Rosier, Amanda2023-02-142023-02-142022https://udspace.udel.edu/handle/19716/32274Plant growth promoting rhizobacteria (PGPR) are comprised of diverse bacterial communities residing on and around plant roots (rhizosphere). PGPRs are critical for plant health and could be utilized as ‘biologicals’, replacing ecologically costly crop applications. Currently there exists a gap in our understanding regarding how bacterial interspecies interactions affect PGPR plant beneficial activities. The University of Delaware patented PGPR Bacillus subtilis strain ‘UD1022’ offers an ideal model for assessing these interactions. This work investigates whether bacterial extracellular signaling, antibiotic and secondary metabolite production of UD1022 influences pathogenic and beneficial organisms found in the rhizosphere of the several species of the legume Medicago. ☐ The findings throughout this work center on the key roles of PGPR extracellular molecules, both as agents instigating plant benefits and as potential agonists interfering with the functions of other PGPRs. Plant beneficial products identified to date include siderophores, enzymes, antibiotic peptides and bacterial quorum signaling molecules. These microbial molecules are generated by bacteria as elements of their own metabolic functions, and not specifically in response to plant-derived elements in the rhizosphere. A growing number of identified bacteria extracellular metabolites enhance plant health through a multitude of direct and indirect mechanisms. Quorum sensing signal molecules known as N-acyl homoserine lactones (AHLs) are used by bacterial communities to coordinate activities. Recent evidence has shown that plants respond to these molecules through increased root growth and by ‘priming’ their defense responses to enhance disease resistance known as induced systemic resistance (ISR). Similarly, the peptide antibiotic known as ‘surfactin’ produced by Bacillus spp. (including UD1022) has been shown to both directly inhibit plant pathogens and to in induce ISR. These and other PGPR plant beneficial molecules are generally understood in isolation, and few studies have considered how these exo-metabolites may interact with each other and whether these interactions could affect their individual plant beneficial functions. ☐ To evaluate UD1022 as an effective PGPR for the related M. sativa (alfalfa), we initially challenged a suite of alfalfa phytopathogens in a series of direct and indirect antagonism assays. We found UD1022 to be strongly antagonistic toward several different ascomycetes and one oomycete pathogen of alfalfa, and these were only observed in direct contact with the PGPR. Further investigations into the probable mechanism(s) of UD1022 antagonism implicated surfactin as contributing to the inhibition response by the ascomycete Ascochyta medicaginicola. Surfactin, however, was not responsible for the antagonism toward the oomycete Phytophthora medicaginis. The inhibition of P. medicaginis may be attributed to factors of biofilm production/motility by UD1022 (likely in conjunction with yet to be verified antimicrobial extracellular molecules). These antagonism activities by UD1022 suggest it would be applicable for use as a PGPR in Medicago spp. such as alfalfa. ☐ Given that Medicago growth is enhanced by forming a symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti, we sought to evaluate whether co-inoculation of the PGPR UD1022 with S. meliloti would yield improved plant growth. Inoculation of S. meliloti strain Rm8530 with UD1022 did not improve plant shoot biomass or number of nodules in the model legume M. truncatula. We proposed that, though the two bacteria demonstrated no direct inhibition of growth, UD1022 may be interfering with the symbiont’s ability to effectively interact with the plant. S. meliloti forms biofilms which have been found to be required for efficient establishment of symbiosis, and this biofilm production is regulated through AHL quorum sensing (QS) signaling. Through evaluating the biofilm production of Rm8530 in co-culture with UD1022 culture filtrate, we established that UD1022 inhibited Rm8530 biofilms. Further investigation revealed the presence of a putative AHL cleaving lactonase enzyme in the genome of UD1022. We next showed that the UD1022 lactonase, ‘YtnP’ is capable of ‘quorum quenching’ (QQ) Rm8530 AHL molecules. To extend our understanding of this interaction in context of M. truncatula plant roots, a novel application of an AHL biosensor was incorporated into plate-based growth of M. truncatula seedlings. Through this method, Rm8530 QS signals and their subsequent QQ by UD1022 were observed to occur in the root zone of M. truncatula. Moreover, this method revealed no detectable production of AHL mimic molecules by the legume and that the legume did not itself QQ Rm8530 AHLs. These QS – QQ interactions between the PGPR UD1022 and the symbiont Rm8530 could be interfering the efficient symbiosis of the Sinorhizobium strain. ☐ Finally, the potential effects of Rm8530 on UD1022 plant beneficial molecules was queried through transcriptome analysis of the PGPR UD1022 co-cultured with the symbiotic N-fixing Rm8530. Intriguingly, many genes related to UD1022 plant growth promotion were significantly differentially expressed in co-culture with Rm8530. These differentially expressed genes (DEGs) include significant downregulation of many antibiotic genes such as those encoding surfactin and plipastatin. Many genes related to late stages of sporulation were also downregulated and when the phenotype was evaluated through sporulation assays, sporulation of UD1022 was significantly increased in co-culture with Rm8530. These responses by UD1022 indicate potential interference of UD1022 plant beneficial activities by Rm8530. ☐ Through our elegant tri-trophic model system, we expand our understanding of PGPR interspecies dynamics to include possibly non-advantageous interactions. These interactions are diverse, often indirect, and not limited to any single bacteria species grown in mixed cultures. Understanding these inhibitory interactions is important for advancing the use of PGPRs in agriculture.Bacillus subtilisBiofilmsMedicagoPlant growth promoting rhizobacteriaQuorumSinorhizobium melilotiThesis1370010388https://doi.org/10.58088/xztt-ff772022-09-21en