Free-living Nitrogen-fixing Microorganisms Research Articles

Black locust coppice stands homogenize soil diazotrophic communities by reducing soil net nitrogen mineralization

Black locust (BL, Robinia pseudoacacia) is considered a promising tree species for reforestation due to its great ability to fix nitrogen. However, after two or three coppice-harvesting rotations, the productivity of BL declines. Whether soil microbial communities are affected and how these groups correlate with the nitrogen mineralization process across multi-generation stands remains unclear. We investigated the composition and structure of free-living nitrogen-fixing microorganisms (diazotrophs) by sequencing the marker gene nifH and compared these results to levels of soil nitrogen mineralization in the bulk soil and rhizosphere in black locust plantations on Mount Tai, China. The results showed multi-generation BL coppice plantations decreased the total soil nitrogen (N), soil phosphorus (P), soil microbial biomass N (MBN), soil microbial biomass C (MBC), soil nitrification rate (Rn), soil ammonification rate (Ra), and net soil N mineralization rate (Rm), but significantly increased the concentration of soil NH4+-N to maintain sufficient NO3−-N. The dominant species in bulk soil and rhizosphere changed from Rhodopseudomonas (22.62% and 15.76%), unclassified_c_Alphaproteobacteria (22.37% and 29.28%), unclassified_o_Rhizobiales (15.40% and 13.31%), Bradyrhizobium (12.00% and 11.74%) in seedling plantations to Bradyrhizobium (45.95% and 47.86%) and Rhodopseudomonas (43.56% and 41.84%) in coppice plantations, respectively. Mantel test and Redundancy analysis (RDA) revealed that Rn, Ra, and Rm were the most important factors shaping the diazotrophic communities. Our results suggest that the multi-generation BL coppice plantation can homogenize soil diazotrophic communities, which is mainly regulated by the available N loss caused by nitrogen mineralization. Strengthening the management technology of coppice plantations will provide more beneficial external consumption.

The Composition of Nitrogen-Fixing Microorganisms Correlates With Soil Nitrogen Content During Reforestation: A Comparison Between Legume and Non-legume Plantations.

Numerous reforestation projects have been conducted to improve soil fertility in degraded forests, often causing alterations to the soil microbial communities. However, it remains unclear whether microbial functional groups are affected and how these groups correlate with an increase in the nutrient contents during reforestation. We investigated the abundance and composition of free-living nitrogen-fixing microorganisms (diazotrophs) by quantifying and sequencing the marker gene nifH in bulk soils from five reforestation approaches, including legumes and non-legumes, in subtropical China. The relationships between diazotrophic community attributes and soil nitrogen (N) content [NO3−, NH4+, and microbial biomass N (MBN)] were examined under various approaches. Abundance of diazotrophs was highest in the native tree plantation (Schima spp. and Michelia macclurei) and Acacia mangium monoculture (AM), and lowest in the Pinus massoniana monoculture. The diazotrophic abundance correlated positively with soil organic matter and water content while there was a negative correlation to pH. The composition of diazotrophic community differed significantly among the five reforestation approaches examined and was closely correlated with variations in soil pH, NH4+ and water content. Diazotrophic community composition was closely related to soil NH4+ content, whereas abundance was not. The AM contained higher NH4+, NO3− and MBN contents than the other reforestation approaches, which may be associated with the indicator species of diazotrophs (Actinobacteria, Proteobacteria, and Firmicutes). However, there were more indicator species of Proteobacteria in the mixed Acacia plantation (Acacia mangium and Acacia crassicarpa) than in AM, which might have contributed to the remarkedly lower N content compared to AM. Overall, the soil N content under reforestation appeared to be more related to the composition of diazotroph community than to the abundance of diazotrophs.

Response of Free-Living Nitrogen-Fixing Microorganisms to Land Use Change in the Amazon Rainforest

The Amazon rainforest, the largest equatorial forest in the world, is being cleared for pasture and agricultural use at alarming rates. Tropical deforestation is known to cause alterations in microbial communities at taxonomic and phylogenetic levels, but it is unclear whether microbial functional groups are altered. We asked whether free-living nitrogen-fixing microorganisms (diazotrophs) respond to deforestation in the Amazon rainforest, using analysis of the marker gene nifH. Clone libraries were generated from soil samples collected from a primary forest, a 5-year-old pasture originally converted from primary forest, and a secondary forest established after pasture abandonment. Although diazotroph richness did not significantly change among the three plots, diazotroph community composition was altered with forest-to-pasture conversion, and phylogenetic similarity was higher among pasture communities than among those in forests. There was also 10-fold increase in nifH gene abundance following conversion from primary forest to pasture. Three environmental factors were associated with the observed changes: soil acidity, total N concentration, and C/N ratio. Our results suggest a partial restoration to initial levels of abundance and community structure of diazotrophs following pasture abandonment, with primary and secondary forests sharing similar communities. We postulate that the response of diazotrophs to land use change is a direct consequence of changes in plant communities, particularly the higher N demand of pasture plant communities for supporting aboveground plant growth.

Evaluation of microbial inoculants as biofertilizers for the improvement of growth and yield of soybean and maize crops in savanna soils

Certain rhizobacteria exert considerable influence on plant growth and development, particularly under limiting conditions. The effects of some indigenous soil microbial isolates and commercially produced microbial inoculants, referred to as bio-inoculants, on the growth and dry matter yield (DMY) of maize (Zea mays) and soybean (Glycine max) crops were assessed under greenhouse conditions. In two sets of experiments, one set comprised of free-living nitrogen-fixing microorganisms (Azospirillum spp.), three soils from Ibadan, Mokwa and Shanono located in different agro-ecological zones, and maize as the test crop. The other set consisted of microbial inoculants that can act as biocontrol agents applied to sterilized and non-sterilized soils; soybean was the test crop. The bio-inoculants were applied separately and also in combination. The treatments included a reference termed ‘mineral N’ where macro- and micro-nutrients were supplied at optimal rates, a control where bio-inoculants were not applied, and four replicates. All treatments, excluding the reference, received only macro-nutrients at suboptimal rates. The crops were grown for eight weeks and growth parameters were measured. The shoot DMY of maize was relatively large (42 to 63 g plant-1) and differed significantly among the soils but the bio-inoculants did not improve the shoot DMY significantly (P > 0.05) in any of the soils when compared with the control. However, sole inoculation of Mazospiriflo-2 enhanced nitrogen uptake significantly in maize grown in Shanono soil. For soybean, the shoot DMY was also not improved by the inoculation or the addition of the microbial products compared with the control. Key words: Biocontrol, cereals and legumes, inoculum, plant growth promoting rhizobacteria, soil.

Diversity of free-living nitrogen-fixing microorganisms in the rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings

The composition of free-living nitrogen-fixing microbial communities in rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings was studied by the presence of nifH genes using Polymerase Chain Reaction-Denatured Gradient Gel Electrophoresis (PCR-DGGE) approach. Eleven rhizosphere tailing samples and nine non-rhizosphere tailing samples from six plant communities were collected from two wastelands with different discarded periods. The nested PCR method was used to amplify the nifH genes from environmental DNA extracted from tailing samples. Twenty-two of 37 nifH gene sequences retrieved from DGGE gels clustered in Proteobacteria (α-Proteobacteria and β-Proteobacteria) and 15 nifH gene sequences in Cyanobacteria. Most nifH gene fragments sequenced were closely related to uncultured bacteria and cyanobacteria and exhibited less than 90% nucleotide acid identity with bacteria in the database, suggesting that the nifH gene fragments detected in copper mine tailings may represent novel sequences of nitrogen-fixers. Our results indicated that the non-rhizosphere tailings generally presented higher diversity of nitrogen-fixers than rhizosphere tailings and the diversity of free-living nitrogen-fixers in tailing samples was mainly affected by the physico-chemical properties of the wastelands and plant species, especially the changes of nutrient and heavy metal contents caused by the colonization of plant community.

Diversity of free-living nitrogen-fixing microorganisms in wastelands of copper mine tailings during the process of natural ecological restoration

Biological nitrogen fixing is an important source of nitrogen input in the natural ecological restoration of mine wastelands. The diversity of nifH genes in tailings samples under different plant communities in Yangshanchong and Tongguanshan wastelands in Tongling, was analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach. The nitrogen-fixing microorganism community in the upper layer of tailings of Tongguanshan wasteland discarded in 1980 showed higher Shannon-Wiener diversity index than that in Yangshanchong wasteland discarded in 1991. The diversity of nifH genes in Yangshanchong wasteland of copper mine tailings did not display a consistent successional tendency with development of plant communities during the process of natural ecological restoration. Phylogenetic analysis of 25 sequences of nifH gene fragments retrieved from the DGGE gels indicated that there were mainly two taxa of free-living nitrogen-fixing microorganisms, Proteobacteria and Cyanobacteria living in the wastelands investigated, most of which were unique and uncultured. Canonical correspondence analysis (CCA) based on the relationship between band patterns of DGGE profile and physico-chemical properties of tailings samples showed that the diversity of nifH genes in different tailing samples was mainly affected by loss of ignition, water content, pH and available Zn contents of wastelands. The dominant plant species and development period of plant communities by ameliorating pH, reducing the toxicity of heavy metals, increasing organic matter and water content affected the diversity and structure of the free-living nitrogen-fixing microorganisms in wastelands of copper mine tailings.

Impact of Strains of Entomopathogenic Fungi On Some Main Groups of Soil Microorganisms

The influence of entomopathogenic fungal strains - one Metarhizium anisopliae (Metsch.) So- rok. and seven Beauveria bassiana (Bals.-Criv.) Vuill. on some main groups of soil microorganisms was studied after introduction of their conidia into the soil. The soil samples were analyzed for densities of bacteria, actinomycetes and fungi by decimal dilutions of soil suspensions grown on selective media. The presence of conidia of fungal strains in the soil after a month from introduction was proved by bait method. Three of the strains of B. bassiana were established to be of the highest persistence, this being expressed by mortality of Galleria mellonella L. (Lepidoptera, Pyralidae) larvae from 70% to 90%, followed by strains 224Re of B. bassiana and 31 of M. anisopliae. The obtained results showed that examined strains of the entomopathogenic fungi manifested dif- ferent in manner and varying in degrees of impact on density of the main groups of soil microor- ganisms. Relatively insignificant changes were established under the influence of the strains 224Re B. bassiana and 31 M. anisopliae. The other strains of B. bassiana caused alterations in microbial balance expressed in different manner - stimulation or suppression on density of free-living nitrogen-fixing microorganisms, mineral nitrogen utilizing bacteria, spore-forming bacteria, cellulose degrading mi- croorganisms, actinomycetes, soil fungi. So each strain could be characterized by specific impact on

Inhibitory effect of petroleum oil on photosynthetic electron transport system in the cyanobacteriumAnabaena doliolum

Virtually nothing is known about the site of action of oil and the mechanism of inhibition of photosynthetic electron transport, a process responsible for the generation of ATP and NADPH, which are essential for carbon fixation. The present study was an attempt to learn something about these aspects. The influence of diesel on photosynthetic O{sub 2}-evolution, {sup 14}CO{sub 2} fixation, and electron transport system has been examined in Anabaena doliolum, a heterocystous cyanobacterium. A. doliolum and other heterocystous cyanobacteria are widely distributed in soil and aquatic ecosystems, and represent an important group of free-living nitrogen fixing microorganisms.

Further Studies on Free-living Nitrogen-Fixing Microorganisms, Other than Azotobacter, Isolated from Egyptian Soils and Waters

Summary Re-examination of the twenty organisms previously isolated by Abd-El-Malek et al. (1979), using the acetylene reduction test, showed that only one isolate, identified as Bacillus sp., had the ability for reducing acetylene. Screening several hundred fresh isolates from Egyptian soil and water samples for N2-fixing ability revealed that 115 organisms were capable of repeated growth on N-deficient medium, showing nitrogen gains by the Kjeldahl analysis. Only five isolates were able to reduce acetylene under aerobic conditions. Addition of 0.01 % Difco yeast extract was essential for growth stimulation and increasing the amount of nitrogen gains by these isolates. Amounts of nitrogen fixed ranged from 4.8 to 10.5 ppm and from 9.6 to 26.7 ppm in the N-deficient medium and the N-poor medium, respectively. On the basis of morphological, cultural, and biochemical characteristics, the N2-fixing cultures were identified as two strains of Bacillus (S15/b and S77/g), two strains of Pseudomonas (S44/b and W70/n), one strain of Klebsiella (S145/m), and one strain of Micrococcus (S171/h).

Field measurements of nitrogenase activity in soils amended with wheat straw

Nitrogenase activity and decomposition of straw were examined in situ in two areas (Gunnedah and Cowra) representative of large areas of the New South Wales wheat belt. Measurements of nitrogenase activity were made by adapting the acetylene reduction assay for use in the field. Evolution of CO2 was monitored as an indirect measure of decomposition of straw. The addition of straw to soil stimulated nitrogenase activity which was related to the amount of straw added and the rate of straw decomposition. There were significant levels of activity provided the soil was moist and warm. Nitrogenase activity increased with mean daily soil temperature (up to at least 30�C) and decreased as the soil dried from field capacity. It is concluded that nitrogen fixation by free-living nitrogen-fixing microorganisms in soils amended with straw may contribute to the nitrogen status of the soil and thus reduce the need for nitrogen fertilizers.

Nitrogen Fixation in a Salt Marsh

Salt marshes appear to be ideal sites for the growth of free-living nitrogen-fixing microorganisms. Each zone of the marsh contains anaerobic mud in which nitrogen-fixing bacteria can develop and there are areas of bare mud ripe for colonization by blue-green algae. The lower zones of salt marshes, in common with most marine habitats, are deficient in available combined nitrogen and therefore any contribution made by nitrogenfixing organisms to the nitrogen status of the marsh is likely to be significant. A number of workers have suggested that nitrogen-fixing blue-green algae contribute to the nitrogen in salt marshes (Stewart & Pugh 1963; Wood 1965; Ranwell 1972; Chapman & Chapman 1973; Fogg 1973) but little experimental work has been carried out.

Studies on the interaction of plants and free-living nitrogen-fixing microorganisms. I. Occurrence of Azotobacter in the rhizosphere of crop plants.

The occurrence of Azotobacter in the rhizosphere of 17 crop plants grown in different types of soil in the field and in the greenhouse has been studied as part of a program of work on the interaction of plants and free-living nitrogen-fixing microorganisms. Azotobacter counts were very low in both rhizosphere and non-rhizosphere soil from the field; they varied with the type and age of the plant and with the type of soil. The organism was usually absent from acidic and poor soils. I n the greenhouse experiments, soil counts of these bacteria were found to be somewhat higher than in the corresponding soil in the field (Granby sandy loam). The root effect was greater with both radish and wheat in the poor soil (Upland sand) than in the fertile Granby soil. Onion exerted no obvious influence on the Azotobacter population in either soil. However, even under optimum conditions of plant growth, numbers of these organisms were very low in comparison with those of other bacteria normally present in soil and in the root zone.