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.     

The carbon source influences the energetic efficiency of the respiratory chain of N2-fixing Acetobacter diazotrophicus

Acetobacter diazotrophicus is a diazotrophic bacterium that colonizes sugarcane tissues. Glucose is oxidized to gluconate in the periplasm prior to uptake and metabolism. A membrane-bound glucose dehydrogenase quinoenzyme [which contains pyrroloquinoline quinone (PQQ) as the prosthetic group] is involved in that oxidation. Gluconate is oxidized further via the hexose monophosphate pathway and tricarboxylic acid cycle. A. diazotrophicus PAL3 was grown in a chemostat with atmospheric nitrogen as the sole N source provided that the dissolved oxygen was maintained at 1.0–2.0% air saturation. The biomass yields of A. diazotrophicus growing with glucose or gluconate with fixed N were very low compared with other heterotrophic bacteria. The biomass yields under N-fixing conditions were more than 30% less than with ammonium as the N source using gluconate as the carbon source but, surprisingly, were only about 14% less with glucose. The following scheme for the metabolism of A. diazotrophicus through the different pathways emerged: (1) the respiratory chain of this organism had a different efficiency of ATP production in the respiratory chain (P:O ratio) under different culture conditions; and (2) N fixation was one (but not the sole) condition under which a higher P:O ratio was observed. The other condition appears to be the expression of an active PQQ-linked glucose dehydrogenase. Received: 6 December 1999 / Received revision: 22 March 2000 / Accepted: 7 April 2000     

Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp.)

We report studies on the possible effects of fertilisation with high level of N (300 kg of N ha^-1) on the occurrence and numbers of the diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicusin sugar cane plants. In the sugar cane genotype SP79-2312, the N fertilised plants generally showed higher concentrations of this element. These same plants also had lower numbers of A. diazotrophicus, while the population of Herbaspirillum spp. was not affected by N application. These differences in the concentration of N and the numbers of A. diazotrophicus due to N application were not shown in the variety SP70-1143. The numbers of A. diazotrophicus were also shown to be influenced by the harvest time, becoming reduced in the harvests that coincided with dry periods of the year.     

Indentification of Acetobacter diazotrophicus,Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans using biochemical and genetic criteria

Nitrogen-fixing Acetobacter diazotrophicus, Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans colonize sugar cane, and are thought to be capable of supplying high levels of fixed nitrogen to this plant. Eight A. diazotrophicus, two H. seropedicae and four H. rubrisubalbicans isolates were identified and compared by complementary biochemical and genetic methods. Utilization of carbon sources and antibiotic resistance patterns allowed differentiation of A. diazotrophicus from Herbaspirillum species. In order to distinguish strains within A. diazotrophicus species, the polymerase chain reaction was employed, using a Rhizobium meliloti dctA primer under low stringency hybridization conditions.     

Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that >10³ cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10⁷/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.The authors are with EMBRAPA-CNPAB, Cx Postal 74.505, Seropédica, Rio de Janeiro, 23851-970, Brazil     

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.     

Comparative study of the response ofAzotobacter vinelandii andAcetobacter diazotrophicus to changes in pH

Summary Curves were established for the pH response of respiration on eleven substrates byAzotobacter vinelandii andAcetobacter diazotrophicus. With every substrate the optimal pH forA. diazotrophicus was lower than forA. vinelandii. The optimal hydrogen ion concentration forA. diazotrophicus was 5 fold to 365 fold greater than forA. vinelandii depending upon the substrate. In general,A. diazotrophicus supports respiration over a wider pH range than doesA. vinelandii.Dedicated to the memory of Professor John G. Torrey     

Effect of trickle fertigation with three forms of nitrogen on soil pH,levels of extractable nutrients below the emitter and plant growth

The effects of application of nitrogen as calcium nitrate, urea or ammonium sulphate at two rates through the trickle irrigation system on pH and nutrient status of the wetted volume of soil below the emitters and on growth and nutrition of courgette (zucchini) plants (Cucurbita pepo L.) was investigated. Soil acidification, caused by nitrification, occurred to a large extent in the volume of soil immediately below the emitters in the urea and ammonium sulphate treatments. Acidification was greater at the high rate of N addition and more pronounced with ammonium sulphate than urea. A significant amount of applied urea appeared to move through the soil as urea and consequently, at the same rate of N addition, levels of ammonium were lower directly below the emitter and those of nitrate were higher further away from the emitters for the urea than ammonium sulphate treatments. Soil acidification below the emitters resulted in significant decreases in levels of exchangeable Ca, Mg and K and increases in levels of exchangeable Al, EDTA-extractable Fe, Mn, Zn and Cu and bicarbonate-extractable P. Vegetative growth and harvestable yields of courgettes were increased by both irrigation and nitrogen applications. Vegetative growth was generally greater at the low rate of N addition than at the high one and generally followed the order calcium nitrate > urea > ammonium sulphate. However, fruit yields followed the order urea > ammonium sulphate > calcium nitrate and were larger at the high rate of N for urea and ammonium sulphate treatments and unaffected by rate for the calcium nitrate treatments. It is suggested that with fertigation, the form of applied N can have significant physiological effects of plant growth and yields because N may be applied into the root zone on numerous occasions during the growing season.     

Importance of myo-inositol,calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts

Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l^-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.Abbreviations BA benzylaminopurine - CaCl₂ calcium chloride, 2,4,-D-2,4-dichlorophenoxyacetic acid - EDTA ethylene diamine tetraacetic acid - KCl potassium chloride - MES-2-N morpholino ethane sulfonic acid - MgCl₂ magnesium chloride - NH₄NO₃ ammonium nitrate - NAA naphthalene acetic acid, p-protoplasts     

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.     

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.     

Different nitrogen sources and growth responses of Spirulina platensis in microenvironments

Spirulina platensis was cultivated, in comparative studies, using several sources of nitrogen. The standard source used (sodium nitrate) was the same as that used in the synthetic medium Zarrouk, whereas the alternative nitrogen sources consisted of ammonium nitrate, urea, ammonium chloride, ammonium sulphate or acid ammonium phosphate. The initial nitrogen concentrations tested were 0.01, 0.03 and 0.05 M in an aerated photobioreactor at 30 °C, with an illuminance of 1900 lux, and 12 h-light/12 h-dark photoperiod over a period of 672 h. Maximum biomass was produced in medium containing sodium nitrate (0.01–0.03–0.05 M), followed by ammonium nitrate (0.01 M) and urea (0.01 M). The final biomass concentrations were 1.992 g l^–1 (0.03 M sodium nitrate), 1.628 g l^–1 (0.05 M sodium nitrate), 1.559 g l^–1 (0.01 M sodium nitrate), 0.993 g l^–1 (0.01 M ammonium nitrate) and 0.910 g l^–1 (0.01 M urea). This suggested that it is possible to utilize nitrogen sources other than sodium nitrate for growing S. platensis, in order to decrease the production costs of scaled up projects.     

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.     

Biological nitrogen fixation associated with sugar cane

A recent¹⁵N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N₂ fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N₂ fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).     

Alleviation of salt-induced stress on seed emergence using soil additives in a greenhouse

Use of sodium chloride (NaCl) as a deicing salt results in high concentrations of ions in roadside soils, which decreases seedling emergence in these areas. Greenhouse experiments performed in soil culture tested the efficacy of three soil fertilizers, gypsum (CaSO₄), potash (potassium chloride, KCl) and potassium nitrate (KNO₃), in alleviating NaCl stress on seedling emergence of three grass species exhibiting a range of salt tolerance, Poa pratensis (Kentucky bluegrass), Bouteloua gracilis (blue grama), and Puccinellia distans (alkali grass). Two-factor factorial designs were utilized for each species-fertilizer combination. Treatments of 5000 mg/L (0.086 M) NaCl with or without fertilizer, in concentrations that were equal to 0.5, 1, and 2 times the molar equivalent of 5000 mg/L NaCl were applied biweekly. Salt stress on Poa pratensis emergence was alleviated by all fertilizers with CaSO₄ having the greatest effect in alleviating NaCl stress and potash and potassium nitrate alleviating stress at lower treatment levels. Emergence of Bouteloua gracilis and Puccinellia distans was in most cases negatively effected by soil amendments.     

Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001

A strain YZN-001 was isolated from swine manure effluent and was identified as Pseudomonas stutzeri. It can utilise not only nitrate and nitrite, but also ammonium. The strain had the capability to fully remove as much as 275.08 mg L⁻¹ NO₃⁻–N and 171.40 mg L⁻¹ NO₂⁻–N under aerobic conditions. Furthermore, At 30 °C, the utilization of ammonium is approximately 95% by 18 h with a similar level removed by 72 h and 2 weeks at 10 and 4 °C, respectively. Triplicate sets of tightly sealed serum bottles were used to test the heterotrophic nitrifying ability of P. stutzeri YZN-001. The results showing that 39% of removed NH₄⁺–N was completely oxidised to nitrogen gas by 18 h. Indicating that the strain has heterotrophic nitrification and aerobic denitrification abilities, with the notable ability to remove ammonium at low temperatures, demonstrating a potential using the strain for future application in waste water treatment.     

Effect of nitrogen form at various levels of potassium,on the Meloidogyne-Fusarium wilt complex in muskmelon

Summary The influence of various potassium concentration and of nitrate or ammonium was evaluated on non inoculated muskmelon plants and on plants inoculated with theFusarium wilt pathogen (F), the root-knot nematode (Meloidogyne javanica) (Nem), or a combination of both pathogens (F+Nem). Increasing potassium concentrations raised top fresh weights (TFW) in all four groups. Nitrate fertilized plants weighed more than plants receiving ammonium, independent of the K⁺ level in the medium. In the Nem+F infected plants TFW values were one and half to two times greater in those receiving the nitrate than in those receiving the ammonium. Number of nematodes in the roots were not affected by nutritional difference. In the ammonium fertilized F and Nem+F plants 30% more wilting was found than after nitrate application. Irrespective of the form of nitrogen that was applied accumulation of N, P and K was found in the roots of the Nem+F and/or Nem plants, while in the shoots Ca, Mg, Na and P accumulated and K was depleted.Contribution from the Agricultural Research Organization, (ARO), No. 944-E, 1983 series.     

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentrationon the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) wascultured in media with different nitrogen sources and concentrations.During the pre-logarithmic phase, the alga grew faster with ammoniumas N source than with nitrate, but the reverse applied during thepost-logarithmic phase. The alga grew poorly in N-free mediumor medium with urea as the sole N source. In the same growth phase,ammonium medium resulted in higher yield of total lipid, but the EPA yielddid not differ significantly different from that using nitrate medium. Themaximum growth rate occurred in medium containing 1.28 mmolL^-1 sodium nitrate, while maximum EPA and total lipid contents werereached at 1.92 mmol L^-1, when EPA accounted for 27.9% totalfatty acids. The growth rate kept stable when NH₄Cl ranged from0.64 to 2.56 mmol L^-1, and the maximum content of total lipidand EPA occurred in the medium with 2.56 mmol L^-1NH₄Cl. The EPA content was higher in the pre- thanpost-logarithmic phase, though the total lipid content was lower. Thehighest EPA content expressed as percent total fatty acid was 27.9% innitrate medium and and 39.0% in ammonium medium.     

Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica (Bacillariophyceae): effect of nitrogen source and concentration

To investigate the nutritional value of the diatom Cyclotella cryptica Reimann, Lewin, and Guillard as an alternative feed for the use in the aquaculture industry, the heterotrophic growth characteristics, total fatty acids, and the resultant fatty acid profile of the microalga were studied when cultivated with sodium nitrate, ammonium chloride, or urea. All three nitrogen sources supported growth under heterotrophic conditions, and their uptake affected the pH of the cultivation medium, even when buffered. The use of sodium nitrate or urea resulted in a significant increase in the pH of the cultivation media, whereas the use of ammonium chloride caused a minor decrease in the pH of the cultivation media. The maximum specific growth rate was highest when urea and ammonium chloride were supplied at a low concentration; however, the total fatty acid content was not significantly affected (P = 0.101) by the nitrogen source when supplied at 10.7 mM nitrogen. The total fatty acid content and fatty acid profile of C. cryptica was more affected by the growth phase (predominately influenced by the initial nitrogen concentration) than by the source of nitrogen.     

Ammonium and urea removal by Spirulina platensis

Different concentrations either of ammonium chloride or urea were used in batch and fed-batch cultivations of Spirulina platensis to evaluate the possibility of substituting nitrate by cheaper reduced nitrogen sources in wastewaters biotreatment. The maximum nitrogen concentration able to sustain the batch growth of this microalga without inhibition was 1.7 mM in both cases. Ammonium chloride was limiting for the growth at lower concentrations, whereas inhibition took place at higher levels. This inhibition effect was less marked with urea, likely because the enzymatic hydrolysis of this compound by urease controlled the ammonia transfer into the cell. Fed-batch experiments carried out by pulse-feeding either ammonium or urea proved that the use of these compounds as nitrogen sources can sustain the long term-cultivation of S. platensis, provided that the conditions for their feeding are accurately optimized.