DOI: 10.5061/dryad.rv15dv4g1
title: Invasibility of a North American soil ecosystem to amphibian-killing fungal pathogens

description: North American salamanders are threatened by the intercontinental spread
 of chytridiomycosis, a deadly disease caused by the fungal
 pathogen Batrachochytrium salamandrivorans (Bsal). To predict the
 potential dispersal of Bsal spores to salamander habitats, we
 evaluated the capacity of soil microbial communities to resist invasion.
 We determined the degree of habitat invasibility using soils from five
 locations throughout the Great Smoky Mountains National Park, a region
 with a high abundance of susceptible hosts. Our experimental design
 consisted of replicate soil microcosms exposed to different propagule
 pressures of the non-native pathogen, Bsal, and an introduced but endemic
 pathogen, B. dendrobatidis (Bd). To compare growth and competitive
 interactions, we used quantitative PCR, live/dead cell viability assays,
 and 16S rRNA amplicon sequencing. We found that soil microcosms with
 intact bacterial communities inhibited both Bsal and Bd growth, but
 inhibitory capacity diminished with increased propagule pressure. Bsal
 showed greater persistence than Bd. Linear discriminant analysis (LDA)
 identified the family Burkolderiaceae as increasing in relative abundance
 with the decline of both pathogens. Although our findings provide evidence
 of environmental filtering in soils, such barriers weakened in response to
 pathogen type and propagule pressure, showing that habitats vary their
 invasibility based on the properties of their local microbial communities.
description: General methods include soil collection in the field,
 experimental microcosm setup with microbially active (non-autoclaved) and
 inactive (autoclaved) soil samples, inoculation with two different
 Batrachochytrid pathogens, and incubating for two weeks while analyzing
 samples at different time points (see manuscript for detailed methods).
 Samples were processed for live/dead cell counts to obtain a proxy of
 microbial activity, and pathogen load via Quantitative PCR to determine
 pathogen response, and sequenced using 16S rRNA methods to understand
 bacterial community structure. Each of these results was then analyzed
 using computational methods to gain insight into the process of microbial
 invasion for this system.
description: # Invasibility of a North American soil ecosystem to amphibian-killing
 fungal pathogens
 [https://doi.org/10.5061/dryad.rv15dv4g1](https://doi.org/10.5061/dryad.rv15dv4g1) These datasets were from soil samples collected in Great Smoky Mountains National Park (July 2020). Soil samples were set up in experimental microcosms in the lab of Dr. Ana Longo at the University of Florida (Gainesville, USA) to determine invasibility to pathogenic microfungi. Microcosms were experimentally invaded by two different concentration levels of the amphibian-killing pathogens *Batrachochytrium dendrobatidis* and *B. salamandrivorans* and incubated across four different time intervals. Datasets resulting from this experiment include: Live/dead cell counts * Number of live cells per microcosm * Number of dead cells per microcosm Quantitative polymerase chain reaction (qPCR) results * Genomic equivalents of microfungus *Batrachochytrium dendrobatidis* for each treatment * Genomic equivalents of microfungus *Batrachochytrium salamandrivorans* for each treatment PacBio long-read 16S rRNA amplicon sequences from metabarcoding * Amplicon sequences from samples to compare bacterial community invasion response across treatments The code provided and deposited on Zenodo shows all steps of data preprocessing, analysis, and visualization. ## Description of the data and file structure The file structure for these data and subsequent code is formatted for use in an R project environment. All data files are included in this repository with .R script files deposited via Zenodo. Below is the recommended folder/file structure to run analyses. Numbered items denote folders, bulleted items represent files. We recommend creating this file structure and then downloading files from the repositories to the Inputs and Scripts folder, respectively. Files and visualizations generated when running the .R files will populate the Outputs and Figures folders. Inputs * 012921_Bd_D1_All_Live Results.csv * 012921_Bd_D14_All_Live Results.csv * 031221_Bsal_D1_All_Live.csv * 031221_Bsal_D7_All_Live.csv * 040221_Bsal_D14_All_Live.csv * Bd_D4_count_data.csv * Bd_D7_count_data.csv * Bd_D14_count_data.csv * Bsal_D1_count_data.csv * Bsal_D4_count_data.csv * Bsal_D14_count_data.csv * Sample_metadata_updated.csv * Concat_file * seqtab_final.rds * tax_final.rds Outputs Scripts * Install_packages.R * Analyses.R * Figure_2_&*_*3_R_code.R * Figure_4_R_code.R * Figure_5_R_code.R * Supplementary_Figure_S2.R * Supplementary_Figure_S3.R * Fake_data_stacked_barplots_Figure_1.R Figures Details for each dataset are included below. ### DATA & FILE OVERVIEW 1\. File List: * 012921_Bd_D1_All_Live Results.csv * 012921_Bd_D14_All_Live Results.csv * 031221_Bsal_D1_All_Live.csv * 031221_Bsal_D7_All_Live.csv * 040221_Bsal_D14_All_Live.csv * Antifungal_isolates_in_soil_samples.csv * Bd_D4_count_data.csv * Bd_D7_count_data.csv * Bd_D14_count_data.csv * Bsal_D1_count_data.csv * Bsal_D4_count_data.csv * Bsal_D14_count_data.csv * Sample_metadata_updated.csv * Concat_file * seqtab_final.rds * tax_final.rds 2\. Relationship between files: qPCR data = files '012921_Bd_D1_All_Live Results.csv', '012921_Bd_D14_All_Live Results.csv', '031221_Bsal_D1_All_Live.csv', '031221_Bsal_D7_All_Live.csv', '040221_Bsal_D14_All_Live.csv' Live/dead cell count data = files 'Bd_D4_count_data.csv', 'Bd_D7_count_data.csv', 'Bd_D14_count_data.csv', 'Bsal_D1_count_data.csv', 'Bsal_D4_count_data.csv', 'Bsal_D14_count_data.csv' 16S amplicon sequencing data = files 'Sample_metadata_updated.csv', 'Concat_file', 'seqtab_final.rds', 'tax_final.rds' BLAST results data = file 'Antifungal_isolates_in_soil_samples.csv' #### DATA-SPECIFIC INFORMATION FOR: 012921\_Bd\_D1\_All\_Live Results.csv These are the qPCR data generated to determine the amount of pathogen in soil samples for each treatment. This file corresponds to samples inoculated with *Batrachochytrium dendrobatidis*, analyzed one day after inoculation, from non-autoclaved (i.e., All_Live) treatments. 1\. Number of variables: 22 2\. Number of cases/rows: 88 3\. Variable List: * Well: alphanumeric well value on qPCR plate * Sample Name: name for a given sample ID * Target Name: target probe used in qPCR * Amp Score: amplification score * Amp Status: qPCR setting for amplification * Task: specifies whether the reading belongs to a sample (UNKNOWN) or a standard (STANDARD) * Cq: PCR cycle number at which the reaction curve intersects the threshold line * Cq Mean: average Cq value * Cq Standard Deviation: standard deviation of Cq value * Quantity: measured quantity of DNA in a sample * Quantity Mean: mean quantity of DNA * Quantity Standard Deviation: standard deviation for quantity of DNA * Intercept: intercept from plotting the standard curve * R-Squared: coefficient of correlation for standard curve * Slope: slope value for standard curve * Efficiency: ratio of target DNA calculated between the end and beginning of the PCR cycle * Auto Threshold: automatic setting for threshold value (TRUE = Yes) * Threshold: level of detection above the baseline where significant results are read * Auto Baseline: automatic setting for baseline detection of fluorescence * Baseline Start: cycle value where baseline detection started (TRUE = Yes) * Baseline End: cycle value where baseline detection ended * Omit: well omitted from analysis (FALSE = No) 4\. Missing data codes: - #### DATA-SPECIFIC INFORMATION FOR: 012921\_Bd\_D14\_All\_Live Results.csv These are the qPCR data generated to determine the amount of pathogen in soil samples for each treatment. This file corresponds to samples inoculated with *Batrachochytrium dendrobatidis*, analyzed fourteen days after inoculation, from non-autoclaved (i.e., All_Live) treatments. 1\. Number of variables: 22 2\. Number of cases/rows: 88 3\. Variable List: * Well: alphanumeric well value on qPCR plate * Sample Name: name for a given sample ID * Target Name: target probe used in qPCR * Amp Score: amplification score * Amp Status: qPCR setting for amplification * Task: specifies whether the reading belongs to a sample (UNKNOWN) or a standard (STANDARD) * Cq: PCR cycle number at which the reaction curve intersects the threshold line * Cq Mean: average Cq value * Cq Standard Deviation: standard deviation of Cq value * Quantity: measured quantity of DNA in a sample * Quantity Mean: mean quantity of DNA * Quantity Standard Deviation: standard deviation for quantity of DNA * Intercept: intercept from plotting the standard curve * R-Squared: coefficient of correlation for standard curve * Slope: slope value for standard curve * Efficiency: ratio of target DNA calculated between the end and beginning of the PCR cycle * Auto Threshold: automatic setting for threshold value (TRUE = Yes) * Threshold: level of detection above the baseline where significant results are read * Auto Baseline: automatic setting for baseline detection of fluorescence * Baseline Start: cycle value where baseline detection started (TRUE = Yes) * Baseline End: cycle value where baseline detection ended * Omit: well omitted from analysis (FALSE = No) 4\. Missing data codes: - #### DATA-SPECIFIC INFORMATION FOR: 031221\_Bsal\_D1\_All\_Live.csv These are the qPCR data generated to determine the amount of pathogen in soil samples for each treatment. This file corresponds to samples inoculated with *Batrachochytrium salamandrivorans*, analyzed one day after inoculation, from non-autoclaved (i.e., All_Live) treatments. 1\. Number of variables: 22 2\. Number of cases/rows: 88 3\. Variable List: * Well: alphanumeric well value on qPCR plate * Sample Name: name for a given sample ID * Target Name: target probe used in qPCR * Amp Score: amplification score * Amp Status: qPCR setting for amplification * Task: specifies whether the reading belongs to a sample (UNKNOWN) or a standard (STANDARD) * Cq: PCR cycle number at which the reaction curve intersects the threshold line * Cq Mean: average Cq value * Cq Standard Deviation: standard deviation of Cq value * Quantity: measured quantity of DNA in a sample * Quantity Mean: mean quantity of DNA * Quantity Standard Deviation: standard deviation for quantity of DNA * Intercept: intercept from plotting the standard curve * R-Squared: coefficient of correlation for standard curve * Slope: slope value for standard curve * Efficiency: ratio of target DNA calculated between the end and beginning of the PCR cycle * Auto Threshold: automatic setting for threshold value (TRUE = Yes) * Threshold: level of detection above the baseline where significant results are read * Auto Baseline: automatic setting for baseline detection of fluorescence * Baseline Start: cycle value where baseline detection started (TRUE = Yes) * Baseline End: cycle value where baseline detection ended * Omit: well omitted from analysis (FALSE = No) 4\. Missing data codes: - #### DATA-SPECIFIC INFORMATION FOR: 031221\_Bsal\_D7\_All\_Live.csv These are the qPCR data generated to determine the amount of pathogen in soil samples for each treatment. This file corresponds to samples inoculated with *Batrachochytrium salamandrivorans*, analyzed seven days after inoculation, from non-autoclaved (i.e., All_Live) treatments. 1\. Number of variables: 22 2\. Number of cases/rows: 88 3\. Variable List: * Well: alphanumeric well value on qPCR plate * Sample Name: name for a given sample ID * Target Name: target probe used in qPCR * Amp Score: amplification score * Amp Status: qPCR setting for amplification * Task: specifies whether the reading belongs to a sample (UNKNOWN) or a standard (STANDARD) * Cq: PCR cycle number at which the reaction curve intersects the threshold line * Cq Mean: average Cq value * Cq Standard Deviation: standard deviation of Cq value * Quantity: measured quantity of DNA in a sample * Quantity Mean: mean quantity of DNA * Quantity Standard Deviation: standard deviation for quantity of DNA * Intercept: intercept from plotting the standard curve * R-Squared: coefficient of correlation for standard curve * Slope: slope value for standard curve * Efficiency: ratio of target DNA calculated between the end and beginning of the PCR cycle * Auto Threshold: automatic setting for threshold value (TRUE = Yes) * Threshold: level of detection above the baseline where significant results are read * Auto Baseline: automatic setting for baseline detection of fluorescence * Baseline Start: cycle value where baseline detection started (TRUE = Yes) * Baseline End: cycle value where baseline detection ended * Omit: well omitted from analysis (FALSE = No) 4\. Missing data codes: - #### DATA-SPECIFIC INFORMATION FOR: 040221\_Bsal\_D14\_All\_Live.csv These are the qPCR data generated to determine the amount of pathogen in soil samples for each treatment. This file corresponds to samples inoculated with *Batrachochytrium salamandrivorans*, analyzed fourteen days after inoculation, from non-autoclaved (i.e., All_Live) treatments. 1\. Number of variables: 22 2\. Number of cases/rows: 88 3\. Variable List: * Well: alphanumeric well value on qPCR plate * Sample Name: name for a given sample ID * Target Name: target probe used in qPCR * Amp Score: amplification score * Amp Status: qPCR setting for amplification * Task: specifies whether the reading belongs to a sample (UNKNOWN) or a standard (STANDARD) * Cq: PCR cycle number at which the reaction curve intersects the threshold line * Cq Mean: average Cq value * Cq Standard Deviation: standard deviation of Cq value * Quantity: measured quantity of DNA in a sample * Quantity Mean: mean quantity of DNA * Quantity Standard Deviation: standard deviation for quantity of DNA * Intercept: intercept from plotting the standard curve * R-Squared: coefficient of correlation for standard curve * Slope: slope value for standard curve * Efficiency: ratio of target DNA calculated between the end and beginning of the PCR cycle * Auto Threshold: automatic setting for threshold value (TRUE = Yes) * Threshold: level of detection above the baseline where significant results are read * Auto Baseline: automatic setting for baseline detection of fluorescence * Baseline Start: cycle value where baseline detection started (TRUE = Yes) * Baseline End: cycle value where baseline detection ended * Omit: well omitted from analysis (FALSE = No) 4\. Missing data codes: - #### DATA-SPECIFIC INFORMATION FOR: Antifungal\_isolates\_in\_soil\_samples.csv The data are the results of running the BLAST algorithm using Geneious Prime version 2022.2.1 to identify 16S rRNA amplicon sequences from our dataset that matched the Antifungal Isolates database. 1\. Number of variables: 12 2\. Number of cases/rows: 114 3\. Variable List: * SampleID: sample name; corresponds to file name from amplicon sequencing data * Sample_Name: sample name; assigned by researchers * Day: the number of days post-incubation the sample was analyzed * Pathogen_Type: whether the sample was inoculated with pathogen *Batrachochytrium dendrobatidis* (Bd), *Batrachochytrium salamandrivorans* (Bsal), or no pathogen (NA) * Site: name of the site where the soil was collected from within the Great Smoky Mountains National Park * Soil_treatment: whether the soil was autoclaved or not before inoculation * No_99.9%_match: the number of reads that aligned at 99.9% from our dataset to the Antifungal Isolates database using BLAST * No_100%_match: the number of reads that aligned at 100% from our dataset to the Antifungal Isolates database using BLAST * Notes: notes on sequences that aligned at 100% threshold; for multiple sequences, it is noted if all sequences matches are the same or the number of matching sequences if reads of different types matched to the same sample * Names: name used in the Antifungal Isolates database to describe the matched read * Taxonomy: taxonomic notation for the matched sequence; from the taxonomy column in the Antifungal Isolates database; hierarchical notation separated by ";" starting with kingdom and going down to the most specific designation * Bd_inhibition: designation of antifungal properties from the Antifungal Isolates database (see below) 4\. Missing data codes: NA (data not available/applicable) 5\. Specialized formats or other abbreviations used: seq/seqs = sequences *from Antifungal Isolates Database ns = non-significant (bacteria had no significant antifungal properties against Bd) inhibitory = inhibition effect against Bd enhancing = enhanced Bd #### DATA-SPECIFIC INFORMATION FOR: Bd\_D4\_count\_data.csv These are the data from counting the number of live and dead cells taken from soil samples inoculated with *Batrachochytrium dendrobatidis* and analyzed four days post-inoculation. Live/dead cell counts serve as a proxy of overall microbial activity. 1\. Number of variables: 10 2\. Number of cases/rows: 144 3\. Variable List: * File_name: file name corresponding to count data * Count: the average number of cells counted * Day: days post-inoculation those samples were analyzed * Fluor_type: the type of fluorescence used in confocal microscopy (green is to visualize live cells; red for dead cells) * Lower_threshold: lower threshold used when analyzing images in ImageJ * Well_no: well number on the plate used for cell visualization * Soil_initials: initials of the soil site samples were collected from * Soil_type: expanded name corresponding to the site the samples were collected from * Sterilization_status: whether that sample had been autoclaved or not before inoculation * Inoc_type: the concentration of pathogen added to the sample 4\. Specialized formats or other abbreviations used: Abbreviations used in the Soil_initials columns are expanded in the Soil_type column #### DATA-SPECIFIC INFORMATION FOR: Bd\_D7\_count\_data.csv These are the data from counting the number of live and dead cells taken from soil samples inoculated with *Batrachochytrium dendrobatidis* and analyzed seven days post-inoculation. Live/dead cell counts serve as a proxy of overall microbial activity. 1\. Number of variables: 10 2\. Number of cases/rows: 144 3\. Variable List: * File_name: file name corresponding to count data * Count: the average number of cells counted * Day: days post-inoculation those samples were analyzed * Fluor_type: the type of fluorescence used in confocal microscopy (green is to visualize live cells; red for dead cells) * Lower_threshold: lower threshold used when analyzing images in ImageJ * Well_no: well number on the plate used for cell visualization * Soil_initials: initials of the soil site samples were collected from * Soil_type: expanded name corresponding to the site the samples were collected from * Sterilization_status: whether that sample had been autoclaved or not before inoculation * Inoc_type: the concentration of pathogen added to the sample 4\. Specialized formats or other abbreviations used: Abbreviations used in the Soil_initials columns are expanded in the Soil_type column #### DATA-SPECIFIC INFORMATION FOR: Bd\_D14\_count\_data.csv These are the data from counting the number of live and dead cells taken from soil samples inoculated with *Batrachochytrium dendrobatidis* and analyzed fourteen days post-inoculation. Live/dead cell counts serve as a proxy of overall microbial activity. 1\. Number of variables: 10 2\. Number of cases/rows: 144 3\. Variable List: * File_name: file name corresponding to count data * Count: the average number of cells counted * Day: days post-inoculation those samples were analyzed * Fluor_type: the type of fluorescence used in confocal microscopy (green is to visualize live cells; red for dead cells) * Lower_threshold: lower threshold used when analyzing images in ImageJ * Well_no: well number on the plate used for cell visualization * Soil_initials: initials of the soil site samples were collected from * Soil_type: expanded name corresponding to the site the samples were collected from * Sterilization_status: whether that sample had been autoclaved or not before inoculation * Inoc_type: the concentration of pathogen added to the sample 4\. Specialized formats or other abbreviations used: Abbreviations used in the Soil_initials columns are expanded in the Soil_type column #### DATA-SPECIFIC INFORMATION FOR: Bsal\_D1\_count\_data.csv These are the data from counting the number of live and dead cells taken from soil samples inoculated with *Batrachochytrium salamandrivorans* and analyzed one-day post-inoculation. Live/dead cell counts serve as a proxy of overall microbial activity. 1\. Number of variables: 10 2\. Number of cases/rows: 144 3\. Variable List: * File_name: file name corresponding to count data * Count: the average number of cells counted * Day: days post-inoculation those samples were analyzed * Fluor_type: the type of fluorescence used in confocal microscopy (green is to visualize live cells; red for dead cells) * Lower_threshold: lower threshold used when analyzing images in ImageJ * Well_no: well number on the plate used for cell visualization * Soil_initials: initials of the soil site samples were collected from * Soil_type: expanded name corresponding to the site the samples were collected from * Sterilization_status: whether that sample had been autoclaved or not before inoculation * Inoc_type: the concentration of pathogen added to the sample 4\. Specialized formats or other abbreviations used: Abbreviations used in the Soil_initials columns are expanded in the Soil_type column #### DATA-SPECIFIC INFORMATION FOR: Bsal\_D4\_count\_data.csv These are the data from counting the number of live and dead cells taken from soil samples inoculated with *Batrachochytrium salamandrivorans* and analyzed four days post-inoculation. Live/dead cell counts serve as a proxy of overall microbial activity. 1\. Number of variables: 10 2\. Number of cases/rows: 144 3\. Variable List: * File_name: file name corresponding to count data * Count: the average number of cells counted * Day: days post-inoculation those samples were analyzed * Fluor_type: the type of fluorescence used in confocal microscopy (green is to visualize live cells; red for dead cells) * Lower_threshold: lower threshold used when analyzing images in ImageJ * Well_no: well number on the plate used for cell visualization * Soil_initials: initials of the soil site samples were collected from * Soil_type: expanded name corresponding to the site the samples were collected from * Sterilization_status: whether that sample had been autoclaved or not before inoculation * Inoc_type: the concentration of pathogen added to the sample 4\. Specialized formats or other abbreviations used: Abbreviations used in the Soil_initials columns are expanded in the Soil_type column #### DATA-SPECIFIC INFORMATION FOR: Bsal\_D14\_count\_data.csv These are the data from counting the number of live and dead cells taken from soil samples inoculated with *Batrachochytrium salamandrivorans* and analyzed fourteen days post-inoculation. Live/dead cell counts serve as a proxy of overall microbial activity. 1\. Number of variables: 10 2\. Number of cases/rows: 144 3\. Variable List: * File_name: file name corresponding to count data * Count: the average number of cells counted * Day: days post-inoculation those samples were analyzed * Fluor_type: the type of fluorescence used in confocal microscopy (green is to visualize live cells; red for dead cells) * Lower_threshold: lower threshold used when analyzing images in ImageJ * Well_no: well number on the plate used for cell visualization * Soil_initials: initials of the soil site samples were collected from * Soil_type: expanded name corresponding to the site the samples were collected from * Sterilization_status: whether that sample had been autoclaved or not before inoculation * Inoc_type: the concentration of pathogen added to the sample 4\. Specialized formats or other abbreviations used: Abbreviations used in the Soil_initials columns are expanded in the Soil_type column #### DATA-SPECIFIC INFORMATION FOR: Sample\_metadata\_updated.csv Sample metadata for all samples; used to construct the phyloseq object with the 16S rRNA amplicon sequencing data. 1\. Number of variables: 8 2\. Number of cases/rows: 105 3\. Variable List: * SampleID: fastq file name for each sample from the sequencing run * Sample_Name: researcher assigned sample name * Day: days post-inoculation * Pathogen_type: the type of pathogen in each sample * Site: site locality where soil samples were collected in Great Smoky Mountains National Park * Soil_treatment: whether each sample was first autoclaved or non-autoclaved * Sample_or_control: whether each sample was considered a true sample or a control in the analysis * Soil_Type: soil type assigned to the location where each soil sample was collected 4\. Missing data codes: NA (not available/applicable) #### DATA-SPECIFIC INFORMATION FOR: Concat\_file The concatenated file of sequences for our mock community standard (ZymoBIOMICS). 1\. Number of variables: 2 2\. Number of cases/rows: 50 3\. Variable List: * Taxonomic ID: taxonomic designation of microbe in mock community * sequence: 16S rRNA sequence 4\. Missing data codes: NA (data not available) 5\. Specialized formats or other abbreviations used: fastq format #### DATA-SPECIFIC INFORMATION FOR: seqtab\_final.rds See Analysis.R file and the DADA2 pipeline for file generation. Contains pre-processed 16S rRNA sequences. File used to generate a phyloseq object for microbiome analysis. This is a formatted .rds file. #### DATA-SPECIFIC INFORMATION FOR: tax\_final.rds See Analysis.R file and the DADA2 pipeline for file generation. Contains taxonomic assignments for 16S rRNA sequences. File used to generate a phyloseq object for microbiome analysis. ## Sharing/Access information Links to other publicly accessible locations of the data: * GitHub link in Related Works * 16S rRNA sequences from this project can be downloaded from the NCBI SRA database under BioProject ID: PRJNA897848 ## Code/Software Run the Analyses.R file code first to have the appropriate datasets loaded in your R environment before running any other script files. Packages are loaded where appropriate in the code but to install and load all necessary packages before running analysis or visualization code see the 'Install_packages.R' script.