The potential for plant growth-promoting bacteria to impact crop productivity in future agricultural systems is linked to understanding the principles of microbial ecology.

Global climate change poses challenges to land use worldwide, and we need to reconsider agricultural practices. While it is generally accepted that biodiversity can be used as a biomarker for healthy agroecosystems, we must specify what specifically composes a healthy microbiome. Therefore, understanding how holobionts function in native, harsh, and wild habitats and how rhizobacteria mediate plant and ecosystem biodiversity in the systems enables us to identify key factors for plant fitness. A systems approach to engineering microbial communities by connecting host phenotype adaptive traits would help us understand the increased fitness of holobionts supported by genetic diversity. Identification of genetic loci controlling the interaction of beneficial microbiomes will allow the integration of genomic design into crop breeding programs. Bacteria beneficial to plants have traditionally been conceived as "promoting and regulating plant growth". The future perspective for agroecosystems should be that microbiomes, via multiple cascades, define plant phenotypes and provide genetic variability for agroecosystems.

Silica Particles Trigger the Exopolysaccharide Production of Harsh Environment Isolates of Growth-Promoting Rhizobacteria and Increase Their Ability to Enhance Wheat Biomass in Drought-Stressed Soils.

In coming decades, drought is expected to expand globally owing to increased evaporation and reduced rainfall. Understanding, predicting, and controlling crop plants' rhizosphere has the potential to manipulate its responses to environmental stress. Our plant growth-promoting rhizobacteria (PGPR) are isolated from a natural laboratory, 'The Evolution Canyon', Israel, (EC), from the wild progenitors of cereals, where they have been co-habituating with their hosts for long periods of time. The study revealed that commercial TM50 silica particles (SN) triggered the PGPR production of exopolysaccharides (EPS) containing D-glucuronate (D-GA). The increased EPS content increased the PGPR water-holding capacity (WHC) and osmotic pressure of the biofilm matrix, which led to enhanced plant biomass in drought-stressed growth environments. Light- and cryo-electron- microscopic studies showed that, in the presence of silica (SN) particles, bacterial morphology is changed, indicating that SNs are associated with significant reprogramming in bacteria. The findings encourage the development of large-scale methods for isolate formulation with natural silicas that ensure higher WHC and hyperosmolarity under field conditions. Osmotic pressure involvement of holobiont cohabitation is also discussed.

Fighting Fusarium Pathogens in the Era of Climate Change: A Conceptual Approach.

Fusarium head blight (FHB) caused by Fusarium pathogens is one of the most devastating fungal diseases of small grain cereals worldwide, substantially reducing yield quality and food safety. Its severity is increasing due to the climate change caused by weather fluctuations. Intensive research on FHB control methods has been initiated more than a decade ago. Since then, the environment has been rapidly changing at regional to global scales due to increasing anthropogenic emissions enhanced fertilizer application and substantial changes in land use. It is known that environmental factors affect both the pathogen virulence as well as plant resistance mechanisms. Changes in CO2 concentration, temperature, and water availability can have positive, neutral, or negative effects on pathogen spread depending on the environmental optima of the pathosystem. Hence, there is a need for studies of plant-pathogen interactions in current and future environmental context. Long-term monitoring data are needed in order to understand the complex nature of plants and its microbiome interactions. We suggest an holobiotic approach, integrating plant phyllosphere microbiome research on the ecological background. This will enable the development of efficient strategies based on ecological know-how to fight Fusarium pathogens and maintain sustainable agricultural systems.

Paenibacillus polymyxa biofilm polysaccharides antagonise Fusarium graminearum.

Fusarium Head Blight (FHB) caused by Fusarium graminearum pathogens constitutes a major threat to agricultural production because it frequently reduces the yield and quality of the crop. The disease severity is predicted to increase in various regions owing to climate change. Integrated management where biocontrol plays an important role has been suggested in order to fight FHB. P. polymyxa A26 is known to be an effective antagonist against F. graminearum. Deeper understanding of the mode of action of P. polymyxa A26 is needed to develop strategies for its application under natural settings in order to effectively overcome the pathogenic effects. This study aims to re-evaluate a former study and reveal whether compounds other than non-ribosomal antibiotic lipopeptides could be responsible for the antagonistic effect, despite what is often reported. Wheat seedlings were grown to maturity and the spikes infected with the pathogen under greenhouse conditions. The development of FHB infection, quantified via the disease incidence severity and 100-kernel weight, was strongly correlated (r > 0.78, p < 0.01) with the content of the polysaccharide component D-glucuronic acid in the biofilm. Furthermore, while increased inoculum density from 106 to 108 cells/ml did not affect wild type performance, a significant increase was observed with the P. polymyxa mutant deficient in nonribosomal lipopeptide synthesis. Our results show that P. polymyxa A26 biofilm extracellular polysaccharides are capable of antagonizing F. graminearum and that the uronate content of the polysaccharides is of critical importance in the antagonism.

Titania (TiO2) 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.

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.

Sfp-type PPTase inactivation promotes bacterial biofilm formation and ability to enhance wheat drought tolerance.

Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are non-ribosomal peptide and polyketide derived metabolites (NRPs/PKs). Modular non-ribosomal peptide synthetases catalyze main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 Sfp-type 4'-phosphopantetheinyl transferase (Sfp-type PPTase). The inactivation of the gene resulted in loss of NRPs/PKs production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. A26Δsfp biofilm promotion is directly mediated by NRPs/PKs, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their Sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type. Challenges with P. polymyxa genetic manipulation are discussed.

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.

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 ß-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 ß-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 ß-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

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.

Paenibacillus polymyxa invades plant roots and forms biofilms.

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.