Analyzing the antagonistic potential of the lichen microbiome against pathogens by bridging metagenomic with culture studies

Abstract

Naturally occurring antagonists toward pathogens play an important role to avoid pathogen outbreaks in ecosystems, and they can be applied as biocontrol agents for crops. Lichens present long-living symbiotic systems continuously exposed to pathogens. To analyze the antagonistic potential in lichens, we studied the bacterial community active against model bacteria and fungi by an integrative approach combining isolate screening, omics techniques, and high resolution mass spectrometry. The highly diverse microbiome of the lung lichen [Lobaria pulmonaria (L.) Hoffm.] included an abundant antagonistic community dominated by Stenotrophomonas, Pseudomonas, and Burkholderia. While antagonists represent 24.5% of the isolates, they were identified with only 7% in the metagenome; which means that they were overrepresented in the culturable fraction. Isolates of the dominant antagonistic genus Stenotrophomonas produced spermidine as main bioactive component. Moreover, spermidine-related genes, especially for the transport, were identified in the metagenome. The majority of hits identified belonged to Alphaproteobacteria, while Stenotrophomonas-specific spermidine synthases were not present in the dataset. Evidence for plant growth promoting effects was found for lichen-associated strains of Stenotrophomonas. Linking of metagenomic and culture data was possible but showed partly contradictory results, which required a comparative assessment. However, we have shown that lichens are important reservoirs for antagonistic bacteria, which open broad possibilities for biotechnological applications.

Keywords: Stenotrophomonas; antagonistic bacteria; lichen; plant growth promotion; spermidine.

FIGURE 1

Distribution of antagonistic bacteria isolated from multiple Lobaria pulmonaria thalli. Dual-culture experiments with 388 bacterial cultures were used to identify antagonists against four different model pathogens. Escherichia coli and Staphylococcus aureus were used as models for human pathogens, Botrytis cinerea and Rhinocladoniella sp. were used as models for plant and lichen pathogens, respectively. Results are presented in a schematic illustration of antagonists, targeting a specific pathogen (indicated by relative numbers and different colors, respectively). Divided circles indicate particular combinations of pathogens, which were inhibited by an assigned percentage of Lobaria-associated isolates. Singular antagonists: Lobaria-associated isolates that inhibited only one model pathogen; Dual antagonists: isolates that inhibited two distinct model pathogens; Triple antagonists: isolates that inhibited three distinct model pathogens; Quadruple antagonists: isolates that inhibited four distinct model pathogens.

FIGURE 2

Summary of identified antagonistic bacteria in a multi-level chart (http://sourceforge.net/p/krona). The displayed taxa were shown to inhibit growth of one or more of the utilized model pathogens: B. cinerea, E. coli, S. aureus, and Rhinocladionella sp. The inner circles represent higher taxonomic ranks, while more detailed taxonomic ranks (up to genus level) are presented in the outer circles.

FIGURE 3

Co-occurrence pattern between taxa of the lichen-associated microbiome. Nodes represent taxa at family level which are colored by phylum affiliation. Node size reflects sequence abundance of each taxon. The edges represent Spearman correlations in green (positive correlation, R > 0.6) and red lines (negative correlation, R < -0.6).

FIGURE 4

Analysis of internal spermidine concentrations and lichen-associated Stenotrophomonas sp. isolates. The isolates were cultivated on solid cultivation media followed by cell disruption and quantification of internal spermidine levels (n = 21). A total of six Lobaria-associated isolates were utilized together with one plant-associated isolate (P69) in a comparative approach. Stenotrophomonas sp. 165P3RAB was shown to contain the highest internal spermidine concentration (p < 0.1) compared to all other isolates, while isolate 329P5R had the lowest concentration (p < 0.01).

FIGURE 5

Greenhouse experiment with Stenotrophomonas-primed tomato seeds and two distinctive controls (P69_Py and Control). Three Lobaria-associated isolates (165P3RAB, 329P5R, and 401P2) were utilized together with a plant-associated isolate (P69). Root (R; n = 64) and stem (S; n = 66) lengths were measured after 2 weeks plant growth under limited water supply. White (root lengths) and gray (stem lengths) plot pairs represent the treatments with specific isolates and complementary controls. Statistical analysis was employed to identify significant (p < 0.05) differences in combined root and stem lengths. Different letters were used to differentiate between statistically discriminative groups.