Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes.

BACKGROUND AND AIMS: Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N(2)-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N(2)-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N(2)-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N(2)-fixing activity is the main cause for the Sub-optimal phenotype. METHODS: Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity. KEY RESULTS: Effective nodules (Nodule Function 83-100 %) contained four developmental zones and N(2)-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24-57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads. CONCLUSIONS: Three major responses to N(2)-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N(2)-fixation of clovers.

Isolation and characterization of rhizosphere bacteria with potential for biological control of weeds in vineyards.

AIMS: Deleterious rhizosphere inhabiting bacteria (DRB) have potential to suppress plant growth. This project focuses on the isolation of DRB with potential for development as commercial products for weed control. METHODS AND RESULTS: Bacteria were isolated from the rhizosphere, rhizoplane, and endorhizosphere of seedlings and mature plants of wild radish (Raphanus raphanistrum), annual ryegrass (Lolium rigidum) and capeweed (Arctotheca calendula) growing in vineyards in the Swan Valley, Western Australia. A majority (81.5%) of the 442 strains was obtained from either rhizospheres or rhizoplanes. Rapid screening techniques were developed to evaluate in the laboratory and glasshouse the effects of bacteria on plants. Strains were screened in the glasshouse for deleterious effects on annual ryegrass, wild radish, grapevine rootlings (Vitis vinifera) and the legume cover crop subterranean clover (Trifolium subterraneum). Three strains were identified using the Biolog system and 16S rRNA gene sequencing as two strains of Pseudomonas fluorescens (WSM3455 and WSM3456) and one strain of Alcaligenes xylosoxidans (WSM3457). One of the P. fluorescens (WSM3455) strain produced hydrogen cyanide, an inhibitor of plant roots and a broad-spectrum antimicrobial compound. CONCLUSIONS: Three strains specifically inhibited wild radish but had no significant deleterious effects on either grapevine rootlings or subterranean clover. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggested manipulation of the weed seedling rhizosphere using identified DRB as a potential biocontrol agent for wild radish.

Phylogenetic relationships of three bacterial strains isolated from the pasture legume Biserrula pelecinus L.

Three bacterial strains (WSM 1283, WSM 1284, WSM 1497) isolated from root nodules of the pasture legume Biserrula pelecinus L. growing in Morocco, Italy and Greece, respectively, were studied in order to determine their phylogenetic relationship to the other members of the family Rhizobiaceae. A polyphasic approach, which included analyses of morphological and physiological characteristics, plasmid profiles, symbiotic performance and 16S rRNA gene sequencing, indicated that these strains belong to the genus Mesorhizobium.

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli.

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acid-adapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.

Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419.

Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6. Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant. A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants. Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance. This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.

Iron-deficiency specifically limits nodule development in peanut inoculated with Bradyrhizobium sp.

Severely iron-deficient peanuts (Arachis hypogaaea L.) grown on calcareous soils in central Thailand failed to nodulate until given foliar iron applications. Glasshouse experiments were conducted on two cultivars (Tainan 9 and Robut 33-1) to identify which stage of the nodule symbiosis was most sensitive to iron-deficiency. Iron-deficiency did not limit growth of soil or rhizosphere populations of peanut liradyrhizobium. Similar numbers of root nodule initials formed in the roots of both control and iron-sprayed plants, showing that iron-deficiency did not directly affect root infection and nodule initiation. Plants sprayed with iron produced greater numbers of excisable nodules and carried a greater nodule mass than untreated plants. Five days after iron application, nodules on sprayed plants of CV. Tainan 9 contained 200-fold higher bacteroid numbers per unit weight and 14-fold higher concentrations of leghaemoglobain. The onset of nitrogenase activity was also delayed by iron deficiency in both cultivars. Tainan 9 appeared more sensitive to iron-deficiency than Robut 33-1 in terms of nodule mass produced, but both cultivars showed the same effect of iron-deficiency on nitrogenase activity per plant. It is concluded that the failure of the infecting rhizobia to obtain adequate amounts of iron from the plant results in arrested nodule development and a failure of nitrogen fixation.

Effect of rhizobial strain and host plant on nitrogen isotopic fractionation in legumes.

Lotus pedunculatus L., Medicago sativa L., Macroptilium atropurpureum, Glycine max, and Trifolium repens L. were grown in a N-free medium and inoculated with one of ten Rhizobium strains. Dry matter, N content, and delta(15)N values were determined for various plant parts.Nodules, with the exception of those from lucerne, were enriched in (15)N relative to atmospheric N. Considerable variation was found in delta(15)N values of plant herbage (-4.5 to +0.8). The extent of isotopic discrimination was dependent on both the host plant and the infecting rhizobial strain. This further complicates, but does not invalidate, the use of small variations in the natural abundance of (15)N to estimate the contribution of symbiotically fixed N(2) to the N in legume herbage. Some other implications of the observed differences are also discussed.

Effect of inoculation of Zea mays with Azospirillum brasilense strains under temperate conditions.

Seven strains of Azospirillum brasilense were compared for their effect on the growth of Zea mays grown under temperate conditions in sand--vermiculite pot cultures. Inoculation with all seven strains tested, including Fix(-) mutant strains, increased dry weight and total nitrogen content of shoots, but nitrogen concentrations were unaffected. Low and variable rates of acetylene reduction activity were observed from excised roots of inoculated plants without preincubation. Estimates of N2-fixing A. brasilense associated with inoculated roots showed differences between strains in establishing themselves in the rhizosphere and endorhizosphere. In some strains enrichment in the endorhizosphere of roots occurred following inoculation, but the relative numbers and location of the strains did not appear to affect the yield response.