Differences in resources use lead to coexistence of seed-transmitted microbial populations

Seeds are involved in the vertical transmission of microorganisms in plants and act as reservoirs for the plant microbiome. They could serve as carriers of pathogens, making the study of microbial interactions on seeds important in the emergence of plant diseases. We studied the influence of biological disturbances caused by seed transmission of two phytopathogenic agents, Alternaria brassicicola Abra43 (Abra43) and Xanthomonas campestris pv. campestris 8004 (Xcc8004), on the structure and function of radish seed microbial assemblages, as well as the nutritional overlap between Xcc8004 and the seed microbiome, to find seed microbial residents capable of outcompeting this pathogen. According to taxonomic and functional inference performed on metagenomics reads, no shift in structure and function of the seed microbiome was observed following Abra43 and Xcc8004 transmission. This lack of impact derives from a limited overlap in nutritional resources between Xcc8004 and the major bacterial populations of radish seeds. However, two native seed-associated bacterial strains belonging to Stenotrophomonas rhizophila displayed a high overlap with Xcc8004 regarding the use of resources; they might therefore limit its transmission. The strategy we used may serve as a foundation for the selection of seed indigenous bacterial strains that could limit seed transmission of pathogens.

asymptomatic and can take place on non-host plants 16,17 , which can then serve as a reservoir 65 of plant pathogens. 66 To control seed transmission of plant pathogens one should either eradicate them 67 on seed-producing crops or perform seed treatments. Fungicide application during plant 68 production or as seed treatment is an effective means of fungal pathogen management 18 . 69 Since these chemical-base methods are unsatisfactory for bacterial plant pathogens and 70 have a potentially harmful environmental impact 19 , alternative control methods have been 71 proposed, including seed health testing, physical seed treatment (e.g thermotherapy) and 72 biological methods such as seed coating of specific biocontrol agents 14,18 . Direct 73 incorporation of biocontrol agents within the seed tissues through inoculation of seed-74 9 of Abra43 and Xcc8004 did not significantly alter (P > 0.01) the observed functional richness, 163 the estimated functional richness and the functional diversity of the seed microbiome (Fig.  164 2a-c). While harvesting year significantly impacted functional membership (P < 0.0001, 165 44.4%) and functional composition (P < 0.0001, 52.6%) of the seed microbiome, no 166 significant differences (P > 0.001) in functional membership or composition ( Fig. 2d and Fig.  167 2e) were observed after seed transmission of Abra43 and Xcc8004. In addition, we did not 168 detect any significant difference in RA of broad functional categories following pathogen 169 transmission (Fig. 2f). Altogether these results highlight that seed transmission of Xcc8004 170 and Abra43 do not modify either the predicted structure or function of the seed microbiome. The absence of shift in structure and function of the seed microbiome following the 180 plant-pathogen transmission suggests that the targeted plant pathogens and the other 181 members of the seed microbiome do not compete for nutrient and space. We first 182 investigated the spatial localization of Xcc8004 within seeds using fluorescence in situ 183 hybridization. Using surface sterilized seeds, Xanthomonas spp. were detected inside 184 inoculated seeds (Fig. 3). They were particularly present in embryonic tissues, especially at 185 the surface of the radicle among other bacteria (Fig. 3a). They were also detected, however, 186 in other embryonic zones of the seeds. Xanthomonas spp. were identified in the same 187 tissues in control samples, but with lower abundance than Xcc8004-seeds (Fig. 3b). Using a 188 photon detection method or analog integration on confocal microscope, similar results were 189 obtained but with brighter signals from the specific probe using analog integration (Fig. 3c  190 and Fig. 3d). Interestingly, cells were single or as packs of several bacteria (Fig. 3a-d). In 191 control and Xcc8004-inoculated seeds hybridized with negative probe, no signal was 192 identified ( Fig. 3e and Fig. 3f). On the internal side of the seed coat, Xanthomonas spp. 193 were further identified in Xcc8004 inoculated seeds, showing different niches of colonization 194 ( Fig. 3g and Fig. 3h). representative bacterial isolates (see materials and methods). Overall, 11 metagenome-204 assembled genomes (MAGs; >50% completeness, <10% contamination) and 21 genomes 205 sequences were obtained (Table 1). These genomic sequences represented the major 206 bacterial species detected with the ParaKraken taxonomic classification approach, and, 207 based on their gyrB sequences, represented the main gyrB amplicon sequences variants 208 (ASVs) of the radish seed microbiomes (Table 1 and Additional file 6 24,35 ). According to the 209 average nucleotide identity based on blast (ANIb) values, these 32 genomic sequences were 210 divided into 22 bacterial species (Additional file 7). The relative abundance of each genome 211 sequence within the seed microbiome was estimated by mapping the metagenomics reads to 212 these sequences and expressed as average coverage. Overall, genomic sequences related 213 to Pantoea agglomerans and Pseudomonas viridiflava were highly abundant in all radish 214 seed samples, with an average coverage of approximately 200X and 80X, respectively 11 217

Assessment of resources overlap among bacterial members of the seed microbiome. 218
Overlap in nutritional resource use between Xcc8004 and bacterial populations from 219 the seed microbiome was assessed by profiling nutrient consumption patterns of Xcc8004 220 and seed bacterial isolates with GEN III MicroPlate. Overall, the bacterial consumption 221 pattern was largely grouped by the phylogenetic relationships between strains (Additional 222 file 9). Overlap in nutritional resources between Xcc8004 and seed-associated bacterial 223 strains ranged from 0.23 to 0.50, with the highest overlap being observed with strains 224 CFBP13503 and CFBP13529 of Stenotrophomonas rhizophila (Fig. 4) (Fig. 4). The highest median overlap was 232 observed with the functional categories E (0.61) and I (0.66), while the median overlap for G 233 and P was below 0.5. In all cases, the most pronounced resource overlap was associated 234 with genome sequences of Xanthomonadales (Fig. 4). 235 To determine if the observed resource overlap was associated with a decrease of 236 Xcc8004 population during competition with these bacterial strains, co-inoculation of 237 Xcc8004 was performed with each seed bacterial isolate on radish seed exudates media 238 (see materials and methods). We observed a significant decrease (P < 0.01) in Xcc8004 239 CFU at 2 3, 4 and 5 dpi during co-inoculation with strains CFBP13503 and CFBP13529 (Fig.  240   5). This effect seems to be dependent on competition for nutritional resources, since no 241 direct antagonism was observed between Xcc8004 and S. rhizophila strains during overlay 242 assay (data not shown). To gain insight into potential competition for nutrients between 243 Xcc8004 and S. rhizophila strains, we compared the set of OGs that were exclusively shared 244 between these strains (Additional file 10). A total of 251 CDSs divided into 219 OGs were 245 specifically shared between Xcc8004 and S. rhizophila. While, most of these OGs have no 246 predicted function, nine CDSs were related to carbohydrate utilization (pectacte lyase, 247 mannosidase and glucosidase). In addition, multiple protein-coding genes involved in rapid 248 utilization of the limiting resource(s), such as TonB-dependent transporters (TBDTs) were 249 also shared between the Xanthomonadales strains (Additional file 10). Xcc8004 and S. rhizophila were also detected in germinating seeds and radish seedlings 261 (Additional file 11). The competition for resources between Xcc8004 and S. rhizophila 262 strains observed on exudates media (Fig. 5) and the co-occurrence of these two species 263 within germinating seeds and seedlings either suggest that resources are not a limiting factor 264 within these habitats or that both species are located in different tissues. In our experimental design, the impact of Xcc8004 and Abra43 transmission on the 293 structure and function of the seed microbiome was assessed during two consecutive years. 294 The changes in taxonomic and functional profiles were mostly driven by the harvesting year, 295 thus confirming previous results obtained through community profiling approaches 35,37,46 . This 296 is perhaps not surprising, as abiotic factors, such as soil or field management practices, have 297 been already observed to have a strong influence in the seed microbita 37 . In contrast, the 298 seed transmission of the two plant pathogens, Xcc8004 and Abra43, did not impact the 299 overall composition of the seed microbiome. 300 Previous reports have shown that Xcc8004 is seed-transmitted through the xylem 301 and the stigma, while Abra43 is seed-transmitted via fruits 30-32 . Hence Xcc8004 is probably 302 one of the primary colonists of the seed microbiota, while Abra43 is a late-arriving species. 303 Although both phytopathogenic agents were efficiently transmitted to radish seeds, they 304 impacted neither the structure nor the function of the seed microbiome, implying that they are 305 not competing with the other members of the microbiome for the same ecological niches in 306 the seed habitat. According to in situ hybridization, Xanthomonas sp. co-occurred with other 307 bacterial taxa within the seed coat and at the surface of the seed embryo. Since no spatial 308 separation was observed between Xcc8004 and other seed-borne bacterial populations, then 309 differences in resources consumption can probably explain this co-existence. Based on our 310 genomics prediction of resource overlap and competition assays performed on radish 311 exudate media, most of the bacterial populations of the seed microbiome are not competing 312 with Xcc8004 for nutritional resources. Although the competition assay performed does not 313 necessary reflect the actual composition and concentration of nutrients that are available for 314 microbial growth during seed development 36,49 , it can serve as a proxy for assessing 315 competition between seed-borne bacterial species. 316 Under our experimental conditions, the only bacterial population that competes for 317 nutritional resources with Xcc8004 belongs to the species S. rhizophila. Competition for a 318 limiting resource can be divided into two categories: exploitative competition that is related to 319 the rapid use of the limiting resource and contest competition that involves antagonistic 320 interactions with production of antimicrobial compounds 50 . As we did not observe an 321 antagonistic relationship with the overlay assay used in this work, the observed competition 322 between S. rhizophila strains and Xcc8004 is likely due to resources use 48 . This contest 323 competition is frequently related to efficient uptake of nutrients by the competing species 50 . 324 Interestingly numerous OGs that are specifically shared between Xcc8004 and S. rhizophila 325 strains CFBP13503 and CFBP13529 are related to TBDTs, which form a specific 326 carbohydrate utilization system 51 . 327 According to the community profiling approach performed on more than 300 radish 328 seed samples 24,35 , it appears that S. rhizophila strains and Xcc8004 co-exist within the seed 329 habitat. Moreover, both bacterial species co-occurred on germinating seeds and seedlings 330 during germination and emergence, suggesting that nutritional resources are not limited 331 enough within these habitats for observing a strong niche preemption. Alternatively, we 332 cannot rule out the hypothesis of both species being not spatially related. In contrast to 333 Xcc8004, S. rhizophila is mainly located at the seed surface of radish, since no strains were 334 recovered after seed surface sterilization (unpublished observations). Seed surface 335 localization is in accordance with the epiphytic localization of S. rhizophila DSM 14405 on 336 leaves and roots tissues of cotton, sweet pepper and tomato 52 . Owing to this predicted 337 localization, it is tempting to postulate that S. rhizophila is a late colonist of the seed 338 microbiome, being transmitted throug the external pathway. 339 Nowadays, sanitary quality of seeds is achieved through chemical methods and 340 prophylaxis measures. Since reducing pesticide usage is an important objective for 341 sustainable agriculture, the search for alternative seed treatments is essential. One of these 342 alternative treatments consists in coating the seeds with microorganisms possessing 343 biocontrol activities. However, biocontrol-based strategies require a better understanding of 344 the colonization abilities of the microbial consortia within the seed habitat and its persistence 345 within the spermosphere 21 . Our data showed that S. rhizophila and Xcc8004 have a high 16 resource overlap and can compete in vitro for resources. An interesting approach to restrict 347 seed transmission of Xcc8004 could be the introduction of S. rhizophila into the seed tissues 348 with EndoSeed TM technology 20 . Inoculation would encourage the co-existence of both taxa 349 within the same ecological niche and maybe limit the size of Xcc8004 population within seed 350 tissues. In addition to this augmentative biological control strategy, the limitation of Xcc8004 351 transmission could be potentially obtained by sowing radish seeds in plots containing a high-352 level of S. rhizophila. Indeed, the composition of the spermosphere-associated microbial 353 assemblages is highly influenced by local horizontal transmission 53 . All things considered, S. 354 rhizophila seems to be a promising candidate to reduce seed transmission of the pathogen 355 Xcc8004, although further seed transmission experiments are needed to corroborate the fate 356 of these taxa in planta. Collectively, these data may serve as a foundation for further 357 research towards the design of novel biocontrol-based strategies on seed samples. 358 359

Conclusions 360
Seed-transmission of phytopathogenic agents is significant for disease emergence and 361 spread. In the present study, we have shown that the presence of Abra43 and Xcc8004 362 within radish seed samples did not modify the structure and function of the seed microbiome. 363 The absence of community shift is explained in part by the low overlap in the use of 364 resources between Xcc8004 and the main seed-borne bacterial populations. However, we 365 have identified potential competitors of Xcc8004 belonging to S. rhizophila species. HiSeq3000. PacBio sequencing was performed on a RSII system (details in Text S1). 396 PacBio reads were used to improve the contigs length of the meta-assembly but were not 397 To investigate changes in the relative abundance between the different seed 431 samples, rare species (those occurring in less than 75% of the samples) were first removed 432 from the species count table. Fcros differential abundance analysis 31 was then performed 433 between each sample pair on all species and just the species with the highest abundance 434 (>0.1% RA). A p-value cutoff of 0.05 and an f-score of 0.9 were used to determine differential 435 abundance.
The species scaled data was also run through DUO 436 gyrB amplicon library preparation were performed on germinating seed (n=6) and seedling 492 (n=8) samples according to the procedure described earlier 46 (see details in Text S1). 493 Libraries were sequenced with a MiSeq reagent kit v2 (500 cycles). Fastq files were 494 processed with DADA2 1.6 73 using the parameters described in the workflow for "Big Data: 495 Paired-end". The only modification made regarding this protocol was a change in the 496 truncLen argument according to the quality of the sequencing run. Species abundance was 497 assessed on germinating seed and seedling samples with the R package phyloseq 60 (ASVs 498 taxonomic affiliation details in Text S1). 499 500

DOPE-FISH and CLSM microscopy of Xcc8004 infected seeds 501
Seeds were surface-sterilized, cut in half and fixed in a paraformaldehyde solution. DOPE-502 FISH was performed with probes from Eurofins (Austria) labeled at both the 5' and 3' end 503 positions according to Glassner et al. 74 using an EUBmix targeting all bacteria (EUB338, 504 EUB338II, EUB338III) coupled with the fluorochrome Cy3 75,76 , and a Xanthomonas spp. 505 targeting probe (5' -TCATTCAATCGCGCGAAGCCCG-3') coupled with Cy5 77 . A NONEUB 506 probe 78 coupled with Cy3 or Cy5 was also used independently as a negative control (further 507 protocol details in Text S1). Samples were observed under a confocal microscope (Olympus 508 Fluoview FV1000 with multiline laser FV5-LAMAR-2 HeNe(G) and laser FV10-LAHEG230-2) 509 (see Text S1 for image processing details). Pictures were cropped, and whole pictures were    The experimental plot from where the bacterial isolate was obtained, gyrB amplicons sequence variant (ASV), genome size, number of contigs,