DOI: 10.5061/dryad.73n5tb333
title: Phyllosphere microbial associations improve plant reproductive success

description: The above-ground (phyllosphere) plant microbiome is increasingly
 recognized as an important component of plant health. We hypothesized that
 phyllosphere bacterial recruitment may be disrupted in a greenhouse
 setting, and that adding a bacterial amendment would therefore benefit the
 health and growth of host plants. Using a newly developed synthetic
 phyllosphere bacterial microbiome for tomato (Solanum lycopersicum), we
 tested this hypothesis across multiple trials by manipulating microbial
 inoculation of leaves and measuring subsequent plant growth and
 reproductive success, comparing results from plants grown in both
 greenhouse and field settings. We confirmed that greenhouse-grown plants
 have a relatively depauperate phyllosphere bacterial microbiome, which
 both makes them an ideal system for testing the impact of phyllosphere
 communities on plant health and important targets for microbial amendments
 as we move towards increased agricultural sustainability. We find that the
 addition of the synthetic microbial community early in greenhouse growth
 leads to an increase in fruit production in this setting, implicating the
 phyllosphere microbiome as a key component of plant fitness and
 emphasizing the role that these bacterial microbiomes likely play in the
 ecology and evolution of plant communities.
description: For 16s Sequencing related data,
 paired-end reads were filtered and trimmed to 230(F) and 160(R) base pairs
 (bps), using DADA2 with default parameters (Callahan et al., 2016).
 Following denoising, merging reads and removing chimeras, DADA2 was used
 to infer amplicon sequence variants (ASVs), which are analogous to
 operational taxonomic units (OTUs), and taxonomy was assigned using the
 DADA2-trained SILVA database. Using DNA extraction and PCR negative
 controls from 16S sequencing, the decontam package was implemented using
 default settings to identify and remove potential contamination from the
 samples (Davis et al., 2018). The assigned ASVs, read count data, and
 sample metadata were combined in a phyloseq object (McMurdie &
 Holmes, 2013) for downstream analyses. The <em>phyloseq</em>
 package was used to calculate beta diversity (using Bray-Curtis distance),
 and a permutational analysis (PERMANOVA) was performed on data rarified to
 400 reads (Weiss et al., 2017) (to account for extraordinarily low read
 counts in untreated greenhouse samples) using the adonisfunction in the
 <em>vegan</em> package (Oksanen et al., 2022).
 Tomatoes were harvested and their number and weight were recorded
 multiple times per plant from onset of fruit production to plant
 termination in the greenhouse, at weeks 17, 18 and 19 in the second trial,
 and weeks 18 and 24 in the third trial. These metrics were measured only
 once after harvest from each individual plant grown in the field, at week
 24. Tomatoes were weighed individually in trial 2, and as total harvested
 weight per plant in trial 3, as described. Data was recorded initially in
 laboratory notebooks, then transferred to `.csv`s before being processed
 in R. 
description: All data is provided as either `.csv` files, or as `.rds` files,
 which can be opened and processed in R.
description: # Phyllosphere microbial associations improve plant reproductive success
 These data represent the processed sequencing data (mostly in the form of
 phyloseq objects) and the greenhouse and field tomato plant harvest
 results associated with the study. Data includes sequencing results, with
 both bacterial absolute and relative abundance, as well as tomato number,
 and tomato weight, either as weight per tomato or weight harvested per
 plant. ## Description of the data and file structure Data is presented as
 one of two file types, either a `.csv` or a `.rds`. Most sequencing
 related data is presented as `.rds` files, which must be loaded into R in
 order to be utilized. Further, these data are formatted as `phyloseq`
 objects, and require the package `phyloseq` in order to be processed. Some
 sequencing related data (ie. absolute,
 `Trial_1_Greenhouse_Bacterial_Abundance.csv`, and relative abundance,
 `Trial_3_Greenhouse_and_Field_Bacterial_Abundance.csv`) are `.csv` files.
 All of the greenhouse and field related data (tomato counts and weights)
 are presented as `.csv` files. Data can be cross referenced with the
 `.html` R code provided with the paper as follows, with information on
 column naming included below: *
 Trial\_1\_Greenhouse\_Bacterial\_Abundance.csv * `Treatment`: Bacterial
 treatment applied; Control -&gt; No bacteria, PhylloStart -&gt;
 PhylloStart Community applied * `Sample_ID`: Unique sample ID per plant *
 `TotalAbundance`: Total calculated abundance from qPCR of bacterial reads
 * Trial\_3\_Greenhouse\_and\_Field\_Bacterial\_Abundance.csv * `Bacteria`:
 Bacterial treatment applied; Control -&gt; No bacteria, Low -&gt;
 PhylloStart Community applied at OD600=0.0002 density, High -&gt;
 PhylloStart Community applied at OD600=0.02 density * `Sample`: Unique
 sample ID per plant * `Location`: Whether plant was sampled from field or
 greenhouse * `TotalSynComAbundance`: Relative abundance of SynCom
 associated ASVs * Trial\_2\_Greenhouse\_Fruit\_Count.csv * `ID`: Unique
 sample ID per plant * `Treatment`: Bacterial and Azomite treatment applied
 (somewhat redundant with next two columns) * `Bacteria`: Bacterial
 treatment; Control -&gt; No bacteria, Bacteria -&gt; PhylloStart
 Community applied * `Azomite`: Azomite treatment (concentration applied in
 grams) * `Total_Fruit`: Number of fruit produced per plant *
 Trial\_3\_Greenhouse\_Fruit\_Count.csv * `Plant_ID`: Unique sample ID per
 plant * `ID`: Bacterial and Azomite treatment applied * `Treatment`:
 Bacterial density applied; Control -&gt; No bacteria, Phyllostart Low
 -&gt; PhylloStart Community applied at OD600=0.0002 density,
 Phyllostart High -&gt; PhylloStart Community applied at OD600=0.02
 density * `Azomite_g`: Azomite treatment (concentration applied in grams)
 * `Total_Fruit`: Number of fruit produced per plant *
 Trial\_3\_Field\_Fruit\_Count.csv * `Plant Number`: Unique sample ID per
 plant * `Bacteria`: Bacterial density applied; No\_Bac -&gt; No
 bacteria, B\_low -&gt; PhylloStart Community applied at OD600=0.0002
 density, B -&gt; PhylloStart Community applied at OD600=0.02 density *
 `Azomite_con`: Azomite treatment (concentration applied in grams) *
 `Total_Fruit`: Number of fruit produced per plant *
 Pathogen\_Invasion\_Experiment\_CFU\_Counts.csv * `fertilizer`: Nutrient
 treatment; Low -&gt; No fertilizer applied, High -&gt; Fertilizer
 applied * `inocula`: PhylloStart/SynCom treatment; Buffer -&gt; No
 bacteria applied, Syncom -&gt; PhylloStart applied * `CFU_1`: First
 replicate CFU count * `CFU_2`: Second replicate CFU count * `CFU_avg`:
 Average of both counts * Trial\_2\_Greenhouse\_Fruit\_Weights.csv * `ID`:
 Unique sample ID per plant * `Treatment`: Bacterial and Azomite treatment
 applied (somewhat redundant with next two columns) * `Bacteria`: Bacterial
 treatment; Control -&gt; No bacteria, Bacteria -&gt; PhylloStart
 Community applied * `Azomite`: Azomite treatment (concentration applied in
 grams) * `Average_Weight`: Average weight of individual fruit produced per
 plant in grams * Trial\_3\_Greenhouse\_Fruit\_Weights.csv * `Plant_ID`:
 Unique sample ID per plant * `ID`: Bacterial and Azomite treatment applied
 * `Treatment`: Bacterial density applied; Control -&gt; No bacteria,
 Phyllostart Low -&gt; PhylloStart Community applied at OD600=0.0002
 density, Phyllostart High -&gt; PhylloStart Community applied at
 OD600=0.02 density * `Azomite_g`: Azomite treatment (concentration applied
 in grams) * `Average_Weight`: Average weight of individual fruit produced
 per plant in grams * Trial\_3\_Field\_Fruit\_Weights.csv * `Plant Number`:
 Unique sample ID per plant * `Tomatoes`: Total number of fruit produced
 per plant * `Full_ID`: All treatments concatenated * `Bacteria`: Bacterial
 density applied; No\_Bac -&gt; No bacteria, B\_low -&gt;
 PhylloStart Community applied at OD600=0.0002 density, B -&gt;
 PhylloStart Community applied at OD600=0.02 density * `Azomite`: Was
 Azomite applied or not?; Azo -&gt; Yes, Control -&gt; No *
 `Azomite_con`: Azomite treatment (concentration applied in grams) * `Row`:
 Row in the field plant was located at * `Total_Weight_kg`: Total weight of
 harvest per plant in kg * `Total_Fruit`: Number of fruit produced per
 plant * Supplemental\_Trial\_Fruit\_Weight.csv * `Plant_ID`: Unique sample
 ID per plant * `Bacteria`: Bacterial treatment, PhylloStart or Control *
 `Azomite`: Was Azomite applied or not?; Azo -&gt; Yes, Control
 -&gt; No * `Weight`: Weight in kg harvested per plant *
 Trial\_1\_AllSamples.rds (All samples from trial one, including other
 Azomite concentrations not used in main figure) * `X`: Formatting
 artifact, please ignore * `samples.out`: Sequencing ID * `Sample_Type`: Is
 this a sample or a sequencing control? All data included are for samples.
 * `Sample_ID`: Sample treatment info; G -&gt; Granular PhylloStart, GU
 -&gt; Granular + Ultrafine Azomite, U -&gt; Ultrafine Azomite, C
 -&gt; No Azomite, any samples with a B have the same as described
 above, but were inoculated with the PhylloStart Community at OD600=0.02
 density * `Project`: Location, for these data, all are from the greenhouse
 * `Treatment`: Expanded treatment information, see above *
 Trial\_1\_Greenhouse\_DataFrame\_AbsAbund.rds (Absolute abundance, used in
 figures section) * `Treatment`: Treatment, either Control or Bacteria
 (PhylloStart) * `Sample_ID`: Unique ID per Sample * `TotalAbundance`:
 Total absolute bacterial 16s rRNA abundance per sample, as calculated by
 qPCR * Trial\_1\_Greenhouse\_PhyloseqObject\_absAbund\_stats.rds (Absolute
 abundance, used in statistics section) * `X`: Formatting artifact, please
 ignore * `samples.out`: Sequencing ID * `Sample_Type`: Is this a sample or
 a sequencing control? All data included are for samples. * `Sample_ID`:
 Sample treatment info; G -&gt; Granular PhylloStart, GU -&gt;
 Granular + Ultrafine Azomite, U -&gt; Ultrafine Azomite, C -&gt;
 No Azomite, any samples with a B have the same as described above, but
 were inoculated with the PhylloStart Community at OD600=0.02 density *
 `Project`: Location, for these data, all are from the greenhouse *
 `Treatment`: Expanded treatment information, see above *
 Trial\_1\_Greenhouse\_PhyloseqObject\_relAbund.rds (Relative abundance) *
 `X`: Formatting artifact, please ignore * `samples.out`: Sequencing ID *
 `Sample_Type`: Is this a sample or a sequencing control? All data included
 are for samples. * `Sample_ID`: Sample treatment info; G -&gt;
 Granular PhylloStart, GU -&gt; Granular + Ultrafine Azomite, U
 -&gt; Ultrafine Azomite, C -&gt; No Azomite, any samples with a B
 have the same as described above, but were inoculated with the PhylloStart
 Community at OD600=0.02 density * `Project`: Location, for these data, all
 are from the greenhouse * `Treatment`: Expanded treatment information, see
 above * Trial\_3\_Greenhouse\_and\_Field\_PhyloseqObject\_ForBeta.rds (Raw
 abundances) * `X`: Formatting artifact, please ignore * `Samples`:
 Sequencing ID * `Sequence_ID`: Unique ID per plant * `Treatment`: Azomite
 treatment; Azo -&gt; Azomite applied, Control -&gt; No Azomite
 applied * `Concentration`: Amount of Azomite applied in grams *
 `Bacteria`: PhylloStart treatment, Community applied at OD600=0.0002
 (Low), or PhylloStart Community applied at OD600=0.02 density (High), or
 no PhylloStart applied (Control) * `Timepoint`: Timepoint for measurement
 (all are T1) * `Location`: Where the plant was grown, field or greenhouse
 * `PlottingCoordinates`: Arbitrary numbers assigned for internal use to
 plot the figures * Trial\_3\_Greenhouse\_and\_Field\_PhyloseqObject.rds
 (Relative abundances) * `X`: Formatting artifact, please ignore *
 `Samples`: Sequencing ID * `Sequence_ID`: Unique ID per plant *
 `Treatment`: Azomite treatment; Azo -&gt; Azomite applied, Control
 -&gt; No Azomite applied * `Concentration`: Amount of Azomite applied
 in grams * `Bacteria`: PhylloStart treatment, Community applied at
 OD600=0.0002 (Low), or PhylloStart Community applied at OD600=0.02 density
 (High), or no PhylloStart applied (Control) * `Timepoint`: Timepoint for
 measurement (all are T1) * `Location`: Where the plant was grown, field or
 greenhouse * `PlottingCoordinates`: Arbitrary numbers assigned for
 internal use to plot the figures ## Sharing/Access information Data was
 generated for the purpose of this study and is not available elsewhere. ##
 Code/Software All data was either processed through R as described in the
 methods of the paper or transferred from lab notebooks into `.xlsx` files,
 which were then converted to `.csv`s and processed in R. Data is made
 available as both `.rds` files, and, when possible, as `.csv`.