Enumeration of rhizobia by enzyme-linked immunosorbent assay (ELISA)

The use of the enzyme-linked immunosorbent assay (ELISA) to enumerate rhizobia in peat carrier and in soil has been investigated. The ELISA technique takes less time than the conventional plant infection technique often used to enumerate rhizobia present in the presence of other micro-organisms. A minimum of 10₂–10₃ cells are required for a detectable ELISA reaction, limiting the use of this technique when the number of rhizobia is low.     

An Ecological Study of Marked Rhizobium trifolii Strains on the Host Plant Trifolium repens var. Huai in an Acidic Peat and a Neutral Mineral Soil

An ecological study of the nodulation of Trifolium repens var. grassland Huai by genetically marked Rhizobium trifolii was carried out in two Irish soils, a neutral mineral and an acidic peat. An indigenous population of 2 x 10⁴ R. trifolii /g was found in the mineral soil. In the peat soil, 4 x 10¹ R. trifolii /g was found in the uninoculated peat. This number increased to 4.5 x 10⁵ R. trifolii /g, however, eight weeks after the peat soil was neutralized, supplemented with nutrients and sown with uninoculated clover seed. Indigenous R. trifolii strains from the mineral soil were effective whereas strains from the peat soil were ineffective on the host plant T. repens under plant room conditions. The introduced strains were inoculated on to clover seed at the rate of 1 x 10⁵ R. trifolii /seed. In the mineral soil, the introduced inoculum failed to establish at any period during the growing season. In the peat soil, the percentage establishment of the introduced inoculum varied from 40-50% of nodules selected eight weeks after sowing to 70-90% of nodules selected at the end of the growing season.     

Production and storage of Rhizobium leguminosarum cell concentrates for use as inoculants

Recovery of Rhizobium leguminosarum cells by centrifugation after growth in an industrial fermenter was 100-fold higher when cells were grown on yeast extract (5 g/1) as sole source of carbon and nitrogen when compared with the yields recovered when cells were grown in standard mannitol-yeast extract medium. Methods of storing concentrated suspensions of R. leguminosarum were investigated. Freeze-drying caused a marked decrease in viable cell numbers. Viable cell numbers of bacterial concentrates stored in peat decreased steadily from 10₁₁-10₁₂ cfu/g to 10₉ cfu/g or less during 26 weeks storage at room temperature or at 4°C. Cell concentrates stored in 40% glycerol at — 20°C maintained viable numbers higher than 10₁₁ cfu/ml during a 76 week storage period.     

Indigenous bradyrhizobia populations in tropical farm-lands

Using the plant-infection technique, and soybeans as test plants, counts of root nodule bacteria in seven farm-lands in Southern Nigeria were estimated. All sites including those without a recent legume cover history contained bradyrhizobia. Abundance, however correlated with the presence of a legume. Populations varied from 0.95 to 3.63 log₁₀ rhizobia per g soil.
In addition to the presence of a legume, soil moisture and pH appeared to influence the rhizobial distribution in these soils. Most of the nodules produced on the test plant were ineffective, indicating that the rhizobial presence alone does not guarantee adequate N₂ fixation in a farming set-up.     

Survival and infectivity of a Rhizobium meliloti strain maintained in water and buffer suspensions

Rhizobium meliloti B323 cells were suspended in deionized water, phosphate buffer pH 6.5 and 5.5 and these buffers supplemented with Ca²⁺, Mg²⁺ (1 mmol/l) and Fe³⁺ (0.1 mmol/l). Initial cell count was 1.10⁸ cells/ml. The viable count of rhizobia suspended in buffer at pH 6.5, with and without salts, remained constant or even increased during storage. Cells suspended in buffer at pH 5.5 with salts, decreased in numbers in the first 5 months, then, until the 10th month, the count remained at 10⁵ cells/ml. Rhizobia suspended in buffer at pH 5.5 and deionized water decreased in viability almost to zero by the 10th month. In those suspensions where viability was maintained, the symbiotic infectivity of cells was also maintained as compared with a control performed with fresh cultured rhizobia. In suspensions in deionized water and buffer at pH 5.5 where the viability diminished during the experiment, the rhizobia lost their ability to infect roots immediately after inoculation but maintained their capacity to form late nodules on the hosts.     

Coat colour inheritance in Soay, Orkney and Shetland sheep

Analysis of the numerical proportions of Soay, Orkney and Shetland sheep of different colours together with test matings, produced results compatible with the hypothesis that these breeds have a multiple allelic series at locus A , white ( A ₁) being dominant to grey ( A ₂) and both being dominant to the gene for self-colour ( A ₅). The alleles at the A locus are epistatic to the alleles for pigment production at locus B , black ( B ₁) being dominant to brown ( B ₂).     

Compensatory growth of juvenile brown flounder Paralichthys olivaceus (Temminck & Schlegel) following thermal manipulation

The compensatory growth of juvenile brown flounder Paralichthys olivaceus (body mass c. 12 g) following different thermal exposure was investigated. Fish were exposed to one of the five temperatures: 8·5 ( T _8·5), 13·0 ( T _13·0), 17·5 ( T _17·5), 22·0 ( T _22·0) and 26·5° C ( T _26·5) for 10 days and fish grew best at 22·0° C. Then the water temperature in all treatments was equably adjusted to 22·0° C over 3 days. At the end of the following 30 days after temperature adjustment, there were no significant differences between body masses of fish in the different treatments (wet body mass at the end of the experiment ranged from 22·13 to 24·56 g). Results indicated that the juvenile P. olivaceus achieved complete compensatory growth. Analysis of the dynamics of the feeding rates and feed conversion efficiencies indicated that compensatory growth of the fish experienced low temperature ( T _8·5, T _13·5 and T _17·5) or high temperature ( T _26·5) exposure was mainly dependent on increasing feed intake (hyperphagia) and possibly by improvement in feed conversion efficiency. The moisture content was not affected by different temperature exposure significantly. The lipid and energy content of juvenile P. olivaceus in T _8·5, however, were significantly lower than other treatment. Results of the current study indicate that a short period of low or high temperature exposure may not affect annual growth, but may affect lipid and energy deposition.     

Temperature responses are a window to the physiology of dark respiration: differences between CO2 release and O2 reduction shed light on energy conservation

We showed that temperature responses of dark respiration for foliage of Pinus radiata could be approximated by Arrhenius kinetics, whereby E ₀ determines shape of the exponential response and denotes overall activation energy of respiratory metabolism. Reproducible and predictable deviation from strict Arrhenius kinetics depended on foliage age, and differed between R _CO2 and R _O2. Inhibition of oxygen reduction ( R _O2) by cyanide (inhibiting COX) or SHAM (inhibiting AOX) resulted in reproducible changes of the temperature sensitivity for R _O2, but did not affect R _CO2. Enthalpic growth – preservation of electrons in anabolic products – could be approximated with knowledge of four variables: activation energies ( E ₀) for both R _CO2 and R _O2, and basal rates of respiration at a low reference temperature ( R _REF). Rates of enthalpic growth by P. radiata needles were large in spring due to differences between R _REF of oxidative decarboxylation and that of oxygen reduction, while overall activation energies for the two processes were similar. Later during needle development, enthalpic growth was dependent on differences between E ₀ for R _CO2 as compared with R _O2, and increased E ₀( R _O2) indicated greater contributions of cytochrome oxidase to accompany the switch from carbohydrate sink to source. Temperature-dependent increments in stored energy can be calculated as the difference between R _CO2▵ H _CO2 and R _O2▵ H _O2.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

The construction, detection and use of bioluminescent Rhizobium leguminosarum biovar trifolii strains

A. CRESSWELL, L. SKØT AND A.R. COOKSON. 1994. The gene encoding the firefly luciferase enzyme ( luc ) was introduced to Rhizobium leguminosarum biovar trifolii strains with a view to using the resulting bioluminescent strains to study the survival of genetically-engineered rhizobia in soil microcosms. The genetically-engineered micro-organisms (GEMs) behaved similarly to their parent strains with respect to growth rate in laboratory media and in their symbiotic performance with their host plants. No gene transfer could be detected in laboratory mating experiments. When inoculated onto a non-sterile soil the population of the GEM declined sharply from an initial cell density of 2 times 107⁷ g^-1 soil to approach a stable cell density of approximately 3 times 10² g^-1 after 150 d. Direct photography of bioluminescent rhizobia enabled the detection of colonies as small as 0.1 mm in diameter without the need for transferring colonies onto filter paper. When a Rhizobium strain carrying the luc marker on a plasmid was used as inoculant it was possible to visualize differences in colonization of the rhizosphere of white clover and ryegrass by contact print and colour transparency films. The photographic detection methods described here demonstrate the possibilities of using bioluminescent rhizobia for assessing their survival in soil, and for looking at rhizosphere populations which may be an important site for potential gene transfer.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

Application of the 'Inoculated Pack Test' method for the determination of the heat resistance of Clostridium sporogenes PA3679 spores in acidified mushrooms

The effect of pH in the range 5.2–6.7 on the thermal destruction of Clostridium sporogenes PA3679 spores suspended in mushrooms in brine acidified with citric acid was examined by the 'inoculated pack test' method. The results indicated that increasing acidity is accompanied by decreasing decimal reduction times at 121.1°C: D _121.1 at pH 6.0 and 5.2 was, respectively, 64% and 17.5% of that at pH 6.7, the pH of natural mushrooms ( D _121.1= 2.22 min). A linear model ( r = 0.988, α= 0.05) was developed where the D _121.1 value was a function of the pH over the range studied. The inoculated pack test seems to be the only method to evaluate the actual microbial heat resistance, whether of spore or of vegetative forms, in order to estimate within reasonably close limits a suitable process time required to eliminate health hazards and to prevent spoilage losses in a given food product.     

Three parameters comprehensively describe the temperature response of respiratory oxygen reduction

Using an exponential model that relies on Arrhenius kinetics, we explored Type I, Type II and dynamic (e.g. declining Q ₁₀ with increasing temperature) responses of respiration to temperature. Our Arrhenius model provides three parameters: R _REF (the base of the exponential model, nmol g⁻¹ s⁻¹), E ₀ (the overall activation energy of oxygen reduction that dominates its temperature sensitivity, kJ mol⁻¹) and δ (that describes dynamic responses of E ₀ to measurement temperature, 10³ K²). Two parameters, E ₀ and δ , are tightly linked. Increases in overall activation energy at a reference temperature were inversely related to changes in δ . At an E ₀ of ca. 45 kJ mol⁻¹, δ approached zero, and respiratory temperature response was strictly Arrhenius-like. Physiologically, these observations suggest that as contributions of AOX to combined oxygen reduction increase, E ₀( REF ) decreases because of different temperature sensitivities for V _max, and δ increases because of different temperature sensitivities for K _1/2 of AOX and COX. The balance between COX and AOX activity helps regulate plant metabolism by adjusting the demand for ATP to that for reducing power and carbon skeleton intermediates. Our approach enables determination of respiratory capacity in vivo and opens a path to development of process-based models of plant respiration.     

In vitro evidence for the ionoregulatory role of rainbow trout mucus in acid, acid/aluminium and zinc toxicity

Rainbow trout body mucus dialysed with acidified distilled water at pH 7,5 and 3 experienced ion depletion which was greatest at pH 3 and minimal between pH 7 and 5. Mucus Na⁺ loss is exacerbated in the presence of 1 mg I⁻¹ aluminium as A1₂(SO₄), at pH 5 and 7. Al₂(SO₄), causes greater depletion of Na⁺ from mucus than A1C1₃. A lethal level of zinc (2 mg 1⁻¹) does not deplete mucus Na⁻ or K⁺, unlike a lethal level of aluminium (1 mg 1⁻¹) at pH 7. The results are discussed in terms of the ionoregulatory role of mucus in heavy metal and acid toxicity.     

Note: Influence of different heating media on thermal resistance of Neosartorya fischeri isolated from papaya fruit

E. RAJASHEKHARA, E.R. SURESH AND S. ETHIRAJ. 1996. A heat-resistant mold identified as a strain of Neosartorya fischeri was isolated from microbiologically spoiled papaya fruits. The optimum heat activation temperature and time for the ascospores of the test mold was found to be 80°C for 15–30 min. The decimal reduction times ( D -values) at 85°, 87° and 89°C in phosphate buffer (pH 7·0) as heating medium were 35·25, 11·1 and 3·90 min respectively and hence the calculated z -value was 4·0°C. In grape and mango juices as heating media, the D _80°C and the D _85°C values were increased as the °Brix level raised from 10 to 45. In commercial fruit juices of mango, orange, pineapple and mango-pineapple blend as heating media D _85°C values were greater than those observed for phosphate buffer.     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

The Occurrence of Bacillus coagulans with High Heat Resistance

S ummary : The heat resistance of spores of Bacillus coagulans strain Sp. 33, isolated from canned chopped spinach, has been determined. It appeared to be higher in 1/40 M phosphate than in 1/15 M phosphate and higher at pH 6°8 than at pH 6°0. In 1/40 M phosphate buffer of pH 6°8 the calculated D ₁₁₀ was 2°4 min. In chopped spinach of pH 6°2 the calculated D ₁₁₀ was 0°84 min. In addition a simple and reliable method of assessing the thermal resistance of micro-organisms in thermal death time tubes is reported.     

Carbon dioxide anaesthesia in rainbow trout: effects of hypercapnic level and stress on induction and recovery from anaesthetic treatment

The physiological and anaesthetic effects of three different levels of air-saturated and buffered CO₂ anaesthesia, P _w co ₂=31, 78, or 125 mmHg, were examined in cannulated rainbow trout Oncorhynchus mykiss . Complete anaesthesia (no opercular movements) was not achieved by these hypercapnic levels after 20 min of CO₂ exposure. Although increasing P _w co ₂ reduced the induction times to the early stages of anaesthesia, it also resulted in increasing hyperventilatory, hypoxaemic, and acid-base disturbances. After a 10-min recovery period, while the respiratory acidosis component of the acid-base disturbance was corrected, there was a significant metabolic acidosis. Recovery time was longest in the high P_w co₂ treatment where 33% of the fish died. Two additional groups ( P _w co ₂=37 and 78 mmHg) were exposed to an acute stress prior to the anaesthetic treatment. Stress reduced the hypoventilatory effects of the low P _w co₂ treatment, increased the recruitment of anaerobic metabolism, and prolonged recovery time. Although the increase in plasma catecholamines elicited by the stress was small relative to the response obtained with the anaesthetic, stress prior to CO₂ anaesthesia impaired the efficiency of the treatment. Overall, our results suggest that P _w co₂ levels above 37 mmHg and/or stress prior to the anaesthesia impair the efficiency of air-saturated and buffered CO₂ anaesthesia by exacerbating the hypoxaemic effects of the hypercapnic treatment.