Rapid Identification of Rhizobia Nodulating Soybean by a High-Resolution Melting Analysis

Abstract

Soybean [Glycine max (L.) Merr.] is one of the most important and oldest crops. Due to its ability to form symbiotic interactions with nitrogen-fixing bacteria, it is a valuable source of nitrogen for agriculture and proteins for humans and livestock. In Europe, for instance, in Poland, the soybean cultivation area is still not large but is gradually increasing due to climate change. The lack of indigenous soybean microsymbionts in Polish soils forces the application of commercial strains to establish effective symbioses. Fast and reliable identification methods are needed to study the persistence, competitiveness, and dispersal of bradyrhizobia introduced as inocula. Our study aimed to apply real-time PCR coupled with high-resolution melting curve (HRM) analysis to detect and differentiate bacterial strains occupying soybean nodules. HRM-PCR was performed on crude extracts from nodules using primers specific for recA, a highly conserved nonsymbiotic gene. By comparing them with the reference strains, we were able to identify and assign Bradyrhiobium strains that had been introduced into field locations in Poland. In conclusion, HRM analysis was proven to be a fast and accurate method for identifying soybean microsymbionts and might be successfully used for identifying other legume-nodulating bacteria.

Keywords

• high-resolution melting PCR (HRM-PCR)
• <i>Glycine max</i>
• soybean
• soybean-nodulating bradyrhizobia (SNB)
• <i>Bradyrhizobium japonicum</i>
• <i>Bradyrhizobium diazoefficiens</i>