Main conclusionPGPRs:P. fluorescensMs9N andS. maltophiliaLl4 inhibit in vitro growth of three legume fungal pathogens from the genusFusarium. One or both trigger up-regulation of some genes (CHIT, GLU, PAL, MYB, WRKY) inM. truncatularoots and leaves in response to soil inoculation.AbstractPseudomonas fluorescens(referred to as Ms9N; GenBank accession No. MF618323, not showing chitinase activity) andStenotrophomonas maltophilia(Ll4; GenBank accession No. MF624721, showing chitinase activity), previously identified as promoting growth rhizobacteria ofMedicago truncatula, were found, during an in vitro experiment, to exert an inhibitory effect on three soil-borne fungi:Fusarium culmorumCul-3,F. oxysporum857 andF. oxysporumf. sp. medicaginisstrain CBS 179.29, responsible for serious diseases of most legumes includingM. truncatula.S. maltophiliawas more active thanP. fluorescensin suppressing the mycelium growth of two out of threeFusariumstrains. Both bacteria showed β-1,3-glucanase activity which was about 5 times higher inP. fluorescensthan inS. maltophilia.Upon soil treatment with a bacterial suspension, both bacteria, but particularlyS. 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 theMYB(MtMYB74,MtMYB102) andWRKY(MtWRKY6,MtWRKY29,MtWRKY53,MtWRKY70) families which encode TFs inM. truncatularoots 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 twoM.truncatulagrowth-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 ofFusariumdirectly and indirectly by up-regulation of some defense priming markers such asCHIT, GLUandPALgenes in plants. This is also the first study of the expression of someMYBandWRKYgenes in roots and leaves ofM. truncatulaupon soil treatment with two PGPR suspensions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00425-023-04145-9.