Influence of light and pH on growth and nitrogenase activity on temperate-grown Azolla

Summary We investigated the influence of light intensity and pH on growth and acetylene reduction in three strains of Azolla filiculoides and one strain of A. pinnata cultivated under ambient conditions in Naples, Italy. A. pinnata showed maximum growth and acetylene reduction activity at 65% of natural light intensity (82 Klux) and at pH 5–7. In contrast, three A. filiculoides strains showed maximum values in a wider range of light intensity (50–100%) and pH (5–9). All optimal growth conditions of light intensity and pH were obtained from the determination of doubling time for dry and fresh weight increases. The lowest doubling time for all strains was in the range 2.7–3.3 days. Under these optimal conditions, the acetylene reduction activity rates were 46 nmol ethylene h^–1 mg^–1 dry weight for two strains of A. filiculoides from California, and 90 nmol for A. filiculoides from Naples and A. pinnata from Tancheng, Peoples Republic of China. These values correspond to those reported for strains of A. filiculoides and A. pinnata cultivated in areas outside Italy where the use of Azolla as biofertilizer has resulted in increased crop production. Therefore, the strains studied here may have a similar potential in Southern Italy as a source of biomass, dry matter and nitrogenous compounds.Research work supported by CNR, Italy. Special grant I. P. R. A. Sub-project 1. Paper No. 814     

Ammonium uptake and excretion in Azolla-Anabaena symbiosis

Summary The processes of NH ₄ ⁺ uptake and induced NH ₄ ⁺ excretion in the Azolla-Anabaena symbiosis were studied. Uptake rates in accessions of Azolla microphylla and A. mexicana were similar. No pH optimum for NH ₄ ⁺ uptake was observed. Rates of N excretion induced by methionine sulfoximine were also similar. When A. caroliniana was subjected to the herbicide Ignite (Hoechst-Roussel), more NH ₄ ⁺ was initially released than with methionine sulfoximine treatment. Glutamine synthetase was not completely suppressed.     

Influence of gibberellic acid on the sporulation of Azolla caroliniana, Azolla microphylla and Azolla pinnata

strains used in the study sporulate throughout the year while Azolla caroliniana and Azolla pinnata strains show sporulation only during November–March, the favourable period for sporulation. Application of gibberellic acid (GA) at a concentration of 2.5–100.0 μg ml^–1 substantially enhanced the sporulation frequency and number of sporocarps (mega plus micro) per plant in Azolla microphylla (two strains) during the unfavourable period (September–October) by 77.3–200.0%. The increase in the number of microsporocarps was more than that of the megasporocarps. The sporulation frequency and number of sporocarps in Azolla pinnata (two strains) and Azolla caroliniana during the favourable season also increased considerably due to GA (2.5–10.0 μg ml^–1) application. The optimum GA concentration for the sporulation of the three Azolla species was 2.5 μg ml^–1, which was used in the subsequent investigations. Efficacy of GA for enhancing sporulation was tested across 20 Azolla pinnata strains. A significant positive response was observed in all the strains. Of them, 17 strains for the sporulation frequency and 15 strains for the sporocarp number showed a >25% increase due to GA treatment. Under field conditions, Azolla microphylla (two strains), Azolla caroliniana and Azolla pinnata exhibited 66.0–88.1% higher sporulation frequency and 42.8–52.6% higher sporocarp number due to GA application. However, the proportion of megasporocarps to total sporocarps did not change. Received: 4 August 1998     

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     

Green manure production of Azolla microphylla and Sesbania rostrata and their long-term effects on rice yields and soil fertility

Summary Azolla spp. and Sesbania spp. can be used as green manure crops for wetland rice. A long-term experiment was started in 1985 to determine the effects of organic and urea fertilizers on wetland rice yields and soil fertility. Results of 10 rice croppings are reported. Azolla sp. was grown for 1 month and then incorporated before transplanting the rice and 3–4 weeks after transplanting the rice. Sesbania rostrata was grown for 7–9 weeks and incorporated only before transplanting the rice. Sesbania sp. grew more poorly before dry season rice than before wet season rice. Aeschynomene afraspera, which was used in one dry season rice trial, produced a larger biomass than the Sesbania sp. The quantity of N produced by the Azolla sp. ranged from 70 to 110 kg N ha^-1. The Sesbania sp. produced 55–90 kg N ha^-1 in 46–62 days. Rice grain yield increases in response to the green manure were 1.8–3.9 t ha^-1, similar to or higher than that obtained in response to the application of 60 kg N ha^-1 as urea. Grain production per unit weight of absorbed N was lower in the green manure treatments than in the urea treatment. Without N fertilizer, N uptake by rice decreased as the number of rice crops increased. For similar N recoveries, Sesbania sp. required a lower N concentration than the Azolla sp. did. Continuous application of the green manure increased the organic N content in soil on a dry weight basis, but not on a area basis, because the application of green manure decreased soil bulk density. Residual effects in the grain yield and N uptake of rice after nine rice crops were found with a continuous application of green manure but not urea.     

Effect of high temperature on plasmid curing of Rhizobium spp. in relation to nodulation of pigeonpea [Cajanus cajan (L.) Millsp.]

Summary Fifty-six isolates of Rhizobium and Bradyrhizobium spp. (Cajanus) were studied for their plasmid profile and N₂-fixation efficacy. One to three plasmids were reproducibly detected in all the Rhizobium spp. strains but no plasmid was detected in the Bradyrhizobium spp. strains. Rhizobium sp. strain P-1 was mutagenized by Tn5 and three nod^– and six nod⁺fix^– were screened for symbiotic parameters. Neomycin-sensitive mutants were isolated by elevated temperatrue (40°C) from tranconjugants carrying Tn5 insertions. The high temperature cured these mutants from the single large plasmid present in the parent strain P-1. All these cured mutants were nod^–, indicating that the genes for nodulation were present on this plasmid, which is readily cured at a high temperature (40°C). The high temperature in the semi-arid zones of Haryana could be responsible for the low nodulation of pigeonpea because the plasmid carrying the nodulation genes is cured at 40°–45°C giving rise to non-nodulating mutants.     

Short-Term Effects of Arsenate-Induced Toxicity on Growth, Chlorophyll and Carotenoid Contents, and Total Content of Phenolic Compounds of Azolla filiculoides

This study evaluated the toxic effects of arsenic (As) on the growth, total antioxidant activity, total content of phenolic compounds, and content of photosynthetic pigments of Azolla filiculoides. The aquatic fern was propagated and exposed to Yoshida nutrient solution contaminated with sodium arsenate (Na₂HAsO₄??7H₂O) at six concentrations (5, 10, 20, 30, 60, and 120???g?As?mL^?1), including the control without As contamination. Azolla cultures were kept under environmental chamber conditions?26??C, 12?h photoperiod and 80% HR for 96?h. Increased As concentrations (>30???g?mL^?1) significantly diminished growth of A. filiculoides and the total content of chlorophyll and total phenolic compounds, but significantly enhanced of total carotenoid?+?xanthophylls content. The concentrations of 5 and 10???g?As?mL^?1 significantly stimulated the growth of A. filiculoides. This aquatic fern tolerates As concentrations lower than 30???g?mL^?1, and its maximum As accumulation (28???g?g^?1 dry weight) was achieved when exposed to 60???g As mL^?1, but showing clear symptoms of As toxicity.     

Degradation of benzoate via benzoyl-coenzyme A and gentisate by Bacillus stearothermophilus PK1, and purification of gentisate 1,2-dioxygenase

The thermophilic Bacillus stearothermophilus PK1 utilized benzoate, 3-hydroxybenzoate, and gentisate as sole source of carbon and energy. 2- and 4-Hydroxybenzoate, 2,3- and 3,4-dihydroxybenzoate, and catechol did not support growth. Degradation of benzoate proceeded via benzoyl-coenzyme A (benzoyl-CoA) and gentisate. The inducible benzoyl-CoA ligase converted benzoate but not 3-hydroxybenzoate to its coenzyme A thioester. Gentisate 1,2-dioxygenase from B. stearothermophilus PK1 was purified to homogeneity. The enzyme is presumed to be a homohexamer with a subunit molecular mass of 40 kDa. It showed maximal activity at 65–70°C. After incubation for 80 min at 65°C, 50% of the original activity was lost. Gentisate 1,2-dioxygenase activity from strain PK1 was strictly dependent on exogenously added Fe²⁺, and it was inhibited by metal-chelating agents, indicating an essential role of Fe²⁺ in catalysis.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday     

Soil chemical and microbiological properties along a chronosequence of Caragana microphylla Lam. plantations in the Horqin sandy land of Northeast China

Caragana microphylla Lam., a leguminous shrub, is a dominant native plant species widely planted to stabilize the moving and semi-moving sand dunes in the semi-arid Horqin sandy land of Northeast China. The objective of this study was to determine how C. microphylla plantations affected the physical, chemical and microbiological properties of a sandy soil. Soil samples at the depths of 0–10, 10–20, 20–30, 30–40, and 40–50 cm were collected from the C. microphylla plantations with an age sequence of 0, 5, 10, and 23 years. The results showed that shrub growth altered microclimate, increased litter input, and hence, improved soil water holding capacity, contents of total carbon, total N and microbial biomass C and N, electrical conductivity, and activities of urease, phosphomonoesterase, protease, dehydrogenase and polyphenol oxidase, and decreased soil bulk density. These trends increased with increasing plantation age but decreased with increasing soil depth. C. microphylla establishment could be an effective and applicable measure to restore vegetation and control desertification in the Horqin sandy land, and recommended for adoption in semi-arid sandy areas on a large scale.     

黄土丘陵半干旱区人工柠条林土壤固碳特征及其影响因素

为了探讨黄土丘陵区不同生长年限的人工柠条林地土壤有机碳含量的变化特征及其影响因素,更好地阐明黄土丘陵区柠条林土壤的固碳机理,本文采用时空替代法,以撂荒2 a的坡耕地为对照,对黄土丘陵半干旱区不同林龄(10 a、17 a、26 a、34 a、40 a、50 a)人工柠条林地土壤有机碳(SOC)、全氮(STN)、全磷(STP)及柠条林的根系生物量和枯落物现存量进行了分析。结果表明:1)在0~60 cm的土层剖面上,0~20 cm土层SOC含量明显高于其他土层,并随土层深度的增加逐层递减,其中柠条林地0~20 cm土层SOC含量变化幅度为2.68~11.44 g·kg-1,而40~60 cm土层SOC含量仅在1.64~2.73 g·kg-1波动;与对照相比,随林龄增加柠条林地0~60 cm土层平均SOC含量先减小后增加最后趋于平稳:10 a和17 a柠条林SOC含量比对照显著降低了34.5%和26.9%,26 a柠条林的SOC含量显著升高,其值是对照的1.43倍,40 a和50 a柠条林SOC含量处于积累与消耗相对稳定的状态。2)对SOC含量与STN、STP含量及根系生物量和枯落物现存量进行相关性分析表明,SOC含量与STN含量、根系生物量及枯落物现存量之间存在极显著线性相关,但与STP含量相关性不明显,说明土壤中氮含量的增加能明显提高土壤的固碳能力,而根系生物量和枯落物现存量的多少能够决定土壤的固碳水平。     

Effect of heavy metals on growth and survival of Macrophomina phaseolina (Tassi) Goid.

Summary The effects of Cd, Co, Ni and Zn on growth and survival of Macrophomina phaseolina were studied in vitro. Cd, Ni and Co at 500 g ml^–1 inhibited growth by 78.8%, 73.6% and 11.8%, respectively, after 4 days at 25 ± 1°C. The mycelial dry weight yield was enhanced by 2.1% at 100 g ml^–1 Zn. The population of the pathogen declined in soil amended separately with Cd, Co, Ni and Zn. Cd (4000 mg kg^–1) proved to be the most toxic, by completely inhibiting the survival of the pathogen in soil after 20 days.     

Effect of low root temperature on symbiotic nitrogen fixation and competitive nodulation of Onobrychis viciifolia (sainfoin) by strains of arctic and temperate rhizobia

Summary Symbiotic effectiveness and competitive nodulation of the temperate forage legume sainfoin (Onobrychis viciifolia cv. Melrose) by two strains of arctic rhizobia (from Astragalus or Oxytropis sp.) and two strains of temperate rhizobia (from sainfoin) were evaluated at temperatures of 9, 12, or 15°C for roots and 20°C (day) and 15°C (night) for shoots. The inocula consisted of effective individual or paired arctic and temperate strains which were identified on the basis of differential growth. At 9°C, the arctic strains were generally more competitive than the temperate strains, whereas at the highest temperature tested, the converse was apparent. Symbiotic effectiveness (shoot dry weight, nitrogenase activity, and number of nodules) was similarly affected by root temperature, except that at 15°C the arctic strains were generally as effective as the temperate strains. The marked interaction between temperature and strain on competitiveness or on effectiveness indicates symbiotic adaptation of the arctic rhizobia to low temperatures. On the basis of these data we suggest that the use of selected, cold-adapted rhizobia in sainfoin inoculants may be beneficial in temperate regions where low soil temperatures occur early in the growing season.Contribution no. 413 and 1334 of the Agriculture Canada Sainte-Foy Research Station and Plant Research Centre, respectively     

Temporal and spatial patterns of denitrification enzyme activity and nitrous oxide fluxes in three adjacent vegetated riparian buffer zones

The aim of this study was to investigate temporal and spatial patterns of denitrification enzyme activity (DEA) and nitrous oxide (N₂O) fluxes in three adjacent riparian sites (mixed vegetation, forest and grass). The highest DEA was found in the surface (0–30 cm) soil and varied between 0.7±0.1 mg N kg^–1 day^–1 at 5°C and 5.9±0.4 mg N kg^–1 day^–1 at 15°C. There was no significant difference (P >0.05) between the DEA in the uppermost (0–30 cm and 60–90 cm) soil depths under different vegetation covers. In the two deepest (120–150 cm and 180–210 cm) soil depths the DEA varied between 0.0±0.0 mg N kg^–1 day^–1 at 5°C and 4.4±0.9 mg N kg^–1 day^–1 at 15°C and was clearly associated with the accumulation of buried organic carbon (OC). Two threshold values of OC were observed before DEA started to increase significantly, namely 5 and 25 g OC kg^–1 soil at 10–15°C and 5°C, respectively. In the three riparian sites N₂O fluxes varied between a net N₂O uptake of –0.6±0.4 mg N₂O-N m^–2 day^–1 and a net N₂O emission of 2.5±0.3 mg N₂O-N m^–2 day^–1. The observed N₂O emission did not lead to an important pollution swapping (from water pollution to greenhouse gas emission). Especially in the mixed vegetation and forest riparian site highest N₂O fluxes were observed upslope of the riparian site. The N₂O fluxes showed no clear temporal trend.     

Biological nitrogen fixation at elevated temperature in different Azospirillum species and strains

Nitrogenase activity, nitrogenase synthesis, and the growth of four species of Azospirillum were examined at 30°C and 42°C. Azospirillum brasilense Sp 7 did not synthesize nitrogenase at 42°C nor was the enzyme stable at this temperature. In A. lipoferum 708 and A. brasilense 12S the nitrogenase activity was stable at 42°C but the synthesis of nitrogenase was reduced. In A. brasilense Sp 9 the nitrogenase activity was stable and showed the highest C₂H₂ reduction activity at 42°C.     

Variation in temperature requirements for germination and early seedling root development in Chamaecrista rotundifolia and three allied species

Two experiments are discussed. In the first, rates of root elongation during the first four days of growth at 25 °C were studied in Chamaecrista rotundifolia (34 accessions), C. nictitans (2 accessions), C. pilosa (1 accession) and C. serpens (3 accessions). In the second, a comparison was made of germination characteristics of 14 accessions of C. rotundifolia and one of C. pilosa at nine constant temperatures (11–27 °C). Entries included accessions from a wide latitudinal range. There were statistically significant differences in root length on all four days of measurement. No accession had significantly longer roots than the commercial cultivar cv. Wynn on day 1, but over the following three days, 4, 10 and 13 accessions had significantly longer roots, respectively. Root length on day 4 was positively correlated with latitude of origin. In the second experiment, maximum germination occurred at 25.5–27 °C, with progressively lower percentages at lower temperatures. There were highly significant differences between accessions at each temperature (P<0.001), and a highly significant accession × temperature interaction. No accession had a higher germination percentage than cv. Wynn at any temperature. Percentage mortality differed significantly (P<0.05) at all temperatures except 27 °C. Mortality at low temperature was negatively correlated with latitude of provenance. Increased temperatures up to 22.5 °C reduced the time taken to reach 50% of maximum germination (T₅₀), and thereafter it was reasonably constant. With one exception (22.5 °C), no accession germinated significantly more quickly than cv. Wynn, and at 13 °C cv. Wynn germinated significantly more rapidly than all other entries. Over all temperatures, there was a significant negative correlation between days to 50% germination and latitude of provenance. Estimated base temperature for germination (T _b ) ranged from 10.6 °C to 13.6 °C. T _b was positively correlated with latitude of provenance. It is argued that accessions from higher latitudes have developed mechanisms for survival of soft seed at low temperatures, low base germination temperature, germination rates and root elongation rates that adapt them to an environment with summer-dominant rainfall and winters which are too cool to support growth.     

Promotion of mycorrhiza formation and growth of willows by the bacterial strain <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. 23L on fly ash

Re-vegetation of fly ash, the principal by-product of coal fired power stations, is hampered by its unfavourable chemical and physical properties for plant growth. In the present study, we evaluated the use of inoculation with a mycorrhiza-associated bacterial strain (Sphingomonas sp. 23L) to promote mycorrhiza formation and plant growth of three willow clones (Salix spp.) on fly ash from an over-burdened dump in a pot experiment. The high pH_H2O (8.7) and low nitrogen content (N_t = 0.1 g kg⁻¹) in combination with hydrophobicity of the particle surfaces caused low plant growth. Inoculation of the willows with Sphingomonas sp. 23L improved the nitrogen uptake by plants, increased plant growth and stimulated formation of ectomycorrhizae with an autochthonous Geopora sp. strain on all three willow clones. The ectomycorrhiza formed by the Geopora sp. was morphologically and anatomically described. The inoculation significantly increased the shoot growth of two Salix viminalis clones and the root growth of a S. viminalis x caprea hybrid clone. We conclude that inoculation with mycorrhiza promoting bacterial strains might be a suitable approach to support mycorrhiza formation with autochtonous site-adapted ectomycorrhizal fungi in fly ash and thereby to improve re-vegetation of fly ash landfills with willows.     

Growth and survival of cowpea bradyrhizobia in various carrier materials

Summary Pakistan does not yet have the technology for commercial production ofRhizobium andBradyrhizobium inoculum. Therefore, investigations were undertaken to evaluate the suitability of different materials like compost, sawdust, rice husks, sugar cane, filter mud, and peat asBradyrhizobium carriers. The growth and survival of bradyrhizobia (strain TAL 441 of the cowpea type) was studied in sterilized and unsterilized carriers mixed with loam and enriched with lucerne meal and sucrose. Three different sterilization methods (autoclaving, gamma irradiation, and dry heating of the carriers) were used. The growth and survival of bradyrhizobia in the inoculants were studied at two different storage temperatures, 4° and 20°C. After 2–21 months of inoculation, maximum survival of rhizobia (7.6 × 10⁹ cells g^–1) was observed in autoclaved filter mud containing loam-lucerne meal and sucrose. The survival of rhizobia in autoclaved peat was 3.4x 109 cells g-t. The maximum viable number of rhizobia per seed of mungbean (Vigna radiata) was 7.7 × 10⁸ in gamma-irradiated compost and least (1 × 10⁷ cells seed^–1) in rice husks.     

Evaluation of some phosphoroamides as soil urease inhibitors

Summary The effectiveness of six phosphoroamides for retardation of urea hydrolysis in soils was studied by determining the effects of 10 g g^–1 soil of each compound on the amounts of urea hydrolyzed when soils treated with urea were incubated at 10°, 20°, 30°, and 40°C for 3, 7, and 14 days. The phosphoroamides used wereN-(diaminophosphinyl)-cyclohexylamine,N-benzyl-N-methyl phosphoric triamide, diethyl phosphoric triamide, trichloroethyl phosphorodiamidate, dimethyl phosphoric triamide, andN-butyl phosphorothioic triamide [N-(n-butyl) thiophosphoric triamide]. The soils used were selected to obtain a range in properties, and the effects of the six phosphoroamides studied were compared with those of two compounds known to be among the most effective compounds thus far proposed for retardation of urea hydrolysis in soils (phenylphosphorodiamidate and hydroquinone). The data obtained showed that all six of the phosphoroamides evaluated compared favorably with hydroquinone as soil urease inhibitors and that two of them [N-butyl phosphorothioic triamide andN-(diaminophosphinyl)-cyclohexylamine] were superior to phenylphosphorodiamidate for retardation of urea hydrolysis in soils at 20°, 30°, or 40°C.     

Fate of Azolla spp. and urea nitrogen applied to wetland rice (Oryza sativa L.)

Summary Using ¹⁵N, the fate of N applied to wetland rice either as Azolla or urea was studied in a field at the International Rice Research Institute (IRRI). In bigger plots nearby, yield response and N uptake were also determined with unlabelled N sources. Azolla microphylla was labelled by repeated application of labelled ammonium sulfate. Labelled and unlabelled N were used alternately in applications of Azolla or urea 0 and 42 days after transplanting, in order to determine the effect of the time of application on the availability of Azolla N. The quantities of Azolla N incorporated were 23% more than those of urea N (30 kg N ha^–1) in the isotope plots or 7% less in the yield response plots. Grain yield and total N uptake by the rice plants in the yield-response plots were higher in the urea-treated plots than in the Azolla-treated plots, but the physiological effect of Azolla N (grain yield response/increase in N uptake) was higher than that of rea. The labelled N balance was studied after the first and second crops of rice. Losses of labelled N after the first crop were higher from urea (30%–32%) than from Azolla (0%–11 %). Losses in N applied as a side dressing 42 days after transplanting were less than those of N applied basally. No further losses of ¹⁵N occurred after the first crop. The recovery of Azolla ¹⁵N in the first crop of rice was 39% from the basal application and 63% from the side dressing. The recovery of urea ¹⁵N was 27% from the basal application and 48% from the side dressing. Recoveries of residual N from both Azolla and urea during the second rice crop were similar. Laboratory incubation of the Azolla used and the changes in labelled exchangeable N in the soil showed that at least 65% of Azolla N (4.7% N content) was mineralized within 10 days.     

Evaluation of nitrogen fixation by different strains of theAzolla-Anabaena symbiosis in the presence of a high level of ammonium

Summary The proportion of N derived from N₂ fixation for 99 strains ofAzolla spp. (comprising all known species) in the presence of ammonium (40 mg/1) was assessed using a¹⁵N-dilution technique. The percentage of N derived from air varied from 29.5% to 79.9%. Although the N concentration ofAzolla spp. was not correlated with fertilizer N, it correlated fairly well with N₂ fixation. Regression analysis suggests that the N yield ofAzolla spp. is more dependent on N₂ fixation than on ammonium assimilation. The high correlation between N yield and isotopically determined, fixed N₂ indicates that the N yield could be used as a parameter in the selection ofAzolla spp. strains that are capable of maintaining high N₂ fixation in the presence of a high level of ammonium.