Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions

A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO₂ content (pCO₂) at the Swiss FACE (Free Air CO₂ Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil–associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas. The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO₂ in the rhizosphere of L. perenne. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO₂ treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO₂. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both narG and napA sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO₂ treatment.     

Nitrate-reducing community in production water of three oil reservoirs and their responses to different carbon sources revealed by nitrate-reductase encoding gene (napA)

The nitrate-reducing microbial community in oil reservoirs was examined by PCR using primers to amplify a segment of napA gene encoding for a subunit of the nitrate reductase, and the effects of different organic carbon additions on the nitrate-reducing community were also evaluated. The orders Rhodocyclales and Burkholderiales within Betaproteobacteria and Pseudomonadales within Gammaproteobacteria were recovered in production water of all three oil reservoirs. Amendment of organic acids promoted Enterobacteriales within Gammaproteobacteria and orders of Rhodocyclales, Pseudomonadales, and Burkholderiales, while alkanes favored the Rhizobiaceae family within Alphaproteobacteria and orders of Rhodocyclales and Pseudomonadales. Results indicated that the functional gene napA can be used as a valuable biomarker in analyzing the diversity of nitrate reducers in oil reservoirs. Nitrate-reducing microbial community shifts following the available carbon sources. Information about napA gene in oil reservoirs environment is scarce. This is the first study that combines molecular and culture-dependent approaches to reveal the diversity of the nitrate-reducing microbial community in production water of oil reservoirs by using the napA gene.     

Dynamics of Inorganic Nitrogen in Nitrate and Glucose-amended Alkaline–saline Soil

Induction of assimilatory NO ₃ ⁻ reduction through the application of an easily decomposable substrate in alkaline–saline soils of the former lake Texcoco (Mexico) resulted in a fast immobilization of NO ₃ ⁻ in excess of N required for metabolic activity and the release of large concentrations of NO ₂ ⁻ and smaller amounts of NH ₄ ⁺ . We postulated that this was regulated by the amounts of NO ₃ ⁻ and glucose added, and affected by the specific characteristics of soil from the former lake Texcoco. This was investigated by spiking soils of different electrolytic conductivity (EC) 56.0 dS m⁻¹ (soil A of Texcoco) and 11.6 dS m⁻¹ (soil B of Texcoco) with different concentrations of NO ₃ ⁻ and glucose while dynamics of CO₂, NH ₄ ⁺ , NO ₂ ⁻ and NO ₃ ⁻ were monitored in an aerobic incubation for 7 days. For comparison reasons (control) an agricultural soil with low EC (0.3 dS m⁻¹) was included as well. In the agricultural soil, 67% of the added glucose mineralized within 7 days, but only 15% in soil A of Texcoco and 20% in soil B of Texcoco. The application of NO ₃ ⁻ to the agricultural soil added with glucose increased cumulative production of CO₂ 1.2 times, 1.5 times in soil A of Texcoco and 1.8 times in soil B of Texcoco. Concentration of NO ₂ ⁻ increased to > 100 mg NO ₂ ⁻ -N kg⁻¹ when 1000 mg glucose-C kg⁻¹ and 500 mg NO ₃ ⁻ -N kg⁻¹ were added to soil A and B of Texcoco, but remained < 3 mg NO ₂ ⁻ -N kg⁻¹ in the agricultural soil. The ratio between the cumulative production of CO₂ and the decrease in concentration of NO ₃ ⁻ was approximately one in soil A and B of Texcoco, but 10 in the agricultural soil after 3 days. It was found that micro-organisms in the alkaline–saline soil of the former lake Texcoco were capable of immobilizing large quantities of NO ₃ ⁻ when an easy decomposable substrate was available in excess of what might be required for metabolic activity while producing large concentrations of NO ₂ ⁻ , but these phenomena were absent in an agricultural soil. In soil of Texcoco, concentrations of NO ₂ ⁻ and NH ₄ ⁺ increased with increased salinity and availability of NO ₃ ⁻ . This ability to remove large quantities of NO ₃ ⁻ under these conditions and then utilize it at a later time might benefit micro-organisms of the N limited alkaline–saline soils of Texcoco.     

Phylogenetic analysis of the archaeal community in an alkaline-saline soil of the former lake Texcoco (Mexico)

The soil of the former lake Texcoco is an extreme environment localized in the valley of Mexico City, Mexico. It is highly saline and alkaline, where Na⁺, Cl⁻, HCO₃⁻ and CO₃²⁻ are the predominant ions, with a pH ranging from 9.8 to 11.7 and electrolytic conductivities in saturation extracts from 22 to 150 dS m⁻¹. Metagenomic DNA from the archaeal community was extracted directly from soil and used as template to amplify 16S ribosomal gene by PCR. PCR products were used to construct gene libraries. The ribosomal library showed that the archaeal diversity included Natronococcus sp., Natronolimnobius sp., Natronobacterium sp., Natrinema sp., Natronomonas sp., Halovivax sp., “Halalkalicoccus jeotgali” and novel clades within the family of Halobacteriaceae. Four clones could not be classified. It was found that the archaeal diversity in an alkaline-saline soil of the former lake Texcoco, Mexico, was low, but showed yet uncharacterized and unclassified species. César Valenzuela-Encinas and Isabel Neria-González contributed equally to this publication.     

Genetic Characterization of the Nitrate Reducing Community Based on <Emphasis Type="Italic">narG</Emphasis> Nucleotide Sequence Analysis

The ability of facultative anerobes to respire nitrate has been ascribed mainly to the activity of a membrane-bound nitrate reductase encoded by the narGHJI operon. Respiratory nitrate reduction is the first step of the denitrification pathway, which is considered as an important soil process since it contributes to the global cycling of nitrogen. In this study, we employed direct PCR, cloning, and sequencing of narG gene fragments to determine the diversity of nitrate-reducing bacteria occurring in soil and in the maize rhizosphere. Libraries containing 727 clones in total were screened by restriction fragment analysis. Phylogenetic analysis of 128 narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria. Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch. All together, our results revealed a more complex nitrate-reducing community than did previous culture-based studies. A significant and consistent shift in the relative abundance of the nitrate-reducing groups within this functional community was detected in the maize rhizosphere. Thus a substantially higher abundance of the dominant clone family and a lower diversity index were observed in the rhizosphere compared to the unplanted soil, suggesting that a bacterial group has been specifically selected within the nitrate-reducing community. Furthermore, restriction fragment length polymorphism analysis of cloned narG gene fragments proved to be a powerful tool in evaluating the structure and the diversity of the nitrate-reducing community and community shifts therein.     

Relative Abundances of Proteobacterial Membrane-Bound and Periplasmic Nitrate Reductases in Selected Environments

Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 × 10² to 6.8 × 10⁴ copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.     

Effect of salinity and inoculation on growth,nitrogen fixation and nutrient uptake ofVigna radiata (L.) Wilczek

This study reports the effect of salinity and inoculation on growth, ion uptake and nitrogen fixation byVigna radiata. A soil EC_e level of 7.5 dS m⁻¹ was quite detrimental causing about 60% decline in dry matter and grain yield of mungbean plants whereas a soil EC_e level of 10.0 dS m⁻¹ was almost toxic. In contrast most of the studied strains of Rhizobium were salt tolerant. Nevertheless, nodulation, nitrogen fixation and total nitrogen concentration in the plant was drastically affected at high salt concentration. A noticeable decline in acetylene reduction activity occurred when salinity level increased to 7.5 dS m⁻¹.     

<Emphasis Type="Italic">Tindallia texcoconensis</Emphasis> sp. nov., a new haloalkaliphilic bacterium isolated from lake Texcoco,Mexico

A new alkaliphilic and moderately halophilic, strictly anaerobic, fermentative bacterium (strain IMP-300^T) was isolated from a groundwater sample in the zone of the former soda lake Texcoco in Mexico. Strain IMP-300^T was Gram-positive, non-sporulated, motile and rod-shaped. It grew within a pH range from 7.5 to 10.5, and an optimum at 9.5. The organism was obligately dependent on the presence of sodium salts. Growth showed an optimum at 35°C with absence of growth above 45°C. It fermented peptone and a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonium. Its fatty acid pattern was mainly composed of straight chain saturated, unsaturated, and cyclopropane fatty acids. The G + C content of genomic DNA was 40.0 mol%. Analysis of the 16S rRNA gene sequence indicated that the new isolate belongs to the genus Tindallia, in the low G + C Gram-positive phylum. Phylogenetically, strain IMP-300^T has Tindallia californiensis, as closest relative with a 97.5% similarity level between their 16S rDNA gene sequences, but the DNA–DNA re-association value between the two DNAs was only 42.2%. On the basis of differences in genotypic, phenotypic, and phylogenetic characteristics, strain IMP-300^T is proposed as a new species of the genus Tindallia, T. texcoconensis sp. nov. (type strain IMP-300^T = DSM 18041^T = JCM 13990^T).     

Dual inoculation withRhizobium sp. andGlomus fasciculatum enhances nodulation,yield and nitrogen fixation in chickpea (Cicer arietinum Linn.)

Summary Seed inoculation with Rhizobium and soil inoculation withGlomus fasciculatum increased nodulation, nitrogen and phosphorus concentration in plants and yield of chickpea (Cicer arietinum) var. BG 212 in pots containing unsterilized soil especially with 50kgP₂O₅ ha⁻¹ in the form of superphosphate. Inoculation with Rhizobium orG. fasciculatum separately or in combination significantly increased the N₂ fixed in straw and grain than uninoculated controls as determined by¹⁵N atom percent excess of plants grown in soil amended with labelled ammonium sulphate (¹⁵NH₄)₂SO₄) at the rate of 20kg N ha⁻¹. These increases were most pronounced when P was applied at 50kgP₂O₅ ha⁻¹.     

The relative contribution of symbiotic N2 fixation and other nitrogen sources to grassland ecosystems along an altitudinal gradient in the Alps

The significance of symbiotic N₂ fixation in legumes (Trifolium alpinum L., T. nivale Sieber, T. pratense L., T. badium Schreber, T. thalii Vill., T. repens L., Lotus alpinus [DC.] Schleicher, L. corniculatus L., Vicia sativa L.) and other N sources for the N budget of grassland ecosystems was studied along an altitudinal gradient in the Swiss Alps. The total annual symbiotic N₂ fixation was compared with other sources of N for plant growth of the total plant community (mineralisation and wet deposition). The contribution of symbiotically fixed N to total above-ground N yield of the swards decreased from at least 16% to 9% with increasing altitude where legumes were present. This decrease was due to a decrease in the yield proportion of legumes from 15% at 900 and 1380 m a.s.l. to 5% at 2100 and 2300 m a.s.l. (no legumes were found above 2750 m a.s.l.) and not to a decline in the activity of symbiotic N₂ fixation. With increasing altitude legumes are more patchily distributed. The high symbiotic N₂ fixation of individual plants up to their altitudinal limit is not primarily the result of low mineral N availability since an addition of NH₄ ⁺ or NO₃ ⁻ fertiliser at 2300 m a.s.l. led either to no decrease or only to a minor decrease in symbiotic N₂ fixation. At 1380 m a.s.l., N mineralisation (13.45 g N m⁻² yr⁻¹) appeared to be the main source of N for growth of the sward; N from symbiosis (at least 1.0 g to 2.6 g N m⁻² yr⁻¹) and wet deposition (0.4 g to 0.6 g m⁻² yr⁻¹) was not a significant N source for plant growth at this altitude. At 2100 m a.s.l., the combined amounts of N from symbiotic N₂ fixation (at least 0.1 g N m⁻² yr⁻¹) and wet deposition (0.3 g N m⁻² yr⁻¹) appeared to be similarly important for plant growth as soil N mineralisation (0.47 g N m⁻² yr⁻¹). At high altitudes, wet N deposition and symbiotic N₂ fixation together represent a significant source of N for the grassland ecosystem while at low altitudes these N inputs appear to be much less important. This revised version was published online in June 2006 with corrections to the Cover Date.     

Removal of <Emphasis Type="Italic">p</Emphasis>-chlorophenol by the marine microalga <Emphasis Type="Italic">Tetraselmis marina</Emphasis>

The ability of Tetraselmis marina, a green coastal microalga, to remove chlorophenols under photoautotrophic conditions was investigated. T.marina was able to grow in the presence of 20 mg L⁻¹ of the phenolic compounds tested. The EC₅₀ (growth rate) value of p-chlorophenol (p-CP) to T.marina was found to be 25.5 mg L⁻¹. The microalga was able to remove chlorophenols, showing higher efficiency for p-CP. The effect of photoregime and NaHCO₃ concentration on p-CP removal was investigated. Under continuous illumination with 1 g L⁻¹ NaHCO₃ initial concentration T.marina removed 65% of 20 mg L⁻¹ in a 10-day cultivation period.     

Endocrinological investigation into the reproductive cycles of two sympatric skate species, Malacoraja senta and Amblyraja radiata, in the western Gulf of Maine

The smooth skate, Malacoraja senta, and thorny skate, Amblyraja radiata, are two commercially exploited batoids found within the Gulf of Maine. During the past five years, we conducted a large study to accurately describe important biological life history parameters previously lacking for these species. As part of that project, the current study reports our findings on the hormonal profiles associated with the reproductive cycles of M. senta and A. radiata. Blood samples were obtained from mature M. senta and A. radiata of both sexes from all months of the year, and plasma testosterone (T), estradiol (E₂) and progesterone (P₄) concentrations were determined using radioimmunoassay (RIA). In female M. senta and A. radiata, monthly T concentrations ranged from 4,522 pg ml⁻¹ to 1,373 pg ml⁻¹ and 31,940 pg ml⁻¹ to 14,428 pg ml⁻¹, E₂ concentrations from 831 pg ml⁻¹ to 60 pg ml⁻¹ and 8,515 pg ml⁻¹ to 2,902 pg ml⁻¹, and P₄ concentrations from 3,027 pg ml⁻¹ to 20 pg ml⁻¹ and 3,264 pg ml⁻¹ to 331 pg ml⁻¹, respectively. No statistical differences were detected between any months for any hormone. Estradiol concentrations were not correlated with ovary weight, shell gland weight, or diameter of the largest follicles in either species. Monthly T concentrations in male M. senta and A. radiata ranged from 23,146 to 12,660 pg ml⁻¹ and from 57,500pg ml⁻¹ to 24,737 pg ml⁻¹, while E₂ concentrations ranged from 7.5 pg ml⁻¹ to undetectable and 103 to 30 pg ml⁻¹, respectively. No statistical differences were observed between months for either steroid. Testosterone concentrations were weakly correlated with testes weight and percent of stage VI spermatocysts in A. radiata, however, no correlation was detected between T and stage VI spermatocysts in M. senta. Collectively, these data support the previous conclusion that M. senta and A. radiata of both sexes are capable of reproducing year round in the western Gulf of Maine.     

Identification of reactive toxicants: Structure–activity relationships for amides

A diverse series of amides were evaluated for aquatic toxicity (IGC₅₀) assessed in the Tetrahymena pyriformis population growth impairment assay and for reactivity (EC₅₀) with the model soft nucleophile thiol in the form of the cysteine residue of the tripeptide glutathione. All alkylamides along with some halo-substituted amides are well predicted by the simple hydrophobicity (log K _ow)–electrophilicity (E _lumo) response-surface model [log(IGC⁻¹ ₅₀) = 0.45(log K _ow) − 0.342(E _lumo) − 1.11]. However, 2-halo amides with the halogen at the end of the molecule and α,β-unsaturated primary amides are among those derivatives identified as being more toxic than predicted by the model. Amides, which exhibit excess toxicity, were capable of forming covalent bonds through an S_N2 displacement or a Michael addition. Moreover, only those amides exhibiting excess toxicity were reactive with thiol, suggesting that the reactivity with model nucleophiles such as the thiol group may provide a means of accurately defining reactive toxicants.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Chronic Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Does Not Induce NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Use by <Emphasis Type="Italic">Acer saccharum</Emphasis> Marsh.

The ability of an ecosystem to retain anthropogenic nitrogen (N) deposition is dependent upon plant and soil sinks for N, the strengths of which may be altered by chronic atmospheric N deposition. Sugar maple (Acer saccharum Marsh.), the dominant overstory tree in northern hardwood forests of the Lake States region, has a limited capacity to take up and assimilate NO₃⁻. However, it is uncertain whether long-term exposure to NO₃⁻ deposition might induce NO₃⁻ uptake by this ecologically important overstory tree. Here, we investigate whether 10 years of experimental NO₃⁻ deposition (30 kg N ha⁻¹ y⁻¹) could induce NO₃⁻ uptake and assimilation in overstory sugar maple (approximately 90 years old), which would enable this species to function as a direct sink for atmospheric NO₃⁻ deposition. Kinetic parameters for NH₄⁺ and NO₃⁻ uptake in fine roots, as well as leaf and root NO₃⁻ reductase activity, were measured under conditions of ambient and experimental NO₃⁻ deposition in four sugar maple-dominated stands spanning the geographic distribution of northern hardwood forests in the Upper Lake States. Chronic NO₃⁻ deposition did not alter the V _max or K _m for NO₃⁻ and NH₄⁺ uptake nor did it influence NO₃⁻ reductase activity in leaves and fine roots. Moreover, the mean V _max for NH₄⁺ uptake (5.15 μmol ¹⁵N g⁻¹ h⁻¹) was eight times greater than the V _max for NO₃⁻ uptake (0.63 μmol ¹⁵N g⁻¹ h⁻¹), indicating a much greater physiological capacity for NH₄⁺ uptake in this species. Additionally, NO₃⁻ reductase activity was lower than most values for woody plants previously reported in the literature, further indicating a low physiological potential for NO₃⁻ assimilation in sugar maple. Our results demonstrate that chronic NO₃⁻ deposition has not induced the physiological capacity for NO₃⁻ uptake and assimilation by sugar maple, making this dominant species an unlikely direct sink for anthropogenic NO₃⁻ deposition.     

Natural 15N abundance of soil N pools and N2O reflect the nitrogen dynamics of forest soils

Natural ¹⁵N abundance measurements of ecosystem nitrogen (N) pools and ¹⁵N pool dilution assays of gross N transformation rates were applied to investigate the potential of δ¹⁵N signatures of soil N pools to reflect the dynamics in the forest soil N cycle. Intact soil cores were collected from pure spruce (Picea abies (L.) Karst.) and mixed spruce-beech (Fagus sylvatica L.) stands on stagnic gleysol in Austria. Soil δ¹⁵N values of both forest sites increased with depth to 50 cm, but then decreased below this zone. δ¹⁵N values of microbial biomass (mixed stand: 4.7 ± 0.8‰, spruce stand: 5.9 ± 0.9‰) and of dissolved organic N (DON; mixed stand: 5.3 ± 1.7‰, spruce stand: 2.6 ± 3.3‰) were not significantly different; these pools were most enriched in ¹⁵N of all soil N pools. Denitrification represented the main N₂O-producing process in the mixed forest stand as we detected a significant ¹⁵N enrichment of its substrate NO₃⁻ (3.6 ± 4.5‰) compared to NH₄⁺ (−4.6 ± 2.6‰) and its product N₂O (−11.8 ± 3.2‰). In a ¹⁵N-labelling experiment in the spruce stand, nitrification contributed more to N₂O production than denitrification. Moreover, in natural abundance measurements the NH₄⁺ pool was slightly ¹⁵N-enriched (−0.4 ± 2.0 ‰) compared to NO₃⁻ (−3.0 ± 0.6 ‰) and N₂O (−2.1 ± 1.1 ‰) in the spruce stand, indicating nitrification and denitrification operated in parallel to produce N₂O. The more positive δ¹⁵N values of N₂O in the spruce stand than in the mixed stand point to extensive microbial N₂O reduction in the spruce stand. Combining natural ¹⁵N abundance and ¹⁵N tracer experiments provided a more complete picture of soil N dynamics than possible with either measurement done separately.     

Effects of kelp (Macrocystis integrifolia) on soil chemical properties and crop response

In 1981 a two-year field plot experiment was established to assess the effects of quantities (0, 7.5, 15, 30, 60 and 120 t ha⁻¹) of fresh kelp (Macrocystis integrifolia) on crop growth and nutritional response and chemical properties of a fine-textured soil. Soil was analyzed for NO₃−N, NH₄−N, electrical conductivity, pH, Cl and exchangeable cations (K, Mg, Ca, Mn and Na). The plots were planted to beans (Phaseolus vulgaris) in the first year and peas (Pisum sativum) in the second year. Marketable bean yields increased in the first year with kelp applications up to 60 t ha⁻¹, with yields, emergence and flowering being reduced by the 120 t ha⁻¹ application. Soluble salts (EC) and Cl concentrations in the soil eight days after application increased linearly and sharply with increasing quantities of kelp. Increased K concentration and moisture content, characteristics of plants growing in a salt-stressed soil environment, were measured. A subsequent companion greenhouse experiment confirmed that the reduced bean emergence and growth with 120 t ha⁻¹ applications of kelp were primarily due to soluble salts. The only growth effects upon peas in the second year was a slight reduction in leaf plus stem yields with increasing applications of kelp.     

Toxic effect of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether on growth of soil isolate <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> T1/1

The toxic and growth inhibiting effects of methyl tert-butyl ether (MTBE) on the hydrocarbon-degrading Pseudomonas veronii T1/1 strain (isolated from gasoline contaminated soil) were studied. In our experiments, the MIC of MTBE was found to be 60 mM and the EC₅₀ was 51.7 mM. In the concentration range 0–30 mM, MTBE did not significantly influence the growth parameters of this bacterium, but at concentrations over 30 mM MTBE exerted a significant growth inhibiting effect. In the presence of 70 mM MTBE, the specific growth rate dropped from 0.4731 to 0.1201 h⁻¹, while the length of the lag period increased from 5.41 to 17.01 h and the yield coefficient declined from 0.2652 to 0.0718 g g⁻¹. MTBE at 100 mM inhibited the growth of this strain completely. These findings may have important environmental implications, as high concentrations of MTBE could influence the efficiency of soil and groundwater bioremediation processes significantly.     

Effects of the novel allelochemical ethyl 2-methylacetoacetate from the reed (<Emphasis Type="Italic">Phragmitis australis</Emphasis> Trin) on the growth of several common species of green algae

Bioassays were performed to investigate the effects of the novel allelochemical, ethyl 2-methylacetoacetate (EMA), isolated from the reed (Phragmitis australis) on the growth of three common species of algae; Scenedesmus obliquus, Selenastrum capricornutum and Chlamydomonas reinhardtii. The results demonstrated that EMA has three quite different types of effect on these three species of algae. The growth of S. capricornutum was significantly inhibited by EMA during the whole cultivation period. The EC₅₀ values of EMA on S. capricornutum was 0.6 mg L⁻¹(7 days). However, the inhibitory effect of EMA on S. obliquus was apparent during the first 4 days of batch cultivation and then the inhibitory effect disappeared, and a stimulating effect was observed instead. The EC₅₀ value of EMA on S. obliquus was 0.43 mg L⁻¹(4 days). In addition, following the addition of EMA, the cells of S. obliquus and S. capricornutum became significantly larger than the normal untreated one and the algal cells changed morphologically. The microstructure of the algal cells was disrupted by the addition of EMA. There was no significant inhibition of the growth of C. reinhardtii by EMA, but cell motility was affected.