Two Medicago truncatula growth-promoting rhizobacteria capable of limiting in vitro growth of the Fusarium soil-borne pathogens modulate defense genes expression

Abstract

PGPRs: P. fluorescens Ms9N and S. maltophilia Ll4 inhibit in vitro growth of three legume fungal pathogens from the genus Fusarium. One or both trigger up-regulation of some genes (CHIT, GLU, PAL, MYB, WRKY) in M. truncatula roots and leaves in response to soil inoculation. Pseudomonas fluorescens (referred to as Ms9N; GenBank accession No. MF618323, not showing chitinase activity) and Stenotrophomonas maltophilia (Ll4; GenBank accession No. MF624721, showing chitinase activity), previously identified as promoting growth rhizobacteria of Medicago truncatula, were found, during an in vitro experiment, to exert an inhibitory effect on three soil-borne fungi: Fusarium culmorum Cul-3, F. oxysporum 857 and F. oxysporum f. sp. medicaginis strain CBS 179.29, responsible for serious diseases of most legumes including M. truncatula. S. maltophilia was more active than P. fluorescens in suppressing the mycelium growth of two out of three Fusarium strains. Both bacteria showed β-1,3-glucanase activity which was about 5 times higher in P. fluorescens than in S. maltophilia. Upon soil treatment with a bacterial suspension, both bacteria, but particularly S. maltophilia, brought about up-regulation of plant genes encoding chitinases (MtCHITII, MtCHITIV, MtCHITV), glucanases (MtGLU) and phenylalanine ammonia lyases (MtPAL2, MtPAL4, MtPAL5). Moreover, the bacteria up-regulate some genes from the MYB (MtMYB74, MtMYB102) and WRKY (MtWRKY6, MtWRKY29, MtWRKY53, MtWRKY70) families which encode TFs in M. truncatula roots and leaves playing multiple roles in plants, including a defense response. The effect depended on the bacterium species and the plant organ. This study provides novel information about effects of two M. truncatula growth-promoting rhizobacteria strains and suggests that both have a potential to be candidates for PGPR inoculant products on account of their ability to inhibit in vitro growth of Fusarium directly and indirectly by up-regulation of some defense priming markers such as CHIT, GLU and PAL genes in plants. This is also the first study of the expression of some MYB and WRKY genes in roots and leaves of M. truncatula upon soil treatment with two PGPR suspensions.

Keywords: Antifungal activity; Bacterial glucanase activity; Expression of genes in response to PGPR; Fabaceae; Plant growth-promoting rhizobacteria.

Fig. 1

Effects of P. fluorescens Ms9N and S. maltophilia L14 on in vitro growth of F. culmorum after 4 days (a), F. oxysporum after 5 days (b) and F. oxysporum f. sp. medicaginis after 9 days (c). Data are means ± SD (n = 3) and the results were replicated in at least 3 independent experiments. Letters denote significance of differences, as determined by one-way ANOVA (P < 0.05), followed by Tukey’s HSD post-hoc test

Fig. 2

β-1,3-glucanase activity in P. fluorescens Ms9N and S. maltophilia Ll4 after 3 days in the peptone medium, supplemented with 0.02% laminarin. Data are means ± SD (n = 3) and the results were replicated in at least 3 independent experiments. Letters denote significance of differences, as determined by one-way ANOVA (P < 0.05), followed by Tukey’s HSD post-hoc test

Fig. 3

Expression of CHIT I, CHIT II, CHIT III, CHIT IV, CHIT V and GLU genes in M. truncatula roots and leaves before, 24 h and 72 h after treating the soil with MgSO₄ solution (a, c, e, g, i, k) and after treatment with P. fluorescens Ms9N and S. maltophilia L14 suspensions (b, d, f, h, j, l). Expression in roots and leaves after treating the soil with MgSO₄ (left side) measured relative to the lowest observed expression taken as 1; expression in roots and leaves after treating the soil with bacterial suspensions (right side) measured relative to the control samples (roots and leaves after soil treatment with MgSO₄ only) taken as 1. At least 3 biological replicates were performed for all experiments and are shown as the mean ± SD. Statistical analyses were performed using two-way ANOVA, followed by Tukey’s HSD post-hoc test

Fig. 3

Expression of CHIT I, CHIT II, CHIT III, CHIT IV, CHIT V and GLU genes in M. truncatula roots and leaves before, 24 h and 72 h after treating the soil with MgSO₄ solution (a, c, e, g, i, k) and after treatment with P. fluorescens Ms9N and S. maltophilia L14 suspensions (b, d, f, h, j, l). Expression in roots and leaves after treating the soil with MgSO₄ (left side) measured relative to the lowest observed expression taken as 1; expression in roots and leaves after treating the soil with bacterial suspensions (right side) measured relative to the control samples (roots and leaves after soil treatment with MgSO₄ only) taken as 1. At least 3 biological replicates were performed for all experiments and are shown as the mean ± SD. Statistical analyses were performed using two-way ANOVA, followed by Tukey’s HSD post-hoc test

Fig. 4

Expression of PAL1, PAL2, PAL3, PAL4 and PAL5 genes in M. truncatula roots and leaves before, 24 h and 72 h after treating the soil with MgSO₄ solution (a, c, e, g) and after treatment with P. fluorescens Ms9N and S. maltophilia L14 suspensions (b, d, f, h). Expression in roots and leaves after treating the soil with MgSO₄ (left side) measured relative to the lowest observed expression taken as 1; expression in roots and leaves after treating the soil with bacterial suspensions (right side) measured relative to the control samples (roots and leaves after soil treatment with MgSO₄ only) taken as 1. At least 3 biological replicates were performed for all experiments and are shown as the mean ± SD. Statistical analyses were performed using two-way ANOVA, followed by Tukey’s HSD post-hoc test

Fig. 5

Phylogenetic trees and domain organization based on deduced amino acid sequences of Medicago truncatula (Mt) and Arabidopsis thaliana (At) WRKY6, WRKY29, WRKY53 and WRKY70 (a, b). Bn Brassica napus, Br Brassica rapa, Ca Cicer arietinum, Cr Capsella rubella, Cs Camelina sativa, Es, Eutrema salsugineum, Gm Glycine max, Nt Nicotiana tabacum, Pv Phaseolus vulgaris, Rc Ricinus communis

Fig. 6

Expression of selected MYB74, MYB102, WRKY6, WRKY29, WRKY53 and WRKY70 genes in M. truncatula roots and leaves before, 24 h and 72 h after soil treatment with MgSO₄ solution (a, c, e, g, i, k) and after treatment with P. fluorescens Ms9N and S. maltophilia L14 suspensions (b, d, f, h, j, l). Expression in roots and leaves after treating the soil with MgSO₄ (left side) measured relative to the lowest observed expression taken as 1; expression in roots and leaves after treating the soil with bacterial suspensions (right side) measured relative to the control samples (roots and leaves after soil treatment with MgSO₄ only) taken as 1. At least 3 biological replicates were performed for all the experiments and are shown as the mean ± SD. Statistical analyses were performed using two-way ANOVA, followed by Tukey’s HSD post-hoc test

Fig. 6

Expression of selected MYB74, MYB102, WRKY6, WRKY29, WRKY53 and WRKY70 genes in M. truncatula roots and leaves before, 24 h and 72 h after soil treatment with MgSO₄ solution (a, c, e, g, i, k) and after treatment with P. fluorescens Ms9N and S. maltophilia L14 suspensions (b, d, f, h, j, l). Expression in roots and leaves after treating the soil with MgSO₄ (left side) measured relative to the lowest observed expression taken as 1; expression in roots and leaves after treating the soil with bacterial suspensions (right side) measured relative to the control samples (roots and leaves after soil treatment with MgSO₄ only) taken as 1. At least 3 biological replicates were performed for all the experiments and are shown as the mean ± SD. Statistical analyses were performed using two-way ANOVA, followed by Tukey’s HSD post-hoc test