Analysis of Plant Growth-Promoting Effects of Fluorescent Pseudomonas Strains Isolated from Mentha piperita Rhizosphere and Effects of Their Volatile Organic Compounds on Essential Oil Composition.

Many species or strains of the genus Pseudomonas have been characterized as plant growth promoting rhizobacteria (PGPR). We used a combination of phenotypic and genotypic techniques to analyze the community of fluorescent Pseudomonas strains in the rhizosphere of commercially grown Mentha piperita (peppermint). Biochemical techniques, Amplified rDNA Restriction Analysis (ARDRA), and 16S rRNA gene sequence analysis revealed that the majority of the isolated native fluorescent strains were P. putida. Use of two Repetitive Sequence-based PCR (rep-PCR) techniques, BOX-PCR and ERIC-PCR, allowed us to evaluate diversity among the native strains and to more effectively distinguish among them. PGPR activity was tested for the native strains and reference strain P. fluorescens WCS417r. Micropropagated M. piperita plantlets were exposed to microbial volatile organic compounds (mVOCs) emitted by the bacterial strains, and plant biomass parameters and production of essential oils (EOs) were measured. mVOCs from 11 of the native strains caused an increase in shoot fresh weight. mVOCs from three native strains (SJ04, SJ25, SJ48) induced changes in M. pierita EO composition. The mVOCs caused a reduction of metabolites in the monoterpene pathway, for example menthofuran, and an increase in menthol production. Menthol production is the primary indicator of EO quality. The mVOCs produced by native strains SJ04, SJ25, SJ48, and strain WCS417r were analyzed. The obtained mVOC chromatographic profiles were unique for each of the three native strains analyzed, containing varying hydrocarbon, aromatic, and alogenic compounds. The differential effects of the strains were most likely due to the specific mixtures of mVOCs emitted by each strain, suggesting a synergistic effect occurs among the compounds present.

Programmed Lab Experiments for Biochemical Investigation of Quorum-Sensing Signal Molecules in Rhizospheric Soil Bacteria.

Biochemistry courses in the Department of Molecular Biology at the National University of Río Cuarto, Argentina, are designed for undergraduate students in biology, microbiology, chemistry, agronomy, and veterinary medicine. Microbiology students typically have previous coursework in general, analytical, and organic chemistry. Programmed sequences of lab experiments allow these students to investigate biochemical problems whose solution is feasible within the context of their knowledge and experience. We previously designed and reported a programmed lab experiment that familiarizes microbiology students with techniques for detection and characterization of quorum-sensing (QS) and quorum-quenching (QQ) signal molecules. Here, we describe a sequence of experiments designed to expand the understanding and capabilities of biochemistry students using techniques for extraction and identification of QS and QQ signal molecules from peanut rhizospheric soil bacteria, including culturing and manipulation of bacteria under sterile conditions. The program provides students with an opportunity to perform useful assays, draw conclusions from their results, and discuss possible extensions of the study. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:256-262, 2016.

Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia.

Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs), in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti) produces two symbiosis-promoting EPSs: succinoglycan (or EPS I) and galactoglucan (or EPS II). Studies of the S.meliloti/alfalfa symbiosis model system have revealed numerous biological functions of EPSs, including host specificity, participation in early stages of host plant infection, signaling molecule during plant development, and (most importantly) protection from environmental stresses. We evaluated functions of EPSs in bacterial resistance to heavy metals and metalloids, which are known to affect various biological processes. Heavy metal resistance, biofilm production, and co-culture were tested in the context of previous studies by our group. A range of mercury (Hg II) and arsenic (As III) concentrations were applied to S. meliloti wild type strain and to mutant strains defective in EPS I and EPS II. The EPS production mutants were generally most sensitive to the metals. Our findings suggest that EPSs are necessary for the protection of bacteria from either Hg (II) or As (III) stress. Previous studies have described a pump in S. meliloti that causes efflux of arsenic from cells to surrounding culture medium, thereby protecting them from this type of chemical stress. The presence of heavy metals or metalloids in culture medium had no apparent effect on formation of biofilm, in contrast to previous reports that biofilm formation helps protect various microorganism species from adverse environmental conditions. In co-culture experiments, EPS-producing heavy metal resistant strains exerted a protective effect on AEPS-non-producing, heavy metal-sensitive strains; a phenomenon termed "rescuing" of the non-resistant strain.

The role of bacterial biofilms and surface components in plant-bacterial associations.

The role of bacterial surface components in combination with bacterial functional signals in the process of biofilm formation has been increasingly studied in recent years. Plants support a diverse array of bacteria on or in their roots, transport vessels, stems, and leaves. These plant-associated bacteria have important effects on plant health and productivity. Biofilm formation on plants is associated with symbiotic and pathogenic responses, but how plants regulate such associations is unclear. Certain bacteria in biofilm matrices have been found to induce plant growth and to protect plants from phytopathogens (a process termed biocontrol), whereas others are involved in pathogenesis. In this review, we systematically describe the various components and mechanisms involved in bacterial biofilm formation and attachment to plant surfaces and the relationships of these mechanisms to bacterial activity and survival.

Systemic induction of monoterpene biosynthesis in Origanumxmajoricum by soil bacteria.

Italian oregano (Origanumxmajoricum) was subjected to root system inoculation with three species of plant growth-promoting rhizobacteria (PGPRs) (Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense), and essential oil (EO) content and plant growth were measured. Composition of monoterpenes, a major EO component, was analyzed qualitative and quantitatively by gas chromatography. Total EO yield for plants inoculated with P. fluorescens or A. brasilense was 3.57 and 3.41 microg/mg fresh weight, respectively, approximately 2.5-fold higher than controls, without change of quantitative oil composition. The major EO compounds, cis- and trans-sabinene hydrate, gamma-terpinene, carvacrol, and thymol, showed increased biosynthesis. Carvacrol was the only terpene showing significant increase of R% in plants inoculated with A. brasilense. Plant growth parameters (shoot and root fresh and dry weights, numbers of leaves and nodes) were evaluated. Shoot fresh weight was significantly increased by all three PGPR species, but only P. fluorescens and A. brasilense increased root dry weight. These two species have clear commercial potential for economic cultivation of O.xmajoricum. Knowledge of the factors affecting yield and accumulation of monoterpenes is essential for improving production of these economically important plant compounds.