Analysis of factors limiting associative N2-Fixation (C2H2 reduction) with two cultivars of Sorghum nutans

A 5–10 times larger nitrogenase activity per plant (max. 8 μmol C₂H₄ h⁻¹ plant⁻¹) was found in Sorghum nutans cultivar CSV 5 compared to the cultivar IS 5218 (max. 0.9 μmol C₂H₄ h⁻¹ plant⁻¹). This significant difference was reproduced for all water regimes from 8 to 21% soil moisture. The number of chemoorganotrophic bacteria (“total cell number”) on a medium with six carbon sources was very similar for both cultivars (3−4 × 10⁸ cells g⁻¹ rhizosphere soil). On the other hand, in the “high fixing” variety CSV 5 the number of N₂-fixing bacteria was greater than in IS 5218 by a factor of 3, the number of actinomycetes was reduced ten-fold and Arthrobacter strains to one-third or one-quarter. The number of aerobic N₂-fixing bacteria growing in an O₂ gradient system was increased 100-fold. Cultivar CSV 5 has a reduced photosynthetic area (20–30%), a reduced root weight (up to 50%, depending on the water regime) and a reduced transpiration rate (30–40%) compared to cv. IS 5218. Grain production however is 10–20% greater. At all soil moisture regimes the pH in the rhizosphere of cv. CSV 5 is 0.5–1.5 pH units below the values for cv. IS 5218.     

Fluorescent antibody technique to identify Azospirillum brasilense associated with roots of grasses

Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N₂-fixing ability was demonstrated by C₂H₂ reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum). These N₂-fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N₂-fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N₂-fixing bacteria associated with grass roots.     

Occurrence and characterization of nitrogen-fixing Azospirilla in some soils of Egypt

The occurrence and characterization of N₂-fixing azospirilla in some Egyptian soils has been investigated. Seven soils, representing a wide range in texture and properties were selected from different localities in Egypt. The highest nitrogenase activity reported for soil samples under investigation were related to numbers of N₂-fixing microorganisms (Azotobacter spp., Azospirillum spp., and Clostridium spp.). Seven strains of azospirilla were isolated and purified. Based on morphological characteristics, three types of cell morphology were distinguished. Cultural and physiological characteristics as well as nitrogenase activity of representative isolates in presence of different concentrations of NaCl were determined. According to the physiological properties studied, all isolates were classified as members of Azospirillum brasilense.     

Isolation,partial identification and application of diazotrophic rhizobacteria from traditional Indian rice cultivars

A diversity of N₂-fixing (diazotrophic) bacteria was isolated from two traditional rice cultivars, Sataria and Kartiki, from the rice growing area of Mithila region of North Bihar, India, where low levels of nitrogen fertilizers are applied. Nitrogen-free semisolid media NFb, JMV and LGI with different carbon sources and pH-values were used for enrichment and isolation of root-associated diazotrophs. The colonization density of roots by diazotrophs, as estimated from positive pellicle formation at highest dilution in nitrogen-free enrichment media, was 10⁶–10⁸ diazotrophic bacteria per g fresh root weight. Roots of the cultivar Kartiki were found to be more densely colonized endophytically by diazotrophs as detected after chloramine T (1%) surface disinfection. To ascertain the phylogenetic affiliation of the isolates, phylogenetic oligonucleotide probes and the Fluorescent in situ Hybridization (FISH) technique were applied. Using group-specific rRNA directed oligonucleotide probes, the majority of the isolates could be identified as alpha-, beta-, or gamma-proteobacteria. Using 16S and 23S rRNA-directed genus- or species-specific probes, Herbaspirillum seropedicae, Azospirillum amazonense, Burkholderia cepacia/vietnamiensis, Rhizobia and Pseudomonas spp. were found to be the most prominent root associated culturable diazotrophs. Diazotrophic Gluconacetobacter spp. were also demonstrated as colonizers of rice roots. Burkholderia cenocepacia, Pseudomonas sp. and three diazotrophic PGPR reference strains were used for the inoculation of axenically grown rice seedlings to determine the plant growth promoting potential. Significant increases in the shoot length (up to 60%), shoot dry weight (up to 33%) and the grain yield (up to 26%) per plant were observed in non-axenic pot and field trials. Using semisolid enrichment media after surface sterilization of field grown inoculated rice roots and oligonucleotide probing of the diazotrophic enrichment cultures, a sustainable colonization with the inoculated bacteria could be demonstrated.     

Effects of Plant Growth Promoting Rhizobacteria on Yield and Some Fruit Properties of Strawberry

Effects of plant growth promoting rhizobacteria (PGPR) [(Pseudomonas BA-8 (biological control agent), Bacillus OSU-142 (N₂-fixing), and Bacillus M-3 (N₂-fixing and phosphate solubilizing)] on yield and some fruit properties of strawberry cultivar ‘Selva’ in the province of Erzurum, Turkey in 2002–2003. Foliar + root application of PGPR strains significantly increased yield per plant as compared with the control. Root application of PGPR strains significantly increased total soluble solids, total sugar and reduced sugar, but decreased titratable acidity. It was also determined that bacteria applications have no important effect on the average fruit weight and pH. The results of this study suggested that Pseudomonas BA-8, Bacillus OSU-142 and Bacillus M-3 have potential for increasing yield in strawberry plant.     

Diversity of diazotroph populations in the rhizosphere of maize (Zea mays L.) growing on different French soils

Summary We studied the dominant diazotrophs associated with maize roots and rhizosphere soil originating from three different locations in France. An aseptically grown maize plantlet, the spermosphere model, was used to isolate N₂-fixing (acetylene-reducing) bacteria. Bacillus circulans was the dominant N₂-fixing bacterium in the rhizosphere of maize-growing soils from Ramonville and Trogny, but was not found in maize-growing sandy soil from Pissos. In the latter soil, Enterobacter cloacae, Klebsiella terrigena, and Pseudomonas sp. were the most abundant diazotrophs. Azospirillum sp., which has been frequently reported as an important diazotroph accociated with the maize rhizosphere, was not isolated from any of these soils. The strains were compared for their acetylene-reducing activity in the spermosphere model. The Bacillus circulans strains, which were more frequently isolated, also exhibited significantly greater acetylene-reducing activity (3100 nmol ethylene day^-1 plant^-1) than the Enterobacteriaceae strains (180 nmol ethylene day^-1 plant^-1). This work indicates for the first time that Bacillus circulans is an important maizerhizosphere-associated bacterium and a potential plant growth-promoting rhizobacterium.     

Plant,Microbial and Soil Factors,Determining Nitrogen Fixation in the Rhizosphere

A genotype effect on associative (rhizosphere) N₂-fixation was observed with two cultivars of Sorghum bicolor (nutans) with a maximum rate of 8 μmol C₂H₄ · h^?1 · plant^?1 in one genotype compared to 0.9 μmol in the other. Characteristics of the high fixing genotype were a reduced transpiration rate, a lower number of stomata and increased root exudate production per gram root dry weight with higher concentration of dicarboxylic acids. The bacterial rhizosphere composition revealed a three times higher number of N₂-fixing bacteria, a tenfold reduction of actinomycetes and a threefold reduction of Arthrobacter associated with the high fixing cultivar compared to the low fixing genotype. From these and other plant rhizospheres two new nitrogen fixing bacteria, Pseudomonas stutzeri and Erwinia herbicola, were characterized. With the N₂-fixing bacteria Azospirillum brasilense and Klebsiella pneumoniae an enhancement of specific nitrogenase activity by aromatic compounds, for example phenolics, the herbicide alachlor and the insecticide carbofuran was demonstrated. An oscillating nitrogenase activity in Azospirillum brasilense under microaerobic conditions was found, resulting from an encystation and deencystation under those conditions. Experiments with wheat roots demonstrated that reduced oxygen tensions, essential for a maximum rhizosphere N₂-fixation, reduced root growth significantly and altered the N-metabolism of the roots.     

Organic acid and sugar contents in sugarcane stem apoplast solution and their role as carbon source for endophytic diazotrophs

We have identified aconitate, malate, and citrate organic acids as well as sucrose, glucose, and fructose sugar compounds from the apoplast solution of the stem of the sugarcane cultivars NiF-8 and RK86-129. The content of all the compounds varied with the cultivars, growth stages, and internode portions. Aconitate showed the highest content among the organic acids and sucrose was the predominant sugar compound. Moreover, Gluconoacetobacter diazotrophicus ATTCC 49037 and Herbaspirillum rubrisubalbicans PA18 differed in their ability to utilize sucrose or aconitate as sole carbon source. The G. diazotrophicus strain preferred sucrose to aconitate while H. rubrisubalbicans preferred aconitate to sucrose as carbon source.     

Hydrogen metabolism and nitrogen fixation of soybean (Glycine max. L.) inoculated with different strains ofRhizobium spp.

Summary Hydrogenase activities and N₂-fixing capacities of soybean nodules (Glycine max. cv. Hodgson), inoculated with strains ofBradyrhizobium japonicum andRhizobium fredii from different geographical regions, were measured after 35 days of culture under controlled conditions. Of the strains tested, 47% induced nodules with bacteroids which recycled H₂. The data obtained suggest that H₂-recycling ability is not a major factor influencing early N₂-fixation which depends essentially on the precocity and intensity of the initial nodulation.     

N2-fixing bacteria in the rhizosphere: Quantification and hormonal effects on root development

The paper summarizes the results of a series of experiments on enumeration of N₂-fixing bacteria (diazotrophs) and hormonal effects of Azospirillum on root development. Numbers of N₂-fixing and N-heterotrophic bacteria were determined on the root (rhizoplane plus “inner” root surface) and in the rhizosphere soil (0–3 mm from the root surface) of Arrhenatherum elatius, other forage grasses and some herbaceous plant species. Pot experiments involved freshly collected soil from an unfertilized grassland area containing its natural population of N₂-fixing bacteria. The MPN (most probable number) of diazotrophs in relation to the MPN of the total bacterial population was always lower on the root than in the rhizosphere soil, suggesting that diazotrophs were not selectively advantaged at the root surface. Supply of mineral nitrogen (NH₄NO₃) decreased the proportion of N₂-fixing bacteria at the rhizoplane as well as in the rhizosphere soil. Similar results were obtained when N was supplied via the leaves. The data suggest that N₂-fixing bacteria in the rhizosphere are poor competitors once they loose their competitive advantage of binding dinitrogen. Correspondingly, the increase in the MPN of the diazotrophs found during plant development was interpreted as a result of decreased available combined N in the rhizosphere. The proportion of N₂-fixing bacteria relative to the total number of bacteria was generally below 1%. Considering the potential amount of substrate released from the roots and the substrate requirement of the bacterial population, N₂-fixation was considered insignificant for plant growth under the given conditions. For the investigations on possible beneficial effects on plant development by bacterial hormones, Azospirillum brasilense was chosen because evidence suggests that amongst the soil bacteria releasing hormones, especially IAA, certain strains of this species are more important than other bacteria. Application of A. brasilense Cd (ATCC 29710) onto the roots of young wheat plants grown in soil increased the number of lateral roots, the total root length and the number of root hairs. Similar results were obtained after application of IAA. This suggests that IAA is an important factor responsible for the effects observed after inoculation with A. brasilense. The increase in root surface may improve acquisition of nutrients and enhance growth of plants. Another hormonal effect of A. brasilense was an increase in nodulation of Medicago sativa grown on agar. Again pure IAA resulted in a similar increase in nodule number. Increases in nodule number were only in part associated with a change in root morphology. Therefore an effect of IAA on the plant immanent regulation system for nodulation is likely.     

Use of NaCN to increase the growth of N2-fixing bacteria in a model spermosphere mimicked by sucrose and amino acids leached by seeds

In order to study the establishment of a spermosphere, the C₂H₂ reduction activity of N₂-fixing bacteria isolated from river sand was examined in a simulated spermosphere in the river sand which contained sucrose, an amino acid mixture, and CN^- released from plant seeds. The sand incubated with 10^-10 to 10^-9 mol CN^- 30 g sand^-1 exhibited higher C₂H₂ reduction activity than that without CN^-. The change in the most probable number of N₂ fixers with increasing quantities of CN^- roughly corresponded to that in C₂H₂ reduction activity. However, the most probable number of non-N₂-fixing bacteria decreased except for CN^--tolerant ones. Both C₂H₂ reduction activity and proliferation of the N₂ fixers isolated on a modified Burk's medium were almost similar to those in the bacteria in the sand. In contrast, the proliferation of some nonfixers decreased with an increasing CN^- concentration. C₂H₂ reduction activity of N₂ fixers cultured in combination with non-fixers exhibited a clear peak at 10^-7 M CN^- as for C₂H₂ reduction activity in the sand. We therefore speculate that cyanide evolved from seeds during a pregermination period may suppress the growth of general bacteria, but may promote the proliferation of N₂ fixers, thus contributing to the establishment of a spermosphere.     

土壤中大豆根瘤菌优势类群以及与寄主共生关系的多样性

我国大豆种植区使用人工选育制成的根瘤菌剂接种效果不稳定。研究指出南方、北方和黄淮流域的6个省土壤中分布的主要Bradrhizobium.japonicum是DH444、USDA110、LL120、005和C224血清型的菌株,主要的Rhizobium.fredii是2048、USDA217、DE1611、2120和2077血清型的菌株。它们占每个取样地点分离物总数的60.3-81.6％。植物感染结瘤法测得大豆种植地点土著根瘤菌菌数在104/克土以上。盆栽试验表明大豆根瘤菌与大豆品种共生时有较强的亲和选择性和共生效应的多样性,其有效结瘤和固氮效率与根瘤菌个体菌株和大豆品种极相关。大多数土著大豆根瘤菌是低或中效固氮的菌株,因而认为选育抗土著根瘤菌而有利于人工接种菌株结瘤的大豆品种和强竞争性的高效菌株仍是提高我国大豆生产的有效途径。     

The "oxygen paradox" of dinitrogen-fixing bacteria

N₂ fixation by aerobic bacteria is a very energy demanding process, requiring efficient oxidative phosphorylation, while O₂ is toxic for the nitrogenase complex. N₂-fixing bacteria have evolved a variety of strategies to cope with this apparent "O₂ paradox". This review compares strategies that azospirilla and other well-known N₂-fixing soil bacteria use to overcome this O₂ paradox. Attention will be given to the relationships between the natural habitat of these soil bacteria and their prevailing adaptations. In view of this knowledge the following questions will be addressed: are the specific adaptations observed in azospirilla sufficient to allow optimal proliferation and N₂ fixation in their natural habitat? Could improving the O₂ tolerance of the N₂-fixing process contribute to the development of more efficient strains for the inoculation of plants?     

Exploration of bacterial N2-fixation systems in association with soil-grown sugarcane,sweet potato,and paddy rice: a review and synthesis

N₂ fixation systems in the nonleguminous crops and bacteria associations have been intensively studied over the last 50 years. Their structure and regulation have been investigated to explore the enhancement of N acquisition in these ecosystems leading to crop-growth with minimum chemical fertilizers. Several lines of important evidence have been accumulated indicating that the magnitudes of associative (nonsymbiotic) N₂ fixation in sugarcane (Saccharum spp.), sweet potato (Ipomoea batatas L.), and paddy rice (Oryza sativa L.) are agronomically significant. In these three crops, unique bacterial N₂-fixation systems may function in addition to the low-level activity (due to the competition in carbon/energy use) of the commonly occurring rhizosphere-associated system by free-living bacteria such as Beijerinckia, Azotobacter, and Klebsiella. Active expressions of the dinitrogenase reductase-encoded gene (nifH) phylogenetically similar to those of Bradyrhizobium spp. and Azorhizobium sp. were abundantly found in the N₂-fixing sugarcane stems, sweet potato stems, and storage tubers. These rhizobia micro-aerobically fix N₂ in the carbon compounds-rich apoplasts. Gluconacetobacter diazotrophicus and Herbaspirillum spp. were previously isolated from inside the sugarcane stems, as the candidates of endophytic N₂ fixers. However, the current molecular and physiological investigations suggest that their major role is production of phytohormonal substances. In paddy rice fields, methane is produced from organic compounds in anoxia and oxidized by contacting with oxygen gas. An active N₂-fixation by methane-oxidizing methanotrophs such as Methylosinus sp. takes place in the root tissues (aerenchyma) and also in the surface soil. This methanotrophic N₂-fixation supports the sustainability of soil fertility although the N₂-fixation and soil fertility are affected by chemical fertilizers. Finally, we discuss the ecological implications of the newly identified rhizobia and methanotroph systems in the N nutrition in nonlegumes and N reservation in field environments.     

Changes in acetylene reduction activities and effects of inoculated rhizosphere nitrogen-fixing bacteria on rice

Acetylene reduction activities (ARAs) of soils and rice plants during rice-growing season were monitored in temperate region in northeast China. This activity was significantly higher in rhizosphere soil than that in inter-row soil after rice seedlings were transplanted. The ARA was high for most of growing season, suggesting that the native N₂-fixing bacteria responded to rice roots very quickly. Sixteen strains of free-living N₂-fixing bacteria were isolated from three different soils. The ARAs of these strains were correlated with the averaged soil ARAs, suggesting that the isolated strains were likely the active flora responsive to rice roots. The strains were inoculated by soaking seedling roots into the liquid culture for 2 h, and the seedlings were transplanted into pots. Most strains tested did not show any growth-promoting effects except Azotobacter armeniacus and Azotobacter nigricans, which showed growth-promoting effects only at late rice growth stage and only when inoculated in combination but not separately. Present data indicated the promising future applications of these two strains in combination in the region, but further research is needed to understand the underlying mechanisms.     

Cultivable heterotrophic N<Subscript>2</Subscript>-fixing bacterial diversity in rice fields in the Yangtze River Plain

Heterotrophic N₂-fixing bacteria are a potentially important source of N₂ fixation in rice fields due to the moist soil conditions. This study was conducted at eight sites along a geographic gradient of the Yangtze River Plain in central China. A nitrogen-free solid malate-sucrose medium was used to isolate heterotrophic N₂-fixing bacteria. Numbers of the culturable N₂-fixing bacteria expressed as CFU (colony forming units) ranged between 1.41ǂ.42᎒⁶ and 1.24ǂ.23᎒⁸ in the sampled paddy field sites along the plain. Thirty strains with high ARA (acetylene reduction activity) were isolated and purified; ARA of the strains varied from 0.9 to 537.8 nmol C₂H₄ culture^-1 h^-1, and amounts of ¹⁵N fixed ranged between 0.008 and 0.4866 mg·culture^-1·day^-1. According to morphological and biochemical characteristics, 14 strains were identified as the genus Bacillus, 2 as Burkholderia, 1 as Agrobacterium, 4 as Pseudomonas, 2 as Derxia, 1 as Alcaligenes, 1 as Aeromonas, 2 as Citrobacter, and 3 strains belonged to the corynebacter-form group.     

IDENTIFICATION OF ENDOPHYTIC BACTERIA ASSOCIATED WITH N2 FIXATION AND INDOLE ACETIC ACID SYNTHESIS AS GROWTH PROMOTERS IN CURCUMA ALISMATIFOLIA GAGNEP.

Endophytic bacteria carrying out dinitrogen (N₂) fixation and indole acetic acid (IAA) synthesis were firstly identified in C. alismatifolia, a globally important flower crop. Their potential as growth promoters to stimulate the rapid growth of host plant was also examined. It will be beneficial to reduce the propagation period of tissue culture plantlets, and also utilize as a biofertilizer for rhizome production in the field. Seven endophytic bacteria were isolated from the leaf, four isolates from the leaf base, and two from the rhizome. ECS203, a gram-negative bacterium with a round shape, showed the highest N₂ fixation at 4.2 nmol C₂H₄/10⁶ cells/hr, and ECS202 showed the highest IAA synthesis at 296 μL μg ^{? 1} protein. Three selected isolates of N₂-fixing and IAA synthesizing endophytic bacteria, i.e., ECS202, ECS203, and ECS204, isolated from the leaf base, were used to reinoculate Curcuma plantlets derived from tissue culture. Then, plants were grown in sterilized sand for 2 months and weekly supplied with N-free nutrient solution. Plant growth, colonization, nitrogen fixation, and IAA synthesis were measured at two months after planting. The inoculated plants clearly showed a better performance of plant growth and yield in terms of the plant height, plant weight, leaf area, and diameter of new rhizomes compared with uninoculated plants. The chlorophyll content and N concentration of leaves and roots also increased in inoculated plants. Endophytic bacteria from inoculated plants colonized the roots, rhizome, and leaf base. Partial sequence analysis using 16S rDNA indicated that the isolate ECS202 corresponded to Sphingomonas pseudosanguinis (99.2% similarity over 1,371 bp), ECS203 to Bacillus drentensis (99.4% similarity over 1,450 bp) and ECS204 to Bacillus methylotrophicus (99.9% similarity over 13,06 bp).     

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

 Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N₂ fixation (BNF) using an agar growth medium containing 5 mg N l^–1of ¹⁵N-labelled (NH₄)₂SO₄. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with ¹⁵N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N₂-fixing bacteria and plants. Received: 4 May 1999     

Free-living nitrogen-fixing bacteria in temperate cropping systems: Influence of nitrogen source

Sustainable cropping systems rely on a minimum of external inputs. In these systems N is largely acquired in animal manures and leguminous green manures. Little is known of how these organic forms of N fertilizer influence the presence and activity of free-living N₂-fixing bacteria. High concentrations of inorganic N in soil inhibit N₂-fixation in cyanobacteria and Azotobacter spp. It is likely that manure and fertilizer applications would result in concentrations of inorganic N capable of inhibiting N₂ fixation and, ultimately, the presence of these organisms. We investigated the effect of synthetic and organic N fertilizer sources on the populations and N₂-fixation potential of free-living N₂-fixing bacteria in the Farming Systems Trial at the Rodale Research Institute. Field plots received the following N treatments prior to corn (Zea mays L.) production: (1) Legume rotations and green manures supplying about 165 kg N ha^-1; (2) beef cattle manure applied at a rate of 220 kg N ha^-1 (plus 60 kg N ha^-1 from 1994 hay plow-down); or (3) fertilizer N (urea and NH₄NO₃) applied at a rate of 145 kg N ha^-1. Soil samples were collected at two depths from corn plots four times during the growing season, and analyzed for soil moisture, soil pH, numbers of N₂-fixing cyanobacteria and Azotobacter spp., extractable NH _inf4 ^sup+ and NO _inf3 ^sup- , and potentially mineralizable N. Soil samples collected in mid-July were analyzed for nitrogenase activity (by C₂H₂ reduction) and total C and N. Populations of Azotobacter spp. and cyanobacteria were influenced only slightly by treatment; however, cyanobacteria species composition was notably influenced by treatment. Nitrogenase activity in surface soils was greatest in legume-N plots and in subsurface plots levels were greatest in fertilizer-N plots. Populations and activity of free-living N-fixing bacteria appeared to be somewhat reduced in all plots as a result of low soil pH levels and high concentrations of inorganic N across all treatments. Annual applications of N to all plots resulted in high levels of potentially mineralizable N that in turn may have reduced non-symbiotic N₂-fixation in all plots.     

Diversity and plant growth promoting evaluation abilities of bacteria isolated from sugarcane cultivated in the South of Brazil

Plant growth promoting (PGP) bacteria associated with sugarcane are a promising alternative for the expansion of this crop in Southern Brazil. In this study bacterial strains from different sugarcane fields were isolated to estimate their diversity, to evaluate some of their PGP activities and to use them as inoculant strains in field experiment. Samples of rhizospheric soil, roots, and stems of sugarcane were collected in six Rio Grande do Sul localities. The isolation of bacteria was made in three different N-free media. DNA from each isolate was subjected to nifH or 16S rDNA PCR-RFLP, and to the 16S rDNA partial sequencing. Five hundred and sixteen strains were isolated and several PGP characteristics were analyzed. Shannon index was used to evaluate the bacterial diversity. Indexes varying from 0.94 to 2.46 were obtained. Soil pH and clay were the characteristics most closely related to bacterial diversity. Achromobacter, Agrobacterium, Burkholderia, Gluconacetobacter, and Stenotrophomonas were the most abundant genera. Concerning the PGP activities, indolic compounds production was detected in 368 isolates; 138 isolates were able to solubilize phosphate; and 390 were siderophores producers. The inoculation of sugarcane with Gluconacetobacter diazotrophicus VI27 strain showed a significant increase in the number of sets germinated, in the amount of soluble solids, and in the yield of sugarcane juice compared with the control. As a conclusion, a diverse population of PGP bacteria was found in the sugarcane samples. These bacteria, especially G. diazotrophicus strain VI27, could be used as biofertilizers of sugarcane as well as other cereal crops under controlled conditions to avoid or reduce the use of standard N fertilizers.