Plant Broth- (Not Bovine-) Based Culture Media Provide the Most Compatible Vegan Nutrition for In Vitro Culturing and In Situ Probing of Plant Microbiota

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 <i>Firmicutes</i>, <i>Alphaproteobacteria</i> and <i>Gammaproteobacteria,</i> and less frequently <i>Bacteroidetes</i> and <i>Actinobacteria</i>. Whereas bovine-based culture media (modified R2A) confined the diversity to <i>Firmicutes</i>, the plant broth-based culture media revealed a wider scope of endophytes beyond rhizobia, i.e., multiple genera such as <i>Chryseobacterium</i>, <i>Cronobacter</i>, <i>Kosakonia</i>, <i>Tsukamurella</i>, 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.