P source (TSP).
Several subgroups of Acidobacteria and also the family Acidobacteriales (representing 120 differentially enriched OTUs) were enriched in the rhizosphere of DOR-364 [P-inefficient] with RPB amendment, highlighting the importance of this group to solubilize inorganic phosphate. This phylum was described to be involved in several transformations of the P cycle, including mobilization and uptake [17, 42]. This fact highlights the better responsiveness of the rhizosphere microbiome of Dor-364 [P-inefficient] to phosphate amendment from both P sources, but with a more pronounced effect when RPB was used for amendments, which demands a higher microbial activity to make it available. This fact suggests that Dor-364 [P-inefficient] structures a more competitive microbiome. Regardless of the directions of these interactions, our results provide fundamentally novel insights into the molecular basis of plant-microbiome interactions for P-uptake in the rhizosphere which, in turn, can be deployed by plant breeders to sustainably enhance bean production.
Due to the functional redundancy and the existence of taxa closely phylogenetic related presenting different metabolic rates and involvement in soil processes , it is important to take into account not only the composition, but also the functional assembly of the bacterial communities. In a previous study, different long-term management of fertilizers in soil showed significant changes in bacterial community structure, despite of not affecting the abundance of genes involved in P cycle . In this context, it is important to analyse the functional potential of the rhizosphere microbiome regarding P mobilization and uptake.
Functional assembly under phosphorus depleted conditions on phosphorus inefficient genotype
The shaping of a microbial community specialized towards P cycling when P is limited was already described for forest soils  and other studies related low P availability to changes in soil microbial communities [52, 53]. Despite of not being responsive to P addition in terms of growth, Dor-364 [P-inefficient] showed an improvement of several functions involved in P metabolism when exposed to P depleted conditions. The abundance of genes potentially involved in the regulation of phosphate transport and uptake (mainly two component systems regulons) were higher under P depleted conditions and, consequently, functions involved in extracellular mobilization and uptake of organic and inorganic P in the soil (alkaline phosphatase, phytase, PQQGDH) were also enriched. Many studies have already reported the overexpression of these genes and activation of enzymes under P depleted conditions [41, 54]. Interestingly, in the rhizosphere of IAC Imperador [P-efficient], this enrichment was not observed in plants growing under P depleted condition. This fact reinforces that the P-efficient common bean uses another strategy to supply its demand for P not being dependent on the microbiome as the P-inefficient common bean, which enriches bacteria potentially involved in the P cycle under depleted P. It is therefore tempting to speculate that, the efficiency of IAC Imperador [P-efficient] occurs due to physiological adaptations, like root development, selected during plant breeding [55–58] at the expense of the establishment of the symbiotic interactions with rhizosphere microorganisms. In this sense, the absence of these phenotypic traits in Dor-364 [P-inefficient] genotype underpinned its higher reliance on its rhizosphere microbiome for P use.
The P-source emerged as another recruitment cue shaping the Dor-364 microbiome. For instance, the amendment with RPB was responsible for a higher number of differentially enriched functions involved mainly in organic P mobilization, further suggesting the ability of Dor-364 [P-inefficient] of engaging with a microbiome better equipped to mobilise organic fertilizers. This occurs mainly due to the assembly a rhizosphere microbial community specialized in exploring organic P from the soil, once even in the presence of insoluble P source the community was shaped to explore organic P. It is possible that this is responsible for this genotype classification as non-responsive to P amendment. Only one function involved in P solubilization, that is PQQGDH, was differentially enriched in the P depleted conditions compared to optimal P amendment (with both TSP and RPB) in P inefficient genotype. Inorganic P is mainly released by the chelation of metallic phosphate ions in the soil [59, 60]. The higher number of functions involved in organic P uptake suggests that Dor-364 [P-inefficient] is more efficient in the use of organic sources of P, but this needs to be further investigated.
Complexity of bacterial community interactions on phosphorus inefficient genotype
Visualising the rhizosphere community structure through network analysis allowed us to observe that Dor-364 [P-inefficient] has significant higher number of organisms (nodes) and associations (edges) under P depleted condition than with P optimal level. However, once the average path length, that determines the average cohesion between nodes  was higher under P depleted conditions, and the diameter, which is the largest distance between a pair of nodes, is higher under P depleted conditions, the community network in low P is looser when compared with bacterial communities assembled under P optimal level.
Modularity, which corresponds to a group of nodes highly connected between which other and with a few links with other collection of nodes , can be proportional to the response of a community during a disturbance . This feature was higher with P amendment, suggesting that these conditions are highly resilient, and therefore any disturbance in the environment could be softened by the rhizosphere bacterial community functional redundancy. This pattern was observed for both P sources.
The betweenness centrality of a node, is responsible for the identification of possible keystone species in a given network [62, 64]. In the rhizosphere of Dor-364, an OTU representing the order Ktedonobacteriales (Chloroflexi) probably consist in a keystone specie to structure the community under P depleted conditions. Very few information of this order is found due to the fact that only few representatives are cultivated , but it was already described as keystone species, being part of a core rhizosphere microbiome in sugarcane . Amendments with different sources of P resulted in different keystone species identified by the betweenness centrality. With TSP amendment, the keystone specie in the rhizosphere of Dor-364 [P-inefficient] was an OTU belonging to the family Caulobacteriaceae (Alphaproteobacteria) and with RBP additions, an OTU from the family Acidobacteriaceae-Subgroup 1 (Acidobacteria). The importance of Caulobacteriaceae in soils and its role in recalcitrant organic matter degradation was recently described [67, 68]. Acidobacteriaceae is ubiquitous in several environments, including soils, and members of this subgroup were already reported as plant growth promotion rhizobacteria with ability to solubilize inorganic phosphate .