Jiahui Shao1, Zhihui Xu1, Nan Zhang1, Qirong Shen1, Ruifu Zhang1,2*

Contribution of indole-3-acetic acid in the plant growth promotion by the rhizospheric strain Bacillus amyloliquefaciens SQR9

Jiahui Shao1, Zhihui Xu1, Nan Zhang1, Qirong Shen1, Ruifu Zhang1,2*

1National Engineering Research Center for Organic-based Fertilizers, and Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization，Nanjing Agricultural University, Nanjing, 210095, P.R. China

2Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China

Running title: Plant growth-promoting by B. amyloliquefaciens SQR9

Corresponding author

Ruifu Zhang, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. E-mail: Tel:86-025-84396477, Fax: 86-25-84396220

Supplementary material

Investigation of plant-growth-promoting factors

Detection of acetoin produced by B. amyloliquefaciens SQR9 was performed by the method of Nicholson (2008) as follows：one hundred forty microliter of creatine (0.5% [w/v] in water), 200 µl of α-naphthol (5% [w/v] in 95% ethanol), and 200 µl of KOH (40% [w/v] in water) were sequentially added to 200 µl of acetoin standard solution or appropriately diluted culture supernatant. The mixed samples were vortexed after each addition. The reaction mixtures were vortexed again after incubation for 15 min at room temperature before measurement of A560.

Cultural supernatants and 2,3-butanediol standard solution were assayed by TLC on precoated silica gel G plates (Analtech, Wilmington, DE)(Nicholson 2008). The silica gel G plates were run in n-heptane–ethyl acetate (1:5). For butanediol visualization, the plates were sprayed with modified Seebach solution (0.05 g ammonium cerium [IV] sulfate dihydrate, 0.1 g phosphomolybdic acid and 0.6 ml concentrated H2SO4 in 9.2 ml water) and incubated in an oven at 150°C. Under these conditions, 2,3-butanediol migrated at an Rf of 0.5.

The phytase activity was measured at 50°C by adding 0.1 ml of culture filtrate in 0.9 ml of 2 mM sodium phytate (0.4% dodecasodium salt from rice, P3168, dissolved with 100 mM Tris-HCl buffer containing 2 mM CaCl2, pH 7.0). After 30 minutes, the enzyme reaction was stopped with 0.75 ml of 5% (w/v) trichloroacetic acid. The liberated phosphate was measured at 700 nm after adding 1.5 ml of color reagent. The color reagent was freshly prepared by mixing four volumes of 2.5% (w/v) ammonium molybdate in 5.5% (w/v) sulfuric acid and one volume of 2.5% (w/v) ferrous sulfate solution (Shimizu 1992). One unit (U) of phytase activity was defined as 1μmol of phosphate per minute liberated under the assay condition.

Quantification of IAA produced by B. amyloliquefaciens SQR9 by HPLC. IAA standard powders (sigma) were prepared in methanol in a gradual concentration: 3, 5, 7, 9, 11, 13 and 15 μg ml-1. Samples were analyzed by HPLC (1200 series, Agilent, USA) at 220 nm using a UV detector. Mobile phase was methanol- 0.1% acetic acid (60 / 40) at a flow rate of 0.4 ml min-1 for 20 min. After incubation in the Landy medium, the B. amyloliquefaciens SQR9 cells were separated by centrifugation (5,000×g, 20 min, 4 °C). Supernatants were adjusted to pH 2.5 with 1.0 M HCl and extracted three times with ethyl acetate (1:3, v/v). The organic solvents were vacuum dried at 37°C and then dissolved in 3 ml methanol. The extracted samples were filtered through a 0.45 µm membrane before HPLC analysis as described above. IAA was determined and quantified by integrating the areas of peaks with the help of standard samples.

Supplementary data

Fig. S1 Detection of IAA produced by B. amyloliquefaciens SQR9 cultured with 3 mM L-tryptophan by HPLC. Broken lines indicated the standard sample of IAA, continued lines indicated IAA in the culture filtrate of B. amyloliquefaciens SQR9.

Fig. S1

References

Nicholson WL (2008) The Bacillus subtilis ydjL (bdhA) gene encodes acetoin reductase/2,3-butanediol dehydrogenase. Appl Environ Microbiol 74: 6832-6838.

Shimizu M (1992) Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Biosci Biotech Bioch 56: 1266-1269

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