Cover crop effects on the fate of N following soil application of swine manure

Biological nitrogen fixation (BNF) in sugarcane is considered one of the principal reasons for the success of the Brazilian Ethanol Program (PRO-ALCOOL) for motor car fuel. The BNF influences positively the energy balance of sugarcane crops for alcohol production. Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium associated with sugarcane, and is particularly abundant and active in the early stages after germination. The objective of this work was to evaluate the effect of the addition of increasing amounts of two sources of mineral nitrogen (ammonium sulphate and calcium nitrate) on the population of G. diazotrophicus and also to evaluate its effect on nitrogenase (acetylene reduction) activity and accumulation of N by two sugarcane hybrids, SP 701143 and SP 792312. The results showed that both varieties differed in the form of nitrogen they prefer to uptake from the soil. The variety SP 701143 preferred ammonium sulphate, whilst the variety SP 792312 preferred N from calcium nitrate. In both varieties, the addition of increased doses of ammonium and nitrate inhibited the population of G. diazotrophicus but in the variety SP 701143 the inhibition was more pronounced in the presence of calcium nitrate. The acetylene reduction activity was inhibited in both varieties, especially in variety SP 792312 in the presence of either of the two nitrogen sources.     

Low recovery frequency of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> from plants and associated mealybugs in Cuban sugarcane fields

This study was aimed to isolate and identify the N₂-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCR-amplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N₂-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.     

Acetobacter diazotrophicus,an indoleacetic acid producing bacterium isolated from sugarcane cultivars of México

Thirteen cane cultivars grown on fields in México were sampled to assess the occurrence of Acetobacter diazotrophicus, a recently identified N₂-fixing bacterium. Results showed that the isolation frequencies extended over a broad range (1.1 to 67%), likely to be related to the nitrogen fertilization level. The lowest isolation frequencies (1.1 to 2.5%) were obtained from plants growing at high nitrogen doses (275–300 kg ha^-1) and the highest values (10–67%) from plants cultivated with 120 kg N ha^-1. All eighteen strains of A. diazotrophicus produced indoleacetic acid (IAA) in defined culture medium. Estimates obtained from HPLC analyses revealed that A. diazotrophicus strains produced from 0.14 to 2.42 g IAA mL^-1 in culture medium. Considering that A. diazotrophicus is found within the plant tissue, the biosynthesis of IAA suggests that the bacteria could promote rooting and improve sugarcane growth by direct effects on metabolic processes, in addition to their role in N₂ fixation.     

Improving Sugarcane Growth and Nutrient Uptake by Inoculating Gluconacetobacter diazotrophicus

Seven Gluconacetobacter diazotrophicus strains from sugarcane roots were screened for their efficiency to promote growth and nutrient uptake in sugarcane at three levels of urea N (0, 75, and 150 kg N ha⁻¹). Inoculation by these strains improved germination, tiller number and plant height. N-uptake and apparent N-recovery increased due to inoculation and the effect was more at N₇₅ level. Gluconacetobacter diazotrophicus isolate IS100 was found to be the most efficient in promoting plant growth and nutrient uptake in sugarcane.     

Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.     

Biological Dinitrogen Fixation in Gramineae and Palm Trees

Biological nitrogen fixation (BNF) in the Gramineae family has been well documented, but a complete understanding of this issue is needed to turn the research into a practical approach. The literature has a long and diverse list of diazotrophic bacteria found colonizing several plant tissues, such as roots, stems, leaves, and trash as well as the rhizosphere. However, only a limited amount of research has focussed on existing associations of N₂-fixing microorganisms with grasses or cereal, especially for BNF inputs and ecological studies under field conditions. The recent discovery of the endophytic diazotroph bacteria such as Acetobacter diazotrophicus, Herbaspirillum spp. and Azoarcus spp. colonizing the interior of sugarcane, rice, Kallar grass (Leptochloa fusca (L.) Kunth), respectively, and other species of grasses as well as cereals has led to a considerable interest in exploring these novel associations. There is a general consensus that plant genotype is a key factor to higher contributions of BNF together with the selection of more efficient bacterial strains. This review summarizes the present data on this field and introduces the discovery of a new group of diazotrophic bacteria colonizing palm trees and therefore opening a future perspective for using these plants, especially African oil palm, to replace diesel as a fuel.     

Ecological Occurrence of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> and Nitrogen-fixing <Emphasis Type="Italic">Acetobacteraceae</Emphasis> Members: Their Possible Role in Plant Growth Promotion

Gluconacetobacter diazotrophicus has a long-standing history of bacterial-plant interrelationship as a symbiotic endophyte capable of fixing atmospheric nitrogen. In low nitrogen fertilized sugarcane fields it plays a significant role and its occurrence was realised in most of the sugarcane growing countries. In this mini review, the association of G. diazotrophicus with sugarcane, other crop plants and with various hosts is discussed. The factors affecting survival in the rhizosphere and the putative soil mode of transmission are emphasized. In addition, other N₂-fixing Acetobacteraceae members, including Gluconacetobacter azotocaptans, Gluconacetobacter johannae and Swaminathania salitolerans, occurring in coffee, corn and rice plants are also covered. Lastly, the plant-growth-promoting traits identified in this group of bacteria, including N₂ fixation, phytohormone synthesis, P and Zn solubilization and biocontrol, are analysed.     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Foliar Iron Fertilization of Peach (Prunus persica (L.) Batsch): Effects of Iron Compounds, Surfactants and Other Adjuvants

During a survey of nitrogen-fixing Burkholderia associated with sugarcane in Tamil Nadu, some endophytes were isolated on PCAT medium. Isolation was based on the use of the selective PCAT medium. Four isolates were studied, all belonging to the genus Burkholderia. One of them, MG43 was consistently more active in reducing acetylene and was identified as Burkholderia vietnamiensis. This isolate was used to inoculate micro-propagated sugarcane plantlets in a comparison with two other diazoptrophs, viz. Gluconacetobacter diazotrophicus^T and Herbaspirillum seropedicae^T. Inoculated plants and uninoculated controls were used in a pot experiment followed by two field experiments under different rates of nitrogen fertilisers. MG43 and G. diazotrophicus performed best in sugarcane, their natural host. Biomass increase due to MG43 inoculation reached 20% in the field. Inoculated plants were heavily colonised by the inoculated bacterium (up to 115,000 CFU g⁻¹ root fresh weight). Inoculation by a combinaison of the three strains performed less well than inoculation by a single MG43 suspension. Ecological implications are discussed, as well as the potential of these bacteria to provide a feasible alternative to higher N fertilisers rates in a low input and long term sustainable rural economy.     

Intracellular colonization of roots of Arabidopsis and crop plants by Gluconacetobacter diazotrophicus

Summary We have investigated the interaction of Gluconacetobacter diazotrophicus, a non-nodulating endophytic nitrogen-fixing bacterium isolated from the intercellular spaces of sugarcane, with Arabidopsis thaliana and the crop plants maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), oilseed rape (Brassica napus), tomato (Lycopersicon esculentum), and white clover (Trifolium repens). Using seedlings grown aseptically in sucrose-containing culture media, we have shown that inoculation with very low numbers of G. diazotrophicus results in extensive intracellular colonization of root meristems and progressive systemic intracellular root colonization. Light microscopic examination of thin sections of resin-embedded root tips of Arabidopsis and these crop plants inoculated with β-glucuronidase (GUS)-labeled and with NifH promoter-GUS-labeled G. diazotrophicus showed blue-stained G. diazotrophicus within the cytoplasm of root cells, indicating that intracellular conditions were suitable for nitrogenase gene expression. Electron microscopy confirmed that these bluestained intracellular G. diazotrophicus were within membrane-bounded vesicles. We discuss whether these novel inoculations with G. diazotrophicus are likely to enable non-nodular endosymbiotic nitrogen fixation and whether these inoculations can also provide a plant system to investigate the endosymbiotic theory of the origin of eukaryotic organelles.     

Survival of endophytic bacteria in polymer-based inoculants and efficiency of their application to sugarcane

Background & aims

Studies have been conducted to evaluate maintenance of cell viability and stability, as well as to select cheap carriers to extend the shelf life of plant beneficial bacterial inoculants for agricultural crops. The purpose of this study was to evaluate the shelf life and the colonization efficiency of novel liquid and gel-based inoculant formulations for sugarcane. The different inoculant formulations were all composed of a mixture of five strains of diazotrophic bacteria (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense and Burkholderia tropica), which are recognized as sugarcane growth promoters.

Methods

Different inoculant formulations containing as carrier the polymers carboxymethylcellulose (CMC) and corn starch (60/40 ratio) at five different concentrations (named PIC, for Polymeric Inoculant Carrier) were supplemented, or not, with 2?% MgO, an interfacial stabilizing agent. Bacterial survival in the different formulations during storage was evaluated under controlled conditions, and two experiments with mini-cuttings of sugarcane variety RB72454 were carried out under greenhouse conditions.

Results

Laboratory tests showed that in the formulation composed of 0.8?g of the polymeric mixture per 100?g of the final product (PIC 0.8), survival of G. diazotrophicus and A. amazonense was around 10⁹?CFU?mL^?1 after 120?days of storage, regardless of the supplementation with MgO. The other formulation (2.2?g of polymeric mixture, PIC 2.2) presented survival levels of 10⁸?CFU?mL^?1 for up to 60?days of storage for all the individual strains. In the greenhouse, sugarcane seedlings showed a positive growth response 50?days after inoculation when inoculated with the mixture of five bacteria, with and without PIC 2.2.

Conclusions

The polymer carriers described here allowed for the long-term survival of the five different bacterial strains tested. In addition, short-term experiments in the greenhouse showed that their application as part of an inoculant on sugarcane cuttings was at least as effective in terms of bacterial colonization and the promotion of plant growth as that of the bacterial mixture without carriers.     

OBPC Symposium: Maize 2004 &; beyond—Intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

Summary The problem of environmental nitrogen enrichment is most likely to be solved by reducing the inputs of synthetic nitrogen fertilizers through the creation of cereals that, like legumes, are able to fix nitrogen. In legumes, rhizobia present intracellularly in vesicles in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Rhizobia within these membrane-bounded compartments are supplied with energy from plant photosynthates and, in return, the bacteria provide the plant with biologically fixed nitrogen. Recently, we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of the root meristems of maize, rice, wheat, and other major non-legume crops can be colonized intracellularly by the non-rhizobial, non-nodulating, nitrogen-fixing bacterium, Gluconacetobacter diazotrophicus, that occurs naturally in sugarcane. G. diazotrophicus expressing nitrogen-fixing genes is present in membrane-bounded compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume species, similar to the intracellular colonization of legume root nodule cells by rhizobia. In order to obtain an indication of the likelihood of adequate growth and yield of maize, for example, with reduced inputs of synthetic nitrogen fertilizers, we are determining the extent to which nitrogen fixation is correlated with systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs of synthetic nitrogen fertilizer.     

Nitrogen use efficiency of sugarcane in relation to its BNF potential and population of endophytic diazotrophs at different N levels

The nitrogen fixing bacterial endophytes Gluconacetobacter diazotrophicus and Herbaspirillum spp. have been proposed to benefit sugarcane (Saccaharum spp. hybrids) growth. Variable populations of these endophytes exist depending upon ontogenic and climatic variations as well. This study investigates the effect of variable chemical nitrogen application in soil on the population of endophytic diazotrophs, acetylene reduction ability of excised roots, plant N-nutrient use efficiency and probable interactions among different parameters in eight commercial sugarcane varieties of subtropical India. Recovery efficiency (RE), agronomic efficiency (AE), partial factor productivity (PFP) and physiologic efficiency (PE) indicators were used for accounting N-nutrient use efficiency. The population of G. diazotrophicus was more at N₇₅ compared to N₀ and N₁₅₀, whereas Herbaspirillum population increased from N₀ to N₁₅₀. ARA was positively correlated with Gluconacetobacter population in rhizosphere and root, whereas it had poor correlation with Herbaspirillum population. Positive correlation of RE and AE with ARA of roots, Gluconacetobacter and Herbaspirillum populations in roots and stems indicate their positive contribution in total nitrogen uptake by the plant per kg of N applied. Average PFP was 808.9 at N₇₅ compared to 408.7 at N₁₅₀ indicating that N was utilized efficiently at low N input status in sugarcane. Strong positive correlations of AE₇₅ (agronomic efficiency from 75 kg N ha⁻¹ to 150 kg N ha⁻¹) with N-uptake (r ² = 0.615), cane yield (r ² = 0.758) and PFP (r ² = 0.758) and other parameters compared to AE (agronomic efficiency from 0 kg N ha⁻¹ to 75 kg N ha⁻¹ or 150 kg N ha⁻¹) correlations with N-uptake (r ² = 0.111), cane yield (r ² = 0.368) and PFP (r ² = 0.190) indicated that the AE of sugarcane was strongly directed towards producing more cane yield per unit of N fertilizer once the sugarcane plant has established using initial dose of nitrogen and thus AE₇₅ seems to be a more appropriate indicator for accounting N-nutrient use efficiency in sugarcane.     

一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

【背景】我国甘蔗生产中氮肥过量施用严重,导致生产成本居高不下,充分发挥甘蔗与内生固氮菌的联合固氮作用,减少氮肥施用量,对促进我国甘蔗产业可持续发展具有重要意义。【目的】筛选优势甘蔗内生固氮菌,对其基本特性、联合固氮效率及促生长功能进行评价。【方法】从甘蔗根系分离到一株内生固氮菌GXS16,利用乙炔还原法测定固氮酶活性,通过PCR扩增nifH基因确定菌株为固氮菌;通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类;通过接种盆栽甘蔗检测菌株的促生长作用,采用¹⁵N同位素稀释法检测菌株相对固氮效率。【结果】菌株GXS16固氮酶活性为2.42μmol-C₂H₄/(h·mL),根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果,菌株GXS16属于伯克氏菌属(Burkholderia);菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶(1-Aminocyclopropane-1-Carboxylate Deaminase,ACC)活性及合成生长素吲哚乙酸...     

The contribution of nitrogen fixation to sugarcane (Saccharum officinarum L.), and the identification and characterization of part of the associated diazotrophic bacterial community

Background and aims

Rhizospheric, epiphytic and endophytic bacteria are associated with several non-legumes, colonizing their surface and inner tissues. Many of these bacteria are beneficial to their hosts, and are collectively termed plant growth-promoting rhizobacteria (PGPR). Recent interest has focused particularly upon PGPR that are endophytic (i.e. PGPE), and which have been reported to be associated with important crops such as rice, wheat and sugarcane. Different mechanisms are involved in bacteria-induced plant growth promotion (PGP), including biological nitrogen fixation (BNF), mineral solubilization, production of phytohormones and pathogen biocontrol. In Uruguay, sugarcane (Saccharum officinarum L.) is considered a strategic multipurpose crop, used for bioenergy, feed, sugar and bioethanol production. The aim of this work was to estimate the BNF contribution to Uruguayan sugarcane cultivars, as well as to identify and characterize the (culturable) putatively endophytic diazotrophic bacteria associated with these varieties.

Methods and results

Results using the ¹⁵N-dilution technique have shown that these sugarcane varieties obtain significant inputs of N from BNF (34.8–58.8% Ndfa). In parallel, a collection of 598 isolates of potentially endophytic diazotrophs was obtained from surface-sterilized stems using standard isolation techniques, and nifH ⁺ isolates from these were the subject of further studies. The bacteria were shown to belong to several genera, including Pseudomonas, Stenotrophomonas, Xanthomonas, Acinetobacter, Rhanella, Enterobacter, Pantoea, Shinella, Agrobacterium and Achromobacter. Additionally, some PGP features were studied in 35 selected isolates. The data obtained in this study represent the initial steps in a program aimed at determining the mechanisms of PGP of non-legume crops in Uruguay (such as sugarcane) with potentially beneficial plant-associated bacteria.     

Antagonistic potential of Gluconacetobacter diazotrophicus against Fusarium oxysporum in sweet potato (Ipomea batatus)

Gluconacetobacter diazotrophicus, an endophytic diazotroph also encountered as rhizosphere bacterium, is reported to possess different plant growth promoting characteristics. In this study, we assessed the biocontrol potential of G. diazotrophicus under in vitro conditions with soil-borne plant pathogenic Fusarium oxysporum. The possible compounds involved in the biocontrol involves 2,4-diacetylphloroglucinol, Pyrrolnitrin and Pyoluteorin. Thin layer chromatography analysis revealed that G. diazotrophicus produced the antibiotic, Pyoluteorin which helped in the suppression of soil-borne pathogenic. The volatile compounds of G. diazotrophicus also inhibited the growth of F. oxysporum. Tests for the production of hydrogen cyanide indicated that G. diazotrophicus does not produced HCN.     

Biological nitrogen fixation in trees in agro-ecosystems

The integration of trees, especially nitrogen fixing trees (NFTs), into agroforestry and silvo-pastoral systems can make a major contribution to sustainable agriculture by restoring and maintaining soil fertility, and in combating erosion and desertification as well as providing fuelwood. The particular advantage of NFTs is their biological nitrogen fixation (BNF), their ability to establish in nitrogen-deficient soils and the benefits of the nitrogen fixed (and extra organic matter) to succeeding or associated crops.The importance of NFTs leads to the question of how we can maximise or optimize their effects and how we can manage BNF and the transfer of nitrogen to associated or succeeding plantings. To be able to achieve these goals, suitable methods of measuring BNF in trees are necessary. The total nitrogen difference (TND) method is simple, but is better suited for low than high soil N conditions. The acetylene reduction assay (ARA), although sensitive and simple, has many technical limitations especially for NFTs, and the estimates of BNF have generally been very low, compared to other methods. For NFTs, the ¹⁵N techniques are still under development, but have already given some promising results (e.g., has been used to measure large genetic variability in BNF within different NFTs).Various factors affect BNF in trees. They include the age of trees, the microbial component, soil moisture, temperature, salinity, pH, soil N level and plant nutrient deficiencies. Some of the factors, e.g. temperature, affect the symbiosis more than plant growth, and differences in the effects of these factors on BNF in different NFT genotypes have been reported. These factors and research needs for improving BNF in trees are discussed.