An endohyphal bacterium impacts growth and metabolism of carbohydrates associated with storage and hemicellulose degradation of its fungal host

In nature, complex organismic communities have evolved for optimal colonization of habitats. Interkingdom interactions between fungi and bacteria can be mutualistic, but also parasitic. Ongoing research reveals an increasing number of fungi inhabited by bacteria, which results in diverse phenotypic alterations in fungi. Therefore, we were interested, how widespread the presence of endofungal bacteria is in strains of the genus Trichoderma, which fulfil a variety of ecological functions – from beneficial plant interaction and mycoparasitism on pathogenic fungi to degradation of cellulosic litter.

We found evidence for the presence of endohyphal bacteria of different species in the majority of Trichoderma strains tested. Interestingly, we did not detect a preference of specific bacterial species for a fungal species or vice versa. In the saprophyte Trichoderma reesei, we detected endohyphal bacteria by confocal microscopy and specific staining. We could confirm the presence of a Methylobacterium species in the hyphae by sequencing of 16S rRNA. After curing T. reesei QM6a from the bacteria using antibiotics, re-sequencing of the 16S rRNA and whole genome sequencing of the isolated bacterium confirmed its identity. Again, the association with Methylobacterium turned out to be strain specific with strains from different tropic habitats than species specific. Isolation of the bacterium from T. reesei QM6a showed that it is not obligate biotroph and both the bacterium and the fungus are viable individually.

In order to evaluate the interrelationship of Methylobacterium and T. reesei, we applied phenotype microarrays to assess metabolic contributions of the bacterium and performed functional assays. Antagonism against pathogenic fungi on plates was not perturbed in the absence of Methylobacterium from T. reesei on no general growth defect was obvious. However, BIOLOG analysis clearly showed a light dependent alteration of growth in the cured strain especially on xylitol, an intermediate of hemicellulose degradation and D-mannitol, a carbohydrate with important roles in stress response and carbon storage.

Accordingly, comparative transcriptome analysis between wild-type and cured fungal strains indicates an influence of the endohyphal Methylobacterium of T. reesei QM6a on diverse metabolic pathways, with different patterns upon growth in light or in darkness. The hypothesis, that the endohyphal bacterium of T. reesei QM6a supports the metabolic adaptation of the fungus to growth in light is corroborated by sequencing the genome of Methylobacterium, which comprises multiple genes with light-response associated protein domains.

In summary, we discovered an intriguing new physiological aspect of T. reesei, which opens up a new field of research with high potential for gaining an in depth understanding of interkingdom interaction of fungi with their prokaryotic inhabitants. On a broader scale, our findings highlight the abundance and important interaction of soil fungi with their endohyphal bacteria for ecosystem functions like carbon degradation, which is currently hardly considered in microbiome research and warrants further studies into the role of such interkingdom interactions in microbial communities.