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.
Conclusion: 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:tut/oai:encore.tut.ac.za:d1001665 |
| Date | 20 July 2009 |
| Creators | Spriggs, AC, Dakora, FD |
| Publisher | BMC Microbiology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | |
| Rights | BMC Microbiology |
| Relation | BioMed Central |
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