Analysis of plant growth-promoting properties of Bacillus amyloliquefaciens UCMB5113 using Arabidopsis thaliana as host plant

Abstract

This study showed that Bacillus amyloliquefaciens UCMB5113 colonizing Arabidopsis roots changed root structure and promoted growth implying the usability of this strain as a novel tool to support sustainable crop production. Root architecture plays a crucial role for plants to ensure uptake of water, minerals and nutrients and to provide anchorage in the soil. The root is a dynamic structure with plastic growth and branching depending on the continuous integration of internal and environmental factors. The rhizosphere contains a complex microbiota, where some microbes can colonize plant roots and support growth and stress tolerance. Here, we report that the rhizobacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5113 stimulated the growth of Arabidopsis thaliana Col-0 by increased lateral root outgrowth and elongation and root-hair formation, although primary root elongation was inhibited. In addition, the growth of the above ground tissues was stimulated by UCMB5113. Specific hormone reporter gene lines were tested which suggested a role for at least auxin and cytokinin signaling during rhizobacterial modulation of Arabidopsis root architecture. UCMB5113 produced cytokinins and indole-3-acetic acid, and the formation of the latter was stimulated by root exudates and tryptophan. The plant growth promotion effect by UCMB5113 did not appear to depend on jasmonic acid in contrast to the disease suppression effect in plants. UCMB5113 exudates inhibited primary root growth, while a semi-purified lipopeptide fraction did not and resulted in the overall growth promotion indicating an interplay of many different bacterial compounds that affect the root growth of the host plant. This study illustrates that beneficial microbes interact with plants in root development via classic and novel signals.

Keywords: Beneficial bacteria; Biocontrol; Growth promotion; Phytohormones; Rhizosphere; Root structure.

Fig. 1

Effects of Bacillus amyloliquefacienes UCMB5113 on A. thaliana Col-0 seedlings. Five-day-old seedlings were placed vertically on 0.2× MS agar plates with 2.5 cm distance in between plants and treated with water, 10 µl (2 × 10⁷ cfu) or 25 µl (5 × 10⁷ cfu) UCMB5113 as a streak at 3 cm distance from the primary root of the seedlings. After 6 days of co-cultivation, the growth pattern of the whole root was analyzed. Overview of the growth promotion aspect of Bacillus treated plants (a), closeup pictures, where arrows indicate increased number of lateral roots (b) and the increased frequency of root hairs on primary and later roots and initiation of root hairs close to root tip after UCMB5113 inoculation (c). Root and shoot fresh weight and image analysis of primary root length, total number of lateral roots, and total root area carried out by the rhizo software (d). Values represent means and standard deviations (n = 16), where samples labelled with identical letters are not significant at p < 0.05

Fig. 2

Effects of Bacillus UCMB5113 on plant growth of some Arabidopsis signaling mutants. Five-day-old seedlings (n = 16) were lined up on 0.2× MS agar plates and treated with water or Bacillus UCMB5113 (3 cm distance from the main root tip). Six days later digital images were taken for the overall root analysis. a Overall pictures of water control, 10 or 25 µl UCMB5113-treated plants. b Root hairs of control, 10 or 25 µl UCMB5113-treated plants. c Image analysis of control (white bars), 10 µl (grey bars) or 25 µl UCMB5113 (black bars) treated plants. Rhizo software was used to determine the primary and lateral root elongation, while root-hair number and total root area were determined with Image J. Values represent means and standard deviations (n = 16), where samples with the same letter are not significantly different at p < 0.05

Fig. 3

Effect of auxin level in Arabidopsis root by UCMB5113 and culture filtrate. Five-day-old seedlings on 0.2× MS agar plates were treated with water or Bacillus UCMB5113 and analyzed 6 days later. a Auxin analysis of indole-3-acetic acid (IAA) level in Arabidopsis roots and shoots. b Expression of auxin responsive DR5:GFP in primary roots or lateral roots. c Production of auxin by UCMB5113 monitored from day 0 to 4 in LB (diamonds) supplemented with tryptophan (squares), or root exudates (triangles) with tryptophan (circles), or tryptophan and root exudates but without UCMB5113 (X). Results shown as mean and standard deviation of three samples

Fig. 4

Root expression of the cytokinin responsive reporter Arr5:Gus. Five-day-old seedlings on 0.2× MS agar plates were treated with water or Bacillus UCMB5113 and analyzed 6 days later for expression of Arr5:Gus in primary or lateral roots

Fig. 5

Brassinosteroid analysis of UCMB5113 treated Arabidopsis. Five-day-old seedlings on 0.2× MS agar plates were treated with water or Bacillus UCMB5113 and analyzed 6 days later for brassinosteroid levels in roots and shoots (striped bars). The brassinosteroids analyzed were brassinolide (BL), castasterone (CS), teasterone (TE), typhasterol (TY), and homocastasterone (homoCS). Values represent means and standard deviations (n = 4), where samples labelled with identical letters are not significant at p < 0.05

Fig. 6

Effect of Bacillus compounds on growth and development of A. thaliana Col-0. a One-week-old Arabidopsis seedlings were lined up on 0.2× MS agar plates. Bacillus UCMB5113 sterile filtrated culture supernatant from day 2 to 6, crude lipopeptide fraction, or auxin were dropped on filter paper at 3 cm distance from the main root tip. At 6 days of post-inoculation, digital images were taken for the overall root analysis. Samples were control (c), filter paper (fp), 25 µl volume of water (w), LB (lb), culture supernatants from day 2 (d2), day 3 (d3), day 4 (d4), day 5 (d5), day 6 (d6), crude lipopeptides (LP), 10× diluted crude lipopeptides (lp) synthetic auxin applied as 800 pg (A). b Analysis of primary root length and total number of later roots deduced by use of the rhizo software. Values represent means and standard deviations (n = 16), where samples labelled with identical letters are not significant at p < 0.05

Fig. 7

Effect of crude lipopeptide compounds on growth and development of A. thaliana Col-0 wild type and mutants. One-week-old seedlings of A. thaliana Col-0 and the mutants coi1-16, jar1, myb72, and npr1 were treated on the roots with water, 5 % methanol (solvent control), or enriched lipopeptide fraction (LP) and grown on 0.5× MS agar plates. Plants were analyzed after 10 days (a) and effects on total number of lateral roots measured after treatment with water (white bars), methanol (grey bars), or enriched lipopeptide fraction (LP) (black bars) (b). Values represent means and standard deviations (n = 16), where samples labelled with identical letters are not significant at p < 0.05