Illumina MiSeq analysis validated the appropriateness of leaf-based culture media as an in situ similis culturomic strategy of plant microbiota

Culturomics of plant microbiota accommodates the use of combinations of plant-based culture media together with various culture conditions to simulate the culture-independent community of plant endophytes. Plant leaves were used in different preparations as sole growth substrates as: a) intact leaf surface- inoculated method and b) leaf strips immersed in plain liquid semi-solid water agar. The leaves as such were rich enough in multiple nutrients required for the growth of tested endophytic population as well as creating specific micro-environments of varying oxygen gradients. In vitro cultivation of sunflower microbiota on homologous sunflower leaf-based culture and/or heterologous maize leaf-based culture media were investigated. Cultivation dependent, CFUs counts, and cultivation independent, PCR-DGGE and Illumina MiSeq amplicon sequencing, were applied. With MiSeq analysis and taking into consideration total OTUs detected, the microbiome profile of homo/hetero-logous cultivation indicated a distinguished clustering pattern of sunflower endorhizosphere bacteria. Two distinctive clusters were obvious at cutoff 75%. The first cluster contained a sub-cluster of the synthetic R2A culture away from the plant culture media based on intact leaf surfaces of either homologous sunflower or heterologous maize. As to the method of leaf strips, the heterologous maize leaf strips clustered rather closer to the mother culture-independent community and far distant to R2A; the homologous sunflower samples scattered among the sub-clusters. As to taxa diversity, microbiome analysis showed that OTUs affiliated to Proteobacteria were the most common and occupied ca. 66 % in mother samples. Upon in vitro, they were particularly enriched in leaf-based culture media (92 - 99%) compared to R2A (79%). Specifically, Beta- and Delta-proteobacteria were particularly exposed in leaf strips-based culture media (> 19-24% and up to 0.14%, respectively). Bacteroidetes, which represent ca. 30% of total community in mother samples, were not generally reproduced by the tested cultivation methods. An exception was the heterologous maize leaf strips that uncovered both Flavobacteriia (ca. 1.73%) and Sphingobacteriia (5.01%). While Firmicutes represent < 3% of total culture independent community, they were specially thrived in R2A (ca. 20%) different to the leaf-based culture media (< 5%). Strikingly, the satellite taxa of acidobacteria, verrucomicrobia, and chloroflexi that were below detection in mother samples, were only resolved by leaf strip-based culture strategy (> 0.01- 0.6%). It is evident that the leaf-based culture media, particularly in the form of leaf strips, furnish a milieu/theatre of complex/ diverse nutritional matrices and oxygen gradient that favor in vitro cultivation of plant microbiota, its simplicity is of practical advantage to facilitate in vitro recovery of diverse members of the plant microbiota serving SynCom applications.