Pyrosequencing Reveals Contrasting Soil Bacterial Diversity and Community Structure In

1 Pyrosequencing reveals contrasting soil bacterial diversity and community structure of two

2 main winter wheat cropping systems in China

5Jun Zhao†1,2, Ruifu Zhang†1,2,3, Chao Xue1,2, Weibing Xun1,2, Li Sun1,2, Yangchun Xu1,2,3 and Qirong

6 Shen*1,2,3

81Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River,

9Ministry of Agriculture, Nanjing, 210095, China

102Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization, Nanjing

11Agricultural University, Nanjing, 210095, China

123Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization，Nanjing,

13210095, China

16†Both authors contributed equally to this paper

18Running title: Wheat soil bacterial communities of two cropping systems.

20*Corresponding author: Qirong Shen, Tel: 86-25-84396477; Fax: 86-25-84396260, and E-mail:

[email protected]

23 Table S1. Soil type of each sampling site based on Chinese Soil Taxonomy (CST) system from

24 two different cropping systems

Sites Soil types CS Clay loamy endogleyic-Fe-leachic-stagnic anthrosol JT Clay loamy Fe-leachic-gleyic-stagnic anthrosol ZJG Clay Fe-leachic-gleyic-stagnic anthrosol DZ Sand loamy calcaric-mottlic-udi-orthic primosol QZ Sand loamy typic-claypani-udic agrosol 3

25Table S2. Relative abundances of bacterial phyla and proteobacterial classes in all samples

26combined, and in each cropping system.

All Winter wheat-rice (WR) Winter wheat-maize (WM) Proteobacteria 35.12 37.76 31.17 Alphaproteobacteria* 6.638 3.415 11.123 Betaproteobacteria* 10.615 13.369 6.782 Deltaproteobacteria* 4.078 5.470 2.142 Gammaproteobacteria* 6.788 6.037 7.833 Acidobacteria 16.127 14.886 17.952 Chloroflexi 4.832 6.700 2.210 Bacteroidetes 8.470 7.065 10.434 Verrucomicrobia 2.368 2.415 2.265 Actinobacteria 5.484 2.516 9.377 Nitrospira 1.023 1.321 0.616 Gemmatimonadetes 1.063 0.891 1.258 Firmicutes 0.885 0.731 1.088 Planctomycetes 0.808 0.459 1.306 Armatimonadetes 0.525 0.540 0.498 TM7 0.378 0.240 0.562 Epsilonproteobacteria < 0.001 < 0.001 < 0.001 BRC1 0.090 0.127 0.039 Spirochaetes 0.048 0.082 < 0.001 OP11 0.019 0.030 0.002 OD1 0.055 0.064 0.041 Lentisphaerae 0.001 0.002 < 0.001 WS3 0.610 0.862 0.266 Chlorobi 0.325 0.567 < 0.001 Unclassified bacteria 22.01 22.74 20.92 27Relative abundances are based on the proportional frequencies of those DNA sequences that could

28be classified at the phylum level and proteobacterial classes.

29*indicate classes of Proteobacteria

33 34 4

35Table S3. Relative abundances of bacterial phyla and proteobacterial classes in all five sampling 36sites Taxa Winter wheat-rice (WR) Winter wheat-maize (WM) CS (%) JT (%) ZJG (%) DZ (%) QZ (%) Proteobacteria 39.98 ± 1.57 a 36.05 ± 0.96 bc 37.24 ± 0.88 ab 33.52 ± 1.55 c 28.81 ± 3.10 d Alphaproteobacteria* 3.1 ± 0.6 b 3.9 ± 1.9 b 3.2 ± 3.3 b 12.4 ± 3.0 a 10.0 ± 3.5 a Betaproteobacteria* 13.8 ± 1.2 a 13.8 ± 1.4 a 12.4 ± 1.3 a 6.4 ±0.4 b 7.1 ± 0.2 b Deltaproteobacteria* 6.1 ± 0.8 a 4.5 ± 0.3 b 5.8 ± 0.8 a 2.4 ± 0.4 c 1.9 ± 0.3 c Gammaproteobacteria* 6.1 ± 0.4 bc 4.9 ± 0.5 c 7.1 ± 1.2 b 8.7 ± 0.8 a 7.0 ± 0.5 b Acidobacteria 13.64 ± 0.92 c 16.02 ± 0.43 b 15.00 ± 0.51 bc 20.73 ± 1.39 a 15.17 ± 0.71 bc Chloroflexi 6.60 ± 0.65 b 8.19 ± 0.41 a 5.31 ± 1.64 b 2.22 ± 0.11 c 2.20 ± 0.58 c Bacteroidetes 6.37 ± 0.83 c 6.89 ± 1.12 bc 7.93 ± 0.32 b 10.68 ± 0.08 a 10.19 ± 0.96 a Verrucomicrobia 2.39 ± 0.25 ab 2.64 ± 0.30 a 2.21 ± 0.73 ab 1.80 ± 0.26 b 2.73 ± 0.31 a Actinobacteria 3.00 ± 0.41 c 3.05 ± 0.24 c 1.50 ± 0.51 d 4.70 ± 0.71 b 14.05 ± 0.65 a Nitrospira 0.89 ± 0.08 bc 1.20 ± 0.14 b 1.87 ± 0.27 a 0.68 ± 0.22 c 0.55 ± 0.26 c WS3 0.89 ± 0.06 a 0.78 ± 0.06 a 0.93 ± 0.35 a 0.45 ± 0.13 b 0.08 ± 0.04 c Gemmatimonadetes 0.69 ± 0.06 c 1.70 ± 0.04 a 0.29 ± 0.06 d 0.85 ± 0.02 b 1.66 ± 0.12 a Chlorobi 0.63 ± 0.06 a 0.24 ± 0.09 b 0.84 ± 0.20 a – – Firmicutes 0.51 ± 0.20 d 0.65 ± 0.06 cd 1.03 ± 0.01 b 0.83 ± 0.13 bc 1.35 ± 0.22 a Armatimonadetes 0.40 ± 0.02 b 0.72 ± 0.09 a 0.50 ± 0.20 ab 0.41 ± 0.13 b 0.58 ± 0.19 ab Planctomycetes 0.38 ± 0.11 c 0.35 ± 0.20 c 0.65 ± 0.23 c 1.47 ± 0.19 a 1.14 ± 0.16 b TM7 0.19 ± 0.01 c 0.32 ± 0.03 b 0.21 ± 0.13 c 0.41 ± 0.03 b 0.72 ± 0.03 a BRC1 0.14 ± 0.04 a 0.11 ± 0.03 ab 0.13 ± 0.10 a 0.06 ± 0.02 ab 0.02 ± 0.01 b Spirochaetes 0.08 ± 0.02 a 0.10 ± 0.02 a 0.06 ± 0.05 a – – OP11 0.07 ± 0.04 a 0.08 ± 0.01 a 0.04 ± 0.02 a – < 0.001 OD1 0.02 ± 0.04 ab 0.05 ± 0.09 a 0.02 ± 0.02 ab 0.06 ± 0.07 a 0.03 ± 0.04 a Lentisphaerae < 0.001 – – – – 37 Value (mean ± S.D., n=3) of each phylum and proteobacterial classes in total bacterial

38community, respectively.

39 Significant statistical differences (ANOVA, P<0.05, Duncan’s test) are denoted with different

40lowercase letters.

41*indicate classes of Proteobacteria

42 43 44 45 46 47 48 5

49Table S4. The detected, percentages of overlap, unique OTUs for each sample per 5,400

50sequences. 51 52 CS_1 CS_2 CS_3 JT_1 JT_2 JT_3 ZJG_1 ZJG_2 ZJG_3 DZ_1 DZ_2 DZ_3 QZ_1 QZ_2 QZ_3 CS_1 20.9* 47.3 49.5 37.4 37.9 37.5 37.3 36.6 36.6 24.9 24.7 25.1 20.7 21.0 20.6 CS_2 20.6 47.7 37.1 37.0 39.4 34.5 35.4 34.8 24.4 25.2 24.5 21.6 20.7 20.8 CS_3 19.8 36.7 38.0 38.9 35.3 35.8 31.1 24.9 24.4 24.6 20.8 21.2 20.1 JT_1 24.3 44.2 45.7 31.1 32.9 31.1 24.7 24.7 25.2 21.0 21.8 21.5 JT_2 24.0 46.1 32.7 32.1 32.3 24.9 24.2 24.3 21.4 20.6 20.7 JT_3 24.7 32.0 32.6 31.3 23.5 24.9 24.3 20.8 21.4 21.4 ZJG_1 24.0 44.3 47.8 29.9 30.0 30.6 23.0 22.3 23.9 ZJG_2 21.3 44.9 29.7 29.2 30.6 24.7 24.0 23.6 ZJG_3 23.4 27.9 28.3 28.6 21.8 21.4 22.1 DZ_1 22.7 46.5 49.3 35.8 36.2 35.8 DZ_2 24.1 49.3 35.4 37.2 35.1 DZ_3 23.4 37.2 37.2 35.6 QZ_1 25.4 50.1 52.1 QZ_2 24.2 49.6 QZ_3 23.4 #OTUs 1831 1763 1739 1866 1861 1824 1885 1883 1849 1504 1549 1498 1436 1432 1425 53 54* Values in boldface type represent unique OTUs in each sample, and italic type represents 55overlapped OTUs between two samples. 56 6

57Table S5. Pearson correlation coefficients between soil characteristics. 58 pH Available P Available K Soil organic C Total N C:N ratio pH 1.000 Available P -0.470 1.000 Available K 0.530* -0.456 1.000 Soil organic C 0.323 0.656 0.111 1.000 Total N -0.594 0.966 -0.344 -0.552 1.000 C:N ratio 0.931 -0.719 -0.474 -0.004 -0.833 1.000 59 60* Significant correlations between soil geochemical characteristics are indicated in bold type when 61P < 0.05. 7

62Fig. S1. Three dimension principal component analysis (PCA) of bacterial communities based on 63 OTUs at a distance of 3% for individual samples from winter wheat-rice (circle) and 64 winter wheat-maize (triangle) cropping sytems.