Background: Symbiotic N2 fixation in legumes is constrained by many factors, including the paucity of suitable soil rhizobia To maximise growth of legume species therefore often requires the application of effective rhizobia as inoculants. But where native strains out-compete introduced rhizobia for nodule formation, it is important that the competitiveness of selected strains is tested in the field and glasshouse prior to their recommendation as commercial inoculants. However the methodology for strain identification inside nodules has often proved difficult and thus limited this field of research. In this study, the suitability of the antibiotic resistance technique (both intrinsic low-resistance fingerprinting and high-resistance marking) and the serological indirect ELISA method were assessed for their ability to detect selected Cyclopia rhizobia under glasshouse and field conditions. The four rhizobial strains that were used, namely PPRICI3, UCT40a, UCT44b and UCT61a, were isolated from wild Cyclopia species growing in the Western Cape fynbos of South Africa.

Nitrogen (N) derived from symbiotic fixation of atmospheric N(2) in wild and cultivated populations of Cyclopia, a woody endemic genus used to make honeybush tea in the Western Cape of South Africa, was quantified by the (15)N natural abundance method. Because Cyclopia species are naturally mycorrhizal, non-N(2)-fixing arbuscular mycorrhizal shrubs of similar phenology to Cyclopia were chosen as reference plants to provide the delta(15)N value of soil-derived N. Isotopic analysis showed that wild populations of Cyclopia were highly dependent on N(2) fixation for their N nutrition, ranging from 70 +/- 4% to 100 +/- 7% (mean +/- SE) at all sites, except for one. Further evidence of the high dependency of wild Cyclopia populations on symbiotic N was provided by their significantly higher foliar N concentrations compared with the non-legume reference plants. However, cultivated Cyclopia exhibited variable amounts of N(2) fixation, with Cyclopia genistoides (L.) R. Br., for example, showing low amounts of N(2) fixation at Sites P2 and P3 (0 +/- 51% and 8 +/- 46%, respectively) as a result of low D values (D is defined as the difference between the mean delta(15)N value of the reference plants and the B value of the test Cyclopia species, where B is the delta(15)N of an inoculated test legume grown in an N-free growth medium), whereas at Sites P1, P2, P5 and P6, up to 89 +/- 2%, 94 +/- 13%, 85 +/- 13% and 100 +/- 18%, respectively, plant N was derived from atmospheric fixation. The high symbiotic N nutrition observed for wild populations of Cyclopia suggests that these populations are major contributors to the N economy of the nutrient-poor soils of the South African fynbos. These data indicate that by breeding for high N(2) fixation rates in Cyclopia cultivars and selecting more efficient rhizobial strains, this legume has the potential to achieve higher N(2) fixation rates under cultivation. The low variability in Cyclopia delta(15)N values within sites, however, suggests that genetic variability is not a major factor influencing N(2) fixation rates in cultivated Cyclopia, and that more benefit may be gained from soil amelioration and the selection of improved rhizobial strains.

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Three newly selected strains of Cyclopia rhizobia together with an inoculant strain, which has never been tested in the field with adequate experimental design, was assessed under both nursery and field conditions for symbiotic performance. The three new test strains were initially selected for their superior Nz-fixing abilities under glasshouse conditions, and then evaluated in this study for field performance. Cyclopia subternata Vogel and Cyclopia genistoides (L.) R. Br., which have the potential for producing high quality honeybush tea, were used as host plants in both the nursery and field studies.The effect of seedling inoculation at the nursery level was also examined for the four test strains under nursery conditions. The inoculation of cuttings under nursery conditions produced.significant increases in shoot biomass, shoot %N and shoot N content. More specifically, inoculating C. subternata with strains UCT44b and UCT61a significantly increased shoot biomass and N content relative to strain PPRICI3. Strains UCT44b and UCT61a also showed better nodulation with C. subternata cuttings compared to strains UCHOa and PPRICI3. Field inoculation of Cyclopia increased all growth parameters relative to the uninoculated control, except for leaf %N. Cyclopia subternata inoculated with strains UCT44b, UCT40a and UCT61a produced significantly lower b 15 N values than the uninoculated C. subternata reference plant. Using the 15N natural abundance method, C. subternata was estimated to be gaining about half of its N from N z fixation, while C. genistoides obtained less than half of its N from symbioticnutrition.

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