Crop diversity increases disease suppressive capacity of soil microbiomes

Microbiomes can aid in the protection of hosts from infection and disease, but the mechanisms 15 underpinning these functions in complex environmental systems remain unresolved. Soils contain microbiomes that influence plant performance, including their susceptibility to disease. For example, some soil microorganisms produce antimicrobial compounds that suppress the 18 growth of plant pathogens, which can provide benefits for sustainable agricultural management. 19 Evidence shows that crop rotations increase soil fertility and tend to promote microbial diversity, 20 and it has been hypothesized that crop rotations can enhance disease suppressive capacity, either 21 through the influence of plant diversity impacting soil bacterial composition or through the increased abundance of disease suppressive microorganisms. In this study, we used a long-term 2 field experiment to test the effects of crop diversity through time (i.e., rotations) on soil 24 microbial diversity and disease suppressive capacity. We sampled soil from seven treatments 25 along a crop diversity gradient (from monoculture to five crop species rotation) and a spring 26 fallow (non-crop) treatment to examine crop diversity influence on soil microbiomes including 27 bacteria that are capable of producing antifungal compounds. Crop diversity significantly 28 influenced bacterial community composition, where the most diverse cropping systems with 29 cover crops and fallow differed from bacterial communities in the 1-3 crop species diversity 30 treatments. While soil bacterial diversity was about 4% lower in the most diverse crop rotation 31 (corn-soy-wheat + 2 cover crops) compared to monoculture corn, crop diversity increased 32 disease suppressive functional group prnD gene abundance in the more diverse rotation by about 33 9% compared to monocultures. Identifying patterns in microbial diversity and ecosystem 34 function relationships can provide insight into microbiome management, which will require 35 manipulating soil nutrients and resources mediated through plant diversity.

2 field experiment to test the effects of crop diversity through time (i.e., rotations) on soil 24 microbial diversity and disease suppressive capacity. We sampled soil from seven treatments 25 along a crop diversity gradient (from monoculture to five crop species rotation) and a spring 26 fallow (non-crop) treatment to examine crop diversity influence on soil microbiomes including 27 bacteria that are capable of producing antifungal compounds. Crop diversity significantly 28 influenced bacterial community composition, where the most diverse cropping systems with 29 cover crops and fallow differed from bacterial communities in the 1-3 crop species diversity 30 treatments. While soil bacterial diversity was about 4% lower in the most diverse crop rotation 31 (corn-soy-wheat + 2 cover crops) compared to monoculture corn, crop diversity increased 32 disease suppressive functional group prnD gene abundance in the more diverse rotation by about 33 9% compared to monocultures. Identifying patterns in microbial diversity and ecosystem 34 function relationships can provide insight into microbiome management, which will require 35 manipulating soil nutrients and resources mediated through plant diversity. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; 7 We sampled soil from six crop diversity treatments, but to eliminate any immediate crop 138 effect all the treatments were sampled in the corn phase and a spring fallow treatment (Table 1)  To characterize bacterial taxonomic diversity, we used barcoded primers (515f/806r primer set) 159 developed by the Earth Microbiome Project to target the V4-V5 region of the bacterial 16S 160 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. We assessed the relative abundance of disease suppressive functional genes by targeting 183 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Statistical analyses 204
We examined microbiome differences among crop diversity treatments by comparing 205 total community diversity and composition as well as disease suppression markers. We tested for 206 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; differences in total bacterial diversity (based on Shannon Diversity Index H¢, bacterial species 207 richness, and Pielou's Evenness Index J¢) and prnD gene abundance in response to crop diversity 208 treatment using analysis of variance (ANOVA). We checked that data met assumptions of 209 analyses, and we treated crop diversity treatment as a fixed factor and block as a random effect. 210 We used Tukey's Honestly Significant Difference (HSD) tests to identify between-group 211 differences in bacterial diversity and prnD gene abundance. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Bacterial community composition and soil function relationships 238
The crop diversity treatment significantly influenced soil microbiomes represented by the 239 bulk soil bacterial community composition (R 2 = 0.37, p < 0.001; Appendix S1: Table S2, Fig.  240 1). Bacterial communities from the fallow plots and the most diverse crop rotations (CSW, 241 CSW 1cov , CSW 2cov ) were more similar to each other than the lower crop diversity treatments 242 (C 1cov , CS) (Fig. 1). The monoculture corn (mC) treatment was more distinct in bacterial 243 community composition than all other crop diversity treatments (Fig. 1). 244 Bacterial diversity, as measured using Shannon Diversity Index (H¢), was surprisingly 245 greater under lower crop diversity systems than higher crop diversity systems, but highest in 246 fallow treatments the most diverse non-cropping system (crop rotation: F 6,20 =10.16, p<0.0001; 247 block: F 1,20 =0.20, p=0.6600; Fig. 2). Among, the corn cropping systems, mC had the highest 248 Shannon Diversity Index in the most diverse rotation of corn-soybean-wheat with two cover 249 crops (CSW 2cov ). In addition, bacterial species richness and Pielou's Evenness Index (J¢) revealed 250 similar patterns across crop diversity treatments (evenness: F 6,18 =2.36, p=0.073; richness: 251 F 6,18 =2.61, p=0.053; Fig. 2). Across all diversity metrics, the longest crop rotation (CSW 2cov ) 252 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; showed the lowest richness and evenness values, and fallow soils generally had the highest 253 values (Fig. 2). 254 Soil physicochemical properties and soil function were related bacterial community 255 composition to varying degrees. A summary of soil attributes is presented in Appendix S1: Table  256 S1 and elsewhere (McDaniel and Grandy 2016). Bacterial community composition was best 257 explained by soil texture, which varied across the experiment site from 9 to 38 % clay 258 (R 2 =0.066, p<0.05, Table 3a). However, bacterial community composition was also marginally 259 affected by soil moisture (R 2 =0.048, p<0.10, Table 2). Labile C as measured with permanganate 260 oxidization was related to bacterial community composition (R 2 =0.074, p<0.05), but potentially 261 mineralizable C did not. Potentially mineralizable nitrogen (PMN), however, which is produced 262 in the same aerobic incubation as PMC and an indicator of nutrient-supplying power of a soil (a 263 biologically available N pool), was significantly correlated with bacterial community 264 composition (R 2 =0.063, p<0.05, Table 3 In cropping systems, the prnD gene in CSW 2cov treatment was the most abundant, and the gene 270 abundance was significantly higher than in CSW and fallow treatments (Fig. 3). Our diversity 271 benchmark, the fallow treatment (i.e., lowest crop diversity), showed the lowest prnD gene 272 abundances (Fig. 3). Based on multiple linear regression analysis, plant and soil factors 273 significantly related to prnD abundance (Adjusted R 2 =0.40, F=4.571, p=0.005). Crop species 274 number (p=0.003), soil carbon (p=0.002), and soil moisture (p=0.0005) appeared to be 275 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; significant predictors of prnD gene abundance (Table 3). We also observed a shift in the 276 composition of disease-suppression microorganisms (represented by phlD gene fingerprint 277 analysis using terminal restriction length polymorphism, T-RFLP) along the crop diversity 278 gradient. The phlD community composition in the fallow treatment was different from other 279 cropping systems (Appendix S1: Fig. S1). 280 281 Soil bacterial-disease suppressive function relationship 282 The bacterial taxa primarily responsible for treatment differences between mC and the 283 other crop diversity treatments were Sphingomonadales spp. and Acidobacteria subgroup Gp6 284 (Appendix S1: Table S3). When we compared a subset of taxa representing broad biocontrol 285 bacterial community (composed of Streptomyces spp. and Pseudomonas spp.), there was no 286 significant pattern in community composition across the crop diversity treatment 287 (PERMANOVA; crop rotation: R 2 =0.321, p=0.132; Appendix S1: Table S4). 288 289 Discussion 290 291 Soil microbiomes represent microbial communities living in close association with host 292 plants and can protect host organisms from infection and disease. In this study, we found that 293 crop rotation history impacted soil microbiomes and altered disease suppression potential in 294 agricultural soils. However, we found some unexpected results that contrasted with our 295 hypothesis. Contrary to our hypothesis, bacterial diversity decreased with increasing cropping 296 diversity (Fig. 2). However, the PPS capability of the soil microbial community increased with 297 crop diversity, but surprisingly the lowest PPS was in the diverse fallow treatments (Fig. 3). We 298 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; observed that without crop plants (as reflected in the no crop fallow treatment), disease 299 suppressive potential was significantly diminished compared to crop treatments, possibly due to 300 reduced selection for soil microorganisms with disease suppression traits. The composition of the 301 soil microbial community may be more important than diversity to soil suppressive function. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; bacterial diversity with increasing crop diversity is not just structural, but also functional, and 322 may indicate carbon resource specialization among bacteria since they are probably the major 323 contributor to C catabolism in these substrate-induced respiration methods (Goldfarb et al reported biocontrol bacterial taxa (e.g., Pseudomonas spp. and Streptomyces spp.) across the 363 crop diversity gradient; however, we did not detect distinct changes in putative biocontrol 364 community composition (Appendix S1: Table S4). 365 Our study revealed that cover crops in combination with corn-soy-wheat rotations 366 increased abundance of the prnD gene, which is responsible for producing antifungal compound 367 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; suggest that the land-use regime, plant diversity, and plant species influence disease suppressive 437 microbial communities. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; and interpretation, preparation of the manuscript, or decision to submit the work for publication. 460 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (1) Continuous monoculture (mC) 1 (2) Continuous monoculture, one cover crop (C 1cov ) 2 (3) Two-crop rotation (CS) 2 (4) Three-crop rotation (CSW) 3 (5) Three-crop rotation, one cover crop (CSW 1cov ) 4 (6) Three-crop rotation, two cover crops (CSW 2cov ) 5 (7) Spring Fallow/early successional field (fallow) 10 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/030528 doi: bioRxiv preprint first posted online Nov. 4, 2015; was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.