Molecular mechanisms of plant growth promotion for methylotrophic Bacillus aryabhattai LAD

Abstract

Plant growth-promoting rhizobacteria (PGPR) can produce hormone-like substances, promote plant nutrient uptake, enhance plant resistance, inhibit the growth of pathogenic bacteria, and induce plant resistance to biotic and abiotic stresses. Bacillus is one of the most studied genera that promote plant root development. Since its discovery in 2009, B. aryabhattai has shown promising properties such as promoting plant growth and improving crop yield. However, the mechanisms of B. aryabhattai promoting plant growth remain to be investigated. In this study, the chromosome of B. aryabhattai strain LAD and five plasmids within the cell were sequenced and annotated. The genome, with a length of 5,194,589 bp and 38.12% GC content, contains 5,288 putative protein-coding genes, 39 rRNA, and 112 tRNA. The length of the five plasmids ranged from 116,519 to 212,484 bp, and a total of 810 putative protein-coding genes, 4 rRNA, and 32 tRNA were predicted in the plasmids. Functional annotation of the predicted genes revealed numerous genes associated with indole-3 acetic acid (IAA) and exopolysaccharides (EPSs) biosynthesis, membrane transport, nitrogen cycle metabolism, signal transduction, cell mobility, stress response, and antibiotic resistance on the genome which benefits the plants. Genes of carbohydrate-active enzymes were detected in both the genome and plasmids suggesting that LAD has the capacity of synthesizing saccharides and utilizing organic materials like root exudates. LAD can utilize different carbon sources of varied carbon chain length, i.e., methanol, acetate, glycerol, glucose, sucrose, and starch for growth and temperature adaptation suggesting a high versatility of LAD for thriving in fluctuating environments. LAD produced the most EPSs with sucrose as sole carbon source, and high concentration of IAA was produced when the maize plant was cultivated with LAD, which may enhance plant growth. LAD significantly stimulated the development of the maize root. The genome-based information and experimental evidence demonstrated that LAD with diverse metabolic capabilities and positive interactions with plants has tremendous potential for adaptation to the dynamic soil environments and promoting plant growth.

Keywords: carbohydrate-active enzymes; growth promoting; indole acetic acid; rhizosphere; sequencing.

Figure 1

Circular chromosome diagram of B. aryabhattai LAD. From the inner circle to the outer circle, the information on each of the seven circles shows genome position, GC skew, GC content, coding sequence on the positive and strands (classified by Cluster of Orthologous Groups of protein), and the position of the coding sequence, tRNA and rRNA on the genome.

Figure 2

(A) Annotation of carbohydrate active enzymes (CAZymes) genes on B. aryabhattai LAD genome. The hmmscan program was used for estimation of the CAZy genes (alignment length > 80 amino acids, E-value <1e-5; alignment length < 80 amino acids, E-value <1e-3). (B) eggNOG annotation. The protein-coding gene sequences were compared to COG database using DIAMOND blastp (cutoff at 1e-6).

Figure 3

Pan-genome analysis of 19 Bacillus strains. The number in the overlapping center represents the number of orthologous coding sequences (core genome) shared by all analyzed strains. The number of coding sequences specific to each strain was shown in the non-overlapping portion of the oval.

Figure 4

Pan genome and core genome profile plot of 19 Bacillus strains. The analysis was performed through the Bacterial Pan Genome Analysis (BPGA) software package.

Figure 5

The growth plots for B. aryabhattai LAD. LAD cells were cultivated in the optimum growth medium with different carbon sources, i.e., methanol (0.5, v v⁻¹), sodium acetate (2%), glycerol (2%), glucose (2%), sucrose (2%), and starch (2%), respectively. The temperature for LAD growth included 20°C and 37°C. Each data point represents the mean value of three replicates.

Figure 6

Detection of IAA production by UPLC. The left figure shows the sample of the control, and the right shows the sample of B. aryabhattai LAD culture cultivated with maize seedlings.

Figure 7

Maize root development in laboratory and field soils impacted by B. aryabhattai LAD. (A) The impacts of LAD on the maize root length. (B) The impacts of LAD on the maize root surface area. (C) The impacts of LAD on the maize root volume. Lab control: the root of the maize seedlings treated with water and cultivated in laboratory soil for 14 d; Lab LAD: the root of the maize seedlings treated with LAD and cultivated in laboratory soil for 14 d; Field control: the root of the maize seedlings treated with water and cultivated in laboratory soil for 60 d; Field LAD: the root of the maize seedlings treated with LAD and cultivated in laboratory soil for 60 d. Each treatment had 10 maize plants and was repeated three times. Student’s t-test was used to compare the root development under LAD treatment with that of the control. Statistical differences were shown with asterisks (i.e., *** representing p < 0.001).