Engineering ecologically complementary rhizosphere probiotics using consortia of specialized bacterial mutants

While bacterial diversity is beneficial for the functioning of rhizosphere microbiomes, multi-species bioinoculants often fail to promote plant growth. One potential reason for this is that competition between inoculated consortia members create conflicts for their survival and functioning. To circumvent this, we used transposon mutagenesis to increase the functional diversity within Bacillus amyloliquefaciens bacterial species and tested if we could improve plant growth-promotion by assembling consortia of closely related but functionally specialized mutants. While most insertion mutations were harmful, some improved strains’ plant growth-promotion traits without increasing antagonism between them. Crucially, plant growth-promotion could be improved by applying these specialist mutants as consortia, leading to clear positive relationships between consortia richness, plant root colonization and protection from bacterial wilt disease. Together, our results suggest that increasing intra-species diversity could be an effective way to increase probiotic consortia multifunctionality, leading to more stable plant growth-promotion throughout growth cycle via insurance effects.


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(IPCR) method as previously described by Le Breton and colleagues (52). First, 5 μg of genomic DNA isolated 192 from each respective transposon mutant was digested with Taq I and circularized using 'Rapid Ligation' kit 193 (Fermentas, Germany). IPCR was carried out with ligated DNA (100 ng), using oIPCR1 and oIPCR2 primers (SI 9 (at three-leaves stage) were then transplanted to seedling trays containing natural, non-sterile soil collected 208 from a tomato field in Qilin Town, Nanjing, China (64). Plants were inoculated with individual B.
soil (82). The R. solanacearum strain was inoculated using the same method one week later at a final concentration of 10 6 CFU g -   antagonistic effects towards each other based on agar overlay assays. These eight mutants were then used to 244 assemble a total of 29 consortia with 2, 4 or 8 mutants following a substitutive design where each mutant was 245 equally often present at each richness level (see Table S5 for detailed consortia assembly). Mutants were 246 mixed in equal proportions in each consortium with final total bacterial density of 10 8 cells mL -1 (e.g., 50:50% 247 or 25:25:25:25% in two and four mutant consortia, respectively). This design has previously been used to

Effects of transposon insertions on B. amyloliquefaciens T-5 traits measured in vitro and in vivo 282
We first quantified the effects of transposon insertions on B. amyloliquefaciens T-5 traits using 479 mutants 283 that were randomly selected across the whole mutant library (2000 mutants in total). Most insertions had 284 negative effects on the four measured traits, with more than half of the mutants showing reduced swarming 285 (58.7%), biomass production (67.2%) and biofilm formation (60.8%) compared to the wild-type strain (Fig. 1A, beneficial mutations resulted mainly in a moderate improvement, while harmful mutations often led to severe 290 reduction in measured bacterial traits (Fig. 1A). Moreover, several insertions caused trade-offs, where improvement regarding one trait led to a reduction in the expression of other traits (Fig. 1B). For example, 292 swarming motility correlated negatively with biofilm production, while biomass production led to a trade-off 293 with both biofilm production and pathogen suppression (Fig. 1B). These results thus further suggest that 294 transposon insertions constrained the simultaneous expression of multiple traits, leading to specialized B.

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amyloliquefaciens T-5 mutants, which could be clustered in three phenotypically distinct groups (Adonis test: Fig. 1C). Relative to the wild-type strain, mutants belonging to the cluster 1 showed 297 increase in biofilm formation and pathogen suppression but reduced biomass and no change in biofilm 298 production ( Fig. 1D, SI Appendix, Table S6). Mutants in the cluster 2 showed improved swarming motility and 299 reduced pathogen suppression but no significant changes in biomass production or biofilm formation (Fig. 1D, Table S6). Finally, mutants grouped in the cluster 3 had poor performance overall, showing highly 301 reduced swarming motility and pathogen suppression, with no changes in biomass production or biofilm 302 formation (Fig. 1D, SI Appendix, Table S6). To test if the mutants grouped in different clusters also differed in 303 their tomato root colonization or ability to protect plants from pathogen infections, 47 mutants representing 304 all three clusters were randomly selected for a greenhouse experiment (the specific effects of insertions on 305 biological processes, cellular components and molecular function for all mutants are shown in SI Appendix, 306 Figure S1 and Dataset S2). Compared to the wild-type, 57% of Bacillus mutants (27/47) reached lower 307 population densities in the rhizosphere (30 days post-pathogen inoculation (dpi)), and this was especially clear 308 for mutants belonging to clusters 2 and 3. In contrast, mutants belonging to the cluster 1 retained efficient 309 root colonization and some of them showed improved root colonization relative to the wild-type. Similarly, 310 while 93% of mutants (44/47) exhibited reduced plant protection relative to the wild-type strain, this was less 311 clear with mutants belonging to cluster 1, whom a few showed even improved plant protection relative to the 312 wild-type (30 dpi, Fig. 1E, F, SI Appendix, Table S7). Together, these results suggest that while most transposon 313 mutants showed reduced performance relative to the wild-type strain, some of them showed improvement in growth-promotion traits measured in vitro (SI Appendix, Table S3, Table S4, Dataset S2; two representative mutants per each measured traits selected). We first confirmed that the mutants did not show direct  Table S8). Crucially, the effect of consortia richness remained significant after 351 sequential removal of each mutant and refitting of the model, which demonstrates that the effect of diversity 352 was relatively more important compared to mutant identity effects (SI Appendix, Table S9). Together, these 353 data suggest that mutant consortia diversity was positively linked with consortia performance in vivo, which 354 was associated with consortia mean performance and pathogen suppression measured in vitro. To explore potential underlying mechanisms between consortia diversity and improved performance, we 358 focused on analyzing the dynamics of root colonization and plant protection using 47 individual B.

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amyloliquefaciens T-5 mutants at 5, 15 and 30 dpi time points. Specifically, we tested the insurance hypothesis 360 (87), which predicts that a community composed of functionally diverse genotypes is likely to perform better 361 because of the likelihood that some mutants will thrive as prevailing conditions change during the plant 362 growth, providing increased stability for the plant-microbe interaction. Trait correlation with the root 363 colonization and plant protection became more significant with time and most significant correlations were 364 observed at the final time point (30 dpi, followed by middle (15 dpi) and early (5 dpi) time points (Table 1, SI 365 Appendix, Figure S5). Specifically, high swarming motility predicted the rhizosphere colonization during the 366 seedling stage (5 dpi, Table 1; Fig 3A, Fig 3I, F 1,46 = 15.65, R 2 = 0.2538, p < 0.001), while biomass production was not significantly associated 374 with either root colonization or plant protection at any time points (Table 1; Fig 3B, 3G). As a result, the mean

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In this work, we tested if increasing intra-species diversity of B. amyloliquefaciens T-5 bacterium via 385 mutagenesis could offer a viable strategy for improving mutant consortia multifunctionality and plant 386 growth-promotion. Our results show that mutations that improved bacterial performance regarding one trait growth-promotion: swarming motility, biomass production, biofilm formation and direct pathogen suppression identified in several genes associated with broad range of functions (SI Appendix, Figure S1, Dataset S2). With eight specialist strains that were used for consortia assembly experiment, increased swarming motility was  log-transformed before the analysis). Table data represent only the most parsimonious models based on the 'df' denotes degrees of freedom and 'R 2 ' denotes total variance explained by regression coefficient of determination. The arrows represent the direction of coefficient values: ↑: coefficient > 0; ↓: coefficient < 0.
Significant effects (p < 0.05) are highlighted in bold.

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Day post pathogen inoculation (dpi)      Linear term   strain and pathogen-only control treatments, respectively. Mean differences between consortia were analyzed 847 using student's t-test: *** denotes for statistical significance at p < 0.001; ** denotes for statistical significance 848 at p < 0.01; * denotes for statistical significance at p < 0.05. Bottom panel shows the 'trait specialism' of eight 849 mutants (colored squares) and their presence (black squares) and absence (white squares) in the tested