Development of molecular diagnostic test to quantify Rhizobium leguminosarum bv. viciae (rhizobia group E/F) in soil

Abstract

Aims Rhizobia are essential for nitrogen fixation in legumes. The current method for enumerating rhizobia in soil by the Most Probable Number (MPN) method is costly and time consuming. This paper describes the development of a qPCR test to quantify Rhizobium leguminosarum bv. viciae (Rlv) in soil. Methods TaqMan probe and primers were developed . Specificity of qPCR was confirmed against Rlv and non-target rhizobia and compared to vetch nodulation. Relationship between qPCR results and Rlv numbers was assessed by comparing qPCR of Rlv strains added into sand at known cell density to plate counts on agar. The reliability of the test was assessed on 41 field soils and compared to quantification by MPN on vetch and symbiotic performance of rhizobia in soil extracts was examined on field pea and faba bean. Results The developed test was specific for Rlv, with 42 strains testing positive for both qPCR and nodulation of vetch and 42 other rhizobia negative for both qPCR and nodulation. A dilution series of two Rlv strains inoculated into sand produced a tight correlation (R 2 = 0.92) between qPCR data and plate counts. Assessment of field soils indicated a significant relationship ( P < 0.001, R 2 = 0.85) between qPCR and MPN and between qPCR and nitrogen fixation capacity with field pea (R 2 = 0.82) and faba bean (R 2 = 0.51). Conclusion The qPCR test developed is specific for Rlv strains and shows high correlation with plate counts, MPN population estimates and symbiotic performance. This test is now delivered as a commercial service to growers and researchers as “PREDICTA® rNod”.

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