In situ similis culturomic strategies based on vegetable (veggie)-discs extend diversity of in vitro-cultivated microbiota of vegetables

Abstract

Background and aim

Realizing that in vitro cultivation of plant microbiota is crucial to access core resources of the microbial members of the holobiont; culturing strategies are currently advanced based on plant-based culture media. Followed was the introduction of “in situ similis” cultivation strategy depending on the use of plant intact organs, e.g. leaves/ roots that finger print plant nutritional composition and expose compartment-affiliated microbiota.

Methods

Here, we advance a practical strategy to in vitro cultivation of tomato microbiota, making use of veggie-discs of homologous tomato and heterologous vegetables (potato and taro), as well as plant broth-based culture medium. Colony forming units (CFUs) are well-developed on water agar plates with veggie-discs as such or immersed with over-lay agar technique and/or membrane filters. The culturable bacteria community (CFUs) was analyzed by DGGE, and representative pure isolates were subjected to morpho-physiological studies and 16S rRNA gene sequencing.

Results

Veggie-discs acted as compatible natural/nutritional mat developing copious/fully-grown CFUs of bacteria, including actinomycetes, and fungi. The strategy uncovered the highly divergent composition of tomato culturable community, being extended to representatives of Actinomycetota, Bacillota, Bacteroidota and Pseudomonadota. Genuinely, the strategy expanded the diversity of tomato microbiota: brought into cultivation additional 18 genera not previously reported; novel cultivation of unique isolates that showed higher similarity to previously-uncultured clones representing Pseudomonadaceae, Oxalobacteraceae and Sphingomonadaceae.

Conclusion

The presented veggie-discs cultivation offers additional tools to in vitro render the hidden compartment-affiliated microbiota (bacteria/actinomycetes/fungi) accessible for future application of synthetic community approach (SynCom) and microbiota-target interventions, towards improved vegetables nutrition, health and quality, especially under soilless cultivation.

Graphical abstract

The graphical abstract that illustrates the idea of in situ-similis cultivation, where tomato microbiota are transferred to friendly and compatible veggie-discs compared to exotic and incompatible chemically-synthetic culture media.