Plant-Based Culture Media: Efficiently Support Culturing Rhizobacteria and Correctly Mirror Their In-Situ Diversity
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Journal of Advanced Research (2016) 7, 305–316 Cairo University Journal of Advanced Research ORIGINAL ARTICLE Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity Hanan H. Youssef a, Mervat A. Hamza a, Mohamed Fayez a, Elhussein F. Mourad a, Mohamed Y. Saleh a, Mohamed S. Sarhan a, Ragab M. Suker a, Asmaa A. Eltahlawy a, Rahma A. Nemr a, Mahmod El-Tahan b, Silke Ruppel c, Nabil A. Hegazi a,* a Environmenal Studies and Research Unit (ESRU), Department of Microbiology, Faculty of Agriculture, Cairo University, Giza, Egypt b Regional Center of Food and Feed (RCFF), Agricultural Research Centre, Giza, Egypt c Leibniz Institute of Vegetable and Ornamental Crops (IGZ), Grossbeeren, Germany ARTICLE INFO ABSTRACT Article history: Our previous publications and the data presented here provide evidences on the ability of plant- Received 8 May 2015 based culture media to optimize the cultivability of rhizobacteria and to support their recovery Received in revised form 25 July 2015 from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. Accepted 28 July 2015 nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, Available online 19 September 2015 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 Keywords: Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth Plant-based culture media on agar plates of such plant-based culture media. Cell growth and biomass production in liquid Rhizobacteria batch cultures were comparable to those reported with the synthetic culture media. In addition, Cultivability the tested plant-based culture media efficiently recovered populations of rhizobacteria associ- À Community structure of rhizobacteria ated to plant roots. Culturable populations of >106–108 cfu g 1 were recovered from the Cactus ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture- Succulent plants 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 dis- tinct 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, * Corresponding author. E-mail address: [email protected] (N.A. Hegazi). Peer review under responsibility of Cairo University. Production and hosting by Elsevier http://dx.doi.org/10.1016/j.jare.2015.07.005 2090-1232 Ó 2015 Production and hosting by Elsevier B.V. on behalf of Cairo University. 306 H.H. Youssef et al. 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. Ó 2015 Production and hosting by Elsevier B.V. on behalf of Cairo University. Introduction to morphophysiological identification, to compare the effect of culture media tested on their community structure. Prokaryotic taxonomy is nowadays based on genome data, which allows classification of non-culturable bacteria, and Material and methods greatly contributes to our understanding of the microbial diversity in the plant-soil system [1,2]. However, culturable Tested plants methods are far from redundant but required, and present a challenge to environmental microbiology specialists [3]. They The tested plants, the cactus Opuntia ficus-indica (prickly provide information about communities that cannot be pears) and the succulent plants Aloe vera and Aloe arborescens, obtained directly from sequencing efforts and/or culture- are cultivated in Orman Botanical Garden, Giza- Egypt, as independent methods alone [4–6], and remain important for ornamental plants for display and research. Such plants were the physiological and genetical characterization of specific bac- chosen for their availability in arid and semi-arid environments terial species containing functionally important traits. In addi- as well as their copious juicy nature. tion, the isolation of individual bacterial species in pure Samples of the full-grown plants were obtained by first cultures allows full assessment of environmental impacts and insertion and separation of the vegetative part of plant into further manipulation for the benefit of natural ecosystems. plastic bags. Then, the root system (intact roots with closely Developments over the last decade have led to the recovery adhering soil) was carefully removed and transferred to plastic of unculturables from various populated habitats, e.g. the bags. Free soil samples were secured from the soil nearby the use of dilute nutrient media, long-term incubation, encapsulat- roots. Samples were kept in the refrigerator until analyses, ing individual cells into gel microdroplets (GMD), diffusion which were conducted within few days of sampling. chambers, and the soil substrate membrane system [6–8]. Through rhizodeposition, small-molecular weight sec- Preparation of plant-based culture media ondary metabolites, amino acids, secreted enzymes, mucilage, and cell lysates, plants may induce selective pressure on the The succulent leaves of A. vera and A. arborescens and mature microbial composition in the root region [9–12]. In an effort stem pads of O. ficus-indica were washed, sliced, and then to better reproduce the natural environment, plant-based cul- blended with equal aliquots of distilled water (w/v) for ca. ture media have been introduced for the culturing of rhizobac- 5 min in a Waring blender. The resulting slurry homogenate teria as a sole growth milieu [13]. From a theoretical point of was used as such or coarse-filtered through cheesecloth to view, plant juices and/or extracts are well suited as culture obtain plant juice; ca. 73–82% of the plant fresh weight was media for microbial growth and fermentations, as they contain recovered as juice. To obtain plant saps, the succulent leaves all the necessary nutrients as well as growth factors such as of Aloe vera and Aloe arborescens were washed, sliced and amino acids, vitamins and minerals. Representatives of patho- manually pressed by a squeezer; the sap recovered represented genic fungi and human pathogens were successfully grown on ca. 24–26% of the plant fresh weight. The pH for saps, juices the extracts/juices of a variety of plants as well as legume and slurry homogenates was in the range of 3.6–5.2. All plant seeds-protein [14,15]. Recovery and isolation of fungal endo- substrates were stored at À20 °C. In addition, a dehydrated phytes of Hordeum murinum were significantly improved by powder was prepared from cactus (O. ficus-indica): stem pads supplementing commercial culture media with the whole host were sliced and sun-dried (>30 °C) for 3–4 days, then further plant extract [16]. Furthermore, microbial metabolites were oven-dried in a hot air (70 °C) for 48 h and mechanically- productively recovered from culture media based on plant sub- grinded to pass through a 2-mm sieve. strates, especially the by-products of agro-industries [17–20]. The plant homogenates, juices and saps obtained from the Green biorefinery of brown and green juices produces varieties tested plants were further diluted with distilled water (v/v); of organic acids, amino acids, feed additives, enzymes, pro- 1:10, 1:20, 1:40, 1:80, and 1:100. Exclusively, such diluted teins, peptides or fungal and bacterial biomass [21]. juices and saps were used as such to prepare the plant-based Data presented here provide further support for our origi- agar culture media (2% agar, w/v). For the dehydrated powder nal approach of the sole use of plant-based culture media to of cactus the liquid and agar (2% agar) culture media were replace the chemically-synthetic standard ones, traditionally prepared by dissolving 4 g in 1 L of distilled water. All media used for culturing of rhizobacteria [13]. A number of cacti were adjusted to pH 7.0 and autoclaved at 121 °C for 20 min. (Opuntia. ficus-indica; prickly pears) and succulent (Aloe vera and Aloe arborescens) plants were used to obtain the nutrient Chemically-synthetic standard culture media juices, saps, slurry homogenates and powders for the prepara- tion of plant-based culture media. Such media were tested for culturing rhizobacteria present in pure cultures (in vitro) and The rich nutrient agar [22] and N-deficient combined carbon- for recovering the rhizobacteria population associated with sources medium (CCM) [23] were used. À1 roots of homologous and heterologous host plants. Secured Nutrient agar [22]: It contains (g l ): beef extract, 3.0; pep- isolates of culturable endophytic rhizobacteria were subjected tone, 5.0; glucose, 1.0; yeast extract, 0.5; agar, 15; pH, 7.2. Plant-based culture media for culturing rhizobacteria 307 N-deficient combined carbon sources medium, CCM [23]: lawn); and 4–5, very good (continued and more dense bacterial It comprises of (g lÀ1): glucose, 2.0; malic acid, 2.0; mannitol, lawn). 2.0; sucrose, 1.0; K2HPO4, 0.4; KH2PO4, 0.6; MgSO4, 0.2; Growth and biomass production of rhizobacteria isolates in NaCl, 0.1; MnSO4, 0.01; yeast extract, 0.2; fermentol (a local liquid batch cultures product of corn-steep liquor), 0.2; KOH, 1.5; CaCl2, 0.02; FeCl3, 0.015; Na2 MoO4, 0.002. In addition, CuSO4, 0.08 mg; ZnSO4, 0.25 mg; sodium lactate, 0.6 ml (50% v/v) The growth of Enterobacter agglomerans and Klebsiella oxy- were added per litre. toca was monitored in liquid plant-based culture media, using cactus powder (4 g/litre) and slurry homogenate (diluted with Growth of rhizobacteria isolates on agar plates distilled water 1:20, v/v). For comparisons, the standard liquid combined carbon sources medium (CCM) was included.