Hans Ljunggren

Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii.

Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.

Structural studies of the Rhizobium trifolii extracellular polysaccharide

Abstract The structure of the extracellular polysaccharide of Rhizobium trifolii has been investigated. Methylation analysis, sequential degradations by oxidation and elimination of oxidized residues, uronic acid degradation, and degradation by oxidation of the acetylated polysaccharide with chromium trioxide in acetic acid were the main methods used. It is proposed that the polysaccharide is composed of heptasaccharide repeating-units having the following structure: An unusual feature is that some of the repeating units are incomplete and lack the terminal β- d -galactopyranosyl group. The polysaccharide contains O -acetyl groups (somewhat more than 1 mol. per unit), linked to O-2 and O-3 of 4- O -substituted d -glucopyranosyl chain-residues. A previous finding that the polysaccharide contains 2-deoxy- d - arabino -hexose (2-deoxy- d -glucose) residues is erroneous.

Microbial numbers and activity during infiltration of septic-tank effluent in a subsurface sand filter

Abstract A subsurface, 4-person sand-filter system for treating septic-tank effluent was subjected to conventional water analysis and to an extended microbial analysis of the filter material. Measured rates of CO 2 production in the filter material suggest that the system has a good microbial capacity to degrade the organic matter in wastewater; the volume effectively treated depends on the BOD (e.g. 951 m −2 day −1 at a BOD of 169 mg l −1 and 285 l m −2 at a BOD of 74 mg l −1 ). The actual load was 40 to 80 l m −2 day −1 . Based on viable counts of bacteria the amounts of P and N bound in the 13 ± 10 g dw m −2 viable biomass were calculated to correspond to the amounts produced by 1 person in m −2 , indicating that the viable biomass also gives an accurate estimate of the active biomass. The levels of ATP in the sand-filter surface revealed that the loading of wastewater occurred unevenly. The high numbers of ammonium-oxidizing bacteria (4 × 10 3 −6 x 10 6 g −1 dw) and denitrifying bacteria (2 × 10 7 −1 =× 10 9 g −1 dw) in the surface layer show that the system was predominantly operating aerobically and had a high potential for removing nitrogen. The microbial techniques used were sensitive enough to detect the decrease in biomass that occurred with increasing depth. Reduction of COD, P and N in the wastewater during infiltration were estimated to be 86, 70 and 60% respectively. These figures are considered to be overestimates, since analysis of Cl showed that groundwater had leaked into the bed, diluting the effluent water. A correction factor for the dilution effect was therefore introduced. In addition, N and P from the surrounding arable land periodically leaked into the bed. The environment in the sand filter was sufficiently stable to ensure high microbial activity; thus the purifying capacity of septic-tank effluent should remain high throughout the year.

A Modified, Highly Sensitive Enzyme-linked Immunosorbent Assay (ELISA) for Rhizobium meliloti Strain Identification

SUMMARY: An evaluation of the ELISA technique was made in order to obtain a very specific and sensitive method for strain identification of Rhizobium meliloti. Antisera against an R. meliloti strain were produced by using as antigen either whole cells or purified lipopolysaccharides from the cell wall. The use of whole cells as antigen gave rise to antibodies which were more sensitive and strain specific than those produced from purified lipopolysaccharides. A further evaluation of the ELISA method was the use of a new type of conjugate consisting of a purified antibody linked to β-galactosidase by using as coupling reagent a hetero-bifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which contains two reactive groups directed towards different functional groups. Substrate for this type of conjugate consisted of o-nitrophenyl-β-D-galactopyranoside. This improved technique makes it possible to detect bacteria in samples containing 1 × 103 cells ml−1 whilst retaining strain specificity. Furthermore, preparations of nodules formed by different R. meliloti strains containing 1 × 105 cells ml−1 could be analysed whilst retaining strain specificity. The technique presented thus offers advantages in higher sensitivity and reliability than conventional ELISA techniques.

Field measurement of symbiotic nitrogen fixation in an established lucerne ley using15N and an acetylene reduction method

SummaryLucerne is an important forage legume in the south and south-east of Sweden on well-drained soils. However, data is lacking on the apparent amount of nitrogen derived through N2 fixation by field-grown lucerne. This report provides basic information on the subject. The experiment was performed in a lucerne ley grown 40 km north of Uppsala. The input of nitrogen through fixation to the above-ground plant material of an established lucerne (Medicago sativa L.) ley was estimate by15N methodology during two successive years. The amount of fixed N was 242 kg N ha−1 in 1982 and 319 kg N ha−1 in 1983. The proportion of N derived from the atmosphere (%Ndfa) was 70% and 80% for the two years respectively. The first harvest in both years contained a lower proportion fixed N. Both N2 fixation and dry matter production were enhanced during the second year, particularly in the first harvest. The Ndfa was 61% in the first harvest in 1982, compared to 72% Ndfa during the same period in 1983. This demonstrates the strong influence of environment on both dry matter production and N2 fixation capacity of the lucerne.In addition anin situ acetylene reduction assay was used in 1982 to measure the seasonal distribution of the N2 fixation and in 1983 to study the effect of soil moisture on the N2 fixation process. The seasonal pattern showed great dependence on physiological development and harvest pattern of the lucerne ley. The maximum rate of N2 fixation occurred at the bud or early flower stage of growth and was followed by a rapid decline as flowering proceeded. After harvest the nitrogenase activity markedly decreased and remained low during at least two weeks until regrowth of new shoots began. Irrigation doubled the nitrogenase activity of the lucerne in late summer 1983, when soil moisture content in the top soil was near wilting point. No changes in nitrogenase activity did occur in response to watering earlier during the summer, when the soil matric potential was around −0.30 MPa.

A comparison between the acetylene reduction method, the isotope dilution method and the total nitrogen difference method for measuring nitrogen fixation in lucerne (Medicago sativa L.)

SummaryIn view of the increasing need for the exact estimation of the input of nitrogen in agroecosystems, an application of the acetylene-reduction technique was developed. The technique, consisting of a plastic bag incubation system, using propane as an internal standard of the apparent volume, made it possible to carry out repeated incubations on the same plant system. The evaluated technique included studies of the diffusion of ethylene and propane in a soil column, as well as studies of the optimal substrate concentration and grade of purity of the substrate. In addition, the conversion factor between amounts of reduced acetylene as compared wtih reduced dinitrogen was determined by15N2 incubations to be 4.41. The developedin situ acetylene-reduction technique was compared with an isotope dilution method and a total nitrogen difference method. By comparing the derived total nitrogen fixation values from each with the value derived from the acetylene-reduction method; it was shown that the values differed significantly. The acetylene-reduction method gave the highest nitrogen fixation values, the isotope dilution the lowest values and the total nitrogen difference method was intermediate. No statistical significant difference existed between the two different reference crops used in the isotope dilution method.

Eficiência simbiótica e caracterização ecológica de uma população nativa de Rhizobium loti no Uruguai

5 ABSTRACT - The objectives of this work were to describe the distribution, density and seasonal variation of the indigenous populations of Rhizobium loti in different Uruguayan soils and to deter- mine the symbiotic effectiveness and stress tolerance factors of different isolates, both with the aim of obtaining selected strains to re-introduce as inoculants in Lotus pastures. R. loti was present in ten soils studied and their densities varied from year to year and within each soil. All the isolates nodulated Lotus corniculatus effectively. The nodules in Lotus pedunculatus and Lotus subbiflorus were small, red on the surface and ineffective in nitrogen fixation. The study of 50 isolates from the ten soils showed high variability in their symbiotic efficiency and tolerance to pH. The indigenous population was acid tolerant in culture medium (pH 4.5), 83% of them could grow at pH 4.5 in 3 days. This work showed that there was a great diversity between the strains of R. loti isolated from Uruguayan soils and supports the importance of selecting among them the most efficient and resistant strains to be included in the inoculants.

Composition of the bacterial population in sand-filter columns receiving artificial wastewater, evaluated by soft independent modelling of class analogy (SIMCA)

Abstract The objective of the present study was to develop a test system for clustering and characterizing the functions of the bacterial populations in wastewater infiltration systems. To create different bacterial ecosystems, sand-filter columns were loaded with a low loading (2.9 ± 0.1 cm d −1 ) and a high loading (8.7 ± 0.2 cm d −1 ) of an artificial wastewater for 114 days. Before loading onto the columns the wastewater was pretreated in a three-chambered container for 24 h. The low loaded filters attained a high and steady rate of mineralization and complete nitrification, whereas the high loaded filters developed a clogging surface, and incomplete nitrification despite a high rate of mineralization. On day 60 the sand surface was sampled, and the bacteria were extracted and enumdrated. From each column system 88 bacterial isolates were collected. Each isolate was subjected to 51 physiological and biochemical tests. The results were analyzed using the SIMCA statistical package. Using partial least-square regression discriminant analysis we developed a model that revealed five major clusters of bacteria among the 176 isolates. Two clusters consisted of bacteria from the high loaded filters, and another two clusters consisted of bacteria from the low loaded filters. The fifth cluster represented a mixed population containing members from both loading levels. The results were used to characterize the bacterial populations, and a hypothesis as to how these populations developed in their respective system was proposed. The model could be used for classifying the bacterial isolates into low or high loaded system. Only six isolates from each loading level were misclassified. The model was stable as indicated by the fact that both the numbers of tests and the numbers of isolates entered in the model could be reduced without any significant loss of information.

Aflatoxin formation and the dual phenomenon in Aspergillus flavus link

Trials were performed with three aflatoxin-forming isolates of Aspergillus flavus from formic acid-treated materials containing aflatoxin, one A. flavus strain isolated from mouldy barley kept for two months in an anaerobic jar and one non-toxic A. flavus strain from the culture collection at our Department. The nontoxic strain and one aflatoxin producer were cultured in salts-sugar-asparagine substrate (SLM) for aflatoxin production and in a specially prepared grass substrate (GS). Formic acid and ammonium formate were added to both substrates, and sucrose in a low amount was added to the grass substrate. The aflatoxin-forming isolate segregated on the grass substrate into two different lines, one with high aflatoxin production and one with very low aflatoxin-forming ability, higher growth rate and reduced sporulation, on the SLM substrate. When exposed to sucrose in grass substrate and ammonium formate in SLM, one toxic and one non-toxic strain were provoked to increased aflatoxin formation. The A. flavus isolate from the anaerobic jar also segregated on the grass substrate, and these segregants were more sensitive to a high dose of formic acid. In these A. flavus strains there seems to be a continuous variation between different lines, depending on cultivation conditions. In the two aflatoxin-forming isolates left, such segregation tendencies were not very marked on any substrate.

Occurrence of storage fungi, especially aflatoxin-forming Aspergillus flavus, in soil, greenstuff and prepared hay

Abstract An investigation of the fungal flora in soil and greenstuff from a hayfield was carried out in order to show whether the occurrence of storage fungi, especially A. flavus, was sufficiently high to cause heavy infection of hay. Samples of the hay were field-dried to 63, 76 and 80% dry matter and stored in 150-kg containers in the barn. Three tons of the hay was field-dried to 60 and 70% dry matter and hayloft-dried. Field-dried and hayloft-dried hay were examined after 4 and 4 1 2 months respectively. For comparison, hayrack-dried hay of an excellent quality of another origin was included and examined after 6 months of storage in a barn. Field-dried hay was heavily infected with Aspergillus and Penicillium, and in one case with Rhizopus. Hayloft-dried hay which was judged to be of good quality contained Aspergillus and Penicillium, but to much lesser extent. Hayloft-drying from 60% dry matter gave the best product. The hayrack-dried hay contained no A. flavus, only small amounts of Penicillium, and showed good agreement with the greenstuff fungal population. The greenstuff on the experimental field contained few A. flavus, with low aflatoxin-forming capability. From both field-dried and hayloft-dried hay, A. flavus with variable aflatoxin-forming capacity was frequently isolated.