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MB Sample ID: SA194041

Local Sample ID:	72269_4
Subject ID:	SU002144
Subject Type:	Plant
Subject Species:	Lithospermum officinale
Taxonomy ID:	475907

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Subject:

Subject ID:	SU002144
Subject Type:	Plant
Subject Species:	Lithospermum officinale
Taxonomy ID:	475907

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
72269_4	SA194041	FL023770	Chitinophaga sp. R-72269	Treatment

Collection:

Collection ID:	CO002137
Collection Summary:	Six glass jars with three plants each were used per treatment. Each jar containing three plants was thus considered as a biological replicate; six biological replicates per treatment were used. The treatments consisted of inoculating bacteria. PBS was used as a negative control treatment. Plants were grown for seven weeks. The root system of each plant was harvested separately and fresh weight was recorded before lyophilisation. The dry weight was then measured and the samples stored at -80 C for analysis of the content of A/Sd by targeted and non-targeted metabolomics
Sample Type:	Plant

Treatment:

Treatment ID:	TR002156
Treatment Summary:	A set of 50 bacteria previously isolated from another A/Sd-producing plant, Alkanna tinctoria L. (Tausch), was selected based on diversity and on phenotypic traits tested in vitro (Rat et al., 2021). The 50 strains were first grown in 35 ml Reasoner’s 2A broth (R2B, Acumedia) at 28°C, 100 rpm for 72 h. Before preparing the inocula, 5 ml of each bacterial culture was sampled and used to estimate bacterial concentrations via counting of colony-forming units (CFU), while the remaining culture was used as inoculum. The inoculum was centrifuged at 4°C, 14000 rpm for 10 min, the supernatant was discarded, and the pellet was preserved in 2 ml of R2B medium supplemented with 10% glycerol. Bacterial pellets were then stored at -20°C until inoculation. To prepare the inoculum, a pellet was suspended in 28 ml of sterile phosphate-buffered saline (PBS) at pH 7.4. The resuspended inoculum was then adjusted to a concentration of 104-106 CFU/ml, and ten µl were used to inoculate each plant. The bacterial suspension was first injected in the MSRmod medium with a micropipette. Then, a shoot tip of L. officinale of 3.5 cm length was selected and the top two-three leaves were removed by cutting. The plant was finally transferred in sterile conditions and inserted at the positions where the bacteria had been injected in the medium. Plants treated with only PBS were used as non-inoculated controls. To avoid light in the root compartment, which can inhibit the production of shikonin (Yazaki et al., 1999), the surface of the medium was covered with sterilized (in an oven at 145°C during 10 h) quartz sand and the lower half of the jar was wrapped with aluminium foil (Figure 1). The jars were then incubated in a growth chamber at 20ºC, 16:8 h light:dark, with a light intensity of 50 µmol m-2s-1. Plants were harvested for analysis after seven weeks of incubation

Treatment ID:

TR002156

Treatment Summary:

A set of 50 bacteria previously isolated from another A/Sd-producing plant, Alkanna tinctoria L. (Tausch), was selected based on diversity and on phenotypic traits tested in vitro (Rat et al., 2021). The 50 strains were first grown in 35 ml Reasoner’s 2A broth (R2B, Acumedia) at 28°C, 100 rpm for 72 h. Before preparing the inocula, 5 ml of each bacterial culture was sampled and used to estimate bacterial concentrations via counting of colony-forming units (CFU), while the remaining culture was used as inoculum. The inoculum was centrifuged at 4°C, 14000 rpm for 10 min, the supernatant was discarded, and the pellet was preserved in 2 ml of R2B medium supplemented with 10% glycerol. Bacterial pellets were then stored at -20°C until inoculation. To prepare the inoculum, a pellet was suspended in 28 ml of sterile phosphate-buffered saline (PBS) at pH 7.4. The resuspended inoculum was then adjusted to a concentration of 104-106 CFU/ml, and ten µl were used to inoculate each plant. The bacterial suspension was first injected in the MSRmod medium with a micropipette. Then, a shoot tip of L. officinale of 3.5 cm length was selected and the top two-three leaves were removed by cutting. The plant was finally transferred in sterile conditions and inserted at the positions where the bacteria had been injected in the medium. Plants treated with only PBS were used as non-inoculated controls. To avoid light in the root compartment, which can inhibit the production of shikonin (Yazaki et al., 1999), the surface of the medium was covered with sterilized (in an oven at 145°C during 10 h) quartz sand and the lower half of the jar was wrapped with aluminium foil (Figure 1). The jars were then incubated in a growth chamber at 20ºC, 16:8 h light:dark, with a light intensity of 50 µmol m-2s-1. Plants were harvested for analysis after seven weeks of incubation

Sample Preparation:

Sampleprep ID:	SP002150
Sampleprep Summary:	The lyophilised roots were ground to a fine powder using a ball mill (Fritsch Pulverisette 0, Germany). For each powdered sample, a subsample of 35 mg was placed into microcentrifuge tubes for SMs extraction with 1.5 ml of methanol (LC-MS grade, Honeywell Riedel de Haën, USA) in an ultrasound bath at 10% power for three hours (Bandelin Sonorex Digital 10P, Berlin, Germany) followed by centrifugation for 10 minutes at 12500 rpm (Hermle Z 216 MK, Wehingen, Germany). The supernatants were collected and subjected to Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analysis, after filtering with 0.22 μm polytetrafluorethylene (PTFE) filters.

Sampleprep ID:

SP002150

Sampleprep Summary:

The lyophilised roots were ground to a fine powder using a ball mill (Fritsch Pulverisette 0, Germany). For each powdered sample, a subsample of 35 mg was placed into microcentrifuge tubes for SMs extraction with 1.5 ml of methanol (LC-MS grade, Honeywell Riedel de Haën, USA) in an ultrasound bath at 10% power for three hours (Bandelin Sonorex Digital 10P, Berlin, Germany) followed by centrifugation for 10 minutes at 12500 rpm (Hermle Z 216 MK, Wehingen, Germany). The supernatants were collected and subjected to Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analysis, after filtering with 0.22 μm polytetrafluorethylene (PTFE) filters.

Combined analysis:

Analysis ID	AN003361
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Waters Acquity UPLC HSS C18 SB (100 x 2.1mm, 1.8um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Focus
Ion Mode	POSITIVE
Units	intensity units

Chromatography:

Chromatography ID:	CH002487
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity UPLC HSS C18 SB (100 x 2.1mm, 1.8um)
Flow Gradient:	0 min 95A/5B, 1 min 50A/50B, 8 min 0A/100B, 13 min 0A/100B, 13.01 min 95A/5B, 16 min 95A/5B.
Solvent A:	100% water; 0.1% formic acid;
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003130
Analysis ID:	AN003361
Instrument Name:	Thermo Q Exactive Focus
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The solvents used were ultrapure water (A) and methanol (B), both with 0.1% (v/v) formic acid. The gradient elution program was as follows: 0 min 95A/5B, 1 min 50A/50B, 8 min 0A/100B, 13 min 0A/100B, 13.01 min 95A/5B, 16 min 95A/5B. Data were acquired in positive ionisation mode, with the capillary temperature set to 320 oC using the instrument's MS/MS discovery feature. The normalised collision energy was set to 35 eV. The instrument control, acquisition and initial processing of the data were conducted by the Xcalibur software (version 4.1, Thermo Scientific, USA). Furthermore, data alignment and feature extraction were performed on the XCMS online platform (Huan et al., 2017). Identification of some detected compounds was performed using the software Compound Discoverer (version 3.2, Thermo Scientific, USA).
Ion Mode:	POSITIVE