Bacterial microbiome associated with the rhizosphere and root interior of crops in Saskatchewan, Canada

Rhizosphere and root associated bacteria are key components of plant-microbiomes and influence crop production. In sustainable agriculture, it is important to investigate bacteria 20 diversity in various plant species and how edaphic factors influence the bacterial microbiome. In 21 this study, we used high-throughput sequencing to assess bacterial communities associated with the rhizosphere and root interior of canola, wheat, field pea, and lentil grown at four locations in 23 Saskatchewan, Canada. Rhizosphere bacteria communities exhibited distinct profiles among 24 crops and sampling locations. However, each crop associated with distinct root endophytic 25 bacterial communities, suggesting that crop species may influence the selection of root bacterial microbiome. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant phyla in the 27 root interior, whereas Gemmatimonadetes, Firmicutes and Acidobacteria were prevalent in the 28 rhizosphere soil. Pseudomonas and Stenotrophomonas were predominant in the rhizosphere and 29 root interior, whereas Acinetobacter , Arthrobacter , Rhizobium , Streptomyces, Variovorax and Xanthomonas were dominant in the root interior of all crops. The relative abundance of specific 31 bacterial groups in the rhizosphere, correlated with soil pH, silt and organic matter contents, however, there was no correlation between root endophytes and analyzed soil properties. These 33 results suggest that the root microbiome may be modulated by plant factors rather than soil 34 characteristics.

Venn diagram also revealed that number of OTUs that were detected only in the rhizosphere of 161 wheat and canola was higher when compared to pea and lentil separately ( Figure 2). Similarly, the 162 number of OTUs detected only in the root interior of canola and wheat was higher when compared 163 to pea and lentil ( Figure 2). Additionally, some OTUs were shared between the rhizosphere and 164 root interior. However, these OTUs represented a high percentage (97-99%) and (77-90%) of the 165 sequence reads in the root interior and rhizosphere, respectively. 166 The diversity of bacterial communities associated with crops was evaluated using Chao 1 167 estimator and Simpson's reciprocal (1/D) index (Table 3) (Table 4). In the root 178 interior, the diversity and number of OTUs were higher in canola, followed by wheat, pea and 179 lentil (Table 3). Growing season influenced the diversity (P<0.05) of the bacteria communities 180 associated with rhe root interior but not the species richness.

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The community structure of bacterial OTUs, determined using PCoA, indicated distinct 182 pattern in the rhizosphere and root endophytic bacterial communities associated with the crops, 183 with the first two axes explaining 33% and 69% of the total variation in the rhizosphere and root 184 interior, respectively (Figures 3 and 4). Permanova analysis confirmed that bacteria community 185 structure differed between the rhizo-compartments (P≤0.001) ( Table 5). Rhizosphere bacteria 186 exhibited high variability in the OTU profiles among all crops and locations studied. As a result, 187 no clustering was identified by PCoA in response to these factors ( Figure 3). Bacterial 188 communities associated with the root interior were clustered in 3 regions using PCoA, 189 corresponding to canola, wheat, and a cluster containing pea and lentil communities ( Figure 4).

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However, no clustering was detected between endophytic communities from different field 191 locations. Permanova analysis also indicated that crop species influenced (P≤0.001) both 192 rhizosphere and root endophytic bacteria communities in all the sampling locations (Table 5). 193 Similarly, bacterial comunities in both rhizo-compartments were influenced (P≤0.001) by 194 sampling locations, except for root endophytes associated with lentil (Table 5)  Rhizosphere soil associated with the crops exhibited similar phyla profiles, characterized by a high 202 abundance of Proteobacteria and Actinobacteria followed by Bacteroidetes, Gemmatimonadetes,

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Firmicutes and Acidobacteria ( Figure S1). The remaining phyla represented less than 1% of the 204 total OTUs detected in the rhizosphere. However, there were notable differences in the phyla 205 profiles of bacteria colonizing the root interior of the four crops studied. For example, 206 Proteobacteria was the predominant phylum in lentil and pea, followed by canola and wheat.

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Actinobacteria and Bacteroides were also detected in wheat and canola, but their relative 208 abundance was very low in pea and lentil (<0.02%). Relative abundance of the remaining phyla 209 inside the roots was low. Furthermore, the phylum Fusobacteria was only observed in the root 210 interior of canola ( Figure S2).

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Within the root endophytic Proteobacteria, the genus Rhizobium was predominant in the two 212 legume species lentil and pea, accounting for 91-99% of the total population. Interestingly,

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Rhizobium was also detected in the interior of wheat and canola roots, accounting for up to 7% of 214 the total endophytic population ( Figure S3). Within the rhizosphere, the abundance of Rhizobium 215 accounted for up to 38% and 10% in pea and lentil, respectively, opposed to only 2% of the total       D r a f t  D r a f t