DOI: 10.25349/d9904v
title: Data from: A fluorometric assay for high-throughput phosphite quantitation in biological and environmental matrices

description: Phosphite, the anion of phosphorus acid, is increasingly recognized as an
 important metabolite in the global biogeochemical phosphorus cycle and as
 a fungicide and fertilizer in agriculture. As such, methods for detecting
 phosphite quantitatively and selectively are critical to evidencing
 phosphorus redox chemistry. Here, we present a fluorescence-based assay
 for phosphite, based on the NAD+-dependent oxidation of phosphite by
 phosphite dehydrogenase and the subsequent reduction of resazurin to
 resorufin. With the application of a thermostable phosphite dehydrogenase,
 a medium-invariant analytical approach, and novel sample preparation
 methods, the assay is capable of rapid and accurate phosphite
 quantification with a 3 mm limit of detection in a wide array of
 biologically- and environmentally-relevant matrices, including bacterial
 and archaeal cell lysate, seawater, anaerobic digester sludge, and plant
 tissue. We demonstrate the utility of the assay by quantitating phosphite
 uptake in a model crop plant in the presence or absence of a
 phosphite-oxidizing strain of Pseudomonas stutzeri as a soil additive,
 establishing this bacterium as an efficient phosphite converting
 biofertilizer.
description: # Data from: A Fluorometric Assay for High-throughput Phosphite
 Quantitation in Biological and Environmental Matrices ### Data Overview: A
 collection of fluorescence data collected using the phosphite (Phi) assay
 method described in "A Fluorometric Assay for High-throughput
 Phosphite Quantitation in Biological and Environmental Matrices".
 Excel files contain the fluorescence output data from the Spark 10m
 microplane reader (Tecan), unless otherwise noted, including the
 instrument's operating conditions, and the organization of
 fluorescence readouts by sample ID. Fluorescence values are averaged in
 triplicate, then the sample containing no 17X-PTDH is subtracted from all
 average values. Every sample has at least 3 points on a standard addition
 curve ranging from 0-5 nmol of spiked Phi (excluding example assays which
 have a wider range). Red boxes with "OVER" were samples that
 saturated the detector and were not used for any analysis. Other supplied
 data include absorbance measurements collected to determine 17X-PTDH and
 LDH* activity. #### Files 1.
 3-1-22_LOD_3_1x_and_0x_PMS_annot.xlsx 2.
 3-11-22_17X-PTDH_Activity_and_concentration.xlsx 3.
 LDH_activity_in_STET_Annot.xlsx 4. 9-23-21_E.coli_LB_LOD_1.xlsx 5.
 11-16-21_e.coli_LB_LOD_2_and_3.xlsx 6.
 12-2-21_Seawater_LOD_1_and_Recovery_Annot.xlsx 7.
 11_9_21_Seawater_LOD_2_and_3_Annot.xlsx 8.
 4-26-22_n.maritimus_pellet_LOD1_Annot.xlsx 9.
 4-24-22_n._maritimus_LOD_2_Annot.xlsx 10.
 3-13-22_pt_assay_kinetics_fig_2_annot.xlsx 11.
 3-6-22_LDH_E._coli_comparison_in_LB_and_Ecoli_LOD_in_LB.xlsx 12.
 3-4-22_Example_Std_Adn_and_LOD_1_annot.xlsx 13.
 2-28-22_1x_PMS_and_LOD_2_annot.xlsx 14.
 3-8-22_50__seawater_recovery_annot.xlsx 15.
 2-16-22_n._maritimus_LOD_3_Annot.xlsx 16.
 2-9-22__Radish_leaf_LOD_1_annot.xlsx 17.
 1-26-22_Ecoli_LDH_comparison_in_MOPS_and_LOD_Annot.xlsx 18.
 1-19-22_Artificial_seawater_Annot.xlsx 19.
 1-14-22_Anaerobic_digester_LODs_and_Recovery_Annot.xlsx 20.
 8-30-22_MS_radish_pt_assays_annot.xlsx 21. 4-1-22_radishes_annot.xlsx 22.
 3-29-22_radish_data_annot.xlsx 23. 3-30-22_radish_analysis_annot.xlsx 24.
 3-13-22_radish_leaves_and_stems_Pt_recovery_annot.xlsx 25.
 2-8-22_2_root_extract_LOD_annot.xlsx ### Data-Specific Information #####
 3-1-22\_LOD\_3\_1x\_and\_0x\_PMS\_annot.xlsx This fluorescence data is for
 two experiments of the Phi assay: the first portion of the plate (A1-C3)
 shows the Phi assay prepared as described in Methods, using 100 uM PMS;
 the second portion of the plate (D1-F3) shows the Phi assay prepared with
 no PMS. Instrument parameters are detailed in the first 57 lines, then an
 initial reading (t = 0) was taken of the samples. Then the kinetic
 experiment begins, and the instrument parameters are detailed in lines
 68-96. Below the instrument acquisition, averages and standard deviation
 (lines 133-164) are taken for the triplicate measurements of the standard
 addition curve as well as the PTDH-free blank. Finally, to determine the
 LOD of the assay, the final time point was used to generate a standard
 addition curve (lines 175-184). Variables: * Fluorescence [A.U.] -
 fluorescence values are reported by the instrument under the column (and
 row, for the t= 0 measurement) representing the plate's well
 location. In the averaged data (133-164), the header is the amount of Phi
 spiked in each sample, to help identify what wells correspond to what
 samples. * Time [s] - time since the instrument began acquisition on the
 kinetics experiment. * Cycle Nr. - Number of times the instrument has
 taken a measurement since the beginning of the kinetics experiment. #####
 3-11-22\_17X-PTDH\_Activity\_and\_concentration.xlsx This data sheet shows
 three UV-Vis absorbance experiments taken on a Cary 60 from purified
 17X-PTDH in order to determine its activity . The reaction mixture
 contained 1.2 ug protein and 3 mL of assay mix (100 mM MOPS, pH 7.25, 10
 mM NAD+, and 10 mM Phi). Columns A and B show the absorbance values at 340
 nm taken in a kinetics experiment with the above reaction mixture stirred
 at 25C. Columns H and I show the absorbance values at 340 nm taken in a
 kinetics experiment with the above reaction mixture stirred at 37C.
 Columns Q and R show the absorbance at wavelengths of 500 to 200 nm.
 Instrumental parameters are reported at the bottom of the reported
 absorbance values for each column. Variables: * Abs [A.U.]- Absorbance
 values reported by the instrument at the given wavelength of incident
 light. * Time [min] - Minutes since data acquisition began. * Wavelength
 [nm] - Wavelength of light incident on the sample. #####
 LDH\_activity\_in\_STET\_Annot.xlsx This data sheet shows two UV-Vis
 absorbance experiments taken on a Cary 60 from purified LDH* in order to
 determine its activity. The reaction mixture contained 1 ug protein and 3
 mL of assay mix (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5% v/v
 Triton X-100, 0.5 mM NADH, 30 mM pyruvate). Columns A and B show the
 absorbance values at 340 nm taken in a kinetics experiment with the above
 reaction mixture stirred at 25C. Columns C and D show the absorbance
 values at 340 nm taken in a kinetics experiment with the above reaction
 mixture stirred at 25C, with the purified enzyme undergoing 1x
 freeze/thaw. Column E shows the absorbance values in column D, adjusted so
 that Abs @ t = 0 is 0, for plotting purposes. Variables: * Abs [A.U.]-
 Absorbance values reported by the instrument at the given wavelength of
 incident light. * Time [min] - Minutes since data acquisition began. #####
 9-23-21\_E.coli\_LB\_LOD\_1.xlsx This fluorescence data shows an
 experiment to determine the LOD of the Phi assay on *E. coli* lysate grown
 in LB without LDH treatment. This fluorescence data was taken on a Duetta
 Fluorescence and Absorbance Spectrometer (Horiba). The excitation
 wavelength was 535 nm (5 nm bandpass) and the emission range was 550 nm to
 700 nm (5 nm bandpass), and the integration time was 0.1 s. Full
 wavelength scans are shown for each sample, and are averaged under the
 column indicating the sample name based on the amount of Phi spiked (e.g.
 3 nmol). In columns CE-CJ, averaged fluorescence values at 585 nm are
 assembled and labeled by the amount of Phi spiked for clarity. The
 propagated error, slope of the standard addition line, and LOD of the
 sample standard addition curve is shown here. Variables: * Wavelength [nm]
 - Wavelength of light incident on the sample. * Fluorescence [A.U.] -
 fluorescence values are reported by the instrument for a given sample. *
 Smoothed Fluorescence [A.U.] - fluorescence values in the column to the
 left at a 5 nm rolling average to smooth the data according to the
 bandpass. ##### 11-16-21\_e.coli\_LB\_LOD\_2\_and\_3.xlsx This
 fluorescence data shows two experiments to determine the LOD of the Phi
 assay on *E. coli* lysate grown in LB without LDH treatment. These two
 runs represent two biological replicates run under the same conditions.
 This fluorescence data was taken on a Genios Pro 96/384 Fluorescence
 Absorbance Microplate Reader (Tecan). The excitation wavelength was 535 nm
 (5 nm bandpass) and the emission wavelength collected was 585 nm (5 nm
 bandpass), and the integration time was 0.1 s. The values of fluorescence
 were recorded from the instrument directly to the provided data sheet. The
 fluorescence values for each well are placed in the row corresponding to
 the amount of Phi spiked for that sample. The triplicate measurements are
 averaged under "Average" and the PTDH-free blank is subtracted
 under "Adjusted", and the standard deviation is shown under
 "Std Dev". The propagated error, slope of the standard addition
 line, and LOD of the sample standard addition curve is shown here.
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument for a given sample. #####
 12-2-21\_Seawater\_LOD\_1\_and\_Recovery\_Annot.xlsx This fluorescence
 data shows two experiments to determine the LOD and Phi recovery of
 seawater in the Phi assay. Seawater from the SB Channel was coarsely
 filtered and spiked with 100 uM Phi prior to analysis. The instrument
 parameters are reported in lines 1-41. The fluorescence values for each
 well are placed in the row corresponding to the amount of Phi spiked for
 that sample. The triplicate measurements are averaged under
 "Average" and the PTDH-free blank is subtracted under
 "Adjusted", and the standard deviation is shown under "Std
 Dev". The % recovery calculation, propagated error and LOD of the
 sample standard addition curve are also shown here. Variables: *
 Fluorescence [A.U.] - fluorescence values are reported by the instrument
 under the column and row representing the plate's well location. In
 the averaged data the "nmol Phi added" is the amount of Phi
 spiked in each sample, to help identify what wells correspond to what
 samples. ##### 11\_9\_21\_Seawater\_LOD\_2\_and\_3\_Annot.xlsx This
 fluorescence data shows two experiments to determine the LOD of seawater
 in the Phi assay. Seawater from the SB Channel was coarsely filtered and
 spiked with 100 uM Phi prior to analysis. The instrument parameters are
 reported in lines 1-41. The fluorescence values for each well are placed
 in the row corresponding to the amount of Phi spiked for that sample. The
 triplicate measurements are averaged under "Average" and the
 PTDH-free blank is subtracted under "Adjusted", and the standard
 deviation is shown under "Std Dev". The propagated error and LOD
 of the sample standard addition curve are also shown here. Variables: *
 Fluorescence [A.U.] - fluorescence values are reported by the instrument
 under the column and row representing the plate's well location. In
 the averaged data the "nmol Phi added" is the amount of Phi
 spiked in each sample, to help identify what wells correspond to what
 samples. ##### 4-26-22\_n.maritimus\_pellet\_LOD1\_Annot.xlsx This
 fluorescence data shows an experiment to determine the LOD of concentrated
 *N. maritimus* culture in the Phi assay. The culture was grown as
 described in Methods, and was concentrated 1000x using tangential flow. To
 lyse the cells, the samples were subjected to three freeze/thaw cycles.
 The instrument parameters are reported in lines 1-45. The fluorescence
 values for each well are placed in the row corresponding to the amount of
 Phi spiked for that sample. The triplicate measurements are averaged under
 "Average" and the PTDH-free blank is subtracted under "Adj
 Intensity", and the standard deviation is shown under "Std
 Dev". The propagated error and LOD of the sample standard addition
 curve are also shown here. Variables: * Fluorescence [A.U.] - fluorescence
 values are reported by the instrument under the column and row
 representing the plate's well location. In the averaged data
 (133-164), the "nmol Phi added" is the amount of Phi spiked in
 each sample, to help identify what wells correspond to what samples. #####
 4-24-22\_n.\_maritimus\_LOD\_2\_Annot.xlsx This fluorescence data shows an
 experiment to determine the LOD of concentrated *N. maritimus* culture in
 the Phi assay. The culture was grown as described in Methods, and was
 concentrated 1000x using tangential flow. To lyse the cells, the samples
 were subjected to three freeze/thaw cycles. The instrument parameters are
 reported in lines 1-45. The fluorescence values for each well are placed
 in the row corresponding to the amount of Phi spiked for that sample. The
 triplicate measurements are averaged under "Average" and the
 PTDH-free blank is subtracted under "Adj Intensity", and the
 standard deviation is shown under "Std Dev". The propagated
 error and LOD of the sample standard addition curve are also shown here.
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument under the column and row representing the plate's well
 location. In the averaged data the "nmol Phi spiked" column is
 the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples. #####
 3-13-22\_pt\_assay\_kinetics\_fig\_2\_annot.xlsx This fluorescence data is
 to determine the kinetics of resorufin fluorescence in the Phi assay
 prepared as described in Methods, using 1 ug 17X-PTDH and 100 uM PMS.
 Instrument parameters are detailed in the first 57 lines, then an initial
 reading (t = 0) was taken of the samples. Then the kinetic experiment
 begins, and the instrument parameters are detailed in lines 65-93. Below
 the instrument acquisition, averages and standard deviation (lines
 130-154) are taken for the triplicate measurements of the standard
 addition curve as well as the PTDH-free blank. Outlier fluorescence values
 in F2, F3, and E10 were removed for clarity (an occasional problem in
 kinetics experiments due to the misalignment of the plate and taking only
 one measurement per well) these conditions were excluded from the analysis
 and the figure presented in the paper. Variables: * Fluorescence [A.U.] -
 fluorescence values are reported by the instrument under the column (and
 row, for the t= 0 measurement) representing the plate's well
 location. In the averaged data (133-164), the header is the amount of Phi
 spiked in each sample, to help identify what wells correspond to what
 samples. * Time [s] - time since the instrument began acquisition on the
 kinetics experiment. * Cycle Nr. - Number of times the instrument has
 taken a measurement since the beginning of the kinetics experiment. #####
 3-6-22\_LDH\_E.\_coli\_comparison\_in\_LB\_and\_Ecoli\_LOD\_in\_LB.xlsx
 This fluorescence data shows an experiment to compare the effect of LDH*
 treatment on *E. coli* lysate. The culture was grown, split into two
 aliquots, and one was treated with LDH* and pyruvate as described in
 Methods. The instrument parameters are reported in lines 1-45. The
 fluorescence values for each well are placed in the row corresponding to
 the amount of Phi spiked for that sample. The triplicate measurements are
 averaged under "Avg Intensity" and the PTDH-free blank is
 subtracted under "Adj Intensity", and the standard deviation is
 shown under "Std Dev". The propagated error and LOD of the
 LDH*-treated sample's standard addition curve are also shown here.
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument under the column and row representing the plate's well
 location. In the averaged data the "nmol Phi added" column is
 the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples. #####
 3-4-22\_Example\_Std\_Adn\_and\_LOD\_1\_annot.xlsx This fluorescence data
 shows an experiment to demonstrate the standard addition curve and how it
 can be used to read the Phi content of a sample. In the first experiment,
 the buffer of the Phi assay mix was spiked with 100 uM Phi. In the second
 experiment, the Phi assay mix was prepared as described with MOPS buffer.
 The instrument parameters are reported in lines 1-45. The fluorescence
 values for each well are placed in the row corresponding to the amount of
 Phi spiked for that sample. The triplicate measurements are averaged under
 "Avg Intensity" and the PTDH-free blank is subtracted under
 "Adj Intensity", and the standard deviation is shown under
 "Std Dev". The propagated error and LOD of the standard addition
 curve is shown here, as well as the read Phi concentration by the standard
 addition curve. * Fluorescence [A.U.] - fluorescence values are reported
 by the instrument under the column and row representing the plate's
 well location. In the averaged data the "nmol Phi added" column
 is the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples. #####
 2-28-22\_1x\_PMS\_and\_LOD\_2\_annot.xlsx This fluorescence data is
 presented to show an example of the Phi assay run with assay buffer with
 100 uM PMS, 2 ug 17X-PTDH, and spiked Phi to determine the kinetics of
 resorufin fluorescence over time. Instrument parameters are detailed in
 the first 57 lines, then an initial reading (t = 0) was taken of the
 samples. Then the kinetic experiment begins, and the instrument parameters
 are detailed in lines 65-93. Well B10 gave outlier fluorescence values for
 cycles 20-25 (an occasional problem in kinetics experiments due to the
 misalignment of the plate and taking only one measurement per well), and
 was excluded from the analysis and the figure presented in the paper.
 Below the instrument acquisition, averages and standard deviation (lines
 133-164) are taken for the triplicate measurements of the standard
 addition curve as well as the PTDH-free blank. Finally, to determine the
 LOD of the assay, the final time point was used to generate a standard
 addition curve (lines 175-184). Variables: * Fluorescence [A.U.] -
 fluorescence values are reported by the instrument under the column (and
 row, for the t= 0 measurement) representing the plate's well
 location. In the averaged data (133-164), the header is the amount of Phi
 spiked in each sample, to help identify what wells correspond to what
 samples. * Time [s] - time since the instrument began acquisition on the
 kinetics experiment. * Cycle Nr. - Number of times the instrument has
 taken a measurement since the beginning of the kinetics experiment. #####
 3-8-22\_50\_\_seawater\_recovery\_annot.xlsx This fluorescence data shows
 an experiment to determine the Phi recovery of spiked seawater. The
 seawater was concentrated to 50% its original volume on a vacuum
 concentrator and was spiked with 190 uM Phi (9.5 nmol detected in 50 uL
 assay sample volume). The instrument parameters are reported in lines
 1-45. The fluorescence values for each well are placed in the row
 corresponding to the amount of Phi spiked for that sample. The triplicate
 measurements are averaged under "Avg Fluor. Intensity" and the
 PTDH-free blank is subtracted under "Adj Intensity", and the
 standard deviation is shown under "Std Dev". The % recovery,
 propagated error, and LOD of the LDH*-treated sample's standard
 addition curve are also shown here. Wells with saturated fluorescence
 ("OVER") were not relevant to the experiment. Variables: *
 Fluorescence [A.U.] - fluorescence values are reported by the instrument
 under the column and row representing the plate's well location. In
 the averaged data the "Phi added" [nmol] column is the amount of
 Phi spiked in each sample, to help identify what wells correspond to what
 samples. ##### 2-16-22\_n.\_maritimus\_LOD\_3\_Annot.xlsx This
 fluorescence data shows an experiment to determine the LOD of concentrated
 *N. maritimus* culture in the Phi assay. The culture was grown as
 described in Methods, and was concentrated 1000x using tangential flow. To
 lyse the cells, the samples were subjected to three freeze/thaw cycles.
 The instrument parameters are reported in lines 1-41. The fluorescence
 values for each well are placed in the row corresponding to the amount of
 Phi spiked for that sample. The triplicate measurements are averaged under
 "Average" and the PTDH-free blank is subtracted under "Adj
 Intensity", and the standard deviation is shown under "Std
 Dev". The propagated error and LOD of the sample standard addition
 curve are also shown here. Variables: * Fluorescence [A.U.] - fluorescence
 values are reported by the instrument under the column and row
 representing the plate's well location. In the averaged data, the
 "nmol Pt added" column is the amount of Phi spiked in each
 sample, to help identify what wells correspond to what samples. #####
 2-9-22\_\_Radish\_leaf\_LOD\_1\_annot.xlsx This fluorescence data shows an
 experiment to determine the LOD of radish leaf extract in the Phi assay.
 Radish leaves were lysed by a mortar and pestle cooled with LN2. Cells
 were extracted using STET buffer (4mL/g wet weight), then diluted 1:100
 prior to the assay. The instrument parameters are reported in lines 1-41.
 The fluorescence values for each well are placed in the row corresponding
 to the amount of Phi spiked for that sample. The triplicate measurements
 are averaged under "Average" and the PTDH-free blank is
 subtracted under "Adj Intensity", and the standard deviation is
 shown under "Std Dev". The propagated error and LOD of the
 sample standard addition curve are also shown here. Variables: *
 Fluorescence [A.U.] - fluorescence values are reported by the instrument
 under the column and row representing the plate's well location. In
 the averaged data, the "nmol Pt added" column is the amount of
 Phi spiked in each sample, to help identify what wells correspond to what
 samples. #####
 1-26-22\_Ecoli\_LDH\_comparison\_in\_MOPS\_and\_LOD\_Annot.xlsx This
 fluorescence data shows an experiment to determine the LOD of the Phi
 assay on *E. coli* lysate grown in MOPS Minimal Medium with and without
 LDH treatment. Cell growth, lysate extraction, and LDH treatment are
 detailed in Methods. The instrument parameters are reported in lines 1-41.
 The two samples were run in two separate runs of acquisition, and are
 shown in two Excel sheets. Note that this was because of the gain
 adjustment, which allowed the fluorescence to be read in the linear range
 for both experiments. The instrument adjusted gain based on a well of the
 relevant experiment's sample with the highest nmol Phi spiked (4.5
 nmol in this case). The fluorescence values for each well are placed in
 the row corresponding to the amount of Phi spiked for that sample. The
 triplicate measurements are averaged under "Avg" and the
 PTDH-free blank is subtracted under "Adj. Int.", and the
 standard deviation is shown under "Std Dev". The propagated
 error and LOD of the sample standard addition curve are also shown here.
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument under the column and row representing the plate's well
 location. In the averaged data, the "nmol Pt added" column is
 the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples. #####
 1-19-22\_Artificial\_seawater\_Annot.xlsx This fluorescence data shows an
 experiment to determine the effect of concentration and individual
 presence of salts in artificial seawater on Phi assay performance. The
 formulation of artificial seawater is described in Methods. The instrument
 parameters are reported in lines 1-55. The fluorescence values for each
 well are placed in the row corresponding to the amount of Phi spiked for
 that sample. The triplicate measurements are averaged under "Avg
 Intensity" and the PTDH-free blank is subtracted under "Adj.
 Int.", and the standard deviation is shown under "Std Dev".
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument under the column and row representing the plate's well
 location. In the averaged data, the "nmol Phi added" column is
 the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples. #####
 1-14-22\_Anaerobic\_digester\_LODs\_and\_Recovery\_Annot.xlsx This
 fluorescence data shows an experiment to determine the LOD and % Phi
 recovery of anaerobic digester sludge. The sludge was stored anaerobically
 and filtered with a 0.22 um syringe filter as described in Methods. The
 instrument parameters are reported in lines 1-55. The fluorescence values
 for each well are placed in the row corresponding to the amount of Phi
 spiked for that sample. The triplicate measurements are averaged under
 "Mean" and the PTDH-free blank is subtracted under "Adj.
 Int.", and the standard deviation is shown under "Std Dev".
 Fluorescence values that were not relevant to the experiment are
 highlighted in grey (Plate row A). The % recovery, propagated error, and
 LOD of the anaerobic digester sludge sample's standard addition curve
 are also shown here. Variables: * Fluorescence [A.U.] - fluorescence
 values are reported by the instrument under the column and row
 representing the plate's well location. In the averaged data, the
 "nmol Pt" column is the amount of Phi spiked in each sample, to
 help identify what wells correspond to what samples. #####
 8-30-22\_MS\_radish\_pt\_assays\_annot.xlsx This fluorescence data shows
 several experiments to determine the Phi content of radish seedlings grown
 with various P sources and the presence or absence of Phi-oxidizing *P.
 stutzeri*. Radish seedlings were grown in MS minimal plant media and lysed
 using a LN2-cooled mortar and pestle, as described in Methods. Cell
 contents were extracted using 4 mL STET buffer/g wet weight, and the
 extract was diluted 1:100 prior to assay. This experiment was run on a
 Tecan M200 Infinite Pro. The experimental parameters are detailed in lines
 1-50. As detailed there, multiple reads were taken per well in a square
 pattern and averaged. This was the case for all samples, except well G4,
 where only one measurement is used for the well, due to its large
 difference from the other three measurements. The triplicate measurements
 are averaged under "Avg Intensity" and the PTDH-free blank is
 subtracted under "Adj. Int.", and the standard deviation is
 shown under "Std Dev". Variables: * Fluorescence [A.U.] -
 fluorescence values are reported by the instrument under the column and
 row representing the plate's well location. In the averaged data, the
 "nmol Phi added" column is the amount of Phi spiked in each
 sample, to help identify what wells correspond to what samples. * Well -
 Well of the plate being measured. * Mean [A.U.] - Average of the four
 fluorescence readings taken per well. * StDev (instrument output, not in
 analysis) - Standard deviation of the four fluorescence readings taken per
 well. * 0;1, 1;1, 1;0, 0;0 - Coordinates of the square pattern that the
 instrument is following to take the four fluorescence readings of the
 well. ##### 4-1-22\_radishes\_annot.xlsx This fluorescence data shows an
 experiment to determine the LOD of radish leaf extract in the Phi assay.
 Radish leaves were lysed by a mortar and pestle cooled with LN2. Cells
 were extracted using STET buffer (4mL/g wet weight), then diluted 1:100
 prior to the assay. The instrument parameters are reported in lines 1-53.
 The fluorescence values for each well are placed in the row corresponding
 to the amount of Phi spiked for that sample. The triplicate measurements
 are averaged under "Avg Inten." and the PTDH-free blank is
 subtracted under "Adj Intensity", and the standard deviation is
 shown under "St Dev". The propagated error and LOD of the sample
 standard addition curve are also shown here. Variables: * Fluorescence
 [A.U.] - fluorescence values are reported by the instrument under the
 column and row representing the plate's well location. In the
 averaged data, the "nmol Phi added" column is the amount of Phi
 spiked in each sample, to help identify what wells correspond to what
 samples. ##### 3-29-22\_radish\_data\_annot.xlsx This fluorescence data
 shows an experiment to determine the LOD of radish leaf and root extract
 in the Phi assay. Radish leaves and roots were lysed by a mortar and
 pestle cooled with LN2. Cells were extracted using STET buffer (4mL/g wet
 weight), then diluted 1:100 prior to the assay. The instrument parameters
 are reported in lines 1-53. The fluorescence values for each well are
 placed in the row corresponding to the amount of Phi spiked for that
 sample. The triplicate measurements are averaged under "Avg Fl.
 Int." and the PTDH-free blank is subtracted under "Adj
 Inten.", and the standard deviation is shown under "St
 Dev". The propagated error and LOD of the sample standard addition
 curve are also shown here. Variables: * Fluorescence [A.U.] - fluorescence
 values are reported by the instrument under the column and row
 representing the plate's well location. In the averaged data, the
 "nmol Phi added" column is the amount of Phi spiked in each
 sample, to help identify what wells correspond to what samples. #####
 3-30-22\_radish\_analysis\_annot.xlsx This fluorescence data shows several
 experiments to determine the Phi content of radish seedlings grown with
 various P sources and the presence or absence of Phi-oxidizing *P.
 stutzeri*. Radish seedlings were grown in MS minimal plant media and lysed
 using a LN2-cooled mortar and pestle, as described in Methods. Cell
 contents were extracted using 4 mL STET buffer/g wet weight, and the
 extract was diluted 1:100 prior to assay. The instrument parameters are
 reported in lines 1-53. The fluorescence values for each well are placed
 in the row corresponding to the amount of Phi spiked for that sample. The
 triplicate measurements are averaged under "Avg Fl. Int." and
 the PTDH-free blank is subtracted under "Adj Inten.", and the
 standard deviation is shown under "St Dev". The propagated error
 and LOD of the sample standard addition curve are also shown here.
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument under the column and row representing the plate's well
 location. In the averaged data, the "nmol Phi added" column is
 the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples. #####
 3-13-22\_radish\_leaves\_and\_stems\_Pt\_recovery\_annot.xlsx This
 fluorescence data shows an experiment to determine the % recovery of
 radish leaf and root extract in the Phi assay. Radish leaves and roots
 were lysed by a mortar and pestle cooled with LN2. Cells were extracted
 using STET buffer (4mL/g wet weight), then diluted 1:100 prior to the
 assay. Samples were then spiked with 100 uM Phi (5 nmol detected in 50 uL
 assay sample volume). The instrument parameters are reported in lines
 1-55. The fluorescence values for each well are placed in the row
 corresponding to the amount of Phi spiked for that sample. The triplicate
 measurements are averaged under "Avg Fluor. Int." and the
 PTDH-free blank is subtracted under "Adj Fluor. Inten.", and the
 standard deviation is shown under "St Dev". The propagated
 error, LOD, and % recovery of the sample standard addition curve are also
 shown here. Variables: * Fluorescence [A.U.] - fluorescence values are
 reported by the instrument under the column and row representing the
 plate's well location. In the averaged data, the "nmol Phi
 added" column is the amount of Phi spiked in each sample, to help
 identify what wells correspond to what samples. #####
 2-8-22\_2\_root\_extract\_LOD\_annot.xlsx This fluorescence data shows an
 experiment to determine the LOD of radish leaf and root extract in the Phi
 assay. Radish leaves and roots were lysed by a mortar and pestle cooled
 with LN2. Cells were extracted using STET buffer (4mL/g wet weight), then
 diluted 1:100 prior to the assay. The instrument parameters are reported
 in lines 1-55. The fluorescence values for each well are placed in the row
 corresponding to the amount of Phi spiked for that sample. The triplicate
 measurements are averaged under "Avg Intensity" and the
 PTDH-free blank is subtracted under "Adj Intensity", and the
 standard deviation is shown under "St Dev". The propagated error
 and LOD of the sample standard addition curve are also shown here.
 Variables: * Fluorescence [A.U.] - fluorescence values are reported by the
 instrument under the column and row representing the plate's well
 location. In the averaged data, the "nmol Phi added" column is
 the amount of Phi spiked in each sample, to help identify what wells
 correspond to what samples.