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-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.