DOI: 10.5061/dryad.xwdbrv19x
title: How do Less-expensive Nitrogen Alternatives Affect Legume Sanctions on Rhizobia?

description: Mutualistic interactions involving multiple partners require ‘sanctioning’
 - the ability to influence the fitness of each partner based on its
 respective contribution. Sanctions must be sensitive to even small
 differences if even slightly less-beneficial partners could gain a fitness
 advantage by diverting resources away from the mutualistic service towards
 their own reproductive fitness. Here, we test whether legume hosts
 sanction even mediocre N2-fixing rhizobial strains by influencing either
 its nodule growth or carbon accumulation (polyhydroxybutryate or PHB) per
 rhizobia cell. We also test if sanctions depend on the availability of
 less-expensive nitrogen alternatives, either as nitrate or co-inoculation
 with a more-efficient isogenic strain. We found that nitrate eliminated
 differences in nodule size between the mediocre and more-efficient
 strains, suggesting that host sanctions were compromised. However, nitrate
 additions also decreased PHB accumulation by the mediocre strain, which
 may eliminate any fitness advantages of lower fixation by this strain.
 Co-inoculation with a more-efficient strain could also compromise host
 sanctions if reduction in fitness from smaller nodules does not offset the
 potential fitness gain from greater PHB accumulation that we observed in
 the mediocre strain. Hence, a host’s ability to sanction mediocre strains
 depends not only on alternative sources of nitrogen but also the relative
 importance of different components of rhizobial fitness.
description: Dataset was collected by two undergraduate students, first into a
 lab notebook, and then added to Microsoft Excel. That data are then
 accessed for analyses by exporting to .csv and importing into R Studio. We
 provide the main dataset .csv file and multiple .R files. 
description: # How do less-expensive nitrogen alternatives affect legume sanctions on
 rhizobia? ## README file generated on 2020-07-31 by Katherine Muller. ###
 Info on the experiment: Growing conditions: KM notebook 7 -Bean plants
 were grown in 2 magenta units filled with a 1:1 mixture of vermiculite and
 sand (see p. 70). N-free nutreint solution (Fujikake) was wicked from the
 bottom with cotton cord. For the first two weeks after germination, 0.5mM
 of KNO3 was added to the nutrient solution to help early growth before
 nodulation. Plants were grown in batches of 8 (four plants per inoculum
 strain). The first batch, started on Dec. 21st, 2016 (see p. 56-58), had
 some issues that were (hopefully) resolved during the second batch, which
 was started on 2017-02-15 (there were a false start on Jan. 30th and Feb.
 15th). THe main issue with the first batch that the PHB knockout (SAM 100)
 did not grow well in the YMB media I used to start inoculum cultures. I
 used TY for the second and third batch of plants (both strains seemed to
 grow similarly). I also measured inoculum density with dilution plates
 after I inoculated plants (I didn't wind up doing this for the
 successful batch two, which was attempt # 3). Another issue with batch 1
 was that a few plants died because the seedlings did not find the hole in
 the top of the box. I resolved this in the second batch by covering the
 top with a piece of tin foil around the hole, leaving a single light
 source for seeds to grow through (didn't work well). The second batch
 took a few tries to start. I ultimately ended up growing the inoculum in
 TY medium and letting the seedlings grow a few days before inoculating
 (all dates recorded in lab notebook). Replanted boxes were accounted for
 in sow date. Number of plants for each strain in each batch: ``` b1 b2 b3
 ``` CE3 4 4 7 SAM100 2 3 7 Dates: Seed surface sterilization: (usually
 took 1-2 days for radicles to emerge) ``` batch 1 = 2016-12-20. planted
 germinated seeds magenta boxes the next day (inoculated the same day as
 planting). batch 2 = 2017-02-15. batch 3 = 2017-10-02 (7 plants),
 2017-10-09 (9 plants--sowed new seeds). ``` inoculation: ``` batch 1:
 cultures started on 2016-12-19, plants inoculated on 2016-12-21 with 5 x
 10^7 cells (determined with OD600) diluted in 10mL sterile water
 immediately before transfer to plant boxes (the bacteria probably
 wouldn't survive long in the sterile water). batch 2: inoculated
 2017-02-20. Used OD600 only. batch 3: inoculated in (2017-10-04, using
 OD600 only) (more inoculum added on 2017-10-12 to boxes that were
 replanted on 10-09). Different planting dates were accounted for in the
 analysis. ``` ## scripts: These three scripts contain the code used to
 analyze raw H2 and CO2 data. There were 3 batches of bean plants singly
 inoculated with CE3 or or SAM100. Batch 1 was excluded due to poor
 performance of the SAM100 plants (I switched to a different culture medium
 for batches 2 and 3). ### Scripts ##### Scripts used to process raw H2 and
 CO2 data and calculate physiological measurements of N2 fixation activity
 and efficiency. - specify_directoriesBatch2.R: runs the analysis for batch
 2 data (sources other scripts) - specify_directoriesBatch3.R: runs the
 analysis for batch 3 data (sources other scripts) -
 analyzeH2assaydata_magentabox.R: main script use to convert raw H2 and CO2
 data into physiological measurements of N2 fixation activity and
 efficiency. - analyzeH2assaydata_magentaboxBatch3.R: same as previous,
 with small changes for timing adjustments in batch 3. -
 H2assayRscriptMasterFunctions.R: Code for curve-fitting functions used for
 O2 steps. ##### Scripts used to edit and combine data: -
 batch2_dataEditing.R: Combines all batch 2 data into an R workspace,
 batch2CombinedData_Edited.RData. - batch3_dataEditing.R: Combines all
 batch 3 data into an R workspace, batch3CombinedData_Edited.RData. -
 editDataAndConvertUnits20190415.R: Combines data from batches 2 and 3 and
 organizes by 'weeks after sowing', into R workspace
 editedH2assayDataForPaper20190603.RDATA. ##### Script used for statistical
 comparisons: - statsForFig1.R. Provides statistical results presented in
 the paper, plus a model and graphs showing batch effects. ### Data
 (starting in rawest form) #### Raw data output from Labview (directories)
 - H2assaydata_batch2 - H2assaydata_batch2 File names in both directories
 include the assay date, side of the chamber (left or right), and plantID
 code. For example, 20171020LC13.txt is for plantID C13 on the left-hand
 chamber assayed on 2017-10-20. See plantCodes_beans2017.csv for plantID
 codes. Each raw data file has one row per 25 second interval. Each row has
 the measurement of ppm H2 and CO2, the gas flow of O2 and N2 (column names
 were switched in the raw data and corrected in the analysis script). The
 column 500vs2000 refers to the sensitivity setting for the CO2 sensor
 (maximum CO2 below or above 500ppm). This was selected before the assay
 based on the baseline CO2 of the root system. #### Data with physiological
 measurements of N2 fixation activity and efficiency calculated from raw H2
 and CO2 data (directory) - H2assayOutput_batch2 - H2assayOutput_batch3
 Each directory contains a set of .png plots showing the analysis for each
 raw data file. The combined output is collected in batch2output.csv and
 batch3output.csv. The points used to calculate efficiency slopes are saved
 in a separate file (batch2slopes.csv, batch3slopes.csv) in case I wanted
 to remove any O2 steps (e.g., if high O2 suppressed fixation in some
 plants). I didn't end up doing that. #### Other raw plant data
 (mainly for batch 2). - plantCodes_beans2017.csv. Provides info on
 anonymized plantIDs, including inoculum strain and germination date. -
 batch2waterUse_beans2017.csv: data on water use in magenta boxes (based on
 volume needed to fill bottom box to a fill-line after a given time
 period). This wasn't measured for batch 3. -
 batch2Chlorophyll_beans2017.csv: Chlorophyll fluorescence measured with an
 atLeaf chlorophyll meter. batch2Chlorophyll_beans2017.csv This time I did
 3 measurements per leaf (chl1, chl2, chl3) instead of putting all
 chlorophyll measurements in the same column. This time I took a
 chlorophyll measurement (3 per leaf) each time I ran an efficiency assay.
 - batch2development_beans2017.csv Growth stage codes: 1 = no buds yet
 (vegetative) 2 = buds present, but no flowers open 3 = some flowers open
 (&lt; half) 4 = most or all flowers open (&gt;half) 5 = pods beginning to
 form 6 = pods elongating 7 = pods filling - batch2podMass_beans2017.txt:
 includes mass of dried pods, seed mass/plant, and the number of
 seeds/plant for each plantID in batch 2. - seedMass_beansbatch2.txt:
 includes the number and mass of dried seeds per plant for each plantID in
 batch 3.