Heidemarie Thierfelder

Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that nodulate soybeans on the asiatic continent.

ABSTRACT A highly supported maximum-likelihood species phylogeny for the genus Bradyrhizobium was inferred from a supermatrix obtained from the concatenation of partial atpD, recA, glnII, and rpoB sequences corresponding to 33 reference strains and 76 bradyrhizobia isolated from the nodules of Glycine max (soybean) trap plants inoculated with soil samples from Myanmar, India, Nepal, and Vietnam. The power of the multigene approach using multiple strains per species was evaluated in terms of overall tree resolution and phylogenetic congruence, representing a practical and portable option for bacterial molecular systematics. Potential pitfalls of the approach are highlighted. Seventy-five of the isolates could be classified as B. japonicum type Ia (USDA110/USDA122-like), B. liaoningense, B. yuanmingense, or B. elkanii, whereas one represented a novel Bradyrhizobium lineage. Most Nepalese B. japonicum Ia isolates belong to a highly epidemic clone closely related to strain USDA110. Significant phylogenetic evidence against the monophyly of the of B. japonicum I and Ia lineages was found. Analysis of their DNA polymorphisms revealed high population distances, significant genetic differentiation, and contrasting population genetic structures, suggesting that the strains in the Ia lineage are misclassified as B. japonicum. The DNA polymorphism patterns of all species conformed to the expectations of the neutral mutation and population equilibrium models and, excluding the B. japonicum Ia lineage, were consistent with intermediate recombination levels. All species displayed epidemic clones and had broad geographic and environmental distribution ranges, as revealed by mapping climate types and geographic origins of the isolates on the species tree.

Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae, inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C2H4·mg protein-1·h-1 to 400 nmol C2H4 by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O2, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce14CO2 from [U-14C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 μmol in 24 h by 2·1012 cells) were metabolized to14CO2, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O2 in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 μM succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix+ and fix-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a14C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of14C in the medium than two species ofPseudomonas and two species ofArthrobacter.

Malonate and Krebs Cycle Intermediates Utilization in the Presence of other Carbon Sources by Rhizobium japonicum and Soybean Bacteroids

Abstract Free living cells of Rhizobium japonicum 61-A-101 and bacteroids from Glycine max var. Mandarin infected with the same strain utilized malonate with a substrate saturation greater than 10-2 mol/l. At low concentrations of malonate (10-5 mol/1) the free living cells were significantly more active in utilizing malonate than bacteroids. In bacteroids two substrate saturation ranges were found, one between 3 x 10-4mol/l and 10-3 mol/l, the other at more than 10-2 mol/l. Utilization of malonate was not affected by 10 to 100 times larger concentrations of either arabinose or xylose. 10-3 mol/l succinate inhibited the utilization of malonate (10-4 mol/l) completely in bacteroids, and by 90% in free living cells. Succinate utilization (10-4 mol/1) was reduced in those cells exposed to 100 times higher malonate concentration only by 20-30% . Utilization and incorporation of pyruvate and 2-oxoglutarate into bacteroids was also only slightly affected by 100 times larger malonate concentration. Citrate utilization by bacteroids however was reduced by more than 70%. The rate of endoxidation of malonate as sole carbon source (14CO2 production from [2-14C] malonic acid)was about 1.5 μmol · h-1 • mg protein-1 and about half the rate with Pseudomonas putida and 70% o f the rate with Pseudomonas fluorescens under the same conditions (pH 6.0, 28 °C).

Production of Biomass of Arbuscular Mycorrhizal Fungi in the Glass Bead Compartment System

A major obstacle to the biochemical and genetic study of arbuscular mycorrhizal (AM) fungi has been the fact that they cannot be cultivated axenically without their plant host. In most cases, fungal material has to be isolated from pot cultures by procedures that are either laborious or yield biomass of low purity.

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

Abstract Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its KM was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn2+ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities.

The Occurrence of Choline Kinase II in the Cytoplasm of Soybean Root Nodules Infected with Various Strains of Bradyrhizobium japonicum

Summary The soluble nodule-specific protein choline kinase II (CK II) accounted for 37% – 49 % of the total choline kinase activity in those symbioses where stable peribacteroid membranes (PBM) are made. The induction of choline kinase II was independent of whether the bacteria were nif + or fix − . A fix − strain which does not build stable PBM (strain 61-A-24) could not induce the production of CK II. The mutant RH 31-Marburg (fix-) which builds PBM which fuse to give larger vacuoles, induced CK II but to a lesser extent (25 % – 43 % of total activity) than the wild type. Choline kinase II induction for each bacterial strain was the same irrespective of soybean cultivar.

Sustainable Agriculture/Forestry and Biological Nitrogen Fixation

The biological nitrogen cycle is closely linked to the carbon cycle and both are affected by human activities. The pools and annual turnover rates of the global carbon cycle (in Gigatonnes C) reveal that the major pools, the atmosphere (750 Gt), the biomass of land plants (590 Gt), and the usable fossil energy sources (1150 Gt), are all of the same order of magnitude. Significantly larger are the pools of organic soil carbon (1560 Gt), the soil carbonates (1740 Gt), and the carbon in the oceans (38,000 Gt). The lithosphere carbon pool (more than 6 x 10 Gt) is several orders of magnitude larger.