Seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of Rhopalosiphum padi

The concentrations and composition of free amino acids in phloem sap from two cultivars of oats and barley, both susceptible to the aphid Rhopalosiphum padi, were determined by means of high performance liquid chromatography. Sap was collected from excised aphid stylets at three developmental stages (seedlings, tillering plants and plants undergoing stem elongation) from plants given or not given fertiliser and grown outdoors. In connection, the growth of individual R. padi nymphs was estimated at the same phenological stages on plants grown in the greenhouse. The content of free amino acids was consistently higher in seedlings than in plants at the early tillering stage. Only in seedlings did the addition of fertiliser increase amino acid levels. Barley phloem sap contained more free amino acids than that of oats when fertiliser was added and at later developmental stages. Phloem sap of oats and barley showed similar patterns in their composition of free amino acids at the seedling stage, but as the plants grew older the patterns became increasingly different. Plants given fertiliser had higher amounts of dicarboxylic amino acids (glutamic and aspartic acid) than unfertilised plants. The concentrations of γ-amino butyric acid, glycine, histidine, and methionine were very low in all treatments. The relative growth rates of R. padi nymphs were low when amino acid content was low and vice versa. The results are discussed in relation to host plant suitability and plant resistance mechanisms.     

Rhizobacteria that produce auxins and contain 1‐amino‐cyclopropane‐1‐carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well‐watered and water‐limited potato (Solanum tuberosum)

Plant‐growth‐promoting rhizobacteria (PGPR) utilise amino acids exuded from plant root systems, but hitherto there have been no direct measurements of rhizosphere concentrations of the amino acid 1‐amino‐cyclopropane‐1‐carboxylic acid (ACC) following inoculation with PGPR containing the enzyme ACC deaminase. When introduced to the rhizosphere of two potato (Solanum tuberosum) cultivars (cv. Swift and cv. Nevsky), various ACC deaminase containing rhizobacteria (Achromobacter xylosoxidans Cm4, Pseudomonas oryzihabitans Ep4 and Variovorax paradoxus 5C‐2) not only decreased rhizosphere ACC concentrations but also decreased concentrations of several proteinogenic amino acids (glutamic acid, histidine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine). These effects were not always correlated with the ability of the bacteria to metabolise these compounds in vitro, suggesting bacterial mediation of root amino acid exudation. All rhizobacteria showed similar root colonisation following inoculation of sand cultures, thus species differences in amino acid utilisation profiles apparently did not confer any selective advantage in the potato rhizosphere. Rhizobacterial inoculation increased root biomass (by up to 50%) and tuber yield (by up to 40%) in pot trials, and tuber yield (by up to 27%) in field experiments, especially when plants were grown under water‐limited conditions. Nevertheless, inoculated and control plants showed similar leaf water relations, indicating that alternative mechanisms (regulation of phytohormone balance) were responsible for growth promotion. Rhizobacteria generally increased tuber number more than individual tuber weight, suggesting that accelerated vegetative development was responsible for increased yield.     

Interaction between barley and mixed cultures of nitrogen fixing and phosphate-solubilizing bacteria

Pot experiments were carried out to investigate the effect of inoculation with pure and mixed cultures of nitrogen fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7 and the phosphate-solubilizing strain Agrobacterium radiobacter 10 on growth and mineral nutrition of two barley cultivars. A significant positive effect on grain yield both of the studied barley cultivars was obtained after inoculation with mixtures of A. lipoferum 137 + A. radiobacter 10 and A. lipoferum 137 + A. mysorens 7 only. The acetylene reduction activity on roots or in batch culture was significantly higher when A. lipoferum 137 and A. radiobacter 10 were combined. Using ¹⁵N isotope dilution technique it was established that these mixed cultures significantly increased the accumulation of nitrogen fertilizer in the plants. The strain A. radiobacter 10 promoted a better accumulation of phosphorus fertilizer by plants and A. mysorens 7 increased the total phosphorus content in plant tissues. The maximum positive effect of joint inoculation on plant development was observed when the combined nitrogen in soil was in short supply. It was concluded that inoculation with bacterial mixtures provided a more balanced nutrition for the plants and the improvement in root uptake of nitrogen and phosphorus was the major mechanism of interaction between plants and bacteria. The introduced bacteria were able to colonize actively the rhizoplane of barley. No interspecific competition or antagonism were established between components of the bacterial mixtures in the rhizoplane. The strains A. mysorens 7 and A. radiobacter 10 improved viability of A. lipoferum 137 when the plants were grown in acid soil. Field experiments carried out on 3 barley cultivars confirmed the assertion that inoculation with mixed cultures significantly increases the grain yield and nitrogenous nutrition of plants as compared with single cultures.     

Zinc hyperaccumulation substitutes for defense failures beyond salicylate and jasmonate signaling pathways of Alternaria brassicicola attack in Noccaea caerulescens

The hypothesis of metal defense as a substitute for a defective biotic stress signaling system in metal hyperaccumulators was tested using the pathosystem Alternaria brassicicola–Noccaea caerulescens under low (2 µM), medium (12 µM) and high (102 µM) Zn supply. Regardless the Zn supply, N. caerulescens responded to fungal attack with the activation of both HMA4 coding for a Zn transporter, and biotic stress signaling pathways. Salicylate, jasmonate, abscisic acid and indoleacetic acid concentrations, as well as biotic stress marker genes (PDF1.2, CHIB, LOX2, PR1 and BGL2) were activated 24 h upon inoculation. Based on the activation of defense genes 24 h after the inoculation an incompatible fungal–plant interaction could be predicted. Nonetheless, in the longer term (7 days) no effective protection against A. brassicicola was achieved in plants exposed to low and medium Zn supply. After 1 week the biotic stress markers were even further increased in these plants, and this compatible interaction was apparently not caused by a failure in the signaling of the fungal attack, but due to the lack of specificity in the type of the activated defense mechanisms. Only plants receiving high Zn exhibited an incompatible fungal interaction. High Zn accumulation in these plants, possibly in cooperation with high glucosinolate concentrations, substituted for the ineffective defense system and the interaction turned into incompatible. In a threshold‐type response, these joint effects efficiently hampered fungal spread and, consequently decreased the biotic stress signaling.     

Augmentation of AM fungi fails to ameliorate the adverse effects of temporal resource variation on a lettuce crop

While agricultural research has traditionally focused on average environmental conditions, environmental variability, independent of the mean, can also have biological consequences. Using lettuce (Lactuca sativa) as a model system, we tested two hypotheses: (1) increased temporal variability in water supply impacts plant growth, yield, photosynthesis, water relations and nutrition and (2) arbuscular mycorrhizal AM fungal associations benefit this agricultural crop, especially when plants experience temporal variability in water supply. The experiment used a randomized complete block design with two blocks and three variables (each with two levels): ± mycorrhizal inoculation, high or low variability in watering intervals, and high or low total watering volume. Temporal variability in water supply, at a time scale similar to what is common in agricultural practices, had negative effects on lettuce production. Inoculation treatments were successful in doubling the extent of AM fungal infection in lettuce roots. There were no main effects of mycorrhizal inoculation on any measured variable, but augmented mycorrhizal associations interacted with variability in water supply to increase root/shoot ratios and decrease tissue concentrations of N and P. Successful application of AM fungi to sustainable agriculture probably requires a general theoretical framework for predicting when effects on plants will be beneficial versus neutral or even detrimental.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Sebacina vermifera</Emphasis> increase growth performance at the expense of herbivore resistance in <Emphasis Type="Italic">Nicotiana attenuata</Emphasis>

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.     

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

Low-level herbivory by root-knot nematodes (Meloidogyne incognita) modifies root hair morphology and rhizodeposition in host plants (Hordeum vulgare)

Low amounts of root infestation by plant parasitic nematodes are suggested to increase nutrient supply and in turn enhance microbial activity and net mineralization rate in the rhizosphere. These effects are generally related to “leakage” of plant-derived metabolites from damaged roots. Besides leakage, the present study examines other nematode–host interactions such as alterations in root exudation and morphology, which were almost not considered yet. This includes undamaged root parts in order to assess systemic plant response. The root-knot nematode Meloidogyne incognita (Kofoid and White 1919; Chitwood 1949) and barley (Hordeum vulgare L. cv. Europa) was used as model system. Host plants were grown in mini-rhizotrons inoculated with 0, 2,000, 4,000 or 8,000 M. incognita for 4 weeks. Root morphology, rhizodeposition (sugars, carboxylates, amino acids), and rhizosphere microbial communities (PLFAs) were assessed. In treatments with 4,000 nematodes, shoot biomass, total N and P content increased by the end of the experiment. Generally, an enhanced release of plant metabolites (sugars, carboxylates, amino acids) from the apical root zone occurred 1 week after inoculation with 4,000 and 8,000 M. incognita, indicating root leakage. Low levels of root herbivory stimulated root hair elongation in both infected and uninfected roots. These systemic changes in root morphology likely contributed to the increased sugar exudation in uninfected roots in all nematode treatments at 3 weeks after inoculation. Root-knots formed a separate microhabitat within the root-system. They were characterised by decreased rhizodeposition and increased fungal to bacterial ratio in the adhering rhizosphere soil. The present study provides the first evidence that, apart from leakage, nematode root herbivory at background levels induces local and systemic effects on root morphology and exudation, which in turn may affect plant performance.     

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Québec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha^-1 P-superphosphate, or with strain 68 plus 70 kg ha^-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha^-1 P-superphosphate, or with strain P31 plus 35 kg ha^-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.     

The Utilization of Carbohydrate and Nitrogen Reserves by Taxus During Its Spring Growth Period

The spring growth and the utilization of carbohydrate and nitrogen reserves in this growth was studied in Taxus media cv. Hicksii plants 0, 2, 4 and 6 weeks after the plants started growing in the spring. The effect of nitrogen applied the previous season on the storage and utilization of the carbohydrate and nitrogen reserves during spring growth was determined. The plants were separated into buds (all new growth), stems, needles (those produced the previous season) and roots and analyzed for changes in total nitrogen, basic and non-basic amino acids, total available carbohydrate, sugars, hemicelluloses, organic acids and chlorophyll. The bulk of the soluble nitrogen reserves were stored as arginine in the stems and old needles. With the onset of spring growth, arginine nitrogen was converted to other amino acids which accumulated in the new growth (buds). The roots, stems and needles of plants grown under high nitrogen levels always contained more total nitrogen than those grown under low nitrogen levels. The bulk of the carbohydrate reserves were stored as hemicelluloses. The plants grown under high nitrogen levels utilized the bulk of the carbohydrate reserves from the roots and smaller amounts from the stems and old needles, while plants grown under low nitrogen levels used only the reserves in the roots. In the low nitrogen plants, carbohydrates accumulated in the needles and stems. Both the carbohydrate and nitrogen reserves were important in the dry weight increase due to spring growth. However, the nitrogen reserves were the limiting factor and the high nitrogen plants grew twice as much, produced more chlorophyll, and utilized more nitrogen and carbohydrate reserve in spring growth than low nitrogen plants. The additional chlorophyll allowed the production of more carbohydrates and these additional carbohydrates were used in increased growth rates, while in the low nitrogen plants the carbohydrate produced was less and accumulated within the plant.     

The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrs1 resistance genotype.

NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product.     

Antioxidative and growth-promoting effect of selenium on senescing lettuce

In human and animal cells, Se plays an essential role in antioxidation and exerts an antiaging function but it is toxic at high dietary intake. To increase its intake in forage and foodstuffs, Se fertilization is adopted in some countries where soils are low in bioavailable Se, even though higher plants are regarded not to require Se. To test its ability to counteract senescence-related oxidative stress in higher plants, a pot experiment was carried out with lettuce (Lactuca sativa) cultivated with increasing amounts of H₂SeO₄. The yields harvested 7 or 14 weeks after sowing revealed that a low Se dosage (0.1 mg kg^–1 soil) stimulated the growth of senescing seedlings (dry weight yield by 14%) despite a decreased chlorophyll concentration. The growth-promoting function was related to diminished lipid peroxidation. In young and senescing plants, the antioxidative effect of Se was associated with the increased activity of glutathione peroxidase (GSH-Px). In the senescing plants, the added Se strengthened the antioxidative capacity also by preventing the reduction of tocopherol concentration and by enhancing superoxide dismutase (SOD) activity. When no Se was added, tocopherols and SOD activity diminished during plant senescence. The higher Se dosage (1.0 mg kg^–1 soil) was toxic and reduced the yield of young plants. In the senescing plants, it diminished the dry weight yield but not the fresh weight yield.     

Growth Enhancement,Amino Acid Synthesis and Reduction in Susceptibility Towards Phytophthora megakarya by Arbuscular Mycorrhizal Fungi Inoculation in Cocoa Plants

The effects of some selected arbuscular mycorrhizal (AM) fungi, Gigaspora margarita and Glomus mossae on the growth and the role of soluble amino acids of two contrasting cocoa cultivars (ICS84 tolerant and SNK10 sensitive) against black pod disease caused by Phytophthora megakarya were investigated. Root colonization by AM fungi is between 50 and 70% 18 weeks after planting. Tested AM fungi significantly increased all the plant growth parameters (height, number of leaves, shoot and root matter) and P uptake as compared to non‐inoculated plants in pot experiments. AM fungi inoculated cocoa reduced the disease severity. Compared to the control, the soluble amino acid levels increased with inoculation of the AM fungi strains in the necrotic stems of disease on inoculated cocoa plants. Significant relationships between amino acids and disease severity observed for two cocoa cultivars imply that the induction of specific amino acids synthesized by leaves, such as arginine, cysteine and glutamic acid, may represent potential candidate molecules for adaptation of such cultivars to P. megakarya disease. Inoculating seedlings with AMF in nurseries could enhance the development of cocoa plants protected against P. megakarya.     

Response of barley grains to the interactive e.ect of salinity and salicylic acid

Effect of grain soaking presowing in 1 mM salicylic acid (SA) and NaCl (0, 50, 100, 150 and 200 mM) on barley (Hordeum vulgare cv Gerbel) was studied. Increasing of NaCl level reduced the germination percentage, the growth parameters (fresh and dry weight), potassium, calcium, phosphorus and insoluble sugars content in both shoots and roots of 15-day old seedlings. Leaf relative water content (RWC) and the photosynthetic pigments (Chl a, b and carotenoids) contents also decreased with increasing NaCl concentration. On the other hand, Na, soluble sugars, soluble proteins, free amino acids including proline content and lipid peroxidation level and peroxidase activity were increased in the two plant organs with increasing of NaCl level. Electrolyte leakage from plant leaves was found to increase with salinity level. SA-pretreatment increased the RWC, fresh and dry weights, water, photosynthetic pigments, insolube saccharides, phosphorus content and peroxidase activity in the stressed seedlings. On the contrary, Na⁺, soluble proteins content, lipid peroxidation level, electrolyte leakage were markedly reduced under salt stress with SA than without. Under stress conditions, SA-pretreated plants exhibited less Ca²⁺ and more accumulation of K⁺, and soluble sugars in roots at the expense of these contents in the plant shoots. Exogenous application (Grain soaking presowing) of SA appeared to induce preadaptive response to salt stress leading to promoting protective reactions to the photosynthetic pigments and maintain the membranes integrity in barley plants, which reflected in improving the plant growth.     

Metabolic pathways regulated by abscisic acid,salicylic acid and γ‐aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera)

Abscisic acid (ABA), salicylic acid (SA) and γ‐aminobutyric acid (GABA) are known to play roles in regulating plant stress responses. This study was conducted to determine metabolites and associated pathways regulated by ABA, SA and GABA that could contribute to drought tolerance in creeping bentgrass (Agrostis stolonifera). Plants were foliar sprayed with ABA (5 μM), GABA (0.5 mM) and SA (10 μM) or water (untreated control) prior to 25 days drought stress in controlled growth chambers. Application of ABA, GABA or SA had similar positive effects on alleviating drought damages, as manifested by the maintenance of lower electrolyte leakage and greater relative water content in leaves of treated plants relative to the untreated control. Metabolic profiling showed that ABA, GABA and SA induced differential metabolic changes under drought stress. ABA mainly promoted the accumulation of organic acids associated with tricarboxylic acid cycle (aconitic acid, succinic acid, lactic acid and malic acid). SA strongly stimulated the accumulation of amino acids (proline, serine, threonine and alanine) and carbohydrates (glucose, mannose, fructose and cellobiose). GABA enhanced the accumulation of amino acids (GABA, glycine, valine, proline, 5‐oxoproline, serine, threonine, aspartic acid and glutamic acid) and organic acids (malic acid, lactic acid, gluconic acid, malonic acid and ribonic acid). The enhanced drought tolerance could be mainly due to the enhanced respiration metabolism by ABA, amino acids and carbohydrates involved in osmotic adjustment (OA) and energy metabolism by SA, and amino acid metabolism related to OA and stress‐defense secondary metabolism by GABA.     

Interactions between Azotobacter chroococcum and vesicular-arbuscular mycorrhiza and their effects on plant growth

B rown , M. E. & G arr , G. R. 1984 Interactions between Azotobacter chroo-coccum and vesicular-arbuscular mycorrhiza and their effects on plant growth. Journal of Applied Bacteriology 56 , 429–437.
The effects of simultaneous inoculation of roots of lettuce seedlings with vesicular-arbuscular endophytes and Azotobacter chroococcum are described. Endophytes alone increased yields of lettuce grown in partially sterilized P-deficient soil, but not in the same unsterile soil. Dual inoculation with endophytes and Azotobacter in both soils produced larger plants than either inoculum alone. In another poorly structured unsterile soil with adequate P for plant growth, endophytes depressed yields of lettuce. There was no association between endophytes and Azotobacter . In none of the experiments did Azotobacter influence the level of endophyte infection in the roots; but numbers of Azotobacter on the root systems were decreased in the presence of the endophytes. Azotobacter alone always increased growth of young plants before the effects of the endophytes were observed.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.