Plant microbiota support the diversity and productivity of plants. Thus, cultivation-dependent approaches are indispensable for in vitro manipulation of hub taxa. Despite recent advances in high-throughput methods, cultivability is lagging behind other environmental microbiomes, notably the human microbiome. As a plant-based culturing strategy, we developed culture media based on a broth of cooked aqueous mixtures of host plants. This improved the in vitro growth of representative isolates of plant microbiota and extended the in situ recovery of plant microbiota. With clover, 16S rRNA gene sequencing of representative isolates confirmed the predominance of Firmicutes, Alphaproteobacteria and Gammaproteobacteria, and less frequently Bacteroidetes and Actinobacteria. Whereas bovine-based culture media (modified R2A) confined the diversity to Firmicutes, the plant broth-based culture media revealed a wider scope of endophytes beyond rhizobia, i.e., multiple genera such as Chryseobacterium, Cronobacter, Kosakonia, Tsukamurella, and a potentially/presumptive novel species. Matrix-assisted laser desorption/ionization time-of-flight (MADI-TOF) analysis clustered isolates according to their plant niches, the endo-phyllosphere/endo-rhizosphere. We recommend the plant broth for simplicity, reproducibility and perdurable storage, supporting future culturomics applications, good laboratory practice (GLP) and good manufacturing practice (GMP). The strategy creates an “in-situ-similis” vegan nutritional matrix to analyze microbial diversity and reveal novel microbial resources pertinent to biotechnological and environmental applications.

Alliances of microbiota with plants are masked by the inability of in vitro cultivation of their bulk. Pure cultures piled in international centers originated from dissimilar environments/hosts. Reporting that plant root/leaf-based culture media support the organ-specific growth of microbiota, it was of interest to further investigate if a plant-based medium prepared from homologous (maize) supports specific/adapted microbiota compared to another prepared from heterologous plants (sunflower). The culture-independent community of maize phyllosphere was compared to communities cross-cultivated on plant broth-based media: CFU counts and taxa prevalence (PCR-DGGE; Illumina MiSeq amplicon sequencing). Similar to total maize phyllospheric microbiota, culture-dependent communities were overwhelmed by Proteobacteria (>94.3–98.3%); followed by Firmicutes (>1.3–3.7%), Bacteroidetes (>0.01–1.58%) and Actinobacteria (>0.06–0.34%). Differential in vitro growth on homologous versus heterologous plant-media enriched/restricted various taxa. In contrast, homologous cultivation over represented members of Proteobacteria (ca. > 98.0%), mainly Pseudomonadaceae and Moraxellaceae; heterologous cultivation and R2A enriched Firmicutes (ca. > 3.0%). The present strategy simulates/fingerprints the chemical composition of host plants to expand the culturomics of plant microbiota, advance real-time in vitro cultivation and lab-keeping of compatible plant microbiota, and identify preferential pairing of plant-microbe partners toward future synthetic community (SynComs) research and use in agriculture.