Potential for plant growth promotion by a consortium of stress-tolerant 2,4-dinitrotoluene-degrading bacteria: isolation and characterization of a military soil

Abstract

The presence of explosives in soils and the interaction with drought stress and nutrient limitation are among the environmental factors that severely affect plant growth on military soils. In this study, we seek to isolate and identify the cultivable bacteria of a 2,4-dinitrotoluene (DNT) contaminated soil (DS) and an adjacent grassland soil (GS) of a military training area aiming to isolate new plant growth-promoting (PGP) and 2,4-DNT-degrading strains. Metabolic profiling revealed disturbances in Ecocarbon use in the bare DS; isolation of cultivable strains revealed a lower colony-forming-unit count and a less diverse community associated with DS in comparison with GS. New 2,4-DNT-tolerant strains were identified by selective enrichments, which were further characterized by auxanography for 2,4-DNT use, resistance to drought stress, cold, nutrient starvation and PGP features. By selecting multiple beneficial PGP and abiotic stress-resistant strains, efficient 2,4-DNT-degrading consortia were composed. After inoculation, consortium UHasselt Sofie 3 with seven members belonging to Burkholderia, Variovorax, Bacillus, Pseudomonas and Ralstonia species was capable to successfully enhance root length of Arabidopsis under 2,4-DNT stress. After 9 days, doubling of main root length was observed. Our results indicate that beneficial bacteria inhabiting a disturbed environment have the potential to improve plant growth and alleviate 2,4-DNT stress.

Fig. 1

A. UPGMA tree and heat map showing the phylogenetic relationships and abundance of the isolated strains for each of the different soils. The identified species correspond to the 68 clusters of different 16S rRNA gene ARDRA profiles, grouped based on 99% nucleotide similarity of the 16S rRNA gene sequences. From left to right: the UPGMA phylogenetic tree constructed with Geneious using the 16S rRNA gene sequences, heat map showing the abundance of the species in cfu g⁻¹ soil for DS and GS obtained on 1/10 rich medium (yellow: 0 cfu g⁻¹; light blue: 0.1–10³ cfu g^-1, blue: 10³–10⁵ cfu g⁻¹, dark blue: 10⁵–10⁶ cfu g⁻¹ and red: 10⁶–10⁷ cfu g⁻¹) and the relative abundances in % of the species in each soil after enrichment cultures (EC) of the soils in the colour codes (yellow: 0; orange: 1–25%; dark-red (25–75%) and purple (75–100%). The corresponding strain number is shown next to the heat map, the most closely related bacterial 16S rRNA gene sequence and Genbank accession number of the most closely related species. B. Pie diagrams showing the bacteria family distribution of the different soils (in percentage %) on 1/10 rich medium (DS-s,w; GS-s,w) and isolated after successive dilution cultures with 2,4-DNT [enrichment culture 2,4-DNT contaminated soil (ECDS)-s,w; ECGS-s,w]. Calculations were performed on the different 16S rRNA gene identified strains.

Fig. 2

A. Number of isolates grouped per genus, scoring positive for auxanography using 2,4-DNT as sole N-source. DS: 2,4-DNT contaminated soil summer and winter combined, GS: grassland soil summer and winter combined. B. Positive auxanography response towards 2,4-DNT as sole N-source by Ralstonia sp. HC90 (a) and negative auxanography response towards 2,4-DNT by Paenibacillus sp. HC2 (b). A sample of bacteria in MgSO₄-buffer (100 μl of 10⁷ cfu ml⁻¹) was spread evenly over the whole surface of the minimal agar plate. After drying overnight, a drop of 2,4-DNT (30 μmol dissolved in DMSO) was spot on the plate and spread-out in half a circle, as shown in the scheme (c). After 5 days of incubation at 30°C, growth of bacteria was observed as white colonies. Ralstonia sp. HC90 grows only on the 2,4-DNT treated zone of the plate (+); Paenibacillus sp. HC2 strictly avoided growth on the 2,4-DNT treated zone (−).

Fig. 3

Metabolization of 2,4-DNT by three consortia in liquid cultures under carbon limiting conditions (500 μM of 2,4-DNT was added as sole energy source). Y-axis: (left) concentration of the explosives in μM; (right) bacterial protein concentration (mg l⁻¹) measured by the Bradford assay; X-axis: time in hours.A. Consortium UHS1 (Pseudomonas mandelii HC88, V. paradoxus VM685 and Burkholderia sp. HC114).B. Consortium UHS2 (V. paradoxus VM685; Burkholderia sp. HC114, Pseudomonas mandelii HC88, Ralstonia sp. HC90 and Burkholderia phytofirmans HC106).C. Consortium UHS3 (V. paradoxus VM685; Burkholderia sp. HC114, Pseudomonas mandelii HC88, Ralstonia sp. HC90, Burkholderia phytofirmans HC106, Bacillus subtilis HC45 and Variovorax sp. HC92).

Fig. 4

A. Root length of A. thaliana seedlings non-exposed (bars in white) and exposed to 1 mg l⁻¹ 2,4-DNT for 9 days (bars in grey) in the absence or presence of consortia UHS1, UHS2, UHS3 (10⁶ cfu ml⁻¹). Values are mean ± standard error of at least 15 biological replicates. (Significance level versus control: *** P < 0.001, non-parametric Kruskal–Wallis test). B. Pictures of root hairs were taken under the binocular (20×) of (a) un-inoculated 2,4-DNT exposed plants and (b) 2,4-DNT exposed plants inoculated with UHS3.