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

Keywords: microbial biomass; plant microbiome/microbiota; plant-only-based culture media; rhizobacteria culturability; wild desert barley.

Fig. 1

Nutritional profiles of tested plant refuse based on multiple chemical analyses: A, Major contents of carbohydrates, proteins, fiber, and ash; B, C/N ratio; C, contents of macro- and micro-nutrients (ppm g⁻¹ dehydrated powder); D, total and individual amino acids contents (mg g⁻¹ dehydrated powder); E, remaining measured chemical parameters of dehydrated powders prepared for tested plants (Trifolium alexandrinum, Paspalum vaginatum, and Opuntia ficus-indica) and used for the preparation of plant-only-based culture media.

Fig. 2

Growth of rhizobacteria isolates on agar plates of plant-only-based culture media prepared from plant slurry homogenates and powders as well as nutrient agar: A, Collective growth indices of tested rhizobacteria isolates, with each depicted value representing the sum of growth indices measured for all 16 isolates; B, varying growth behavior in terms of collective growth indices of all tested rhizobacteria families; C, radar presentation of all tested rhizobacteria isolates on plant-only-based culture media (CP, clover powder; HP, turfgrass powder; FP, cactus powder; CS, clover slurry; FS, cactus slurry) and nutrient agar (NA). Tested isolates: B1, B. circulans B43; B2, B. circulans 3B; B3, B. licheniformis En17/3; B4, B. macerans 21B; B5, B. Polymyxa 1E; B6, B. polymyxa 30B; B7, B. subtilis NA20; E1, E. agglomerans K4; E2, E. agglomerans K3; K1, Klebsiella sp. 31Sh; P1, Pantoea sp. En5/1; A1, Azotobacter chroococcum B4; Ps1, P. aeruginosa Arbo17; Ps2, P. cepacia 34Sh; Ps3, P. fluorescens Arbo4; Ps4, Pseudomonas sp. B6.

Fig. 3

A, Growth and cell biomass production of Klebsiella oxytoca, Enterobacter agglomerans, and Azospirillum brasilense in liquid batch cultures prepared from plant only-based culture media based on slurries and/or powders of clover, cactus, and turfgrass compared to the artificial CCM culture medium; B, calculated doubling times in min; each figure represents 2 points at the log phase for 2 replicates of batch cultures (n=4); significant differences (P<0.05) are indicated by different letters. (CP, clover powder; HP, turfgrass powder; FP, cactus powder; CS, clover slurry; FS, cactus slurry)

Fig. 4

PCR-DGGE cluster analyses (based on Euclidean distance) of rhizobacteria communities associated with barley roots and CFU harvest of different agar culture media plates: A, UPGMA cluster analysis; B, principal coordinates analysis generated from unweighted DGGE banding data; C, Deviations of different culture media calculated for diversity indices as percentages from the total deviation from barley roots. NA, nutrient agar; CCM, N-deficient combined carbon-source medium; SE, soil extract agar; CS, clover slurry; FP, cactus powder; CP, clover powder; HP, turfgrass powder; MC, mother culture (surface-sterilized barley roots).