How do Less-expensive Nitrogen Alternatives Affect Legume Sanctions on Rhizobia?
Data files
Mar 05, 2024 version files 219.32 KB
Abstract
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.
README: 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 (< half)
4 = most or all flowers open (>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.
Methods
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.