Soil salinity is a widespread problem that limits crop yield throughout the world. The accumulation of soluble salts in the soil can inhibit plant growth by increasing the osmotic potential of interstitial water, inducing ion toxicity and nutrient imbalances in plants. Over the last decade, considerable effort has been put into developing economical and effective methods to reclaim these damaged soils. Phytoremediation is a technique that uses plants to extract, contain, immobilize and degrade contaminants in soil. The most common process for salt bioremediation is phytoextraction which uses plants to accumulate salt in the shoots, which is then removed by harvesting the foliage. As developing significant plant biomass in saline soils is an issue, a group of free-living rhizobacteria, called plant growth promoting rhizobacteria (PGPR), can be applied to plant seeds to aid plant growth by alleviating salt stress. The principle objective of this research was to test the efficacy of PGPR in improving the growth of plants on salt-impacted soils through greenhouse and field studies. In this research, previously isolated PGPR strains of Pseudomonas putida. UW3, Pseudomonas putida UW4, and Pseudomonas corrugata CMH3 were applied to barley (Hordeum valgare C.V. AC ranger), oats (Avena sativa C.V. CDC baler), tall wheatgrass (Agropyron elongatum), and tall fescue (festuca arundinacea C.V. Inferno). PGPR effects on plant growth, membrane stability, and photosynthetic activity under salt stress were examined. Greenhouse studies showed that plants treated with PGPR resulted in an increase in plant biomass by up to 500% in salt-impacted soils. Electrolyte leakage assay showed that plants treated with PGPR resulted in 50% less electrolyte leakage from membranes. Several chlorophyll a fluorescence parameters, Fv/Fm, effective quantum yield, Fs, qP, and qN obtained from pulse amplitude modulation (PAM) fluorometry showed that PGPR-treated plants resulted in improvement in photosynthesis under salt stress. Field studies showed that PGPR promoted shoot dry biomass production by 27% to 230%. The NaCl accumulation in plant shoots increased by 7% to 98% with PGPR treatment. The averaged soil salinity level at the CMS and CMN site decreased by 20% and 60%, respectively, during the 2008 field season. However, there was no evidence of a decrease in soil salinity at the AL site. Based on the improvements of plant biomass production and NaCl uptake by PGPR observed in the 2008 field studies, the phytoremediation efficiency on salt-impacted sites is expected to increase by 30-60% with PGPR treatments. Based on the average data of 2007 and 2008 field season, the time required to remove 25% of NaCl of the top 50 cm soil at the CMS, CMN and AL site is estimated to be six, twelve, and sixteen years, respectively, with PGPR treatments. The remediation efficiency is expected to accelerate during the remediation process as the soil properties and soil salinity levels improve over time.

Soil salinity is a widespread problem that limits crop yield throughout the world. The accumulation of soluble salts in the soil can inhibit plant growth by increasing the osmotic potential of interstitial water, inducing ion toxicity and nutrient imbalances in plants. Over the last decade, considerable effort has been put into developing economical and effective methods to reclaim these damaged soils. Phytoremediation is a technique that uses plants to extract, contain, immobilize and degrade contaminants in soil. The most common process for salt bioremediation is phytoextraction which uses plants to accumulate salt in the shoots, which is then removed by harvesting the foliage. As developing significant plant biomass in saline soils is an issue, a group of free-living rhizobacteria, called plant growth promoting rhizobacteria (PGPR), can be applied to plant seeds to aid plant growth by alleviating salt stress. The principle objective of this research was to test the efficacy of PGPR in improving the growth of plants on salt-impacted soils through greenhouse and field studies. In this research, previously isolated PGPR strains of Pseudomonas putida. UW3, Pseudomonas putida UW4, and Pseudomonas corrugata CMH3 were applied to barley (Hordeum valgare C.V. AC ranger), oats (Avena sativa C.V. CDC baler), tall wheatgrass (Agropyron elongatum), and tall fescue (festuca arundinacea C.V. Inferno). PGPR effects on plant growth, membrane stability, and photosynthetic activity under salt stress were examined. Greenhouse studies showed that plants treated with PGPR resulted in an increase in plant biomass by up to 500% in salt-impacted soils. Electrolyte leakage assay showed that plants treated with PGPR resulted in 50% less electrolyte leakage from membranes. Several chlorophyll a fluorescence parameters, Fv/Fm, effective quantum yield, Fs, qP, and qN obtained from pulse amplitude modulation (PAM) fluorometry showed that PGPR-treated plants resulted in improvement in photosynthesis under salt stress. Field studies showed that PGPR promoted shoot dry biomass production by 27% to 230%. The NaCl accumulation in plant shoots increased by 7% to 98% with PGPR treatment. The averaged soil salinity level at the CMS and CMN site decreased by 20% and 60%, respectively, during the 2008 field season. However, there was no evidence of a decrease in soil salinity at the AL site. Based on the improvements of plant biomass production and NaCl uptake by PGPR observed in the 2008 field studies, the phytoremediation efficiency on salt-impacted sites is expected to increase by 30-60% with PGPR treatments. Based on the average data of 2007 and 2008 field season, the time required to remove 25% of NaCl of the top 50 cm soil at the CMS, CMN and AL site is estimated to be six, twelve, and sixteen years, respectively, with PGPR treatments. The remediation efficiency is expected to accelerate during the remediation process as the soil properties and soil salinity levels improve over time.

There is accumulating evidence that arbuscular mycorrhizal fungi (AMF) produce a glycoprotein called glomalin, which has the potential to increase soil carbon (C) and nitrogen (N) storage, thereby reducing soil emissions of carbon dioxide (CO2) and nitrous oxide (N2O) into the atmosphere. However, other soil microorganisms such as plant growth-promoting rhizobacteria (PGPR) that interact with AMF could indirectly influence glomalin production. The objectives of this study were to determine the effects of AMF and PGPR interactions on glomalin production and identify possible combinations of these organisms that could enhance C and N storage in the rhizosphere. The effects of AMF and PGPR interactions on pea (Pisum sativum L.) growth and correlations between glomalin production and plant growth also were assessed.<p> A series of growth chamber and laboratory experiments were conducted to examine the effect of fungal and host plant species on glomalin production by comparing the amounts of glomalin produced by Glomus clarum, G. intraradices, and G. mosseae in association with corn (Zea mays L.), in addition to examining differences in the ability of corn, pea, and wheat (Triticum aestivum L.) to support glomalin production by G. intraradices. There were no significant differences in glomalin production [measured in the rhizosphere as Bradford-reactive soil protein (BRSP)] by the three AMF species, whereas host plant significantly affected glomalin production. Specifically, higher BRSP concentrations were found in the rhizosphere of corn as compared to pea and wheat.<p> Additionally, the effect of long-term storage on the growth promoting traits of the PGPR strains selected; namely, Pseudomonas cepacia R55 and R85, P. aeruginosa R75, P. putida R105, and P. fluorescence R111 were investigated. These bacterial strains previously had been identified as PGPR, but had since undergone approximately twenty years of storage at -80¢ªC; thus, it was necessary to confirm that these strains had retained their plant growth promoting characteristics. Apparently, long-term storage had no significant adverse effect on the PGPR strains as all strains increased the total biomass of wheat significantly and demonstrated antagonism against fungal pathogens.<p> The possibility that spore-associated bacteria (SAB) could influence AMF associations, thereby affecting glomalin production, and subsequent crop yield potential was assessed. This was achieved by first isolating bacteria from disinfested spores of the AMF species and determining their potential as PGPR for wheat. According to fatty acid methyl ester (FAME) profiles, four genera of bacteria were isolated from AMF spores namely; Arthrobacter, Bacillus, Micrococcus, and Paenibacillus, of which Bacillus species were the most common SAB. None of these isolates, however, showed growth promoting abilities on wheat.<p> Based on the preliminary findings, the combined effects of the three AMF species and the five PGPR strains were examined on plant growth and glomalin production under gnotobiotic conditions using pea as the host plant. Interactions between G. intraradices and R75, R85, or R105 resulted in increased BRSP concentration in the mycorrhizosphere of pea. Additionally, significant interactions were observed between the AMF species and PGPR strains on BRSP concentration in pea rhizosphere under non-sterile conditions. As observed under sterile conditions, the co-inoculation of pea with G. intraradices and R75 or R85 increased BRSP concentrations in the rhizosphere of pea grown in non-sterile soil, although interaction effects were not significantly different from the control or when G. intraradices was applied alone. Significant AMF and PGPR interactions were observed to affect AMF colonization; however, the combination of these organisms did not significantly affect pea growth, nutrient uptake, and C and N storage in the plant rhizosphere. No correlations were detected between glomalin-related soil protein (GRSP), pea growth, nutrient concentrations in the plant tissue, and soil organic C and N content. This study demonstrated that although the potential exists to manipulate certain AMF and PGPR to enhance glomalin production, co-inoculation of AMF and PGPR did not enhance plant growth or C and N storage beyond that achieved by inoculation of either organism.

Soybean (Glycine max (L.) Merr.) is a subtropical legume that requires root zone temperatures (RZTs) in the 25 to 30$ sp circ$C range for optimal symbiotic activity. The inability of soybean to adapt to cool soil conditions limits its development and yield in short season areas. In particular, nodulation and N$ sb2$ fixation by this subtropical crop species is sensitive to cool (RZT). The objectives of this thesis were to determine whether or not PGPR could be used to help overcome the low RZT inhibition of soybean nodulation, to improve soybean nitrogen fixation and yield under field conditions and to determine the methods by which such increases occurred. The work reported in this thesis has demonstrated that PGPR can increase early season nodulation and total seasonal nitrogen fixation and yield of soybean growing in an area with cool spring soils. The ability of PGPR to stimulate soybean nodulation and growth was shown to be related to their ability to colonize soybean roots, and this was shown to be related to RZT. All steps in early nodulation were stimulated by the presence of PGPR. The beneficial effects of PGPR are exerted through a diffusible molecule excreted into the growth medium. The addition of genistein, a plant-to-bacteria signal molecule already shown to stimulate soybean N$ sb2$ fixation at low RZT, plus PGPR causes increases in soybean nodulation, N$ sb2$ fixation, and growth that were greater than those caused by the addition of PGPR alone, but only at 25 and 17.5$ sp circ$C, and not at 15$ sp circ$C RZT.

Thirty-two isolates of rhizosphere bacteria, selected for their ability to inhibit zoospore germination and/or mycelial growth of Pythium aphanidermatum (Pa) in vitro, were evaluated in a test tube bioassay using cucumber c.v. 'Straight 8'. These isolates were identified as Psedudomonas corrugata (Pc13 or 35) and P. fluorescens (Pf15, 16 or 27). All but one of the five isolates effectively colonised the roots of cucumber plants in short term studies. Isolates 15 and 35 were found to maintain high population densities throughout the time period. Pa-inoculated plants treated with the Pc13 or Pf15 produced fruit yields equal to 92 and 74% respectively of the control (no Pa, no bacteria). Pa-inoculated plants without bacteria yielded only 46% of the control. In the fall crop, Pa-inoculated plants treated with Pc13 or Pf15 yielded 52 and 47% of the control compared to Pa-only treatment, which yielded 12.5% of the control. In both crops, treatment with any of the bacterial isolates resulted in significantly reduced cull rates compared to the Pa-only treatment.

Plant roots are central to C- and N-cycling in soil. However, (i) plants differ strongly in tissue recalcitrance (e.g. lignin content) affecting their mineralization in soil, and (ii) rhizodeposits also vary strongly in terms of the metabolites that they contain. Therefore, (i) we used 13C labelled ryegrass root and shoot residues as substrates to investigate the impact of tissue recalcitrance on soil processes through controlled incubation of soil, (ii) we assessed variations in root C-deposition between barley genotypes and their respective impacts on soil processes using 13CO2 labelled plants, (iii) using 13C/15N enriched ryegrass root residues as tracer material, we investigated the impacts of barley genotypes on mineralization of recently incorporated plant residues in soil and plant uptake of the residue-derived N, and (iv) we applied a quantitative trait loci analysis approach to identify barley chromosome regions affecting soil microbial biomass and other soil and root related traits. In the first study, addition of root residues resulted in reduced C-mineralization rates, soil microbial activity and soil organic matter (SOM) priming relative to shoot residues. Planted experiments revealed (i) genotype effects on plant-, SOM- and residuederived surface soil CO2-C efflux and showed that incorporation of plant derived-C to the silt-and-clay soil fraction varied between genotypes, indicating relative stabilization of root derived-C as a result of barley genotype, (ii) that plant uptake of residue released N between genotypes was linked to genotype impacts on residue mineralization, and (iii) barley chromosome regions that influence plant-derived microbial biomass C. These results (i) suggest that greater plant tissue recalcitrance can lower soil C-emissions and increase C-storage in soil, and (ii) demonstrate the barley genetic influence on soil microbial communities and C- and N-cycling, which could be useful in crop breeding to improve soil microbial interactions, and thus promote sustainable crop production systems.

Salt stress is a severe environmental challenge in agriculture, limiting the quality and productivity of the crops around the globe. Plant growth promoting rhizobacteria (PGPR) is proposed as a friendly solution to overcome those challenges. The desert plant endophytic bacterium, Enterobacter sp. SA187 has shown plant growth promotion and salt stress tolerance beneficial effect on the model plant Arabidopsis thaliana in vitro as well as under the field conditions on different crops. SA187 has a distinguished morphology of yellow colonies (SA187Y) that could be due to carotenoid biosynthesis. However, the bacteria tend to lose the yellow color upon incubation with the plants and the colonies turn to white (SA187W). In comparison to SA187Y, SA187W shows 50% reduction on the beneficial impact on A. thaliana fresh and dry weight of root and shoot system. By counting the CFU/plant, we showed that SA187Y and SA187W both have similar colonization rate in both shoots and roots. Under non-salt conditions, optimal bacterial colonization was observed on day 8 after inocubation, however, under the salt stress condition, the optimal colonization was observed at day 4. Moreover, during the time period of the incubation of the SA187Y with the plants, there was a consistent noticeable loss of the yellow color of the colonies. This change in color is only observed eight days after transfer and the number of white colonies increases with the increase of the incubation time. In addition, SA187W was GFP-tagged by Tn7 transposon system and visualized by confocal laser scanning microscopy. The SA187W-GFP colonies have shown a similar colonization pattern as SA187Y-GFP, bacteria were colonizing the differentiation zone and cell elongation zone in the roots. Finally, the gene expression of the carotenoid biosynthesis pathways genes in SA187Y showed an overall higher gene expression compared to SA187W. In conclusion, the color loss seems to affect the beneficial impact of the bacteria on plants. However, the reduced beneficial impact is not due to the colonization efficiency of bacteria on the plant roots but could be due to a regulation of gene expression of carotenoid biosynthesis.

Active disease resistance in plants is induced during the pathogen infection process that triggers multiple defense-related genes to establish broad-spectrum resistance. Several biotic and abiotic agents can mimic natural induced resistance (IR), categorized as systemic acquired (SAR) or induced systemic resistance (ISR). IR, triggered by acibenzolar-S-methyl (ASM) or plant growth-promoting rhizobacteria (PGPR), was evaluated on two-to-three types of tobacco in greenhouse and field studies. Tobacco mosaic virus (TMV) local lesion assays monitored induction and maintenance of ASM-induced SAR over a 21 day period via proportional reduction in the number of TMV local lesions between an untreated control and ASM-treated plants. Intraspecific variation in SAR was found among tobacco types; burley and flue-cured tobaccos responded by day 3, while oriental tobacco responded between day 3 and 6. The SAR signal was greatest between 6 and 15 days following ASM application, but IR was slightly evident even at 21 days after ASM application in all three tobacco types. Bottom and middle leaves responded similarly on all sample dates, but top leaves showed the weakest SAR response. Tobacco cyst nematode (TCN; Globodera tabacum solanacearum) is one of the most destructive pathogens of tobacco in Virginia. Among four PGPR combinations tested, a mixture of Bacillus amyloliquefaciens IN937a (GB99) and B. subtilis A13 (GB122) most consistently suppressed TCN reproduction in flue-cured and oriental tobacco. Application of ASM similarly reduced final numbers of TCN cysts, but also resulted in chlorosis, stunting, and lower plant fresh weight. GB99+GB122 also suppressed TCN development and reproduction in susceptible and resistant flue-cured cultivars, but reductions by ASM were less consistent. In a split-root trial, soil amendment with GB99+GB122 in one half of an oriental tobacco root system lowered final numbers of TCN more than did ASM. ASM exhibited undesirable effects in phytotoxicity trials in flue-cured and oriental tobacco, but GB99+GB122 was not phytotoxic. When oriental tobacco seedlings were grown in a GB99+GB122-treated soil-less media, a single application of 200 mg ASM/L one week after transplanting significantly suppressed TCN reproduction in the field without phytotoxicity. Further field research is needed to confirm this effect in flue-cured tobacco. / Ph. D.

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Ce projet explore l’hypothèse selon laquelle les gènes et les métabolites végétaux communément régulés joueraient un rôle majeur dans l’interaction riz-PGPR et constituerait une signature moléculaire de la perception des PGPR par le riz. Dans cet objectif, une analyse intégrant le suivi de l’expression d’une sélection de gènes ainsi que le profilage des métabolites secondaires a été conduite sur les racines d’un unique cultivar de riz (Nipponbare) en réponse à l’inoculation de dix souches de PGPR appartenant à divers genres bactériens (Azospirillum, Herbaspirillum, Paraburkholderia). Nos résultats ont permis l’identification de quatre gènes de riz pouvant être considérés comme marqueurs de l’association riz-PGPR, avec notamment deux gènes impliqués dans la biosynthèse de phytoalexines et un gène codant pour une protéine PR (pathogenesis-related). De plus, une signature métabolique commune, constituée de neuf composés, a été mise en évidence, dont la réduction de l’accumulation de trois alkylrésorcinols et l’augmentation de l’accumulation de deux amides d’acides hydroxycinnamiques (HCAA) : la N-p-coumaroylputrescine et la N-féruloylputrescine. Cette signature métabolique a été corrélée avec l’augmentation de l’expression de deux gènes impliqués dans la biosynthèse de la N-féruloylputrescine. Il est intéressant d’observer que la confrontation du riz à un pathogène bactérien entraine une réduction de l’accumulation de ces HCAA dans les racines. Cette accumulation d’HCAA, qui sont des composés antimicrobiens potentiels, pourrait être considérée comme une réaction primaire de la perception de bactéries par le riz / Besides, a common metabolomic signature of nine compounds was highlighted, with the reduced accumulation of three alkylresorcinols and increased accumulation of two hydroxycinnamic acid amides (HCAA), identified as N-p-coumaroylputrescine and N-feruloylputrescine. This coincided with the increased transcription of two genes involved in the N-feruloylputrescine biosynthetic pathway. Interestingly, exposure to a rice bacterial pathogen triggered a reduced accumulation of these HCAA in roots. Accumulation of HCAA, that are potential antimicrobial compounds, might be considered as a primary reaction of rice to bacterial perception