Culture Dependent and Independent Analysis of Potential Probiotic Bacterial Genera and Species Present in the Phyllosphere of Raw Eaten Produce.

The plant phyllosphere is colonized by a complex ecosystem of microorganisms. Leaves of raw eaten vegetables and herbs are habitats for bacteria important not only to the host plant, but also to human health when ingested via meals. The aim of the current study was to determine the presence of putative probiotic bacteria in the phyllosphere of raw eaten produce. Quantification of bifidobacteria showed that leaves of Lepidium sativum L., Cichorium endivia L., and Thymus vulgaris L. harbor between 103 and 106 DNA copies per gram fresh weight. Total cultivable bacteria in the phyllosphere of those three plant species ranged from 105 to 108 CFU per gram fresh weight. Specific enrichment of probiotic lactic acid bacteria from C. endivia, T. vulgaris, Trigonella foenum-graecum L., Coriandrum sativum L., and Petroselinum crispum L. led to the isolation of 155 bacterial strains, which were identified as Pediococcus pentosaceus, Enterococcus faecium, and Bacillus species, based on their intact protein pattern. A comprehensive community analysis of the L. sativum leaves by PhyloChip hybridization revealed the presence of genera Bifidobacterium, Lactobacillus, and Streptococcus. Our results demonstrate that the phyllosphere of raw eaten produce has to be considered as a substantial source of probiotic bacteria and point to the development of vegetables and herbs with added probiotic value.

Anhydrobiotic engineering for the endophyte bacterium Kosakonia radicincitans by osmoadaptation and providing exogenously hydroxyectoine.

This study presents an anhydrobiotic engineering approach aiming at conferring a high degree of desiccation tolerance to the Gram-negative endophyte Kosakonia radicincitans. In particular, pre-conditioning of bacteria under high salinities provides a remarkable positive influence on drying survival. The endophytic bacteria accumulate exogenous hydroxyectoine > 500 µmol g-1 dry weight cells exerted by osmotic stress at 4% NaCl. Microfermentation research demonstrated that hydroxyectoine provides positive effects on reducing the lag phase duration and alleviates the dissolved oxygen consumption under high salinity conditions. Beyond the amassing of hydroxyectoine, this work provides evidence supporting the notion that hydroxyectoine can produce significant changes in the endogenous bacterial metabolome during the exponential growth phase at high-osmolarity. Metabolome changes include alterations on tricarboxylic acid cycle, novo-synthesis of specific intracellular metabolites such as mannitol, myo-inositol and trehalose, and fold changes on amino acids such as L-leucine, L-asparagine, L-serine, L-methionine and L-proline. The significant fold change of L-aspartate suggests a potential acidic proteome at high-osmolarity environments, extending the knowledge of salt-stressed bacterial endophytes. Thus, these findings place the metabolic salt stress response and the hydroxyectoine accumulation by K. radicincitans into a physiological context, paving the way into the interaction between cellular phenotype associated with salt stress tolerance and drying survival capacity of Gram-negative endophytes.

Plant growth-promoting bacteria Kosakonia radicincitans mediate anti-herbivore defense in Arabidopsis thaliana.

MAIN CONCLUSION: This study demonstrates that the application of the PGPB strain, Kosakonia radicincitans enhances a plant's resistance against phloem-feeding and chewing insects in Arabidopsis thaliana. The plant growth-promoting bacterial strain K. radicincitans DSM 16656 applied to A. thaliana reduced the number of phloem-feeding insects of both the specialist Brevicoryne brassicae and the generalist Myzus persicae. While weight gain of the generalist chewing insect Spodoptera exigua was reduced by 30% on A. thaliana plants treated with K. radicincitans, growth of the specialist caterpillar Pieris brassicae was not affected when compared with caterpillars from control plants. Since generalist and specialist chewing insects responded differentially to PGPB application, the implication of signaling pathways in PGPB mediated changes in plant defense was studied using two signaling pathway mutants impaired in their salicylic acid (npr1-1 mutant) or jasmonic acid (coi1-1 mutant) pathway. We found that the jasmonic acid pathway is relevant for upregulation of aliphatic glucosinolates and suppression of the chewing generalist S. exigua larval growth. Chewing from generalist P. brassicae increased glucosinolate content in A. thaliana leaves mediated via both signaling pathways. However, only in the npr1-1 mutant, which contains the highest aliphatic glucosinolate content, the P. brassicae induced further enrichment of glucosinolates, resulting in a reduction of larval growth. Effects of K. radicincitans on plant resistance could not be explained by changes in glucosinolate contents or composition. Our results demonstrate the distinct role played by K. radicincitans in suppressing insect performance in A. thaliana.

Site-directed mutagenesis to deactivate two nitrogenase isozymes of Kosakonia radicincitans DSM16656T.

Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron-molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15N2-labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and 15N incorporation revealed that BNF is impaired in K. radicincitans DSM16656T ΔnifH and ΔanfHΔnifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.

The plant growth-promoting bacterium Kosakonia radicincitans improves fruit yield and quality of Solanum lycopersicum.

BACKGROUND: Production and the quality of tomato fruits have a strong economic relevance. Microorganisms such as the plant growth-promoting bacterium (PGPB) Kosakonia radicincitans (DSM 16656) have been demonstrated to improve shoot and root growth of young tomato plants, but data on yield increase and fruit quality by K. radicincitans are lacking. RESULTS: This study investigated how K. radicincitans affects tomato fruits. After inoculation of tomato seeds with K. radicincitans or a sodium chloride buffer control solution, stalk length, first flowering and the amount of ripened fruits produced by inoculated and non-inoculated plants were monitored over a period of 21 weeks. Inoculation of tomato seeds with K. radicincitans accelerated flowering and ripening of tomato fruits. Sugars, acidity, amino acids, volatile organic compounds and carotenoids in the fruits were also analyzed. CONCLUSION: It was found that the PGPB K. radicincitans affected the amino acid, sugar and volatile composition of ripened fruits, contributing to a more pleasant-tasting fruit without forfeiting selected quality indicators. © 2017 Society of Chemical Industry.

Plant powder teabags: a novel and practical approach to resolve culturability and diversity of rhizobacteria.

We have developed teabags packed with dehydrated plant powders, without any supplements, for preparation of plant infusions necessary to develop media for culturing rhizobacteria. These bacteria are efficiently cultivated on such plant teabag culture media, with better progressive in situ recoverability compared to standard chemically synthetic culture media. Combining various plant-based culture media and incubation conditions enabled us to resolve unique denaturing gradient gel electrophoresis (DGGE) bands that were not resolved by tested standard culture media. Based on polymerase chain reaction PCR-DGGE of 16S rDNA fingerprints and sequencing, the plant teabag culture media supported higher diversity and significant increases in the richness of endo-rhizobacteria, namely Gammaproteobacteria (Enterobacteriaceae) and predominantly Alphaproteobacteria (Rhizobiaceae). This culminated in greater retrieval of the rhizobacteria taxa associated with the plant roots. We conclude that the plant teabag culture medium by itself, without any nutritional supplements, is sufficient and efficient for recovering and mirroring the complex and diverse communities of rhizobacteria. Our message to fellow microbial ecologists is: simply dehydrate your plant canopy, teabag it and soak it to prepare your culture media, with no need for any additional supplementary nutrients.

Fluorescent Pseudomonads in the Phyllosphere of Wheat: Potential Antagonists Against Fungal Phytopathogens.

Fluorescent pseudomonads isolated from wheat leaves were characterized regarding their antagonistic potential and taxonomy in relation to protect crop plants from infestation by Fusarium and Alternaria fungi causing diseases in wheat. Using a dual culture assay, inhibition of fungal growth was found for 40 isolates of 175 fluorescent pseudomonads. Twenty-two of the antagonists were able to suppress strains of Fusarium as well as Alternaria. By means of real-time qPCR, the phlD gene encoding the antibiotic 2,4-diacetylphloroglucinol was detected in 20 isolates. On the basis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectral patterns, the isolates with antagonistic activity were assigned to the phylogenetic subgroup Pseudomonas fluorescens and the closely related Pseudomonas gessardii subgroup. The results of the study suggest that pseudomonads in the phyllosphere of crop plants may possibly contribute to natural plant protection.

Unveiling the influence of salinity on bacterial microbiome assembly of halophytes and crops.

BACKGROUND: Climate change and anthropogenic activities intensify salinity stress impacting significantly on plant productivity and biodiversity in agroecosystems. There are naturally salt-tolerant plants (halophytes) that can grow and withstand such harsh conditions. Halophytes have evolved along with their associated microbiota to adapt to hypersaline environments. Identifying shared microbial taxa between halophyte species has rarely been investigated. We performed a comprehensive meta-analysis using the published bacterial 16S rRNA gene sequence datasets to untangle the rhizosphere microbiota structure of two halophyte groups and non-halophytes. We aimed for the identification of marker taxa of plants being adapted to a high salinity using three independent approaches. RESULTS: Fifteen studies met the selection criteria for downstream analysis, consisting of 40 plants representing diverse halophyte and non-halophyte species. Microbiome structural analysis revealed distinct compositions for halophytes that face high salt concentrations in their rhizosphere compared to halophytes grown at low salt concentrations or from non-halophytes. For halophytes grown at high salt concentrations, we discovered three bacterial genera that were independently detected through the analysis of the core microbiome, key hub taxa by network analysis and random forest analysis. These genera were Thalassospira, Erythrobacter, and Marinobacter. CONCLUSIONS: Our meta-analysis revealed that salinity level is a critical factor in affecting the rhizosphere microbiome assembly of plants. Detecting marker taxa across high-halophytes may help to select Bacteria that might improve the salt tolerance of non-halophytic plants.

Impact of the PGPB Enterobacter radicincitans DSM 16656 on growth, glucosinolate profile, and immune responses of Arabidopsis thaliana.

Plant growth-promoting bacteria (PGPB) affect plant cellular processes in various ways. The endophytic bacterial strain Enterobacter radicincitans DSM 16656 has been shown to improve plant growth and yield in various agricultural and vegetable crops. Besides its ability to fix atmospheric nitrogen, produce phytohormones, and solubilize phosphate compounds, the strain is highly competitive against native endophytic organisms and colonizes the endorhizosphere in high numbers. Here, we show that E. radicincitans inoculation of the noncrop plant Arabidopsis thaliana promotes plant growth. Furthermore, high performance liquid chromatography (HPLC) analysis revealed that bacterial inoculation slightly decreased amounts of aliphatic glucosinolates in plant leaves in a fast-growing stage but increased these compounds in an older phase where growth is mostly completed. This effect seems to correlate with developmental stage and depends on the nitrogen requirement. Additionally, nitrogen deficiency studies with seedlings grown on medium containing different nitrogen concentrations suggest that plant nitrogen demand can influence the intensity of plant growth enhancement by E. radicincitans. This endophyte seems not to activate stress-inducible mitogen-activated protein kinases (MAPKs). Analyzing transcription of the defense-related genes PR1, PR2, PR5, and PDF1.2 by quantitative real time polymerase chain reaction (qPCR) revealed that E. radicincitans DSM 16656 is able to induce priming via salicylic acid (SA) or jasmonate (JA)/ethylene (ET) signaling pathways to protect plants against potential pathogen attack.

The Combined Applications of Microbial Inoculants and Organic Fertilizer Improve Plant Growth under Unfavorable Soil Conditions.

The performance of two bio-inoculants either in single or in combined applications with organic fertilizer was tested to determine their effect on plant growth and yield under normal and unfavorable field conditions such as low pH value and low content of P. Arbuscular Mycorrhiza Fungi (AMF; three species of Glomus) and the plant-growth-promoting bacterial strain Kosakonia radicincitans DSM16656 were applied to barley in a two-year field experiment with different soil pH levels and available nutrients. Grain yield; contents of P, N, K, and Mg; and soil microbial parameters were measured. Grain yield and the content of nutrients were significantly increased by the applications of mineral fertilizer, organic fertilizer, AMF, and K. radicincitans, and the combined application of organic fertilizer with AMF and with K. radicincitans over the control under normal growth conditions. Under low-pH and low-P conditions, only the combined application of the organic fertilizer with K. radicincitans and organic fertilizer with AMF could increase the grain yield and content of nutrients of barley over the control.

Genome sequence of Enterobacter radicincitans DSM16656(T), a plant growth-promoting endophyte.

Enterobacter radicincitans sp. nov. DSM16656(T) represents a new species of the genus Enterobacter which is a biological nitrogen-fixing endophytic bacterium with growth-promoting effects on a variety of crop and model plant species. The presence of genes for nitrogen fixation, phosphorous mobilization, and phytohormone production reflects this microbe's potential plant growth-promoting activity.

Inoculating plant growth-promoting bacteria and arbuscular mycorrhiza fungi modulates rhizosphere acid phosphatase and nodulation activities and enhance the productivity of soybean (Glycine max).

Soybean [Glycine max (L.) Merrill] cultivation is important for its dual role as rich source of dietary protein and soil fertility enhancer, but production is constrained by soil nutrient deficiencies. This is often resolved using chemical fertilizers that exert deleterious effects on the environment when applied in excess. This field study was conducted at Nkolbisson-Yaoundé in the agro-ecological zone V of Cameroon to assess the performance of soybean when inoculated with plant growth-promoting bacteria (PGPB) and arbuscular mycorrhiza fungi (AMF), with or without NPK fertilizer addition. Ten treatments (Control, PGPB, AMF, PGPB+AMF, PGPB+N, PGPB+PK, PGPB+N+PK, PGPB+AMF+N, PGPB+AMF+PK, and PGPB+AMF+N+PK) were established in a randomized complete block design with three replicates. Mycorrhizal colonization was only observed in AMF-inoculated soybean roots. In comparison to control, sole inoculation of PGPB and AMF increased the number of root nodules by 67.2% and 57%, respectively. Co-application of PGPB and AMF increased the number of root nodules by 68.4%, while the addition of NPK fertilizers significantly increased the number of root nodules by 66.9-68.6% compared to control. Acid phosphatase activity in soybean rhizosphere ranged from 46.1 to 85.1 mg h-1 kg-1 and differed significantly across treatments (p < 0.001). When compared to control, PGPB or AMF or their co-inoculation, and the addition of NPK fertilizers increased the acid phosphatase activity by 45.8%, 27%, 37.6%, and 26.2-37.2%, respectively. Sole inoculation of PGPB or AMF and their integration with NPK fertilizer increased soybean yield and grain contents (e.g., carbohydrate, protein, zinc, and iron) compared to the control (p < 0.001). Soil phosphorus correlated significantly (p < 0.05) with soybean grain protein (r = 0.46) and carbohydrate (r = 0.41) contents. The effective root nodules correlated significantly (p < 0.001) with acid phosphatase (r = 0.67) and soybean yield (r = 0.66). Acid phosphatase correlated significantly (p < 0.001) with soybean grain yield (r = 0.63) and carbohydrate (r = 0.61) content. Effective root nodules correlated significantly with carbohydrate (r = 0.87, p < 0.001), protein (r = 0.46, p < 0.01), zinc (r = 0.59, p < 0.001), and iron (r = 0.77, p < 0.01) contents in soybean grains. Overall, these findings indicate strong relationships between farm management practices, microbial activities in the rhizosphere, and soybean performance.

Clay chips and beads capture in situ barley root microbiota and facilitate in vitro long-term preservation of microbial strains.

Capturing the diverse microbiota from healthy and/or stress resilient plants for further preservation and transfer to unproductive and pathogen overloaded soils, might be a tool to restore disturbed plant-microbe interactions. Here, we introduce Aswan Pink Clay as a low-cost technology for capturing and storing the living root microbiota. Clay chips were incorporated into the growth milieu of barley plants and developed under gnotobiotic conditions, to capture and host the rhizospheric microbiota. Afterward, it was tested by both a culture-independent (16S rRNA gene metabarcoding) and -dependent approach. Both methods revealed no significant differences between roots and adjacent clay chips in regard total abundance and structure of the present microbiota. Clay shaped as beads adequately supported the long-term preservation of viable pure isolates of typical rhizospheric microbes, i.e. Bacillus circulans, Klebsiella oxytoca, Sinorhizobium meliloti, and Saccharomyces sp., up to 11 months stored at -20°C, 4°C, and ambient temperature. The used clay chips and beads have the capacity to capture the root microbiota and to long-term preserve pure isolates. Hence, the developed approach is qualified to build on it a comprehensive strategy to transfer and store complex and living environmental microbiota of rhizosphere toward biotechnological application in sustainable plant production and environmental rehabilitation.

It Takes Two to Tango: A Bacterial Biofilm Provides Protection against a Fungus-Feeding Bacterial Predator.

Fungus-bacterium interactions are widespread, encompass multiple interaction types from mutualism to parasitism, and have been frequent targets for microbial inoculant development. In this study, using in vitro systems combined with confocal laser scanning microscopy and real-time quantitative PCR, we test whether the nitrogen-fixing bacterium Kosakonia radicincitans can provide protection to the plant-beneficial fungus Serendipita indica, which inhabits the rhizosphere and colonizes plants as an endophyte, from the fungus-feeding bacterium Collimonas fungivorans. We show that K. radicincitans can protect fungal hyphae from bacterial feeding on solid agar medium, with probable mechanisms being quick hyphal colonization and biofilm formation. We furthermore find evidence for different feeding modes of K. radicincitans and C. fungivorans, namely "metabolite" and "hyphal feeding", respectively. Overall, we demonstrate, to our knowledge, the first evidence for a bacterial, biofilm-based protection of fungal hyphae against attack by a fungus-feeding, bacterial predator on solid agar medium. Besides highlighting the importance of tripartite microbial interactions, we discuss implications of our results for the development and application of microbial consortium-based bioprotectants and biostimulants.

The interaction between Rhizoglomus irregulare and hyphae attached phosphate solubilizing bacteria increases plant biomass of Solanum lycopersicum.

The synergistic interaction between arbuscular mycorrhizal fungi (AMF) and phosphate solubilizing bacteria (PSB) can enhance growth and phosphorous uptake in plants. Since PSBs are well known hyphal colonizers we sought to understand this physical interaction and exploit it in order to design strategies for the application of a combined microbial inoculum. Phosphate-solubilizing bacteria strongly attached to the hyphae of Rhizoglomus irregulare were isolated using a two compartment system (root and hyphal compartments), which were separated by a nylon mesh through which AMF hyphae could pass but not plant roots. Allium ampeloprasum (Leek) was used as the host plant inoculated with R. irregulare. A total of 128 bacteria were isolated, of which 12 showed stable phosphate solubilizing activity. Finally, three bacteria belonging to the genus Pseudomonas showed the potential for inorganic and organic phosphate mobilization along with other plant growth promoting traits. These PSBs were further evaluated for their functional characteristics and their interaction with AMF. The impact of single or co-inoculations of the selected bacteria and AMF on Solanum lycopersicum was tested and we found that plants inoculated with the combination of fungus and bacteria had significantly higher plant biomass compared to single inoculations, indicating synergistic activities of the bacterial-fungal consortium.

First Report of Kosakonia radicincitans Bacteraemia from Europe (Austria) - Identification and Whole-Genome Sequencing of Strain DSM 107547.

Kosakonia radicincitans is a species within the new genus Kosakonia. Many strains of this genus have been isolated from plants, but some strains are assumed to act as facultative human pathogens. In this study, an in-depth analysis of a Kosakonia isolate from human blood was performed. The strain was originally isolated from blood and identified as a member of the Enterobacter cloacae complex, exhibiting an atypical result in susceptibility testing. Therefore, the genetic background was examined, including phylogenetic classification and screening for virulence factors. Using whole-genome sequencing, the isolate was identified as a K. radicincitans strain, revealing a virulence gene cluster for yersiniabactin biosynthesis in contrast to all other strains of the species. Whole-genome sequencing was the perfect method for identifying putative virulence factors of a particular Kosakonia strain and will help distinguish beneficial strains from pathogenic strains in the future. To our knowledge, this is the first report of Kosakonia-related bacteraemia from Europe.

Selected Rhizosphere Bacteria Help Tomato Plants Cope with Combined Phosphorus and Salt Stresses.

Plants are often challenged by multiple abiotic stresses simultaneously. The inoculation of beneficial bacteria is known to enhance plant growth under these stresses, such as phosphorus starvation or salt stress. Here, for the first time, we assessed the efficiency of selected beneficial bacterial strains in improving tomato plant growth to better cope with double stresses in salty and P-deficient soil conditions. Six strains of Arthrobacter and Bacillus with different reservoirs of plant growth-promoting traits were tested in vitro for their abilities to tolerate 2-16% (w/v) NaCl concentrations, and shown to retain their motility and phosphate-solubilizing capacity under salt stress conditions. Whether these selected bacteria promote tomato plant growth under combined P and salt stresses was investigated in greenhouse experiments. Bacterial isolates from Cameroonian soils mobilized P from different phosphate sources in shaking culture under both non-saline and saline conditions. They also enhanced plant growth in P-deficient and salt-affected soils by 47-115%, and their PGP effect was even increased in higher salt stress conditions. The results provide valuable information for prospective production of effective bio-fertilizers based on the combined application of local rock phosphate and halotolerant phosphate-solubilizing bacteria. This constitutes a promising strategy to improve plant growth in P-deficient and salt-affected soils.

The Response of Maize to Inoculation with Arthrobacter sp. and Bacillus sp. in Phosphorus-Deficient, Salinity-Affected Soil.

Salinity and phosphorus (P) deficiency are among the most serious soil factors constraining crop productivity. A proposed strategy for alleviating these stresses is supporting plants by inoculation with growth-promoting rhizobacteria (PGPR). Here, a comparison of the ability of two maize composite and two F1 hybrid varieties to tolerate a P deficiency in either a saline or a non-saline environment showed that the uptake of nutrients by all four entries was significantly reduced by the imposition of both soil salinity and P deficiency, and that their growth was compromised to a similar extent. Subsequently, the ameliorative effect of inoculation with three strains of either Arthrobacter sp. or Bacillus sp. in an environment, which suffered simultaneously from salinity and P deficiency, was investigated. Inoculation with each of the strains was found to limit the plants' uptake of sodium cations, to increase their uptake of potassium cations, and to enhance their growth. The extent of the growth stimulation was more pronounced for the composite varieties than for the F1 hybrid ones, although the amount of biomass accumulated by the latter, whether the plants had been inoculated or not, was greater than that of the former varieties. When the bacterial strains were cultured in vitro, each of them was shown as able to produce the phytohormones auxin, abscisic acid, gibberellins, and cytokinins. The implication is that since the presence in the rhizospere of both Arthrobacter sp. and Bacillus sp. strains can support the growth of maize in salinity-affected and P deficient soils in a genotype-dependent fashion, it is important to not only optimize the PGPR strain used for inoculation, but also to select maize varieties which can benefit most strongly from an association with these bacteria.

Formulating bacterial endophyte: Pre-conditioning of cells and the encapsulation in amidated pectin beads.

Despite the benefits of bacterial endophytes, recent studies on the mostly Gram-negative bacteria lack of regard for formulation strategies. The encapsulation into biopolymeric materials such as amidated pectins hydrogels is a suitable alternative. Here, this research aimed at supporting the capability of the plant growth-promoting bacteria Kosakonia radicincitans DSM16656T to endophytically colonize plant seedlings. In this approach, the pre-conditioned cells through osmoadaptation and hydroxyectoine accumulation were used. In general, pre-osmoadapted and hydroxyectoine-supplemented bacteria cells formulated in amidated pectin dried beads increased the endophytic activity by 10-fold. Moreover, plant promotion in radish plants enhanced by 18.9% and 20.7% for a dry matter of tuber and leaves. Confocal microscopy studies with GFP-tagged bacteria revealed that bacterial aggregates formed during the activation of beads play an essential role in early colonization stages. This research encourages the integration of fermentation and formulation strategies in a bioprocess engineering approach for exploiting endophytic bacteria.

"In situ similis" Culturing of Plant Microbiota: A Novel Simulated Environmental Method Based on Plant Leaf Blades as Nutritional Pads.

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.