Conclusions
Interaction between soil fungi and bacteria in the plant root microbiota
(rhizomicrobiota) is becoming a central topic in understanding the
ecology of the rhizosphere and exploiting the potential of combined
manipulation of the fungal and bacterial component in order to improve
plant nutrition and growth and resistance to biotic and abiotic stress
(for examples see
This interaction can span from almost random microbial assemblies to
specific symbiotic associations of fungal hyphae and bacterial cells
Concerning Trichoderma spp., most of the studies have focused on
the antagonistic interaction toward pathogenic bacteria, while positive
effect on mutualistic bacteria have been poorly investigated. A recent
work on mycorrhizal fungi , showed the alignment of fungal/rhizobial
mutualism toward eliciting plant gene expression, but no direct
observation has been made on the modulation of rhizobial mutualism.
Evidence presented in this work indicates that Trichodermastrains (belonging to four species) have an impact on S. melilotiphenotypes, at either physiological and host plant growth promotion
levels, and alter gene expression of a large fraction of the genome.
Additionally, such effects are different for the differentTrichoderma species and for the single S. melilotistrains, clearly showing a strong component of genotype-by-genotype
interaction. In particular, we found that the various combinations ofS. meliloti strains and Trichoderma species elicit the
differential expression of a variable fraction of genes, spanning from
6% to 25% of total S. meliloti genes. The same fungal species
can determine up to 3-fold differences in the extent of the stimulon
among strains (e.g. T. velutinum from 6.6% to 25.0%), as well
as the same S. meliloti strain can vary on a similar extent its
stimulon among different fungal species treatments (e.g. 1021 from 6.0%
to 25.0%). Under a condition simulating the first step of symbiosis,
i.e. the recognition of the host plant by the perception of root
exudates, the same S. meliloti strains displayed stimulons not
higher than 19% of total genome and average values around 1%-8% in
most strains and conditions . The larger stimulons observed here, with
fungal spent media, led to speculate that several functions encoded by
the S. meliloti genome could involve interaction with members of
the soil and root microbiota rather than simply the symbiosis with
plant. A metabolic model reconstruction done on S. melilotishowed the importance of many genes residing on the chromosome and the
pSymB chromid (accounting roughly for 2/3 of the genome) for metabolic
adaptation to soil and rhizosphere, while relatively few were relevant
for symbiosis .
Within these large stimulons, ca. 23% of shared DEGs display a
differential expression which is significantly modelled by rhizobium x
fungus interaction, again supporting the hypothesis that a relatively
important fraction of the S. meliloti genome and of its variation
could be associated to the interaction with fungi in the
rhizomicrobiota. Concerning the functional groups of the overall DEGs
contributing to differences among stimulons, relevant contributions of
COG categories K (transcription) and S (unknown function) were found.
From one side this result highlights that at least some of the genes
with still unknown functions may be related to functions normally not
assessed in laboratory conditions, and involved in social interaction in
the natural environment, from another side it emphasizes the role of
transcriptional regulators (e.g. two-component systems) and gene x gene
(i.e. epistatic) interactions in social interactions. The relevance of
epistatic interaction is further confirmed by comparing the results from
the cis -hybrid strain to the two parental strains 1021 and
BL225C. Unexpectedly, these results indicate that the swapping of the
symbiotic megaplasmid pSymA affects global stimulons in conditions
(fungal spent media) apparently unrelated to symbiosis with plant.
Indeed, stimulons of the cis -hybrid strain do not overlap with
those of the two parental strains. Epistatic interactions between genes
residing on the megaplasmid pSymA and the rest of genome were already
shown, though very few in terms of number of genes involved, by genome
reduction experiments and Tn-seq analyses . On the samecis -hybrid strain we showed that stimulons from root exudates
are, as expected, displaying nonadditive (epistatic) effects . The
finding that the transcriptome of the same strain reports similar
evidence strongly suggests the presence on pSymA of genes related to
social interaction in the rhizomicrobiota.
Moreover, the reported data on the symbiotic quality of the S.
meliloti - Trichoderma combinations disclose the possibility
that the rhizomicrobiota may modulate rhizobium-plant mutualistic
interactions (synergistically or antagonistically, depending on the
genotypic combinations, again emphasizing a genotype x genotype effect).
Indeed, also results obtained in culture with spent media indicated
effects, including the genotype-species specific response of
plant-growth promoting related phenotypes of S. meliloti , which
could modulate symbiotic quality, as the production of indole-3-acetic
acid (IAA). IAA production in S. meliloti has been in fact
demonstrated to positively modulate symbiotic quality . Moreover, within
some stimulons (e.g. the cis -hybrid strain treated with T.
velutinum ) we found the presence of some nod genes, which are
required to produce the lipochito-oligosaccharide molecule (Nod Factor),
the first molecular signal directed toward the host plant by rhizobia .
Modulation, e.g. synergistic effect, by fungi on rhizobium symbiotic
quality has been observed for mycorrhizal fungi but until now not for
non-mycorrhizal fungi as Trichoderma and for the effect of fungi
on rhizobium physiology and genome-wide transcriptional patterns. Data
obtained on symbiotic tests with S. meliloti - Trichodermacombinations demonstrated that alfalfa growth performances are indeed
affected by S. meliloti - Trichoderma mix. However, a
plant variety dependency over the effect of the different combinations
was observed, which points out to the importance of the tripartite
interaction between host plant genotype and the genotypes of the
rhizomicrobiota, here oversimplified with with S. meliloti -Trichoderma combinations.
We may conclude hypothesizing that rhizobium fitness in symbiosis, and
consequently rhizobial genome evolution and genomic diversity, could be
modulated by rhizospheric microbes, which may have favoured or
antagonized the overall differentiation pathway leading to
nitrogen-fixing root nodules. Under this hypothesis we propose novel
models for studying rhizobium-plant interaction, which should include
other components of the rhizomicrobiota, in the present proof-of-concept
exemplified by Trichoderma . Such novel models should clarify the
role of genes, many of them still with unknown function, in the
tripartite social interaction between rhizobium, host plant, and
microbiota, leading to understanding the multifaceted selective
pressures over rhizobium in soil, rhizosphere and the benefits of
symbiosis.
In perspective, such models would allow to translate laboratory evidence
into the application of novel bioinoculant consortia due to high
relevance for agriculture of the nitrogen-fixation operated by rhizobia
and the biocontrol by Trichoderma , considering that for both
formulations and regulation on their use already exist , as well
application to alfalfa cultivation .