Plant beneficial bacterium promotes plant growth by altering social networks of bacteria in the rhizosphere

doi:10.21203/rs.3.rs-2491444/v1

Download PDF

Article

Plant beneficial bacterium promotes plant growth by altering social networks of bacteria in the rhizosphere

https://doi.org/10.21203/rs.3.rs-2491444/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Plant growth-promoting rhizobacteria (PGPR) represent a green solution to emerging food production challenges but the knowledge of their role in shaping phylogenetic and social relationships of bacterial rhizosphere community is lacking. We here address this gap in knowledge by following effects of Bacillus velezensis SQR9, the commercially important PGP rhizobacterium, on rhizosphere community focusing on Bacillus species. Results show that SQR9 dramatically shifts phylogenetic and social relationships in the rhizosphere enriching moderately related and swarming compatible Bacillus species with decreased carbon resource niche overlap. By combining Bacillus isolates from the SQR9 treated rhizosphere with SQR9 into multispecies consortia we confirm in hydroponic systems and soil experiments that relatedness-based sociality and competition for carbon resources represent fundamental knowledge for the intelligent design of efficient PGP consortia. The work contributes toward sustainable agriculture by providing the first insight in PGPR’s sociality effects in the rhizosphere and their importance for PGP consortia design.

Biological sciences/Ecology/Microbial ecology

Biological sciences/Microbiology/Environmental microbiology/Soil microbiology

The rhizosphere microbiome, also referred to as the second genome of the plant, can have a variety of beneficial effects on plant growth, nutrition and health^{1, 2}. In the agricultural context, plant microbiomes have been proposed as the cornerstone of the next green revolution by improving crop performance while reducing undesired chemical remnants³. Moreover, microbial inoculants, commonly referred to as plant growth-promoting rhizobacteria (PGPR), have shown promise for improving plant growth, nutrition, and stress resistance^4,5. In general, PGPR function by directly affecting the synthesis of phytohormones, enhancing nutrient uptake from soil and suppressing soil-borne plant pathogens^{6, 7}. They also act indirectly by impacting the resident soil microbiome. To date, however, little research has been conducted on the effects of PGPR inoculants on the indigenous plant microbiota^{8, 9}, and thus, the extent to which introduced PGPR inoculants affect the composition and function of the rhizosphere microbiome is largely unknown.

Rhizosphere competence and root colonization are two essential traits for the successful application of PGPRs as probiotics and biocontrol agents^{10, 11}. For example, Bacillus and Pseudomonas, widely used as microbial inoculants, usually form robust biofilms on the plant root surface in the early stages after application¹². Due to the intensive colonization of roots, these bacterial species can influence the composition of indigenous microbial communities by enriching microorganisms that are beneficial to host plant health^{8, 13}, but empirical evidence is scarce. The application of PGPR is theorized to have the greatest negative impact on microbial species whose metabolic niches overlap^{14, 15}. However, ecological interactions with other species may also alter the niche space of a given species¹⁶. According to theory and experiments, competition and antagonistic interactions reduce the niche space available to organisms¹⁷. In contrast, the theory predicts that mutualistic interactions expand the species’ niche space. However, empirical evidence is lacking on how the use of beneficial bacteria in the rhizosphere affects social trends in a community and its metabolic niche space¹⁸.

Social interactions are widespread in the microbial world, and cells can interact with each other in a variety of ways, resulting in either competition or cooperation between neighbours^{19, 20}. PGPRs produce a variety of secondary metabolites, including bacteriocins^{1, 21}, which can antagonize surrounding bacteria²⁰, but less is known about their cooperative behaviors. For example, Bacillus spp., one of the soil organisms that exhibit complex social behaviors, such as cooperative swarming, preferentially merge their swarms with close relatives^{22, 23}, recognized as a differential behavior toward relatives, known as kin discrimination. Specifically, within B. subtilis, kin soil isolates, defined as strains with at least 99.5% sequence identity at the housekeeping gene level, mostly merged their swarms and invaded a new territory as co-operators. In contrast, isolates with a nucleotide identity below 99.5% formed a visible boundary line at the site of encounter and did not enter a common swarm^24–26 or mixed intimately in a biofilm²⁷. In the coincubation experiments, kin cells formed mixed biofilms on the plant root, whereas non-kin cells engaged in antagonistic interactions with one strain primarily colonizing the plant root surface²⁵. Interestingly, associations of B. subtilis swarms with other species within the genus did not strictly follow this pattern, antagonizing closely related species and sometimes merging with more distantly related Bacillus spp.²⁶. Hence, relating phylogenetic shifts in the indigenous rhizo-Bacillus community to social interactions and community function remains a challenge, despite the importance of this information for a more efficient design of microbial inoculants.

Here, we fill this knowledge gap by using the well-known gram-positive PGPR strain Bacillus velezensis SQR9, a commercially available product (BIO™, China)²⁸ with an outstanding ability to suppress soil-borne diseases and promote plant growth, especially cucumber growth^{13, 28–33}. Through carefully designed pot experiments supported by bioinformatics of the bacterial community in the rhizosphere and in-depth functional analyses of the rhizosphere isolates, we show that B. velezensis SQR9 strongly affects the cooccurrence of the indigenous rhizosphere bacterial community and shifts the Bacillus community in the rhizosphere toward lower diversity and increased cooperativity. These new findings combined with the knowledge of relatedness, sociality, metabolic niche, and PGPR traits of Bacillus rhizosphere isolates, guided the recommendation for the design of plant beneficial Bacillus communities.

B. velezensis SQR9 alters the bacterial community in cucumber rhizospheres

B. velezensis SQR9 (hereafter referred to as SQR9) is a biostimulant that promotes the growth of cucumber (Cucumis sativus) through various plant growth promoting (PGP) mechanisms^{28, 29, 33}. However, it is not known if and how this beneficial bacterium interacts with the resident rhizosphere community. To unravel this mystery, we grew cucumber seedlings with and without SQR9 in a pot experiment using sterile (SS) and natural soil (NS) as described in Fig. 1 (for details, see Methods section). As expected, inoculation with B. velezensis SQR9 (SS_SQR9 and NS_SQR9) significantly (p < 0.01) stimulated the growth of cucumber seedlings in both natural and sterile soil compared with the noninoculated control (Fig. S1A and S1B). Furthermore, the growth of cucumber plants in the sterile soil (SS treatment) was slightly slower than in the natural soil treatment (Fig. S1A and S1B).

To obtain detailed information on how strain SQR9 influences the resident bacterial community in the rhizosphere, we then used 16S rRNA amplicon sequencing, which provided comprehensive insight into the diversity of the bacterial community. In addition, we applied primers that amplify the gyrase-encoding gene gyrA of Bacillus species and relatives³⁴. Compared to 16S rRNA-specific primers, the gyrA primers showed better phylogenetic resolution for the diversity of the Bacillus community³⁴. By using both, more general 16S rRNA and more specific gyrA primers, we show here that the addition of SQR9 to sterile soil (SS_SQR9) significantly decreased the Shannon diversity index (H-index) of bacterial communities in the rhizosphere (p < 0.001) and Bacillus related communities (p < 0.001) (Fig. 2A and 2B). In contrast, in nonsterile soils (NS_SQR9) we did not detect a reduction in the H-index for the total bacteria but only for the Bacillus community and related species (Fig. 2A and 2B). Nonmetric multidimensional scaling (NMDS) analysis of 16S rRNA amplicons showed no significant separation of communities due to SQR9 treatment (Fig. 2C). In contrast, gyrA NMDS analysis confirmed the separation between SQR9-treated and untreated rhizosphere (Fig. 2D, Supplemental results 1). Overall, these results support the conclusion that SQR9 alters the composition of the indigenous bacterial community in the rhizosphere and that these shifts are more pronounced at lower taxonomic levels than at higher taxonomic levels, with more broadly distributed diversity.

Next, we applied network analyses of the gyrA gene co-occurrence patterns to further investigate the interactions between SQR9 and the indigenous rhizosphere bacterial community and their impact on community assembly. In the SQR9-treated rhizosphere, we observed a reduction in the total number of nodes and an increase in the number of links (including both positive and negative links) (Spearman’s correlation coefficient R > 0.80, p < 0.01, two-sided tests, Table S1). This result indicated that the addition of SQR9 strongly affects the cooccurrence of the indigenous rhizosphere bacterial community (Table S1, Fig. 2E, and 2F, Supplemental results 2). Specifically, the color-coded nodes, which represent the degree of relatedness between the SQR9-gyrA and other gyrA sequences, confirmed a shift in community structure and enrichment of specific bacterial taxa in the rhizosphere (Fig. 2E and 2F). For example, in the untreated soil network, blue nodes representing phylogenetically distant members (50% ≤ D < 60%) dominated (Fig. 2E). In contrast, in the SQR9-treated soil, we observed enrichment of both the highly phylogenetically related (Module 1- red, 98% ≤ D < 100%) and moderately phylogenetically related members (Module 2- green, 70% ≤ D < 80%) with a concomitant increase in node connections (Fig. 2F). Moreover, the number of distantly related members (blue nodes) decreased in the SQR9-inoculated sample (Fig. 2F, Tables S2, Supplemental results 2). These results reconfirm that SQR9 interacts with and shapes the structure of the Bacillus community and promotes the enrichment of more closely related members in the rhizosphere bacterial community.

B. velezensis SQR9 enriches swarm merging interactions between isolates of the rhizosphere Bacillus sp. community

Given the results of bioinformatics analyses suggesting that SQR9 increases the connectedness of the Bacillus rhizosphere community, we set up experiments to test the hypothesis of their increased compatibility. Hence, we isolated spore-forming Bacillus strains and their relatives from the cucumber rhizosphere that had developed in sterile soil (SS) and sterile soil treated with SQR9 (SS_SQR9) (Fig. 1) and then tested their compatibility by a swarm encounter assay. When a boundary formed at the site of the swarm encounter, we assumed that the two strains were antagonistic or noncompatible, and if the two swarms merged, they were assumed to be compatible^{24, 35}.

We obtained more than 200 spore-forming isolates and then selected 30 isolates from each treatment (SS and SS_SQR9) based on taxonomic criteria (Bacillus and related species), their swarming ability on 0.7% agar and their biofilm-forming activity (Supplemental methods 1). We then examined the phenotype of swarm interaction for 435 pairwise strain combinations (excluding self-self-pairs) from each treatment. Next, we compared the frequency of swarm phenotypes (merging, intermediate or boundary, Fig. 3A) from sterile untreated (SS) and SQR9-treated (SS_SQR9) rhizosphere soil and found that inoculation of the cucumber rhizosphere with SQR9 increased the frequency of the swarm merging phenotype of Bacillus sp. isolates. During swarming, only 29.7% of pairwise combinations of isolates obtained from sterile treatment (SS) merged, while 58.9% of pairs of isolates obtained from cucumber rhizospheres treated with SQR9 merged their swarms. Consistent with this, the frequency of Bacillus sp. boundary formation was lower in SQR9-treated soils (18.4% pairwise combinations) than in sterile soil (52.9%) (Fig. 3B, Table S3). These results are consistent with the bioinformatics data presented in Fig. 2E and 2F, and suggest that strain SQR9 alters the Bacillus community toward more compatible and potentially cooperative behavior.

Phylogeny and compatibility of rhizosphere isolates

To associate different swarming patterns from the SS and SS_SQR9 rhizosphere to the phylogenetic relatedness of interacting strains within each treatment, we determined the gyrA nucleotide identity between strains and constructed phylogenetic trees of isolated strains from treatments SS and SS_SQR9 (Fig. 4A and 4C, Supplemental results 3), which are referred to as isolated communities. Strains isolated from the SS treatment in most cases showed more than 90% identity at the gyrA gene and a lower frequency of the swarm merging phenotype (Fig. 4B). In contrast, the SS_SQR9 treatment strains were on average less related (down to 84%) but more compatible with a higher frequency of the merging phenotype (Fig. 4D). Merging was a predominant phenotype between SS_SQR9 treatment strains, with gyrA identity ranging from 96 to 99.5% (Fig. 4C and 4D).

For strains in the SS treatment, the boundary phenotype was predominant within the arbitrary clusters (boundaries (B) = 125, merging (M) = 8), with merging and boundary formation between clusters present almost equally (B = 105, M = 121) (Fig. 4B). Among strains isolated from the SQR9-treated rhizosphere soil (SS_SQR9), the merging phenotype dominated within species clusters and between arbitrary clusters (B = 28, M = 92). Although some strains of two closely related species within arbitrary clusters formed boundaries (e.g. P. polymyxa and P. barcinonensis or B. safensis and B. pumilus), some also merged (e.g., B. licheniformis and B. amyloliquefaciens). Moreover, we observed an enrichment of merging between strains from different arbitrary clusters (B = 52, M = 164) (Fig. 4D). Overall, and in line with our bioinformatics data (Fig. 2), these observations suggest that SQR9 rhizosphere inoculation reduces the frequency of antagonists and makes the rhizosphere bacterial community more compatible.

Learning from nature: SQR9 enrichment of moderately related swarm mergers promotes plant growth

Thus far, our results have shown that B. velezensis SQR9 added to the rhizosphere changes the composition and social interactions of the bacterial community. However, how this information can be used to improve the design of PGP consortia has yet to be determined. It has been shown previously that highly related swarm compatible strains coexist on plant roots³⁵ and that moderately related strains also merge swarms²³. Hence, we reasoned that moderately related compatible strains will coexist on plant roots and exhibit less resource competition than closely related strains, which will improve the activity of moderately related PGP consortia. To test this hypothesis, we first determined the resource competition among the candidate PGPR strains in our consortia. We measured the carbon source utilization of 30 strains from the rhizosphere treated with SQR9 using the GEN III MicroPlate test assay performed by the Biolog system. Principal component analysis (PCA) showed that the patterns of carbon source utilization correlated strongly with the phylogenetic relatedness of the Bacillus isolates (Fig. 5A and 4C). In conjunction with information on swarming patterns and relatedness of strains to SQR9 (Fig. S2), this information provided the basis for consortia design.

As the final objective was to enhance plant growth, we applied the insights gained to mimic the consequences of SQR9 soil inoculation in the rhizosphere. We hypothesized that phylogenetic relatedness-based sociality and competition for carbon resources represent fundamental knowledge for the rational design of PGP consortia. To test this prediction, we compared two sets of consortia: the highly related swarming consortia (HR, 100% identity of the gyrA gene, isolates 5, 35, 73, SQR9) and the moderately related swarming consortia (MR, 70% ≤ D < 80%, isolates 2, 37, 43, SQR9) (strain traits indicated in Fig. 5A, 4C and S2). As it has been previously shown that increasing the richness of PGP consortia also positively affects PGP activity on tomato plants³⁶, we tested the effect of MR and HR consortia with increasing richness on cucumber growth in an experimental hydroponic system and potting experiments with natural soil.

As predicted, we did not detect an increase in PGP activity, an improvement in cucumber root colonization or an increase in IAA and siderophore production by combining HR strains, regardless of whether we used one or multiple strain consortia (Fig. 5B, S3A and S3B). MR consortia, on the other hand, resulted in an increase in root colonization and IAA production with increased MR richness (R²= 0.458, p ＜ 0.0001; R²= 0.623, p ＜ 0.0001) (Fig. 5C). Concerning siderophore production, the effect was significant (p ＜ 0.15), but the correlation with MR richness was less strong (R² = 0.165) (Fig. 5C). Additionally, the MR consortia improved cucumber growth in hydroponic (Fig. S3C and S3D) and potting experiments with nonsterile natural soil (Fig. S4A), again with significant increases in shoot height and dry shoot weight with increasing richness (Fig. S4B and S4C). Although individual MR isolates showed low PGP potential (Fig. S5), their ability to promote plant growth increased when multiple strains were combined (Fig. 5C, Supplemental results 4). Our results indicate that mixing moderately related swarm-merging strains significantly promotes PGP activity in a richness-dependent manner, highlighting the importance of relatedness-dependent ecological compatibility and niche breadth for consortia design.

Next, we constructed two types of consortia (HR and MR) with increasing richness to test our prediction further in a hydroponic system. Specifically, each consortium contained a mixture of one to eight strains from our collection of 60 cucumber rhizosphere strains. These gave 150 combinations for each consortia type: 60 with a single strain, 30 with two strains, 30 with four strains, and 30 with eight strains (Table S4). The HR consortia contained selected strains that exhibited a merging phenotype between their swarms and high competition for carbon sources (Table S4); the MR consortia contained selected strains exhibiting a merging phenotype between their swarms and lower competition for carbon sources (Table S4). We hypothesized that the HR and MR consortia would affect strain abundance, IAA production and siderophore production differently and that MR consortia would have more plant growth beneficial properties. Consistent with the trends shown in Fig. 5, in the hydroponic experimental system, the MR consortia showed improved performance in the hydroponic system in terms of PGP activity (Bacillus abundance, IAA production; siderophore production), which was evident with increasing richness (Fig. S6). Although in experiments with eight different strains tested in 150 combinations, the scattering of results was too high for us to predict significant correlations, the trend of increasing PGP activity with increasing richness was clear (Fig. S6). These richness dependent effects were not observed for HR consortia. We concluded that the positive effect of MR consortia richness on PGP activity may be due to low competition among individuals and the potential synergies created by complementary niches and thus more effective use of available resources (Fig. S6).

Plant-associated beneficial microorganisms show great promise for improving crop quality and productivity^{37, 38}. However, their use is hampered by our limited understanding of the relationships between the effects of microbial inoculants on the plant and its rhizosphere microbiome³⁸. Here, we provide evidence that B. velezensis SQR9 changes the composition of indigenous rhizosphere bacteria, and Bacillus species in particular, toward a less competitive and more cooperative community in which highly and moderately related strains are enriched (Fig. 6). Furthermore, our results provide the first evidence that the associated sociality shifts community-level functionality, as reflected in the improved PGP activities of moderately related consortia, which is important for developing more reliable PGP inoculants.

The application of SQR9 alters the composition of the Bacillus community and related species in the rhizosphere

Although Bacillus species are known for their PGP activities and are commonly applied in agroecosystems to improve plant health^{39, 40}, their impact on the rhizosphere bacterial community is still poorly understood. Previous studies examining the effects of inoculants, such as B. amyloliquefaciens FZB42, BNM122 and B. subtilis PTS-394, by amplicon sequencing of 16S rRNA genes did not detect a significant impact on the overall bacterial community composition^41–43. However, in this study, we complemented 16S rRNA gene analysis with gyrA amplicon sequencing. Although we also detected a decrease in 16S rRNA diversity in the SQR9-treated SS-rhizosphere, the gyrA targeting approach provided a means to unravel previously undetected dramatic changes in the composition of Bacillus species and closely related genera (Paenibacillus, Brevibacillus, Lysinibacillus), emphasizing the importance of intra- and interspecific competition. This approach required the development of a new tool – the gyrA3 primer pair, which covers the broad diversity of Bacillus species and relatives³⁴. In this work, we provide evidence that this primer amplifies gyrA genes of a rhizosphere bacterial community associated with an agriculturally important plant and that by targeting lower taxonomic units one can improve the detection and resolution of the rhizosphere microbiome dynamics by amplicon sequencing. Additionally, this work shows that SQR9 causes a dramatic shift toward a less diverse community with a higher proportion of highly interconnected strains that increased in frequency and were either highly related (98–100% identity, red group) or moderately related (70–80% identity, green group in Fig. 2F). In contrast, the genotypes with 50–60% gyrA identity (blue group) decreased in frequency. SQR9 also increased the number of connections within each group, revealing a positive correlation among the close relationship nodes (Fig. 2E and 2F).

Strain SQR9 changes social interactions in the rhizosphere bacterial community

Although the importance of the rhizosphere microbiome to plant health⁴³ and the potential benefits of bioinoculants are widely recognized^{5, 37}, the effects of bioinoculants on bacterial social interactions in the rhizosphere have been largely ignored. This work shows that strain SQR9 causes a dramatic shift in the community, with a higher proportion of strains that are related and thus more interconnected with SQR9 (Fig. 2E and 2F). Furthermore, compatibility among isolates from the rhizosphere treated with SQR9 dramatically increased (Fig. 3B). Moreover, the patterns of the gyrA gene co-occurrence and the results of swarming assays provided the first evidence that Bacillus and its closely related species are important targets of SQR9 activity in the rhizosphere (Fig. 2E, 2F and S2, Table S2).

What could be the mechanisms behind these effects? It seems that strain SQR9 outcompetes closely and more distantly related species. For example, the untreated isolated rhizosphere community comprised 15 Bacillus species and only one isolate of Lysinibacillus (Fig. 4A). In contrast, the SQR9-treated isolated community comprised four Bacillus species and three more moderately related species (Fig. 4C), confirming the strong antagonism of SQR9 against closely related species in the rhizosphere. In fact, B. velezensis strains, including SQR9, are known to produce a diverse range of bioactive secondary metabolites^45–48. Therefore, intense competition may lead to niche emptying and consequently provide an opportunity for compatible strains to grow, which is confirmed by our results.

The enrichment of compatible strains (Fig. 3) can also be explained by kin discrimination (KD)²⁰. KD is mediated in Bacillus species by mechanisms involving intercellular attack and defense molecules with extensive variation in genes and unique combinations in different strains²³. Antagonistic actions can be identified by the appearance of visible boundaries (boundaries, clearing zones) between swarms²⁵, with strains forming a boundary on semisolid media unable to coexist on plant roots, while swarm mergers join to form a plant root biofilm. Killing competitors can be advantageous, and indeed, we observed that strain SQR9 promoted the enrichment of compatible strains within specific clusters (Fig. S2) and reduced diversity within species clusters (compare Fig. 4A and 4B). Although swarm merging has been generally associated with Bacillus intraspecific sociality (including all intraspecies references) and some more distantly related Bacillus species²³, we found that moderately related species in the rhizosphere, including Bacillus, Paenibacillus and Lysinibacillus species, can also merge their swarms.

By looking at the community from two perspectives, first, how the addition of strain SQR9 changes community relatedness and compatibility in relation to SQR9, and second, how the strains in the rhizosphere community interact with each other after SQR9 inoculation (Fig. 4), we conclude that strain SQR9 shifts the community toward cooperativity. This raises a question: whether this impact is long-lasting or transient is unclear. Future work is needed to determine how these introductions affect the resident microbial communities. Overall, our results demonstrate that SQR9 via antagonism provided an opportunity for compatible strains to grow and consequently decrease competition. This is consistent with the theory that a certain level of competition is particularly important for the development of cooperation in bacteria^{18, 49}.

Smart design of synthetic Bacillus consortia with defined ecological (social) interactions

Plant-associated microbial communities have numerous potential applications in biotechnology, especially in agriculture⁵⁰. Recently, microbial consortia with lower complexity have been studied and used as model systems for the controlled assessment of ecological, structural, and functional properties of microbial communities⁵¹. However, a major challenge is to rationally engineer beneficial consortia with robust survivability and activity for functionally reliable applications under field conditions⁵². Addressing this challenge requires the integration of multiple approaches, including the reverse engineering of natural communities (e.g., inference-based co-occurrence analysis) and the rational engineering of beneficial consortia with desirable interactions for community-level functionality and robustness⁵³. Despite the fact that several underlying factors could directly or indirectly affect the performance of synthetic Bacillus consortia in the rhizosphere⁵⁴, we show that sociality and competition for carbon among these strains are important parameters that should be considered in the development of efficient plant probiotics (PGP inoculants). The results show that mixing compatible strains that do not compete for the same resources leads to more efficient PGP inoculants, and we confirmed their PGP activity in both hydroponic and natural soil systems (Fig. 5C and S4).

Overall, our work provides a general ecological framework for the intelligent assembly of Bacillus consortia for more efficient and reliable applications. First, the work provides evidence that the application of the beneficial strain SQR9 shifts the bacterial community in the rhizosphere and, in particular the Bacillus community toward increased cooperativity. Second, the results suggest that by including the ecological mechanisms (sociality and competition for carbon) used by microbial inoculants, we can improve guidelines and develop more effective products for sustainable agriculture.

Bacterial strains, isolation and culture conditions

We used Bacillus velezensis SQR9 (CGMCC accession no. 5808, China General Microbiology Culture Collection Center) as our model strain to assess its ecological impact on the resident bacterial community in the rhizosphere. Additional natural isolates of Bacillus and other spore formers were obtained from two kinds of cucumber rhizosphere soils, where cucumber seedlings grown in natural soil were transferred to sterile soil only (SS) or additionally inoculated with SQR9 (SS_SQR9) (see experimental design Fig. 1). Two hundred spore-forming isolates were obtained by heat treatment of rhizosphere soil samples and then 30 representative isolates for both SS and SS_SQR9 treatments were selected for further experiments based on the following criteria: a, three metabolic tests (the catalase test, the Voges-Proskauer test and anaerobic growth on agar) and the 16S rRNA nucleotide identity identified them as Bacillus⁵⁵; b, the ability to form pellicles (floating biofilm) in MSgg medium; and c, the ability to swarm on 0.7% agar. Bacillus strains were routinely grown in LB medium and, where appropriate, in B medium prepared as described³⁵.

Experimental design

To test whether the application of SQR9 to the rhizosphere shapes the overall bacterial community structure, we designed a cucumber greenhouse experiment, as presented in Fig. 1. Two-week-old cucumber seedlings from a hydroponic culture system⁵⁶ were transferred to pots filled with 200 g natural soil and incubated for one week, and then DNA from rhizosphere soil was extracted from 9 out of 45 treatments (NS_1w) (blue panel, Fig. 1). The remaining seedlings (orange panel, Fig. 1) were transferred to pots filled with 200 g natural or sterile soil (NS, SS) or both types of soils inoculated with 10 mL suspensions (10⁷ CFU mL^− 1) of B. velezensis SQR9 (NS_SQR9 and SS_SQR9) and incubated for an additional two weeks. At this point, DNA was extracted from the rhizosphere soil for 16S rRNA and gyrA amplicon sequencing. The rhizosphere soil from SS and SS_SQR9 variants (indicated by the orange arrow, Fig. 1) was also used for Bacillus spp. strain isolation. Strains were tested for social interactions in a swarming assay (Fig. 1) as described below. Plant shoot height and shoot dry weight were measured after four weeks of incubation.

DNA extraction and sequencing

Cucumber rhizosphere soils were collected as described by Chaparro et al.⁵⁷. Briefly, cucumber roots were gently removed from the soil, and the loose soil (༞1 mm, not the rhizosphere soil) was manually removed with sterile rubber gloves, leaving approximately 1 mm of soil on the roots. Rhizosphere soils were collected, and total DNA was extracted from 0.25 g of rhizosphere soil using the PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA). To minimize DNA extraction bias, successive DNA extractions of each sample were pooled before performing polymerase chain reaction (PCR). A NanoDrop ND-2000 spectrophotometer (NanoDrop, ND-2000, Thermo Scientific, 111 Wilmington, DE, USA) was used to assess DNA quality according to the 260/280 nm and 260/230 nm absorbance ratios⁵⁸.

Amplification of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed to assess the bacterial community using the primers 338F: 5’-CCTACGGRRBGCASCAGKVRVGAAT-3’ and 806R: 5’-GGACTACNVGGGTWTCTAATCC-3’. For Bacillus and its close relatives, amplification of the gyrA housekeeping gene was performed using the primers 243F: 5’- GCDGCHGCNATGCGTTAYACTC-3’ and 736R: 5’-CGGACAAGMTCWGCKATTTTTTC-3’ to assess the community composition. Nine 16S rRNA samples and three gyrA gene samples were sequenced per treatment. In comparison to 16S rRNA, these primers target Bacillus species, and show better phylogenetic resolution for Bacillus community diversity than 16S rRNA primers³⁴. PCR amplifications were combined in equimolar ratios and sequenced on an Illumina MiSeq instrument.

Bioinformatics analysis

The relative abundance of all gyrA genes was used for network analysis. The network analysis was performed using the Molecular Ecological Network Analyses Pipeline (MENAP) (http://ieg2.ou.edu/MENA/main.cgi)⁵⁹. According to the pipeline, network analysis contains two steps: network construction and network analysis. The network construction includes data updates, data standardization, pairwise similarity of relative abundance across different samples and determination of the adjacency matrix by an RMT-based approach. The network analysis includes network module detection, network overall topological structure, and topological role identification of individual nodes⁶⁰. The Shannon diversity index calculation and Bray-Curtis dissimilarity-based NMDS analysis were performed based on the rarefied table of the sequencing data using the vegan R package (v.2.5-2) (https://cran.r-project.org/package=vegan).

Swarm boundary assay

To test the social interaction between approaching swarms of different Bacillus spp. isolates, 9 cm plates containing B-medium with 0.7% agar were prepared fresh. Bacillus spp. strains were grown on solid Luria-Bertani plates at 30°C for 16 h. Cells were transferred into 3 mL of liquid B-medium and shaken overnight at 30°C. Overnight cultures were then diluted to an optical density (OD₆₀₀) of 0.5, and 2 µL was spotted on the plates at each side of the agar plate. The plates were dried in a laminar flow hood for 20 min, incubated for 2 days at 30°C, and photographed. Three phenotypes (merging, intermediate and boundary) were assigned to 870 pairs of swarms as described previously³⁵.

Establishing a Bacillus community richness gradient for two kinds of consortia to test the effects of PGP in vitro and greenhouse experiments

A probiotic Bacillus community richness gradient was created by establishing four richness levels (1, 2, 3 and 4) using Bacillus strains isolated from rhizosphere soil inoculated with SQR9 (SS_SQR9). We used a substitutive design where the total probiotic community biomass was kept constant at all richness levels. The same experimental design was followed in both the greenhouse and in vitro experiments. For further experiments, a representative Bacillus community for highly related (HR) consortia was built by using our collections with designated numbers: Bacillus velezensis (5), Bacillus velezensis (35), Bacillus velezensis (73), and Bacillus velezensis (SQR9); a representative Bacillus community for moderately related (MR) consortia was built by using isolates Bacillus licheniformis (2), Bacillus pumilus (37), Bacillus pumilus (43), and Bacillus velezensis (SQR9). These two kinds of consortia with varying richness were used to test the PGP effects in vitro and in greenhouse experiments.

To test the PGP effects of different Bacillus consortia in vivo, two forms of greenhouse experiments were performed. (1) The first was a hydroponic culture system, where we tested consortia with 4 Bacillus strains. The planting system was carried out in an aseptic conical flask containing 50 mL ¼ sterile Murashige Skoog (MS) medium, as described by Qiu et al.⁶¹. Briefly, cucumber seeds of the cultivar Jinchun 4 were surface disinfected in 2% NaClO solution for 15 min, washed thoroughly with distilled sterile water and planted in sterile vermiculite in a tissue-culture container. The seeds were allowed to germinate and grow for approximately 4 days in a growth chamber at 23°C with a 16 h light and 8 h dark photoperiod. When two cotyledons appeared, the seedlings were individually transplanted into an aseptic conical flask containing 50 mL ¼ sterile MS medium. After 4 days of growth, plants were inoculated to a final OD₆₀₀ = 0.02 with the Bacillus community grown for 4 h and placed on an orbital shaker at 50 rpm in the growth chamber at 70% humidity with natural light and 28 ± 2°C during the day and 22 ± 2°C at night for another two weeks. Each conical flask was treated as one biological replicate, with nine replicates set up for each treatment and nine replicates for the noninoculated control. Plant shoot height and shoot dry weight were measured after three weeks of incubation. (2) The second was a natural soil system, where the plant growth-promoting efficiency of each Bacillus community (composed of one to eight strains) was assessed in a greenhouse assay with natural, nonsterile soil. The trial was conducted from August 17 to November 8 in 2019 in the greenhouse at Nanjing Agricultural University. The soils used for the greenhouse assay were collected from a field site with a history of cucumber cultivation. The field site was located in Nanjing, Jiangsu Province, China and the soil had the following properties: pH 6.4, organic matter 18.6 g kg^− 1, available N 121 mg kg^− 1, available P 56 mg kg^− 1, and available K 89 mg kg^− 1. Two-week-old cucumber seedlings were transplanted to seedling pots with 5 kg of soil. After 7 days of growth, plants were inoculated with assembled consortia by drenching the pots to a final concentration of 10⁷ CFU g^− 1 soil. Each treatment was replicated 18 times, and the experimental plan included three blocks in a completed randomized design (9 plants for each block). The pots were incubated in a growth chamber at 70% humidity with natural light and 28 ± 2°C during the day and 22 ± 2°C at night. Plants were irrigated regularly with ½ Hoagland medium as described by Qiu et al.⁶¹. Nine randomly selected plants for each treatment were harvested after 55 days. Plant shoot height and shoot dry weight were measured.

Root colonization assay

Two-week-old cucumber seedlings were transferred to 30 mL of 1/4 Murashige and Skoog (MS) culture medium in an aseptic conical flask. The medium hosting the plant was then inoculated with Bacillus spp. strains at the final OD₆₀₀ = 0.02 in medium (approximately 1 × 10⁶ CFU mL^− 1), and placed on an orbital shaker at 100 rpm in the growth chamber at 30°C. After 2 days, the bacterial cells that colonized the cucumber root surfaces were collected and quantified using the method described by Qiu et al.⁶². Briefly, cucumber roots with colonized cells were aseptically removed from the container, washed three times in sterile water, and placed on axenic filter paper to remove the planktonic cells. Then, the roots were placed into a 250 mL sterile flask containing 45 g of glass beads (6 mm in diameter) and 100 mL of sterile water, which was shaken vigorously with a vortex mixer for 10 min to separate the attached cells from the roots.

Carbon source utilization assay

The carbon source utilization patterns of 60 Bacillus isolates from cucumber rhizosphere soils were recorded by the GEN III MicroPlate test assay performed with the Biolog system (Biolog, CA, USA). Briefly, bacterial suspensions were prepared and inoculated in a Biolog GEN III MicroPlate according to the manufacturer’s instructions (http://www.biolog.com). The utilization of carbon sources was determined after 12 h using Protocol A of Biolog’s MicroStation™ System⁶³.

Multiple combinations analysis the correlation between social interactions among community members and PGP phenotypic characterization

We assessed consortia functionality using the same methods as for the single isolates. In this experiment, we focused on three PGP phenotypic traits: colonization abundance and IAA and siderophore production. Bacteria from frozen stock were pregrown overnight in liquid LB at 30°C, pelleted by centrifugation (4,000 × g, 3 min), washed three times in 0.85% NaCl, and the cell density was adjusted to an OD₆₀₀ of 1.0 prior to further experiments. We then built Bacillus consortia using a substitutive design with richness levels of 1, 2, 4, and 8 strains from the collection of 60 Bacillus isolates from the cucumber rhizosphere and tested their effect in natural soil system. There were 150 combinations with each consortia type, 60 with a single strain, 30 with two strains, 30 with four strains, and 30 with eight strains (Table S4). The consortia design was conceived to ensure that each isolate was present at comparable frequency at each diversity level, allowing for separation of the effects of bacterial richness and composition³⁶. Detailed information on the components of the two kinds of Bacillus consortia is shown in Table S4, and the selection criteria were as follows: for HR consortia, we selected strains with a merging phenotype and high carbon resource competition; for MR consortia, we selected strains with a merging phenotype and lower carbon resource competition. Bacillus consortia were grown under the same conditions as the single isolates, after which the different traits were measured at the community level.

Statistical analysis

Figures were produced using the GraphPad Prism 8 or ggplot2 R package. Detailed statistical analysis is described in the figure legends. The data on all individual Bacillus in vitro performances (five PGP traits) were standardized between 0 (minimum value across all treatments) and 1 (maximum value across all treatments) and used in subsequent calculations and analysis as described above.

Data availability

Amplicon sequencing reads from the 16S rRNA gene and gyrA gene are available at NCBI Sequence Read Archive under accession number, PRJNA879238.

Trivedi P., Leach J.E., Tringe S.G.M., Sa T. & Singh B.K. Plant-microbiome interactions: from community assembly to plant health. Nat. Rev. Microbiol. 18, 607–621 (2020).
Finkel O.M. et al. A single bacterial genus maintains root growth in a complex microbiome. Nature 587, 103–108 (2020).
Cordovez V., Din-Andreote F., Carrión V.J. & Raaijmakers J.M. Ecology and evolution of plant microbiomes. Annu Rev. Microbiol. 73, 69–88 (2019).
de Vries F.T., Griffiths R.I., Knight C.G., Nicolitch O. & Williams A. Harnessing rhizosphere microbiomes for drought-resilient crop production. Science 368, 270–274 (2020).
Finkel O.M., Castrillo G., Herrera P.S., Salas G.I. & Dangl J.L. Understanding and exploiting plant beneficial microbes. Curr. Opin. Plant Biol. 38, 155–163 (2017).
Koprivova A. et al. Root-specific camalexin biosynthesis controls the plant growth-promoting effects of multiple bacterial strains. Proc. Natl Acad. Sci. USA 116, 15735–15744 (2019).
Kwak M.J. et al. Rhizoshere microbiome structure alters to enable wilt resistance in tomato. Nat. Biotechnol. 36, 1100–1109 (2018).
Tao C.Y. et al. Bio-organic fertilizers stimulate indigenous soil Pseudomonas populations to enhance plant disease suppression. Microbiome 8, 137 (2020).
Cipriano M.A.P. et al. Lettuce and rhizosphere microbiome responses to growth promoting Pseudomonas species under field conditions. FEMS Microbiol. Ecol. 92, fiw197 (2016).
Chowdury S.P., Hartmann A., Gao X. & Borriss R. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42- a review. Front Microbiol. 6, 780 (2015).
Molina-Santiago C. The extracellular matrix protects Bacillus subtilis colonies from pseudomonas invasion and modulates plant co-colonization. Nat. Commun. 10, 1919 (2019).
Chowdhury S.P. et al. Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community. Plos One 8, e68818 (2013).
Sun X., et al. Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions. ISME J 16 (3), 774–787 (2022).
Hutchinson G.E. Concluding remarks. Cold Spring Harb. Symp. Quant. Biol. 22, 415–457 (1957).
Hutchinson G.E. An introduction to population biology. New Haven CT: Yale University Press (1978).
Levin S.A. & Carpenter S.R. The princeton guide to ecology. Princeton University Press (2009).
Hardin G. The competitive exclusion principle. Science 131, 1292–1297 (1960).
Bruno J.F., Stachowicz J.J. & Bertness M.D. Inclusion of facilitation into ecological theory. Trends Ecol. Evol. 18, 119–125 (2003).
Wall D. Kin recognition in bacteria. Annu. Rev. Microbiol. 70, 143–160 (2016).
Strassmann J.E., Gilbert O.M. & Queller D.C. Kin discrimination and cooperation in microbes. Annu. Rev. Microbiol. 65, 349–367 (2011).
Mendes R., Garbeva P. & Raaijmakers J.M. The rhizosphere microbiome: significance of plant beneficial plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol. Rev. 37, 634–663 (2013).
Kalamara M., Spacapan M., Mandic-Mulec I., & Stanley-Wall N.R. Social behaviours by Bacillus subtilis: quorum sensing, kin discrimination and beyond. Mol. Microbiol. 110, 863–878 (2018).
Lyons N.A. & Kolter R. Bacillus subtilis protects public goods by extending kin discrimination to closely related species. mBio 8, e00723-17 (2017).
Kraigher B., Butolen M., Stefanic P. & Mandic-Mulec I. Kin discrimination drives territorial exclusion during Bacillus subtilis swarming and restrains exploitation of surfactin. ISME J 16 (3), 883–841 (2022).
Stefanic P. et al. Kin discrimination promotes horizontal gene transfer between unrelated strains in Bacillus subtilis. Nat. Commun. 12, 3457 (2021).
Lyons N.A., Kraigher B., Stefanic P., Mandic-Mulec I. & Kolter R. A combinatorial kin discrimination system in Bacillus subtilis. Curr. Biol. 26, 733–742 (2016).
Bolješić, M., Kraigher, B., Dogsa, I., Jerič Kokelj, B., & Mandic-Mulec, I. Kin discrimination modifies strain distribution, spatial segregation, and incorporation of extracellular matrix polysaccharide mutants of Bacillus subtilis strains into mixed floating biofilms. Appl Environ Microbiol, 88(18), e0087122 (2022).
Shao J., Xu Z., Zhang N., Shen Q. & Zhang R. Contribution of indole-3-3acetic acid in the plant growth promotion by the rhizospheric strain Bacillus amyloliquefaciens SQR9. Biol. Fertil. Soils 51, 321–330 (2015).
Qiu M. et al. Application of bio-organic fertilizer can control Fusarium wilt of cucumber plants by regulation microbial community of rhizosphere soil. Biol. Fertil Soils 48, 807–816 (2012).
Xu Z. et al. Enhanced control of cucumber wilt disease by Bacillus amyloliquefaciens SQR9 by altering the regulation of its degU phosphorylation. Appl Environ Microbiol. 80, 2941–2950 (2014).
Xu Z. et al. Antibiotic bacillomycin D affects iron acquisition and biofilm formation in Bacillus velezensis through a Btr-mediated FeuABC-dependent pathway. Cell Rep. 29, 1192–1202 (2019).
Xu Z. et al. Enhanced control of plant wilt disease by a xylose-inducible degQ gene engineered into Bacillus velezensis strain SQR9XYQ. Phytopathology 109(1), 36–43 (2019).
Li Y. et al. Volatile compounds from beneficial rhizosbacteria Bacillus spp. Promote periodic lateral root development in Arabidopsis. Plant Cell Environ. 44, 1663–1678 (2021).
Liu Y. et al. Housekeeping gene gyrA, a potential molecular marker for Bacillus ecology study. AMB Express. 12(1), 133. (2022).
Stefanic P., Kraigher B., Lyons N.A., Kolter R. & Mandic-Mulec I. Kin discrimination between sympatric Bacillus subtilis isolates. Proc. Natl Acad. Sci. USA 112, 14042–14047 (2015).
Hu, J. et al. Probiotic diversity enhances rhizosphere microbiome function and plant disease suppression. mBio 7, e01790-16 (2016).
Kwak M.J. et al. Rhizosphere microbiome structure alters to enable wilt resistance in yomato. Nat. Biotechnol. 36, 1100–1109 (2018).
Philippot, L., Raaijmakers, J.M., Lemanceau, P. & van der Putten, W.H. Going back to the roots: the microbial ecology of the rhizosphere. Nat. Rev. Microbiol. 11, 789–799 (2013).
Beauregard, P.B., Chai, Y., Vlamakis, H., Losick, R., Kolter, R. Bacillus subtilis biofilm induction by plant polysaccharides. Proc. Natl Acad. Sci. USA 110, E1621-E1630 (2013).
Santoyo G., Orozco-Mosqueda M.D.C. & Govindappa M. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and e: a review. Biocontrol Sci. Technol. 22, 855–872 (2012).
Correa, O.S. et al. Bacillus amyloliquefaciens BNM122, a potential microbial biocontrol agent applied on soybean seeds, causes a minor impact on the rhizosphere and soil microbial communities. Appl. Soil Ecol. 41, 185–194 (2009).
Krober, M. et al. Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing. Front Microbiol. 5, 252 (2014).
Chowdury S.P. et al. Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community. Plos One 8, e68818 (2013).
Roeland L.B., Corné M.J.P. & Peter A.H.M.B. The rhizosphere microbiome and plant health. Trends Plant Sci. 17, 478–486 (2012).
Wang D. et al. A genomic island in a plant beneficial rhizobacterium encodes novel antimicrobial fatty acids and a self-protection shield to enhance its competition. Environ Microbiol. 21, 3455–3471 (2019).
Scholz, R. et al. Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42. J Bacteriol. 196, 1842–1852 (2014).
Chen, X.H. et al. Comparative analysis of the complete genome sequence of the plant rowth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol. 25, 1007–1014 (2007).
Shank, E.A., Klepac-Ceraj, V., Collado-Torres, L., Powers, G.E., Losick, R. & Kolter, R. Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. Proc. Natl Acad. Sci. USA 108, E1236-E1243 (2011).
Foster, K.R., Wenseleers, T. & Ratnieks F.L.W. Kin selection is the key to altruism. Trends Ecol. Evol. 21, 57–60 (2006).
Toju, H. et al. Core microbiomes for sustainable agroecosystems. Nat. Plants 4, 247–257 (2018).
Kong, W., R. Meldgin, D., Collins, J.J. & Lu, T. Designing microbial consortia with defined social interactions. Nat. Chem. Biol. 14, 821–829 (2018).
Eng, A. & Borenstein, E. Microbial community design: methods, applications, and opportunities. Curr. Opin. Biotech. 58, 117–128 (2019).
Kehe, J. et al. Massively parallel screening of synthetic microbial communities. Proc. Natl Acad. Sci. USA 116, 12804–12809 (2019).
Bittihn, P., Din, M.O., Tsimring, L.S. & Hasty J. Rational engineering of synthetic microbial systems: from single cells to consortia. Curr. Opin. Microbiol. 45, 92–99 (2018).
Stefanic, P. & Mandic-Mulec, I. Social interaction and distribution of Bacillus subtilis phenotypes at microscale. J. Bacteriol. 191, 1756–1764 (2009).
Qiu, M. et al. Comparative proteomics analysis of Bacillus amyloliquefaciens SQR9 revealed the key proteins involved in in situ root colonization. J. Proteome Res. 13, 5581–5591 (2014).
Chaparro, J.M., Badri, D.V. & Vivanco, J.M. Rhizosphere microbiome assemblage is affected by plant development. The ISME Journal 8, 790–803 (2014).
Xun, W. et al. Diversity-triggered deterministic bacterial assembly constrains community functions. Nat Commun. 10, 3833 (2019).
Zhou, J., Deng, Y., Luo, F., He, Z. & Yang, Y. Phylogenetic Molecular Ecological Network of Soil Microbial Communities in Response to Elevated CO₂. mBio 2, e00122–11 (2011).
Zhou, J., Deng, Y., Luo, F., He, Z., Tu, Q. & Zhi, X. Functional molecular ecological networks. mBio 1, e00169–10 (2010).
Qiu, M. et al. De-coupling of root-microbiome associations followed by antagonist inoculation improves rhizosphere soil suppressiveness. Biol Fertil. Soils. 50, 217–224 (2014).
Qiu, M. et al. Comparative proteomics analysis of Bacillus amyloliquefaciens SQR9 revealed the key proteins involved in in situ root colonization. J Proteome Res. 13, 5581–5591 (2014).
Stefanic P. Environment shapes the intra-species diversity of Bacillus subtilis isolates. Microb. Ecol. 79: 853–864 (2020).

ACKNOWLEDGMENTS

This work was financially supported by the National Natural Science Foundation of China (31972512, 42090064 and 32072675), the National Key Research and Development Program (2021YFD1900300), the National Scientific and Technological Program on Basic Resources Investigation (No. 2019FY102000) and Slovenian Research Agency national program grant P4-0116 and research projects J4-9302 and J4-8228.

AUTHOR CONTRIBUTIONS

ZX, IMM designed the study, YL, TY performed the experiments. YL, ZX, WX, YM, PS, IMM analyzed the data. ZX, WX created the figures. ZX wrote the first draft of the manuscript, ZX, IMM, PS, NZ, RZ and QS revised the manuscript.

DECLARATION OF INTERESTS

The authors declare that they have no conflicts of interest.

There is NO Competing Interest.

Figure4Adistance.xlsx
Figure 4A_distance
Figure4Cdistance.xlsx
Figure 4C_distance
TableS1.docx
Table S1
TableS2.xlsx
Table S2
TableS3.xlsx
Table S3
TableS4.xlsx
Table S4
FigureS1.pdf
FigureS2.pdf
FigureS3.pdf
FigureS4.pdf
FigureS5.pdf
FigureS6.pdf
Supplementalresultsandmethods.docx
SupplementaryLegends.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Plant beneficial bacterium promotes plant growth by altering social networks of bacteria in the rhizosphere

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Strain SQR9 changes social interactions in the rhizosphere bacterial community

Materials And Methods

Bacterial strains, isolation and culture conditions

Experimental design

DNA extraction and sequencing

Bioinformatics analysis

Swarm boundary assay

Root colonization assay

Carbon source utilization assay

Multiple combinations analysis the correlation between social interactions among community members and PGP phenotypic characterization

Statistical analysis

Data availability

References

Declarations

Additional Declarations

Supplementary Files

Status:

Version 1