BACKGROUND : Soil microorganisms are key determinants of soil fertility and plant health. Soil phytopathogenic fungi
are one of the most important causes of crop losses worldwide. Microbial biocontrol agents have been extensively
studied as alternatives for controlling phytopathogenic soil microorganisms, but molecular interactions between
them have mainly been characterised in dual cultures, without taking into account the soil microbial community.
We used an RNA sequencing approach to elucidate the molecular interplay of a soil microbial community in
response to a plant pathogen and its biocontrol agent, in order to examine the molecular patterns activated by the
RESULTS : A simplified soil microcosm containing 11 soil microorganisms was incubated with a plant root pathogen
(Armillaria mellea) and its biocontrol agent (Trichoderma atroviride) for 24 h under controlled conditions. More than
46 million paired-end reads were obtained for each replicate and 28,309 differentially expressed genes were
identified in total. Pathway analysis revealed complex adaptations of soil microorganisms to the harsh conditions of
the soil matrix and to reciprocal microbial competition/cooperation relationships. Both the phytopathogen and its
biocontrol agent were specifically recognised by the simplified soil microcosm: defence reaction mechanisms and
neutral adaptation processes were activated in response to competitive (T. atroviride) or non-competitive (A. mellea)
microorganisms, respectively. Moreover, activation of resistance mechanisms dominated in the simplified soil
microcosm in the presence of both A. mellea and T. atroviride. Biocontrol processes of T. atroviride were already
activated during incubation in the simplified soil microcosm, possibly to occupy niches in a competitive ecosystem,
and they were not further enhanced by the introduction of A. mellea.
CONCLUSIONS : This work represents an additional step towards understanding molecular interactions between plant
pathogens and biocontrol agents within a soil ecosystem. Global transcriptional analysis of the simplified soil
microcosm revealed complex metabolic adaptation in the soil environment and specific responses to antagonistic
or neutral intruders.
Additional file 1: Concentration of the soil microorganism in the
simplified soil microcosm.
Additional file 2: Primer sequences of the microcosm genes analysed
by real-time RT-PCR.
Additional file 3: RNA-Seq sequencing and mapping results for each
Additional file 4: Distribution of read pair alignments to the genomes
of the soil microorganisms. (A, B) Distribution of read pair alignments to
the 13 soil microorganisms calculated with the Samtools software 
and expressed as a percentage (%) of total alignments to the microcosm
genome. (C, D) Distribution of unique read pairs mapping to genes of
the 13 soil microorganisms, counted using HTSeq  and expressed as a
percentage (%) of total unique read pairs mapping to genes in the
microcosm genome. (E, F) Percentage (%) of expressed genes (more
than one read pair) calculated as compared to the total predicted genes
for each soil microorganism. Mean and standard error values of three
replicates are reported for each condition: the simplified soil microcosm
collected at the beginning of the experiment (SSM0) and 24 h after
incubation either without exogenous fungi (SSM), with the biocontrol
agent Trichoderma atroviride (SSM+T), with the plant pathogen Armillaria
mellea (SSM+A) or with both (SSM+T+A).
Additional file 5: Expression levels of genes of the simplified soil
Additional file 6: Pearson’s correlation coefficients among replicates
and conditions for RNA-Seq analysis.
Additional file 7: Clustering and functional annotation results of
differentially expressed genes.
Additional file 8: Proportion of expressed and differentially expressed
genes for each soil microorganism.
Additional file 9: Distribution of differentially expressed genes of each soil
microorganism in 18 clusters, based on the expression profiles.
Additional file 10: Metabolic pathways of the simplified soil microcosm,
modulated by incubation in the soil matrix. Metabolic pathways
deactivated (left panels) and activated (right panels) by 24 h incubation
in the soil matrix (A) without reinforced modulation (cluster 1) and (B)
with reinforced modulation (cluster 15) in the presence of Armillaria
mellea and Trichoderma atroviride combined. Metabolic pathways
modulated by the introduction of (C) T. atroviride (cluster 3), (D) A. mellea
(cluster 5), or (E) both (cluster 7). KEGG pathways were visualised using
the iPath2 tool , the pathways of upregulated (green) and
downregulated (red) genes were highlighted, and a section of the most
relevant pathways is reported for each panel.
Additional file 11: Biological networks of Gene Ontology (GO) terms.
GO biological process terms of the simplified soil microcosm,
upregulated by incubation in the soil matrix with similar expression
profiles in the presence or absence of Armillaria mellea and Trichoderma
atroviride (cluster 1). Significantly enriched GO terms (P < 0.001) were
identified using the BiNGO tool  and visualised with Cytoscape
software . The colour scale legend indicates the level of significance
for enriched GO terms. White nodes indicate not significantly
Additional file 12: Key differentially expressed genes discussed in the
manuscript, based on their functional categories and expression profiles.
Each sheet contains the genes in each cluster discussed.