De-Coupling of Root-Microbiome Associations Followed by Antagonist Inoculation Improves

De-coupling of root-microbiome associations followed by antagonist inoculation

improves rhizosphere soil suppressiveness

Meihua Qiu1,2†, Shuqing Li1,2†, Xuan Zhou1,2, Xiaoshuang Cui1,2, Jorge M. Vivanco3*, Nan

Zhang1,2,Qirong Shen1,2, Ruifu Zhang1,2*

1Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River,

Ministry of Agriculture, 210095, Nanjing, PR. China

2Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization, College of

Resources & Environmental Science, Nanjing Agricultural University, 210095, Nanjing, PR.

3Center for Rhizosphere Biology, Colorado State University, Fort Collins, Colorado 80523-1173

†These authors contributed equally to this paper

Running title: Manipulation of root microbiome to improve soil suppressiveness

Corresponding authors

Ruifu Zhang, College of Resources & Environmental Science, Nanjing Agricultural University,

210095, Nanjing, China, Tel: 86-25-84396477; Fax: 86-25-84396260; E-mail: [email protected] or Jorge M. Vivanco, Department of Horticulture and Landscape

Architecture, Colorado State University, Fort Collins, CO 80523 USA, Tel: 1-970-491-7170; Fax:

1-970-491-7745; Email: [email protected] Genomic DNA extraction

Total genomic DNA of the strains (Bacillus amyloliquefaciens SQR9, Bacillus amyloliquefaciens

FZB42, Bacillus amyloliquefaciens T-5, Bacillus amyloliquefaciens LH-23, Bacillus anyloliquefaciens R1, Bacillus subitilis 168, Bacillus subitilis HJ5, Bacillus subitilis LH-36,

Bacillus subitilis R4, Bacillus methylotrophicus B3, Bacillus methylotrophicus N5, Paenibacillus polymyxa SQR-21, Escherichia coli DH5α, Ralstonia solanacearum QL) was obtained using the

AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen Inc., USA) according to the manufacturer’s instruction. All samples were stored at -20 °C.

Determination of the specificity and sensitivity of primer pair SQR9F (F)/ SQR9R (R)

The specificity of the designed primers was tested by real-time PCR using the optimized reaction conditions and 10 ng of purified DNA from each of the strains as templates. Assays were performed in triplicate. Fig.S1A showed the sensitivity of primer pair SQR9F (F)/ SQR9R (R).

The interval of amplification curve between each two adjacent concentrations of plasmid DNA of pMD-unknown was almost even at the range of 9.99 × 103 to 9.99 × 107 copies, however which was unequal between 9.99 × 102 to 9.99 × 103 copies. This result suggested that poor linear relationship between Ct values and the pMD-unknown copies number occurred from the plasmid concentration of 9.99 × 102 copies (Ct values being above 30). SQR9 strain amplified with Ct values were in the range of 8-10, whereas all the other strains had Ct values above 30, which corresponded to unspecific amplifications (Fig.S1B). The presence or absence of amplified DNA products was confirmed by agarose gel electrophoresis (data not shown). Figure Caption

Fig. S1 Estimation the specificity and sensitivity of primer pair SQR9F (F)/ SQR9R (R). A:

Amplification plots of 10-fold diluted purified plasmid DNA of pMD-unknown copies ranging from 9.99×102 to 9.99×107 copies. B: Detection of the specificity of the designed primers by the optimized real-time PCR assay. 10 ng of purified DNA from the 14 strains listed was subjected to the real-time PCR.

Fig. S1 Fig. S2 Appearance of cucumbers plants in different treatment (2 months after transplanting)

CK FOC FOC+SQR9 SQR9 Car- Car- Car- Car- CK FOC FOC+SQR9 SQR9

Note: CK-untreated control, FOC-soil inoculated with FOC at a concentration of 105 spore g-1 soil,

SQR9-soil inoculated with SQR9 at a concentration of 108 cfu g-1 soil, FOC+SQR9-both agents were inoculated as the concentration above. The prefix “Car” means that soil in these treatments was pretreatment with carbendazim, at a final concentration of 10 mg kg-1 soil.

Fig. S2