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

Abstract

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.

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

“…To gain insights into their unknown functions and exploit their potentials, different approaches were recently introduced to culture such not-yet-cultured bacteria [2,3]. In this respect, the plantonly-based culture media were presented as natural culture media to replace myriad formulas of synthetic culture media, and strongly recommended to increase the cultivability of the plant microbiota [4][5][6][7][8][9][10]. To alleviate the stress of disproportionate nutrients, present in common culture media, we aimed at culturing maize and sunflower microbiota on the natural nutrients present in the plant inoculum itself, compared to highly diluted plant-only-based culture media and standard R2A.…”

Plant Pellets: A Compatible Vegan Feedstock for Preparation of Plant-Based Culture Media and Production of Value-Added Biomass of Rhizobia

“…Our previous research introduced and documented the challenge of plant-only-based culture media for increasing in vitro cultivability of rhizobacteria [32][33][34][35]37]. Here, the developed plant pellets represent a novel application of plant-only-based culture media potentiating the use of plant materials for value-added biomass production of rhizobacteria on a large scale.…”

“…In view of our previous publications [32][33][34][35]37], we set up a number of preliminary experiments to assess the growth of Rhizobium leguminosarum and biomass production on the expense of increasing concentrations of dehydrated clover plant powder and boosting effects of supplementation with rich and available agro-byproducts of local molasses and glycerol in various concentrations. Then, the main experiments were designed to measure the suitability of supplemented concentrations of molasses and glycerol not only to enrich the plant material but also to formulate stable and consistent plant pellets.…”

“…In particular, our recent work demonstrated that culture media prepared from the juices or slurries of Trifolium alexandrinum, Paspalum vaginatum, and Opuntia ficus-indica support in vitro growth and biomass production of a number of model rhizobacteria [32]. In addition, innovations to facilitate the application of plant-only-based culture in the form of plant powder teabags resulted in improving in vitro cultivability of rhizobacteria and also allowed investigations on their diversity and, to a lesser extent, the biomass potential [33][34][35][36]. Taken together, utilization of plant materials for culture media preparation offers a promising and relatively inexpensive strategy towards biomass production of agro-biopreparates.…”

“…While plant materials in the forms of juices, slurries, and powders in teabags have been used for the application of plant-only-based culture media to improve in vitro cultivability of rhizobacteria [33][34][35][36], no studies have (1) directly assessed the potential of such media to produce biomass and (2) integrated the rich nutrient stores of plant materials with agro-byproducts to produce value-added biomass.…”