Seeds offer an internal microbial niche, termed the endosphere, colonized by communities of endophytic bacteria. To elucidate the functions of seed endophytes during germination and early plant growth, studies with culturable isolates are essential. Conventional growth media favor few fast-growing taxa, while micro organisms with restricted nutrient requirements are usually outcompeted prior to isolation. Consequently, current knowledge of the interaction between seeds and their endophytes remains limited to only few bacterial taxa, despite a "black box" of unculturable isolates colonizing the endosphere. Here, we designed various solid media to mimic the endosphere of germinating soybean (Glycine max L.) seeds and assessed their effect on the diversity of culturable endophytic bacteria. The embryonic axis (i.e., the future plant) possessed higher richness and harbored more unique genera (i.e., Brevundimonas, Methylobacterium, Microbacterium, Pseudoclavibacter, and Rathayibacter) than cotyledons (i.e., seed storage organs). Overall, media containing germinating and ground seeds enabled culturing and isolation of the broadest diversity of endophytic bacteria, viewed through the molecular identification of 246 isolates. The use of multiple tailored media helped uncover trophic adaptation of the core taxa. Furthermore, comparison of seeds from four lots of distinct cultivars and origin revealed few overlapping taxa, indicating that the parental environment, including soil and fertilization regime, influenced seed endophytic diversity. Extended diversity of native seed endophytic bacteria revealed the functional relevance of unique Arthrobacter, Bacillus, and Curtobacterium strains to seed germination under salt stress, exemplifying the importance of enhanced culturing approaches to elucidate the role of microbiota in seed germination. IMPORTANCE Plant growth-promoting endophytic isolates that appear to advance seed germination are often obtained from plant niches other than the seed endosphere. Isolating pure cultures of native endophytes from seeds during germination is crucial to investigate their function during early plant growth. Here, the diversity of endophytic bacteria isolated from seeds during soybean germination was enhanced by combining media tailored to the nutritional composition of the seed endosphere, including pregerminated seeds themselves. Our results show that isolation from distinct soybean seed compartments affected such diversity, with the embryonic axis harboring more unique taxa while displaying higher endophytic richness. Furthermore, using pools of seeds from separate lots, each corresponding to a certain cultivar and field site, supported isolation of further unique strains that often unveiled substantial effects on germination performance. Such findings are relevant to assist studies on the interactions between seeds and their native endophytic bacteria.
Keywords: 16S rRNA; biodiversity; cotyledons; embryonic axis; endophytes; germination; gyrB; holobionts; microbiota; parental environment; soybean; trophic niche.