Salme Timmusk

The Plant-Growth-Promoting Rhizobacterium Paenibacillus polymyxa Induces Changes in Arabidopsis thaliana Gene Expression: A Possible Connection Between Biotic and Abiotic Stress Responses

This paper addresses changes in plant gene expression induced by inoculation with plant-growth-promoting rhizobacteria (PGPR). A gnotobiotic system was established with Arabidopsis thaliana as model plant, and isolates of Paenibacillus polymyxa as PGPR. Subsequent challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) indicated that inoculated plants were more resistant than control plants. With RNA differential display on parallel RNA preparations from P. polymyxa-treated or untreated plants, changes in gene expression were investigated. From a small number of candidate sequences obtained by this approach, one mRNA segment showed a strong inoculation-dependent increase in abundance. The corresponding gene was identified as ERD15, previously identified to be drought stress responsive. Quantification of mRNA levels of several stress-responsive genes indicated that P. polymyxa induced mild biotic stress. This suggests that genes and/or gene classes associated with plant defenses against abiotic and biotic stress may be co-regulated. Implications of the effects of PGPR on the induction of plant defense pathways are discussed.

Cytokinin production by Paenibacillus polymyxa

Abstract The production of hormones has been suggested to be one of the mechanisms by which plant growth-promoting rhizobacteria (PGPR) stimulate plant growth. To evaluate whether the free-living soil bacterium, Paenibacillus polymyxa, releases the hormone group cytokinins and, if so, their identity, the content of cytokinins in the growth media, before and after cultivation of this bacterium, was determined by immunoaffinity chromatography (IAC). This method allows the isolation of almost all known cytokinins and their metabolites. Separation and characterization were done by high performance liquid chromatography (HPLC) with on-line ultraviolet (UV) detection, and final identification was by gas chromatography-mass spectrometry. Iso-pentenyladenine (iP) was identified in the two defined media used for the cultivation of P. polymyxa, but not earlier than at its late stationary growth. A third medium, supplemented with yeast extract, contained iso-pentenyladenine riboside (iPR) and some additional cytokinin-like substances before inoculation. When the same medium was sampled after the cultivation of P. polymyxa up to its logarithmic growth phase, the cytokinin concentration had decreased. After prolonged cultivation of P. polymyxa, small amounts of iP appeared in all three media, and iPR had disappeared from the yeast-containing medium, which indicates that the bacterium can metabolize cytokinins.

Paenibacillus polymyxa Invades Plant Roots and Forms Biofilms

ABSTRACT Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.

Bacterial distribution in the rhizosphere of wild barley under contrasting microclimates.

Background All plants in nature harbor a diverse community of rhizosphere bacteria which can affect the plant growth. Our samples are isolated from the rhizosphere of wild barley Hordeum spontaneum at the Evolution Canyon (‘EC’), Israel. The bacteria which have been living in close relationship with the plant root under the stressful conditions over millennia are likely to have developed strategies to alleviate plant stress. Methodology/Principal Findings We studied distribution of culturable bacteria in the rhizosphere of H. spontaneum and characterized the bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACCd) production, biofilm production, phosphorus solubilization and halophilic behavior. We have shown that the H. spontaneum rhizosphere at the stressful South Facing Slope (SFS) harbors significantly higher population of ACCd producing biofilm forming phosphorus solubilizing osmotic stress tolerant bacteria. Conclusions/Significance The long-lived natural laboratory ‘EC’ facilitates the generation of theoretical testable and predictable models of biodiversity and genome evolution on the area of plant microbe interactions. It is likely that the bacteria isolated at the stressful SFS offer new opportunities for the biotechnological applications in our agro-ecological systems.

Drought-Tolerance of Wheat Improved by Rhizosphere Bacteria from Harsh Environments: Enhanced Biomass Production and Reduced Emissions of Stress Volatiles

Water is the key resource limiting world agricultural production. Although an impressive number of research reports have been published on plant drought tolerance enhancement via genetic modifications during the last few years, progress has been slower than expected. We suggest a feasible alternative strategy by application of rhizospheric bacteria coevolved with plant roots in harsh environments over millions of years, and harboring adaptive traits improving plant fitness under biotic and abiotic stresses. We show the effect of bacterial priming on wheat drought stress tolerance enhancement, resulting in up to 78% greater plant biomass and five-fold higher survivorship under severe drought. We monitored emissions of seven stress-related volatiles from bacterially-primed drought-stressed wheat seedlings, and demonstrated that three of these volatiles are likely promising candidates for a rapid non-invasive technique to assess crop drought stress and its mitigation in early phases of stress development. We conclude that gauging stress by elicited volatiles provides an effectual platform for rapid screening of potent bacterial strains and that priming with isolates of rhizospheric bacteria from harsh environments is a promising, novel way to improve plant water use efficiency. These new advancements importantly contribute towards solving food security issues in changing climates.

Perspectives and Challenges of Microbial Application for Crop Improvement

Global population increases and climate change pose a challenge to worldwide crop production. There is a need to intensify agricultural production in a sustainable manner and to find solutions to combat abiotic stress, pathogens, and pests. Plants are associated with complex microbiomes, which have an ability to promote plant growth and stress tolerance, support plant nutrition, and antagonize plant pathogens. The integration of beneficial plant-microbe and microbiome interactions may represent a promising sustainable solution to improve agricultural production. The widespread commercial use of the plant beneficial microorganisms will require a number of issues addressed. Systems approach using microscale information technology for microbiome metabolic reconstruction has potential to advance the microbial reproducible application under natural conditions.

Paenibacillus polymyxa A26 Sfp-type PPTase inactivation limits bacterial antagonism against Fusarium graminearum but not of F. culmorum in kernel assay.

Fusarium graminearum and F. culmorum are the causing agents of a destructive disease known as Fusarium head blight (FHB). FHB is a re-emerging disease in small grain cereals which impairs both the grain yield and the quality. Most serious consequence is the contamination of grain with Fusarium mycotoxins that are severe threat to humans and animals. Biological control has been suggested as one of the integrated management strategies to control FHB. Paenibacillus polymyxa is considered as a promising biocontrol agent due to its unique antibiotic spectrum. P. polymyxa A26 is an efficient antagonistic agent against Fusarium spp. In order to optimize strain A26 production, formulation and application strategies traits important for its compatibility need to be revealed. Here we developed a toolbox, comprising of dual culture plate assays and wheat kernel assays, including simultaneous monitoring of FHB causing pathogens, A26, and mycotoxin production. Using this system we show that, besides generally known lipopeptide antibiotic production by P. polymyxa, biofilm formation ability may play a crucial role in the case of stain A26 F. culmorum antagonism. Application of the system for effective strain selection and maintenance is discussed.

Plant Root Associated Biofilms: Perspectives for Natural Product Mining

In natural systems microbial cells exist predominantly as biofilms. Cells in biofilms are physiologically and sometimes even morphologically distinct of planktonic cells of the same organism. Biofilm formation is regulated by variety of environmental signals, which may include nutrient sources, pH, temperature, and surface properties. The study locations where plants have coevolved with microbial representatives under stress over long period of time, facilitate the generation of theoretical testable, and predictable models of biodiversity and genome evolution. It is likely that microorganisms isolated from these environments offer new opportunities for the biotechnological applications. Physiological characteristics of the biofilms from contrasting environmental regions are discussed. In order to understand the complexity and potential of biofilm, sensitive analytical techniques are required. The range of techniques which can be used in biofilm studies are discussed.

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa

Background Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds. Methods and Principal Findings To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes. Conclusions We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Titania (TiO 2 ) nanoparticles enhance the performance of growth-promoting rhizobacteria

A novel use of nanotitania (TNs) as agents in the nanointerface interaction between plants and colonization of growth promoting rhizobacteria (PGPR) is presented. The effectiveness of PGPRs is related to the effectiveness of the technology used for their formulation. TNs produced by the Captigel patented SolGel approach, characterized by the transmission and scanning electron microscopy were used for formulation of the harsh environment PGPR strains. Changes in the biomass of wheat seedlings and in the density of single and double inoculants with and without TNs were monitored during two weeks of stress induced by drought salt and by the pathogen Fusarium culmorum. We show that double inoculants with TNs can attach stably to plant roots. Regression analysis indicates that there is a positive interaction between seedling biomass and TN-treated second inoculant colonization. We conclude that TN treatment provides an effectual platform for PGPR rational application via design of root microbial community. Our studies illustrate the importance of considering natural soil nanoparticles for PGPR application and thereby may explain the generally observed inconsistent behavior of PGPRs in the field. These new advancements importantly contribute towards solving food security issues in changing climates. The model systems established here provide a basis for new PGPR nanomaterials research.