Siderophore and indolic acid production by Paenibacillus triticisoli BJ-18 and their plant growth-promoting and antimicrobe abilities

Bacterial strain

The bacterial strain Paenibacillus triticisoli BJ-18 (=DSM 25425^T = CGMCC 1.12045^T) was used in our study.

Untargeted metabolomics by LC-MS

The equipment and raw data for untargeted metabolomics were provided by Beijing Novogene Technology Co., Ltd. P. triticisoli BJ-18 was cultured in Lysogeny Broth (LB) (10 g tryptone, 5 g yeast, 10 g NaCl, 15 g agar per 1 L H₂O) at 30 °C for 3 days. A single colony was inoculated in 20 mL CH medium (30 g sucrose, 6.4 g tryptone, 7 g yeast, 0.6 g MgSO₄ ⋅ 7H₂O, 3.5 g NaCl, 0.1 g K₂HPO₄, 0.4 g KH₂PO₄ per 1 L H₂O) and cultured at 30 °C 200 rpm for 48 h. The seed solution was inoculated into 150 mL of CH medium at an inoculation amount of 2% and cultured at 30 °C 200 rpm for 48 h. Cells were harvested by centrifugation at 6,000 rpm at 4 °C for 10 min, ground with liquid nitrogen, and resuspended with 500 µL of 80% methanol solution containing 0.1% formic acid. The homogenate was incubated on ice for 5 min and was centrifuged at 15,000 rpm at 4 °C for 10 min. 200 µL of supernatant was subsequently transferred to a fresh Eppendorf tube with a 0.22 µM filter and was centrifuged at 15,000 rpm at 4 °C for 10 min. LC-MS/MS analyses were performed using the Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher) in a Hyperil Gold column (100 × 2.1 mM, 1.9 µM) with a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% formic acid in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min;100–2% B, 14.1 min; 2% B, 16 min. The Q exactive mass series spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, sheath gas flow rate of 35 arb, aux gas flow rate of 10 arb, and a capillary temperature of 320 °C. Compound discoverer 3.0 (CD 3.0, Thermo Fisher) was used for processing the data files to match results from the mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases. Accurate qualitative and relative quantitative results were obtained through statistical analysis.

Reagents and instruments for separation and purification of compounds 1-6

HPLC data were acquired using a Waters 2695 instrument. HRESIMS and HPLC-ESI-MS data were obtained using an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Agilent, USA) in positive ion mode. ¹H and ¹³C NMR data were obtained by Bruker Avance-500 spectrometers (Rheinstetten, Germany) with solvent signals (DMSO-d ₆, δ_H 2.500/ δ_C 39.520) as references, and HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. The optical rotations measurement was conducted on an Anton Paar MCP200 polarimeter (Anton Paar, Austria). Optical rotations were conducted using a Perkin-Elmer 241 polarimeter, and UV data were detected by a Shimadzu Biospec-1601 spectrophotometer. The absorbance of 96-well clear plate was measured on a microplate reader (Molecular Devices, SpectraMax^® Paradigm^®). Analytically pure solvents including methanol, dichloromethane, and ethyl acetate were used for extraction and chromatographic separation. TLC was carried out on silica gel HSGF₂₅₄ plates and spots were visualized by UV at 254 nm or sprayed with 10% H₂SO₄ and heated. Silica gel (150 −250 µM, Qingdao Haiyang Chemical Co., Ltd.), octadecylsilyl (ODS, 50 µM, YMC CO., LTD), and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC). HPLC separation was performed on an Agilent 1200 HPLC system with a Diode Array Detector (DAD) detector using an ODS column (C₁₈, 250 × 9.4 mM, YMC Pak, 5 µM) at a flow rate of 2.0 mL/min.

Extraction and isolation of the secondary metabolites

P. triticisoli BJ-18 was grown in a 500 L fermenter (Biotech, Shanghai Baoxing Biological Equipment Engineering Co., Ltd.) filled with 200 L of CH medium at pH 5.0 with 40% to 60% dissolved oxygen and shaking at 100 rpm and at 30 °C for 48 h. For doing this, this bacterium was inoculated into 20 mL of CH medium and then scaled-up to 20 L. The seed culture was inoculated into 200 L of CH medium with an inoculation amount of 1% in a 500 L fermenter.

The fermented culture was passed through a 200 mesh macroporous adsorption resin following 3 periods of 30-minute ultrasonic cell-breaking and was eluted with analytically pure methanol. The organic solvent was collected and evaporated using rotary evaporators to obtain the crude extract (7 g). The aqueous phase was discarded after being extracted and separated three times between the ethyl acetate and the aqueous phases, and the organic phase was retained and evaporated using rotary evaporators. The final product was 4 g of EtOAc (ethyl acetate) extract.

The EtOAc extract was separated into 20 subfractions (Pt-1 to Pt-20) after being subjected to ODS column chromatography with a gradient of methanol-water (5–100%). The fraction Pt-10 was further partitioned by Sephadex LH-20 CC separated by 50% methanol in water to create 26 subfractions (Pt-10-1 to Pt-10-26). Compound 4 (4.2 mg, t_R 31.2 min) was obtained from fraction Pt-10-24 (21.3 mg) by RP-HPLC using 55% methanol in acidic water. Fraction Pt-13 was divided into 16 fractions, Pt-13-1 to Pt-13-16, after being subjected to ODS column chromatography with a gradient of MeOH-H₂O (40–80%). Compounds 1-3 (2 mg, 3.1 mg and 8 mg, t_R 62.5 min, 58.4 min and 37.4 min, respectively) were obtained from fractions Pt-13-6 (42.5 mg) by RP-HPLC using 20% acetonitrile in acidic water. Fraction Pt-15 was further partitioned by Sephadex LH-20 CC eluted with 90% methanol in water to cut to 6 subfractions (Pt-15-1 to Pt-15-6). Compound 5 (214 mg, t_R 42.5 min) was obtained from fraction Pt-15-4 (830 mg) by RP-HPLC using 72% methanol in acid water. Compound 6 (37.6 mg, t_R 62.5 min) was isolated from fraction Pb-17 (928 mg) by RP-HPLC (C8) using 80% methanol in acid water.

Deshydroxylferritriacetylfusigen (compound 1): yellow powder; [α]²⁵_D = −14.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 214 (2.44) nm; NMR data (500 MHz, DMSO-d₆) is shown in Table 1; positive HRESIMS m/z 837.4242 [M + H]⁺ (calculated for C₃₉H₆₁N₆O₁₄, 837.4246 Δ = 0.0004).

Desferritriacetylfusigen (compound 2): yellow powder; [ α]²⁵_D = −8.0 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) = 214 (2.43) nm; positive HRESIMS m/z 853.4203 [M + H]⁺, (calculated for C₃₉H₆₀N₆O₁₅, 853.4195 Δ = 0.0008).

Triacetylfusigen (compound 3): brown powder; [α]²⁵_D = −216.0 (c 0.025, MeOH); UV (MeOH) λ_max (log ε) 211 (2.35) nm; positive HRESIMS m/z 906.3316 [M + H]⁺ (calculated for C₃₉H₅₈N₆O₁₅Fe, 906.3309 Δ = 0.0007).

Paenibacillic acid A (compound 4): white powder; [α]²⁵_D= −39.99 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 211 (2.33) nm; NMR data (500 MHz, DMSO-d ₆) is shown in Table 2; positive HRESIMS m/z 303.1704 [M + H]⁺ (calculated for C₁₇H₂₃N₂O₃ 303.1708 Δ = 0.0004).

3-Indoleacetic acid (IAA) (compound 5): white amorphous powder; UV (MeOH) λ_max (log ε) 234 (4.22), 272 (3.77), 280 (3.80), 290 (3.72) nm; positive HRESIMS m/z 176.0709 [M + H]⁺ (calculated for C₁₀H₁₀NO₂ 176.0701 Δ = 0.0008).

3-Indolepropionic acid (IPA) (compound 6): white amorphous powder; UV (MeOH) λ_max (log ε) 235(4.25), 270 (3.77), 280 (3.80), 290 (3.72) nm; positive HRESIMS m/z 190.0871 [M + H]⁺ (calculated for C₁₁H₁₂NO₂, 190.0868 Δ = 0.0003).

Conversion of compound 3 into desferritriacetylfusigen

The removal of the ferric ion was conducted as reported by Kodani et al. (2015) with additional changes. Compound 3 (4 mg) was dissolved in 1.5 mL of water and then 1.5 mL of 1M 8-quinolinol was added. The solution was stirred for 25 min and mixed with three mL of CH₂Cl₂, then left to stand until bilayer separation occurred. The water layer was evaporated using a freeze-dryer vacuum. This was repeated 3 times to eliminate ferri-8-quinolinol and obtain the total dry material. The sample was then dissolved in 100 µL of methanol and HPLC purification was performed with the C18 column (7.8 × 300 mM, Waters, 7 µM; detector: UV), eluted with 20% acetonitrile in water at a flow rate of one mL/min, and monitored at OD₂₁₀ to create 2.0 mg of desferri-compound 3. Positive HRESIMS and NMR of desferri-compound 3 was the same as compound 2.

ECD computations

Systematic conformation analysis of 4a were conducted with CONFLEX (version 7 Rev. A; CONFLEX Corporation) using the MMFF94 molecular mechanics force field. Optimization with DFT calculation at the B3LYP/6-31G(d) level in MeOH in methanol with the PCM model using the Gaussian 09 program (Revision C.01, Gaussian Inc.) afforded the MMFF minima. The 40 lowest electronic transitions were calculated using time-dependent density-functional theory (TDDFT) methodology at the B3LYP/6-31G(d) level. The overall ECD spectra were then produced based on Boltzmann weighting of each conformer as described in literature(Liu et al., 2018a; Liu et al., 2018b).

Determination of plant growth promoting capacity of indolic compounds

The seeds of Arabidopsis thaliana var. Columbia were sterilized using 75% ethanol for 45 s, 5% NaClO for 15 min, and were washed in sterile water 3 times. The seeds were then placed horizontally on solid 1/2 Murashige and Skoog (MS) (Murashige & Skoog, 1962) medium supplemented with 1.5% sucrose, 0.35% (w/v) agar, and different concentrations and combinations of 1 mg/L paenibacillic acid A, IAA, and IPA for direct regeneration. At least ten Arabidopsis strains were cultured in each group of treatments. The cultures were incubated at 25 ± 2  °C under cool fluorescent light (2000 lux 16 h/day photoperiod) for 14 days. The ability of the three indolic acids to promote plant growth was evaluated by measuring the dry weight of the stems and roots of Arabidopsis thaliana.

Detection of indolic compounds

The Salkowski Reagent (PC technique) was used to determine concentrations of indolic compounds (Glickmann & Dessaux, 1995). IAA standard solutions with concentrations of 0, 5.0, 10.0, 15.0, 20.0, and 25.0 µg /mL were used to react with an equal volume of PC reagent in the dark for half an hour and the OD₅₃₀ value was measured to draw the Salkowski calibration curve. The R² value should be greater than 0.99.

P. triticisoli BJ-18 was grown overnight in 50 mL of LB medium at 200 rpm 30 °C. The cells were collected by centrifugation, washed three times with deionized water, and resuspended in one mL of deionized water. 100 µL of the bacterial suspension was added to 20 mL nitrogen-deficient medium (prepared with deionized water and supplemented with 100 mM NH₄Cl as a nitrogen source) with 36 mg/L iron citrate or no iron citrate. After three days of incubation at 200 rpm and 30  °C, all samples were treated with the same method to measure the corresponding OD₅₃₀ value. The content of indolic compounds of each sample was calculated using the Salkowski calibration curve. The nitrogen-deficient medium with deionized water was used as a blank control. Each sample was treated three times.

Antimicrobial assay

The antimicrobial assay was performed according to the recommended guidelines of the National Center for Clinical Laboratory Standards (NCCLS) (Li et al., 2008). The bacterial cells of Escherichia coli (ATCC1.0090), Staphylococcus aureus (ATCC6538), and Bacillus subtilis (ATCC6663) were grown in Mueller-Hinton Broth (MHB) medium (5.0 g beef powder, 1.5 g starch, 17.5 g acid hydrolyzed casein, and 1 L H₂O) at 37 °C for 24 h. The fungus (yeast), Candida albicans (CGMCC2.2086), was grown in Saurer’s Dextrose Broth (SDB) medium (5 g casein trypsin digest, 5 g gastric enzyme digest, 20 g dextrose and 1 L ddH₂O) at 28 °C for 48 h.

The cells of bacteria and yeast cultivated in MHB and SDB media as described above were seeded onto each well of a 96-well clear plate, then the gradient concentrations of test compounds 1-6 were added to each well and mixed with the targeted microbes to reach a final volume of 100 µL. The inoculation concentrations of bacteria and yeast were 0.5–2.5 × 10⁵ cfu / mL and 0.5–2. 5 × 10³cfu / mL, respectively. The suspension was cultured at 37 °C for 1 day and 28 °C for 2 days, respectively. Gentamicin, ampicillin, sulphate streptomycin, and amphotericin B were used as positive controls. A microplate reader was employed to perform at the OD_595. The IC₅₀ of compounds 1-6 were plotted, calculated, and obtained. Each antimicrobial assay was tested in triplicate.

Siderophores detection by blue agar CAS assay

Siderophores were detected using a blue agar CAS medium as described by Schwyn & Neilands (1987). The solid CAS medium was composed of one mL 20% sucrose solution, three mL sterilized 10% casamino acid, 100 µL one mmol/L CaCl₂, five mL CAS dye solution (a mixture of 0.012 g of chrome azurol S in 10 mL of ddH₂O, two mL one mmol/L FeCl_3, and 0.015 g hexadecyltrimethylammonium bromide in eight mL of deionized H₂O), PIPES buffer (pH 6.8–7.0), and 2 g agar powder per 100 mL H₂O.

P. triticisoli BJ-18 was inoculated in 20 mL of liquid LB medium with shaking at 200 rpm at 30 °C for 48 h. one mL of the bacterial solution was centrifuged, washed with sterile deionized water, and then suspended with 200 µL of sterile deionized H₂O. 10 µL of the suspension was inoculated on a CAS plate. The color changed from blue to light orange, indicating the presence of iron carriers.

Untargeted metabolomics profiling of P. triticisoli BJ-18

The untargeted metabolomics profiling of P. triticisoli BJ-18 were analyzed by using LC-MS and the data were comparatively analyzed with the databases of mzCloud, ChemSpider and mzVault. A total of 101 compounds were measured, the majority of which were common compounds involved in the basic metabolism (Table S1), including carbohydrates, amino acids, peptides, alcohols, aldehydes, ketones, fatty acids, lipids, nucleic acids, vitamins, alkaloids, cyclics and their respective derivatives (Fig. 1A). Of the 101 compounds, 46 have the molecular weights of 100–200 Da, 34 have the molecular weights of 200–300 Da, 18 have the molecular weights of more than 300 Da and 3 have the molecular weights of less than 100 Da (Fig. 1B).

Notably, N²-acetyl ornithine, a precursor to fusarinines, is included among these compounds. Also, plant growth regulators such as trehalose 6-phosphate (T6P, resistant to drought and salt stress. Wingler et al., 2012; Prasad et al., 2014; Kretzschmar et al., 2015), betaine (a non-toxic osmotic regulator. (Cho et al., 2003; Tramontano & Jouve, 1997) and trigonelline (resistant to salt stress. Minorsky, 2002; Tramontano & Jouve, 1997), and other active molecules such as oxymatrine (a drug used to treat hepatitis B and tumors. Lu et al., 2003; Song et al., 2006), diosmetin (in food or medicine with anti-oxidant properties, anti-infective, and anti-shock functions Pallab et al., 2019), luotonin A (anti-tumor drug. González-Ruiz et al., 2010), α-humulene (hippone, sesquiterpene, with anti-inflammatory effect. Rogerio et al., 2009), (-)-caryophyllene oxide (anti-tumor and antifungal drug. Yang et al., 1999; Park et al., 2011), tetrahydrocurcumin (hepatotoxicity prevention drug and natural whitening ingredients. Pari & Murugan, 2004; Trivedi et al., 2017) were also included among these compounds.

Structural determination of secondary metabolites from P. triticisoli BJ-18

The cells of P. triticisoli BJ-18 were fermented and concentrated, and 4 g of EtOAc extracts was obtained. The extracts were separated using a combination of silica gel chromatography, sephadex LH-20, ODS column chromatography, and HPLC. Six compounds (compounds 1-6) were ultimately obtained and were further analyzed using NMR and MS to establish their structures. Further the structure and characters of the six compounds (compounds 1-6) were compared with the corresponding compounds in the literature. Among the six compounds, compounds 1-3 were identified as fusarinines that were classical siderophores, while compounds 4-6 were identified as indolic acids (Fig. 2). Both compound 1 and 4 have new structures.

Compound 2 was a cyclic tripolymer which has three same monomers (m/z 284.1366) identified as desferritriacetylfusigen (Anke, 1977). It was characterized by ¹H NMR (500 MHz, DMSO-d ₆), δ_H 9.70 (s), 8.21 (d, J = 7.5 Hz), 6.32 (s), 4.30–4.03 (m, 3H), 3.33 (s), 2.81 (m), 1.88 (s), 1.85 (s), 1.75–1.39 (m, 4H) (Fig. S1) and by ¹³C NMR (125 MHz, DMSO-d ₆) δ_C 22.7, 23.5, 25.8, 28.5, 32.4, 46.8, 52.3, 63.5, 117.9, 150.1, 167.4, 170.0, 172.6 (Fig. S2). Compound 5 was identified as 3-indoleacetic acid (IAA) (Gathungu et al., 2014). As shown in Fig. S2 , it was characterized by ¹H NMR (500 MHz, DMSO-d₆), δ_H 12.14 (s), 10.90 (s), 7.49 (d, J = 7.9 Hz), 7.34 (d, J = 8.1 Hz), 7.22 (d, J = 2.3 Hz), 7.07 (t, J = 7.6 Hz), 6.98 (t, J = 7.4 Hz), 3.63 (s, 2H). Compound 6 was identified as 3-indolepropionic acid (IPA) (Rustamova et al., 2019). As shown in Fig. S4, it was characterized by ¹H NMR (500 MHz, acetone-d ₆), δ_H 9.97 (s), 7.59 (d, J = 7.9 Hz), 7.37 (d, J = 8.1 Hz), 7.16 (s), 7.09 (t, J = 7.5 Hz), 7.02 (t, J = 7.5 Hz), 3.06 (t, J = 7.7 Hz), 2.70 (t, J = 7.7 Hz).

Compound 1 was a new member of fusarinines (siderophores) and compound 4 was a new member of the indolic acids. Their structures were further determined by extensive spectroscopic experiments. The structure of compound 3 was determined by NMR after iron was removed and it was identified as triacetylfusigen. Overall, the six compounds include three fusarinines (siderophores) and three indolic acids. (Fig. 2).

Compounds 1, 3, and 4, are described in greater detail as follows:

Deshydroxylferritriacetylfusigen (compound 1) was a yellow powder with a molecular formula of C₃₉H₆₀N₆O₁₄ (thirteen degrees of unsaturation) by HRESIMS m/z 837.4242 [M + H]⁺ (calculated for C₃₉H₆₁N₆O_{14 ,} 837.4246). Its ¹H, ¹³C and HSQC NMR spectroscopic data (Figs. S5– S7) suggested the existence of two methyl groups (δ_C∕H22.3/1.84 and 25.3/1.87), five methylenes, including one O-methylene (δ_C∕H63.1/4.17 and 4.12), one double bond (δ_C∕H117.5/6.31 and δ_C 149.6), three carboxylic carbons (δ_C 166.0, 169.5 and 172.1), one N-methine (δ_C∕H 51.8/4.19), one amide N-H proton (δ_H 8.23), and one amide N-OH proton (δ_H9.92). Thirteen carbon signals in the ¹³C NMR and thirty-nine in the molecular formula indicated that compound 1 was a cyclic tripolymer similar to compound 2. Comparison of MS-MS spectrum of compound 1 (Fig. S8) and compound 2 (Fig. S9) revealed that unlike compound 2, two of the three monomers of compound 1 have the same molecular weight as the monomers of compound 2 (m/z 284.1366), and the other was different (m/z 268.1566). The molecular weight of this unique monomer is 16 less than that of others, which is the molecular weight of an oxygen atom. Although the NMR of compound 1 was similar to compound 2, the integration of N-OH protons in ¹H NMR of compound 1 was less than compound 2. The molecular formula also suggested that there were only two hydroxyls in compound 1 rather than three. The planar structure was confirmed by combining ¹H-¹H COSY and HMBC (Figs. S10 & S11, Fig. 3). The ROESY spectrum (Fig. S12) showed that CH₃-11 had the NOE correlation with the olefinic proton (δ_H6.31) indicating that the double bond was in a Z- configuration. Compound 2 was deduced to come from ornithine (Schrettl et al., 2007), and the absolute configuration of compound 1 was determined to be 5S. Our data suggested that compound 1 has a new structure and we proposed the name deshydroxylferritriacetylfusigen for the new number of fusarinines (Table 1).

Compound 3 was a brown powder with a molecular formula of C₃₉H₅₇N₆O₁₅Fe by HRESIMS ([M + H]⁺ at m/z 906.3316, calculated for C₃₉H₅₈N₆O₁₅Fe, 906.3309). Compound 3 had no signal of NMR (Fig. S13) due to its metal-shielding characteristics. A strong complexing agent (Kodani et al., 2015) desferri-compound 3 was a yellow powder with a molecular formula of C₃₉H₆₀N₆O₁₅ (thirteen degrees of unsaturation) by HRESIMS ([M + H]⁺ at m/z 853.4203, calculated for C₃₉H₆₀N₆O₁₅, 853.4195) obtained following the precipitation and separation of the metal ions by 8-quinolinol. Analysis of its HRESIMS and ¹H NMR (Fig. S14) data revealed that desferri-compound 3 had the same structure as compound 2. Compound 3 was determined to be a ferri-complex compound, identified as triacetylfusigen.

Compound 4 was isolated as white amorphous powder with the molecular formula of C₁₇H₂₂N₂O₃ (eight degrees of unsaturation) determined by HRESIMS m/z 303.1704 [M + H]⁺ (calculated for C₁₇H₂₃N₂O₃ 303.1708). The ¹H and ¹³C NMR combining with HSQC (Figs. S15– S17) revealed one methyl group [δ_C∕H13.6/0.78 (t, J = 7.4Hz)], six methylenes, including two O-methylene [δ_C∕H 67.1/3.30 (m) and 72.3/5.46 (s)], one ethane [δ_C 56.1/3.63 (dd, J = 10.3, 5.0)], eight aromatic/olefinic carbons (δ_C 108.2, 109.8, 118.1, 119.7,121.9, 126.2, 129.4 and 137.2) and one carboxylic carbons (δ_C170.0). These data suggested that compound 4 was similar to lycoperodine-1 (Yahara et al., 2004). The ¹H–¹H COSY data (Fig. S18) revealed four isolated proton spin-systems of C-1-N-C-3-C-4, C-5–C-6, C-7–C-8, and C-3′–C-4′–C-5′–C-6′, respectively. The HMBC spectrum (Fig. S19) showed correlations from H-2′ to C-3′, C-8a and C-9a, from H-1 to C-3 and C-9a, from H-3 and H-4 to C-1, from H-4 to C-9a and C-4a, from H-5 to C-5a and C-4a, and from H-7 and H-8 to C-8a. The planar structure of compound 4 was established by linking these fragments (Fig. 3 & Table 2). The ECD calculation method (Ma et al., 2014) was used to determine the absolute configurations. The structure of compound 4 was simplified into two stereoisomers 4a (3S) and 4b (3R). The calculated ECD curve of compound 4a correlated with the experimental CD spectrum of 4 (Fig. 4). The absolute configuration of compound 4 was established as 3S using the time-dependent density functional theory (TDDFT) at the B3LYP/6-311G (d,p) level (Fig. S20). Compound 4 was the derivative of IPA (here named paenibacillic acid A).

Effects of indolic acids compounds on plant growth promotion

The effects of indolic acids compounds on growth of Arabidopsis thaliana var. Columbia were investigated. Paenibacillic acid A, IAA, and IPA were shown to promote the growth of plant shoots and roots (Fig. 5) as well as the dry weight of the plants (Fig. 6). The growth-promoting ability increased as the concentration of paenibacillic acid A, IAA, and IPA increased until their highest growth-promoting ability was attained, and then their plant growth-promoting ability decreased with increasing concentration. The significant efficiencies of plant-growth promotion were obtained when the concentrations of paenibacillic acid A, IAA and IPA were at 100 nM, 250 nM and 50 nM, respectively, suggesting that IPA has the strongest ability of plant growth promotion among the three indolic acids compounds.

We further determined the content of indolic compounds produced by P. triticisoli BJ-18 when it was measured in the nitrogen-deficient medium with and without iron. The results showed that P. triticisoli BJ-18 produced 37.03 ± 1.21 µg/mL of the indolic compounds in a medium with an iron ion concentration of 4.409 × 10⁻⁴ mM and 13.457 ± 0.78 µg/mL of the indolic compounds in a medium without iron, suggesting that the content of indolic compounds in P. triticisoli BJ-18 is positively related to the iron in the environment.

Antimicrobial activity

The antimicrobial properties of siderophores and indolic acids were assayed against the indicator strains (bacteria E. coli, S. aureus and B. subtilis, and yeast C. albicans) (Table 3). Both compounds 1 and 2 that are siderophores showed antimicrobial activity against E. coli, S. aureus and B. subtilis, but did not show obvious inhibitory activity against yeast C. albicans.

Gentamicin, ampicillin, streptomycin sulfate and amphotericin B were used as positive controls against E. coli, S. aureus, B. subtilis and C. albicans, respectively. The compounds were tested at concentrations of 5 µM, 10 µM and 20 µM. The IC₅₀ was calculated using the Spearman-Karber’s method.

Siderophore detection by blue agar CAS assay

Siderophore production by P. triticisoli BJ-18 was determined by blue agar CAS assay as described in materials and methods. A yellow ring appeared around each colony after seven days of P. triticisoli BJ-18 growth on a blue agar plate, indicating that P. triticisoli BJ-18 had the ability to secrete siderophores and transfer iron from the environment to the bacterial cells (Fig. S21). The data are consistent with the above results that compounds 1-3 are siderophores.