Isolation of disease-tolerant cotton (Gossypium hirsutum L. cv. SVPR 2) plants by screening somatic embryos with fungal culture filtrate

We report an in vitro selection method that has led to isolation of Fusarium wilt and Alternaria leaf spot disease-tolerant plantlets in cotton (Gossypium hirsutum L. cv. SVPR2). Embryogenic callus was isolated from hypocotyl explants of cotton cultured on 5–50% Fusarium oxysporum culture filtrate-fortified callus induction medium. Somatic embryos tolerant to fungal culture filtrate (FCF) were isolated from this embryogenic callus on somatic embryo regeneration medium fortified with 40% FCF. Sixteen plantlets were selected as FCF-tolerant from 34 somatic embryos tested, which corresponds to about 47% success rate. The FCF-tolerant plants were analyzed for disease tolerance by challenging them with spores of F. oxysporum and Alternaria macrospora. Four plants were selected as F. oxysporum tolerant from a total of 24 plants tested. The selected plants showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1 × 10⁵ spores/mL of F. oxysporum. From the FCF-tolerant plants, another nine randomly selected plantlets were challenged with spores of A. macrospora in order to test their tolerance to Alternaria leaf spot disease. The number of lesions per leaf significantly decreased from 8.2 to 0.9 and the lesion lengths were also reduced from 2.8 to 1.2 mm per leaf spot in these plants. Electrophoresis analysis of extracellular proteins from the FCF-tolerant plants showed enhanced secretion of proteins in the range of 24–36 kDa. Isozyme analysis by of FCF-tolerant plants by using native gels showed the presence of chitinase. Quantitative analysis showed that there was 13-fold increase in a chitinase activity in the selected FCF-tolerant plants compared to the control plants. Our results show that over-expression of chitinase enzyme leads to enhanced disease resistance against F. oxysporum and A. macrospora.     

Increased Growth and Yield of Hydroponically Grown Greenhouse Tomato Plants Inoculated with Arbuscular Mycorrhizal Fungi and Fusarium oxysporum f. sp. radicis-lycopersici

The arbuscular mycorrhizal fungi Glomus monosporum, G. vesiculiferum, G. deserticola, G. intraradices, G. mosseae, and two unidentified species were tested to determine their effect on plant growth and fruit production of tomato (Lycopersicon esculentum Mill.) cv. Trust inoculated with Fusarium oxysporum f. sp. radicis-lycopersici (FORL) under near-commercial greenhouse conditions. Inoculation with G. monosporum and G. mosseae significantly increased fruit yield and fruit number of tomato plants grown hydroponically in sawdust. Plant height and plant dry weight increased significantly when inoculated with G. monosporum and G. mosseae. Further, plants inoculated with G. monosporum and G. mosseae showed significantly lower FORL root infection than the untreated control plants.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f. sp.dianthi (Prill and Del.) Snyd. and Hans

Summary Fusarium oxysporum f. sp.dianthi, pathogenic on carnation plants is very sensitive toBacillus subtilis M51 inhibition.Fusarium oxysporum disease (fusariosis) is prevented for a period of two months after treatment of plants withBacillus subtilis M51. The persistence ofB. subtilis M51, marked for selenomycin resistance (MZ51) and inoculated on the roots of carnation cuttings was studied. Soil used was two types: naturally infested withFusarium oxysporum and free from this pathogen. Bacterial cells presence on the roots was detected by direct plating and the presence of the pathogen in the roots was investigated by histological assays. Evidence gathered by these procedures suggest that plant protection is dependent on the physical presence ofB. subtilis M51 cells on the roots.     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.     

A novel Agrobacterium rhizogenes-mediated transformation method of soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings

A novel Agrobacterium rhizogenes-mediated transformation method using a primary-node explant from Dairyland cultivar 93061 was developed for soybean using the disarmed Agrobacterium strain SHA17. Transformed plants regenerated from explants inoculated with SHA17 were fertile and phenotypically normal. In a comparative experiment, regeneration frequencies were not significantly different between explants inoculated with A. rhizogenes strain SHA17 and Agrobacterium tumefaciens strain AGL1; however, a 3.5-fold increase in transformation efficiency [(number of Southern or TaqMan-positive independent events/total number of explants inoculated) × 100] was found for explants cocultured with SHA17 compared to AGL1 (6.6 and 1.64%, respectively). Southern analysis of 48 T₀ plants suggested that 37.5, 23, and 39.6% of the T₀ plants contained 1, 2, and 3 or more T-DNA fragments integrated into the genome, respectively. Additionally, T₁ progeny analysis of 8 independent events resulted in typical Mendelian inheritance of T-DNA genes. Of seven T₀ plants that had two or more T-DNA fragments, six contained multiple loci segregating in T₁ progenies. Further analysis of four lines confirmed the presence of PAT, GUS, and/or DsRED2 proteins in transgenic plants that were encoded on the T-DNA into the T₂ generation.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f.sp.dianthii (Prill and Del.) Snyd. and Hans.

Summary Thirty two bacteria antagonistic to a number of phytopathogenic fungi were isolated from soil samples. One bacterial strain, designated as M 51, appeared to be particularly active towardsF. oxysporum f. sp.dianthii, in vitro andin vivo and it was inhibitoryin vitro to three otherFusarium spp. used. Tests to find if there was protection against fusarium wilt were carried out by three different methods of inoculation of the cuttings: a) dipping of cuttings for ten minutes in bacterial suspension; b) spraying of suspension on perlite where the rooted cuttings were planted; c) spraying the greenhouse bench rooting boxes, where the non-rooted cuttings were planted, with bacterial suspension. Following this all the cuttings were transplanted into soil naturally highly infested withFusarium oxysporum f. sp.dianthii (3000 units/g). Good protection against fusarium wilt was obtained for cuttings inoculated by method (b). However protection decreased gradually about 60 days after they were transplanted; both control and inoculated cuttings showed a comparable mortality rate. Method of inoculation and the development of the protective effect are discussed.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

Effects of supplementary UV-B radiation on development of damping-off in spinach caused by the soil-borne fungusFusarium oxysporum

The effects of UV-B radiation (290–320 nm) on development of damping-off of spinach (Spinacia oleracea) caused by the fungusFusarium oxysporum were examined in a growth cabinet. The incidence of disease greatly increased when experimental plants were grown in visible radiation with supplementary UV-B radiation. This increase was suppressed by increasing the irradiation of visible radiation.Fusarium oxysporum was isolated from the roots of all damping-off plants and the roots of some unwilted plants, indicating that spinach infected with the pathogen did not necessarily suffer from damping-off in 15d. Supplementary UV-B radiation suppressed the increase in growth components such as the number of leaves, the plant height and the fresh weight of aboveground plant parts, but did not affect the fresh weight of roots. The ratio of the number of plants infected with pathogen to the total number of plants was over 80% irrespective of light conditions. It was suggested that the defense response of spinach to this pathogen was greatly influenced by the physiological state of aboveground plant parts resulting from supplementary UV-B radiation.     

Colonization of root tissues and protection against Fusarium wilt of rye (Secale cereale) by nonpathogenic rhizosphere strains of Fusarium culmorum

Colonization of rye (Secale cereale) tissues by nonpathogenic rhizosphere Fusarium culmorum isolates DEMFc2 and DEMFc5 and a pathogenic strain DEMFc37, and their effect on plant fresh weight were studied in pot experiments. Both rhizosphere isolates colonized the epidermis and the cortex but were not found in vessels, while the pathogen colonized all three layers of root cells. The numbers of pathogen CFU isolated from plant tissues were much higher than those of the rhizosphere isolates in spite of the same number of macroconidia used as inoculum (1 × 10⁵ g⁻¹ of soil). Inoculation of seedlings with DEMFc2 resulted in a 20% increase, with DEMFc5 in more than a 20% reduction, and with DEMFc37 in a 38% reduction of shoot fresh weight of 14-day-old plants. Pre-colonization of plants with (either of) the rhizosphere isolates and subsequent inoculation with the pathogen resulted in plant weights the same as those observed in plants inoculated with the rhizosphere strain alone. The disease severity index for shoots of plants pre-colonized with DEMFc2 was reduced from class 4 (86% diseased plants) observed for plants inoculated with the pathogen alone to class 2 (average of 8% diseased plants) when pre-treated with the rhizosphere strain. The CFU number of the pathogen isolated from the interior of roots of plants pre-colonized with the rhizosphere isolates was as low as 10% of the number isolated from plants inoculated with the pathogen alone. A study of in vitro interactions between the rhizosphere isolates and the pathogen suggests that changes in plant colonization by the pathogen and its effect on fresh weight of plants pre-colonized with the rhizosphere isolates were not connected with inhibition of its growth by a direct action of the rhizosphere isolates. The results suggest that strain DEMFc2 can be considered as a potential biocontrol agent.     

Effect of Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii on the pathogenicity of Meloidogyne incognita race 2 to soybean

Screenhouse studies were conducted to investigate the effects of Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii on the pathogenicity of Meloidogyne incognita race 2 on soybean and the influence of the nematode on wilt incidence and growth of soybean. The interaction of each fungus with the nematode resulted in reduced shoot and root growth. Final nematode population was also reduced with concomitant inoculation of nematode and fungus or inoculation of fungus before nematode. While M. incognita suppressed wilt incidence in two nematode-susceptible cultivars of soybean (TGX 1485-2D and TGX 1440-IE), it had limited effect on wilt incidence in the nematode resistant cultivar of soybean (TGX 1448-2E). When F. oxysporumwas inoculated with the nematode, the mean number of nematodes that penetrated soybean roots decreased by 75% in TGX 1448-2E, 68% in TGX 1485-1D and 65% in TGX 1440-1E. Similarly when the soil was treated with S. rolfsii, the number decreased by 78% in TGX 1448-2E, 77% in TGX 1485-1D and 68% in TGX 1440-1E. The nematode did not develop beyond second-stage juvenile in TGX-1448-2E.     

Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action

The bacterial strain E1R-j, isolated as an endophyte from wheat roots, exhibited high antifungal activity to Gaeumannomyces graminis var. tritici (Ggt). Strain E1R-j was identified as Bacillus subtilis based on morphological, physiological and biochemical methods as well as on 16S rDNA analysis. This strain inhibited mycelium growth in vitro of numerous plant pathogenic fungi, especially of Ggt, Coniothyrium diplodiella, Phomopsis sp. and Sclerotinia sclerotiorum. In greenhouse experiments, soil drenches with cell densities of 10⁶, 10⁹ and 10¹² CFU ml⁻¹ E1R-j reduced significantly take-all disease, caused by Ggt, in wheat seedling by 62.6%, 68.6% and 70.7%, respectively, compared to the inoculated control, 4 weeks after sowing. Growth parameters such as lengths and fresh weights of roots and shoots of Ggt-inoculated control plants were significantly lower compared to Ggt-inoculated and E1R-j treated plants. Field experiments in the season 2006/2007, heights of wheat plants in the Ggt inoculated plots were significantly reduced compared to the non inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in inoculated control plants were lower compared to the other treatments. In the experimental year 2007/2008, independent treatments with the bacterial strain E1R-j and the fungicide Triadimefon reduced take-all disease in wheat roots by 55.3% and 61.9%, compared to the inoculated control plants. In this season plant height in inoculated control was significantly lower and also the yield parameters seeds per head and especially TKW were drastically reduced compared to the other treatments. E1R-j treatment alleviated the detrimental effects of take-all on grain yield parameters to a similar extent as Triadimefon application. SEM studies revealed that in the presence of E1R-j, hyphae of Ggt showed leakage, appeared ruptured, swollen and shriveled. Following root drench, strain E1R-j was able to colonize endophytically roots and leaves of wheat seedlings. While the population of the bacterial strain in wheat roots steadily increased from the second to the fourth leaf stage, in the leaf tissue the population of the strain rapidly declined. TEM studies also showed that cells of E1R-j were present in roots of wheat seedlings and effectively retarded infection and colonization of Ggt in root tissue; suppression of Ggt by E1R-j was accompanied by disintegration of hyphal cytoplasm. In addition, in the presence of E1R-j cells in Ggt-infected root tissue morphological defense reactions were triggered such as formation of wall appositions and papillae. The results presented indicate that the endophytic strain E1R-j of B. subtilis meets demands required for biocontrol of take-all.     

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

Long-term effects of nitrate on lucerne (Medicago sativa L.) nitrogen fixation is not influenced by the denitrification status of the microsymbiont

Studies on the inhibitory effects of combined nitrogen on biological nitrogen fixation in legume crops have been usually carried out after short-term nitrate treatments at high concentrations. As these treatments are quite different from field conditions, a study was conducted to evaluate the effects of the continuous presence of nitrate (0, 1, 5 and 10 mM) throughout three months on lucerne (Medicago sativa L.). Plants were grown in a greenhouse with perlite as substrate and were inoculated with a denitrifying Sinorhizobium meliloti strain (102-F-51) and a non-denitrifying strain (102-F-65). During the first 60 days of growth, the highest nitrate treatment resulted in a complete inhibition of the main symbiotic parameters (nodule initiation and development and specific nitrogen fixation) in plants inoculated with either strain. However, after 3 months of growth in the presence of nitrate, this inhibition was partly abolished, with a high number of new functioning nodules being formed. Acetylene reduction activity (ARA) of these plants was 70% of the control plants. As this process was observed in plants nodulated with either strain, it is concluded that this was not related to the denitrifying ability of the strain, but is an intrinsic property of the lucerne nitrogen fixing system. As legume plants usually grow under natural field conditions in the continuous presence of nitrate, the ability to use simultaneously nitrate and atmospheric nitrogen could be of adaptive and agronomic importance. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense on Induction of Defense Enzymes and Phenolics in Banana

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, -1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 – 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 – 9 d after P. fluorescens treatment. PAL, chitinase and -1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.     

Selection of Rhizobium leguminosarum bv. viceae strains for inoculation of Pisum sativum L. cultivars: Analysis of symbiotic efficiency and nodulation competitiveness

From an analysis of 481 Rhizobium leguminosarum bv. viceae strains with 7 pea cultivars in pot and field experiments, we demonstrated that effective strains could be isolated from a rich medium-acid grey forest soil of the Oröl area (Central region of the European part of Russia) but not from a poor acid podzolic soil of the St. Petersburg area (North-West Russia). The proportion of the isolates significantly increasing N accumulation in pea plants (10.2%) is higher than that of strains increasing the shoot dry mass (4.6%) in the pot experiments. The mean values of the increase for N accumulation (33.8%) upon inoculation are also higher than for shoot mass (27.0%) in these experiments. N accumulation in the inoculated pea plants in the pot experiments was significantly correlated with seed yield and seed N accumulation in field experiments, while for shoot dry mass these correlations were either weak or not significant. Two-factor analysis of variance demonstrated that the contribution of plant cultivars to the variation of the major symbiotic efficiency parameters is higher (30.8–31.6%) and contributions of cultivar-strain specificity is lower (5.4–8.8%) than the contributions of strain genotypes (13.4–14.9%). We identified an ineffective R. leguminosarum bv. viceae strain 50 which can be used as a tester for assessing the nodulation competitiveness of the effective strains by an indirect method (analysis of dry mass and N accumulation in pea plants inoculated with the mixture of the tested effective strains and the tester strain). The relative competitive ability (RCA) determined by this method was 75.7–82.8% for strain 52 but only 10.5–13.8% for strain 250a; this difference was confirmed by a direct method (use of the streptomycin-resistant mutants). Results of screening of the diverse collection of 53 effective R. leguminosarum bv. viceae strains by the indirect method permits us to divide them into 3 groups (32 high-competitive, 10 medium-competitive and 11 low-competitive strains) but reveals no correlation between the competitiveness and symbiotic efficiency. N accumulation in the pea shoots is demonstrated to be a much more suitable criterion than the shoot mass for selection either of the highly-effective or of highly-competitive (by the indirect estimation) R. leguminosarum bv. viceae strains in the pot experiments.     

Effect of the saprophytic fungus Fusarium oxysporum on arbuscular mycorrhizal colonization and growth of plants in greenhouse and field trials

Effects of the saprophytic fungus Fusarium oxysporum on arbuscular mycorrhizal (AM) colonization and plant dry matter were studied in greenhouse and field experiments. Host plants: maize (Zea mays L.), sorghum (Sorghum vulgare L.), lettuce (Lactuca sativa L.), tomato (Lycopersicum esculentum L.), wheat (Triticum vulgare L), lentil (Ervum lens L.) and pea (Pisum sativum L.), the AM fungi: Glomus mosseae, G. fasciculatum, G. intraradices, G. clarum, and G. deserticola and the carriers for F. oxysporum inoculum: aqueous solution, thin agar slices, and pellets of agar and alginate were tested under greenhouse conditions. Greatest plant growth and AM colonization responses in sterilized and unsterilized soils were observed with pea, Glomus deserticola and sodium alginate pellets as the carrier for F. oxysporum inoculum. Under field conditions, adding F. oxysporum increased the survival of transplanted pea, possibly through a beneficial effect on AM fungi. Application of F. oxysporum increased shoot dry matter, N and P concentrations of pea and sorghum plants, and the level of AM colonization attained by indigenous or introduced AM fungi. These parameters were similar in plants inoculated with either G. deserticola or with the indigenous AM fungi. Application of the saprophytic fungus increased the number of propagules of AM fungi in field plots in which pea was grown, but this increase was not sufficient to increase AM colonization of sorghum after the pea crop. This revised version was published online in June 2006 with corrections to the Cover Date.     

Formation of nodular structures on the non-legumes Brassica napus,B. campestris,B. juncea and Arabidopsis thaliana with Bradyrhizobium and Rhizobium isolated from Parasponia spp. or legumes grown in tropical soils

Strains of Bradyrhizobium formed nodule-like structures on Arabidopsis and species of Brassica in pots with sandvermiculite and in glass tubes on a nitrogen-free mineral salts agar. Broad-host-range Rhizobium strains NGR234 from Lablab purpureus and NGR76 from Phaseolus vulgaris formed similar nodule-like structures on Brassica spp. The size of these structures on plants in pots were large, often reaching 10 mm in diameter.The frequency of inoculated Brassica plants in pots with nodule-like structures was 25–50%, depending on the inoculum strain. The inheritable nature of factors involved in the formation of the nodule-like structures was demonstrated when the structures occurred on 100% of inoculated B. napus seedlings derived from plants with the nodule-like structures.Nodule-like structures occurred without, but not with, the application of a cellulase-pectolyase-PEG treatment to the roots. Attempts to isolate Bradyrhizobium or Rhizobium from the nodule-like structures failed. Internal infection of these structures could not be detected using either the light or electron microscope. The inoculum strains of root-nodule bacteria were detected in high numbers in the rhizosphere of plants 5 months after inoculation. On agar plates bacterial colonies could be seen, with undiminished growth, over the surface of the agar extending to the root surface. However, ground root tissue of Brassica was toxic to Bradyrhizobium strains. This suggested that Bradyrhizobium strains would not survive after infecting the roots of Brassica spp. Nitrogen fixation was associated with high rhizosphere populations of Azospirillum and not with Bradyrhizobium induced nodule structures of Brassica spp.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.