Knockout of an Azorhizobial dTDP-L-Rhamnose Synthase Affects Lipopolysaccharide and Extracellular Polysaccharide Production and Disables Symbiosis with Sesbania rostrata

A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans. oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase. The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity. Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase. The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis. Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata. There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present. Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads. Infection centers emitted a blue autofluorescence under UV light. The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A. caulinodans and S. rostrata.

EFFECTS OF CaCO3 AND INOCULUM LEVEL ON NODULATION AND GROWTH OF ALFALFA IN AN ACID SOIL

The effects of CaCO3 and inoculum level on nodulation and growth of alfalfa (Medicago sativa L.) in an acid soil were studied by measuring alfalfa yield, nodule numbers and nodule weights in relation to various levels of CaCO3 amendment and to the number of Rhizobium meliloti applied per seed. When sufficient CaCO3 was applied to the soil (4 mg CaCO3/g soil) to reduce soluble Al to sub-toxic levels, the relative yield (yield without N/yield with N) of alfalfa increased from 0.7 with no inoculum to 1.0 with 3 × 107 rhizobia per seed. Increasing the inoculum level from no inoculum to 3 × 107 rhizobia per seed decreased the amount of ineffective nodule tissue from 0.65 to 0.27 mg/pot without CaCO3 added, and from 0.60 to 0.06 mg/pot with the addition of 4.0 mg CaCO3/g soil. When no inoculum was applied, the amount of effective nodule tissue was increased from 0 to 0.91 mg/pot by adding 4.0 mg CaCO3/g soil. Application of inoculum increased the amount of effective nodule tissue at all levels of CaCO3. The results demonstrate the importance of liming to decrease the soluble Al content of acid soils for alfalfa production, and show that high inoculum levels can be used to improve alfalfa yields on acid soils.

Nodulation Studies in Legumes III. The Effects of Delaying Inoculation on the Seedling Symbiosis of Barrel Medic, Medicago Tribuloides Desr.

The technique of delayed inoculation with effective, ineffective, or mixtures of effective + ineffective nodule bacteria was used to study seedling symbiosis of barrel medic, M. tribuloides, in sand culture.