Metagenomic Approaches to Determine Soil Microbial
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Metagenomic Approaches to Determine Soil Microbial Communities Associated with Armillaria Root Disease Bradley Lalande1, Zaid Abdo1, John Hanna2, Deborah Page-Dumroese2, Marcus Warwell2, Joanne Tirocke2, Mee-Sook Kim3 Ned B. Klopfenstein2, and Jane Stewart1 1Colorado State University, Fort Collins, CO, 2Rocky Mountain Research Station, USDA Forest Service, Moscow, ID, 3Kookmin University, Seoul, South Korea Introduction Results A C Data was collected at the Priest River Experimental Forest in Species identification found that 56 trees were associated with P = 0.081 P = 0.005 (Dead vs. Healthy) P = 0.107 P = 0.003 northern Idaho within a western white pine (Pinus monticola) A. altimontana; whereas, only three trees were associated with seed provenance study. Six-hundred trees remain from the A. solidipes. A. altimontana and healthy trees were associated original 2,400 planted in 1971 (Fig. 1). The research objective is with more diverse bacterial communities, both in richness and to provide a baseline for soil fungal and bacterial communities, Shannon’s diversity, compared with A. solidipes, less healthy which are associated with two types of Armillaria species, A. trees, and dead trees; however, this difference was only solidipes (high virulence) and A. altimontana (low virulence). significant for tree health (Fig. 2 A,C). Interestingly, A. solidipes Determinations of differences in microbial communities can be and dead trees were associated with more diverse fungal B applied to develop novel management techniques to reduce communities compared to A. altimontana and less healthy or D P = 0.72 P = 0.261 damage by virulent Armillaria species. healthy trees, although this also was only significant for tree P = 0.68 P = 0.22 health (Fig. 2 B,D). Based on the 712 unique bacterial OTUs identified, more Pseudomonadaceae and Spartobacteria were associated with healthy trees, and more Acidobacteria were associated with dead trees (Fig. 3). In respect to Armillaria species, more Pseudomonadaceae and Rhizobiales were associated with A. altimontana; whereas, more Acidobacteria and Figure 2. Average observed values and Operational Taxonomic Unit (OTU) richness for Enterobacteriaceae were associated with A. solidipes (Fig. 3). bacterial communities (A) and fungal communities (B) Average Shannon’s diversity and Photo Credit: G.I. McDonald Based on the 3,383 unique fungal OTUs identified, more inverse Simpson’s values for bacterial (C) and fungal communities (D). Figure 1. A) map of western white pine planting Cortinariceae and Hypocreaceae (Trichoderma) associated and associated Armillaria spp. B) Plot location in northern Idaho. with healthy trees, but more Inocybaceae were associated with dead trees (Fig. 3). More Trichocomaceae, Cortinariaceae, and Rhizopogonaceae were found in association with A. altimontana, and more Mortierellaceae were found in association with A. solidipes (Fig. 3). 1.001.00 1.00 1.00 Materials & Methods 1.00 A B C D OTU Sampling was completed during OTU OTU Acidomicrobiaceae OTU Acidomicrobiaceae Acidomicrobiales 0.75 Tremellomycetes unclassified Acidomicrobiales 0.750.75 0.75 Tremellomycetes unclassified Acidobacteria Gp1 Atheliaceae 0.75 Acidobacteria Gp1 Atheliaceae Acidobacteria Gp16 Cortinariaceae late June. From the remaining ca. Acidobacteria Gp16 Cortinariaceae Acidobacteria Gp2 Cunninghamellaceae Acidobacteria Gp2 Cunninghamellaceae Acidobacteria Gp3 Heliotiales Acidobacteria Gp3 Heliotiales Acidobacteria Gp4 Hypocreaceae Acidobacteria Gp4 Hypocreaceae Acidobacteria Gp6 Hypocreales 600 trees, 63 trees were were Acidobacteria Gp6 Hypocreales Acidobacteria Gp7 Inocybaceae Acidobacteria Gp7 Inocybaceae 0.50 0.500.50 Actinobacteria 0.50 Mortierellaceae Actinobacteria Mortierellaceae 0.50 Bacteria unclassified Myxotrichaceae Bacteria unclassified Myxotrichaceae Betaproteobacteria Piskurozymaceae selected, based by health status Betaproteobacteria Piskurozymaceae Bradyrhiziobiaceae Pucciniomycotina Pucciniomycotina Bradyrhiziobiaceae Enterobacteriaceae Abundance Relative Rhizopogonaceae Rhizopogonaceae Enterobacteriaceae Abundance Relative Gammaproteobacteria Relative Abundance Relative Trichocomaceae Relative Abundance Relative Trichocomaceae Gammaproteobacteria Gemmatimonadaceae Armillaria Fungi unclassified Fungi unclassified and previous association. Gemmatimonadaceae 0.25 Planctomycetaceae 0.25 0.25 Leotiomycetes Leotiomycetes 0.25 Planctomycetaceae Pseudomonadaceae Acomycota unclassified Acomycota unclassified Pseudomonadaceae Rhizobiales Rhizobiales Spartobacteria Rhizomorphs, bulk density soil core, Spartobacteria 0.00 DBH, and tree health status were 0.000.00 0.00 0.00 A. altimontana A. solidipes A. altimontana A. solidipes Dead Healthy Moderate collected from each sampled tree. Dead Healthy Moderate Tree Health Armillaria Tree Health Armillaria Soil RNA and DNA were extracted, 16S Tree Health NMDS Ordination Plot ITS Tree Health NMDS Ordination Plot and tag-amplicon sequencing of the E 16S Armillaria NMDS Ordination Plot F G ITS Armillaria NMDS Ordination Plot H 0.3 0.3 0.3 rDNA ITS2 (fungal) and 16S 0.3 (bacterial) was completed. 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.1 Rhizomorph-derived cultures were 0.2 0.1 0.0 0.0 0.0 0.0 NMDS2 NMDS2 NMDS2 established. DNA was extracted and NMDS2 0.1 0.1 - 0.1 - 0.1 - the translation elongation factor-1α - 0.2 0.2 - 0.2 - 0.2 - (tef1) was amplified and sequenced - 0.3 0.3 - 0.3 - 0.3 - for species identification. Illumina - -0.4 -0.2 0.0 0.2 0.4 -0.4 -0.2 0.0 0.2 0.4 -0.4 -0.2 0.0 0.2 0.4 -0.4 -0.2 0.0 0.2 0.4 fastq files were cleaned using NMDS1 NMDS1 NMDS1 NMDS1 Trimmomatic and aligned to Silva Figure 3. A & B ) Stacked bar graph identifying most prevalent bacterial communities; Tree Health (A), Armillaria species (B). C & D) Bar graph identifying most prevalent and UNITE reference databases for fungal communities; Tree health (C), Armillaria species (D). E & F) Ordination plot for bacterial communities ; Tree health (E), Armillaria species (F). G & H) Ordination plot identification. OTU tables were for fungal communities; Tree Health (G), Armillaria species (H). referenced to microbial communities using R. Richness and Discussion diversity samples were analyzed. Potentially higher bacterial diversity is associated with healthy trees and A. altimontana; whereas, higher fungal diversity may be associated with dead trees and A. solidipes. When examining OTUs within communities, we found higher levels of Pseudomonadaceae References Kim, M.S. et al. 2015. Can Metagenetic Studies of Soil Microbial Communities Provide Insights Toward Developing Novel Management Trichoderma A. altimontana. Approaches for Armillaria Root Disease?, 2015 WIFDWC Proceedings and species associated with healthy trees and These organisms are known to be important in biocontrol Mesanza, N. 2016. Native rhizobacteria as biocontrol agents of Heterobasidion annosum s.s. and Armillaria mellea infection of Pinus radiata. Biological Control. (101). 8-16 against pathogens in disease-suppressive soils. Preliminary results suggest novel approaches could be developed for managing Ross-Davis, A.L. 2013. Forest Soil Microbial Communities: Using Metagenomic Approaches to Sample Permanent Plots. 2013 WIFDWC Proceedings Ross-Davis, A.L. 2014. Using Metagenomic Approach to Improve Our Understanding of Armillaria Root Disease. 2014 WIFDWC Armillaria root disease by fostering soil conditions to favor microbial communities that suppress Armillaria root disease. Results will be Proceedings Ross-Davis, A.L. 2015. Fine-Scale Variability of Forest Soil Fungal Communities in Two Contrasting Habitat Type Series in Northern Idaho, USA Identified with Microbial Metagenomics. 2015 WIFDWC Proceedings correlated to soil physical/chemical properties and efforts are underway to replicate these results using artificial inoculations. Tedersoo, L. Lindahl, B. 2016. Fungal Identification Biases in Microbiome Projects. Environmental Microbiology Reports (00)..