title: Assessing the suitability of antibiotic resistance markers and the indirect ELISA technique for studying the competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions in South Africa.
abstract: 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.
  
  
  RESULTS
  The test strains formed two distinct groups with regard to their intrinsic resistance to the antibiotics streptomycin sulphate and spectinomycin dihydrochloride pentahydrate, making it impossible to use intrinsic antibiotic resistance to distinguish strains from within the same intrinsic resistance group. The use of strains marked with double antibiotic resistance was also investigated. A number of these strains lost their antibiotic marker tags after one plant passage; and some also lost their competitive ability. The indirect ELISA technique provided a more satisfactory method of identifying selected Cyclopia strains under both field and glasshouse conditions. The primary antibodies raised against strains UCT40a, UCT61a and UCT44b gave absorbance readings that were unambiguously negative (0.30 OD405), while those of strain PPRICI3 were ambiguous (0.50 OD405) with many false positive readings (1.0 A405). The indirect ELISA method showed a high level of analytical sensitivity in glasshouse experiments and there were no cross-reactions between the four test strains. The method was also suitable for detecting three of the four test strains in competition studies under field conditions, and can also be used to identify some strains under field conditions.
  
  
  CONCLUSIONS
  The antibiotic marker method was found unsuitable for identifying Cyclopia rhizobia in competition experiments in both glasshouse and field conditions. However, the indirect ELISA technique was found suitable for identifying these strains in glasshouse studies. The method was also appropriate for identifying strains UCT40a, UCT44b and UCT61a, but not strain PPRICI3, in field competition studies.
authors.affiliation: Chemistry Department, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa. amy.spriggs@sanbi.org
authors.forename: Amy C
authors.initials: AC
authors.lastname: Spriggs
authors.forename: Felix D
authors.initials: FD
authors.lastname: Dakora
mesh_terms: D053998:Cyclopia Plant / Q000382:microbiology*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D009586:Nitrogen Fixation; D055170:Plant Root Nodulation; D012231:Rhizobium / Q000235:genetics*; D053204:Root Nodules, Plant / Q000382:microbiology; D012680:Sensitivity and Specificity; D013019:South Africa