High-throughput cultivation methods have recently been developed to accelerate the recovery of microorganisms reluctant to cultivation. They simulate in situ environmental conditions for the isolation of environmental microbiota through the exchange of growth substrates during cultivation. Here, we introduce leaf-based culture media adopting the concept of the plant being the master architect of the composition of its microbial community. Pre-physical treatments of sunflower plant leaves, namely punching, freezing, and/or autoclavation, allowed the diffusion of electrolytes and other nutrients to configure the leaf surface as a natural pad, i.e., creating an "in situ similis" environment suitable for the growth of rarely isolated microbiota. We used surface inoculation and membrane-filtration methods to assess the culturability of endophytic bacteria from the sunflower phyllosphere and rhizosphere. Both methods supported excellent colony-forming unit (CFU) development when compared to standard R2A medium, with a special affinity to support better growth of epiphytic and endophytic populations of the phyllosphere compared with the rhizosphere. A 16S rRNA gene analysis of >122 representative isolates indicated the cultivation of a diverse set of microorganisms by application of the new methods. It indicated the predominance of 13 genera of >30 potential species, belonging to Firmicutes, Proteobacteria, and Actinobacteria, and especially genera not commonly reported for sunflower, e.g., Rhizobium, Aureimonas, Sphingomonas, Paracoccus, Stenotrophomonas, Pantoea, Kosakonia, and Erwinia. The strategy successfully extended diversity and richness in the endophyllosphere compared to the endorhizosphere, while CFUs grown on the standard R2A medium mainly pertain to Firmicutes, especially Bacillus spp. MALDI-TOF MS analysis clustered the isolates according to their niche and potential functions, where the majority of isolates of the endorhizosphere were clustered away from those of the endophyllosphere. Isolates identified as Gammaproteobacteria and Alphaproteobacteria were distinguishably sub-clustered, which was in contrast to the heterogeneous isolates of Firmicutes (Bacillus spp.). In conclusion, leaf in situ similis cultivation is an effective strategy to support the future application of culturomics of plant microbiota. This is an effort to access novel isolates that are more adapted and competitive in their natural environments, especially those subjected to abiotic stresses like those prevailing in arid/semi-arid zones, and, consequently, to support the application of agro-biotechnologies, among other technologies, to improving agriculture in such zones.

中文翻译：

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植物微生物群的“原位模拟”培养：一种基于植物叶片作为营养垫的新型模拟环境方法。

最近已经开发出高通量的培养方法以加速不希望培养的微生物的回收。他们模拟了通过在培养过程中交换生长底物来分离环境微生物的原位环境条件。在这里，我们介绍以植物为主要概念的叶基培养基，该植物是其微生物群落组成的主要设计师。向日葵植物叶片的物理前处理，即打孔，冷冻和/或高压灭菌，可以使电解质和其他营养物质扩散，从而将叶片表面配置为天然垫，即创造一个适合植物生长的“原位相似”环境。很少分离的微生物群的生长。我们使用了表面接种和膜过滤方法来评估向日葵叶球根和根际中内生细菌的可培养性。与标准R2A培养基相比，这两种方法均支持出色的菌落形成单位（CFU）发育，并且与根际相比，具有特殊的亲和力以支持叶球的附生和内生种群更好的生长。对> 122个代表性分离株的16S rRNA基因分析表明，通过应用新方法可以培养出多种微生物。它显示了超过30个潜在物种中的13个属的优势种，它们属于Firmicutes，Proteobacteria和Actinobacteria，尤其是向日葵中不常见的属，例如，根瘤菌，Aureimonas，Sphingomonas，副球菌，Stenotrophomonas，Pantoea，Kosakonia，和欧文尼亚。与根际内相比，该策略成功地扩展了叶内层的多样性和丰富性，而在标准R2A培养基上生长的CFU主要属于Firmicutes，尤其是芽孢杆菌。MALDI-TOF MS分析根据分离物的利基和潜在功能对其进行了聚类，其中根际内层的大多数分离物与叶内层中的分离物聚集在一起。与丙酸菌属（Bacillus spp。）的异质分离株相反，被鉴定为丙种细菌和丙种细菌的分离株被明显地亚簇化。总之，叶原位相似栽培是支持植物微生物群培养物未来应用的有效策略。