On the production of legume inoculants in a mixture of peat and soil

Summary A study was made of the growth of rhizobia in mixtures of peat, heavy loam and CaCO₃, with or without addition of salts and of organic nutrients. Extremely high numbers (4000–20.000 millions per g of moist material) were obtained by using alder bog peat and 10–20% lucerne meal in these mixtures and by growing the rhizobia in pure condition with access of air. When lucerne meal is present in these quantities, addition of salts, yeast and other nutrients is superfluous. Good results have been obtained by using dispensing-bottles as containers, plugged with cotton-wool. The media in the bottles are inoculated with a liquid inoculum by means of a hollow needle, which is stuck between the cotton-wool plug and the wall of the neck of the bottle. By this simple procedure contamination of the media with air-borne organisms is reduced to a minimum. The numbers remain high for a long period when the cultures are stored at 2°C. with access of some air.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Acer grandidentatum</Emphasis> Nutt.

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single- and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P = 0.0242) but the zone of explant origin (P = 0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P = 0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.     

Mineral composition and growth of Colorado spruce (Picea pungens) seedlings under calcareous soil conditions

Summary Field studies were conducted to assess the mineral nutrition and growth of Colorado spruce (Picea pungens Engln) seedlings (2–4 yr) from provenances selected for superior growth on calcareous prairie soils. Tissue nutrient concentrations and response to nitrogen were determined by use of foliar analysis and growth pattern studies. Soil conditions ranged from 7.6–7.8 for pH, 12–23% for total CaCO₃, and 5–6% for active CaCO₃. Foliage mineral composition showed relatively low phosphorus (0.09–0.15%) and high calcium (0.45–1.52%) assimilation. Seasonal growth and seedling response to added nitrogen was not adversely affected by the calcareous soil condition. Levels of nitrogen in the foliage required for optimum growth ranged from 1.5–2.0% and were similar to that of other conifer species.     

Inoculation with an enhanced N2‐fixing Bradyrhizobium japonicum strain (USDA110) does not alter soybean (Glycine max Merr.) response to elevated [CO2]

This study tested the hypothesis that inoculation of soybean (Glycine max Merr.) with a Bradyrhizobium japonicum strain (USDA110) with greater N₂ fixation rates would enhance soybean response to elevated [CO₂]. In field experiments at the Soybean Free Air CO₂ Enrichment facility, inoculation of soybean with USDA110 increased nodule occupancy from 5% in native soil to 54% in elevated [CO₂] and 34% at ambient [CO₂]. Despite this success, inoculation with USDA110 did not result in greater photosynthesis, growth or seed yield at ambient or elevated [CO₂] in the field, presumably due to competition from native rhizobia. In a growth chamber experiment designed to study the effects of inoculation in the absence of competition, inoculation with USDA110 in sterilized soil resulted in nodule occupation of >90%, significantly greater ¹⁵N₂ fixation, photosynthetic capacity, leaf N and total plant biomass compared with plants grown with native soil bacteria. However, there was no interaction of rhizobium fertilization with elevated [CO₂]; inoculation with USDA110 was equally beneficial at ambient and elevated [CO₂]. These results suggest that selected rhizobia could potentially stimulate soybean yield in soils with little or no history of prior soybean production, but that better quality rhizobia do not enhance soybean responses to elevated [CO₂].     

Effects of some chemical factors on flagellation and swarming ofVibrio alginolyticus

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0–9.0% (w/v) NaCl, cells with polar tufts of 2–4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3–9.8% (w/v) Na₂SO₄ had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1–0.3% boric acid or 0.05–0.1% aluminium hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na-deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01–0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05–0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.     

Influence of Rhizobial inoculation on yield of wheat (Triticum aestivum L.)

Summary Soil + charcoal (1∶3) carrier based and liquid cultures of Rhizobia were used to inoculate wheat seed cv. HD2329. The plants received 100 kg N in two equal splits and 60 kg P₂O₅ and 40 kg K₂₀ ha⁻¹. Inoculation with rhizobia had little effect on grain yield of wheat. Significant increase in straw yield and N-uptake occurred due to inoculation. A comparison of results of a similar experiment conducted during 1983–84, showed that inoculation with the same strains of rhizobia and application 50 kg N ha⁻¹ as basal dressing, was more effective in increasing yield and N-uptake in wheat cv. HD2329. It appears reasonable to assume occurrence of nitrogen fixation by root nodule bacteria in rhizosphere of wheat.     

Carbon isotope evidence for recent climate‐related enhancement of CO 2 assimilation and peat accumulation rates in Antarctica

Signy Island, maritime Antarctic, lies within the region of the Southern Hemisphere that is currently experiencing the most rapid rates of environmental change. In this study, peat cores up to 2 m in depth from four moss banks on Signy Island were used to reconstruct changes in moss growth and climatic characteristics over the late Holocene. Measurements included radiocarbon dating (to determine peat accumulation rates) and stable carbon isotope composition of moss cellulose (to estimate photosynthetic limitation by CO ₂ supply and model CO ₂ assimilation rate). For at least one intensively ¹⁴C‐dated Chorisodontium aciphyllum moss peat bank, the vertical accumulation rate of peat was 3.9 mm yr^?1 over the last 30 years. Before the industrial revolution, rates of peat accumulation in all cores were much lower, at around 0.6–1 mm yr^?1. Carbon‐13 discrimination (Δ), corrected for background and anthropogenic source inputs, was used to develop a predictive model for CO ₂ assimilation. Between 1680 and 1900, there had been a gradual increase in Δ, and hence assimilation rate. Since 1800, assimilation has also been stimulated by the changes in atmospheric CO ₂ concentration, but a recent decline in Δ (over the past 50–100 years) can perhaps be attributed to documented changes in temperature and/or precipitation. The overall increase in CO ₂ assimilation rate (¹³C proxy) and enhanced C accumulation (¹⁴C proxy) are consistent with warmer and wetter conditions currently generating higher growth rates than at any time in the past three millennia, with the decline in Δ perhaps compensated by a longer growing season.     

Survival of several <Emphasis Type="Italic">Rhizobium/Bradyrhizobium</Emphasis> strains on different inoculant formulations and inoculated seeds

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 °C), or (b) at room temperature (25 °C). The effect of water content (30–50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage. Electronic Publication     

Spring-associated limestones of the Eastern Alps: overview of facies,deposystems, minerals,and biota

In the Eastern Alps, both fossil spring limestones and actively limestone-depositing springs are common. The geological context and a few radiometric age data of fossil spring-associated limestones (SAL) mentioned herein indicate that they accumulated subsequent to the Last Glacial Maximum in the Eastern Alps (24–21 ka BP). Prevalent facies of the SAL deposits, active and fossil, including phytoclastic tufa, microbialites s.l., springstone, and moss tufa form, or formed, from (a) waterfall/creek systems, (b) hillslope-paludal systems, (c) moss-tufa systems, and from (c) foreland-type systems. Precipitated minerals include calcite and, at springs of elevated Mg/Ca ratio, magnesian calcite and aragonite. In a few limestone-depositing, oxygen-deficient springs with dissolved Fe²⁺, downstream, iron oxide precipitates ahead of CaCO₃ (mineralogical zonation). Biota associated with calcium-carbonate deposition include cyanobacteria, green micro-algae, macro-algae, and mosses. Calcium-carbonate precipitation may be speeded by biological mediation, but mineralogy and polymorphy of precipitated CaCO₃ are not biotically controlled. In the Eastern Alps, SAL deposits in total range from 190 to 2,520 m a.s.l., corresponding to mean annual temperatures of 10°C to less than 0°C. In altitudes below the continuous permafrost line (about 2,600–3,000 m a.s.l., depending on location), SAL deposition is chiefly controlled by proper balance between water supply and sufficient supersaturation for CaCO₃, rather than by mean annual temperature.     

The effect of foliar nutrition on yield of greenhouse tomatoes and quality of the crop

Tomatoes grown in traditional mediums such as peat moss are usually supplied with essential nutrients by preplant fertilization and in the later growth stages by top dressing application to the root zone. According to the data from literature the efficient method of nutrient supply during the intensive stage of growth may be estimated as by foliar spraying. In the greenhouse experiment conducted in 1998–1999 a preplant fertilization by multicomponential fertilizer MIS-4 was applied to peat moss growing medium in full (4 kg per m³) and half (2 kg per m³) of recommended rate. Supplemental fertilization during the growing period was provided to the root zone or by using liquid multicomponential fertilizers Ekolist S and Mikrosol U in 5 sprays conducted at two week intervals. Results of the study proved that advantageous effect of foliar tomato nutrion grown in eat substrate was observed only in treatments with restricted supply of fertilizers to the growing medium. The maximum fruit yield was received from plots provided by the reduced preplant MIS-4 dose to 50 % of recommended rate combined with foliar sprays by Ekolist S. Such system of fertilization enhanced the marketable yield of fruit by 9.8 % and early yield by 11.3 % as compared to that obtained with preplant and top dressing soil application. Foliar nutrion did not change dry matter and vitamin C content and increased total and reducing sugars accumulation in tomato fruits.     

Soil surface CO2 flux in a Minnesota peatland

Soil surface CO₂ flux was measured in hollow and hummock microhabitats in a peatland in north central Minnesota from June to October in 1991. We used a closed infrared gas exchange system to measure soil CO₂ flux. The rates of CO₂ evolution from hummocks (9.8 ± 3.5 g m⁻² d⁻¹, [mean ± SE]) were consistently higher than those from hollows (5.4 ± 2.9 g m⁻² d⁻¹) (the hummock values included the contribution of moss dark respiration, which may account for 10–20% of the total measured flux). The soil CO₂ flux was strongly temperature-dependent (Q₁₀ ≈ 3.7) and appeared to be linearly related to changes in water table depth. An empirical multiplicative model, using peat temperature and water table depth as independent variables, explained about 81% of the variance in the CO₂ flux data. Using the empirical model with measurements of peat temperature and estimates of hollow/hummock microtopographic distribution (relative to water table elevation), daily rates of “site-averaged” CO₂ evolution were calculated. For the six-month period (May–October), the total soil CO₂ released from this ecosystem was estimated to be about 1340 g CO₂ m⁻². Published as Paper No. 9950, Journal Series, Nebraska Agricultural Research Division, University of Nebraska, Lincoln, NE, USA.     

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23°C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO₃ was added to the growth media. There were strong interactions between CaCO₃ and media, CaCO₃ and pH level, and CaCO₃, media, and pH level. In media with CaCO₃, all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO₃ increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 × 10¹⁰/g). In CaCO₃-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.     

In vitro regeneration of Sophora toromiro from seedling explants

Embryonic axes with cotyledons, shoot-tips of embryonic axes, isolated cotyledons, as well as axillary buds and leaves from 20-year-old trees of Sophora toromiro, were evaluated for their capacity to trigger organogenesis and to regenerate plantlets under in vitro conditions. Embryonic shoot-tips were the only explants capable of regenerating plants. They developed rapidly in vitro in the presence of NAA and BA while in subculture roots were induced at the proximal end in the presence of 0.49 μM IBA within 40–60 days. Development was completed with a subculture phase under non-sterile conditions using a mixture of equal parts of sterilized vermiculite/sand/soil in growth chambers, before final acclimation in the greenhouse. In the presence of NAA, BA and GA₃, whole embryonic axes formed multiple shoots that branched when grown in 2.27 or 11.35 μM TDZ in subculture. Similarly, callus was initiated at the embryo axis base, developing into several new shoots in the presence of TDZ. Because of the relatively high shoot induction rate along the embryonic axis, this axis presents a valuable source of new juvenile explants. Growth and rhizogenesis was satisfactory only when organs from seed pods of the year or from the previous season were used. Experiments with isolated cotyledons produced callus only, while axillary buds and leaves did not show any responses in the presence of several growth regulators assayed. Inoculation of seedlings with various strains of rhizobia under in vitro conditions resulted in root outgrowths, but not in nodules that are typical of rhizobia infection. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synergistic interactions of arbuscular mycorrhizal fungi and rhizobia promoted the growth of <Emphasis Type="Italic">Lathyrus sativus</Emphasis> under sulphate salt stress

In order to examine the influence of microsymbionts on plants, arbuscular mycorrhizal (AM) fungi and rhizobia were used to examine the growth of Lathyrus sativus under sulphate salt stress. Seedlings of L. sativus were inoculated with a combination of selected microsymbionts. Plants were grown under greenhouse conditions with five Na₂SO₄ concentrations (0, 1%, 2%, 3% and 4% (weight : weight)). The inoculations combinations used were the AM fungus, Glomus mosseae and/or the rhizobium, Mesorhizobium mediterraneum. The results showed that sulphate salinity inhibited plant growth and biomass production. However, compared with the control treatments, dual-inoculation of G. mosseae and M. mediterraneum reduced the harmful influence of sulphate salinity. Parmeters including plant height, the extent of AM colonization, total biomass, nodules biomass, P concentration, N concentration and proline concentration confirmed that dual inoculation plays a vital role in promoting the growth of L. sativus under sulphate salt stress. The results suggested that the use of this dual inoculation could be exploited in grassland plantation establishment and in pastoral ecosystem reclamation programmes in arid and semi-arid areas subject to moderate salt contamination.     

Requirement for particular seed-borne fungi for seed germination and seedling growth of <Emphasis Type="Italic">Xyris complanata</Emphasis>, a pioneer monocot in topsoil-lost tropical peatland in Central Kalimantan,Indonesia

Hawaii yellow-eyed grass (Xyris complanata: Xyridaceae) inhabits infertile, acidic peat soil in the rainy tropical zone in Southeast Asia. This monocot plant produces a large number of dormant seeds in order to make a large deposit to seed bank in the soil. Under laboratory conditions, surface-sterilized X. complanata seeds are rarely able to germinate on sterilized peat moss bed; they require inoculation with either seed epiphytic or soil fungi to facilitate active seed germination. In the present study, three different genera of seed epiphytic fungi were isolated, and two common fungal genera, Fusarium sp. (strain R-1) and Penicillium sp. (strain Y-1), were found to promote seed germination of X. complanata. In sterile peat moss beds, the germination-stimulating fungi also showed growth-promoting effects on X. complanata seedlings. These results suggest that the seed germination-promoting fungi likely function as genuine partners for X. complanata in tropical open peat lands.     

Productivity correlated to photobiochemical performance of <Emphasis Type="Italic">Chlorella</Emphasis> mass cultures grown outdoors in thin-layer cascades

This work aims to: (1) correlate photochemical activity and productivity, (2) characterize the flow pattern of culture layers and (3) determine a range of biomass densities for high productivity of the freshwater microalga Chlorella spp., grown outdoors in thin-layer cascade units. Biomass density, irradiance inside culture, pigment content and productivity were measured in the microalgae cultures. Chlorophyll-fluorescence quenching was monitored in situ (using saturation-pulse method) to estimate photochemical activities. Photobiochemical activities and growth parameters were studied in cultures of biomass density between 1 and 47 g L⁻¹. Fluorescence measurements showed that diluted cultures (1–2 g DW L⁻¹) experienced significant photostress due to inhibition of electron transport in the PSII complex. The highest photochemical activities were achieved in cultures of 6.5–12.5 g DW L⁻¹, which gave a maximum daylight productivity of up to 55 g dry biomass m⁻² day⁻¹. A midday depression of maximum PSII photochemical yield (F _v/F _m) of 20–30% compared with morning values in these cultures proved to be compatible with well-performing cultures. Lower or higher depression of F _v/F _m indicated low-light acclimated or photoinhibited cultures, respectively. A hydrodynamic model of the culture demonstrated highly turbulent flow allowing rapid light/dark cycles (with frequency of 0.5 s⁻¹) which possibly match the turnover of the photosynthetic apparatus. These results are important from a biotechnological point of view for optimisation of growth of outdoor microalgae mass cultures under various climatic conditions.     

A dry granular inoculant of Rhizobium for soil application

A dry granular inoculant of Rhizobium was prepared from sodium alginate and peralite. High numbers of two groundnut (Arachis hypogaea) Rhizobium strains, NC 92 and TAL 1000 used to prepare inoculants survived in dry granules beyond 180 days. The viable counts were 9.72 and 9.91 log₁₀ rhizobia g^-1 of dry granules for NC 92 and TAL 1000, respectively compared to 8.0 log₁₀ rhizobia g^-1 of peat inoculant for NC 92 at the end of six months storage. The granular inoculant was free from contaminants. In a pot culture experiment the granular inoculant applied to the soil gave similar results when seeds were dressed with a peat inoculant; nodulation and growth of groundnut were similar. The major advantage of this inoculant is that, it can be stored in a dry state without losing much viability.     

Ectomycorrhizal fungi promote growth of <Emphasis Type="Italic">Shorea balangeran</Emphasis> in degraded peat swamp forests

Shorea balangeran is an important component of peat swamp forests in Southeast Asia and is an important source of timber. However, S. balangeran has been decreasing in number due to overexploitation. The objective of this study was to investigate the effect of inoculation of native ectomycorrhizal (ECM) fungi on growth of S. balangeran in degraded peat swamp forest. Spores of Boletus sp., Scleroderma sp., and Strobilomyces sp. were collected from natural peat swamp forest in Indonesia. Seedlings of S. balangeran were inoculated with or without (control) spores and grown in sterilized peat soil under nursery conditions for 6 months. Then, the seedlings were transplanted into a degraded peat swamp forest and grown for 40 months. ECM colonization was 59–67% under nursery conditions and increased shoot height and weight. Shoot height, stem diameter, and survival rates were higher in inoculated seedlings than in control 40 months after transplantation. The results suggest that inoculation of native ECM fungi onto native tree species is useful for reforestation of degraded peat swamp forests.     

A simplein situ method for the characterization of porosity in growth media

Summary The method presented was based on a very simple approach of the forces operating in the pores of porous media. The standardized time (t_v) needed to infiltrate a given volume of a liquid into a porous medium in a defined state was throught to be an integrated measure of pore geometry and continuity. The state of the pore system was defined by external suction (S) and medium porosity characterized by the parameters k₁ and k₂ in the equation t_v=k₁+k₂ S^−0.5. The method theory was not rejected by experiments with glass beads and selected peat based growth media. The method ranked the media with respect to the probability for satisfactory gas exchange in the order of peat, peat and 26% perlite, peat and 34% perlite, and peat and 44% mineral wool. This ranking was achieved 95 days after the media were filled in containers and exposed to a daily watering procedure. Before this time, the ranking of the media was slightly different, if at all possible. Five days after the containers were filled, only peat and 44% mineral wood was significantly different from the other media. Judged by the standardized time method, the probability for satisfactory gas exchange decreased significantly during the 95 days experiment. This aggravation was supported by measurements of the volume fraction of pores filled with gas. The changes with time were least marked in the medium containing mineral wool.     

Differential medium for revelation of bacterial producer strains of L-asparaginases

A specific, fast, and easy method for revelation of active plate producers of L-asparaginase using differential medium on the basis of LB or M9 with 1.5% agar was developed. Each 100 ml of LB or M9 medium additionally contained 6–7 ml of glycerol, 4 g of L-asparagine, 0.2 g of CaCO₃, and diagnostic components: 3 ml of 0.2 M CuSO₄ · 5H₂O and 2.5 ml of 0.1 M K₃Fe(CN)₆, pH 7.6–7.8. The results were counted 12–20 or 24–48 h after strain growth at 37°C in corresponding mediums. Red color of colonies and colored zone around them showed the ability of the strain under study to destroy asparaginic complexes. The recommended method allows revealing bacterial strains producing L-asparaginase with specific activity of not less than 0.1–3.0 MU/mg of protein.