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.

"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.

An inoculum-dependent culturing strategy (IDC) for the cultivation of environmental microbiomes and the isolation of novel endophytic Actinobacteria.

The recent introduction of plant-only-based culture media enabled cultivation of not-yet-cultured bacteria that exceed 90% of the plant microbiota communities. Here, we further prove the competence and challenge of such culture media, and further introduce "the inoculum-dependent culturing strategy, IDC". The strategy depends on direct inoculating plant serial dilutions onto plain water agar plates, allowing bacteria to grow only on the expense of natural nutrients contained in the administered inoculum. Developed colonies are successively transferred/subcultured onto plant-only-based culture media, which contains natural nutrients very much alike to those found in the prepared plant inocula. Because of its simplicity, the method is recommended as a powerful tool in screening programs that require microbial isolation from a large number of diverse plants. Here, the method comfortably and successfully recovered several isolates of endophytic Actinobacteria represented by the six genera of Curtobacterium spp., Plantibacter spp., Agreia spp., Herbiconiux spp., Rhodococcus spp., and Nocardioides spp. Furthermore, two of the isolates are most likely novel species belonging to Agreia spp. and Herbiconiux spp.

Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media - A review.

Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel procedures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to unprecedented levels.

G3 PhyloChip Analysis Confirms the Promise of Plant-Based Culture Media for Unlocking the Composition and Diversity of the Maize Root Microbiome and for Recovering Unculturable Candidate Divisions/Phyla.

The rapid development of high-throughput techniques and expansion of bacterial databases have accelerated efforts to bring plant microbiomes into cultivation. We introduced plant-only-based culture media as a successful candidate to mimic the nutritional matrices of plant roots. We herein employed a G3 PhyloChip microarray to meticulously characterize the culture-dependent and -independent bacterial communities of the maize root compartments, the endo- and ecto-rhizospheres. An emphasis was placed on the preference of the growth of unculturable candidate divisions/phyla on plant-only-based culture media over standard culture media (nutrient agar). A total of 1,818 different operational taxonomic units (OTUs) were resolved representing 67 bacterial phyla. Plant-only-based culture media displayed particular affinity towards recovering endophytic over ectophytic rhizobacteria. This was shown by the slightly higher recovery of CFUs for endophytes on plant-only-based culture media (26%) than on standard culture media (10%) as well as the higher taxa richness and numbers of exclusive families of unculturable divisions/phyla. Out of 30 bacterial phyla (comprising >95% of the whole population), 13 were of a significantly higher incidence on plant-only-based culture media, 6 phyla of which were not-yet-cultured (Atribacteria, OP9; Dependentiae, TM6; Latescibacteria, WS3; Marinimicrobia, SAR406; Omnitrophica, OP3; BRC1). Furthermore, plant-only-based culture media significantly enriched less abundant and/or hard-to-culture bacterial phyla (Acidobacteria, Gemmatimonadetes, and Tenericutes). These results present conclusive evidence of the ability of plant-only-based culture media to bring the plant-fed in situ microbiome into the status of plant-fed in vitro cultures, and to widen the scope of cultivation of heretofore-unculturable bacterial divisions/phyla.

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota.

In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S', H', and D') based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

A novel plant-based-sea water culture media for in vitro cultivation and in situ recovery of the halophyte microbiome.

The plant-based-sea water culture medium is introduced to in vitro cultivation and in situ recovery of the microbiome of halophytes. The ice plant (Mesembryanthemum crystallinum) was used, in the form of juice and/or dehydrated plant powder packed in teabags, to supplement the natural sea water. The resulting culture medium enjoys the combinations of plant materials as rich source of nutrients and sea water exercising the required salt stress. As such without any supplements, the culture medium was sufficient and efficient to support very good in vitro growth of halotolerant bacteria. It was also capable to recover their in situ culturable populations in the phyllosphere, ecto-rhizosphere and endo-rhizosphere of halophytes prevailing in Lake Mariout, Egypt. When related to the total bacterial numbers measured for Suaeda pruinosa roots by quantitative-PCR, the proposed culture medium increased culturability (15.3-19.5%) compared to the conventional chemically-synthetic culture medium supplemented with (11.2%) or without (3.8%) NaCl. Based on 16S rRNA gene sequencing, representative isolates of halotolerant bacteria prevailed on such culture medium were closely related to Bacillus spp., Halomonas spp., and Kocuria spp. Seed germination tests on 25-50% sea water agar indicated positive interaction of such bacterial isolates with the germination and seedlings' growth of barley seeds.

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.

Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity.

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >10(6)-10(8) cfu g(-1) were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium.

Bio-preparates support the productivity of potato plants grown under desert farming conditions of north Sinai: Five years of field trials.

Organic agriculture as well as good agricultural practices (GAPs) intrigues the concern of both consumers and producers of agricultural commodities. Bio-preparates of various rhizospheric microorganisms (RMOs) are potential sources of biological inputs supporting plant nutrition and health. The response of open-field potatoes to the application of RMO bio-preparates, the biofertilizer "Biofertile" and the bioagent "Biocontrol", were experimented over 5 successive years under N-hunger of north Sinai desert soils. Both vegetative and tuber yields of a number of tested cultivars were significantly improved due to rhizobacterial treatments. In the majority of cases, the biofertilizer "Biofertile" did successfully supply ca. 50% of plant N requirements, as the yield of full N-fertilized plants was comparable to those received 50% N simultaneously with bio-preparates treatment. The magnitude of inoculation was cultivar-dependent; cvs. Valor and Oceania were among the most responsive ones. Bio-preparate introduction to the plant-soil system was successful via soaking of tubers and/or spraying the plant canopy. The "Biocontrol" formulation was supportive in controlling plant pathogens and significantly increased the fruit yields. The cumulative effect of both bio-preparates resulted in tuber yield increases of ca. 25% over control.