Effect of mineral fertilizer,pig manure,and <Emphasis Type="Italic">Azospirillum rugosum</Emphasis> on growth and nutrient contents of <Emphasis Type="Italic">Lactuca sativa</Emphasis> L.

Benefits from the application of plant growth-promoting bacteria in agriculture largely depend on the complex interactions between several factors including the nature of fertilizers selected. This study was designed to determine the fine tuning between the inoculated bacteria and different fertilizers and their effect on the growth of lettuce plants (Lactuca sativa L.). Plant growth promotion by a novel species of the genus Azospirillum, namely A. rugosum IMMIB AFH-6, was tested by biochemical, bioassay, and greenhouse studies. The treatments used in the greenhouse study were; unfertilized control (Blank), half recommended dose of chemical fertilizer (1/2CF), full recommended dose of chemical fertilizer (1CF), pig manure fertilizer (PMF), pig manure fertilizer + half recommended dose of chemical fertilizer (PMF + 1/2CF), and pig manure fertilizer + full recommended dose of chemical fertilizer (PMF + 1CF). All these treatments when inoculated with A. rugosum IMMIB AFH-6 inoculation were, respectively, In-Blank, In-1/2CF, In-1CF, In-PMF, In-PMF + 1/2CF, and In-PMF + 1CF. Significant increase in plant biomass and shoot N, P, Ca, and Fe was shown in the In-Blank treatment. Plant growth in soil amended with PMF and A. rugosum IMMIB AFH-6 was significantly lower than in soil treated with the chemical fertilizer, but inoculation combined with chemical fertilizer significantly elevated the plant biomass. The In-PMF + 1/2CF treatment showed the highest yield. A. rugosum IMMIB AFH-6 facilitated the accumulation of trace minerals in higher concentrations when PMF was combined with 1CF. To examine the benefits of inoculation by A. rugosum IMMIB AFH-6, we have proposed a new type of data analysis which considers both biomass and nutrient content of plants. This new type of analysis has shown the importance of the mineral content of plant.     

Development of two culture media for mass cultivation of <Emphasis Type="Italic">Azospirillum</Emphasis> spp. and for production of inoculants to enhance plant growth

High yield culture medium is fundamental for production of inoculants for plant growth-promoting bacteria. Based on substitution of glucose in tryptone–yeast extract–glucose medium by Na-gluconate or glycerol, two new culture media were developed for mass cultivation of the commonly used plant growth-promoting bacterium Azospirillum sp. After 18 h of incubation, these modifications increased populations of different strains of Azospirillum (to ∼10¹¹ cells ml⁻¹ [single cell count] and ∼5 × 10⁹ CFU ml⁻¹ [plate count method]), significantly reduced generation time, and were also suitable for production of common synthetic inoculants.     

Performance of the Giant Reed (<Emphasis Type="Italic">Arundo donax</Emphasis>) in Experimental Wetlands Receiving Variable Loads of Industrial Stormwater

Two emergent macrophytes, Arundo donax and Phragmites australis, were established in experimental subsurface flow, gravel-based constructed wetlands (CWs) and challenged by untreated stormwater collected from the hard-pan and other surfaces of a dairy processing factory in south-west Victoria, Australia. The hydraulic loading rate was tested at two levels, sequentially, 3.75 and 7.5 cm day⁻¹. Some of the monitored variables were removed more efficiently by the planted beds in comparison to unplanted CWs (biochemical oxygen demand (BOD), total nitrogen (TN) and total phosphorus (TP); p < 0.007) but there was no significant difference between the A. donax and P. australis CWs in removal of BOD, suspended solids (SS) and TN (p > 0.007) at 3.75 cm day⁻¹ or SS and TN at 7.5 cm day⁻¹. At 3.75 cm day⁻¹, BOD, SS, TN and TP removal in the A. donax and P. australis CWs was 71%, 61%, 78% and 75% and 65%, 60%, 73% and 41%, respectively. Nutrient removal at 7.5 cm day⁻¹ in the A. donax and P. australis beds was 87%, 91%, 84% and 71% and 96%, 94%, 87% and 55%, respectively. As expected, the A. donax CWs produced considerably more biomass (10 ± 1.2 kg wet weight) than the P. australis CWs (2.7 ± 1.2 kg wet weight). This equates to approximately 107 and 36 tonnes ha⁻¹ year⁻¹ biomass (dry weight) for A. donax and P. australis, respectively (assuming 250 days of growing season and single-cut harvest). The performance similarity of the A. donax- and P. australis-planted CWs indicates that either may be used in HSSF wetlands treating dairy factory stormwater, although the planting of A. donax provides additional opportunities for secondary income streams through utilisation of the biomass produced.     

Life-cycle traits of the dimorphic earthworm species <Emphasis Type="Italic">Allolobophora chlorotica</Emphasis> (Savigny, 1826) under controlled laboratory conditions

Allolobophora chlorotica exists as two colour morphs, pink and green. Field observations have indicated that the two morphs have ecological preferences linked to soil moisture: the green morph dominating in wet soils and the pink morph in dry soils. The aim of this laboratory-based research was to investigate the potential differences in fitness and adaptation to soil-moisture conditions of the two morphs measured in terms of growth rate, reproductive output and cocoon viability. An initial experiment maintained hatchlings of both morphs individually under standard culture conditions. On maturation, these were paired (intra-morph), and cocoon production, viability and incubation time were determined. The green morph had significantly faster (P < 0.01) growth rates than the pink morph. Cocoon production was also significantly greater in the green compared with the pink morph (3.2 and 1.5 cocoons worm⁻¹ 28 days⁻¹, respectively) with corresponding viabilities of 87 and 58%. In a second experiment, the growth rates of pink and green hatchlings were assessed under wet and dry soils (29 and 21% soil moisture, respectively). The growth of the pink morph was not influenced significantly (P > 0.05) by soil moisture. In contrast, lower soil-moisture content significantly (P < 0.05) slowed growth and maturation of the green morph. These results support field observations relating to distribution of the two A. chlorotica colour morphs. We suggest that soil-moisture content may act to isolate these morphs, providing, in extremes, a barrier to inter-morphic mating.     

Assessing the Toxic Effects of Nickel,Cadmium and EDTA on Growth of the Plant Growth-Promoting Rhizobacterium <Emphasis Type="Italic">Pseudomonas brassicacearum</Emphasis>

Plant growth-promoting rhizobacteria (PGPR) play an important role in the biodegradation of natural and xenobiotic organic compounds in soil. They can also alter heavy metal bioavailability and contribute to phytoremediation in the presence or absence of synthetic metal chelating agents. In this study, the inhibitory effect of Cd²⁺ and Ni²⁺ at different concentrations of Ca²⁺ and Mg²⁺, and the influence of the widely used chelator EDTA on growth of the PGPR Pseudomonas brassicacearum in a mineral salt medium with a mixture of four main plant exudates (glucose, fructose, citrate, succinate) was investigated. Therefore, the bacteriostatic effect of Cd²⁺, Ni²⁺ and EDTA on the maximum specific growth rate and the determination of EC50 values was used to quantify inhibitory impact. At high concentrations of Ca²⁺ (800 μmol L^-1) and Mg²⁺ (1,250 μmol L^-1), only a small inhibitory effect of Cd²⁺ and Ni²⁺ on growth of P. brassicacearum was observed (EC50 Cd²⁺, 18,849 ± 80 μmol L⁻¹; EC50 Ni²⁺, 3,578 ± 1,002 μmol L⁻¹). The inhibition was much greater at low concentrations of Ca²⁺ (25 μmol L⁻¹) and Mg²⁺ (100 μmol L⁻¹) (EC50 Cd²⁺, 85 ± 0.5 μmol L⁻¹ and EC Ni²⁺, 62 ± 1.8 μmol L⁻¹). For the chosen model system, a competitive effect of the ions Cd²⁺ and Ca²⁺ on the one hand and Ni²⁺ and Mg²⁺ on the other hand can be deduced. However, the toxicity of both, Cd²⁺ and Ni²⁺, could be significantly reduced by addition of EDTA, but if this chelating agent was added in stoichiometric excess to the cations, it also exhibited an inhibitory effect on growth of P. brassicacearum.     

Control of cotton <Emphasis Type="Italic">Verticillium</Emphasis> wilt and fungal diversity of rhizosphere soils by bio-organic fertilizer

Cotton Verticillium wilt is a destructive soil-borne disease affecting cotton production. In this study, application of bio-organic fertilizer (BIO) at the beginning of nursery growth and/or at the beginning of transplanting was evaluated for its ability to control Verticillium dahliae Kleb. The most efficient control of cotton Verticillium wilt was achieved when the nursery application of BIO was combined with a second application in transplanted soil, resulting in a wilt disease incidence of only 4.4%, compared with 90.0% in the control. Denaturing gradient gel electrophoresis patterns showed that the consecutive applications of BIO at nursery and transplanting stage resulted in the presence of a unique group of fungi not found in any other treatments. Humicola sp., Metarhizium anisopliae, and Chaetomium sp., which were considered to be beneficial fungi, were found in the BIO treatment, whereas some harmful fungi, such as Alternaria alternate, Coniochaeta velutina, and Chaetothyriales sp. were detected in the control. After the consecutive applications of BIO at nursery and transplanting stage, the V. dahliae population in the rhizosphere soil in the budding period, flowering and boll-forming stage, boll-opening stage, and at harvest time were 8.5 × 10², 3.1 × 10², 4.6 × 10², and 1.7 × 10² colony-forming units per gram of soil (cfu g⁻¹), respectively, which were significantly lower than in the control (6.1 × 10³, 3.4 × 10³, 5.2 × 10³, and 7.0 × 10³ cfu g⁻¹, respectively). These results indicate that the suggested application mode of BIO could effectively control cotton Verticillium wilt by significantly changing the fungal community structure and reducing the V. dahliae population in the rhizosphere soil.     

<Emphasis Type="Italic">Erigeron annuus</Emphasis> (L.) Pers., as a green manure for ameliorating soil exposed to acid rain in Southern China

Background, aims, and scope  Increasing soil acidification is a growing concern in southern China. The traditional green manures applied in the fields mostly comprise legumes that tend to accelerate soil acidification. Moreover, acid deposition can act as a source of nitrogen. Hence, we looked for new plant species that would enhance nutrient concentrations when used as green manure and would reduce soil acidity or at least not worsen it. Materials and methods  We studied the use of Erigeron annuus (L.) Pers. for ameliorating acid soil in a pot experiment with simulated acid rain (SAR) treatments (pH 5.8 to 3.0) in an open area in Guangzhou City. The pots were divided into two groups named A and B groups. On day 0, pots of A group were filled with soil and planted with Erigeron annuus seedlings. Pots of B group were only filled with soil as the control. On day 40, seedlings of E. annuus were harvested and buried in the corresponding pots. On day 54, two seeds of Phaseolus vulgaris L. were sown in each pot in both groups. The growth and bean yield of P. vulgaris seedlings were then used to evaluate the effects of E. annuus on acid soil. Plant and/or soil samples were collected on days 0, 40, 54, and 150; corresponding parameters were measured. Results  Results showed that E. annuus could maintain a good growth even on very acid soil. On day 40, the pH decreased significantly (P < 0.0001) in the B group pots without E. annuus compared with the A group. On day 54, after E. annuus was buried as a manure, the soil pH of all A group treatments except the pH 4.0 treatment showed a significant increase compared to day 40 (P < 0.01). At the same time, the application of E. annuus as a manure produced a significant increase of soil K and P (P < 0.001), Ca (P < 0.05), and Mg (P < 0.001) concentrations of all A group SAR treatments compared to their B group counterparts (except control pots for Ca). The soil exchangeable K and available P concentration doubled, and Ca and Mg increased by around 25% in the presence of the E. annuus manure application. Discussion  The higher soil pH in the A group than B group on the day 40 was due to a great absorption of NO₃ ⁻ by the roots of E. annuus. The soil pH increase after E. annuus was applied to the soil of A group was attributed to the release of high amount of K, the mineralization of organic N, and the oxidation of organic acid anions. Nutrient increase in the A group after E. annuus application was mostly the result of the nutrient release during the residue decomposition. The amelioration of the soil was effective as demonstrated by the enhanced growth and bean yield of P. vulgaris seedlings on the manured soil compared to the seedlings grown on a control that was not manured. Conclusions   E. annuus could maintain a good growth in the acid lateritic field soil. Cultivating this plant and applying it to the soil with a rate of 1.6 ton ha⁻¹ doubled the soil K and P concentrations and increased soil exchangeable Ca and Mg concentrations by around 25%. This species would be a good green manure candidate for growing in the acid soils of southern China. Application of E. annuus also has beneficial effects on crop growth through reduced Al toxicity and cation leaching. Recommendations and perspectives  Since E. annuus would improve soil pH and nutrient concentrations with minimum care, it is recommended for treating acid soils with poor yield whenever a low-cost solution is required.     

A diazotrophic, indole-3-acetic acid-producing endophyte from wild cottonwood

An endophytic bacterium, wild poplar strain B (WPB), isolated from stems of wild cottonwood (Populus trichocarpa) was identified to Burkholderia vietnamiensis by analyzing the recA and rDNA genes. Phylogenetic analysis of the nifHDK cluster indicates that the WPB isolate shares high sequence similarity with known B. vietnamiensis strains. The nitrogenase activity of WPB was determined by a ¹⁵N₂ incorporation assay and an acetylene reduction assay. WPB was also monitored for production of indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. In addition, its plant growth promotion capacity was assessed by inoculating the WPB strain onto Kentucky bluegrass in nitrogen-free medium. Compared to uninoculated control plants, the plants inoculated with WPB gained more dry weight (42%, p = 0.01) and more nitrogen content (37%, p = 0.04) in 50 days.     

N<Subscript>2</Subscript> fixation by faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) in a gypsum-amended sodic soil

The response of faba bean to the application of four rates of gypsum (0, 2.5, 5.0, 10.0 t ha⁻¹) to a non-saline, alkaline sodic soil was measured in terms of grain yield, dry matter (DM) production, N accumulation and the proportional dependence of the legume on symbiotic N₂ fixation (P _atm). A yield-independent, time-integrated ¹⁵N-dilution model was used to estimate symbiotic dependence. A significant decrease in the exchangeable sodium percentage and significant increases in exchangeable Ca⁺⁺ and the Ca⁺⁺:Mg⁺⁺ ratio in the 0–10-cm soil layer were measured 30 months after application of 10 t ha⁻¹ gypsum. Despite low and erratic rainfall during crop growth, faba bean DM and N uptake responded positively to gypsum application. The symbiotic dependence of the legume at physiological maturity was little affected by sodicity (P _atm = 0.74 at zero gypsum and 0.81–0.82 at 2.5–10 t ha⁻¹ gypsum). The increase in fixed N due to gypsum application was mainly due to increases in legume DM and total N uptake. At 10 t ha⁻¹ of gypsum, faba bean fixed more than 200 kg N ha⁻¹ in above-ground biomass.     

Production of B-group vitamins by plant growth-promoting Pseudomonas fluorescens strain 267 and the importance of vitamins in the colonization and nodulation of red clover

 We have estimated the production of water-soluble B vitamins by plant growth-promoting rhizobacterium Pseudomonas fluorescens strain 267 in a minimal medium with different C sources and at different pH values. In the minimal medium, strain 267 produced large amounts of niacin (0.92 μg ml^–1) and pantothenic acid (0.75 μg ml^–1), but also other vitamins such as biotin, thiamine, cobalamine and pyridoxine. The production of B vitamins was dependent on the C source and pH of the growth medium. By random Tn5 mutagenesis, thiamine and niacin auxotrophs were isolated from P. fluorescens strain 267 and mutants were used to evaluate the vitamin production on colonization of clover roots under controlled conditions. Red clover root colonization decreased by about 1 order of magnitude in the case of the niacin auxotroph. The vitamin auxotrophs of P. fluorescens in a mixed inoculation of clover with R. leguminosarum bv. trifolii strain 24.1 showed no plant growth-promotion activity. Received: 23 May 2000     

Natural compounds enhancing growth and survival of rhizobial inoculants in vermicompost-based formulations

The present study demonstrates the usefulness of natural microbial growth-promoting compounds for improving the stability and life of vermicompost-based (both granular and its aqueous extract) bioformulations. Granular vermicompost maintained the number of cells of Rhizobium meliloti Rmd 201 up to 5.9 × 10⁸ after 180 days at 28°C compared with 2.1 × 10⁸ in charcoal (powdered), while aqueous extract of the vermicompost supported the 5.6 × 10⁷ rhizobia numbers even after 270 days. The addition of 25 μL/mL cow urine and 0.01 mM calliterpinone, a natural plant growth promoter, increased the rhizobia number significantly in granular vermicompost and its aqueous extract, respectively.     

Phosphate solubilization potentials of soil Acinetobacter strains

Many phosphate solubilizing microorganisms (PSM) require external pyrroloquinoline quinone (PQQ) for strong phosphorus (P) solubilization in vitro. The objective of this study was to isolate efficient and PQQ-independent PSM. A total of 21 PSM were isolated from the rhizosphere soil of wheat and maize grown in the pots. Acinetobacter strains were the only PQQ-independent and most effective solubilizers of tricalcium phosphate containing agar. The mean P dissolved in liquid cultures of Acinetobacter strains in a 5-day incubation ranged from 167 to 888 μg/ml P. The pH dropped to below 4.7 from 7.8 in six isolates, which produced gluconic acid in concentrations ranging between 27.5 and 37.5 mM. There was a linear regression between soluble P and gluconic acid concentrations in the bacterial cultures (P < 0.05; R ² = 0.59). Inoculation with Acinetobacter sp. WR922 significantly (P < 0.05) increased wheat (Triticum aestivum L.) P content by 27% at 15 days after emergence (DAE) and dry matter by 15% at 30 DAE compared to the control. The plant P content in inoculated plants at 30 DAE was linearly correlated with soluble P of the bacterial cultures (P < 0.05; R ² = 0.69). Gluconic acid production directly affected phosphate solubilization in vitro, which in turn influenced plant P content of inoculated plants in PQQ-independent P-solubilizing Acinetobacter strains.     

Variability in Azolla sporulation in response to phosphorus application

 Phosphorus application decreased the sporulation frequency and number of sporocarps per plant in all the three Azolla species and 21 A. pinnata strains evaluated in this study. The number of megasporocarps tended to be more depressed than the number of microsporocarps. Nevertheless, the sporulation of A. caroliniana was less sensitive to P than that of A. pinnata and A. microphylla. Its sporulation frequency in the mineral medium did not decrease at 2.5 μg P ml^–1 and remained unaffected between 5 and 20 μg P ml^–1. The sporulation frequency and sporocarp number in this species in the soil culture also were not significantly affected by an increase in the dose of P from 10.7 to 21.4 or 21.4 to 32.1 mg pot^–1. Large variations in the degree of inhibition of sporulation due to the application of P (21.4 mg pot^–1) also occurred among the A. pinnata strains tested. Received: 20 October 1999     

Attenuation of Nitrogen,Phosphorus and <Emphasis Type="Italic">E. coli</Emphasis> Inputs from Pasture Runoff to Surface Waters by a Farm Wetland: the Importance of Wetland Shape and Residence Time

Water quantity and quality were monitored for 3 years in a 360-m-long wetland with riparian fences and plants in a pastoral dairy farming catchment. Concentrations of total nitrogen (TN), total phosphorus (TP) and Escherichia coli were 210–75,200 g N m⁻³, 12–58,200 g P m⁻³ and 2–20,000 most probable number (MPN)/100 ml, respectively. Average retentions (±standard error) for the wetland over 3 years were 5 ± 1%, 93 ± 13% and 65 ± 9% for TN, TP and E. coli, respectively. Retentions for nitrate–N, ammonium–N, filterable reactive P and particulate C were respectively −29 ± 5%, 32 ± 10%, −53 ± 24% and 96 ± 19%. Aerobic conditions within the wetland supported nitrification but not denitrification and it is likely that there was a high conversion rate from dissolved inputs of N and P in groundwater, to particulate N and P and refractory dissolved forms in the wetland. The wetland was notable for its capacity to promote the formation of particulate forms and retain them or to provide conditions suitable for retention (e.g. binding of phosphate to cations). Nitrogen retention was generally low because about 60% was in dissolved forms (DON and NO_X–N) that were not readily trapped or removed. Specific yields for N, P and E. coli were c. 10–11 kg N ha⁻¹ year⁻¹, 0.2 kg P ha⁻¹ year⁻¹ and ≤10⁹ MPN ha⁻¹ year⁻¹, respectively, and generally much less than ranges for typical dairy pasture catchments in New Zealand. Further mitigation of catchment runoff losses might be achieved if the upland wetland was coupled with a downslope wetland in which anoxic conditions would promote denitrification.     

Perchlorate Depositional History as Recorded in North American Ice Cores from the Eclipse Icefield, Canada, and the Upper Fremont Glacier, USA

Temporal depositional rates are important in order to understand the production and occurrence of perchlorate (ClO₄⁻) as limited information exists regarding the impact of anthropogenic production or atmospheric pollution on ClO₄⁻ deposition. Perchlorate concentrations in discrete ice core samples from the Eclipse Icefield (Yukon Territory, Canada) and Upper Fremont Glacier (Wyoming, USA) were analyzed using ion chromatography tandem mass spectrometry to evaluate temporal changes in the deposition of ClO₄ ⁻ in North America. The ice core samples cover a time period from 1726 to 1993 and 1970 to 2002 for the Upper Fremont Glacier (UFG) and Eclipse ice cores, respectively. The average ClO₄ ⁻ concentration in the Eclipse ice core for the time period from 1970 to 1973 was 0.6 ± 0.3 ng L⁻¹, with higher values of 2.3 ± 1.7 and 2.2 ± 2.0 ng L⁻¹ for the periods 1982–1986 and 1999–2002, respectively. All pre-1980 ice core samples from the UFG had ClO₄ ⁻ concentrations <0.2 ng L⁻¹, and the post-1980 samples ranged from <0.2 ng L⁻¹ to a maximum of 2.6 ng L⁻¹ for the year 1992. A significant positive correlation (R = 0.75, N = 15, p < 0.001) of ClO₄⁻ with SO₄²⁻ was found for the annual UFG ice core layers and of ClO₄ ⁻ with SO₄²⁻ and NO₃⁻ in sub-annual Eclipse ice samples (R > 0.3, N = 121, p < 0.002). The estimated yearly ClO₄⁻ depositional flux for the Eclipse ice core ranged from 0.6 (1970) to 4.7 μg m⁻² year⁻¹ (1982) and the UFG from <0.1 (pre-1980) to 1.4 μg m⁻² year⁻¹ (1992). There was no consistent seasonal variation in the ClO₄⁻ depositional flux for the Eclipse ice core, in contrast to a previous study on the Arctic region. The presence of ClO₄⁻ in these ice cores might correspond to an intermittent source such as volcanic eruptions and/or any anthropogenic forcing that may directly or indirectly aid in atmospheric ClO₄⁻ formation.     

Growth,P uptake in grain legumes and changes in rhizosphere soil P pools

In soils with low P availability, several legumes have been shown to mobilise less labile P pools and a greater capacity to take up P than cereals. But there is little information about the size of various soil P pools in the rhizosphere of legumes in soil fertilised with P although P fertiliser is often added to legumes to improve N₂ fixation. The aim of this study was to compare the growth, P uptake and the changes in rhizosphere soil P pools in five grain legumes in a soil with added P. Nodulated chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), white lupin (Lupinus albus L.), yellow lupin (Lupinus luteus L.) and narrow-leafed lupin (Lupinus angustifolius L.) were grown in a loamy sand soil low in available P to which 80 mg P kg⁻¹ was added and harvested at flowering and maturity. At maturity, growth and P uptake decreased in the following order: faba bean > chickpea > narrow-leafed lupin > yellow lupin > white lupin. Compared to the unplanted soil, the depletion of labile P pools (resin P and NaHCO₃-P inorganic) was greatest in the rhizosphere of faba bean (54% and 39%). Of the less labile P pools, NaOH-P inorganic was depleted in the rhizosphere of faba bean while NaOH-P organic and residual P were most strongly depleted in the rhizosphere of white lupin. The results suggest that even in the presence of labile P, less labile P pools may be depleted in the rhizosphere of some legumes.     

Ecological pre-release risk assessment of two genetically engineered, bioluminescent Rhizobium meliloti strains in soil column model systems

In order to identify potential ecological risks associated with the environmental release of two Rhizobium meliloti strains, genetically engineered with the firefly-derived luciferase gene (luc), a pre-release greenhouse investigation was conducted. The upper 4 cm of soil columns (30 cm diameter; 65 cm depth), which were filled according to the horizons of an agricultural field (loamy sand), were inoculated with seeds of Medicago sativa (alfalfa) and R. meliloti cells at approximately 5×10⁶ cells·g^–1 soil. Four treatments were tested: inoculation with a non-engineered wild type strain (2011), strain L33 (luc ⁺), strain L1(luc ⁺, recA^–) and non-inoculated controls. The fate of the engineered strains was followed by two methods: (1) selective cultivation and subsequent detection of bioluminescent colonies and (2) PCR detection of the luc gene in DNA, directly extracted from soil. Strain R. meliloti L33 declined to 9.0×10⁴ cfu·g^–1 soil within 24 weeks and to 2.8×10³ cfu·g^–1 soil within 85 weeks in the upper 25 cm of the soil columns. Decline rates for R. meliloti L1 were not significantly different. Vertical distribution analysis of the recombinant cells after 37 weeks revealed that in three of four columns tested, the majority of cells (>98%) remained above 10 cm soil depth and no recombinant cells occurred below 20 cm depth. However, in one column all horizons below 20 cm were colonized with 2.2×10⁴ to 6.8×10⁴ cfu g^–1 soil. Ecological monitoring parameters included organic substance, total nitrogen, ammonium and nitrate, microbial biomass, culturable bacteria on four different growth media and the immediate utilization of 95 carbon sources (BiologGN) by soil-extracted microbial consortia. None of the parameters was specifically affected by the genetically engineered cells. Received: 6 December 1996     

Effects of Azospirillum brasilense on root morphology of common bean (Phaseolus vulgaris L.) under different water regimes

 The effects of inoculation with Azospirillum brasilense Cd on root morphology and growth of common bean (Phaseolus vulgaris L.) were studied under different growth systems and water regimes. The root systems were evaluated by image analysis. In a PVC-tube growth system, inoculation with A. brasilense at 10⁷ colony forming units (CFU) ml^–1 increased root length, root projection area, specific root length (m g^–1) and specific root area (cm² g^–1), as compared with non-inoculated controls, resulting in root systems with longer and thinner roots. Water stress induced similar root responses to those observed after inoculation with A. brasilense. No increase in plant biomass was observed in inoculated plants, suggesting that under the tested growth conditions, a relatively larger amount of resources is required for the maintenance of the thinner roots. In water-stressed potted plants, the effect of A. brasilense on tap root length was inoculum-concentration dependent. At 10⁷ CFU ml^–1 this effect was significant as compared to non-inoculated controls. In a pouch system without water stress, inoculation with A. brasilense at a concentration of 10⁵–10⁷ CFU ml^–1 2 days after germination resulted initially (2 days after inoculation) in an increase in root length (95%) and root fresh weight (66%), but reduced root diameter (20%), compared to controls. At this early stage of growth the distribution of root length among the different root diameter classes changed: the thinner-root classes had the largest proportion of longer roots. Received: 3 January 2000     

Crop residues and fertilizer nitrogen influence residue decomposition and nitrous oxide emission from a Vertisol

Crop residues with high C/N ratio immobilize N released during decomposition in soil, thus reducing N losses through leaching, denitrification, and nitrous oxide (N₂O) emission. A laboratory incubation experiment was conducted for 84 days under controlled conditions (24°C and moisture content 55% of water-holding capacity) to study the influence of sugarcane, maize, sorghum, cotton and lucerne residues, and mineral N addition, on N mineralization–immobilization and N₂O emission. Residues were added at the rate of 3 t C ha⁻¹ to soil with, and without, 150 kg urea N ha⁻¹. The addition of sugarcane, maize, and sorghum residues without N fertilizer resulted in a significant immobilization of soil N. Amended soil had significantly (P < 0.05) lower NO₃⁻–N, which reached minimum values of 2.8 mg N kg⁻¹ for sugarcane (at day 28), 10.3 mg N kg⁻¹ for maize (day 7), and 5.9 mg N kg⁻¹ for sorghum (day 7), compared to 22.7 mg N kg⁻¹ for the unamended soil (day 7). During 84 days of incubation, the total mineral N in the residues + N treatments were decreased by 45 mg N kg⁻¹ in sugarcane, 34 mg kg⁻¹ in maize, 29 mg kg⁻¹ in sorghum, and 16 mg kg⁻¹ in cotton amended soil compared to soil + N fertilizer, although soil NO₃⁻–N increased by 7 mg kg⁻¹ in lucerne amended soil. The addition of residues also significantly increased amended soil microbial biomass C and N. Maximum emissions of N₂O from crop residue amended soils occurred in the first 4–5 days of incubation. Overall, after 84 days of incubation, the cumulative N₂O emission was 25% lower with cotton + N fertilizer, compared to soil + N fertilizer. The cumulative N₂O emission was significantly and positively correlated with NO₃⁻–N (r = 0.92, P < 0.01) and total mineral N (r = 0.93, P < 0.01) after 84 days of incubation, and had a weak but significant positive correlation with cumulative CO₂ in the first 3 and 5 days of incubation (r = 0.59, P < 0.05).     

Evaluating N2 fixation by food grain legumes in farmers’ fields in three agro-ecological zones of Zambia, using 15N natural abundance

A survey of N₂ fixation in farmers’ fields of Northern (>1,000 mm rainfall), Central (800–1,000 mm rainfall), and Southern (<800 mm rainfall) Zambia revealed some significant differences in plant growth and symbiotic performance of different food grain legumes. Of the three grain legumes (i.e., Bambara groundnut (Vigna subterranea L. Verdc.), cowpea (Vigna unguiculata L. Walp.), and groundnut (Arachis hypogaea L.)) grown in Southern Zambia, cowpea showed greater shoot biomass and significantly lower shoot δ¹⁵N values than groundnut and Bambara groundnut. The lower shoot δ¹⁵N resulted in greater %Ndfa (59%) in shoots and higher amounts of N-fixed, whether per square meters (6,394.0 mg N), per plant (650.8 mg N), or per hectare (63.9 kg N) relative to groundnut and Bambara groundnut, even though the number of cowpea plants per square meter was significantly lower than that of groundnut or Bambara groundnut. Although the shoot δ¹⁵N values of cowpea, Bambara groundnut and common bean (Phaseolus vulgaris L.) were significantly lower than those of groundnut in Central Zambia and their %Ndfa values, therefore, greater, the higher number of groundnut plants per square meter resulted in significantly greater shoot N content, as well as N-fixed per square meter and per hectare relative to the other species. Despite having similar plant density as cowpea in Central Zambia, common bean could fix only 6.0 kg N ha⁻¹ compared with 35.4 kg N ha⁻¹ by cowpea. In Northern Zambia, Bambara groundnut showed the lowest mean shoot δ¹⁵N value (0.54 ± 0.3‰), followed by groundnut (1.59 ± 1.0‰), and then common bean (the three grain legumes grown in that region). As a result, %Ndfa and N-fixed were significantly greater in groundnut (69.7% and 566.0 mg N per plant) and Bambara groundnut (62.9% and 440.1 mg N per plant) than in common bean (2.6% and 2.4 mg N per plant). In Northern Zambia, groundnut, Bambara groundnut and common bean fixed 78.7, 67.6, and 0.9 kg N ha⁻¹, respectively, even though the plant density per square meter of common bean (which fixed the lowest amount of N per hectare) was twice that of groundnut and Bambara groundnut. A species × site analysis showed that cowpea fixed relatively greater amounts of N per plant, per square meter, and per hectare in Southern than Central Zambia. Bambara groundnut and common bean also had significantly lower δ¹⁵N values and higher %Ndfa in Central than Northern Zambia.